Plant Physiology and Biochemistry

Published by Elsevier
Print ISSN: 0981-9428
A phosphate-solubilizing bacterial strain NII-0909 isolated from the Western ghat forest soil in India was identified as Micrococcus sp on the basis of phenotypic characteristics, carbon source utilization pattern, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The strain exhibited the plant growth-promoting attributes of phosphate solubilization, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, and siderophore production. It was able to solubilize (122.4μg of Ca(3)PO(4) ml(-1)), and produce IAA (109μgml(-1)) at 30°C. P-solubilizing activity of the strain NII-0909 was associated with the release of organic acids and a drop in the pH of the NBRIP medium. HPLC analysis detected two organic acids in the course of P-solubilization. A significant increase in the growth of cow pea was recorded for inoculations under controlled conditions. Scanning electron microscopic study revealed the root colonization of strain on cow pea seedlings. These results demonstrate that isolates NII-0909 has the promising PGPR attributes to be develop as a biofertilizer to enhance soil fertility and promote the plant growth.
Barley plants (cultivars Embrapa 127, 128 and 129) treated with xanthan gum, and with different time intervals between the administration of the inducer and the pathogen. demonstrated induction of resistance against Bipolaris sorokiniana. Induction was shown to have local and systemic action. In order to prove the resistance effect, biochemical analyses were performed to quantify proteins and the enzymatic activity of beta-1,3 glucanase. Results demonstrated that barley plants treated with the inducer, showed an increase in the concentration of proteins, as well as in the activity of the enzyme beta-1,3 glucanase, when compared with the extract from healthy plants. In infected plants, protein concentrations decreased and enzymatic activity was lower than in healthy plants. Results suggest that barley plants treated with xanthan gum developed mechanisms responsible for induced resistance, which are still unknown. The most important macromolecule in the defense mechanism was demonstrated to be PR-protein, due to its accumulation and concentration of proteins. However, it may not be the only macromolecule responsible for the resistance effect.
The COP9 signalosome (CSN) plays important roles in multifaceted cellular processes. Study has shown that inositol 1,3,4-trisphosphate 5/6 kinase (5/6 kinase) interacts with CSN in mammalian cells. However, the biological function of the interaction still remains unknown. Here, we report that the Arabidopsis inositol 1,3,4-trisphosphate 5/6 kinase (AtItpk-1) is also associated with CSN and involved in photomorphogenesis under red light (RL) conditions, as demonstrated by co-immunoprecipitation of AtItpk-1 with CSN and characterization of the atitpk-1 mutants. Expression analysis showed that AtItpk-1 had the same sub-cellular localization and organ expression pattern as CSN. Furthermore, autophosphorylation analysis showed that AtItpk-1 has protein kinase activity. Under RL, the atitpk-1 mutants exhibited phenotype slightly similar with that of the csn mutants, indicating that 5/6 kinase might be involved in the same developmental pathway as CSN. This study suggests that AtItpk-1 may function as a protein kinase that is involved in photomorphogenesis possibly via interaction with COP9 signalosome under red light.
The mitochondrial antioxidant homeostasis was investigated in Arabidopsis ppr40-1 mutant, which presents a block of electron flow at complex III. The activity of the ascorbate biosynthetic enzyme, L-galactono-1,4-lactone dehydrogenase (EC (GLDH) was elevated in mitochondria isolated from mutant plants. In addition increased activities of the enzymes of Foyer-Halliwell-Asada cycle and elevated glutathione (GSH) level were observed in the mutant mitochondria. Lower ascorbate and ascorbate plus dehydroascorbate contents were detected at both cellular and mitochondrial level. Moreover, the more oxidized mitochondrial redox status of ascorbate in the ppr40-1 mutant indicated that neither the enhanced activity of GLDH nor Foyer-Halliwell-Asada cycle could compensate for the enhanced ascorbate consumption in the absence of a functional respiratory chain.
Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.
ScORK11 primary structure and phylogeny. A. Illustration of ScORK11 primary structure. The dots correspond to the cysteine pairs. SP, signal peptide; LRR, leucine-rich repeat; TM, transmembrane domain; JM, intracellular juxtamembrane domain; KINASE, kinase catalytic domain. Putative predicted phosphorylation sites in the cytoplasmic domain are also illustrated. Sequences for these sites are listed in Table S1. B. The five LRRs are aligned to emphasize the most frequent amino acids found at regularly spaced positions. Dark gray boxes represent conserved residues in more than half the sequences while light gray boxes represent conservative substitutions. C. Section of an LRR III RK phylogenetic tree showing the most closely related putative orthologs of ScORK11 in A. thaliana.  
Expression profile of ScORK11. A. RNA gel blot analysis of ScORK11 mRNA from vegetative and reproductive tissues. The radioactive probe used covered the ectodomain of ScORK11. Equal loading of each sample was verified by ethidium bromide staining prior to gel transfer (not shown) and by 18S rRNA hybridization. B. Western blot analysis on vegetative and reproductive tissues. Equal loading of the protein from various tissues was determined using a Bradford protein assay and verified by Coomassie Brilliant Blue staining (not shown). C. RNA gel blot analysis of ScORK11 mRNA in anthers and petals isolated from developing flower buds (upper panel). 18S rRNA loading control for mRNA amount (middle panel). Immunodetection of ScORK11 in the same tissues (lower panel). D. Immunodetection of ScORK11 following 2-D electrophoresis of proteins extracted from ovaries or anthers.  
Cellular localization of ScORK11. A. Epidermal onion cells expressing the ScORK11eGFP construct under the control of the 35S CaMV promoter after microparticle bombardment and observed under fluorescent illumination before (ii) and after plasmolysis (iv). Differential interference contrast (DIC) microscopy, and fluorescence of the same cells after plasmolysis showing protoplast shrinkage (iii, iv). Scale bar 100 mm. B. Two-phase partitioning of cell membranes and immunolocalization of  
Cellular localization of ScORK11. A. Epidermal onion cells expressing the ScORK11eGFP construct under the control of the 35S CaMV promoter after microparticle bombardment and observed under fluorescent illumination before (ii) and after plasmolysis (iv). Differential interference contrast (DIC) microscopy, and fluorescence of the same cells after plasmolysis showing protoplast shrinkage (iii, iv). Scale bar 100 mm. B. Two-phase partitioning of cell membranes and immunolocalization of ScORK11 and organelle markers (ER marker, BIP; vacuole marker, PPiase; chloroplast marker, PsbA; plasma membrane marker, H þ ATPase). IM (intracellular membranes) and PM (plasma membrane). 
