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Characterization of Two Novel Aldo-Keto Reductases from Arabidopsis: Expression Patterns, Broad Substrate Specificity, and an Open Active-Site Structure Suggest a Role in Toxicant Metabolism Following Stress
Abstract
Aldo-keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions. In this study, we have screened the Arabidopsis thaliana genomic sequence for genes with significant homology to members of the mammalian AKR1 family and identified four homologues for further study. Following alignment of the predicted protein sequences with representatives from the AKR superfamily, the proteins were ascribed not to the AKR1 family but to the AKR4C subfamily, with the individual designations of AKR4C8, AKR4C9, AKR4C10, and AKR4C11. Expression of two of the genes, AKR4C8 and AKR4C9, has been shown to be coordinately regulated and markedly induced by various forms of stress. The genes have been overexpressed in bacteria, and recombinant proteins have been purified and crystallized. Both enzymes display NADPH-dependent reduction of carbonyl compounds, typical of the superfamily, but will accept a very wide range of substrates, reducing a range of steroids, sugars, and aliphatic and aromatic aldehydes/ketones, although there are distinct differences between the two enzymes. We have obtained high-resolution crystal structures of AKR4C8 (1.4 A) and AKR4C9 (1.25 A) in ternary complexes with NADP(+) and acetate. Three extended loops, present in all AKRs and responsible for defining the cofactor- and substrate-binding sites, are shorter in the 4C subfamily compared to other AKRs. Consequently, the crystal structures reveal open and accommodative substrate-binding sites, which correlates with their broad substrate specificity. It is suggested that the primary role of these enzymes may be to detoxify a range of toxic aldehydes and ketones produced during stress, although the precise nature of the principal natural substrates remains to be determined.
... Among these members, AKR4C8 is potentially involved in brassinosteroid metabolism (Simpson et al., 2009). Under stress conditions, both AKR4C8 and AKR4C9 transcripts show significant elevation (Simpson et al., 2009). ...
... Among these members, AKR4C8 is potentially involved in brassinosteroid metabolism (Simpson et al., 2009). Under stress conditions, both AKR4C8 and AKR4C9 transcripts show significant elevation (Simpson et al., 2009). In our dataset, AKR4C9 abundance is increased in both ftshi3-1 and pFtsHi3-OE lines. ...
... In our dataset, AKR4C9 abundance is increased in both ftshi3-1 and pFtsHi3-OE lines. In watered conditions, the WT exhibits reduced AKR4C9 abundance, while under drought, the enzyme's abundance is somewhat elevated, consistent with the drought-responsive nature of AKR4C9 transcripts (Simpson et al., 2009). Although the exact role it plays in conferring drought resistance remains unclear, AtFtsHi3 might have some implications for the downstream effect of AKR4C9. ...
With climate change, droughts are expected to be more frequent and severe,
severely impacting plant biomass and quality. Here, we show that overexpres�sing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phe�notypes without compromising plant growth. AtFtsHi3 encodes a chloroplast
envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be
drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression
therefore leads to drought tolerance, while only diminished expression of this
gene leads to growth retardation. To understand the underlying mechanisms of
the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and
pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and
drought conditions. Drought-related processes like osmotic stress, water trans�port, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1
mutants following their enhanced drought response compared to wild-type. The
knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93,
and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chlo�roplast pre-protein import. Mathematical modeling was performed to understand
how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead
to drought tolerance in both overexpression and knock-down lines, yet, on the
other hand, affect plant growth so differently. The results led us to hypothesize
that AtFtsHi3 may form complexes with at least two other protease subunits,
either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9,
AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation
mechanism for these proteases in the hexamer.
... AKR family members are widely distributed throughout vertebrates, yeasts, bacteria, and plants. They have numerous functions in the metabolism of steroids, sugars and other carbonyls, in addition to diverse stress responses, including osmotic, drought and heat stress in plants (Sree et al., 2000;Gavidia et al., 2002;Simpson et al., 2009;Saito et al., 2011;Éva et al., 2014;Pan et al., 2019;Songsiriritthigul et al., 2020). Typically, AKRs catalyze NADPH/NADP + -dependent reduction of aldehydes and ketones to their corresponding alcohols. ...
... Additionally, AKR4C14 from rice is highly efficient in metabolizing MG and malondialdehyde (Songsiriritthigul et al., 2020). In Arabidopsis, both AKR4C8 and AKR4C9 can reduce a range of aliphatic aldehydes, and AKR4C9 exhibits a preference for MG and glyceraldehyde (Simpson et al., 2009). AKR4C9-11 can also catalyze the reduction of DHAP, GAP, Glu and Fru (Saito et al., 2013), and Fru can be converted to sorbitol by AKR4C9 (Éva, et al., 2014). ...
... Sl-AKR9 Hap2 clusters with four Arabidopsis AKRs (AKR8-11) and a Digitalis AKR4C6 with similarities of 74.52%, 82.80%, 73.57%, 67.52%, and 68.79%, respectively (Fig. S6). All four Arabidopsis AKRs can catalyze reduction of various aldehydes and sugar-derived RCs (Simpson et al., 2009;Saito et al., 2013;Éva, et al., 2014). AKR4C9/10 can reduce multiple sugars, although the apparent Km values are high. ...
The unique flavors of different fruits depend upon complex blends of soluble sugars, organic acids and volatile organic compounds (VOCs). 2-Phenylethanol and phenylacetaldehyde are major contributors to flavor in many foods, including tomato. In the tomato fruit, glucose and fructose are the chemicals that most positively contribute to human flavor preferences. We identified a gene encoding a tomato aldo/keto reductase, Sl-AKR9, that is associated with phenylacetaldehyde and 2-phenylethanol contents in fruits. Two distinct haplotypes were identified; one encodes a chloroplast-targeted protein while the other encodes a transit peptide-less protein that accumulates in the cytoplasm. Sl-AKR9 effectively catalyzes reduction of phenylacetaldehyde to 2-phenylethanol. The enzyme can also metabolize sugar-derived reactive carbonyls, including glyceraldehyde and methylglyoxal. CRISPR-Cas9-induced loss-of-function mutations in Sl-AKR9 significantly increased phenylacetaldehyde and lowered 2-phenylethanol content in ripe fruit. Reduced fruit weight and increased soluble solids, glucose and fructose contents were observed in the loss-of-function fruits. These results reveal a previously unidentified
... Notably, the four AKR4C proteins are the most welldescribed plant AKRs with respect to their structure and in vitro oxidoreductase activity on a variety of substrates, though not including GSNO or SNO-CoA (Simpson et al., 2009;Saito et al., 2013). Their high degree of sequence identity is illustrated in Figure 3B, and atomic structures are available for both A. thaliana AKR4C8 ( Figure 3C) and AKR4C9 (Simpson et al., 2009). ...
... Notably, the four AKR4C proteins are the most welldescribed plant AKRs with respect to their structure and in vitro oxidoreductase activity on a variety of substrates, though not including GSNO or SNO-CoA (Simpson et al., 2009;Saito et al., 2013). Their high degree of sequence identity is illustrated in Figure 3B, and atomic structures are available for both A. thaliana AKR4C8 ( Figure 3C) and AKR4C9 (Simpson et al., 2009). They share the native fold that is typical of the AKR superfamily, consisting of a (α/β) 8barrel (TIM barrel) with two additional α-helices on the periphery of the enzyme (Jez et al., 1997;Simpson et al., 2009). ...
... Their high degree of sequence identity is illustrated in Figure 3B, and atomic structures are available for both A. thaliana AKR4C8 ( Figure 3C) and AKR4C9 (Simpson et al., 2009). They share the native fold that is typical of the AKR superfamily, consisting of a (α/β) 8barrel (TIM barrel) with two additional α-helices on the periphery of the enzyme (Jez et al., 1997;Simpson et al., 2009). Three flexible, surface loops (A, B, and C, Figures 3B,C) are involved in defining the specificity of these enzymes toward the substrate, and the flexibility of these loops appears to allow interaction with multiple substrates (Simpson et al., 2009). ...
Nitric oxide (NO) is a short‐lived gas that acts as a signaling molecule in all higher organisms, including plants. Despite the involvement of NO in multiple plant processes, including germination, root growth and fertility, a basic understanding of the mechanisms by which NO exerts its effects is lacking. NO and its derivatives impact these physiological processes through reversible S‐nitrosation of critical protein cysteines. In cells, regulation of NO‐levels is predominantly achieved by reaction of reactive nitrogen species (RNS) with glutathione (GSH), thereby forming S‐nitrosoglutathione (GSNO), a principal NO reservoir. Mutation of Arabidopsis thaliana S‐nitrosoglutathione reductase (GSNOR) leads to higher intracellular concentrations of S‐nitrosothiols, confirming that the GSNOR reduction of GSNO is a major route of GSNO catabolism in plants and other eukaryotes ¹ . We demonstrate in Arabidopsis that absence of GSNOR results in differential regulation of proteins involved in chlorophyll metabolism, the general stress response and photosynthesis ² . In addition, our proteomic analysis identified a significant increase in proteins that belong to the aldo‐keto reductase (AKR) protein superfamily, AKR4C8 and 9. Since specific AKRs have been linked to NO metabolism in mammals, we expressed and purified Arabidopsis AKR4C8 and 9 and close homologues AKR4C10 and 11 and determined that they have NADPH‐dependent activity in GSNO and S‐nitroso‐coenzyme A (SNO‐CoA) reduction. Plants lacking GSNOR also show increased activity of NADPH‐dependent GSNO reduction, consistent with increased AKR activity ² . To address the in vivo role of these AKRs, CRISPR/Cas9‐mediated mutant lines will be generated, and their capacity to metabolize RNS assessed. In addition, by subjecting AKR mutant plants to different abiotic and nitrosative stresses, results will uncover the importance of AKR proteins in NO homeostasis during both optimal growth and stress conditions. Taken together, these data define a new, NADPH‐dependent component of NO metabolism that may be integrated with NADH‐dependent GSNOR activity to control NO homeostasis in plants and other organisms.
1. Lee, U., Wie, C., Fernandez, B. O., Feelisch, M. & Vierling, E. Modulation of nitrosative stress by S‐nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis. Plant Cell 20 , 786–802 (2008).
2. Treffon, P., Rossi, J., Gabellini, G., Trost, P., Zaffagnini, M., Vierling, E. Quantitative proteome profiling of a S‐nitrosoglutathione reductase (GSNOR) null mutant reveals a new class of enzymes involved in nitric oxide homeostasis in plants. Accepted. Front. Plant Sci . (2021).
... Notably, the four AKR4C proteins are the most welldescribed plant AKRs with respect to their structure and in vitro oxidoreductase activity on a variety of substrates, though not including GSNO or SNO-CoA (Simpson et al., 2009;Saito et al., 2013). Their high degree of sequence identity is illustrated in Figure 3B, and atomic structures are available for both A. thaliana AKR4C8 ( Figure 3C) and AKR4C9 (Simpson et al., 2009). ...
... Notably, the four AKR4C proteins are the most welldescribed plant AKRs with respect to their structure and in vitro oxidoreductase activity on a variety of substrates, though not including GSNO or SNO-CoA (Simpson et al., 2009;Saito et al., 2013). Their high degree of sequence identity is illustrated in Figure 3B, and atomic structures are available for both A. thaliana AKR4C8 ( Figure 3C) and AKR4C9 (Simpson et al., 2009). They share the native fold that is typical of the AKR superfamily, consisting of a (α/β) 8barrel (TIM barrel) with two additional α-helices on the periphery of the enzyme (Jez et al., 1997;Simpson et al., 2009). ...
... Their high degree of sequence identity is illustrated in Figure 3B, and atomic structures are available for both A. thaliana AKR4C8 ( Figure 3C) and AKR4C9 (Simpson et al., 2009). They share the native fold that is typical of the AKR superfamily, consisting of a (α/β) 8barrel (TIM barrel) with two additional α-helices on the periphery of the enzyme (Jez et al., 1997;Simpson et al., 2009). Three flexible, surface loops (A, B, and C, Figures 3B,C) are involved in defining the specificity of these enzymes toward the substrate, and the flexibility of these loops appears to allow interaction with multiple substrates (Simpson et al., 2009). ...
Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form S-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme S-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an Arabidopsis thaliana GSNOR null mutant (hot5-2/gsnor1-3). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in hot5-2/gsnor1-3 plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Because specific AKRs have been linked to NO metabolism in mammals, we expressed and purified A. thaliana AKR4C8 and 9 and close homologs AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Further, we found an increase of NADPH-dependent GSNO reduction activity in hot5-2/gsnor1-3 mutant plants. These data uncover a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants.
... The AKR superfamily is widespread in a variety of organisms, and encompasses 190 annotated proteins divided into 16 families by sequence identity [23,24]. Each AKR enzyme has three large loops on the surface of the α/β-barrel; moreover, owing to their high flexibility and low conservation, AKR enzymes have broad substrate specificity, ranging from the glucose, glucocorticoids and small carbonyl compounds to glutathione conjugates and phospholipid aldehydes, et al. [25,26]. For instance, AKR1D family members catalyze the Δ 4 -3-ketosteroids double-bond reduction to form 5β-dihydrosteroids [27][28][29]. ...
... Moreover, the Kcat/Km ration was calculated for evaluating the catalytic efficiency and the result was similar to Km result, in which GmAKR4 had the highest activity among all AKRs, and OsAKR4-1 and OsAKR4-2 in OsAKRs were the most efficient OsAKR for metabolizing benzaldehyde ( Figure 3C and S3). Unlike benzaldehyde to be hydrolyzed, glyphosate is typically oxidized by AKR proteins, with a similar degradation mechanism to that of 5α-dihydro-testosterone and xylitol [25,31,40]. This reaction induced the conversion of NADP + to NADPH, leading to an increase in the A340nm value. ...
