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The de novo Biosynthesis of Vitamin B6 Is Required for Disease Resistance Against Botrytis cinerea in Tomato
Abstract
Vitamin B6 (VB6), an essential cofactor for numerous metabolic enzymes, has recently been shown to act as a potent antioxidant and play important roles in developmental processes and stress responses. However, little is known about the possible function of VB6 in plant disease resistance response against pathogen infection. In the present study, we explored the possible involvement of VB6 in defense response against Botrytis cinerea through functional analysis of tomato VB6 biosynthetic genes. Three de novo VB6 biosynthetic genes, SlPDX1.2, SlPDX1.3 and SlPDX2, and one salvage pathway gene SlSOS4 were identified and the SlPDX1.2, SlPDX1.3 and SlPDX2 genes were shown to encode functional enzymes involved in de novo biosynthesis of VB6, as revealed by complementation of the VB6 prototrophy in yeast snz1 and sno1 mutants. Expression of SlPDX1.2, SlPDX1.3 and SlSOS4 genes was induced by infection with B. cinerea. Virus-induced gene silencing (VIGS)-mediated knockdown of SlPDX1.2 or SlPDX1.3 but not SlPDX2 and SlSOS4 led to increased severity of disease caused by B. cinerea, indicating that the VB6 de novo biosynthetic pathway but not the salvage pathway is involved in tomato defense response against B. cinerea. Furthermore, the SlPDX1.2- and SlPDX1.3-silenced tomato plants exhibited reduced levels of VB6 contents and reactive oxygen species scavenging capability, increased levels of superoxide anion and H2O2 generation, and increased activity of superoxide dismutase after infection by B. cinerea. Our results suggest that VB6 and its de novo biosynthetic pathway play important roles in regulation of defense response against B. cinerea through modulating cellular antioxidant capacity.
... There are three PDX1 homologs in Arabidopsis, two (PDX1.1 and PDX1.3) of which are catalytically active, and they encode PDX (Titiz et al., 2006;Tambasco-Studart et al., 2007). PDX1.3 is required for disease resistance to bacterial and fungal diseases in Arabidopsis (Moccand et al., 2014;Zhang et al., 2014Zhang et al., , 2015. In contrast to Arabidopsis, potato (Solanum tuberosum L.) has only two PDX1 homologs (PDX1.1 and PDX1.2), and only PDX1.1 is catalytically active (Mooney et al., 2013). ...
... These results imply that VB6 vitamers might contribute to disease resistance in modulating plant defense responses against different types of pathogens. Zhang et al. (2014) provided direct evidence supporting this notion by examining Arabidopsis mutant ecotypes that are defective in the de novo, and the salvage pathways of VB6 biosynthesis. Mutations in the Arabidopsis PDX1.2 and PDX1.3 genes involved in the de novo pathway increased disease levels caused by Botrytis cinerea and decreased the content level of VB6 (Zhang et al., 2014(Zhang et al., , 2015. ...
... Zhang et al. (2014) provided direct evidence supporting this notion by examining Arabidopsis mutant ecotypes that are defective in the de novo, and the salvage pathways of VB6 biosynthesis. Mutations in the Arabidopsis PDX1.2 and PDX1.3 genes involved in the de novo pathway increased disease levels caused by Botrytis cinerea and decreased the content level of VB6 (Zhang et al., 2014(Zhang et al., , 2015. Despite these studies on the regulation and co-expression of VB6 genes in the host and the pathogen of the de novo and salvage biosynthetic pathways during host-pathogen interactions remain limited to the best of our knowledge to only one study (Samsatly et al., 2018). ...
Vitamin B6 is well recognized as an essential antioxidant and plays a role in stress responses. Co-expression of plant and pathogen-derived vitamin B6 genes is critical during disease development of R. solani. However, little is known about the functionality of vitamin B6 vitamers during plant-R. solani interactions and their involvement in disease tolerance. Here, we explored the possible involvement of vitamin B6 during disease progression of potato cultivars of different susceptibility levels to R. solani. A distinct pattern of gene expression, pyridoxine (PN) concentration, and fungal biomass was found in the susceptible cv. Russet Burbank and tolerant cv. Chieftain. Accumulation of reactive oxygen species (ROS) in R. solani mycelia or plant tissues applying non-fluorescence or fluorescence methods was related to up-regulation in the vitamin B6 pathway and is indicative of oxidative stress. Russet Burbank was susceptible to R. solani, which was linked to reduced amounts of VB6 content. Prior to infection, constitutive PN levels were significantly higher in Russet Burbank by 1.6-fold compared to Chieftain. Upon infection with R. solani, PN levels in infected tissues increased more in Chieftain (1.7-fold) compared to Russet Burbank (1.4-fold). R. solani AG3 infection of potato sprouts in both cultivars significantly activates the fungal and plant vitamin B6 and glutathione-S-transferase (GST) genes in a tissue-specific response. Significant fold increases of transcript abundance of the fungal genes ranged from a minimum of 3.60 (RsolSG3GST) to a maximum of 13.91 (RsolAG3PDX2) in the surrounding necrotic lesion tissues (zone 1). On the other hand, PCA showed that the top plant genes STGST and STPDX1.1 were linked to both tissues of necrotic lesions (zone 2) and their surrounding areas of necrotic lesions. Functional characterization of Arabidopsis pdx1.3 mutants challenged with R. solani provided evidence into the role of the vitamin B6 pathway in the maintenance of plant tolerance during disease progression. Overall, we demonstrate that the production of vitamin VB6 is under tight control and is an essential determinant of disease development during the interaction of R. solani with potato cultivars.
... Furthermore, gene expression analyses showed that Arabidopsis pdx3 mutants display a strong upregulation of genes related to plant defense . In addition, increased severity of Botrytis cinerea symptoms have been observed in tomato, in which expression of PDX1.2 and PDX1.3 have been knocked-down (Zhang et al., 2014). ...
... oryzae did not reveal significant differences between control lines and a transgenic line accumulating high levels of vitamin B 6 . This contrasts with the previously reported positive contribution of vitamin B 6 to plant responses to environmental stress in various other species, which were notably members of the eudicot group (Arabidopsis, tomato) (Raschke et al., 2011;Zhang et al., 2014Zhang et al., , 2015. However, it should be noted that the evidence for vitamin B 6 contribution to plant performance under biotic and abiotic stresses mostly comes from mutant lines or virus induced gene-silenced lines impaired in vitamin B 6 biosynthesis (Titiz et al., 2006;Zhang et al., 2014Zhang et al., , 2015. ...
... This contrasts with the previously reported positive contribution of vitamin B 6 to plant responses to environmental stress in various other species, which were notably members of the eudicot group (Arabidopsis, tomato) (Raschke et al., 2011;Zhang et al., 2014Zhang et al., , 2015. However, it should be noted that the evidence for vitamin B 6 contribution to plant performance under biotic and abiotic stresses mostly comes from mutant lines or virus induced gene-silenced lines impaired in vitamin B 6 biosynthesis (Titiz et al., 2006;Zhang et al., 2014Zhang et al., , 2015. It remains to be determined whether the positive contribution of vitamin B 6 to stress responses in planta is due to a direct effect based on its reported antioxidant properties and/or an indirect effect based on its role as a coenzyme, possibly for an enzyme contributing to antioxidant capacities (Fitzpatrick, 2011). ...
Vitamin B6 (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B6 content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B6 in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B6 biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B6 levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B6 biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B6 in leaves (up to 28.3‐fold) and roots (up to 12‐fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B6 did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B6 content could also be achieved in rice seeds (up to 3.1‐fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B6‐enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.
... A previous study has accompanied the participation of VitB6 in HR and disease development [11]. Recently, Zhang et al. [26] showed molecular evidence that the de novo vitB6 biosynthetic pathway but not the salvage pathway was involved in tomato defense response against, Botrytis cinerea because silencing of tomato SlPDX1.2 and SlPDX1.3 but not SlSOS4 led to increased disease severity. This led us to study about E. carotovora may interfere with the VitB6 biosynthesis in tomato plants during its infection process. ...
... Our results supported by Denslow et al. [9] and Tambasco-Studart et al. [6] for PDX2 expression but conflict with those of Denslow et al. [9] and Tambasco-Studart et al. [6] for PDX1.2 and PDX1.3 expression. Similar results were found for low-level expression of SOS4 [26]. However, previous studies with Arabidopsis mutants have shown that the de novo pathway is essential and critical in biosynthesis and maintaining the balance of VitB6 content in plants and that the salvage pathway cannot compensate for the defect of the de novo pathwaydeficient plants [6,8]. ...
... Without inoculation with E. carotovora, the similar activity of SOD and CAT whereas the activity of SOD and CAT was increased after inoculation with E. carotovora. Similar results were also found in tomato, against B. cinerea [26]. Interestingly, as major defensive enzymes, SOD and CAT showed significantly increased activity. ...
Vitamin B6 (VitB6) is an essential cofactor for >140 biochemical reactions. Also, VitB6 is a potent antioxidant and helps plants cope with both biotic and abiotic stress conditions. However, the role of VitB6 in plant disease resistance has yet to be confirmed using molecular biology approaches. Here, we analyzed the expression patterns of VitB6 biosynthetic genes, including the de novo (PDX1 [PDX1.2 and 1.3] and PDX2) and the salvage (SOS4) pathways during the response to Erwinia carotovora subsp. carotovora. By quantitative PCR, we found that the most significant upregulation in the transcript profile of PDX2, which showed a 9.2-fold increase in expression at 12 h post inoculation (hpi) compared to 24-48 hpi. We also detected significant upregulation of PDX1.2 and PDX1.3, which were 6.6- and 4.3-fold upregulated at 24 hpi compared to 12 hpi, while SOS4 showed only low-level expression. Also, at 24 hpi, a significant increase in superoxide dismutase, catalase, peroxidase, and polyphenol oxidase activities was observed in plants. Our findings confirm that the expression of de novo and salvage pathway genes is induced by E. carotovora and that this plays an important role in the regulation of defense response by modulating cellular antioxidant capacity.
... Notably, Met, Tyr, Gln, and Glu were also induced in BBAM-infected plants. Vitamins such as riboflavin and pyridoxal phosphate (PLP) have been also related to defense priming in Arabidopsis against B. cinerea and other pathogens (Zhang et al., 2009(Zhang et al., , 2014(Zhang et al., , 2015. Remarkably, the N required for PLP biosynthesis appears to be supplied by the hydrolysis of GLn to Glu through the salvage biosynthetic pathway of vitamin B6 (Zhang et al., 2014); thus, elevated Glu and PLP levels may be related. ...
... Vitamins such as riboflavin and pyridoxal phosphate (PLP) have been also related to defense priming in Arabidopsis against B. cinerea and other pathogens (Zhang et al., 2009(Zhang et al., , 2014(Zhang et al., , 2015. Remarkably, the N required for PLP biosynthesis appears to be supplied by the hydrolysis of GLn to Glu through the salvage biosynthetic pathway of vitamin B6 (Zhang et al., 2014); thus, elevated Glu and PLP levels may be related. Glu participates in signal transduction from systemic tissues upon wounding (Christmann and Grill, 2013). ...
... Previous reports describe the antifungal and defensive function of many of these compounds, particularly the flavonoid quercetin and chlorogenic acid (Jia et al., 2010). The protective roles of malic acid and vitamin B6, mainly on its vitamer piridoxal phosphate, are also well known (Zhang et al., 2014). Both compounds have been described to participate in induced defense and priming mechanisms (Pastor et al., 2014a;Zhang et al., 2014 respectively). ...
Mycorrhizal plants are generally quite efficient in coping with environmental challenges. It has been shown that the symbiosis with arbuscular mycorrhizal fungi (AMF) can confer resistance against root and foliar pathogens, although the molecular mechanisms underlying such mycorrhiza-induced resistance (MIR) are poorly understood. Tomato plants colonized with the AMF Rhizophagus irregularis display enhanced resistance against the necrotrophic foliar pathogen Botrytis cinerea. Leaves from arbuscular mycorrhizal (AM) plants develop smaller necrotic lesions, mirrored also by a reduced levels of fungal biomass. A plethora of metabolic changes takes place in AMF colonized plants upon infection. Certain changes located in the oxylipin pathway indicate that several intermediaries are over-accumulated in the AM upon infection. AM plants react by accumulating higher levels of the vitamins folic acid and riboflavin, indolic derivatives and phenolic compounds such as ferulic acid and chlorogenic acid. Transcriptional analysis support the key role played by the LOX pathway in the shoots associated with MIR against B. cinerea. Interestingly, plants that have suffered a short period of nitrogen starvation appear to react by reprogramming their metabolic and genetic responses by prioritizing abiotic stress tolerance. Consequently, plants subjected to a transient nitrogen depletion become more susceptible to B. cinerea. Under these experimental conditions, MIR is severely affected although still functional. Many metabolic and transcriptional responses which are accumulated or activated by MIR such NRT2 transcript induction and OPDA and most Trp and indolic derivatives accumulation during MIR were repressed or reduced when tomato plants were depleted of N for 48 h prior infection. These results highlight the beneficial roles of AMF in crop protection by promoting induced resistance not only under optimal nutritional conditions but also buffering the susceptibility triggered by transient N depletion.
... It has been previously shown that H 2 O 2 induced in plant cells, accompanied by O − 2 generation, can promote programmed cell death in the host and expansion of disease lesions to facilitate B. cinerea infection (Govrin and Levine, 2000;Patykowski, 2006;Asai and Yoshioka, 2009;Simon et al., 2013;Zhang et al., 2014). Other studies with A. thaliana, tomato and other plants species (Asselbergh et al., 2007;L'Haridon et al., 2011;Windram et al., 2012;Serrano et al., 2014) have also suggested the importance of increased ROS levels in defense against B. cinerea. ...