In situ expression profile of ScORK11 in ovary and anther tissues. Ovaries were harvested 4 DAP while anthers were from 4 mm flower buds. In situ hybridization was performed using either sense (C, D, I) or antisense (E, F, G, H, J) probes from the ScORK11 cDNA clone. Digoxigenin labeling is seen as bluish purple staining. Hybridizations were  
Using a subtraction screen to isolate weakly expressed transcripts from ovule and ovary libraries, we uncovered 30 receptor-like kinases that were predominantly expressed in ovary and fruit tissues following fertilization [1]. Here we describe the analysis of Solanum chacoense ovule receptor kinase 11 (ScORK11), a member of the large LRR III receptor kinase subfamily that localizes to the plasma membrane. In situ analyses demonstrated that ScORK11 gene expression was mainly restricted to the ovule integument, the embryo sac and the pericarp of the fruit. Tight regulation of ScORK11 expression at the mRNA level was also accompanied by both translational and post-translational regulation of protein levels.
Vacuolar sorting of seed storage proteins is a very complex process since several sorting pathways and interactions among proteins of different classes have been reported. In addition, although the C-terminus of several 7S proteins is important for vacuolar delivery, other signals seem also to be involved in this process. In this work, the ability of two sequences of the Amaranthus hypochondriacus 11S globulin (amaranthin) to target reporter proteins to vacuoles was studied. We show that the C-terminal pentapeptide (KISIA) and the GNIFRGF internal sequence fused at the C terminal region of genes encoding secretory versions of green fluorescent protein (GFP) and GFP-beta-glucuronidase (GFP-GUS) were sufficient to redirect these reporter proteins to the vacuole of Arabidopsis cells. According to the three-dimensional structure of 7S and 11S storage globulins, this internal vacuolar sorting sequence corresponds to the alpha helical region involved in trimer formation, and is conserved within these families. In addition, these sequences were able to interact in vitro, in a calcium dependent manner, with the sunflower vacuolar sorting receptor homolog to pea BP-80/AtVSR1/pumpkin PV72. This work shows for the first time the role of a short internal sequence conserved among 7S and 11S proteins in vacuolar sorting.
The unicellular green alga Chlamydomonas reinhardtii has long served as model organism for studies on the circadian clock. This clock is present in all eukaryotes and some prokaryotes allowing them to anticipate and take advantage of the daily oscillations in the environment. Although much is known about the circadian clock in C. reinhardtii, the photoreceptors mediating entrainment of the clock to the daily changes of light remain obscure. Based on its circadian rhythm of phototaxis as a reporter of the clock's phase, we show here that C. reinhardtii strain CC-124 is highly sensitive to blue light of 440 nm when resetting its circadian clock upon light pulses. Thus, CC-124 differs in this respect from what was previously reported for a cell wall-deficient strain. An action spectrum analysis revealed that CC-124 also responds with high sensitivity to green (540 nm), red (640-660 nm), and possibly UV-A (≤400 nm) light, and therefore shows similarities as well to what has been reported for the cell wall-deficient strain. We also investigated two RNA interference strains with reductions in the level of the blue light photoreceptor plant cryptochrome (CPH1). One of them, the strain with the greater reduction, surprisingly showed an increased sensitivity in clock resetting upon blue light pulses of 440 nm. This increase in sensitivity reverted to wild-type levels when the RNA interference strain reverted to wild-type protein levels. It suggests that plant cryptochrome in C. reinhardtii could function as negative rather than positive modulator of circadian clock resetting.
The red alga Grateloupia doryphora Montagne (Howe) (Cryptonemiales, Halymeniaceae) was used as a model to investigate the effects of changes in seawater salinity on the intracellular low-molecular-weight organic compounds. Carbon-partitioning into major organic solutes was followed by 13C nuclear magnetic resonance (NMR) spectroscopy on living algae incubated in NaH13CO3-enriched seawater, and by high resolution 1H and 13C NMR experiments performed on 13C-enriched algal extracts. NMR and high performance liquid chromatography (HPLC) analyses both demonstrated that floridoside level was the most affected by changes in salinity: it rose under the hypersaline treatment and decreased under hyposaline one. Moreover, at low salinity, the high labeling of floridoside (45.3% 13C-enrichment for C1) together with its low concentrations both provided evidence of great increase in the de novo biosynthesis and turnover rate. Our experiments also demonstrated a high incorporation of photosynthetic carbon into amino acids, especially glutamate, under hypoosmotic conditions. On the other hand, isethionic acid and N-methyl-methionine sulfoxide were only partly labeled, which indicates they do not directly derive from carbon photoassimilation. In algae exposed to high salinity, elevated concentrations of floridoside coupled to a low labeling (9.4%) were observed. These results suggest that hyperosmotic conditions stimulated floridoside biosynthesis from endogen storage products rather than from carbon assimilation through photosynthesis.
The seedless grapes BRS Clara and BRS Morena, developed in Brazil, are currently growing in popularity due to their premium texture and taste. However, there are no reports on the polyphenoloxidase (PPO) from these cultivars. In this paper, active and latent PPO from BRS Clara and BRS Morena seedless grapes were extracted using the non-ionic detergents Triton-X-100 (active) and Triton-X-114 (latent), and their catecholase activities were characterized. The PPO extracted using Triton-X-110 exhibited maximum activities at pH 6.0 and at 25 °C. Above 30 °C, a gradual decline in activities was noted, with complete inactivation at 60 °C. The PPO from grapes extracted with Triton-X-114 was activated with 0.2% of the ionic detergent sodium dodecyl sulfate (SDS), and exhibited maximum activities at pH 5.5 and at 30 °C. It was stable until the temperature reached 60 °C.