Genes that participate in the degradation or isolation of glyphosate in plants are promising, for they endow crops with herbicide tolerance with a low glyphosate residue. Recently, the aldo-keto reductase (AKR4) gene in Echinochloa colona (EcAKR4) was identified as a naturally evolved glyphosate-metabolism enzyme. Here, we compared the glyphosate-degradation ability of theAKR4 proteins from maize, soybean and rice, which belong to a clade containing EcAKR4 in the phylogenetic tree, by incubation of glyphosate with AKR proteins both in vivo and in vitro. The results indicated that, except for OsALR1, the other proteins were characterized as glyphosate-metabolism enzymes, with ZmAKR4 ranked the highest activity, and OsAKR4-1 and OsAKR4-2 exhibiting the highest activity among the AKR4 family in rice. Moreover, OsAKR4-1 was confirmed to endow glyphosate-tolerance at the plant level. Our study provides information on the mechanism underlying the glyphosate-degradation ability of AKR proteins in crops, which enables the development of glyphosate-resistant crops with a low glyphosate residue, mediated by AKRs.
... Moreover, AKRs function in primary and secondary metabolic pathways during the growth and development of the plant. With the continuous study of functions, increasing plant AKR families, such as A. thaliana [10], rice [11], H. vulgare [12], and M. truncatula [13], have been identified and excavated. However, the genome-wide analysis of the AKR gene family has not been performed in tomatoes yet. ...
... Plant AKRs play multiple roles in abiotic and biotic stress defenses, including ozone, drought, salinity, hypoxia, and P. syringae inoculation [18,[27][28][29]. MsALR, AKR4C8, and AKR4C9 are reported to be highly induced under abiotic stress [10,30] because AKR mediates stress tolerance by detoxification and ROS clearance in cell-damaging reactions [25]. Moreover, the overexpression of barley AKR4C9 enhances their tolerance in response to freezing, oxidative, and cadmium stresses [8,31]. ...
Tomato is one of the most popular and nutritious vegetables worldwide, but their production and quality are threatened by various stresses in the environment in which they are grown. Thus, the resistance and tolerance of tomatoes to various biotic and abiotic stresses should be improved. Aldo-keto reductases (AKR) are a superfamily of NAD(P)(H)-dependent oxidoreductases that play multiple roles in abiotic and biotic stress defenses by detoxification and reactive oxygen species (ROS) clearance pathways. Here, 28 identified AKR family genes of tomatoes were identified genome-wide, and their characteristics, including chromosomal location, gene structures, protein motifs, and system evolution, were analyzed. Furthermore, the phylogenetic and syntenic relationships in Arabidopsis thaliana, rice, and tomatoes were compared. Expression patterns at different tissues and in response to abiotic stresses, such as drought and salt, were monitored to further explore the function of SlAKRs. Finally, three SlAKRs candidate genes were silenced by Virus induced gene silencing (VIGS) systems in Solanum lycopersicum, showing sensitivity to drought and salt stresses with low contents of proline (Pro) and peroxidase (POD) and high content of malonaldehyde (MDA). This study provides the characteristics and potential functions of SlAKRs in response to abiotic stresses that will be helpful for further studies in S. lycopersicum.
... In this study, we further investigate the function of FaAKR23. To analyze the evolutionary relationship among these reported AKR members in Arabidopsis thaliana, Oryza sativa L., Prunus persica, and Fragaria × ananassa [32,33,[38][39][40], a maximum likelihood (ML) tree was constructed using MEGA 7. The results showed that all AKRs were divided into five subgroups, and FaAKR23, FaAKR24, AtA6PR1, and AtA6PR2 were located in the same group, implying a partly similar function among them ( Figure 1A). Furthermore, the protein sequence alignment of AtA6PR1, AtA6PR2, FaAKR23, and FaAKR24 suggest that these four proteins are harboring the conserved motif of the Aldo-ket red domain ( Figure 1B); another study has indicated that AtA6PR1 and AtA6PR2 are cytosolically localized and respond differentially to cold and salt stresses [40]. ...
... The AKR superfamily is a large enzyme group of oxidoreductases that mainly use NADP(H) as a cofactor; this group is widely distributed in animals, plants, yeast, and bacteria and regulates cell oxidation reduction under multiple stress conditions. Although AKRs play numerous roles in the metabolism of steroids, sugars, and other carbonyls, many AKR superfamily members remain uncharacterized [39]. ...
Strawberry (Fragaria × ananassa) fruits are rich in ascorbic acid (AsA) and anthocyanin, which are essential antioxidants for human health. However, the underlying regulatory mechanism of these antioxidant accumulation, especially AsA accumulation in strawberry fruits, remains largely unknown. In this study, we identified FaAKR23 was a regulator of AsA and anthocyanin accumulation. We transiently expressed FaAKR23 in strawberry fruits and conducted metabolic and molecular analyses to explore the role of FaAKR23 in AsA and anthocyanin accumulation. Transient silencing of FaAKR23 (FaAKR23-RNAi) in strawberry fruits significantly decreased the AsA and anthocyanin contents compared with control (empty vector-RNAi, EV-RNAi). Correspondingly, expression of some structural genes and regulatory factors involved in these two antioxidants’ accumulation was dramatically repressed. In addition, transcriptome analysis of EV-RNAi and FaAKR23-RNAi fruits suggested that FaAKR23 was also involved in starch and sucrose metabolism as well as plant–pathogen interaction. Overall, these results not only provide the coordinated regulatory function of FaAKR23 on AsA and anthocyanin accumulation but also offer a promising candidate gene for strawberry breeding with high antioxidants.
... AKRs comprise a superfamily of generally monomeric 34-37 kDa oxidoreductases that share a common (α/β)8-barrel structural motif and act to decompose a broad range of reactive carbonyl substrates produced during stress [134][135][136]. The substrate specificity is determined by three structural loops and a conserved catalytic tetrad consisting of Asp, Tyr, Lys and His, which is essential for the enzymatic activity of these proteins [137]. In contrast to GSNOR, which is NADH-dependent, the GSNO and S-nitrosated coenzyme A (SNO-CoA) reduction activity of AKR1A1 is dependent on NADPH as cofactor [135,138]. ...
... These data support the hypothesis that these AKR4C proteins are additional components regulating the NO homeostasis in plants. However, it should be noted the AKR4C proteins have also been characterized as having significant activity with other substrates [137,141]. Defining the most significant in vivo substrates of these diverse and typically promiscuous enzymes requires further analysis, and whether other AKRs participate in NO homeostasis through reduction of GSNO is an open question. The catalytic tetrad is mostly conserved among all Arabidopsis AKRs ( Figure 6), with the exception of the ALKR group, where the positively charged Lys residue is exchanged with a negatively charged Glu. ...
Protein cysteines (Cys) undergo a multitude of different reactive oxygen species (ROS), reactive sulfur species (RSS), and/or reactive nitrogen species (RNS)-derived modifications. S-nitrosation (also referred to as nitrosylation), the addition of a nitric oxide (NO) group to reactive Cys thiols, can alter protein stability and activity and can result in changes of protein subcellular localization. Although it is clear that this nitrosative posttranslational modification (PTM) regulates multiple signal transduction pathways in plants, the enzymatic systems that catalyze the reverse S-denitrosation reaction are poorly understood. This review provides an overview of the biochemistry and regulation of nitro-oxidative modifications of protein Cys residues with a focus on NO production and S-nitrosation. In addition, the importance and recent advances in defining enzymatic systems proposed to be involved in regulating S-denitrosation are addressed, specifically cytosolic thioredoxins (TRX) and the newly identified aldo-keto reductases (AKR).
... Most plant AKRs previously studied belong to the AKR4C subfamily (Simpson et al. 2009), while all structures of plant AKRs solved so far are monomeric (Bomati et al. 2005, Olsen et al. 2008, Simpson et al. 2009, Giuseppe et al. 2016, Songsiriritthigul et al. 2020. Hence, there is a lack of structural information regarding multimeric plant AKRs involved in synthesizing sugar alcohols. ...
... Most plant AKRs previously studied belong to the AKR4C subfamily (Simpson et al. 2009), while all structures of plant AKRs solved so far are monomeric (Bomati et al. 2005, Olsen et al. 2008, Simpson et al. 2009, Giuseppe et al. 2016, Songsiriritthigul et al. 2020. Hence, there is a lack of structural information regarding multimeric plant AKRs involved in synthesizing sugar alcohols. ...
Sugar-alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases, celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo-form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the aldo/keto reductase superfamily. Analysis of the Man6PRase holo-form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring from NADP+, where the hydride transfer to the sugar-phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar-phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose 6-phosphate but higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar-alcohols in plants.
... One of the enzyme groups that can potentially catalyze such reduction reactions in xenobiotics are the aldo-keto reductase (AKR) enzymes (Jin & Penning, 2007;Penning, 2015). The AKR superfamily of enzymes are classified as oxidoreductase enzymes that are widely distributed in prokaryotes and eukaryotes (Barski et al., 2008;Simpson et al., 2009) New Phytologist catalyze NAD(P)(H)-dependent reduction of aldehydes and ketones to primary and secondary alcohols, respectively, under both normal or stress conditions. AKRs are encoded by stressregulated genes with broad substrate specificity (Jin & Penning, 2007) and play a central role in cellular responses to osmotic, electrophilic and oxidative stress. ...
... Although AKRs in plants have not been widely explored compared to other Phase I metabolic enzymes, several studies on plant AKRs have been reported. However, since they are known Phase I metabolism enzymes for xenobiotics (Jin & Penning, 2007;Barski et al., 2008;Simpson et al., 2009;Penning, 2015), AKRs are candidate enzymes for SA3 reduction to M424 (Fig. 7). To date, the only case of herbicide detoxification associated with AKR activity in resistant weeds involves glyphosate (Nisarga et al., 2017;Vemanna et al., 2017;Pan et al., 2019;McElroy & Hall, 2020;Ramu et al., 2020). ...
Metabolic resistance to 4‐hydroxyphenylpyruvate dioxygenase (HPPD)‐inhibiting herbicides is a threat in controlling waterhemp (Amaranthus tuberculatus) in the USA. We investigated resistance mechanisms to syncarpic acid‐3 (SA3), a nonselective, noncommercial HPPD‐inhibiting herbicide metabolically robust to Phase I oxidation, in multiple‐herbicide‐resistant (MHR) waterhemp populations (SIR and NEB) and HPPD inhibitor‐sensitive populations (ACR and SEN).
Dose–response experiments with SA3 provided ED50‐based resistant : sensitive ratios of at least 18‐fold. Metabolism experiments quantifying parent SA3 remaining in excised leaves during a time course indicated MHR populations displayed faster rates of SA3 metabolism compared to HPPD inhibitor‐sensitive populations.
SA3 metabolites were identified via LC‐MS‐based untargeted metabolomics in whole plants. A Phase I metabolite, likely generated by cytochrome P450‐mediated alkyl hydroxylation, was detected but was not associated with resistance. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,β‐unsaturated carbonyl resembling a Michael acceptor site. A Phase II glutathione–SA3 conjugate was associated with resistance.
Our results revealed a novel reduction–dehydration–GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD‐inhibiting herbicides. This previously uncharacterized detoxification mechanism presents a unique opportunity for future biorational design by blocking known sites of herbicide metabolism in weeds.
... Among the 10 putative metabolites, 6 were annotated as putative ketones. In Arabidopsis, toxic ketones have been associated with stress responses and have been identified as constituents of cuticular wax [53][54][55]. In grapevines, aldehydes, ketones, and alcohols have been found in leaves and their cuticular wax [56,57]. ...
Background
Priming plants with natural products is extensively studied in the agricultural field to reduce the use of synthetic and copper-based pesticides. Previous studies have shown that Oregano essential oil vapour (OEOV) is an effective priming agent against downy mildew (DM) in grapevine (Vitis vinifera L. cv. Chasselas), activating different transcriptomic regulated defence mechanisms.
Results
In the present study, we complement transcriptomic data with metabolomic insights, confirming some previous regulating patterns and highlighting new mechanisms underlying OEOV-induced resistance. A significant modulation of the phenylpropanoid pathway was noted. The data also confirmed the induction of an oxidative stress response indicated by an up-regulation of reactive oxygen species (ROS)-related genes and a congruent depletion of putative L-glutathione. Interestingly, OEOV promoted the accumulation of organic metabolites such as terpenes and other potential phytoalexins, which could potentially contribute to grapevine innate immune response to Plasmopara viticola.
Conclusion
Overall, this study uncovered a diverse influence of OEOV on V. vinifera defence mechanisms against DM, enhancing our comprehension of the mode of action of essential oils. This insight offers various prospects for crafting innovative biocontrol products, fostering a more dynamic and sustainable approach to agriculture.
... The SEVEN TRANSMEMBRANE MLO family is a large family of membrane proteins of which members have been shown to confer broad resistance to fungal pathogens (Büschges et al., 1997). The NAD(P) OXIDOREDUCTASE is orthologous to the Arabidopsis AKR4C9, a nuclear-encoded, plastid-localized aldo-keto reductase with broad substrate specificity that is responsive to abiotic stresses including cold, salt, and dehydration (Simpson et al., 2009). T A B L E 2 "Strictly conserved" circular RNA (circRNA) present in all samples (19 from Figure S4A). ...
Circular RNAs (circRNAs) are closed‐loop RNAs forming a covalent bond between their 3′ and 5′ ends, the back splice junction (BSJ), rendering them resistant to exonucleases and thus more stable compared to linear RNAs. Identification of circRNAs and distinction from their cognate linear RNA is only possible by sequencing the BSJ that is unique to the circRNA. CircRNAs are involved in the regulation of their cognate RNAs by increasing transcription rates, RNA stability, and alternative splicing. We have identified circRNAs from C. sativa that are associated with the regulation of germination, light response, and lipid metabolism. We sequenced light‐grown and etiolated seedlings after 5 or 7 days post‐germination and identified a total of 3447 circRNAs from 2763 genes. Most circRNAs originate from a single homeolog of the three subgenomes from allohexaploid camelina and correlate with higher ratios of alternative splicing of their cognate genes. A network analysis shows the interactions of select miRNA:circRNA:mRNAs for regulation of transcript stabilities where circRNA can act as a competing endogenous RNA. Several key lipid metabolism genes can generate circRNA, and we confirmed the presence of KASII circRNA as a true circRNA. CircRNA in camelina can be a novel target for breeding and engineering efforts.