... Antioxidative systems are critical for controlling timing and strength of ROS production to maintain redox homeostasis (Torres et al., 2006;Mittler et al., 2011) and for protecting cells from ROS damage (Pallavi Sharma et al., 2012). It has been reported that after B. cinerea infection, A. thaliana (Govrin and Levine, 2000;Simon et al., 2013) and tomato (Asselbergh et al., 2007;Zhang et al., 2014) and Phaseolus vulgaris (Muckenschnabel et al., 1954) continuously accumulate ROS and lesions develop for their insufficient antioxidative systems, and it is nessecery that plants timely modulated its own FIGURE 7 | Activities of catalase (CAT; A), superoxide dismutase (SOD; B) and peroxidases (POD; C) in proteins extracts from "Red Globe" and " 8,12,18,24,36,48,72, and 96 h post-inoculation (hpi) with B. cinerea and sterile water as the control. The means and standard deviations of three independent experiments are shown. ...
... Much attention has been paid to H 2 O 2 induction in plants, which has been conflictingly found to contribute to either increased resistance or susceptibility toward B. cinerea, and on the other hand, O − 2 has generally been suggested to act as a primary substrate to form H 2 O 2 (Govrin and Levine, 2000;Torres et al., 2006;van Kan, 2006;Asselbergh et al., 2007;Serrano et al., 2014). Some reports have suggested that O − 2 plays a role in promoting B. cinerea invasion (Urbanek et al., 1996;Patykowski, 2006;Zhang et al., 2014); however in studies of infected and mock infected tomato leaves, no O − 2 accumulation was observed (Asselbergh et al., 2007). In bean, the induction of O − 2 production in leaves is thought to be one of the key factors that differentiate the interactions with the compatible and incompatible pathogens: B. fabae and B. cinerea, respectively (Urbanek et al., 1996). ...
The necrotrophic fungus Botrytis cinerea is a major threat to grapevine cultivation worldwide. A screen of 41 Vitis genotypes for leaf resistance to B. cinerea suggested species independent variation and revealed 18 resistant Chinese wild Vitis genotypes, while most investigated V. vinifera, or its hybrids, were susceptible. A particularly resistant Chinese wild Vitis, “Pingli-5” (V. sp. [Qinling grape]) and a very susceptible V. vinifera cultivar, “Red Globe” were selected for further study. Microscopic analysis demonstrated that B. cinerea growth was limited during early infection on “Pingli-5” before 24 h post-inoculation (hpi) but not on Red Globe. It was found that reactive oxygen species (ROS) and antioxidative system were associated with fungal growth. O2- accumulated similarly in B. cinerea 4 hpi on both Vitis genotypes. Lower levels of O2- (not H2O2) were detected 4 hpi and ROS (H2O2 and O2-) accumulation from 8 hpi onwards was also lower in “Pingli-5” leaves than in “Red Globe” leaves. B. cinerea triggered sustained ROS production in “Red Globe” but not in “Pingli-5” with subsequent infection progresses. Red Globe displayed little change in antioxidative activities in response to B. cinerea infection, instead, antioxidative activities were highly and timely elevated in resistant “Pingli-5” which correlated with its minimal ROS increases and its high resistance. These findings not only enhance our understanding of the resistance of Chinese wild Vitis species to B. cinerea, but also lay the foundation for breeding B. cinerea resistant grapes in the future.
... H 2 O 2 higher or lower levels increase either the susceptibility or resistance respectively to B. cinerea, while, O 2 − serves as a first substrate for H 2 O 2 formation [13,30,31]. Some reports have suggested that O 2 − plays a role in supporting B. cinerea invasion [32,33]. H 2 O 2 production is induced in plant cells, accompanied by O 2 − generation, which can promote programmed cell death and disease lesion development, thereby increasing B. cinerea infection [27]. ...
... We propose that the high and low levels of ROS in 'Riesling' and 'Zi Qiu' contribute to their susceptibility and resistance to B. cinerea infection, respectively [34]. We evaluated ROS accumulation and antioxidant enzyme activities during the interactions with B. cinerea [33]. Low ROS production and a timely increase in the activity of anti-oxidative enzymes were associated with the strong pathogen resistance of 'Pingli-5' and the HS cultivar 'Red Globe', which suffers from severe infection and sustained ROS production correlated with comparatively unchanged anti-oxidative activities [11]. ...
Botrytis cinerea is a necrotrophic fungal phytopathogen with devastating effects on many Vitis genotypes. Here, a screening of 81 Vitis genotypes for leaf resistance to B. cinerea revealed two highly resistant (HR), twelve resistant (R), twenty-five susceptible (S) and forty-two highly susceptible (HS) genotypes. We focused on the HR genotype, ‘Zi Qiu’ (Vitis davidii), and the HS genotype ‘Riesling’ (V. vinifera), to elucidate mechanisms of host resistance and susceptibility against B. cinerea, using detached leaf assays. These involved a comparison of fungal growth, reactive oxygen species (ROS) responses, jasmonic acid (JA) levels, and changes in the anti-oxidative system between the two genotypes after inoculation with B. cinerea. Our results indicated that the high-level resistance of ‘Zi Qiu’ can be attributed to insignificant fungal development, low ROS production, timely elevation of anti-oxidative functions, and high JA levels. Moreover, severe fungal infection of ‘Riesling’ and sustained ROS production coincided with relatively unchanged anti-oxidative activity, as well as low JA levels. This study provides insights into B. cinerea infection in grape, which can be valuable for breeders by providing information for selecting suitable germplasm with enhanced disease resistance.
... H 2 O 2 higher or lower levels increase either the susceptibility or resistance respectively to B. cinerea, while, O 2 − serves as a first substrate for H 2 O 2 formation [13,30,31]. Some reports have suggested that O 2 − plays a role in supporting B. cinerea invasion [32,33]. H 2 O 2 production is induced in plant cells, accompanied by O 2 − generation, which can promote programmed cell death and disease lesion development, thereby increasing B. cinerea infection [27]. ...
... We propose that the high and low levels of ROS in 'Riesling' and 'Zi Qiu' contribute to their susceptibility and resistance to B. cinerea infection, respectively [34]. We evaluated ROS accumulation and antioxidant enzyme activities during the interactions with B. cinerea [33]. Low ROS production and a timely increase in the activity of anti-oxidative enzymes were associated with the strong pathogen resistance of 'Pingli-5' and the HS cultivar 'Red Globe', which suffers from severe infection and sustained ROS production correlated with comparatively unchanged anti-oxidative activities [11]. ...
... Nine SlTPSs (SlTPS1, SlTPS3, SlTPS4, SlTPS5, SlTPS6, SlTPS7, SlTPS8, SlTPS9, and SlTPS10), 4 SlTPPs (SlTPP2, SlTPP3, SlTPP4, and SlTPP8) and SlTRE1, which have EST or full-length cDNA supports (Table 1), were selected for further functional analysis. As a first step, we examined the expression of the selected SlTPS, SlTPS and SlTRE genes in tomato plants at 48 or 36 h after inoculation with B. cinerea or Pst DC3000, as the pathogens normally colonize and proliferate in the inoculated leaves at these time points (Li et al., 2014bZhang et al., 2014). At 48 h after inoculation with B. cinerea, the expression of SlTPS4, SlTPS6, and SlTPS10 was significantly upregulated, leading to 3.7∼6.3-fold ...
... In our VIGS assays, the silencing efficiency for individual target gene of the 14 selected SlTPSs, SlTPPs and SlTRE1 was estimated to be 61-72% (Figure 3A), which is similar to those observed in our previous studies (Li et al., 2014a,b;Liu et al., 2014;Zhang et al., 2014). Silencing specificity of SlTPS3, SlTPS4, SlTPS5, SlTPS7, or SlTPP2 ( Figure 3B) demonstrates that the altered phenotypes in growth and disease resistance observed in the present study were the consequences of the silencing of specific individual SlTPS or SlTPP genes. ...
Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development, and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs, and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs, and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 as well as to defense signaling hormones (e.g., salicylic acid, jasmonic acid, and a precursor of ethylene). Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4, or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7, or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7, and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato.
... Vitamin B6, as an endogenous growth regulator, is considered an antioxidant in plants, influencing physiological metabolism, growth, development, and stress resistance (Colinas et al., 2016;Moccand et al., 2014;Zhang et al., 2014;Huang et al., 2013). The PDX family genes, key players in the vitamin B6 synthesis pathway, have been shown to be involved in responding to various environmental stresses in plants. ...
The PDX gene is a key gene in the vitamin B6 synthesis pathway, playing a crucial role in plant growth, development, and stress tolerance. To explore the family characteristics of the PDX gene in Brassica napus (B. napus) and its regulatory function under waterlogging stress, this study used five PDX genes from Arabidopsis thaliana as the basis for sequence analysis. Thirteen, eight, and six PDX genes were identified in B. napus, Brassica oleracea (B. oleracea), and Brassica rapa (B. rapa), respectively. Bioinformatics study reveals high conservation of PDX subfamily genes during evolution, and PDX genes in B. napus respond to waterlogging stress.In order to further investigate the effect of the PDX gene on waterlogging tolerance in B. napus, expression analysis was conducted on BnaPDX1.3 gene overexpressing B. napus plants and wild-type plants. The study showed that overexpressing plants could synthesize more VB6 under waterlogging stress, exhibit stronger antioxidant enzyme activity, and have a more effective and stable ROS scavenging system, thus exhibiting a healthier phenotype. These findings suggested that the BnaPDX1.3 gene can enhance the waterlogging tolerance of B. napus, which is of great significance for its response to waterlogging stress. Our study provides a basic reference for further research on the regulation mechanism of the PDX gene and waterlogging tolerance in B. napus.
... A class of interconverting vitamins, comprising pyridoxine, pyridoxamine, pyridoxal, and its phosphorylated derivatives, is collectively referred to as vitamin B6 (Vincent and Munné-Bosch, 2022). According to Zhang et al. (2014Zhang et al. ( , 2015, vitamin B6 has been shown to contribute to plant disease resistance against Pseudomonas syringae and Botrytis cinerea. Plants lacking in vitamin B6 produce more reactive oxygen species (ROS) and eventually experience oxidative damage (Bagri et al., 2018). ...
The purpose of this study was to determine the proximate composition and nutritional profile of ripe and unripe Persea americana (Mill) (avocado pears), grown in Imo State, Nigeria. The P. americana (Zutano variation) was purchased from Ihiagwa Market, Owerri West, Imo State, Nigeria. Some of the P. americana fruit was allowed to ripe within three days and the pulps were dried in the laboratory. Proximate composition (ash, crude protein, fat, fibre, total carbohydrate and moisture content) of both the ripe and unripe avocado pear were determined. Furthermore, the nutritional profile was established, including the levels of minerals (Ca and Mg) and vitamins (A, B3, B6, C, D, and K). From the findings, ash, crude protein, fat, fibre, total carbohydrate and moisture content for the unripe avocado pear were: 0.46, 2.73, 9.94, 4.90, 11.74, and 70.23 %, respectively, while the ripe sample were: 1.05, 2.99, 15.98, 4.43, 7.96, and 67.59 % respectively. The Mg and Ca in the ripe avocado were: 30.36 and 28.87 mg/kg respectively, while for unripe sample are: 16.47 and 15.42 mg/kg). The vitamin A, B3, B6, C, D, and K for the unripe sample were 10.05, 0.65, 0.24, 71.91, 5.46, and 2.70 mg/100g respectively, while the ripe avocado pear were 7.68, 0.44, 0.24, 65.69, 3.82, and 3.47 mg/100g respectively. In conclusion, ripe P. americana provides a rich source of nutritional benefits for magnesium, calcium, and/or lipids. Compared to ripe P. americana, the unripe variety is higher in moisture, fiber, vitamins B3, D, C, and A.
... Vitamin B 6 encompasses a group of vitamins, namely, pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), along with their corresponding phosphate esters, 5'-pyridoxal phosphate (PLP), 5'-pyridoxine phosphate (PNP), and 5'-pyridoxamine phosphate (PMP) [9][10][11]. Vitamin B 6 , also known as VB 6 , plays a crucial role as a cofactor for numerous proteins and enzymes, making it one of the most significant vitamins [12,13]. Most enzymes that rely on pyridoxal phosphate (PLP) as a cofactor [14] are involved in various biochemical processes, such as amino acid biosynthesis, decarboxylation, racemic reactions, Cα-Cβ bond cleavage, elimination, and the replacement of α, β, and γ [15]. ...
Background
Microbial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory.
Results
In this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation.
Conclusion
Our results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.
... VB6 contents in Arabidopsis were lower in response to Pst DC3000 or B. cinerea infection93 . In contrast, B. cinerea infection resulted in 53% increase in VB6 content in tomato plants91 . Despite being contradictory, these results suggest an important role for VB6 in plant ROS management and pathogenicity; the up or down regulation of VB6 might be plant species specific. ...