Pea abscisic acid responsive (ABR17) protein is a member of the pathogenesis-related 10 (PR10) family of proteins and its ribonuclease (RNase) activity has been reported previously. In order to investigate the amino acids important for the demonstrated ribonuclease activity of ABR17, site-directed mutants H69L and E148A were generated, expressed in Escherichia coli and purified to homogeneity. These mutations affected RNase activity differently; the H69L mutant exhibited a decreased RNase activity whereas E148A exhibited an elevated activity. A structural model for pea ABR17 has been generated using the three dimensional structure of Lupinus luteus PR10 protein in order to explain the possible effects of the H69L and the E148A mutations on substrate binding and catalysis.
Glycine-rich proteins (GRPs) belong to a large family of heterogenous proteins that are enriched in glycine residues. The expression of two GRP genes of Arabidopsis thaliana, AtGRP5 and AtGRP23, was induced by 16-hydroxypalmitic acid (HPA), a major component of cutin. The expression of AtGRP3, which encodes a GRP protein that is structurally different from AtGRP5 and AtGRP23, was not responsive to HPA application. Treatment with HPA also induced expression of the pathogen-related PR-1 and PR-4 genes. Abscisic acid and salicylic acid treatments enhanced the transcript levels of AtGRP5 and AtGRP23 as well as those of AtGRP3. It was also demonstrated that HPA effectively elicited the accumulation of H2O2 in rosette leaves of Arabidopsis. Results suggest the possible role of some species of GRPs, such as AtGRP5 and AtGRP23, in response to the pathogenic invasion mediated by cutin monomers in plants.
The biochemical and ultrastructural changes in "green islands" (GIs) on detached Avena sterilis leaves caused by the macrodiolide (8R,16R)-(-)-pyrenophorin in the dark were examined. In the absence of light, leaf segments retained their photosynthetic pigments for 96 h after treatment with (8R,16R)-(-)-pyrenophorin (70 muM), whereas in the untreated leaves complete senescence, loss of photosynthetic pigments and cell disorganization were observed 72 h after detachment. Proteolytic enzyme activity in treated tissues with pyrenophorin remained at low levels for 96 h after treatment and protein dissipation was lower in the treated than in the untreated. Although tissues in "GIs" seem macroscopically healthy, electron microscopy observations revealed structurally disorganized cells filled with granular, electron-dense material. Chloroplasts were severely damaged and contained a large number of plastoglobuli. Similar ultrastructural changes were also observed in A. sterilis tissues treated with the phytotoxin under illumination, indicating a mechanism operating both under illumination and in the dark.
As long as 130 years ago Rissmüller reported substantial retranslocation of iron (Fe) from beech leaves (Fagus sylvatica L.) shortly before leaf fall. This rather limited report on Fe retranslocation via the phloem in plants was the reason for this research to study changes in Fe content in individual beech leaves in more detail during the vegetative period. Besides Fe, other micronutrients and particularly Ca and K, well known to differ substantially in phloem mobility, were analysed as mineral nutrient markers. In addition to beech, other deciduous and evergreen species of Angiosperms and Gymnosperms were also studied. As expected, there was no evidence of Ca retranslocation from senescent leaves, while K as a phloem mobile mineral nutrient was retranslocated in fall in deciduous but not in evergreen trees. There was no indication to support Rissmüller's finding of Fe retranslocation in any of the different species studied. From these results, we conclude that natural leaf senescence of trees during late season does not induce retranslocation of Fe and other micronutrients. Possible reasons for the absence of a distinct retranslocation of Fe in the species studied during late season senescence are the lack of a sink activity, as for example the development of seeds in annual plant species (e.g., cereals), or the presence of a root system still active enough to provide Fe and other mineral nutrients for plant demand, and both factors have to be considered in further studies. Reviewing the data in the literature on Fe and Zn retranslocation during senescence, we conclude that in principle both micronutrients are potentially phloem mobile. However, various prerequisites are needed for the occurrence of phloem mobility which were absent in the plant species studied. Regardless of this conclusion, we recommend that in general early published research data need a critical re-evaluation.
UDP-Glc:protein transglucosylase (UPTG) (EC is an autocatalytic glycosyl-transferase previously postulated as a protein that primes starch biosynthesis. Polyclonal antibodies raised against UPTG purified from potato (Solanum tuberosum L.) tubers were used to screen a potato swelling stolon tip cDNA expression library. The isolation, cloning and sequencing of two cDNAs corresponding to UPTG are described. Recombinant UPTG was labelled after incubation with UDP-[(14)C]-Glc and Mn(2+), indicating that it was enzymatically active. It was determined that purified as well as recombinant UPTG can be reversibly glycosylated by UDP-Glc, UDP-Xyl or UDP-Gal. RNA hybridization studies and western blot analysis indicate that UPTG mRNA and protein are expressed in all potato tissues. Databank searches revealed a high degree of identity between UPTG and several plant sequences that encode for proteins with apparent localization at the cytoplasmic face of the Golgi apparatus and at plasmodesmata. The biochemical properties of UPTG and the apparent lack of a signal peptide that could allow its entrance into plastids argue against the postulated role of UPTG in starch synthesis and point towards a possible role of the protein in the synthesis of cell wall polysaccharides.
Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp interacting with multiple partners in DNA transactions such as DNA replication/repair and recombination as well as chromatin assembly. We previously detected and purified by chromatographic procedures a 31 kDa PCNA from cultured wheat cells (Triticum monococcum L). Here we report the complete sequence of the wheat 31 kDa PCNA showing a very high aminoacid identity with its plant counterparts (maize and rice). This recombinant PCNA has been used as a bait in an affinity chromatography procedure, in order to capture PCNA interacting proteins. We detected by liquid chromatography, tandem mass spectrometry and search in plant protein databases, several specific bands from wheat cell lysates in fractions bound to wheat PCNA-affinity column. One of them is the wheat elongation factor 1A. Its putative regulatory role in DNA replication/repair is discussed.