... The Seven transmembrane MLO family is a large family of membrane proteins of which members have been shown to confer broad resistance to fungal pathogens (Büschges et al., 1997). The NAD(P) oxidoreductase is orthologous to the Arabidopsis AKR4C9, a nuclear encoded, plastid-localized aldo-keto reductase with broad substrate specificity that is responsive to abiotic stresses including cold, salt and dehydration (Simpson et al., 2009). ...
Circular RNAs (circRNAs) are closed-loop RNAs forming a covalent bond between their 3’ and 5’ ends, the backsplice junction (BSJ), rendering them resistant to exonucleases and thus more stable compared to linear RNAs. Identification of circRNAs and distinction from its cognate linear RNA is only possible by sequencing the BSJ that is unique to the circRNA. CircRNAs are involved in regulation of their cognate RNAs by increasing transcription rates, RNA stability and alternative splicing. We have identified circRNAs from Camelina sativa that are associated with the regulation of germination, light response, and lipid metabolism. We sequenced light-grown and etiolated seedlings after 5 or 7 days post-germination and identified a total of 3,447 circRNAs from 2,763 genes. Most circRNAs originate from a single homeolog of the three subgenomes from allohexaploid camelina and correlates with higher ratios of alternative splicing of their cognate genes. A network analysis shows the interactions of select miRNA:circRNA:mRNAs for regulation of transcript stabilities where circRNA can act as a competing endogenous RNA. Several key lipid metabolism genes can generate circRNA and we confirmed the presence of KASII circRNA as a true circRNA. CircRNA in camelina can be a novel target for breeding and engineering efforts.
Core ideas
First discovery of 3,447 genic and 307 intergenic unique putative circRNAs from Camelina sativa .
We identified circRNAs that were regulated in response to seedling de-etiolation.
Most circRNAs originate from only one homeolog of the three subgenomes in this allohexaploid Camelina.
Alternative splicing of exon skipping and intron retention positively correlate with circRNA occurrence.
Validation of KASII circRNAs as an example of lipid metabolism pathways potentially regulated by circRNA.
... For example, in A. thaliana, affinity of AER to smaller RCS, like acrolein, is lower than that of longer-chain RCS [8]. AKR isozymes have relatively broad substrate specificity for various carbonyls [27]. ALDH uses broad range of substrates, such as sinapaldehyde and coniferaldehyde for ALDH2C4, acetyl-CoA for ALDH3F1, and succinic semialdehyde for ALDH5 [28,29]. ...
Abiotic and biotic stress conditions lead to production of reactive carbonyl species (RCS) which are lipid peroxide derivatives and have detrimental effects on plant cells especially at high concentrations. There are several molecules that can be classified in RCS; among them, 4-hydroxy-(E)-2-nonenal (HNE) and acrolein are widely recognized and studied because of their toxicity. The toxicity mechanisms of RCS are well known in animals but their roles in plant systems especially signaling aspects in metabolism need to be addressed. This chapter focuses on the production mechanisms of RCS in plants as well as how plants scavenge and modify them to prevent irreversible damage in the cell. We aimed to get a comprehensive look at the literature to summarize the signaling roles of RCS in plant metabolism and their interaction with other signaling mechanisms such as highly recognized reactive oxygen species (ROS) signaling. Changing climate promotes more severe abiotic stress effects on plants which also decrease yield on the field. The effects of abiotic stress conditions on RCS metabolism are also gathered in this chapter including their signaling roles during abiotic stresses. Different methods of measuring RCS in plants are also presented in this chapter to draw more attention to the study of RCS metabolism in plants.
... All subunits of cofactor-bound tetramers behave similarly to the first purified state structure in which the functional loops are clear and ordered (Fig. 2b and Supplementary Fig. 14). However, cofactor-free tetramers exhibit a higher degree disordered conformation in every subunit (Fig. 2c, Supplementary Fig. 13g-i and Supplementary Fig. 14), including loops for NADP(H)-binding (residues 21-28, 230-241), the loop for substrate binding (residues 87-95) and the loop where the catalytic residue Tyr53 is located in (residues [50][51][52][53][54][55][56][57][58][59]. Moreover, the side chain of the catalytic residue His130, faces the opposite direction of the active center, and the side chain of Trp214 twists. ...
Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.
... Both WT and COS-AER plants showed a basal level of HNE-reducing activity. Because HNE contains both an aldehyde moiety and the α,β-unsaturated bond, the activity value determined here represents the total activities of AER and other aldehydereducing enzymes such as aldo-keto reductases (Simpson et al., 2009). ...
Salt stress on plants induces an increase in reactive oxygen species (ROS), which then leads to the formation of reactive carbonyl species (RCS) such as acrolein and 4‐hydroxy‐( E )‐2‐nonenal (HNE), potent cytotoxins generated from lipid peroxides. We recently showed that salt‐stress treatment of Arabidopsis thaliana plants increased RCS levels, and exogenously added RCS‐scavenging chemicals alleviated the stress symptoms, indicating that RCS were responsible for the tissue damage in salt‐stressed plants. To obtain deeper insights into the role of RCS in stressed plants, we here analyzed changes in the levels of various RCS in the roots and shoots of A. thaliana . NaCl (90 mM) addition to the culture medium as a salt‐stress treatment caused growth inhibition and leaf chlorosis. Carbonyl analysis using HPLC revealed that the stress treatment induced a 2‐fold increase in the root levels of RCS, including acrolein, HNE and 4‐hydroxy‐( E )‐2‐hexenal (HHE). In the shoots, basal levels and stress‐induced increases of the RCS were lower than in roots. In the transgenic A. thaliana plants that overexpress the RCS‐scavenging enzyme 2‐alkenal reductase (AER) cDNA under the β ‐estradiol ( β ‐ED)‐responsive promoter, salt stress induced less damage than in the wild‐type under β ‐ED supplementation. The AER overexpression suppressed the stress‐induced increases in HNE, acrolein, HHE and ( E )‐2‐hexenal in roots and in HNE in leaves, but not the ROS increase. These results suggest that the RCS increase in roots was the primary cause of salt‐induced damages. Enhancing RCS‐scavenging abilities, such as by AER overexpression, could be a new strategy to confer salt‐stress tolerance to plants.
... The salt-sensitive phenotypes of the fio1 mutant, as evidenced by significantly less fresh weight and lower survival rate compared to wild type upon salt stress treatment (Figures 1 and 7), together with the highly induced expression patterns of FIO1 in response to salt stress ( Figure S1), indicate that FIO1-mediated m 6 A modification contributes to salt tolerance. Importantly, the observa- Dutilleul et al., 2003;Giraud et al., 2008;Ortega-Amaro et al., 2015;Patwari et al., 2019;Simpson et al., 2009;Yalcinkaya et al., 2019), as well as with the increased stability of a salt stress negative effector such as GSTU17 (Chen et al., 2012) (Figure 6 and S7). A previous study showed that the salt-sensitive phenotypes of the vir mutant is associated with the increased stability of salt stress negative effectors, including GSTU17, ATAF transcription factor 1 (ATAF1), GIGANTEA (GI), and clade E growth-regulating type 2C protein phosphatase 1 (EGR1) . ...
N ⁶ ‐methyladenosine (m ⁶ A) is an mRNA modification widely found in eukaryotes and plays a crucial role in plant development and stress responses. FIONA1 (FIO1) is a recently identified m ⁶ A methyltransferase that regulates Arabidopsis ( Arabidopsis thaliana ) floral transition; however, its role in stress response remains unknown. In this study, we demonstrate that FIO1‐mediated m ⁶ A methylation plays a vital role in salt stress response in Arabidopsis. The loss‐of‐function fio1 mutant was sensitive to salt stress. Importantly, the complementation lines expressing the wild‐type FIO1 exhibited the wild‐type phenotype, whereas the complementation lines expressing the mutant FIO1 m , in which two critical amino acid residues essential for methyltransferase activity were mutated, did not recover the wild‐type phenotype under salt stress, indicating that the salt sensitivity is associated with FIO1 methyltransferase activity. Furthermore, FIO1‐mediated m ⁶ A methylation regulated ROS production and affected the transcript level of several salt stress‐responsive genes via modulating their mRNA stability in an m ⁶ A‐dependent manner in response to salt stress. Importantly, FIO1 is associated with salt stress response by specifically targeting and differentially modulating several salt stress‐responsive genes compared with other m ⁶ A writer. Collectively, our findings highlight the molecular mechanism of FIO1‐mediated m ⁶ A methylation in the salt stress adaptation.
... The catalytic efficiency (k cat /K m ) of AvAKR is comparable with the previously The substrate specificity of AvAKR towards glyceraldehyde was found to be 14-fold higher as compared to H. vulgare aldehyde reductase (4C1, K m : 56 mM, k cat /K m 29 s −1 M −1 ) with around 2.5-fold higher catalytic efficiency. It is comparable to the monocot (rice) AKR4C(s), mammalian aldose reductase, and some dicot AKR4C(s), which could metabolize reactive aldehydes and sugars (de Sousa et al. 2009;Simpson et al. 2009;Narawongsanont et al. 2012). Apparently, the discrepancy of low turnover number and high catalytic efficiency could be explained by considering the interplay of substrate affinity, specificity, accuracy, and regulatory factors that influence the overall performance of the enzyme. ...
The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics.
Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo–keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.
... Plants encounter a variety of stresses during growth, which will decrease the quality and yield of plants and directly lead to plant death in severe cases [61,62]. FKBP is a relatively conserved gene family comprised of proteins with PPlase activity, which plays an important role in response to stress during plant growth and development [25]. ...
The FKBP (FK506-binding protein) gene family is an important member of the PPlase protease family and plays a vital role during the processes of plant growth and development. However, no studies of the FKBP gene family have been reported in cucumber. In this study, 19 FKBP genes were identified in cucumber, which were located on chromosomes 1, 3, 4, 6, and 7. Phylogenetic analysis divided the cucumber FKBP genes into three subgroups. The FKBP genes in the same subgroup exhibited similar structures and conserved motifs. The cis-acting elements analysis revealed that the promoters of cucumber FKBP genes contained hormone-, stress-, and development-related cis-acting elements. Synteny analysis of the FKBP genes among cucumber, Arabidopsis, and rice showed that 12 kinds of syntenic relationships were detected between cucumber and Arabidopsis FKBP genes, and 3 kinds of syntenic relationships were observed between cucumber and rice FKBP genes. The tissue-specific expression analysis showed that some FKBP genes were expressed in all tissues, while others were only highly expressed in part of the 10 types of tissues. The expression profile analysis of cucumber FKBP genes under 13 types of stresses showed that the CsaV3_1G007080 gene was differentially expressed under abiotic stresses (high temperature, NaCl, silicon, and photoperiod) and biotic stresses (downy mildew, green mottle mosaic virus, Fusarium wilt, phytophthora capsica, angular leaf spot, and root-knot nematode), which indicated that the CsaV3_1G007080 gene plays an important role in the growth and development of cucumber. The interaction protein analysis showed that most of the proteins in the FKBP gene family interacted with each other. The results of this study will lay the foundation for further research on the molecular biological functions of the cucumber FKBP gene family.
... Reduced levels of H 2 O 2 was observed in the AtOTS1 transgenic lines (Fig. 3A,B), significantly so for the highly expressing AtOST1 sugarcane line (T1.2), which could be attributed to elevated activity of SOD, CAT and reduced GSH, antioxidants ( Fig. 3C-E) detected under severe water deficit stress. Furthermore, proteins identified in the proteome of the AtOTS1 genotypes (Table S5) include HSP90 protein, which in this context responds to H 2 O 2 accumulation as a result of stress and activates expression of ascorbate peroxidase (Nishizawa-Yokoi et al. 2010), monodehydroascorbate reductase and GDPmannose 3,5-epimerase 1 and 2 proteins, which are involved in the biosynthesis of ascorbate and serve as electron donor to ascorbate peroxidases in H 2 O 2 neutralisation (Xiao et al. 2021); RAN-GTPase activating protein 2, which promotes tolerance to stress by binding to the YCHF1 gene, restricting its expression, and functions to suppress antioxidant enzyme activities, leading to peroxidation of lipids and accumulation of ROS (Cheung et al. 2013); adenylate kinase 4, which plays a role in cellular response to H 2 O 2 -induced oxidative stress and cell death by regulating ATP homeostasis (Liu et al. 2009b); aldoketo reductase 1 (probable), which is an aldehyde reductase that functions as a detoxifying enzyme by reducing a range of toxic aldehydes and ketones produced during stress (Simpson et al. 2009); and 60S ribosomal protein L23, which is a ribosome structural constituent that responds to oxidative stress caused by light-induced depression of the photosynthesis capacity (Degenhardt & Bonham-Smith 2008). ...
Sugarcane is an economically important crop plant across the globe as it is the primary source of sugar and biofuel. Its growth and development are greatly influenced by water availability; therefore, in periods of water scarcity, yields are severely compromised. Small Ubiquitin‐Like Modifier (SUMO) proteases play an important role in stress responses by regulating the SUMO‐related post‐translational modification of proteins.
In an attempt to enhance drought tolerance in sugarcane, this crop was genetically transformed with a cysteine protease (OVERLY TOLERANT TO SALT‐1; OTS1) from Arabidopsis thaliana using particle bombardment. Transgenic plants were analysed in terms of photosynthetic capacity, oxidative damage, antioxidant accumulation and the SUMO‐enrich protein profile was assessed.
Sugarcane transformed with the AtOTS1 gene displayed enhanced drought tolerance and delayed leaf senescence under water deficit compared to the untransformed wild type (WT). The AtOTS1 transgenic plants maintained a high relative moisture content and higher photosynthesis rate when compared to the WT. In addition, when the transgene was expressed at high levels, the transformed plants were able to maintain higher stomatal conductance and chlorophyl content under moderate stress compared to the WT. Under severe water deficit stress, the transgenic plants accumulated less malondialdehyde and maintained membrane integrity. SUMOylation of total protein and protease activity was lower in the AtOTS1 transformed plants compared to the WT, with several SUMO‐enriched proteins exclusively expressed in the transgenics when exposed to water deficit stress.
SUMOylation of proteins likely influenced various mechanisms contributing to enhanced drought tolerance in sugarcane.