Aphanomyces euteiches causes root rot in pea, leading to significant yield losses. However, the metabolites involved in this pathosystem have not been thoroughly studied. This study aimed to fill this gap and explore mechanisms of bacterial suppression of A. euteiches via untargeted metabolomics using pea grown in a controlled environment. Chemical isotope labeling (CIL), followed by liquid chromatography-mass spectrometry (LC–MS), was used for metabolite separation and detection. Univariate and multivariate analyses showed clear separation of metabolites from pathogen-treated pea roots and roots from other treatments. A three-tier approach positively or putatively identified 5249 peak pairs or metabolites. Of these, 403 were positively identified in tier 1; 940 were putatively identified with high confidence in tier 2. There were substantial changes in amino acid pool, and fatty acid and phenylpropanoid pathway products. More metabolites, including salicylic and jasmonic acids, were upregulated than downregulated in A. euteiches-infected roots. 1-aminocyclopropane-1-carboxylic acid and 12-oxophytodienoic acid were upregulated in A. euteiches + bacterium-treated roots compared to A. euteiches-infected roots. A great number of metabolites were up- or down-regulated in response to A. euteiches infection compared with the control and A. euteiches + bacterium-treated plants. The results of this study could facilitate improved disease management.
... VB6 contents in Arabidopsis were lower in response to Pst DC3000 or B. cinerea infection (Zhang et al. 2015). In contrast, B. cinerea infection resulted in 53% increase in VB6 content in tomato plants (Zhang et al. 2014). Despite being contradictory, these results suggest an important role for VB6 in plant ROS management and pathogenicity; the up or down regulation of VB6 might be plant species speci c. ...
Aphanomyces euteiches causes root rot in pea, leading to significant yield losses. However, the metabolites involved in this pathosystem have not been thoroughly studied. This study aimed to fill this gap and explore mechanisms of bacterial suppression of A. euteiches via untargeted metabolomics using pea grown in a controlled environment. Chemical isotope labeling (CIL), followed by liquid chromatography-mass spectrometry (LC-MS), was used for metabolite separation and detection. Univariate and multivariate analyses showed clear separation of metabolites from pathogen-treated pea roots and roots from other treatments. A three-tier approach positively or putatively identified 5249 peak pairs or metabolites. Of these, 403 were positively identified in tier 1; 940 were putatively identified with high confidence in tier 2. There were substantial changes in amino acid pool, and fatty acid and phenylpropanoid pathway products. More metabolites, including salicylic and jasmonic acids, were upregulated than downregulated in A. euteiches -infected roots. 1-aminocyclopropane-1-carboxylic acid and 12-oxophytodienoic acid were upregulated in A. euteiches + bacterium-treated roots compared to A. euteiches -infected roots. Most of the metabolites upregulated in response to A. euteiches infection were not different from control levels in A. euteiches + bacterium-treated plants. The results of this study could facilitate improved disease management.
... The loss of GRXs reduces the expression of stress-reactive proteins, resulting in an increased accumulation of ROS in cells [28]. The gene Snz1 encoding the phosphosynthase subunit is associated with vitamin B6 (VB6) biosynthesis, a potent antioxidant, and plays a vital role in development and stress response [29]. In the present study, the up-regulated expression of GRP2, NPY1, PST2, CFL1, FLX1, PEX5, GRX2, GRX3, and Snz1 indicated that M. guilliermondii eliminated intracellular ROS and prevented cell damage caused by PAT toxicity by up-regulating the expression of genes related to the redox process ( Figure 6). ...
Patulin (PAT), mainly produced by Penicillium expansum, is a potential threat to health. In recent years, PAT removal using antagonistic yeasts has become a hot research topic. Meyerozyma guilliermondii, isolated by our group, produced antagonistic effects against the postharvest diseases of pears and could degrade PAT in vivo or in vitro. However, the molecular responses of M. guilliermondii over PAT exposure and its detoxification enzymes are not apparent. In this study, transcriptomics is used to unveil the molecular responses of M. guilliermondii on PAT exposure and the enzymes involved in PAT degradation. The functional enrichment of differentially expressed genes indicated that the molecular response mainly includes the up-regulated expression of genes related to resistance and drug-resistance, intracellular transport, growth and reproduction, transcription, DNA damage repair, antioxidant stress to avoid cell damage, and PAT detoxification genes such as short-chain dehydrogenase/reductases. This study elucidates the possible molecular responses and PAT detoxification mechanism of M. guilliermondii, which could be helpful to further accelerate the commercial application of antagonistic yeast toward mycotoxin decontamination.
... In addition, MoPdx1 was not found in this study to be involved in the oxidative (mainly H 2 O 2 ) stress response of M. oryzae ( Figure S4). A previous study indicated that Pdx1 in Arabidopsis, tomatoes, and C. nicotianae might be an antioxidant (Ehrenshaft et al., 1999;Chen and Xiong, 2005;Zhang et al., 2014). The reason for these differences might be the difference in species. ...
B vitamins are essential micro-organic compounds for the development of humans and animals. Vitamin B6 comprises a group of components including pyridoxine, pyridoxal, and pyridoxamine. In addition, vitamin B6 acts as the coenzymes in amino acid biosynthesis, decarboxylation, racemic reactions, and other biological processes. In this study, we found that the expressions of a gene encoding pyridoxine biosynthesis protein (PDX1) were significantly upregulated in the early infectious stages in M. oryzae. Furthermore, deletion of MoPDX1 slowed vegetative growth on different media, especially on MM media, and the growth defect was rescued when MoPdx1-protein was expressed in mutants strains and when commercial VB6 (pyridoxine) was added exogenously. However, VB6 content in different strains cultured in CM media has no significant difference, suggested that MoPdx1 was involved in de novo VB6 biosynthesis not in uptake process, and VB6 regulates the vegetative growth of M. oryzae. The ΔMopdx1 mutants presented abnormal appressorium turgor, slowed invasive growth and reduced virulence on rice seedlings and sheath cells. MoPdx1 was located in the cytoplasm and present in spore and germ tubes at 14 hours post inoculation (hpi) and then transferred into the appressorium at 24 hpi. Addition of VB6 in the conidial suspentions could rescue the defects of appressorium turgor pressure at 14 hpi or 24 hpi, invasive growth and pathogenicity of the MoPDX1 deletion mutants. Indicated that MoPdx1 affected the appressorium turgor pressure, invasive growth and virulence mainly depended on de novo VB6, and VB6 was biosynthesized in conidia, then transported into the appressorium, which play important roles in substances transportation from conidia to appressorium thus to regulate the appressorium turgor pressure. However, deletion of MoPDX1 did not affect the ability that scavenge ROS produced by rice cells, and the mutant strains were unable to activate host defense responses. In addition, co-immunoprecipitation (Co-IP) assays investigating potential MoPdx1-interacting proteins suggested that MoPdx1 might take part in multiple pathways, especially in the ribosome and in biosynthesis of some substances. These results indicate that vitamins are involved in the development and pathogenicity of M. oryzae.
... Gene annotation showed that most candidate genes were related to disease resistance in plant (Table 3). For qRDI-A01-1, one positively regulated gene and two negatively regulated genes were identified (Fig. 7), the positive regulated gene Ghir_A01G022140 encoding a pyridoxal 5′-phosphate synthase subunit PDX1 involved in vitamin B6 biosynthesis, which is important for disease resistance in tomato and Arabidopsis thaliana (Zhang et al. 2014b(Zhang et al. , 2015b, the negative regulated gene Ghir_A01G022110 coding for 2-oxoglutarate-dependent dioxygenase involved in the flavonoid biosynthesis pathway, which is demonstrated to be related to VW resistance by the fact that the enrichment of flavonoids in a spontaneous cotton mutant with red coloration results in a significantly increase in resistance to Verticillium dahliae (Long et al. 2019), and the negative regulated gene Ghir_A01G022270 coding for an ammonium transporter, which is a negative regulator of Arabidopsis defense responses (Pastor et al. 2014). For qRDI-A05-1, one positive regulated gene and one negative regulated gene were identified (Fig. 7), the positive regulated gene Ghir_A05G008530 encoding a suppressor of K + transport growth defect-like protein (SKD1) able to facilitate K + uptake, which confers enhanced salt and drought stress tolerance in transgenic tobacco (Xia et al. 2013). ...
Key message
Combining GWAS, QTL-seq and transcriptome sequencing detected basal defense-related genes showing gDNA sequence variation and expression difference in diverse cotton lines, which might be the molecular mechanisms of VW resistance in G. hirsutum.
Abstract
Verticillium wilt (VW), which is caused by the soil-borne fungus Verticillium dahliae, is a major disease in cotton (Gossypim hirsutum) worldwide. To facilitate the understanding of the genetic basis for VW resistance in cotton, a genome-wide association study (GWAS), QTL-seq and transcriptome sequencing were performed. The GWAS of VW resistance in a panel of 120 core elite cotton accessions using the Cotton 63K Illumina Infinium SNP array identified 5 QTL from 18 significant SNPs meeting the 5% false discovery rate threshold on 5 chromosomes. All QTL identified through GWAS were found to be overlapped with previously reported QTL. By combining GWAS, QTL-seq and transcriptome sequencing, we identified eight candidate genes showing both gDNA sequence variation and expression difference between resistant and susceptible lines, most related to transcription factors (TFs), flavonoid biosynthesis and those involving in the plant basal defense and broad-spectrum disease resistance. Ten KASP markers were successfully validated in diverse cotton lines and could be deployed in marker-assisted breeding to enhance VW resistance. These results supported our inference that the gDNA sequence variation or expression difference of those genes involving in the basal defense in diverse cotton lines might be the molecular mechanisms of VW resistance in G. hirsutum.
... In our experiment, the SlSKIP target gene silencing efficiency was predicted as around 65% (Figure 3A), close to that obtained in our prior works Liu et al., 2014;Zhang Y. et al., 2014;Zhang et al., 2016). It is previously reported that pathogen infection may induce SKIP expression FIGURE 8 | Silencing of SlSKIP1b affected H 2 O 2 accumulation and SA signaling-responsive DRG expression following Pst DC3000 inoculation. ...
SKIP, a component of the spliceosome, is involved in numerous signaling pathways. However, there is no direct genetic evidence supporting the function of SKIP in defense responses. In this paper, two SKIPs, namely, SlSKIP1a and SlSKIP1b, were analyzed in tomato. qRT-PCR analysis showed that the SlSKIP1b expression was triggered via Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea (B. cinerea), together with the defense-associated signals. In addition, the functions of SlSKIP1a and SlSKIP1b in disease resistance were analyzed in tomato through the virus-induced gene silencing (VIGS) technique. VIGS-mediated SlSKIP1b silencing led to increased accumulation of reactive oxygen species (ROS), along with the decreased expression of defense-related genes (DRGs) after pathogen infection, suggesting that it reduced B. cinerea and Pst DC3000 resistance. There was no significant difference in B. cinerea and Pst DC3000 resistance in TRV-SlSKIP1a-infiltrated plants compared with the TRV-GUS-silencing counterparts. As suggested by the above findings, SlSKIP1b plays a vital role in disease resistance against pathogens possibly by regulating the accumulation of ROS as well as the expression of DRGs.
... H 2 O 2 production is induced in plant cells and accompanied by O2-generation, which can encourage programmed cell death and disease lesion expansion, thus increasing B. cinerea infection [30]. H 2 O 2 in higher or lower contents increase either the susceptibility or resistance, respectively, to B. cinerea, while O2-serves as a first substrate for H 2 O 2 formation [14,29,31] and supports B. cinerea attack [32]. "Summer black" leaves showed minor differences in POD activity with lesion development post-inoculation. ...
The necrotrophic fungus Botrytis cinerea causes devastating pre- and post-harvest yield losses in grapevine (Vitis vinifera L.). Although B. cinerea has been well-studied in different plant species, there is limited information related to the resistance and susceptibility mechanisms of Vitis genotypes against B. cinerea infection. In the present study, leaves and berries of twenty four grape genotypes were evaluated against B. cinerea infection. According to the results, one genotype (Ju mei gui) was highly resistant (HR), one genotype (Kyoho) was resistant (R), eight genotypes were susceptible (S), and fourteen genotypes were highly susceptible (HS) against infection of B. cinerea in leaves. Whereas in the case of B. cinerea infection in grape berry, three genotypes were found to be highly resistant, three resistant, eleven genotypes susceptible, and seven were highly susceptible. To further explore the mechanism of disease resistance in grapevine, we evaluated “Ju mei gui” and “Summer black” in terms of B. cinerea progression, reactive oxygen species reactions, jasmonic acid contents, and the activities of antioxidant enzymes in leaf and fruit. We surmise that the resistance of “Ju mei gui” is due to seized fungal growth, minor reactive oxygen species (ROS) production, elevated antioxidant enzyme activity, and more jasmonic acid (JA) contents. This study provides insights into the resistance and susceptibility mechanism of Vitis genotypes against B. cinerea. This will help for the selection of appropriate germplasm to explore the molecular basis of disease resistance mechanisms in grapevine.
... Nonetheless, recent studies of PDX1.2 have revealed a number of interesting features that include its restriction to eudicota and its important contribution to plant fitness [17]. Studies in which expression of PDX1.2 was knocked down by RNA interference have rendered the plants more susceptible to disease, e.g. in tomato [32] and in Arabidopsis [31]; or more sensitive to abiotic stress, e.g. in Arabidopsis [17]. Interestingly, one study has reported that PDX1.2 is essential for embryogenesis in Arabidopsis [14], based on the analysis of a T-DNA insertion mutant line of the SAIL collection [25]. ...