We previously reported on a de novo designed protein "milk bundle-1Trp" (MB-1Trp) as a source of selected essential amino acids (EAA) for ruminant feeding. Here, we attempt to express this de novo designed protein in alfalfa. The microbial version of the gene encoding the protein was modified in order to achieve two expression strategies in transgenic alfalfa plants. Chimeric MB-1Trp genes alone or fused to a signal peptide and an endoplasmic reticulum retention sequence were introduced into alfalfa via Agrobacterium-mediated transformation. Polymerase chain reaction and reverse transcriptase polymerase chain reaction analysis performed on individual transgenic lines demonstrated that the MB-1Trp gene was correctly integrated and transcribed into mRNA. However, under our conditions, it was impossible to detect MB-1Trp protein expression in any of the transgenic plants analyzed. In order to assess MB-1Trp stability in alfalfa, Escherichia coli-derived MB-1Trp was incubated with proteins extracted from leaves of a non-transgenic plant. This study revealed a high susceptibility of mature MB-1Trp to alfalfa proteases, which may have contributed to its lack of accumulation.
Leaf senescence induced by 2,4-dichlorophenoxyacetic acid (2,4-D) and senescence inhibition caused by supplementation with silver (Ag(+)) ions in the form of silver nitrate (AgNO(3)) or silver nanoparticles (AgNPs) were investigated in 8-day-old mung bean (Vigna radiata L. Wilczek) seedlings. Inhibition of root and shoot elongation were observed in mung bean seedlings treated with 500μM 2,4-D. Concomitantly, the activity of 1-aminocyclopropane-1-carboxylic acid synthase was significantly induced in leaf tissue. Leaf senescence induced by 2,4-D was closely associated with lipid peroxidation as well as increased levels of cytotoxic hydrogen peroxide (H(2)O(2)) and superoxide radicals (O(2)(·-)). Despite decreased catalase activity, the activities of peroxidase, superoxide dismutase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase were increased during 2,4-D-induced leaf senescence. Further, the levels of reduced ascorbate, oxidized ascorbate, and reduced glutathione were markedly decreased, whereas the level of oxidized glutathione increased. 2,4-D-induced leaf senescence in mung bean was accompanied by an increase in positive terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, nuclear DNA fragmentation, and the activity of a 15-kDa Ca(2+)-dependent DNase. Supplementation with 100μM AgNO(3) or AgNPs inhibited 2,4-D-induced leaf senescence. The present results suggest that increased oxidative stress (O(2)(·-) and H(2)O(2)) led to senescence in mung bean leaves. Furthermore, significantly induced antioxidative enzymes are not sufficient to protect mung bean cells from 2,4-D-induced harmful ROS.
2,4-dichlorophenoxyacetic acid applied to excised leaves of Mimosa pudica L. inhibited in a dose-dependent manner the shock-induced pulvinar movement. This inhibition was negatively correlated with the amount of [(14)C] 2,4-dichlorophenoxyacetic acid present in the vicinity of the motor cells. Although 2,4-dichlorophenoxyacetic acid is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. This observation opens the question of its mode of action which may be through external signaling or following internal transport by a specific anionic form transporter. The effect was related to molecular structure since 2,4-dichlorophenoxyacetic acid>3,4-dichlorophenoxyacetic acid>2,3-dichlorophenoxyacetic acid. An essential target of 2,4-dichlorophenoxyacetic acid action lies at the plasmalemma as indicated by the induced hyperpolarization of the cell membrane. Compared to indole-3-acetic acid and fusicoccin, it induced a complex effect on H(+) fluxes. Applied to plasma membrane vesicles purified from motor organs, 2,4-dichlorophenoxyacetic acid enhanced proton pumping, but, unlike fusicoccin, it did not increase the H(+)-ATPase catalytic activity in our experimental conditions. Taken together, the data suggest that 2,4-dichlorophenoxyacetic acid acts on cell turgor variation and the concomittant ion migration, in particular K(+), by a mechanism involving specific steps compared to indole-3-acetic acid and fusicoccin.
The aim of this study is to investigate the effect of a well-characterized purified humic acid (non-measurable concentrations of the main plant hormones were detected) on the transcriptional regulation of the principal molecular agents involved in iron assimilation. To this end, non-deficient cucumber plants were treated with different concentrations of a purified humic acid (PHA) (2, 5, 100 and 250 mg of organic carbon L⁻¹) and harvested 4, 24, 48, 76 and 92 h from the onset of the treatment. At harvest times, the mRNA transcript accumulation of CsFRO1 encoding for Fe(III) chelate-reductase (EC; CsHa1 and CsHa2 encoding for plasma membrane H⁺-ATPase (EC; and CsIRT1 encoding for Fe(II) high-affinity transporter, was quantified by real-time RT-PCR. Meanwhile, the respective enzyme activity of the Fe(III) chelate-reductase and plasma membrane H⁺-ATPase was also investigated.
Brassinosteroids are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the biochemical characterization of two brassinosteroid-biosynthetic P450s from rice: CYP90D2 and CYP90D3. A rice dwarf mutant, ebisu dwarf (d2), which contains a defective copy of CYP90D2, is known to be a brassinosteroid-deficient mutant, and CYP90D2 has been considered to act as a C-3 dehydrogenase. However, in vitro biochemical assays using baculovirus/insect cell-produced proteins revealed that both CYP90D2 and CYP90D3 catalyze C-23 hydroxylation of various 22-hydroxylated brassinosteroids, but with markedly different catalytic efficiencies. Both enzymes preferentially convert (22S,24R)-22-hydroxyergost-4-en-3-one, (22S,24R)-22-hydroxy-5α-ergostan-3-one, and 3-epi-6-deoxocathasterone to the corresponding 23-hydroxylated products, but are less active in the conversion of (22S)-22-hydroxycampesterol and 6-deoxocathasterone, in vitro. Consistently, the levels of 23-hydroxylated products of these intermediates, namely, 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone, and 6-deoxotyphasterol were decreased in d2 mutants. These results indicate that CYP90D2 and CYP90D3 can act as brassinosteroid C-23 hydroxylases in rice.