... Changes in environmental conditions will strongly affect plant productivity, and the accumulation of toxic compounds produced by the interaction between ROS and polyunsaturated fatty acids in the membrane lipids will significantly damage plant cells. Plant aldo-keto reductase (AKR) and other enzymes have been shown to be effective in detoxifying reactive aldehydes produced by lipid peroxidation [55,56]. These findings are consistent with the up-regulated expression of aldo-keto reductase 4 observed in this study in response to low-N treatment. ...
Maize (Zea mays L.) is one of the major staple crops providing human food, animal feed, and raw material support for biofuel production. For its growth and development, maize requires essential macronutrients. In particular, nitrogen (N) plays an important role in determining the final yield and quality of a maize crop. However, the excessive application of N fertilizer is causing serious pollution of land area and water bodies. Therefore, cultivating high-yield and low-N-tolerant maize varieties is crucial for minimizing the nitrate pollution of land and water bodies. Here, based on the analysis of the maize leaf transcriptome and proteome at the grain filling stage, we identified 3957 differentially expressed genes (DEGs) and 329 differentially abundant proteins (DAPs) from the two maize hybrids contrasting in N stress tolerance (low-N-tolerant XY335 and low-N-sensitive HN138) and screened four sets of low-N-responsive genes and proteins through Venn diagram analysis. We identified 761 DEGs (253 up- and 508 down-regulated) specific to XY335, whereas 259 DEGs (198 up- and 61 down-regulated) were specific to HN138, and 59 DEGs (41 up- and 18 down-regulated) were shared between the two cultivars under low-N-stress conditions. Meanwhile, among the low-N-responsive DAPs, thirty were unique to XY335, thirty were specific to HN138, and three DAPs were shared between the two cultivars under low-N treatment. Key among those genes/proteins were leucine-rich repeat protein, DEAD-box ATP-dependent RNA helicase family proteins, copper transport protein, and photosynthesis-related proteins. These genes/proteins were involved in the MAPK signaling pathway, regulating membrane lipid peroxidation, and photosynthesis. Our results may suggest that XY335 better tolerates low-N stress than HN138, possibly through robust low-N-stress sensing and signaling, amplified protein phosphorylation and stress response, and increased photosynthesis efficiency, as well as the down-regulation of ‘lavish’ or redundant proteins to minimize N demand. Additionally, we screened glutathione transferase 42 (ZmGST42) and performed physiological and biochemical characterizations of the wild-type (B73) and gst42 mutant at the seedling stage. Resultantly, the wild-type exhibited stronger tolerance to low N than the mutant line. Our findings provide a better understanding of the molecular mechanisms underlying low-N tolerance during the maize grain filling stage and reveal key candidate genes for low-N-tolerance breeding in maize.
... The characterization of AKRs in C. auris has been largely unexplored, despite extensive studies on model organisms and human pathogens (Simpson et al., 2009;Lapthorn et al., 2013;Penning et al., 2021). Homologs of AKRs have been widely found in other microorganisms, such as Saccharomyces cerevisiae AKR, Sporidiobolus salmonicolor AKR, Aspergillus niger AKR, Phanerochaete chrysosporium AKR, Bacillus subtilis AKR, Escherichia coli AKR (Ellis, 2002). ...
... ortholog, PBS1-LIKE 35 (PBL35, AT3G01300), participates in shoot apical meristem homeostasis and plant immunity, while the Potri.006G090600 ortholog, ALDO-KETO REDUCTASE FAMILY 4 MEMBER C11 (AKR4C11, AT3G53880), participates in abiotic stress tolerance through detoxification of reactive carbonyls [65][66][67][68]. Thus, Potri.017G077300 and Potri.006G090600 may play a role in leaf and vein development through such processes. ...
Plant phenotyping is typically a time-consuming and expensive endeavor, requiring large groups of researchers to meticulously measure biologically relevant plant traits, and is the main bottleneck in understanding plant adaptation and the genetic architecture underlying complex traits at population scale. In this work, we address these challenges by leveraging few-shot learning with convolutional neural networks to segment the leaf body and visible venation of 2,906 Populus trichocarpa leaf images obtained in the field. In contrast to previous methods, our approach (a) does not require experimental or image preprocessing, (b) uses the raw RGB images at full resolution, and (c) requires very few samples for training (e.g., just 8 images for vein segmentation). Traits relating to leaf morphology and vein topology are extracted from the resulting segmentations using traditional open-source image-processing tools, validated using real-world physical measurements, and used to conduct a genome-wide association study to identify genes controlling the traits. In this way, the current work is designed to provide the plant phenotyping community with (a) methods for fast and accurate image-based feature extraction that require minimal training data and (b) a new population-scale dataset, including 68 different leaf phenotypes, for domain scientists and machine learning researchers. All of the few-shot learning code, data, and results are made publicly available.
... G1369 is an additional biosynthetic gene encoding the Aldo/keto reductase of M. bicuspidata. The Aldo/keto reductase proteins are an NADPdependent oxidoreductase superfamily whose members catalyse redox transformations involved in detoxification, intermediary metabolism and biosynthesis, among other physiological functions [40][41][42]. G01374 encodes the MFS general substrate transporter, a transport-related gene. MFS transporters selectively transport a wide range of substrates across membranes, are involved in a variety of physiological processes in organisms [43], and indirectly regulate the internal pH and stress response mechanisms in fungi [44]. ...
Background
Metschnikowia bicuspidata is a pathogenic yesst that can cause disease in many different economic aquatic animal species. In recent years, there was a new disease outbreak in ridgetail white prawn (Exopalaemon carinicauda) in coastal areas of Jiangsu Province China that was referred to as zombie disease by local farmers. The pathogen was first isolated and identified as M. bicuspidata. Although the pathogenicity and pathogenesis of this pathogen in other animals have been reported in some previous studies, research on its molecular mechanisms is still very limited. Therefore, a genome-wide study is necessary to better understand the physiological and pathogenic mechanisms of M. bicuspidata.
Result
In this study, we obtained a pathogenic strain, MQ2101, of M. bicuspidata from diseased E. carinicauda and sequenced its whole genome. The size of the whole genome was 15.98 Mb, and it was assembled into 5 scaffolds. The genome contained 3934 coding genes, among which 3899 genes with biological functions were annotated in multiple underlying databases. In KOG database, 2627 genes were annotated, which were categorized into 25 classes including general function prediction only, posttranslational modification, protein turnover, chaperones, and signal transduction mechanisms. In KEGG database, 2493 genes were annotated, which were categorized into five classes, including cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems. In GO database, 2893 genes were annotated, which were mainly classified in cell, cell part, cellular processes and metabolic processes. There were 1055 genes annotated in the PHI database, accounting for 26.81% of the total genome, among which 5 genes were directly related to pathogenicity (identity ≥ 50%), including hsp90, PacC, and PHO84. There were also some genes related to the activity of the yeast itself that could be targeted by antiyeast drugs. Analysis based on the DFVF database showed that strain MQ2101 contained 235 potential virulence genes. BLAST searches in the CAZy database showed that strain MQ2101 may have a more complex carbohydrate metabolism system than other yeasts of the same family. In addition, two gene clusters and 168 putative secretory proteins were predicted in strain MQ2101, and functional analysis showed that some of the secretory proteins may be directly involved in the pathogenesis of the strain. Gene family analysis with five other yeasts revealed that strain MQ2101 has 245 unique gene families, including 274 genes involved in pathogenicity that could serve as potential targets.
Conclusion
Genome-wide analysis elucidated the pathogenicity-associated genes of M. bicuspidate while also revealing a complex metabolic mechanism and providing putative targets of action for the development of antiyeast drugs for this pathogen. The obtained whole-genome sequencing data provide an important theoretical basis for transcriptomic, proteomic and metabolic studies of M. bicuspidata and lay a foundation for defining its specific mechanism of host infestation.
... For example, after NaCl or ABA treatment, the expression level increase of the BGLU24, a member of the glycoside hydrolase family, which was reported to respond to salt tolerance, in the WT plants was more obvious than that in the mutants. Similarly, the expression levels of another five salt-responsive genes, AKR4C8, ANN1, GSTU17, SIGE and EGR1 [54][55][56][57][58], also increased with the same trends. The stress-responsive gene LEA3 was up-regulated in the two CgZFP1 transgenic lines that conferred the salinity and drought tolerance under drought stress conditions [59], but its expression was down-regulated in the Atwrky66 knockdown plants under the NaCl or ABA treatment (Table 4). ...
Group Ⅲ WRKY transcription factors (TFs) play pivotal roles in responding to the diverse abiotic stress and secondary metabolism of plants. However, the evolution and function of WRKY66 remains unclear. Here, WRKY66 homologs were traced back to the origin of terrestrial plants and found to have been subjected to both motifs’ gain and loss, and purifying selection. A phylogenetic analysis showed that 145 WRKY66 genes could be divided into three main clades (Clade A–C). The substitution rate tests indicated that the WRKY66 lineage was significantly different from others. A sequence analysis displayed that the WRKY66 homologs had conserved WRKY and C2HC motifs with higher proportions of crucial amino acid residues in the average abundance. The AtWRKY66 is a nuclear protein, salt- and ABA- inducible transcription activator. Simultaneously, under salt stress and ABA treatments, the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, as well as the seed germination rates of Atwrky66-knockdown plants generated by the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system, were all lower than those of wild type (WT) plants, but the relative electrolyte leakage (REL) was higher, indicating the increased sensitivities of the knockdown plants to the salt stress and ABA treatments. Moreover, RNA-seq and qRT-PCR analyses revealed that several regulatory genes in the ABA-mediated signaling pathway involved in stress response of the knockdown plants were significantly regulated, being evidenced by the more moderate expressions of the genes. Therefore, the AtWRKY66 likely acts as a positive regulator in the salt stress response, which may be involved in an ABA-mediated signaling pathway.
... Additionally, there are other enzymes known to be capable of converting both methylglyoxal and glyoxal by non-GSH-dependent redox reactions which might complement the repertoire of detoxification pathways and can provide relief pressure on the GSH pool when cytotoxic compounds start depleting cellular GSH levels (Simpson et al. 2009;Yamauchi et al. 2011). Interestingly, among them are the enzymes AKR4C8, AKR4C9 and AtChlADR which are partially strongly induced in apocarotenoid-accumulating roots as we showed recently (Koschmieder et al. 2020). ...
Key message
Analysis of carotenoid-accumulating roots revealed that oxidative carotenoid degradation yields glyoxal and methylglyoxal. Our data suggest that these compounds are detoxified via the glyoxalase system and re-enter primary metabolic pathways.
Abstract
Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation. We recently identified redox enzymes previously known to be involved in the detoxification of fatty acid-derived reactive carbonyl species which were able to convert apocarotenoids into corresponding alcohols and carboxylic acids. However, their subsequent metabolization pathways remain unresolved. Interestingly, we found that carotenoid-accumulating roots have increased levels of glutathione, suggesting apocarotenoid glutathionylation to occur. In vitro and in planta investigations did not, however, support the occurrence of non-enzymatic or enzymatic glutathionylation of β-apocarotenoids. An alternative breakdown pathway is the continued oxidative degradation of primary apocarotenoids or their derivatives into the shortest possible oxidation products, namely glyoxal and methylglyoxal, which also accumulated in carotenoid-accumulating roots. In fact, combined transcriptome and metabolome analysis suggest that the high levels of glutathione are most probably required for detoxifying apocarotenoid-derived glyoxal and methylglyoxal via the glyoxalase pathway, yielding glycolate and d -lactate, respectively. Further transcriptome analysis suggested subsequent reactions involving activities associated with photorespiration and the peroxisome-specific glycolate/glyoxylate transporter. Finally, detoxified primary apocarotenoid degradation products might be converted into pyruvate which is possibly re-used for the synthesis of carotenoid biosynthesis precursors. Our findings allow to envision carbon recycling during carotenoid biosynthesis, degradation and re-synthesis which consumes energy, but partially maintains initially fixed carbon via re-introducing reactive carotenoid degradation products into primary metabolic pathways.
... Members of AKR2A involve in the sugar alcohol metabolism (Everard et al., 1997;Kanayama et al., 1992); AKR6C involve in potassium ion efflux (Tang et al., 1995); and AKR13D involve in the biosynthesis of indole alkaloid (Sun et al., 2012). The AKR4 family is specific to plants and associated with various functions ranging from the detoxification of reactive aldehydes/ketones (Simpson et al., 2009) and xenobiotics (Pan et al., 2019) to the biosynthesis of isoflavonoids (Mameda et al., 2018), morphine (Unterlinner et al., 1999), cocaine (Jirschitzka et al., 2012), ascorbic acid (Agius et al., 2003) and metal chelators (Bashir et al., 2006). Notably, many of AKR4 genes are stress inducible and overexpression of them confers tolerance against multiple stresses in transgenic plants (Sengupta et al., 2015). ...
Aldo-keto reductase-domain (PF00248) containing proteins (AKRs) are NAD(P)(H)-dependent oxidoreductases of a multigene superfamily that mediate versatile functions in plants ranging from detoxification, metal chelation, potassium ion efflux to specialized metabolism. To uncover the complete repertoire of AKR gene superfamily in plants, a systematic kingdom-wide identification, phylogeny reconstruction, classification and synteny network clustering analyses were performed in this study using 74 diverse plant genomes. Plant AKRs were omnipresent, legitimately classified into 4 groups (based on phylogeny) and 14 subgroups (based on the ≥60% of protein sequence identity). Species composition of AKR subgroups highlights their distinct emergence during plant evolution. Loss of AKR subgroups among plants was apparent and that various lineage-, order/family- and species-specific losses were observed. The subgroups IA, IVB and IVF were flourished and diversified well during plant evolution, likely related to the complexity of plant’s specialized metabolism and environmental adaptation. About 65% of AKRs were in genomic synteny regions across the plant kingdom and the AKRs relevant to important functions (e.g. vitamin B6 metabolism) were in profoundly conserved angiosperm-wide synteny communities. This study underscores the evolutionary landscape of plant AKRs and provides a comprehensive resource to facilitate the functional characterization of them.
... et AKR4C9 et AKR4C10 d'Arabidopsis thaliana(Simpson et al., 2009) ont été caractérisées de manière fonctionnelle. ...