Background:
PDX1.2 has recently been shown to be a regulator of vitamin B6 biosynthesis in plants and is implicated in biotic and abiotic stress resistance. PDX1.2 expression is strongly and rapidly induced by heat stress. Interestingly, PDX1.2 is restricted to eudicota, wherein it behaves as a non-catalytic pseudoenzyme and is suggested to provide an adaptive advantage to this clade. A first report on an Arabidopsis insertion mutant claims that PDX1.2 is indispensable for viability, being essential for embryogenesis. However, a later study using an independent insertion allele suggests that knockout mutants of pdx1.2 are viable. Therefore, the essentiality of PDX1.2 for Arabidopsis viability is a matter of debate. Given the important implications of PDX1.2 in stress responses, it is imperative to clarify if it is essential for plant viability.
Results:
We have studied the previously reported insertion alleles of PDX1.2, one of which is claimed to be essential for embryogenesis (pdx1.2-1), whereas the other is viable (pdx1.2-2). Our study shows that pdx1.2-1 carries multiple T-DNA insertions, but the T-DNA insertion in PDX1.2 is not responsible for the loss of embryogenesis. By contrast, the pdx1.2-2 allele is an overexpressor of PDX1.2 under standard growth conditions and not a null allele as previously reported. Nonetheless, upregulation of PDX1.2 expression under heat stress is impaired in this mutant line. In wild type Arabidopsis, studies of PDX1.2-YFP fusion proteins show that the protein is enhanced under heat stress conditions. To clarify if PDX1.2 is essential for Arabidopsis viability, we generated several independent mutant lines using the CRISPR-Cas9 gene editing technology. All of these lines are viable and behave similar to wild type under standard growth conditions. Reciprocal crosses of a subset of the CRISPR lines with pdx1.2-1 recovers viability of the latter line and demonstrates that knocking out the functionality of PDX1.2 does not impair embryogenesis.
Conclusions:
Gene editing reveals that PDX1.2 is dispensable for Arabidopsis viability and resolves conflicting reports in the literature on its function.
... Thus, in plants, the relatively newly discovered antioxidant activity of VitB6 has increased the interest of understanding the VitB6 function under different types of environmental stresses (Vanderschuren et al., 2013). This includes salt stress (Chen and Xioang, 2005;Titiz et al., 2006), osmotic stress (Chen and Xiong, 2005), photoinhibition ( Titiz et al., 2006;Havaux et al., 2009), and biotic stress and disease resistance ( Zhang et al., 2014;2015). Furthermore, VitB6 has been shown to play an important role in plant responses to ultraviolet-B radiation (UV-B; 280-315 nm), which is a naturally occurring part of the solar spectrum. ...
Pyridoxine (vitamin B6) and its vitamers are used by living organisms both as enzymatic cofactors and as antioxidants. We used Arabidopsis pyridoxine biosynthesis mutant pdx1.3-1 to study the involvement of the PLP-synthase main polypeptide PDX1 in plant responses to ultraviolet radiation of two different qualities, one containing primarily UV-A (315–400 nm) and the other containing both UV-A and UV-B (280–315 nm). The antioxidant capacity and the flavonoid and glucosinolate (GS) profiles were examined. As an indicator of stress, Fv/Fm of photosystem II reaction centers was used. In pdx1.3-1, UV-A + B exposure led to a significant 5% decrease in Fv/Fm on the last day (day 15), indicating mild stress at this time point. The antioxidant capacity of Col-0 wildtype increased significantly (50–73%) after 1 and 3 days of UV-A + B. Instead, in pdx1.3-1, the antioxidant capacity significantly decreased by 44–52% over the same time period, proving the importance of a full complement of functional PDX1 genes for the detoxification of reactive oxygen species. There were no significant changes in the flavonoid glycoside profile under any light condition. However, the GS profile was significantly altered, both with respect to Arabidopsis accession and exposure to UV. The difference in flavonoid and GS profiles reflects that the GS biosynthesis pathway contains at least one pyridoxine-dependent enzyme, whereas no such enzyme is used in flavonoid biosynthesis. Also, there was strong correlation between the antioxidant capacity and the content of some GS compounds. Our results show that vitamin B6 vitamers, functioning both as antioxidants and co-factors, are of importance for the physiological fitness of plants.
... The literature available for most of the crop plants shows the manifestation of both de novo and salvage pathways in VitB6 genes for enhanced disease resistance upon phytopathogens infection. Recently, Zhang et al. 52 proved that Botrytis cinerea-infected tomato plants show the involvement of the de novo vitB6 biosynthetic pathway and not salvage pathway by affirming SlPDX1.2 and SlPDX1.3 genes but not SlSOS4 through gene silencing studies based on disease severity. ...
Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.
... This gene has been reported previously as OsAAP6, which affects rice nutritional quality by enhancing grain protein content ( Peng et al., 2014). Moreover, numerous mQTL for metabolites that may confer disease resistance or plant defense activities, such as flavonoids (Treutter, 2006;Luo et al., 2009;Saito et al., 2013), Ala ( Park et al., 2009), His ( Seo et al., 2016), serotonin ( , vitamin B6 ( Zhang et al., 2014Zhang et al., , 2015, methyl salicylate ( Park et al., 2007), and zeatin (Choi et al., 2011;Grosskinsky et al., 2013), were mapped with possible candidate genes assigned or validated (Supplemental Table S8). ...
Metabolomic analysis coupled with advanced genetic populations represents a powerful tool to investigate the plant metabolome. However, genetic analyses of the rice (Oryza sativa) metabolome have been conducted mainly using natural accessions or a single biparental population. Here, the flag leaves from three inter-connected chromosome segment substitution line (CSSL) populations with a common recurrent genetic background were used to dissect rice metabolic diversity. We effectively used multiple inter-connected biparental populations, constructed by introducing genomic segments into Zhenshan 97 from ACC10 (A/Z), Minghui 63 (M/Z), and Nipponbare (N/Z), to map metabolic quantitative trait loci (mQTL). A total of 1,587 mQTL were generated, of which 684, 479, and 722 were obtained from the A/Z, M/Z, and N/Z CSSL populations, respectively, and we designated 99 candidate genes for 367 mQTL. In addition, 1,001 mQTL were specifically generated from joint linkage analysis with 25 candidate genes assigned. Several of these candidates were validated, such as LOC_Os07g01020 for the in vivo content of pyridoxine and its derivative, and LOC_Os04g25980 for cis-zeatin glucosyltransferase activity. We propose a novel biosynthetic pathway for O-methylapigenin C-pentoside, and demonstrated that LOC_Os04g11970 encodes a component of this pathway through fine mapping. We postulate that the methylated apigenin may confer plant disease resistance. This study demonstrates the power of using multiple inter-connected populations to generate a large number of veritable mQTL. The combined results are discussed in the context of functional metabolomics and possible features of assigned candidates underlying respective metabolites.
... Vitamin B 6 is a potent antioxidant and has been demonstrated in plants to be critical for ameliorating high-light stress (Havaux et al., 2009). In addition, several groups reported that the vitamin contributes to pathogen resistance in plants (Denslow et al., 2007;Herrero et al., 2007;Titiz et al., 2006;Zhang et al., 2014Zhang et al., , 2015. For example, tomato and Arabidopsis mutants with reduced PLP biosynthesis become hyper-susceptible to infection with the fungus Botrytis cinerea or the bacterium P. syringae, respectively . ...
... The B6 vitamers' protective role against biotic stress has also been reported . The modulation in mycorrhizal plants of B6 vitamer accumulation upon infection with the foliar pathogen Botrytis cinerea (Sanchez-Bel et al., 2016) is remarkable, as vitB6 itself can induce defense priming against this pathogen (Zhang et al., 2014). All these results suggest a central role of B6 vitamers in the defense priming associated with the AM symbiosis that deserves further investigation. ...
Arbuscular mycorrhizal ( AM ) symbioses can improve plant tolerance to multiple stresses. We compared three AM fungi ( AMF ) from different genera, one of them isolated from a dry and saline environment, in terms of their ability to increase tomato tolerance to moderate or severe drought or salt stress. Plant physiological parameters and metabolic profiles were compared in order to find the molecular mechanisms underlying plant protection against stress.
Mycorrhizal growth response was determined, and ultrahigh‐performance LC‐MS was used to compare the metabolic profile of plants under the different treatments.
All AMF increased plant tolerance to stress, and the positive effects of the symbiosis were correlated with the severity of the stress. The AMF isolated from the stressful environment was the most effective in improving plant tolerance to salt stress. Differentially accumulated compounds were identified and the antistress properties of some of them were confirmed.
We demonstrate that AM symbioses increase plant metabolic plasticity to cope with stress. Some responses were common to all AMF tested, while others were specifically related to particular isolates. Important metabolism reprograming was evidenced upon salt stress, and we identified metabolic pathways and compounds differentially accumulated in mycorrhizas that may underlie their enhanced tolerance to stress.
... These results are supported by the over expression of the antioxidant VB6 genes (i.e., PDX) in Arabidopsis that led to increased tolerance to oxidative stress [24,56]. Equally Arabidposis mutants with defects in vitamin B6 de novo biosynthetic pathway (PDX1.2 or PDX1.3) showed increased levels of disease of gray mold caused by Botrytis cinerea [57,58]. Taken together, these results support the novel function of vitamin B6 genes as antioxidant stress protector against ROS to prevent oxidative stress. ...
Biotic stress, as a result of plant-pathogen interactions, induces the accumulation of reactive oxygen species in the cells, causing severe oxidative damage to plants and pathogens. To overcome this damage, both the host and pathogen have developed antioxidant systems to quench excess ROS and keep ROS production and scavenging systems under control. Data on ROS-scavenging systems in the necrotrophic plant pathogen Rhizoctonia solani are just emerging. We formerly identified vitamin B6 biosynthetic machinery of R. solani AG3 as a powerful antioxidant exhibiting a high ability to quench ROS, similar to CATALASE (CAT) and GLUTATHIONE S-TRANSFERASE (GST). Here, we provide evidence on the involvement of R. solani vitamin B6 biosynthetic pathway genes; RsolPDX1 (KF620111.1), RsolPDX2 (KF620112.1), and RsolPLR (KJ395592.1) in vitamin B6 de novo biosynthesis by yeast complementation assays. Since gene expression studies focusing on oxidative stress responses of both the plant and the pathogen following R. solani infection are very limited, this study is the first coexpression analysis of genes encoding vitamin B6, CAT and GST in plant and fungal tissues of three pathosystems during interaction of different AG groups of R. solani with their respective hosts. The findings indicate that distinct expression patterns of fungal and host antioxidant genes were correlated in necrotic tissues and their surrounding areas in each of the three R. solani pathosystems: potato sprout-R. solani AG3; soybean hypocotyl-R. solani AG4 and soybean leaves-R. solani AG1-IA interactions. Levels of ROS increased in all types of potato and soybean tissues, and in fungal hyphae following infection of R. solani AGs as determined by non-fluorescence and fluorescence methods using H2DCF-DA and DAB, respectively. Overall, we demonstrate that the co-expression and accumulation of certain plant and pathogen ROS-antioxidant related genes in each pathosystem are highlighted and might be critical during disease development from the plant’s point of view, and in pathogenicity and developing of infection structures from the fungal point of view.
... A number of genes encoding receptor-like protein kinase TPK1b, transcriptional factors SHINE3, AIM1, SlDRW1, SlSRN1, SlSR1, and SlSR3L (Abuqamar et al., 2008(Abuqamar et al., , 2009Buxdorf et al., 2014;Liu et al., 2014a,b), histone H2B monoubiquitination enzymes SlHUB1 and SlHUB2 , mitogen-activated protein kinase kinase SlMKK2 and SlMKK4 , phosphatidylinositolphospholipase SlPLC2 (Gonorazky et al., 2016), NADPH oxidase SlRbohB , 12-oxophytodienoate reductase SlOPR3 and matrix metalloproteinase Sl3-MMP have been identified to play important roles in tomato immunity against B. cinerea. Enzymes involved in biosynthesis of vitamin B6 and trehalose-6-phosphate as well as concurrent over-activation of cytosolic glutamine synthetase and γ-aminobutyric acid shunt are also involved in tomato immune response to B. cinerea (Seifi et al., 2013;Zhang et al., 2014Zhang et al., , 2016. Furthermore, simultaneous suppression of both polygalacturonase and expansin or accumulation of anthocyanin decreased the susceptibility of ripening fruits to B. cinerea (Cantu et al., 2008;Zhang et al., 2013). ...
The Ethylene-Responsive Factors (ERFs) comprise a large family of transcriptional factors that play critical roles in plant immunity. Gray mold disease caused by Botrytis cinerea, a typical necrotrophic fungal pathogen, is the serious disease that threatens tomato production worldwide. However, littler is known about the molecular mechanism regulating the immunity to B. cinerea in tomato. In the present study, virus-induced gene silencing (VIGS)-based functional analyses of 18 members of B3 group (also called Group IX) in tomato ERF family were performed to identify putative ERFs that are involved in disease resistance against B. cinerea. VIGS-based silencing of either SlERF.B1 or SlERF.C2 had lethal effect while silencing of SlERF.A3 (Pit4) significantly suppressed vegetative growth of tomato plants. Importantly, silencing of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 resulted in increased susceptibility to B. cinerea, attenuated the B. cinerea-induced expression of jasmonic acid/ethylene-mediated signaling responsive defense genes and promoted the B. cinerea-induced H2O2 accumulation. However, silencing of SlERF.A3 also decreased the resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 but silencing of SlERF.A1, SlERF.B4 or SlERF.C3 did not affect the resistance to this bacterial pathogen. Expression of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 was induced by B. cinerea and by defense signaling hormones such as salicylic acid, methyl jasmonate, and 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor). SlERF.A1, SlERF.B4, SlERF.C3, and SlERF.A3 proteins were found to localize in nucleus of cells and possess transactivation activity in yeasts. These data suggest that SlERF.A1, SlERF.B4, and SlERF.C3, three previously uncharacterized ERFs in B3 group, and SlERF.A3, a previously identified ERF with function in immunity to Pst DC3000, play important roles in resistance against B. cinerea in tomato.