The objective of this study was to explore the response of 24-epibrassinolide to improve the biological yield of Ni-tolerant and Ni-sensitive varieties of Vigna radiata and also to test the propositions that 24-epibrassinolide induced up-regulation of antioxidant system protects the efficiency of V. radiata, grown under Ni-stress. Surface sterilized seeds of var. T-44 (Ni-tolerant) and PDM-139 (Ni-sensitive) were soaked in DDW (control), 10(-10), 10(-8), or 10(-6) M of 24-epibrassinolide for 8 h (shotgun approach). These treated seeds were then inoculated with specific Rhizobium grown in sandy loam soil supplemented with different levels of Ni 0, 50, 100, or 150 mg Ni kg(-1) of soil and were allowed to grow for 45-days. At this stage of growth, plants were sampled to assess the various growths and nodule related traits as well as selected biochemical characteristics. The remaining plants were allowed to grow to maturity to study the yield characteristics. The results indicated that plant-fresh and dry mass, number of nodules, their fresh and dry mass, leghemoglobin content, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, activities of nitrate reductase and carbonic anhydrase decreased proportionately with the increasing concentrations of soil nickel. However, the application of 24-epibrassinolide as shotgun approach (pre-sowing seed soaking) to the nickel-stressed or non-stressed plants improved growth, nodulation and enhanced the activity of various antioxidant enzymes (viz. catalase, peroxidase and superoxide dismutase) and also the content of proline. The up-regulation of antioxidant enzymes as well as proline (osmolyte) triggered by 24-epibrassinolide could have conferred tolerance to the Ni-stressed plants resulting in improved growth, nodulation and yield attributes.
Ca(NO(3))(2) accumulation is a major factor that limits greenhouse production in China. The present investigation was carried out to study the effect of 24-epibrassinolide (EBL) on nitrogen metabolism (including contents of NO(3)(-), NH(4)(+) and amino acids and related enzymes activities) in cucumber seedlings (Cucumis sativus L. cv. Jinyou No. 4) under 80 mM Ca(NO(3))(2) stress. This study found that exogenous EBL significantly reduced the accumulation of NO(3)(-) and NH(4)(+) by Ca(NO(3))(2), and enhanced the inactivated enzymes activities involved in the nitrogen metabolism. In addition, EBL alleviated the inhibition of photosynthesis nitrogen-use efficiency by Ca(NO(3))(2). Increased total amino acids by EBL under stress increased the precursor of proteins biosynthesis, thus promoting the biosynthesis nitrogen containing compounds. The presence of Ca(NO(3))(2) increased polyamines level, which might result from the increased content of free putrescine that is harmful to plant growth. However, exogenous EBL induced a further increase in total polyamines. The increase is likely caused by the elevated contents of conjugated and bound forms of polyamines. In summary, exogenously EBL compensated for the damage/losses by Ca(NO(3))(2) stress to some extent through the regulation of nitrogen metabolism and metabolites.
Fast and stable 2-fold accumulation of cytokinins (CKs) was detected initially in roots and then in shoots of 4-day-old wheat (Triticum aestivum L.) seedlings in the course of their treatment with 0.4μM 24-epibrassinolide (EBR). Elevated cytokinin level has been maintained only in the presence of EBR, while the hormone removal has led to return of cytokinin concentration to the control level initially in the roots and then in the shoots. EBR-induced accumulation of cytokinins was accompanied by inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC activity and expression of the gene coding for this enzyme, and on the contrary the decline in CKs level resulted in increase in these characteristics up to the control level in roots and then in shoots. Sharp accumulation of cytokinin O-glucosides has been discovered in response to EBR-treatment suggesting fast EBR-induced activation of production of cytokinins, which excessive amounts were transferred into the storage forms. The obtained data provide evidence for the involvement of EBR in regulation of cytokinin level in wheat seedlings.
DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10(-5)) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200x more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.
The ubiquitin-mediated 26S proteasome pathway (UPS) is of great importance to plant growth and development. Previously research showed that a subunit of the 26S proteasome, named RPN1a, was involved in trichome's branching in Arabidopsis. Mutation in RPN1a give rise to more trichome branches on leaves. Here, we found that T-DNA insertion mutation in RPN1a resulted in increased trichome branches on main stem, and trichome number on rosette leaves and the main stem compared with the wild type plant. Expression analysis results showed that the transcription levels of ZFP6, ZFP5, GIS, GL1, GL2, GL3, TTG1 and MYB23, which promote trichome initiation, were up-regulated in the rpn1a mutant, and expression of FRC4, which is responsible for increased trichome branching, was also increased in the rpn1a mutant. Moreover, the mRNA expression level of RPN1a was significantly repressed by GA (gibberellin) and CK (cytokinin) treatment, which are two important phytohormones that play essential roles in trichome development. These results demonstrate that RPN1a may be involved in trichome development through the GA and CK signaling pathways. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Among various environmental stresses, salt stress is extensively damaging to major crops all over the world. An experiment was conducted to explore the role of exogenously applied 28 homobrassinolide (HBL) and salicylic acid (SA) on growth, photosynthetic parameters, transpiration and proline content of Brassica juncea L. cultivar Varuna in presence or absence of saline conditions (4.2 dsm(-1)). The leaves of 29d old plants were sprayed with distilled water, HBL and/or SA and plant responses were studied at 30 days after sowing (24 h after spray) and 45 days after sowing. The salinity significantly reduced the plant growth, gas exchange parameters but increased proline content and electrolyte leakage in the leaves. The effects were more pronounced at 30 DAS than 45 DAS. Out of the two hormones (HBL/SA) HBL excelled in its effects at both sampling stages. Toxic effects generated by salinity stress were completely overcome by the combination of the two hormones (HBL and SA) at 45 DAS.