Les changements climatiques mondiaux ont déjà affecté et continueront d’affecter la physiologie de la vigne, en particulier le rendement et la composition des baies à la récolte, et donc, en définitive, la qualité et la typicité des vins produits. Parmi les différentes stratégies possibles d’adaptation de la viticulture au changement climatique (modifications de l’encépagement, création de nouvelles variétés mieux adaptées aux conditions futures,...), le contrôle de la composition des baies à la récolte par le biais d’une modification des pratiques culturales et notamment par des manipulations de la canopée (rapport feuilles/fruits (Fe/Fr), manipulation de l’exposition des grappes, échardage) est certainement celle qui serait la plus rapide à mettre en œuvre.Ma thèse a pour but de rechercher le meilleur point de compromis entre diminution du taux de sucres et maintien de l’acidité, de l’accumulation des composés phénoliques et d’une complexité aromatique proche de l’actuelle, en réponse aux manipulations de canopée (rapport Fe/Fr, échardage) couplées ou non à l’application d’acide abscissique (ABA).Différents ratio Fe/Fr ont ainsi été étudiés sur le cépage d’étude Cabernet-Sauvignon en conditions semi-contrôlées en serre sur boutures fructifères puis au vignoble au sein d’une parcelle de production. L’analyse de la composition des baies en métabolites primaires et secondaires, réalisée lors de ces deux expériences, a permis de déterminer que le ratio 6 feuilles restantes par grappe était la valeur seuil en dessous de laquelle un effet sur les métabolites primaires est visible. Une diminution du taux de sucres a notamment été observée, également associée à la diminution de la concentration en anthocyanes totales qui sont particulièrement importantes dans la définition de la typicité des vins de Bordeaux. L’application d’ABA exogène sur les baies avant la véraison a permis de diminuer les effets négatifs de la modification du ratio Fe/Fr sur l’accumulation des anthocyanes et n’a pas montré d’impact sur la typicité des vins lors des dégustations des microvinifications réalisées au cours de cette étude. Ces résultats analytiques obtenus ont été couplés à des analyses transcriptomiques de type RNAseq et PCR quantitative en temps réel (Fluidigm) afin d’identifier les gènes clefs associés au voies métaboliques primaires et secondaires réagissant aux modulations du ratio Fe/Fr couplées ou non à l’ajout d’ABA. Ainsi, le couplage modulation du ratio Fe/Fr et application d’ABA exogène sur les baies serait une méthode potentiellement utilisable afin de réduire le taux de sucres et de maintenir une typicité bordelaise proche de l’actuelle.La caractérisation fonctionnelle d'un gène potentiellement associé à la voie de biosynthèse des méthoxypyrazines, impliquées dans le caractère végétal du raisin et du vin, a également été mise en œuvre. Différentes approches ont été testées et ont fourni des indices utiles, cependant les résultats obtenus sont encore en cours de confirmation.
... It was confirmed that the SNP-3 HRM marker related to the AKR gene can also select gray mold disease-resistant lines from 'Seeds & People' Co. and 'Asia Seed' Co. AKR gene expression was higher in the gray mold disease-resistant line group than in the -susceptible line group. AKR has been mentioned in previous studies and is known to increase the resistance at high expression levels [44,45]. ...
Onions (Allium cepa L.) are one of the most consumed vegetable crops worldwide and are damaged by several fungal diseases in the field or during storage. Gray mold disease caused by the necrotrophic pathogens Botrytis cinerea and Botrytis squamosa is a disease that reduces the productivity and storage life in onions. However, it is difficult to control gray mold disease in onions by using physical and chemical methods. Breeding resistant onions against gray mold disease can reduce the damage caused by pathogens, reduce the labor required for control, and reduce environmental pollution caused by fungicides. However, onions have a large genome size (16Gb), making them difficult to analyze, and have a biennial cycle, resulting in a very long breeding period. Therefore, in this study, markers were developed to shorten the onion breeding period. First, random amplified polymorphic DNA (RAPD) was performed to confirm the genetic relationship between the gray mold disease-resistant and -susceptible lines through a dendrogram. In addition, the sequence characterized amplified region (SCAR)-OPAN1 marker to select resistant lines was developed using a polymorphic RAPD fragment. Second, the RNA-seq of the gray mold-resistant and -susceptible onion lines were analyzed using NGS technology. Using the RNA-seq results and DEG and GO analyses were performed, and the variants, such as SNPs and indels, were analyzed to develop a selectable marker for the resistant line. This study developed the SNP-3 HRM marker for selecting gray mold disease-resistant lines by using the SNPs present in the aldo-keto reductase (AKR) gene with high expression levels in these lines. The SCAR-OPAN1 and SNP-3 HRM markers developed in this study could be used to select gray mold disease-resistant onions in breeding programs to reduce the damage caused by gray mold disease.
... MG acts as a signalling molecule at a lower concentration but becomes extremely toxic at higher concentration (Mostofa et al., 2018) due to the formation of reactive oxygen species as well as advanced glycation end-products (AGEs) with proteins and nucleotides Thornalley, 2008). MG toxicity inside cell is mitigated by a host of MG detoxifying enzymes such as glyoxalase, aldo-keto reductase, aldehyde dehydrogenase and carbonyl reductase (Agrawal et al., 2017;Simpson et al., 2009;Vander-Jagt et al., 2001). Among these, glyoxalase system occupies a pivotal role in the MG detoxification via catalytic conversion of toxic MG into non-toxic lactic acid. ...
23 24 25 J o u r n a l P r e-p r o o f 2 Regulation of antioxidant defense and glyoxalase systems in cyanobacteria 26 27 Abstract 28 Oxidative stress is common consequence of abiotic stress in plants as well as cyanobacteria 29 caused by generation of reactive oxygen species (ROS), an inevitable product of respiration 30 and photosynthetic electron transport. ROS act as signalling molecule at low concentration 31 however, when its production exceeds the endurance capacity of antioxidative defence 32 system, the organisms suffer oxidative stress. A highly toxic metabolite, methylglyoxal (MG) 33 is also produced in cyanobacteria in response to various abiotic stresses which consequently 34 augment the ensuing oxidative damage. Taking recourse to the common lineage of eukaryotic 35 plants and cyanobacteria, it would be worthwhile to explore the regulatory role of glyoxalase 36 system and antioxidative defense mechanism in combating abiotic stress in cyanobacteria. 37 This review provides comprehensive information on the complete glyoxalase system (GlyI, 38 GlyII and GlyIII) in cyanobacteria. Furthermore, it elucidates the recent understanding 39 regarding the production of ROS and MG, noteworthy link between intracellular MG and 40 ROS and its detoxification via synchronization of antioxidants (enzymatic and non-41 enzymatic) and glyoxalase systems using glutathione (GSH) as common co-factor. 42
... MG acts as a signalling molecule at a lower concentration but becomes extremely toxic at higher concentration (Mostofa et al., 2018) due to the formation of reactive oxygen species as well as advanced glycation end-products (AGEs) with proteins and nucleotides Thornalley, 2008). MG toxicity inside cell is mitigated by a host of MG detoxifying enzymes such as glyoxalase, aldo-keto reductase, aldehyde dehydrogenase and carbonyl reductase (Agrawal et al., 2017;Simpson et al., 2009;Vander-Jagt et al., 2001). Among these, glyoxalase system occupies a pivotal role in the MG detoxification via catalytic conversion of toxic MG into non-toxic lactic acid. ...
Oxidative stress is common consequence of abiotic stress in plants as well as cyanobacteria caused by generation of reactive oxygen species (ROS), an inevitable product of respiration and photosynthetic electron transport. ROS act as signalling molecule at low concentration however, when its production exceeds the endurance capacity of antioxidative defence system, the organisms suffer oxidative stress. A highly toxic metabolite, methylglyoxal (MG) is also produced in cyanobacteria in response to various abiotic stresses which consequently augment the ensuing oxidative damage. Taking recourse to the common lineage of eukaryotic plants and cyanobacteria, it would be worthwhile to explore the regulatory role of glyoxalase system and antioxidative defense mechanism in combating abiotic stress in cyanobacteria. This review provides comprehensive information on the complete glyoxalase system (GlyI, GlyII and GlyIII) in cyanobacteria. Furthermore, it elucidates the recent understanding regarding the production of ROS and MG, noteworthy link between intracellular MG and ROS and its detoxification via synchronization of antioxidants (enzymatic and non-enzymatic) and glyoxalase systems using glutathione (GSH) as common co-factor.
... Q9SQ68.1 (40); Papaver sonmiferum, reticuline epimerase (REPI), AKO60181.1; Erythroxylum coca, methylecgonine reductase (MecgoR), ADK94763.1 (18); Medicago sativa, CHR, AAB41556.1 (41); Arabidopsis thaliana, AKR4C9, ABH07515.1 (42); Oryza sativa Indica Group, AKR4C14, ACS92968.1; Homo sapiens, 3-⍺-HDS, 1J96_A (43). ...
Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the β1α1 loop adjacent to the BIA binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.
... Nevertheless, the number of specific genes highlight initial milder differences between the two responses (Figure 4), while discrepancies become more evident in the two later stages. Indeed, self and nonself treatments, respectively, show along the three stages after treatment In particular, genes related to heat, wounding and chitin response were downregulated, while responses to oxidative stress, toxic substances and ions were upregulated in self, involving genes encoding detoxification and anti-oxidation protective enzymes [69][70][71][72]. Such results clearly indicate that self-DNA triggered a response to oxidative stress and detoxification, while downregulating typical stress responsive genes, like HSPs, as it resulted evident at 8 h. ...
The inhibitory effect of extracellular DNA (exDNA) on the growth of conspecific individuals was demonstrated in different kingdoms. In plants, the inhibition has been observed on root growth and seed germination, demonstrating its role in plant–soil negative feedback. Several hypotheses have been proposed to explain the early response to exDNA and the inhibitory effect of conspecific exDNA. We here contribute with a whole-plant transcriptome profiling in the model species Arabidopsis thaliana exposed to extracellular self- (conspecific) and nonself- (heterologous) DNA. The results highlight that cells distinguish self- from nonself-DNA. Moreover, confocal microscopy analyses reveal that nonself-DNA enters root tissues and cells, while self-DNA remains outside. Specifically, exposure to self-DNA limits cell permeability, affecting chloroplast functioning and reactive oxygen species (ROS) production, eventually causing cell cycle arrest, consistently with macroscopic observations of root apex necrosis, increased root hair density and leaf chlorosis. In contrast, nonself-DNA enters the cells triggering the activation of a hypersensitive response and evolving into systemic acquired resistance. Complex and different cascades of events emerge from exposure to extracellular self- or nonself-DNA and are discussed in the context of Damage- and Pathogen-Associated Molecular Patterns (DAMP and PAMP, respectively) responses.
... 10,11 Apart from these, the cofactor-dependent enzyme families like nicotinamide adenine dinucleotide phosphate (NADPH)-dependent AKRs, aldehyde dehydrogenases, and GSH-dependent glyoxalases have shown a potential impact on enzymatic detoxification of the RCCs. 9,12,13 The AKRs bind to RCCs like aldehydes and ketones and convert them into simpler alcohols using NADPH as the cofactor. Similarly, alcohol dehydrogenases bind to specific aldehydes and convert them into alcohol. ...
Reactive carbonyl compounds (RCCs) such as hydroxynonenol, malondialdehyde, acrolein, crotonaldehyde, methylglyoxal, and glyoxal accumulate at higher levels under stress in plants and damage the cell metabolic activities. Plants have evolved several detoxifying enzymes such as aldo–keto reductases (AKRs), aldehyde/alcohol dehydrogenases (ALDH/ADH), and glyoxalases. We report the phylogenetic relationship of these proteins and in silico analysis of rice-detoxifying protein structures and their substrate affinity with cofactors using docking and molecular simulation studies. Molecular simulations with nicotinamide adenine dinucleotide phosphate or glutathione cofactor docking with commonly known reactive substrates suggests that the AKRs, ALDH, and ADH proteins attain maximum conformational changes, whereas glyoxalase has fewer conformational changes with cofactor binding. Several AKRs showed a significant binding affinity with many RCCs. The rice microarray studies showed enhanced expression of many AKRs in resistant genotypes, which also showed higher affinity to RCCs, signifying their importance in managing carbonyl stress. The higher expression of AKRs is regulated by stress-responsive transcription factors (TFs) as we identified stress-specific cis-elements in their promoters. The study reports the stress-responsive nature of AKRs, their regulatory TFs, and their best RCC targets, which may be used for crop improvement programs.
N6‐methyladenosine (m ⁶ A), the most prevalent modification found in eukaryotic mRNAs, is recognized and interpreted by m ⁶ A‐binding proteins called m ⁶ A readers.
The EVOLUTIONARILY CONSERVED C‐TERMINAL REGION (ECT) proteins have increasingly been identified as m ⁶ A readers in plants. A recent study has demonstrated that the loss‐of‐function ect8 mutant is sensitive to salt stress by enhancing the stability of negative salt stress regulators in Arabidopsis ( Arabidopsis thaliana ). In this study, we generated and analyzed the ECT8‐overexpressing transgenic Arabidopsis plants to further explore the function of ECT8 in salt stress response. The electrophoretic mobility shift assay in vitro showed that ECT8 binds to the m ⁶ A‐modified synthetic RNAs, preferring UGUm ⁶ AA and UACm ⁶ AGA motifs over the GGm ⁶ ACU motif. Contrary to the ect8 mutant exhibiting salt hypersensitivity by enhancing the stability of salt stress negative regulators, the ECT8‐overexpressing transgenic Arabidopsis plants displayed salt tolerance by increasing the stability and expression levels of salt stress positive regulators. Moreover, RNA‐immunoprecipitation assay demonstrated that ECT8 binds to the m ⁶ A‐modified salt stress‐responsive mRNAs in planta . Collectively, our current and previous findings highlight that ECT8‐mediated stabilization and destabilization of the genes encoding salt stress positive or negative regulators, respectively, contribute to the salt stress tolerance of Arabidopsis.