... Pyridoxine biosynthesis was important to mediate defence activation against B. cinerea in tomato plants. Pyridoxine biosynthetic gene expressions were induced in the tomato leaves inoculated by B. cinerea, whilst tomato plants silencing these gene expressions exhibited lowered pyridoxine content followed by highly accumulated reactive oxygen species (ROS) in response to B. cinerea infection (Zhang et al., 2014). The increased ROS may facilitate the necrotrophic fungal invasion into the host tissues much easier (Lehmann et al., 2015). ...
Bacterial wilt and grey mould in tomato plants are economically destructive bacterial and fungal diseases caused by Ralstonia solanacearum and Botrytis cinerea, respectively. Various approaches including chemical and biological controls have been attempted to arrest the tomato diseases so far. In this study, in vitro growths of bacterial R. solanacearum and fungal B. cinerea were evaluated using four different vitamins including thiamine (vitamin B1), niacin (vitamin B3), pyridoxine (vitamin B6), and menadione (vitamin K3). In planta efficacies of the four vitamin treatments on tomato protection against both diseases were also demonstrated. All four vitamins showed different in vitro antibacterial activities against R. solanacearum in dose-dependent manners. However, treatment with 2 mM thiamine was only effective in reducing bacterial wilt of detached tomato leaves without phytotoxic-ity under lower disease pressure (10⁶ colony-forming unit [cfu]/ml). Treatment with the vitamins also differ-entially reduced in vitro conidial germination and mycelial growth of B. cinerea. The four vitamins slightly reduced the conidial germination, and thiamine, pyridoxine and menadione inhibited the mycelial growth of B. cinerea. Menadione began to drastically suppress the conidial germination and mycelial growth by 5 and 0.5 mM, respectively. Grey mould symptoms on the inoculated tomato leaves were significantly reduced by pyridoxine and menadione pretreatments one day prior to the fungal challenge inoculation. These findings suggest that disease-specific vitamin treatment will be integrated for eco-friendly management of tomato bacterial wilt and grey mould.
Berries of 41 Vitis genotypes were evaluated for resistance to B. cinerea. Evaluation revealed that four genotypes were highly resistant (HR), eight resistant (R), eighteen susceptible (S) and eleven highly susceptible (HS). We further evaluated HR genotype 'Dong fang zhi xing' and HS genotype 'Gold finger' by comparing the fungal growth, reactive oxygen species (ROS) responses , jasmonic acid (JA) levels, anti-oxidants, e.g., Peroxidase (POD), Superoxide dismutase (SOD) and Malondialhydrate (MDA) content changes after infection with B. cinerea. Our results confirmed that the elevated resistance of 'Dong fang zhi xing' was due to weak fungal development, low ROS production, timely elevation of anti-oxidative functions, and high JA levels. Moreover, HS 'Gold finger' infection was severe and sustained ROS production which may be due to its relatively unchanged anti-oxidative activities and low JA level. Our results could help grape breeders to select suitable germplasm for future research work. K e y w o r d s : Vitis vinifera L.; resistant levels; jasmonic acid; ROS; Anti-oxidant; microscopy.
Xanthomonas oryzae pv. oryzicola (Xoc), causing rice bacterial leaf streak, invades leaves mainly through stomata, which are often closed as a plant immune response against pathogen invasion. How Xoc overcomes stomatal immunity is unclear. Here, we show that the effector protein AvrRxo1, an ATP-dependent protease, enhances Xoc virulence and inhibits stomatal immunity by targeting and degrading rice OsPDX1 (pyridoxal phosphate synthase), thereby reducing vitamin B6 (VB6) levels in rice. VB6 is required for the activity of aldehyde oxidase, which catalyzes the last step of abscisic acid (ABA) biosynthesis, and ABA positively regulates rice stomatal immunity against Xoc. Thus, we provide evidence supporting a model in which a major bacterial pathogen inhibits plant stomatal immunity by directly targeting VB6 biosynthesis and consequently inhibiting the biosynthesis of ABA in guard cells to open stomata. Moreover, AvrRxo1-mediated VB6 targeting also explains the poor nutritional quality such as vitamin B6 of Xoc-infected rice grains.
Vitamin B6 plays a crucial role in plant metabolism. It acts as a cofactor in over 140 reactions and has a potent antioxidant function, as described in plant growth and development related to root and leaf tissues, but not yet in fruit ripening. Here, we aimed to explore simple practices to improve endogenous content of vitamin B6 in avocados, measured by high-performance liquid chromatography (HPLC), and to evaluate to what extent and through which mechanisms pyridoxal-5’-phosphate (PLP) treatment influences fruit quality during postharvest ripening and cold storage. Emphasis was put on evaluating a possible link between vitamin B6 and phytohormones by performing hormonal profiling analyses using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). We hypothesized that PLP could improve quality parameters and prolong the shelf life of fruit. Results showed that Bacon avocados had a low vitamin B6 content compared to Hass and other varieties. However, basic techniques like short-term cold storage increased the vitamin B6 content 2.5 fold. Exogenous application of vitamin B6 slightly delayed avocado ripening at room temperature and caused reduced softening in the mesocarp, which was related to a hormonal response that was most likely mediated by cytokinins. In long-term cold-stored avocados, PLP treatment triggered γ-tocotrienol and plastochromanol-8 accumulation, which improved the tocochromanol composition in the fruit. In conclusion, the vitamin B6 content of Bacon avocados can be improved through simple practices. Exogenous applications of PLP can be used either to delay ripening of avocados at room temperatures or to increase tocochromanol content in cold-stored fruit.
Microbes are natural chemical factories and their metabolome comprise diverse arrays of chemicals. The genus Xanthomonas comprises some of the most important plant pathogens causing devastating yield losses globally and previous studies suggested that species in the genus are untapped chemical minefields. In this study, we applied an untargeted metabolomics approach to study the metabolome of a globally spread important xanthomonad, X. perforans. The pathogen is difficult to manage, but recent studies suggest that the small molecule carvacrol was efficient in disease control. Bacterial strains were treated with carvacrol, and samples were taken at time intervals (1 and 6 h). An untreated control was also included. There were five replicates for each sample and samples were prepared for metabolomics profiling using the standard procedure. Metabolomics profiling was carried out using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) and an autosampler. Annotation of significant metabolites using the Metabolomics Standards Initiative level 2 identified an array of novel metabolites that were previously not reported in Xanthomonas perforans. These metabolites include methoxybrassinin and cyclobrassinone, which are known metabolites of brassicas; sarmentosin, a metabolite of the Passiflora-heliconiine butterfly system; and monatin, a naturally occurring sweetener found in Sclerochiton ilicifolius. To our knowledge, this is the first report of these metabolites in a microbial system. Other significant metabolites previously identified in non-Xanthomonas systems but reported in this study include maculosin; piperidine; β-carboline alkaloids, such as harman and derivatives; and several important medically relevant metabolites, such as valsartan, metharbital, pirbuterol, and ozagrel. This finding is consistent with convergent evolution found in reported biological systems. Analyses of the effect of carvacrol in time-series and associated pathways suggest that carvacrol has a global effect on the metabolome of X. perforans, showing marked changes in metabolites that are critical in energy biosynthesis and degradation pathways, amino acid pathways, nucleic acid pathways, as well as the newly identified metabolites whose pathways are unknown. This study provides the first insight into the X. perforans metabolome and additionally lays a metabolomics-guided foundation for characterization of novel metabolites and pathways in xanthomonad systems.
Pseudoenzymes have emerged as key regulatory elements in all kingdoms of life despite being catalytically nonactive. Yet many factors defining why one protein is active while its homologue is inactive remain uncertain. For pseudoenzyme-enzyme pairs, the similarity of both subunits can often hinder conventional characterization approaches. In plants, a pseudoenzyme, PDX1.2, positively regulates vitamin B6 production by association with its active catalytic homologues such as PDX1.3 through an unknown assembly mechanism. Here we used an integrative experimental approach to learn that such pseudoenzyme-enzyme pair associations result in heterocomplexes of variable stoichiometry, which are unexpectedly tunable. We also present the atomic structure of the PDX1.2 pseudoenzyme as well as the population averaged PDX1.2-PDX1.3 pseudoenzyme-enzyme pair. Finally, we dissected hetero-dodecamers of each stoichiometry to understand the arrangement of monomers in the heterocomplexes and identified symmetry-imposed preferences in PDX1.2-PDX1.3 interactions. Our results provide a new model of pseudoenzyme-enzyme interactions and their native heterogeneity.
Upon fungal and bacterial pathogen attack, plants launch pattern‐triggered immunity (PTI) by recognizing pathogen‐associated molecular patterns (PAMPs) to defend against pathogens. Although PTI‐mediated response has been widely studied, a systematic understanding of the reprogrammed cellular processes during PTI by multi‐omics analysis is lacking. In this study, we generated metabolome, transcriptome, proteome, ubiquitome, and acetylome data to investigate rice (Oryza sativa) PTI responses to two PAMPs, the fungi‐derived chitin and the bacteria‐derived flg22. Integrative multi‐omics analysis uncovered convergence and divergence of rice responses to these PAMPs at multiple regulatory layers. Rice responded to chitin and flg22 in a similar manner at the transcriptome and proteome levels, but distinct at the metabolome level. We found that this was probably due to post‐translational regulation including ubiquitination and acetylation, which reshaped gene expression by modulating enzymatic activities, and possiby led to distinct metabolite profiles. We constructed regulatory atlas of metabolic pathways, including the defence‐related phenylpropanoid and flavonoid biosynthesis and linoleic‐acid derivative metabolism. The multi‐level regulatory network generated in this study sets the foundation for in‐depth mechanistic dissection of PTI in rice and potentially in other related poaceous crop species.
DEK involves in the modulation of cell proliferation, differentiation, apoptosis, migration and cell senescence. However, direct genetic evidence proving the functions of DEK in disease resistance against pathogens is still deficient. In the present study, four DEKs were identified in tomato genome and their roles in disease resistance in tomato were analyzed. The expression levels of DEKs were differently induced by Botrytis cinerea, Pseudomonas syringae pv. tomato (Pst) DC3000 and defense-related signaling molecules (such as jasmonic acid, aethylene precursor and salicylic acid). The DEKs' silencing by virus induced gene silencing led to decreased resistance against B. cinerea or Pst DC3000. The underlying mechanisms may be through the upregulation of the accumulation of reactive oxygen species (ROS) and the changed expression levels of defense-related genes by pathogen inoculation. These results indicate that DEKs involve in disease resistance against different pathogens and thus broaden the knowledge of DEK genes' function in tomato.
Berries of 41 Vitis genotypes were evaluated for
resistance to B. cinerea. Evaluation revealed that four
genotypes were highly resistant (HR), eight resistant (R),
eighteen susceptible (S) and eleven highly susceptible
(HS). We further evaluated HR genotype 'Dong fang
zhi xing' and HS genotype 'Gold fnger' by comparing
the fungal growth, reactive oxygen species (ROS) responses, jasmonic acid (JA) levels, anti-oxidants, e.g.,
Peroxidase (POD), Superoxide dismutase (SOD) and
Malondialhydrate (MDA) content changes after infection
with B. cinerea. Our results confrmed that the elevated
resistance of 'Dong fang zhi xing' was due to weak fungal
development, low ROS production, timely elevation of
anti-oxidative functions, and high JA levels. Moreover,
HS 'Gold fnger' infection was severe and sustained ROS
production which may be due to its relatively unchanged
anti-oxidative activities and low JA level. Our results
could help grape breeders to select suitable germplasm
for future research work.
Stress-associated proteins (SAPs) are A20 and AN1 domain-containing proteins, some of which play important roles in plant stress signaling. Here, we report the involvement of tomato SlSAP family in immunity. SlSAPs responded with different expression patterns to Botrytis cinerea and defense signaling hormones. Virus-induced gene silencing of each of the SlSAP genes and disease assays revealed that SlSAP4 and SlSAP10 play roles in immunity against B. cinerea. Silencing of SlSAP4 resulted in attenuated immunity to B. cinerea, accompanying increased accumulation of reactive oxygen species and downregulated expression of jasmonate and ethylene (JA/ET) signaling-responsive defense genes. Transient expression of SlSAP4 in Nicotiana benthamiana led to enhanced resistance to B. cinerea. Exogenous application of methyl jasmonate partially restored the resistance of the SlSAP4-silenced plants against B. cinerea. SlSAP4 interacted with three of four SlRAD23 proteins. The A20 domain in SlSAP4 and the Ub-associated domains in SlRAD23d are critical for SlSAP4-SlRAD23d interaction. Silencing of SlRAD23d led to decreased resistance to B. cinerea, but silencing of each of other SlRAD23s did not affect immunity against B. cinerea. Furthermore, silencing of SlSAP4 and each of the SlRAD23s did not affect immunity to Pseudomonas syringae pv. tomato DC3000. These data suggest that SlSAP4 contributes positively to tomato immunity against B. cinereal through affecting JA/ET signaling and may be involved in the substrate ubiquitination process via interacting with SlRAD23d.