The rubber particle is a specialized organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis (para rubber tree). It has been demonstrated that the small rubber particles (SRPs) has higher rubber biosynthesis ratio than the large rubber particles (LRPs), but the underlying molecular mechanism still remains unknown. In this study, LRPs and SRPs were firstly separated from the fresh latex using differential centrifugation, and two-dimensional difference in-gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF was then applied to investigate the proteomic alterations associated with the changed rubber biosynthesis capacity between LRPs and SRPs. A total of 53 spots corresponding to 22 gene products, were significantly altered with the |ratio|≥2.0 and T value ≤0.05, among which 15 proteins were up-regulated and 7 were down-regulated in the SRPs compared with the LRPs. The 15 up-regulated proteins in the SRPs included small rubber particle protein (SRPP), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), phospholipase D alpha (PLD α), ethylene response factor 2, eukaryotic translation initiation factor 5A isoform IV (eIF 5A-4), 70-kDa heat shock cognate protein (HSC 70), several unknown proteins, etc., whereas the 7 up-regulated proteins in the LRPs were rubber elongation factor (REF, 19.6kDa), ASR-like protein 1, REF-like stress-related protein 1, a putative phosphoglyceride transfer family protein, β-1,3-glucanase, a putative retroelement, and a hypothetical protein. Since several proteins related to rubber biosynthesis were differentially expressed between LRPs and SRPs, the comparative proteome data may provide useful insights into understanding the mechanism involved in rubber biosynthesis and latex coagulation in H. brasiliensis.
Benzofurans are bioactive compounds isolated from the Asteraceae family. Benzofuran derivatives have been extensively studied because of their toxic effects on humans and animals. The phytotoxic activity of the benzofuran derivative (2R)-6-hydroxytremetone was studied on germination, seedling development, and cytotoxic and genotoxic effects on monocotyledoneous (onion and wheat) and dicotyledoneous (lettuce and tomato) species. Results of these assays demonstrated that (2R)-6-hydroxytremetone is a potent germination inhibitor of onion, lettuce, and tomato seeds. Germination reductions of approximately 80% were measured when seeds were exposed to 100 mg l(-1) of the compound, and showed considerably effects on the posterior development of the sprouts, including rootlets and hypocotyl elongations. Moreover, this benzofuran derivative also significantly reduced the root length and mitotic division of Allium cepa bulbs, although DNA damages were not observed. Our findings suggest that a mechanism of mitosis inhibition may play a role in the phytotoxic effects of plants producing these compounds.
A crude 2S albumin fraction was separated from peanut (Arachis hypogaea L.) cotyledons. Untreated 2S albumin had little inhibitory activity against trypsin, spore germination, or hyphal growth of Aspergillus flavus. However, following treatment of 2S albumin with SDS, increased inhibitory activity was demonstrated. We further purified 2S albumin using Sephadex G-100 and DEAE cellulose (DE-32) chromatography. HPLC analysis showed that the partially pure 2S albumin consisted of two polypeptides, whereas SDS-PAGE analyzes exhibited six polypeptides. One of the polypeptides, 2S-1, was found to contain the same molecular weight and enzymatic properties as the peanut protease inhibitor (PI); however, the N-terminal amino acid sequence of 2S-1 differed from that of PI. An NCBI database search revealed that the 2S-1 polypeptide is homologous to the pathogenesis-related proteins from soybean, cowpea, chickpea, and Lupinus luteus. We hypothesize that the 2S-1 polypeptide might represent a novel antifungal protein.
Transcriptional initiation site of the gPm2S1 gene. Products of the primer extension reaction (lane P) were electrophoresed on an 8% polyacrylamide containing 7 M urea. The gPm2S1 sequencing reactions (lanes CTAG) performed using the same primer were used as ladders. The corresponding nucleotide positions of the primer extension product are shown by asterisks. 
Transient expression analysis of the gPm2S1 promoter. Douglas-fir megagametophytes (stage 5 MG), and zygotic embryos (stage 6 ZE) were bombarded with the plasmid pBI101 (A) or the gPm2S1/uidA construct (p2SSP1.2; B). Blue spots represent cells expressing GUS activity. Bars equal 1 mm.
Histochemical localization of gPm2S1 promoter activity in transgenic tobacco seeds. Seeds of transgenic tobacco were collected 30 d after flowering and stained for GUS activity as described in Methods. Bars in the two left panels and the right panel equal 1 mm and 0.25 mm, respectively. 
To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.
Photosystem II (PSII) is a homodimeric protein-cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the water splitting reaction during oxygenic photosynthesis. In the first part of this review, we describe the current state of the crystal structure at 3.0 A resolution of cyanobacterial PSII from Thermosynechococcus elongatus [B. Loll et al., Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-1044] with emphasis on the core antenna subunits CP43 and CP47 and the small membrane-intrinsic subunits. The second part describes first the general theory of optical spectra and excitation energy transfer and how the parameters of the theory can be obtained from the structural data. Next, structure-function relationships are discussed that were identified from stationary and time-resolved experiments and simulations of optical spectra and energy transfer processes.
Strictosidine synthase (STR; EC plays a key role in the biosynthesis of monoterpenoid indole alkaloids by catalyzing the Pictet-Spengler reaction between tryptamine and secologanin, leading exclusively to 3alpha-(S)-strictosidine. The structure of the native enzyme from the Indian medicinal plant Rauvolfia serpentina represents the first example of a six-bladed four-stranded beta-propeller fold from the plant kingdom. Moreover, the architecture of the enzyme-substrate and enzyme-product complexes reveals deep insight into the active centre and mechanism of the synthase highlighting the importance of Glu309 as the catalytic residue. The present review describes the 3D-structure and function of R. serpentina strictosidine synthase and provides a summary of the strictosidine synthase substrate specificity studies carried out in different organisms to date. Based on the enzyme-product complex, this paper goes on to describe a rational, structure-based redesign of the enzyme, which offers the opportunity to produce novel strictosidine derivatives which can be used to generate alkaloid libraries of the N-analogues heteroyohimbine type. Finally, alignment studies of functionally expressed strictosidine synthases are presented and the evolutionary aspects of sequence- and structure-related beta-propeller folds are discussed.
Flavanone 3beta-hydroxylase (F3H; EC is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC and anthocyanidin synthase (EC, can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.