Upon infection with non-pathogenic microorganisms or treatment with natural or synthetic compounds, plants exhibit a more rapid and potent response to both biotic and abiotic stresses. However, the molecular mechanisms behind this phenomenon, known as defense priming, are poorly understood. β-aminobutyric acid (BABA) is an endogenous stress metabolite that enhances plant tolerance to various abiotic stresses and primes plant defense responses, providing the ability to resist a variety of pathogens (broad-spectrum resistance). In this study, we identified an aspartyl-tRNA synthetase (AspRS), StIBI1 (named after Arabidopsis IMPAIRED IN BABA-INDUCED IMMUNITY 1; IBI1), as a BABA receptor in Solanum tuberosum. We elucidated the regulatory mechanisms by which StIBI1 interacts with two NAC (NAM, ATAF1, 2, and CUC2) transcription factors (TFs), StVOZ1 and StVOZ2 (VASCULAR PLANT ONE ZINC FINGER, VOZ), to activate BABA-induced resistance (BABA-IR). StVOZ1 represses, whereas StVOZ2 promotes, immunity to the late blight pathogen Phytophthora infestans. Interestingly, BABA and StIBI1 influence StVOZ1- and StVOZ2-mediated immunity. StIBI1 interacts with StVOZ1 and StVOZ2 in the cytoplasm, reducing the nuclear accumulation of StVOZ1 and promoting the nuclear accumulation of StVOZ2. Our findings indicate that StVOZ1 and StVOZ2 finely regulate potato resistance to late blight through distinct signaling pathways. In summary, our study provides insights into the interaction between the potato BABA receptor StIBI1 and the TFs StVOZ1 and StVOZ2, which affects StVOZ1 and StVOZ2stability and nuclear accumulation to regulate late blight resistance during BABA-IR. This research advances our understanding of the primary mechanisms of BABA-IR in potato and contributes to a theoretical basis for the prevention and control of potato late blight using BABA-IR.
As one of the most salt-tolerant grasses, characterizing salt-tolerance genes of Zoysia matrella [L.] Merr. not only broaden the theoretical information of salt tolerance, but also provide new salt-resistant genetic resources for crop breeding. The salt-inducible protein disulfide isomerase (ZmPDI) of Zoysia matrella [L.] Merr. was proved to enhance salt tolerance in homologous overexpression transgenic plants. In order to evaluate its potential application in crops, we conducted the salt tolerance evaluation in heterologous overexpression transgenic rice (OX-ZmPDI), Wild-type (WT) rice, and LOC_Os11g09280 (OsPDI, homologous gene of ZmPDI in rice) knock-out rice generated by CRISPR-Cas9 system (CR-OsPDI). Our findings revealed that OX-ZmPDI rice was higher and exhibited longer main root length, more proline (Pro) and malondialdehyde (MDA) and peroxidase (POD) activity than WT control after salt treatment, while CR-OsPDI resulted in contrary phenotypes. These results indicated that ZmPDI can significantly enhance the salt tolerance in rice, whereas loss-of-function of OsPDI reduces the salt tolerance. To further investigate these differences at the molecular level, we collected roots from OX-ZmPDI transgenic, CR-OsPDI transgenic, and wild-type (WT) plants at 0 and 24 h after salt treatment for RNA-seq and data-independent acquisition (DIA) proteome sequencing. Combined analysis of the transcriptome and proteome revealed that ZmPDI has the potential to enhance the salt tolerance of rice by modulating the expression of laccase-6, zingipain-2, WIP3, FKBP65, AKR4C10, GBSSII, Pho1, and TRXf1. Those results provided new information for the molecular regulation mechanism by which ZmPDI improves salt tolerance, and prove the potential of ZmPDI for application in crop breeding.
Defence priming of plants with natural products is extensively studied in the agricultural field to reduce the use of synthetic and copper-based pesticides. Previous studies have shown that Oregano essential oil vapour (OEOV) is an effective priming agent against downy mildew (DM) in grapevine ( Vitis vinifera L. cv. Chasselas), activating different transcriptomic regulated defence mechanisms. In the present study, we complement transcriptomic data with metabolomic insights, confirming some previous regulating patterns and highlighting new mechanisms underlying OEOV-induced resistance. A significant modulation of the phenylpropanoid pathway was noted. The data also confirmed the induction of an oxidative stress response indicated by an up-regulation of reactive oxygen species (ROS)-related genes and a congruent depletion of putative L-glutathione. Interestingly, OEOV promoted the accumulation of organic metabolites such as terpenes and other potential phytoalexins, which could potentially contribute to grapevine innate immune response to Plasmopara viticola . Overall, this study uncovered a diverse influence of OEOV on V. vinifera defence mechanisms against DM, enhancing our comprehension of the mode of action of essential oils. This insight offers various prospects for crafting innovative biocontrol products, fostering a more dynamic and sustainable approach to agriculture.
Dendrogenomics is a new interdisciplinary approach that allows joint analysis of dendrological and genomic data and opens up new ways to study the temporal dynamics of forest treelines, delineate spatial and temporal population structures, decipher individual tree responses to abiotic and biotic stresses, and evaluate the adaptive genetic potential of forest tree populations. These data are needed for the prediction of climate change effects and mitigation of the negative effects. We present here an association analysis of the variation of 27 individual tree traits, including adaptive dendrophenotypes reflecting the individual responses of trees to drought stress, such as the resistance (Rt), recovery (Rc), resilience (Rs), and relative resilience (RRs) indexes measured in 136 Siberian larch trees in 5 populations in the foothills of the Batenevsky Ridge (Kuznetsk Alatau, Republic of Khakassia, Russia), with variation of 9742 SNPs genotyped using ddRADseq in the same trees. The population structure of five closely located Siberian larch populations was relatively weak (FST = 0.018). We found that the level of individual heterozygosity positively correlated with the Rc and RR indices for the five studied drought periods and partly with the Rs indices for three drought periods. It seems that higher individual heterozygosity improves the adaptive capabilities of the tree. We also discovered a significant negative relationship between individual heterozygosity and the Rt index in four out of five periods, which means that growth slows down during droughts more in trees with higher individual heterozygosity and is likely associated with energy and internal resource reallocation toward more efficient water and energy usage and optimization of larch growth during drought years. We found 371 SNPs with potentially adaptive variations significantly associated with the variation of adaptive dendrophenotypes based on all three different methods of association analysis. Among them, 26 SNPs were located in genomic regions carrying functional genes: 21 in intergenic regions and 5 in gene-coding regions. Based on the obtained results, it can be assumed that these populations of Siberian larch have relatively high standing adaptive genetic variation and adaptive potential underlying the adaptations of larch to various climatic conditions.
D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites. Semi-saturation, single and combined-site mutagenesis was performed on all six residues, and several mutants with improved enzymatic activities were obtained. Among them, the mutant SceCPRS158A/Y298H exhibited the highest catalytic efficiency in which the kcat/Km value is 2466.22s-1·mM-1, 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPRS158A/Y298H had an expanded and increased hydrophilicity catalytic pocket, and an enhanced π-π interaction which could contribute to faster conversion efficiency and higher catalytic rate. The whole cell system containing SceCPRS158A/Y298H and glucose dehydrogenase (GDH), under the optimized condition, could reduce 490.21mM D-PL with e.e.≧99%, conversion rate = 98%, and the space-time yield = 382.80g·L-1·d-1, which is the highest level reported so far.
Arthrobacter simplex is extensively used for cortisone acetate (CA) biotransformation in industry, but the Δ1-dehydrogenation molecular fundamental remains unclear. Herein, the comparative proteome revealed several proteins with the potential role in this reaction, which were mainly involved in lipid or amino acid transport and metabolism, energy production and conversion, steroid degradation, and transporter. The influences of six proteins were further confirmed, where pps, MceGA, yrbE4AA, yrbE4BA, and hyp2 showed positive impacts, while hyp1 exhibited a negative effect. Additionally, KsdD5 behaved as the best catalytic enzyme. By the combined manipulation in multiple genes under the control of a stronger promoter, an optimal strain with better catalytic enzyme activity, substrate transportation, and cell stress tolerance was created. After biotechnology optimization, the production peak and productivity were, respectively, boosted by 4.1- and 4.0-fold relative to the initial level. Our work broadens the understanding of the Δ1-dehydrogenation mechanism, also providing effective strategies for excellent steroid-transforming strains.
Plant phenotyping is typically a time-consuming and expensive endeavor, requiring large groups of researchers to meticulously measure biologically relevant plant traits, and is the main bottleneck in understanding plant adaptation and the genetic architecture underlying complex traits at population scale. In this work, we address these challenges by leveraging few-shot learning with convolutional neural networks (CNNs) to segment the leaf body and visible venation of 2,906 P. trichocarpa leaf images obtained in the field. In contrast to previous methods, our approach (i) does not require experimental or image pre-processing, (ii) uses the raw RGB images at full resolution, and (iii) requires very few samples for training (e.g., just eight images for vein segmentation). Traits relating to leaf morphology and vein topology are extracted from the resulting segmentations using traditional open-source image-processing tools, validated using real-world physical measurements, and used to conduct a genome-wide association study to identify genes controlling the traits. In this way, the current work is designed to provide the plant phenotyping community with (i) methods for fast and accurate image-based feature extraction that require minimal training data, and (ii) a new population-scale data set, including 68 different leaf phenotypes, for domain scientists and machine learning researchers. All of the few-shot learning code, data, and results are made publicly available.
Glyphosate is a dominant organophosphate herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the shikimate pathway. Glyphosate is extensively applied since manufactured, which has led to the emergence of various glyphosate-resistant crops and weeds. However, the molecular mechanism of many glyphosate-resistance machineries remains unclear. Recently, the upregulated expression of two homologous aldo-keto reductases (AKRs), designated as AKR4C16 and AKR4C17, were found to contribute to the glyphosate resistance in Echinochloa colona. This represents the first naturally evolved glyphosate-degrading machinery reported in plants. Here, we report the three-dimensional structure of these two AKR enzymes in complex with cofactor by performing X-ray crystallography. Furthermore, the binding-mode of glyphosate were elucidated in a ternary complex of AKR4C17. Based on the structural information and the previous study, we proposed a possible mechanism of action of AKR-mediated glyphosate degradation. In addition, a variant F291D of AKR4C17 that was constructed based on structure-based engineering showed a 70% increase in glyphosate degradation. In conclusion, these results demonstrate the structural features and glyphosate-binding mode of AKR4C17, which increases our understanding of the enzymatic mechanism of glyphosate bio-degradation and provides an important basis for the designation of AKR-based glyphosate-resistance for further applications.
With the development of global warming and greenhouse effect, heat stress (HS) has become a major stress factor affecting cellular metabolism, plant growth, development, productivity, and even survival. Therefore, understanding the mechanisms of heat injury and thermotolerance in plants is important for agricultural and ecological industry. In general, heat injury leads to protein denaturation, biomembrane damage, oxidative stress, osmotic stress, methylglyoxal (MG) stresses, and ion and nutrient imbalance. Correspondingly, MG as signalling molecule can trigger plant thermotolerance, which is implicated in modification of heat shock proteins (HSPs), biomembrane repair, enhancement of antioxidant system, accumulation of osmolytes, stimulation of MG-detoxification system, and biosynthesis of transporters. In this review, based on the current progress in methylglyoxal (MG) as glycating agent and signalling molecule as well as its detoxification system, MG homeostasis in plants, the mechanisms of heat injury induced by MG as glycating agent and thermotolerance triggered by MG as signalling molecule were summarised. The review is designed to further expound the mechanisms of heat injury and thermotolerance in plants, to stir up the rapid development of MG signalling in plant biology. In addition, the paper lays the foundation of acquiring transgenic crop plants with thermotolerance.
Global climate change and abiotic stresses, like waterlogging, salinity, heavy metals, high temperature, etc., greatly affect plant growth, development, and ultimately crop yield. Oxygen radicals and their derivatives produced by plant cells, known as ROS, result in abiotic stress. Plants contain complicated antioxidative defense mechanism, consisting of nonenzymatic and enzymatic components, which check ROS accumulation and induce plant defense. This chapter focuses on deleterious effects of ROS and antioxidant defense mechanism under various abiotic stresses responsible for ROS detoxification and transcription factors associated with ROS and micro-RNA production under abiotic stress. In addition, it also focuses on crop engineering for abiotic stress resistance in relation to antioxidant machinery and reactive species.
The obesity rate in the United States is 42.4% and has become a national epidemic. Obesity is a complex condition that is influenced by socioeconomic status, ethnicity, genetics, age, and diet. Increased consumption of a Western diet, one that is high in processed foods, red meat, and sugar content, is associated with elevated obesity rates. Factors that increase obesity risk, such as socioeconomic status, also increase consumption of a Western diet because of a limited access to healthier options and greater affordability of processed foods. Obesity is a public health threat because it increases the risk of several pathologies, including atherosclerosis, diabetes, and cancer. The molecular mechanisms linking obesity to disease onset and progression are not well understood, but a proposed mechanism is physiological changes caused by altered lipid peroxidation, glycolysis, and protein metabolism. These metabolic pathways give rise to reactive molecules such as the abundant electrophile methylglyoxal (MG), which covalently modifies nucleic acids and proteins. MG-adducts are associated with obesity-linked pathologies and may have potential for biomonitoring to determine the risk of disease onset and progression. MG-adducts may also play a role in disease progression because they are mutagenic and directly impact protein stability and function. In this review, we discuss how obesity drives metabolic alterations, how these alterations lead to MG production, the association of MG-adducts with disease, and the potential impact of MG-adducts on cellular function.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms.
There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly
conserved genes provides evidence for a regional organization of the chromosomes.