Plants sense temperature changes and respond by altering growth and metabolic activity to acclimate to the altered environmental conditions. The B vitamins give rise to vital coenzymes that are indispensable for growth and development but their inherent reactive nature renders them prone to destruction especially under stress conditions. Therefore, plant survival strategies would be expected to include mechanisms to sustain B vitamin supply under demanding circumstances. Here, using the example of vitamin B6, we investigate the regulation of biosynthesis across eudicot and monocot species under heat stress. Most eudicots carry a pseudoenzyme PDX1.2 that is a non-catalytic homolog of the PDX1 subunit of the vitamin B6 biosynthesis protein machinery, PYRIDOXINE SYNTHASE. Using Arabidopsis and tomato as models, we show that PDX1.2 is transcriptionally regulated by the HSFA1 transcription factor family. Monocots only carry catalytic PDX1 homologs but do not respond to heat stress as demonstrated for rice and maize, suggesting fundamental differences in the regulation of vitamin B6 biosynthesis across the two lineages. Investigation of the molecular mechanism of PDX1.2 transcription reveals two alternative transcriptional start sites one of which is exclusive to heat stress. Further data suggest that PDX1.2 leads to stabilization of the catalytic PDX1s under heat stress conditions, which would serve to maintain vitamin B6 homeostasis in times of need in eudicots that carry this gene. Our analyses indicate an important abiotic stress tolerance strategy in several eudicots, which has not been evolutionarily adapted (or is not required) by monocots such as grasses.
Vitamin B6 (VB6) is an important cofactor for numerous enzymatic reactions and plays an important role in abiotic stress tolerance. However, direct molecular evidence supporting a role for VB6 in plant disease resistance remains lacking. In this study, we explored the possible function of VB6 in disease resistance by analyzing disease phenotypes of Arabidopsis mutants with defects in de novo biosynthetic pathway and salvage pathway of VB6 biosynthesis against Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea. Mutations in AtPDX1.2 and AtPDX1.3 genes involved in the de novo pathway, and in AtSOS4 gene involved in the salvage pathway led to increased levels of diseases caused by Pst DC3000 and B. cinerea. The pdx1.2 and pdx1.3 plants had reduced VB6 contents and showed a further reduction in VB6 contents after infection by Pst DC3000 or B. cinerea. Our preliminary results suggest an important role for VB6 in plant disease resistance against different types of pathogens.
Copyright © 2014 Elsevier GmbH. All rights reserved.
Vitamin B6 is one of the most versatile cofactors in plants and an essential phytonutrient in the human diet that benefits a variety of human health aspects. Although biosynthesis of the vitamin has been well resolved in recent years, the main research is currently based on Arabidopsis thaliana with very little work done on major crop plants. Here we provide the first report on interactions and expression profiles of PDX genes for vitamin B6 biosynthesis in potato and how vitamin B6 content varies in tubers of different genotypes. The results demonstrate that potato is an excellent resource for this vitamin and that strong natural variation in vitamin B6 content among the tested cultivars indicates high potential to fortify vitamin B6 nutrition in potato-based foods.
Vitamin B6 has an essential role in cells as a cofactor for several metabolic enzymes. It has also been shown to function as a potent antioxidant molecule. The recent elucidation of the vitamin B6 biosynthesis pathways in plants provides opportunities for characterizing their importance during developmental processes and exposure to stress. Humans and animals must acquire vitamin B6 with their diet, with plants being a major source, because they cannot biosynthesize it de novo. However, the abundance of the vitamin in the edible portions of the most commonly consumed plants is not sufficient to meet daily requirements. Genetic engineering has proven successful in increasing the vitamin B6 content in the model plant Arabidopsis. The added benefits associated with the enhanced vitamin B6 content, such as higher biomass and resistance to abiotic stress, suggest that increasing this essential micronutrient could be a valuable option to improve the nutritional quality and stress tolerance of crop plants. This review summarizes current achievements in vitamin B6 biofortification and considers strategies for increasing vitamin B6 levels in crop plants for human health and nutrition.
Shelf life is an important quality trait for many fruit, including tomatoes. We report that enrichment of anthocyanin, a natural pigment, in tomatoes can significantly extend shelf life. Processes late in ripening are suppressed by anthocyanin accumulation, and susceptibility to Botrytis cinerea, one of the most important postharvest pathogens, is reduced in purple tomato fruit. We show that reduced susceptibility to B. cinerea is dependent specifically on the accumulation of anthocyanins, which alter the spreading of the ROS burst during infection. The increased antioxidant capacity of purple fruit likely slows the processes of overripening. Enhancing the levels of natural antioxidants in tomato provides a novel strategy for extending shelf life by genetic engineering or conventional breeding.
Plants inhabit environments crowded with infectious microbes that pose constant threats to their survival. Necrotrophic pathogens are notorious for their aggressive and wide-ranging virulence strategies that promote host cell death and acquire nutrients for growth and reproduction from dead cells. This lifestyle constitutes the axis of their pathogenesis and virulence strategies and marks contrasting immune responses to biotrophic pathogens. The diversity of virulence strategies in necrotrophic species corresponds to multifaceted host immune response mechanisms. When effective, the plant immune system disarms the infectious necrotroph of its pathogenic arsenal or attenuates its effect, restricting further ingress and disease symptom development. Simply inherited resistance traits confer protection against host-specific necrotrophs (HSNs), whereas resistance to broad host-range necrotrophs (BHNs) is complex. Components of host genetic networks, as well as the molecular and cellular processes that mediate host immune responses to necrotrophs, are being identified. In this review, recent advances in our understanding of plant immune responses to necrotrophs and comparison with responses to biotrophic pathogens are summarized, highlighting common and contrasting mechanisms.
Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre-reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.
In plants, vitamin B₆ biosynthesis requires the activity of PDX1 and PDX2 proteins. Arabidopsis thaliana encodes for three PDX1 proteins, named PDX1.1, 1.2, and 1.3, but only one PDX2. Here, we show in planta complex assembly of PDX proteins, based on split-YFP and FPLC assays, and can demonstrate their presence in higher complexes of around 750 kDa. Metabolic profiling of plants ectopically expressing the different PDX proteins indicates a negative influence of PDX1.2 on vitamin B₆ biosynthesis and a correlation between aberrant vitamin B6 content, PDX1 gene expression, and light sensitivity specifically for PDX1.3. These findings provide first insights into in planta vitamin B₆ synthase complex assembly and new information on how the different PDX proteins affect plant metabolism.
Vitamin B6 is a collective term for a group of six interconvertible compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives. Vitamin B6 plays essential roles as a cofactor in a range of biochemical reactions. In addition, vitamin B6 is able to quench reactive oxygen species in vitro, and exogenously applied vitamin B6 protects plant cells against cell death induced by singlet oxygen (1O2). These results raise the important question as to whether plants employ vitamin B6 as an antioxidant to protect themselves against reactive oxygen species.
The pdx1.3 mutation affects the vitamin B6 biosynthesis enzyme, pyridoxal synthase (PDX1), and leads to a reduction of the vitamin B6 concentration in Arabidopsis thaliana leaves. Although leaves of the pdx1.3 Arabidopsis mutant contained less chlorophyll than wild-type leaves, we found that vitamin B6 deficiency did not significantly impact photosynthetic performance or shoot and root growth. Chlorophyll loss was associated with an increase in the chlorophyll a/b ratio and a selective decrease in the abundance of several PSII antenna proteins (Lhcb1/2, Lhcb6). These changes were strongly dependent on light intensity, with high light amplifying the difference between pdx1.3 and the wild type. When leaf discs were exposed to exogenous 1O2, lipid peroxidation in pdx1.3 was increased relative to the wild type; this effect was not observed with superoxide or hydrogen peroxide. When leaf discs or whole plants were exposed to excess light energy, 1O2-mediated lipid peroxidation was enhanced in leaves of the pdx1.3 mutant relative to the wild type. High light also caused an increased level of 1O2 in vitamin B6-deficient leaves. Combining the pdx1.3 mutation with mutations affecting the level of 'classical' quenchers of 1O2 (zeaxanthin, tocopherols) resulted in a highly photosensitive phenotype.
This study demonstrates that vitamin B6 has a function in the in vivo antioxidant defense of plants. Thus, the antioxidant activity of vitamin B6 inferred from in vitro studies is confirmed in planta. Together with the finding that chloroplasts contain vitamin B6 compounds, the data show that vitamin B6 functions as a photoprotector that limits 1O2 accumulation in high light and prevents 1O2-mediated oxidative damage.
In recent years vitamin B6 has become a focus of research describing the compound’s critical function in cellular metabolism and stress response. For many years the sole function of vitamin B6 was considered to be that of an enzymatic cofactor. However, recently it became clear that it is also a potent antioxidant that effectively quenches reactive oxygen species and is thus of high importance for cellular well-being. In view of the recent findings, the current review takes a look back and summarizes the discovery of vitamin B6 and the elucidation of its structure and biosynthetic pathways. It provides a detailed overview on vitamin B6 both as a cofactor and a protective compound. Besides these general characteristics of the vitamin, the review also outlines the current literature on vitamin B6 derivatives and elaborates on recent findings that provide new insights into transport and catabolism of the compound and on its impact on human health.
The biosynthesis of vitamin B6 was studied in an Escherichia coli mutant, blocked between pyridoxol and pyridoxal. Specific incorporation of glycerol, pyruvate, serine, and glucose was shown by chemical degradation of labeled samples of pyridoxol isolated from the medium of cultures which had been incubated with various ¹⁴C-labeled radiomers of these substrates.
The observed pattern of incorporation of ¹⁴C shows that pyridoxol is derived from 3 triose units. One of these is incorporated by way of pyruvate, as a 2-carbon unit at the oxidation level of acetaldehyde. The other 2 triose units are incorporated intact.
A hypothetical sequence for the biogenesis of pyridoxol is advanced on the basis of the tracer evidence. 5-Deoxyxylulose 1-phosphate and a branched chain 8-carbon sugar are postulated as intermediates.
In spite of the rather simple structure of pyridoxal 5'-phosphate (I), a member of the vitamin B6 group, the elucidation of its de novo biosynthesis remained largely unexplored until recently. Experiments designed to investigate the formation of the vitamin B6 pyridine nucleus mainly concentrated on Escherichia coli. The results of tracer experiments with radioactive and stable isotopes, feeding experiments, and molecular biological studies led to the prediction that 4-hydroxy-L-threonine (VIII, R = H) and 1-deoxy-D-xylulose (VII, R = H) are precursors which are assembled to yield the carbon-nitrogen skeleton of vitamin B6. At this point, the involvement of the phosphorylated forms of these precursors in this assembly seems quite clear. However, vitamin B6 biosynthesis in organisms other than E. coli remains largely unknown. Toxic derivatives of vitamin B6, such as ginkgotoxin, occurring in higher plants may be suitable targets to gain further insight into this tricky problem.
Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis. We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene. sos4 mutant plants are hypersensitive to Na(+), K(+), and Li(+) ions. Under NaCl stress, sos4 plants accumulate more Na(+) and retain less K(+) compared with wild-type plants. SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses. Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels. Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na(+) and K(+) homeostasis in plants. We propose that pyridoxal-5-phosphate regulates Na(+) and K(+) homeostasis by modulating the activities of ion transporters.
Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.
This study deals with the activity of various vitamins against the radical-mediated oxidative damage in human whole blood. We have used a biological method that allows both the evaluation of plasma and that of red blood cell resistance against the free radicals induced by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Spin trapping measures using mainly 5-(diethoxyphosphoryl)-5-methyl-1-pyrolline N-oxide nitrone (DEPMPO) were carried out under several conditions to identify the free radicals implicated in this test. Only the oxygenated-centred radical generated from AAPH was found highly reactive to initiate red blood cell lysis. With DEPMPO only alkoxyl radicals were observed and no evidence was found for alkylperoxyl radicals. The antioxidant activity of several lipid- and water-soluble vitamins has been assessed by the biological assay and through two chemical methods. We have noticed high antioxidant activities for tocopherols (in the order delta>gamma>alpha) in the biological test but not through chemical methods. At 1 microM, the delta-tocopherol efficiency in inhibiting radical-induced red blood cell hemolysis was three times as high as the alpha-tocopherol efficiency. For beta-carotene no significant activity even in whole blood was shown. Highly surprising antioxidant activities were observed for acid folic and pyridoxine, compared to ascorbic acid. At 10 microM, the effectiveness of folic acid was almost three times as high as vitamin C. The biological test seems clinically more relevant than most other common assays because it can detect several classes of antioxidants.
The molecular and cellular mechanisms involved in plant resistance to the necrotrophic fungal pathogen Botrytis cinerea and their genetic control are poorly understood. Botrytis causes severe disease in a wide range of plant species, both in the field and in postharvest situations, resulting in significant economic losses. We have isolated the BOS1 (BOTRYTIS-SUSCEPTIBLE1) gene of Arabidopsis based on a T-DNA insertion allele that resulted in increased susceptibility to Botrytis infection. The BOS1 gene is required to restrict the spread of another necrotrophic pathogen, Alternaria brassicicola, suggesting a common host response strategy against these pathogens. In the case of the biotrophic pathogens Pseudomonas syringae pv tomato and the oomycete parasite Peronospora parasitica, bos1 exhibits enhanced disease symptoms, but pathogen growth is similar in bos1 and wild-type plants. Strikingly, bos1 plants have impaired tolerance to water deficit, increased salinity, and oxidative stress. Botrytis infection induces the expression of the BOS1 gene. This increased expression is severely impaired in the coi1 mutant, suggesting an interaction of BOS1 with the jasmonate signaling pathway. BOS1 encodes an R2R3MYB transcription factor protein, and our results suggest that it mediates responses to signals, possibly mediated by reactive oxygen intermediates from both biotic and abiotic stress agents.
Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen ((1)O(2)), a non-radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of (1)O(2). Vitamin B6 that quenches (1)O(2) in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of (1)O(2), indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild-type level. The flu mutant was also crossed with the jasmonic acid (JA)-depleted mutant opr3, and with the JA, OPDA and dinor OPDA (dnOPDA)-depleted dde2-2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA-induced suppression of H(2)O(2)/superoxide-dependent cell death reported earlier, JA promotes singlet oxygen-mediated cell death in flu, whereas other oxylipins such as OPDA and dnOPDA antagonize this death-inducing activity of JA.
Vitamin B6 is an essential metabolite in all organisms. It can act as a coenzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have a de novo biosynthetic pathway for vitamin B6, but animals must obtain it from dietary sources. In Escherichia coli, it is known that the vitamin is derived from deoxyxylulose 5-phosphate (an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis) and 4-phosphohydroxy-l-threonine. It has been assumed that vitamin B6 is synthesized in the same way in plants, but this hypothesis has never been experimentally proven. Here, we show that, in plants, synthesis of the vitamin takes an entirely different route, which does not involve deoxyxylulose 5-phosphate but instead utilizes intermediates from the pentose phosphate pathway, i.e., ribose 5-phosphate or ribulose 5-phosphate, and from glycolysis, i.e., dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. The revelation is based on the recent discovery that, in bacteria and fungi, a novel pathway is in place that involves two genes (PDX1 and PDX2), neither of which is homologous to any of those involved in the previously doctrined E. coli pathway. We demonstrate that Arabidopsis thaliana has two functional homologs of PDX1 and a single homolog of PDX2. Furthermore, and contrary to what was inferred previously, we show that the pathway appears to be cytosolic and is not localized to the plastid. Last, we report that the single PDX2 homolog is essential for plant viability.
• Arabidopsis
• isoprenoid
• pyridoxine
• deoxyxylulose 5-phosphate
Plant resistance to disease is controlled by the combination of defense response pathways that are activated depending on the nature of the pathogen. We identified the Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) gene that is transcriptionally regulated by Botrytis cinerea infection. Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens but enhances resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae pv tomato. The response to an avirulent bacterial strain is unchanged, limiting the role of BIK1 to basal defense rather than race-specific resistance. The jasmonate- and ethylene-regulated defense response, generally associated with resistance to necrotrophic fungi, is attenuated in the bik1 mutant based on the expression of the plant defensin PDF1.2 gene. bik1 mutants show altered root growth, producing more and longer root hairs, demonstrating that BIK1 is also required for normal plant growth and development. Whereas the pathogen responses of bik1 are mostly dependent on salicylic acid (SA) levels, the nondefense responses are independent of SA. BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response. Our data suggest that BIK1 modulates the signaling of cellular factors required for defense responses to pathogen infection and normal root hair growth, linking defense response regulation with that of growth and development.
Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.
PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.
The tomato protein kinase 1 (TPK1b) gene encodes a receptor-like cytoplasmic kinase localized to the plasma membrane. Pathogen infection, mechanical wounding, and oxidative stress induce expression of TPK1b, and reducing TPK1b gene expression through RNA interference (RNAi) increases tomato susceptibility to the necrotrophic fungus Botrytis cinerea and to feeding by larvae of tobacco hornworm (Manduca sexta) but not to the bacterial pathogen Pseudomonas syringae. TPK1b RNAi seedlings are also impaired in ethylene (ET) responses. Notably, susceptibility to Botrytis and insect feeding is correlated with reduced expression of the proteinase inhibitor II gene in response to Botrytis and 1-aminocyclopropane-1-carboxylic acid, the natural precursor of ET, but wild-type expression in response to mechanical wounding and methyl-jasmonate. TPK1b functions independent of JA biosynthesis and response genes required for resistance to Botrytis. TPK1b is a functional kinase with autophosphorylation and Myelin Basis Protein phosphorylation activities. Three residues in the activation segment play a critical role in the kinase activity and in vivo signaling function of TPK1b. In sum, our findings establish a signaling role for TPK1b in an ET-mediated shared defense mechanism for resistance to necrotrophic fungi and herbivorous insects.
In spite of the rather simple structure of pyridoxal 5′-phosphate (I), a member of the vitamin B6 group, the elucidation of its de novo biosynthesis remained largely unexplored until recently. Experiments designed to investigate the formation of the vitamin B6 pyridine nucleus mainly concentrated on Escherichia coli. The results of tracer experiments with radioactive and stable isotopes, feeding experiments, and molecular biological studies led to the prediction that 4-hydroxy-l-threonine (VIII, R = H) and 1-deoxy-d-xylulose (VII, R = H) are precursors which are assembled to yield the carbon-nitrogen skeleton of vitamin B6. At this point, the involvement of the phosphorylated forms of these precursors in this assembly seems quite clear. However, vitamin B6 biosynthesis in organisms other than E. coli remains largely unknown. Toxic derivatives of vitamin B6, such as ginkgotoxin, occurring in higher plants may be suitable targets to gain further insight into this tricky problem.
Generation of superoxide anion (O2−) by potato tuber protoplasts following treatment with hyphal wall components (HWC) of Phytophthora infestans was demonstrated by the superoxide dismutase (SOD)-sensitive reduction of extracellular cytochrome c or nitroblue tetrazolium (NBT). In the presence of NADPH ability of protoplasts to reduce cytochrome c was enhanced within 2 min after adding HWC and then proceeded as a sigmoidal curve. Addition of SOD during the course of the reaction prevented further reduction of cytochrome c. In the reaction mixture without NADPH or HWC, little reducing activity was detected. A similar reduction of NBT was also found in the protoplasts treated with HWC.Water-soluble glucans from compatible races of P. infestans, which are known as suppressors of hypersensitivity, inhibited the activation of reducing activity when they were applied before elicitation of the reaction with HWC. The glucans from incompatible races caused little inhibition.Of the tested inhibitors of hypersensitive cell death, SH-binding reagent and NADP+inhibited the reducing activity whereas respiratory inhibitors, NaN3, antimycine A, and 2,4-dinitrophenol were ineffective.These results indicate that O2− generation and NADPH oxidation occur on the plasma-membrane upon incompatible recognition of host cells leading to hypersensitive cell death. Suppression of the NADPH-dependent OZ- generation may be involved in compatible relationships in the potato—P. infestans system.
Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1–4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 × 107 M−1 s−1 for 1; 7.5 × 107 M−1 s−1 for 2, 6.2 ×107 M−1 s−1 for 3 and 7.5 × 107 M−1 s−1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Vitamin B(6) (pyridoxal 5'-phosphate and its vitamers) is an important cofactor in numerous enzymatic reactions. In spite of its importance, the consequences of altering vitamin B(6) content on plant growth and development are not well understood. This study compares two mutants for vitamin B(6)-metabolizing enzymes in Arabidopsis thaliana: a pdx1.3 mutant in the de novo synthesis pathway and a salvage pathway sos4 mutant that accumulates more vitamin B(6). We show that despite a difference in total B(6) content in leaf tissue, both mutants share similar phenotypes, including chlorosis, decreased size, altered chloroplast ultrastructure, and root sensitivity to sucrose. Assay of B(6) vitamer content from isolated chloroplasts showed that, despite differing B(6) vitamer content in whole leaf tissue, both mutants share a common deficiency in total and phosphorylated vitamers in chloroplasts. One of the splice variants of the SOS4 proteins was shown to be located in the chloroplast. Our data indicate that some of the phenotypic consequences shared between the pdx1.3 and sos4 mutants are due to B(6) deficiency in chloroplasts, and show that SOS4 is required for maintenance of phosphorylated B(6) vitamer concentrations in chloroplasts. Further, our data are consistent with a diffusion model for transport of vitamin B(6) into chloroplasts.
Vitamin B6, an essential cofactor in enzymatic reactions, has only recently been linked to cellular oxidative stress. We investigated the role of this vitamin as an antioxidant in oxidative responses linked to plant defense. B6 vitamers effectively quenched superoxide and had antioxidant activity when assayed in vitro. The de novo B6 biosynthetic genes (PDX1 and PDX2) were identified in Nicotiana tabacum cv. ‘Burley 21’ and their transcript abundance was assayed during defense responses. PDX1 and PDX2 transcript levels decreased following inoculation with the incompatible pathogen Pseudomonas syringae pv. phaseolicola and transiently increased in response to salicylic acid and methyl jasmonate. Excess vitamin B6 in tobacco leaves interfered with the development of a hypersensitive response caused by P. syringae pv. phaseolicola and increased disease severity caused by the compatible bacterium P. syringae pv. tabaci. Our findings indicate that during plant defense responses, vitamin B6 functions and its synthesis is regulated in a manner consistent with this vitamin's activity as an antioxidant and modulator of active oxygen species in vivo.
Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.
Ultraviolet-B radiation regulates plant growth and morphology at low and ambient fluence rates but can severely impact on plants at higher doses. Some plant UV-B responses are related to the formation of reactive oxygen species (ROS) and pyridoxine (vitamin B(6)) has been reported to be a quencher of ROS. UV-B irradiation of Arabidopsis Col-0 plants resulted in increased levels of PDX1 protein, compared with UV-A-exposed plants. This was shown by immunoblot analysis using specific polyclonal antibodies raised against the recombinant PDX1.3 protein and confirmed by mass spectrometry analysis of immunoprecipitated PDX1. The protein was located mainly in the cytosol but also to a small extent in the membrane fraction of plant leaves. Immunohistochemical analysis performed in pea revealed that PDX1 is present in UV-B-exposed leaf mesophyll and palisade parenchyma but not in epidermal cells. Pyridoxine production increased in Col-0 plants exposed to 3 days of UV-B, whereas in an Arabidopsis pdx1.3 mutant UV-B did not induce pyridoxine biosynthesis. In gene expression studies performed after UV-B exposure, the pdx1.3 mutant showed elevated transcript levels for the LHCB1*3 gene (encoding a chlorophyll a/b-binding protein of the photosystem II light-harvesting antenna complex) and the pathogenesis-related protein 5 (PR-5) gene, compared with wild type.
Pyridoxal phosphate (PLP), a vitamin B(6) vitamer, is an essential cofactor for numerous enzymes. Pyridoxine/pyridoxamine phosphate oxidase (PPOX) catalyzes the synthesis of pyridoxal phosphate from pyridoxine phosphate (PNP) and/or pyridoxamine phosphate (PMP). The At5g49970 locus in Arabidopsis thaliana encodes an AtPPOX, a PNP/PMP oxidase. The expression of the AtPPOX gene varied in different tissues of Arabidopsis examined, being up-regulated by light, heat shock, ABA, and ethylene treatments, and down-regulated by exposure to drought and NaCl. Monoclonal antibodies raised against two different domains of AtPPOX recognized different sizes of AtPPOX, suggesting that AtPPOX proteins are produced as splice variants of the AtPPOX gene in Arabidopsis. Phylogenetic analysis of AtPPOX across all domains of life demonstrated that plant AtPPOX homologs have an additional Yjef_N domain preceding the Pyridox_Oxidase domain at the C-terminal end of the protein, while AtPPOX homologs from bacteria, fungi and animals have only Pyridox_Oxidase domain. The presence of the Yjef_N domain in plant AtPPOX homologs suggests that acquisition of this domain, and its fusion with the pyridox_oxidase domain began with the endosymbiotic acquisition of the chloroplast. Bioinformatic analysis suggested that AtPPOX is localized in chloroplast, but the monoclonal antibody could not be used for subcellular localization of this protein. A GFP-AtPPOX fusion construct introduced into the Arabidopsis protoplast confirmed localization of AtPPOX into the chloroplast. An RNAi mutant of AtPPOX showed sensitivity to high light suggesting a role for PPOX in resistance to photooxidative damage, and alteration in root growth in the presence of sucrose suggests a role for PPOX in root development.
Virus-induced gene silencing (VIGS) enables high-throughput analysis of gene function in plants but is not universally applicable and requires optimization for each species. Here a VIGS system is described for Solanum nigrum, a wild relative of tomato and potato and a valuable model species for ecogenomics. The efficiency of the two most widely used Tobacco rattle virus (TRV) vectors to silence phytoene desaturase (PDS) in S. nigrum was tested. Additionally, the infiltration method and growth temperatures for gene silencing were optimized and the suitability of different control vectors evaluated. Using leucine aminopeptidase (LAP), a herbivore-induced protein, silencing efficiency and the applicability of silenced plants for herbivore feeding assays were assessed. Vacuum infiltration of seedlings with Agrobacterium carrying the vector, pYL156, proved the most efficient means of silencing genes. Empty-vector controls decreased plant growth but control vectors carrying a piece of noncoding sequence did not. Silencing LAP significantly increased the larval mass of Manduca sexta that fed on silenced plants. This VIGS protocol proved highly successful for S. nigrum, which should include control vectors carrying noncoding sequence as control treatments. Silencing LAP provided the first experimental evidence that LAP has a defensive function against herbivores.