The hydrolytic activity of phospholipase D (PLD) yielding phosphatidic acid from phosphatidylcholine and other glycerophospholipids is known to be involved in many cellular processes. In contrast, it is not clear whether the competitive transphosphatidylation activity of PLD catalyzing the head group exchange of phospholipids has a natural function. In poppy seedlings (Papaver somniferum L.) where lipid metabolism and alkaloid synthesis are closely linked, five isoenzymes with different substrate and hydrolysis/transphosphatidylation selectivities have been detected hitherto. A membrane-bound PLD, found in microsomal fractions of poppy seedlings, is active at micromolar concentrations of Ca(2+) ions and needs phosphatidylinositol 4,5-bisphosphate (PIP2) as effector in the hydrolysis of phosphatidylcholine (PC). The optimum PIP2 concentration at 1.2 mol% of the concentration of the substrate PC indicates a specific activation effect. Transphosphatidylation with glycerol, ethanolamine, l-serine, or myo-inositol as acceptor alcohols is also activated by PIP2, however, with an optimum concentration at 0.6-0.9 mol%. In contrast to hydrolysis, a basic transphosphatidylation activity occurs even in the absence of PIP2, suggesting a different fine-tuning of the two competing reactions.
In order to characterize the effects of increasing phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) on nuclear function, we expressed the human phosphatidylinositol (4)-phosphate 5-kinase (HsPIP5K) 1α in Nicotiana tabacum (NT) cells. The HsPIP5K-expressing (HK) cells had altered nuclear lipids and nuclear functions. HK cell nuclei had 2-fold increased PIP5K activity and increased steady state PtdIns(4,5)P(2). HK nuclear lipid classes showed significant changes compared to NT (wild type) nuclear lipid classes including increased phosphatidylserine (PtdSer) and phosphatidylcholine (PtdCho) and decreased lysolipids. Lipids isolated from protoplast plasma membranes (PM) were also analyzed and compared with nuclear lipids. The lipid profiles revealed similarities and differences in the plasma membrane and nuclei from the NT and transgenic HK cell lines. A notable characteristic of nuclear lipids from both cell types is that PtdIns accounts for a higher mol% of total lipids compared to that of the protoplast PM lipids. The lipid molecular species composition of each lipid class was also analyzed for nuclei and protoplast PM samples. To determine whether expression of HsPIP5K1α affected plant nuclear functions, we compared DNA replication, histone 3 lysine 9 acetylation (H3K9ac) and phosphorylation of the retinoblastoma protein (pRb) in NT and HK cells. The HK cells had a measurable decrease in DNA replication, histone H3K9 acetylation and pRB phosphorylation.
Arabidopsis cysteine-rich receptor-like protein kinase 45 (CRK45) was found to be involved in ABA signaling in Arabidopsis thaliana previously. Here, we reported that it also positively regulates disease resistance. The CRK45 overexpression plants increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae whereas the crk45 mutant were more sensitive to P. syringae and weakened expression of the defense genes, compared to the wild type. We also found that treatment with P. syringae leads to a declined expression of CRK45 in the npr1 mutant and the NahG transgenic plants. At the same time, significantly decreased expression of CRK45 transcript in the wrky70 mutant than that in the wild type was also detected. Our results suggested that CRK45 acted as a positive regulator in Arabidopsis disease resistance, and was regulated downstream of NPR1 and WRKY70 at the transcriptional level.
The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,(2)NCED5,(3)ABA2,(4) and AAO3(5) were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes.
HAHB4 is a sunflower gene encoding a homeodomain-leucine zipper (HD-Zip) transcription factor. It was previously demonstrated that this gene is regulated at the transcriptional level by several abiotic factors and hormones. A previous analysis in the PLACE database revealed the presence of four putative ABREs. In this work these four elements and also one W-box and two root-specific expression elements were characterized as functional. Site-directed mutagenesis on the promoter, stable transformation of Arabidopis plants as well as transient transformation of sunflower leaves, were performed. The analysis of the transformants was carried out by histochemistry and real time RT-PCR. The results indicate that just one ABRE out of the four is responsible for ABA, NaCl and drought regulation. However, NaCl induction occurs also by an additional ABA-independent way involving another two overlapped ABREs. On the other hand, it was determined that the W-box located 5' upstream is responsive to ethylene and only two root-specific expression elements, among the several detected, are functional but redundant. Conservation of molecular mechanisms between sunflower and Arabidopsis is strongly supported by this experimental work.
Regulation of lignin biosynthesis affects plant growth and wood properties. Transgenic downregulation of 4-coumarate:coenzyme A ligase (4CL, EC may reduce lignin content in cell walls, which could improve the qualities of pulp in papermaking and increase the efficiency of bioenergy applications. To determine the effects of Ptc4CL1 on lignin biosynthesis and plant growth, Populus tomentosa Carr. was transformed using sense-, antisense-, and RNAi-4CL1 genes. The growth properties, gene expression, enzyme activity, lignin content and composition and content of soluble phenolic acids were investigated in 1-year-old field-grown transgenic poplar trees. Transgenic up- and down-regulation of 4CL1 altered lignin content and composition in transgenic poplars, but there were no negative effects on the growth of transgenic plants. In addition, the severe changes in auxin observed in transgenic lines led to significantly enhanced growth performance. Furthermore, lignin content was tightly correlated with the alteration of 4CL1 enzymatic activity, which was correlated with 4CL1 gene expression. A significant increase in S units in lignin with a slight increase in sinapic acid was observed in 4CL1 down-regulated transgenic poplars. These results suggest that 4CL1 is a traffic control gene in monolignol biosynthesis and confirm that 4CL1 activity has been implicated with sinapoyl activation. Finally, our data demonstrate that there is cross-correlation among 4CL1 gene expression, 4CL1 enzyme activity, soluble phenolic acid, lignin monomer biosynthesis, and lignin content.
Seven 4-desmethylsterols, five triterpene alcohols and three 4α-monomethylsterols were identified by GC-MS during the development of wild peanut, which is Arbi (AraA), and cultivars peanut, which are Trabelsia (AraT) and Chounfakhi (AraC). Our results showed that the maximum level of 4-desmethylsterols (881.07 mg/100 g of oil) was reached at 12 days after flowering (DAF) date of peanut plant in AraA, as well as the highest level of triterpene alcohols (31.51 mg/100 g of oil) was reached at 23 DAF in AraA, whilst, the highest level of 4α-monomethylsterols (15.11 mg/100 g of oil) was reached at 41 DAF in AraC. Herein, the level of triterpene alcohols and 4α-monomethylsterols was overwhelmed by the amount of 4-desmethylsterols at each stage of peanut maturity. Differences were observed in each sterol contents among the studied cultivars and wild one especially in immature stage.