The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3 alpha-HSD (AKRIC4), type 2 3 alpha(17 beta)-HSD (AKR1C3), type 3 3 alpha-HSD (AKR1C2) and 20 alpha(3 alpha)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3 alpha-, 17 beta- and 20 alpha-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with k(cat)/K-m values for substrates that exceeded those obtained with other isoforms by 10-30-fold. In the reduction direction, all isoforms inactivated 5 alpha-dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one; 5 alpha-DHT) to yield 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-androstanediol). However, only AKR1C3 reduced Delta(4)-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17 beta-oestradiol, and progesterone to 20 alpha-hydroxy-pregn-4-ene-3,20-dione (20 alpha-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3a-androstanediol to the active hormone 5 alpha-DHT. AKR1C3 and AKR1C4 oxidized testosterone to Delta(4)-androstene-3,17-dione. All isoforms oxid ized 17 beta-oestradiol to oestrone, and 20 alpha-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high k(cat)/K-m allows this enzyme to form 5 alpha/5 beta-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with Delta(4)-androstene-3,17-dione, but to inactivate 5 alpha-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert Delta(4)-androstene-3,17-dione to testosterone (a substrate aromatizable to 17 beta-oestradiol), oestrone to 17 beta-oestradiol, and progesterone to 20 alpha-hydroxyprogesterone, and this concerted reductive activity may yield a. pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3 alpha-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 sand AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereospecificity for 3 alpha-hydroxysteroids exists.
An aldose reductase homologue (ALDRXV4) was cloned from the resurrection plant Xerophyta viscosa Baker using complementation by functional sufficiency in Escherichia coli. A cDNA library constructed from X. viscosa leaves dehydrated to 85%, 37% and 5% relative water contents (RWC) was converted into an infective phagemid library. Escherichia coli (srl::Tn10) cells transformed with ds-pBluescript phagemids were selected on minimal medium plates supplemented with 1 mM
isopropyl β-d-thiogalactopyranoside and 1.25 M sorbitol. Nine cDNA clones that conferred tolerance to the osmotically stressed E. coli cells were selected. The phagemid from one clone contained the ALDRXV4 insert. The E. coli cells expressing ALDRXV4 were capable of tolerating the osmotic stress, whereas control cultures were not. The ALDRXV4 insert contained an open reading
frame that can code for 319 amino acids, and the predicted protein had a calculated Mr of 35,667. Amino acid sequence comparisons revealed significant similarity to several aldose reductases, with the highest
similarity to aldose reductase proteins from Hordeum vulgare, Bromus inermis and Avena fatua, in the order of 66%, 65% and 65% respectively. Northern blot analysis revealed that ALDRXV4 was expressed only under dehydration conditions in X. viscosa leaves. Western blot analysis detected a protein of 36 kDa under dehydration conditions only. Aldose reductase activity levels
in X. viscosa leaves increased as the leaf RWC decreased, whereas there was no significant change in aldose reductase activity in Sporobolus stafianus as the leaf RWC decreased.
The Web-based software tool Genevestigator provides powerful tools for biologists to explore gene
expression across a wide variety of biological contexts. Its first releases, however, were limited by the scaling
ability of the system architecture, multiorganism data storage and analysis capability, and availability of
computationally intensive analysis methods. Genevestigator V3 is a novel meta-analysis system resulting
from new algorithmic and software development using a client/server architecture, large-scale manual
curation and quality control of microarray data for several organisms, and curation of pathway data for mouse
and Arabidopsis. In addition to improved querying features, Genevestigator V3 provides new tools to analyze
the expression of genes in many different contexts, to identify biomarker genes, to cluster genes into
expression modules, and to model expression responses in the context of metabolic and regulatory networks.
Being a reference expression database with user-friendly tools, Genevestigator V3 facilitates discovery
research and hypothesis validation.
Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.
Plant K+ channel proteins have been previously characterized as tetramers of membrane-spanning alpha subunit polypeptides. Recent studies have identified a 39-kD, hydrophilic polypeptide that is a structural component of purified animal K+ channel proteins. We have cloned and sequenced an Arabidopsis thaliana cDNA encoding a 38.4-kD polypeptide that has a sequence homologous to the animal K+ channel beta subunit. Southern and northern analyses indicate the presence of a gene encoding this cDNA in the Arabidopsis genome and that its transcription product is present in Arabidopsis cells. To our knowledge, this is the first report to document the presence of K+ channel beta subunits in plants.
that the two enzymes are induced coordinately after treat- ment of the cell cultures with elicitor-type molecules. Moreover, Welle et al. (1991) isolated the encoding cDNA. This reductase can play a fundamental role in planta in the balancing of the production of 5-deoxyflavonoids or 5-hy- droxyflavonoids, the synthesis of which depends on the co-action of CHS and CHR or on CHS alone, respectively. Despite this pivotal role and except for the pioneering work already cited, the CHR has not been extensively studied, either at the enzymatic or gene expression levels. We have previously shown that during an incompatible interaction, Medicago sativalPseudomonas syringae pv pisi, severa1 genes involved in phytoalexin production (CHS and isoflavone reductase) were induced (Esnault et al., 1993). This led us to address the question of an involve- ment of CHR. Our strategy was based first on the isolation of cDNA encoding CHR in alfalfa as well as its genomic organization, and second on the analysis of its expression. In this communication, we report the isolation of three cDNAs encoding CHR. The soybean CHR cDNA clone (Welle et al., 1991) was used to screen a AgtlO cDNA library made from poly(A)+ RNA extracted from leaves harvested 6 h after infection with the incompatible bacterium P. syringae pv pisi. After three rounds of screening and subcloning in the pBS SK- plasmid, seven clones were obtained and sequenced. Three of them, MsCHRla, MsCHRlc, MsCHRZa, were full length and their features are described in Table I. They contain an insert of 1202,1115, and 1134 bp, respectively, with an open reading frame of 936 nucleotides. The predicted size of the reductase is 312 amino acids with a molecular mass of around 35 kD. The deduced amino acid sequences are more than 90% homologous to the soybean CHR, and they are related to the aldo-keto reductase family. No signal or ~~
The physiological alpha-oxoaldehyde methylglyoxal binds and modifies arginine, lysine, and cysteine residues in proteins. The kinetics and mechanism of these reactions were investigated with N alpha-acetylamino acids and bovine serum albumin at pH 7.4 and 37 degrees C. The reaction of methylglyoxal with N alpha-acetylarginine involved the initial reversible formation of glycosylamine and 4,5-dihydroxy-5-methylimidazolidine derivatives, with further slow irreversible conversion to an imidazolone, N alpha-acetyl-N delta- (5-methyl-4-imidazolon-2-yl)ornithine. The imidazolone was fluorescent with an excitation lambda max value of 320 nm and an emission lambda max value of 398 nm. Methylglyoxal reacted reversibly with N alpha-acetyllysine to form glycosylamine and bisglycosylamine derivatives. Further reaction of these glycosylamines occurred to form brown, fluorescent oligomers that were not characterized. Methylglyoxal reacted rapidly and reversibly with N alpha-acetylcysteine to form the hemithioacetal adduct. The reaction of methylglyoxal with bovine serum albumin (BSA) at pH 7.4 and 37 degrees C involved the reversible and irreversible formation of methylglyoxal-BSA adducts. Irreversible modification of BSA occurred mainly on arginine residues to form imidazolone. The formation of methylglyoxal-modified proteins involves glycoxidation leading to advanced glycation end product-like fluorescence. It is expected to be increased in diabetes mellitus and may be linked to the development of diabetic complications.
Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.
Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket.
The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase.
The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.
The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (α/β)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation of a single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5β-steroid 4-dehydrogenases (Δ4-3-ketosteroid 5β-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17β-hydroxysteroid dehydrogenase and rat ovary 20α-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.
In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.
Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions.
Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.
Rapid accumulation of toxic products from reactions of reactive oxygen species (ROS) with lipids and proteins significantly contributes to the damage of crop plants under biotic and abiotic stresses. Here we have identified a stress-activated alfalfa gene encoding a novel plant NADPH-dependent aldose/aldehyde reductase that also exhibited characteristics of the homologous human enzyme. The recombinant alfalfa enzyme is active on 4-hydroxynon-2-enal, a known cytotoxic lipid peroxide degradation product. Ectopic synthesis of this enzyme in transgenic tobacco plants provided considerable tolerance against oxidative damage caused by paraquat and heavy metal treatment. These transformants could also resist a long period of water deficiency and exhibited improved recovery after rehydration. We found a reduced production of lipid peroxidation-derived reactive aldehydes in these transformed plants under different stresses. These studies reveal a new and efficient detoxification pathway in plants.
An essential step in macromolecular refinement is the selection of model parameters which give as good a description of the experimental data as possible while retaining a realistic data-to-parameter ratio. This is particularly true of the choice of atomic displacement parameters, where the move from individual isotropic to individual anisotropic refinement involves a sixfold increase in the number of required displacement parameters. The number of refinement parameters can be reduced by using collective variables rather than independent atomic variables and one of the simplest examples of this is the TLS parameterization for describing the translation, libration and screw-rotation displacements of a pseudo-rigid body. This article describes the implementation of the TLS parameterization in the macromolecular refinement program REFMAC. Derivatives of the residual with respect to the TLS parameters are expanded in terms of the derivatives with respect to individual anisotropic U values, which in turn are calculated using a fast Fourier transform technique. TLS refinement is therefore fast and can be used routinely. Examples of TLS refinement are given for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a transcription activator GerE, for both of which there is data to only 2.0 Å, so that individual anisotropic refinement is not feasible. GAPDH has been refined with between one and four TLS groups in the asymmetric unit and GerE with six TLS groups. In both cases, inclusion of TLS parameters gives improved refinement statistics and in particular an improvement in R and free R values of several percent. Furthermore, GAPDH and GerE have two and six molecules in the asymmetric unit, respectively, and in each case the displacement parameters differ significantly between molecules. These differences are well accounted for by the TLS parameterization, leaving residual local displacements which are very similar between molecules and to which NCS restraints can be applied.
An account is given of the molecular replacement method as implemented in the package AMoRe. The overall strategy of the method is presented and the main functions used in the package are described. The most important features of AMoRe are the quality of the fast rotation and translation functions and the facility of multiple inputs to translation and rigid-body refinement functions, which allow for a fast multiple exploration of crystal configurations with a high level of automation.
Aldo-keto reductases (AKRs) are a gene superfamily whose members catalyze the nicotinamide adenine dinucleotide (phosphate) (reduced) (NAD(P)(H))-dependent interconversion of aldehydes and ketones with primary and secondary alcohols. AKRs by functionalizing carbonyl groups for subsequent conjugation reactions can be considered Phase I enzymes. There are 13 human AKR isoforms which are pluripotent. Endogenous substrates include sugar aldehydes, lipid aldehydes, prostaglandins, and steroid hormones. Xenobiotic substrates include carbonyl-containing drugs and carcinogen metabolites (polycyclic aromatic trans-dihydrodiols, nicotine-derived nitrosamino ketones, and aflatoxin dialdehyde); and they have been implicated in cancer chemotherapeutic drug resistance. Human isoforms are regulated by primordial signals (osmotic, electrophilic, and oxidative stress) so that a counterresponse to the stressor may ensue. Many contain an antioxidant response element in their gene promoters suggesting that cancer chemopreventive strategies that target this element could also affect endogenous substrate utilization.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Plant K+ channel proteins have been previously characterized as tetramers of membrane-spanning [alpha] subunit polypeptides. Recent studies have identified a 39-kD, hydrophilic polypeptide that is a structural component of purified animal K+ channel proteins. We have cloned and sequenced an Arabidopsis thaliana cDNA encoding a 38.4-kD polypeptide that has a sequence homologous to the animal K+ channel [beta] subunit. Southern and northern analyses indicate the presence of a gene encoding this cDNA in the Arabidopsis genome and that its transcription product is present in Arabidopsis cells. To our knowledge, this is the first report to document the presence of K+ channel [beta] subunits in plants.
Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
Arabidopsis det2 mutants are small dark-green dwarfs displaying pleiotropic defects in light-regulated development during multiple stages
of the plant life cycle. The DET2 gene encodes a protein that shares ≈40% sequence identity with mammalian steroid 5α-reductases and is implicated in the synthesis
of a class of plant steroids, the brassinosteroids. Here we show that the DET2 protein, when expressed in human embryonic
kidney 293 cells, catalyzes the 5α-reduction of several animal steroid substrates and has similar kinetic properties to the
mammalian steroid 5α-reductase enzymes. Moreover, human steroid 5α-reductases expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis. These data indicate that DET2 is an ortholog of the
mammalian steroid 5α-reductases and provide further evidence that brassinosteroids play an essential role in light-regulated
plant development. The structural and functional conservation between DET2 and human steroid 5α-reductases raise interesting
issues concerning the evolutionary origin of the steroid hormone signaling system.
The effect of 24-epibrassinolide (EBS), a brassinosteroid, on the response of smooth bromegrass (Bromus inermis Leyss) cell suspension cultures under low and high temperature stress was evaluated. EBS minimally increased freezing tolerance of bromegrass cells by 3–5°C, but markedly enhanced cell viability following exposure to high temperature stress. The net effect on hardening was, however, less than that obtained with abscisic acid (ABA). Treatment of cells with EBS increased the accumulation of a subset of ABA-inducible heat-stable proteins, but unlike ABA, EBS did not induce the expression of dehydrin transcripts which hybridized to a barley dehydrin clone. EBS slightly increased the hsp90 transcript levels, while ABA reduced the level of expression. These results confirm that brassinosteroids confer some stress tolerance to plant cells and further suggest that the mechanisms by which these compounds exert anti-stress effects may only in part be similar to that of ABA.
Rapid accumulation of toxic products from reactions of reactive oxygen species (ROS) with lipids and proteins significantly contributes to the damage of crop plants under biotic and abiotic stresses. Here we have identified a stress-activated alfalfa gene encoding a novel plant NADPH-dependent aldose/aldehyde reductase that also exhibited characteristics of the homologous human enzyme. The recombinant alfalfa enzyme is active on 4-hydroxynon-2-enal, a known cytotoxic lipid peroxide degradation product. Ectopic synthesis of this enzyme in transgenic tobacco plants provided considerable tolerance against oxidative damage caused by paraquat and heavy metal treatment. These transformants could also resist a long period of water deficiency and exhibited improved recovery after rehydration. We found a reduced production of lipid peroxidation-derived reactive aldehydes in these transformed plants under different stresses. These studies reveal a new and efficient detoxification pathway in plants.
The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine. In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli. Heterologously expressed codeinone reductase-calmodulin-binding peptide fusion protein was purified from E. coli using calmodulin affinity column chromatography in a yield of 10 mg enzyme l-1. These four isoforms demonstrated very similar physical properties and substrate specificity. As least six alleles appear to be present in the poppy genome. A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6′-deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis. By sequence comparison, both codeinone reductase and 6′-deoxy- chalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases, and thereby possibly arise from primary metabolism.