Biosynthesis of pyridoxal 5'-phosphate (PLP) depends upon the relatively specific action of two consecutive enzymes, viz. pyridoxal (pyridoxine, pyridoxamine) kinase and pyridoxine (pyridoxamine) phosphate oxidase. Less specific phosphatases catalyze hydrolyses of the 5'-phosphates of the vitamers pyridoxal, pyridoxamine, and pyridoxine. From the recognition a generation ago of these processes by which the three forms of vitamin B-6 and their 5'-phosphates are interconverted, more recent studies have provided a fairly sophisticated understanding of the molecular characteristics of the enzymes involved. The evolutionary retention of homologous portions of pyridoxal kinase in humans as well as bacteria and the most recent finding of a highly conserved region of the pyridoxine (pyridoxamine) phosphate oxidase, also from both prokaryotic and eukaryotic organisms, emphasize the importance of these catalysts in the formation of a coenzyme that is essential for most organisms. Both kinase and oxidase involved in B-6 metabolism are potential targets for pharmacologic agents.
Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1-4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 x 10(7) M-1 s-1 for 1; 7.5 x 10(7) M-1 s-1 for 2, 6.2 x 10(7) M-1 s-1 for 3 and 7.5 x 10(7) M-1 s-1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased approximately 10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased approximately 10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1-4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.
Vitamin B6 in its active form pyridoxal phosphate is an essential coenzyme of many diverse enzymes. Biochemistry, enzymology and genetics of de novo vitamin B6 biosynthesis have been primarily investigated in Escherichia coli. Database searches revealed that the key enzymes involved in ring closure of the aromatic pyridoxin ring (PdxA; PdxJ) are present mainly in genomes of bacteria constituting the gamma subdivision of proteobacteria. The distribution of DXS, a transketolase-like enzyme involved in vitamin B6 biosynthesis as well as in thiamine and isoprenoid biosynthesis and the distribution of vitamin B6 modifying enzymes (PdxH: oxidase; PdxK: kinase) was also analyzed. These enzymes are also present in the genomes of animals. Two recent papers (Ehrenshaft et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 9374-9378; Osmani et al., 1999, J. Biol. Chem. 274: 23565-23569) show the involvement of an extremely conserved protein (a member of the UPF0019 or SNZ family) found in all three domains of life (bacteria, archaea, eukarya) in an alternative vitamin B6 biosynthesis pathway. Members of this family were previously identified as a stationary phase inducible protein in yeast, as an ethylene responsible protein in plants and in a marine sponge, as a singlet oxygen resistance protein in Cercospora nicotianae and as a cumene hydroperoxide and H2O2 inducible protein in Bacillus subtilis. In yeast, the SNZ protein interacts with another protein called SNO which also represents a member of a highly conserved protein family (called UPF0030 or SNO family). Phylogenetic trees for the DXS, PdxA, PdxJ, PdxH, PdxK, SNZ and SNO protein families are presented and possible implications of the two different vitamin B6 biosynthesis pathways in cellular metabolism are discussed. A radically different view of bacterial evolution (Gupta, 2000, Crit. Rev. Microbiol. 26: 111-131) which proposes a linear rather than a treelike evolutionary relationship between procaryotic species indicates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. This proposal might help to explain why the PdxA/PdxJ pathway is largely restricted to this subdivision.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
We have previously demonstrated that a tobacco rattle virus (TRV)-based vector can be used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up-regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction- and ligation-free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large-scale functional analysis of tomato ESTs.
Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B6 metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn2+ instead of the commonly used Mg2+ as the divalent cation for catalysis. Under optimal conditions, the V
max of the enzyme was 604 nmol pyridoxal 5′-phosphate (PLP) mg–1 min–1, and the K
m values for pyridoxal and ATP were 688 µM and 98 µM, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.
In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ1-3 and SNO1-3. In media lacking vitamin B(6), SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required. Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell. We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family. Whereas copy 1 is required for conditions in which B(6) is essential for growth, copies 2 and 3 seem more related with B(1) biosynthesis during the exponential phase.
Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to clarify their function, the two genes SNO1 and SNZ1 were expressed in Escherichia coli either individually or simultaneously and with or without a His-tag. When expressed simultaneously, the two protein products formed a complex and showed glutaminase activity. When purified to homogeneity, the complex exhibited a specific activity of 480 nmol.mg(-1).min(-1) as glutaminase, with a Km of 3.4 mm for glutamine. These values are comparable to those for other glutamine amidotransferases. In addition, the glutaminase activity was impaired by 6-diazo-5-oxo-L-norleucine in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine. These data suggest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone. The function of Snz1p and the amido acceptor for ammonia remain to be identified.
The pathway for de novo vitamin B(6) biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2, have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae. Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B(6) pathway encodes a protein responsible for synthesis of the pyridoxine ring.
Changes in AA-GSH cycle activity following Botrytis cinerea infection were studied in tomato whole-leaf extracts as well as in chloroplasts, mitochondria, and peroxisomes. The oxidative
effect of infection affected all cellular compartments although mitochondria and peroxisomes underwent the most pronounced
changes. Apart from organelle-specific variations, a general shift of the cellular redox balance towards the oxidative state
was found. It was manifested by the significant decline in concentrations and redox ratios of the ascorbate and glutathione
pools as well as by the insufficient activity of MDHAR, DHAR, and GR needed for antioxidant regeneration. There was no compatibility
between the ascorbate- and glutathione-mediated changes in different compartments. It was concluded that B. cinerea was able to break down the protective antioxidant barrier of the AA-GSH cycle at both the cellular and organellar levels.
The changes in the AA-GSH cycle activity could partly be related to the B. cinerea-induced promotion of senescence that favoured disease progress.
Peroxisomes, being one of the main organelles where reactive oxygen species (ROS) are both generated and detoxified, have been suggested to be instrumental in redox-mediated plant cell defence against oxidative stress. We studied the involvement of tomato (Lycopersicon esculentum Mill.) leaf peroxisomes in defence response to oxidative stress generated upon Botrytis cinerea Pers. infection. The peroxisomal antioxidant potential expressed as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.19) as well as the ascorbate-glutathione (AA-GSH) cycle activities was monitored. The initial infection-induced increase in SOD, CAT and GSH-Px indicating antioxidant defence activation was followed by a progressive inhibition concomitant with disease symptom development. Likewise, the activities of AA-GSH cycle enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) as well as ascorbate and glutathione concentrations and redox ratios were significantly decreased. However, the rate and timing of these events differed. Our results indicate that B. cinerea triggers significant changes in the peroxisomal antioxidant system leading to a collapse of the protective mechanism at advanced stage of infection. These changes appear to be partly the effect of pathogen-promoted leaf senescence.
Pyridoxine (vitamin B6) is a cofactor required by numerous enzymes in all cellular organisms. Plants are the major source of vitamin B6 for animals, yet the biosynthesis pathway and the function of vitamin B6 in plants are not well elucidated. In this study, an Arabidopsis pyridoxine synthase gene PDX1 was characterized and its in vivo functions were investigated. The PDX1 gene was expressed in all plant parts examined and its expression level was not significantly regulated by abiotic stress or the phytohormone abscisic acid. In roots, PDX1 was highly expressed in a defined region behind the root tips that undergoes rapid cell division. The PDX1 protein was mainly associated with the plasma membrane and endomembranes, implying a potential involvement of vitamin B6 in membrane function. To reveal the in vivo role of PDX1, Arabidopsis insertional mutants were isolated. Strikingly, the pdx1 knockout mutants were impaired in root growth and early seedling development. The stunted roots resulted from both reduced cell division and elongation. Supplementation of the growth media with pyridoxine or reintroduction of the wild-type PDX1 gene into the mutants completely restored the mutant growth, demonstrating that PDX1 is required for pyridoxine biosynthesis in planta. In addition to the developmental defects, pdx1 mutants are hypersensitive to osmotic stress and oxidative stress. These mutant seedlings had increased peroxidation of membrane lipids following UV treatment. Our study establishes a critical role of vitamin B6 in plant development and stress tolerance and suggests that vitamin B6 may represent a new class of antioxidant in plants.
Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. It has been demonstrated to alleviate oxidative stress and in its phosphorylated form participates as a cofactor in >100 biochemical reactions. By means of a genetic approach, we have identified a novel mutant, rsr4-1 (for reduced sugar response), with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Cloning of the mutated gene revealed that rsr4-1 carries a point mutation in a member of the PDX1/SOR1/SNZ (for Pyridoxine biosynthesis protein 1/Singlet oxygen resistant 1/Snooze) family that leads to reduced vitamin B6 content. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers. Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants.
Pyridoxine (pyridoxamine) 5'-phosphate oxidase (PPOX) catalyzes the oxidative conversion of pyridoxamine 5'-phosphate (PMP) or pyridoxine 5'-phosphate (PNP) to pyridoxal 5'-phosphate (PLP). The At5g49970 gene of Arabidopsis thaliana shows homology to PPOX's from a number of organisms including the Saccharomyces cerevisiae PDX3 gene. A cDNA corresponding to putative A. thaliana PPOX (AtPPOX) was obtained using reverse transcriptase-polymerase chain reaction and primers landing at the start and stop codons of At5g49970. The putative AtPPOX is 530 amino acid long and predicted to contain three distinct parts: a 64 amino acid long N-terminal putative chloroplast transit peptide, followed by a long Yjef_N domain of unknown function and a C-terminal Pyridox_oxidase domain. Recombinant proteins representing the C-terminal domain of AtPPOX and AtPPOX without transit peptide were expressed in E. coli and showed PPOX enzyme activity. The PDX3 knockout yeast deficient in PPOX activity exhibited sensitivity to oxidative stress. Constructs of AtPPOX cDNA of different lengths complemented the PDX3 knockout yeast for oxidative stress. The role of the Yjef_N domain of AtPPOX was not determined, but it shows homology with a number of conserved hypothetical proteins of unknown function.
Vitamin B6 is an essential coenzyme for numerous metabolic enzymes and is a potent antioxidant. In plants, very little is known about its contribution to viability, growth and development. The de novo pathway of vitamin B6 biosynthesis has only been described recently and involves the protein PDX1 (pyridoxal phosphate synthase protein). Arabidopsis thaliana has three homologs of PDX1, two of which, PDX1.1 and PDX1.3, have been demonstrated as functional in vitamin B6 biosynthesis in vitro and by yeast complementation. In this study, we show that the spatial and temporal expression patterns of PDX1.1 and PDX1.3, investigated at the transcript and protein level, largely overlap, but PDX1.3 is more abundant than PDX1.1. Development of single pdx1.1 and pdx1.3 mutants is partially affected, whereas disruption of both genes causes embryo lethality at the globular stage. Detailed examination of the single mutants, in addition to those that only have a single functional copy of either gene, indicates that although these genes are partially redundant in vitamin B6 synthesis, PDX1.3 is more requisite than PDX1.1. Developmental distinctions correlate with the vitamin B6 content. Furthermore, we provide evidence that in addition to being essential for plant growth and development, vitamin B6 also plays a role in stress tolerance and photoprotection of plants.
Vitamin B(6) (pyridoxine and its vitamers) plays an essential role as a co-factor for enzymatic reactions and has also recently been implicated in defense against cellular oxidative stress. The biosynthetic pathway was thoroughly characterized in Escherichia coli, however most organisms, including plants, utilize an alternate pathway involving two genes, PDX1 and PDX2. Arabidopsis thaliana contains one copy of PDX2, but three full-length copies of PDX1, one each on chromosomes 2, 3, and 5 (referred to as PDX1.1, PDX1.2, and PDX1.3, respectively). Phylogenetic analysis of the PDX1 homologues in A. thaliana showed that PDX1.1 and PDX1.3 clustered with the homologues from the other dicots, whereas PDX1.2 was more divergent, and did not cluster with either the dicots or monocots. Expression analysis using quantitative PCR showed that PDX1.1 and PDX1.3 were highly expressed in A. thaliana rosettes, while PDX1.2 showed only low level expression. All three PDX1 genes and PDX2 were responsive to abiotic stressors including high light, chilling, drought, and ozone, however, the response of PDX1.2 was disparate from that of the other PDX genes, showing a lessened response to high light, chilling, and drought, but an increased response to ozone. Green fluorescent protein fusion studies demonstrated that PDX2 localizes in the nucleus and membranes of cells, consistent with recent published data for PDX1. Insight into regulation of the biosynthetic genes during abiotic stress could have important applications in the development of stress-tolerant crops.
Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non‐enzymatic components of the ascorbate–glutathione cycle as well as in
superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), glutathione transferase (GST), and l‐galactono‐γ‐lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly
induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total
SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate
reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied
by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios
decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd
days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome
the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity
of l‐galactono‐γ‐lactone dehydrogenase (GLDH). The activities of two glutathione‐dependent enzymes: GSH‐Px and GST were increased
from day 1 to day 4. These results demonstrated that in B. cinerea–tomato interaction mitochondria could be one of the main targets for infection‐induced oxidative stress.
A long known compound of surprising complexity
- Vitamin
- B
Vitamin B6: A long known compound of surprising complexity. Mole-cules 14:329-351.
Analysis of the Arabidopsis rsr4-1/pdx1-3 mutant reveals the critical function of the PDX1 protein family in metabolism, development, and vitamin B6 biosynthesis
- S Wagner
- A Bernhardt
- J E Leuendorf
- C Drewke
- A Lytovko
- N Mujahed
- C Gurgui
- W B Frommer
- E Leistner
- A R Fernie
- H Hellmann
Wagner, S., Bernhardt, A., Leuendorf, J. E., Drewke, C., Lytovko, A.,
Mujahed, N., Gurgui, C., Frommer, W. B., Leistner, E., Fernie, A. R.,
and Hellmann, H. 2006. Analysis of the Arabidopsis rsr4-1/pdx1-3
mutant reveals the critical function of the PDX1 protein family in
metabolism, development, and vitamin B6 biosynthesis. Plant Cell
18:1722-1735.