4-Hydroxy-2-alkenale verschiedener Kettenlnge (C5–C10) werden in einer mehrstufigen Synthese hergestellt: Alkanal 2-Bromalkanaldimethylacetal 2-Alkenaldimethylacetal 4-Brom-2-alkenaldimethylacetal 4-Hydroxy-2-alkenaldimethylacetal 4-Hydroxy-2-alkenal.
Plant somatic embryos usually lack desiccation tolerance. They may acquire such a tolerance upon preculture in the presence of abscisic acid (ABA), followed by slow drying, but not fast drying. ABA causes torpedo-shaped somatic embryos to lose their chlorophyll, suspend growth, exhibit low rates of respiration, and maintain elevated sucrose contents. The subsequent slow drying leads to a partial conversion of sucrose into oligosaccharides and the expression of dehydrin transcripts. Slow-dried, desiccation-tolerant somatic embryos have stable membranes, retain their native protein secondary structure, and have a densely packed cytoplasmic glassy matrix. Fast-dried, desiccation-sensitive somatic embryos experience some loss of phospholipids and an increase in free fatty acids. Their proteins show signs of denaturation and aggregation, and the glassy matrix has reduced hydrogen bonding. The reduced conversion of sucrose into oligosaccharides appears not to underlie dehydration injury. Proteins in slow-dried somatic embryos, not pretreated with ABA, also show signs of denaturation, which might be attributed to low sugar contents. We conclude that by reducing cellular metabolism, ABA maintains high sugar contents. These sugars contribute to the stability of membranes, proteins, and the cytoplasmic glassy matrix, whereas slow drying permits a further fine tuning of this stability. Partitioning of endogenous amphiphiles from the cytoplasm into membranes during drying may cause membrane perturbance, although it might confer protection to membranes in the case of amphiphilic antioxidants. The perturbance appears to be effectively controlled in desiccation-tolerant systems but not in sensitive systems, for which we suggest dehydrins are responsible. In this context, the low desiccation tolerance in the presence of ample sugars is discussed.
A serious factor limiting the engineering of stress tolerance has been our ignorance about the function of stress-induced genes. A stress-activated novel aldose–aldehyde reductase was cloned from alfalfa. The ectopic expression of this gene in tobacco resulted in tolerance to oxidative stress and dehydration. Physiological analysis suggested that aldose reductase probably functions by reducing the level of reactive aldehydes. This provides a promising perspective for the development of crop plants with improved stress tolerance.
The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.
Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, E.C. 1.1.1.213, AKR1C9) is a member of the aldo-keto reductase (AKR) superfamily which inactivates circulating steroid hormones. We have proposed a catalytic mechanism in which Tyr 55 acts as a general acid with its pK value being lowered by a hydrogen bond with Lys 84 which is salt-linked to Asp 50. To test this mechanism, residues at the active site were mutated and the mutant enzymes (Y55F, Y55S, K84M, K84R, D50N, D50E, and H117A) were purified to homogeneity from an Escherichia coli expression system. Spectrophotometric assays showed that mutations of Tyr 55 and Lys 84 gave enzymes that were apparently inactive for steroid oxidation and reduction. All mutants appeared inactive for steroid reduction. The catalytic efficiencies for steroid oxidation were reduced 4-10-fold for the Asp 50 mutants and 300-fold for the H117A mutant. Fluorescence titration with NADPH demonstrated that each mutant bound cofactor unimpeded. Equilibrium dialysis indicated that the competitive inhibitor testosterone formed E.NADH.testosterone complexes only with the Y55F, Y55S, and D50N mutants with Kd values 10-fold greater than those for wild-type. Therefore the loss of steroid oxidoreductase activity observed for the Tyr 55 mutants cannot be attributed simply to an inability to bind steroid. Using a highly sensitive radiometric assay in which the conversion of [14C]-5 alpha-dihydrotestosterone (DHT) to [14C]-3 alpha-androstanediol (3 alpha-Diol) was measured, the rate enhancement (kcat/knoncat) for the reaction was estimated to be 2.6 x 10(9). Using this assay, all mutants formed steroid product with decreases in an overall rate enhancement of 10(1)-10(4). It was found that Tyr 55 made the single largest contribution to rate enhancement. This is the first instance where point mutations in the conserved catalytic tetrad of an AKR yielded enzymes which were still catalytically active. This enabled the construction of pH vs kcat profiles for the reduction of [14C]-5 alpha-DHT catalyzed by the tetrad mutants. These profiles revealed that the titratable group assigned to the general acid (pK = 6.50 +/- 0.42) was eliminated in the Y55F and H117A mutants. The pH-independent value of kcat was decreased in the H117A and Y55F mutants, by 2 and 4 log units, respectively. pH vs kcat(app) profiles for the oxidation of [3H]-3 alpha-Diol showed that the same titratable group (pK = 7.50 +/- 0.30) was eliminated in both the Y55F and K84M mutants but was retained in the H117A mutant. Since only the Y55F mutant eliminated the titratable group in both the reduction and oxidation directions it is assigned as the catalytic general acid/base. The differential effects of His 117 and Lys 84 on the ionization of Tyr 55 are explained by a "push-pull" mechanism in which His 117 facilitates proton donation and Lys 84 facilitates proton removal by Tyr 55.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Aldose reductase, which catalyzes the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction
of a wide variety of aromatic and aliphatic carbonyl compounds, is implicated in the development of diabetic and galactosemic
complications involving the lens, retina, nerves, and kidney. A 1.65 angstrom refined structure of a recombinant human placenta
aldose reductase reveals that the enzyme contains a parallel beta 8/alpha 8-barrel motif and establishes a new motif for NADP-binding
oxidoreductases. The substrate-binding site is located in a large, deep elliptical pocket at the COOH-terminal end of the
beta barrel with a bound NADPH in an extended conformation. The highly hydrophobic nature of the active site pocket greatly
favors aromatic and apolar substrates over highly polar monosaccharides. The structure should allow for the rational design
of specific inhibitors that might provide molecular understanding of the catalytic mechanism, as well as possible therapeutic
agents.
In most higher plants a period of desiccation is the terminal event in embryogenesis. Excised barley embryos acquire desiccation tolerance at a precise developmental stage and cDNA clones have been isolated which are temporally linked with desiccation tolerance. One such clone (pG22-69) with a putative gene product of 34 kd displays high structural homology to mammalian genes encoding an NADPH dependent aldose reductase involved in the synthesis of sorbitol. This first aldose reductase gene of plants is expressed constitutively during embryo maturation and is modulated by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). Immunohistochemistry showed that the protein is preferentially expressed in tissues formed at early stages in embryogenesis. Measurements of enzymatic activity indicate that pG22-69 encodes an active aldose reductase. The finding of this reductase activity and the cloning of the corresponding gene supports the existence of a metabolic pathway in plants playing a role in the synthesis of osmolytes like sorbitol. The significance of this work is that genes of related structure and functions are being used in diverse organisms to fulfil stress related biological requirements.
The expression of a barley gene homologous to aldose reductase and aldehyde reductase is restricted to the embryo and temporally correlated with its acquisition of desiccation tolerance. In the work presented, two aspects of this barley gene were investigated: its transcriptional regulation and the initial characterization of the enzymatic function. The transcriptional regulation of the gene was studied in transgenic tobacco by analysing the expression of chimeric genes containing 5′ sequences of the barley gene transcriptionally fused to the GUS reporter gene. This functional analysis of the promoter revealed that a 1364 bp 5′ fragment confers the appropriate pattern of expression to the reporter gene in tobacco and that a short promoter fragment (−114 to +75) containing the sequence TACGTGGC, homologous to plant G-box elements, is sufficient for developmental expression during embryogenesis.
To investigate the enzymatic properties of the gene product the wild-type protein and a mutant carrying a lysine 259 to methionine substitution were overexpressed in a procaryotic system and purified to homogeneity. The wild-type protein exhibits aldose reductase activity in the reduction of dl-glyceraldehyde and d-erythrose specifically using NADPH as co-factor whereas the mutant shows markedly reduced activity. However, the barley protein possesses some properties different to those of animal aldose and aldehyde reductases and its biological target still needs to be identified.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Abscisic acid (ABA) increases the freezing tolerance of bromegrass (Bromus inermis Leyss) cell-suspension cultures at 23 degrees C and elicits many metabolic changes similar to those observed during cold acclimation. Induction and maintenance of freezing tolerance by ABA is accompanied by the expression of novel polypeptides and translatable RNAs. The objective of this study was to isolate and characterize ABA-responsive cDNAs associated with ABA-induced freezing tolerance in bromegrass cell cultures. Among the 16 ABA-responsive cDNA clones isolated, 9 were expressed only with ABA treatment, 7 showed increased transcript level, and 1 was transiently expressed. Cold responsiveness was determined in three clones with increased transcript levels and in the transiently expressed clone. Deacclimation of ABA-hardened cells was a relatively slow process, because all of the novel transcripts persisted for at least 7 d after cells were cultured in ABA-free medium. Preliminary sequencing of cDNAs has identified several clones that share high sequence homology with genes associated with sugar metabolism, osmotic stress, and protease activity. Clone pBGA61 was fully sequenced and tentatively identified as an NADPH-dependent aldose reductase. The predicted amino acid sequence of the coding region shared 92% similarity with that predicted for barley aldose reductase cDNA. It is proposed that expression of genes related to sugar metabolism and osmotic stress may be required for ABA-induced hardening.
To analyze the patterns of gene expression associated with seed dormancy in wild oat (Avena fatua), we have isolated cDNA clones corresponding to genes that are differentially expressed in dormant and afterripened line M73 embryos. Gene transcripts of these clones were maintained in embryos of imbibed dormant caryopses, but declined rapidly in afterripened embryos after imbibition. GA3 treatment of dormant caryopses, which breaks dormancy, could lower the transcript levels in dormant embryos. When the germination of afterripened caryopses was inhibited by high temperature (35 degrees C), the decline in abundance of the transcripts in afterripened embryos was arrested. These genes were expressed to various degrees in water-stressed, but not in unstressed, 7-day-old seedlings. The expression of the genes was also ABA-inducible in afterripened embryos. The expression patterns in non-dormant line SH430 wild oat were similar to those of afterripened M73. DNA sequence analyses indicated that some of the cDNA clones encode LEA (late embryogenesis-abundant) proteins and aldose reductase. The significance of the expression of these genes in maintaining seed dormancy or longevity is discussed.
Arabidopsis has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by ACT2 and ACT8, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene-specific RNA gel blot hybridization and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays showed that ACT2 and/or ACT8mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most Arabidopsis organs. The 5' flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a beta-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The ACT2/GUS construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the ACT8/GUS construct was weaker. It was observed only in a subset of the organs and tissues expressing ACT2/GUS and was not significantly expressed in the flower. ACT2, ACT8, and ACT8/GUS mRNAs were present at moderate to high levels in pollen, and yet neither ACT2/GUS nor ACT8/GUS enzyme expression could be detected in pollen. This suggested a mechanism of translational control affecting ACT2 and ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.
1. Methylglyoxal is a reactive alpha-oxoaldehyde and physiological metabolite formed by the fragmentation of triose-phosphates, and by the metabolism of acetone and aminoacetone. 2. Methylglyoxal modifies guanylate residues to form 6,7-dihydro-6,7-dihydroxy-6-methyl-imidazo[2,3-b]purine-9(8)one and N2-(1-carboxyethyl)guanylate residues and induces apoptosis. 3. Methylglyoxal modifies arginine residues in proteins to form N(delta)-(4,5-dihydroxy-4-methylimidazolidin-2-yl) ornithine, N(delta)-(5-hydro-5-methylimidazol-4-on-2-yl)ornithine and N(delta)-(5)methylimidazol-4-on-2-yl)ornithine residues. 4. Methylglyoxal-modified proteins undergo receptor-mediated endocytosis and lysosomal degradation in monocytes and macrophages, and induce cytokine synthesis and secretion. 5. Methylglyoxal is detoxified by the glyoxalase system. Decreased detoxification of methylglyoxal may be induced pharmacologically by glyoxalase I inhibitors which have anti-tumor and anti-malarial activities. 6. The modification of nucleic acids and protein by methylglyoxal is a signal for their degradation and may have a role in the development of diabetic complications, atherosclerosis, the immune response in starvation, aging and oxidative stress.
Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.
Human 3α-hydroxysteroid dehydrogenase exists in four isoforms, which belong to the aldo-keto reductase (AKR) superfamily and
are named AKR1C1-AKR1C4. The properties of AKR1C3 have not been fully characterized compared to the other isoforms. In addition,
a cDNA that shows more than 99% homology with AKR1C3 cDNA has been cloned from human myeloblasts. We have here expressed and
purified a recombinant enzyme (designated as DBDH) from this cDNA. DBDH oxidized xenobiotic alicyclic alcohols and 3a- or
17 β-hydroxy-5β-androstanes, and catalyzed the reversible conversion between prostaglandin D2 and 9α, 11β-prostaglandin F2
more efficiently than that of 3α- or 17β-hydroxy-steroids: the respective Km values were 0.6 and 6.8 μM, and kcat/Km values were about 1, 000 min−1.mM−1. Anti-inflammatory drugs highly inhibited the enzyme. The recombinant AKR1C3 prepared by site-directed mutagenesis of DBDH
also showed the same properties as the wild-type DBDH. Analyses of expression of mRNAs for DBDH and AKR1C3 by reverse transcription-PCR
indicated that only one mRNA species for DBDH is expressed in 33 human specimens of liver, kidney, lung, brain, heart, spleen,
adrenal gland, small intestine, placenta, prostate, and testis. These results suggest that AKR1C3 acts as prostaglandin D2 11-ketoreductase, and that its principal gene in the human has a coding region represented by DBDH cDNA.
Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.
The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Delta4-3-ketosteroid 5beta-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68-80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa. A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs are encoded by a family of at most five genes. RNA-blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves. The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat-shock treatment. The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified. The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor. These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity. The function of the enzymes in cardenolide biosynthesis is discussed.