Desiccation tolerance is one of the most important traits determining seed survival during storage and under stress conditions. However, the mechanism of seed desiccation tolerance is still unclear in detail. In the present study, we used a combined model system, desiccation-tolerant and -sensitive maize embryos with identical genetic background, to investigate the changes in desiccation tolerance, malonyldialdehyde (MDA) level, hydrogen peroxide (H2O2) content and antioxidant enzyme activity during seed development and germination in 0, -0.6 and -1.2 MPa polyethylene glycol (PEG)-6000 solutions. Our results indicated that maize embryos gradually acquired and lost desiccation tolerance during development and germination, respectively. The acquirement and loss of desiccation tolerance of embryos during development and germination were related to the ability of antioxidant enzymes including superoxide dismutase (SOD, EC, ascorbate peroxidase (APX, EC, catalase (CAT, EC, glutathione reductase (GR, EC and dehydroascorbate reductase (DHAR, EC to scavenge reactive oxygen species (ROS) and to control MDA content. Compared with treatment in water, PEG-6000 treatment could markedly delay the loss of desiccation tolerance of germinating embryos by delaying water uptake and time course of germination, increasing GR activity and decreasing MDA content. Our data showed the combination of antioxidant enzyme activity and MDA content is a good parameter for assessing the desiccation tolerance of maize embryos. In addition, H2O2 accumulated in mature embryos and PEG-treated embryos after drying, which was at least partially related to a longer embryo/seedling length in rehydration and the physiological mechanisms of priming.
Two phosphoenolpyruvate carboxykinase (PEPCK, EC isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.
The role of sucrose in cyanobacteria is still not fully understood. It is generally considered a salt-response molecule, and particularly, in Synechocystis sp. strain PCC 6803, it is referred as a secondary osmolyte. We showed that sucrose accumulates transiently in Synechocystis cells at early stages of a salt shock, which could be ascribed to salt activation of sucrose-phosphate synthase (SPS, UDP-glucose: D-fructose-6-phosphate 2-alpha-D-glucosyltransferase; EC, the key enzyme in sucrose synthesis pathway, and to an increase of the expression of the SPS encoding gene. Experiments with a mutant strain impaired in sucrose biosynthesis showed that sucrose is essential in stationary phase cells to overcome a later salt stress. Taken together, these results led us to suggest a more intricate function for sucrose than to be an osmoprotectant compound.
Active NADPH dehydrogenase super- and medium-complexes were newly identified in cyanobacteria and are essential to cyclic photosystem I (PSI) activity and respiration and to CO(2) uptake, respectively. Synechocystis sp. strain PCC 6803 cells were treated with exogenous glucose (Glc) for different times. Active staining of NADPH-nitroblue tetrazolium oxidoreductase and western blot were conducted, and the initial rate of P700(+) dark reduction was measured. The expression and enzyme activity of the NADPH dehydrogenase super-complex were gradually inhibited and were found to be closely associated with the decrease in cyclic PSI activity, as reflected by the initial rate of P700(+) dark reduction. By contrast, those of the NADPH dehydrogenase medium-complex and the activity of CO(2) uptake reflected by the expression levels of NdhD3 and NdhF3 were not significantly affected by the addition of exogenous Glc to the cultures; however, the expression and enzyme activity of this medium-complex were found to be significantly influenced by the changes in CO(2) concentration. These results indicated that (1) the responses of the 2 cyanobacterial NADPH dehydrogenase complexes to exogenous Glc in terms of their expression and activity differed and that (2) these responses were closely associated with their respective physiological roles.
Effect of substrate/co-substrate concentration on the DmrH6H catalyzed reaction velocity. The DmrH6H activity was assayed at varying concentrations of substrate, hyoscyamine (A) and co-substrate, 2-oxoglutarate (B). The velocity of the reaction is shown plotted against the substrate/co-substrate concentrations giving a typical Michaelis e Menten curve in each case. 
Effect of hyoscyamine and 2-oxoglutarate on the fl uorescence emission spectra (A, B) and secondary structure (C) of DmrH6H. The fl uorescence emission spectra of DmrH6H are given as a function of hyoscyamine and 2-oxoglutarate concentrations. Stern e Volmer plots are shown in the inset. See Materials and methods for details. Panel C shows the CD spectra of DmrH6H-hyoscyamine and DmrH6H-2-oxoglutarate complexes as ellipticity at different wavelengths showing a clear effect of hyoscyamine and 2-oxoglutarate on the protein secondary structure. 
Hyoscyamine 6β-hydroxylase (H6H; EC, an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K(m) values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14M(-1) and 0.56M(-1), respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.
Steviol glycosides, extracted from the leaves of Stevia rebaudiana (Bert) Bertoni, are calorie-free sugar substitute of natural origin with intensely sweet (Boileau et al., 2012). Stevioside and rebaudioside A are the two main kinds of the diterpenic glycosides. We analyzed the concentration of stevioside and rebaudioside A in Stevia leaves of about 500 samples (hybrid progenies) and discovered a mutation plant "Z05" with very low levels of rebaudioside A. Because UGT76G1, a uridinediphosphate-dependent glycosyltransferases, is responsible for the conversion from stevioside to rebaudioside A (Richman et al., 2005), so mutation identification was done by sequencing the candidate gene, UGT76G1. In this study molecular analysis of two strains revealed a heterozygotic nonsense mutation of c.389T > G (p.L121X) in UGT76G1. Meanwhile, we found some amino acid substitutions significant change the protein structure. And the difference of enzyme activity between two strains proved the lack of functionality of UGT76G1 of the mutation "Z05". So the nonsense mutation and amino acid substitution mutation resulted in the low levels of rebaudioside A.
Quantum dots (QDs) are a novel type of small, photostable and bright fluorophores that have been successfully applied to mammalian and human live cell imaging. In this study, highly dispersive water-soluble mercaptoacetic acid (MAA)-coated CdSe/ZnS QDs were synthesized, which were suitable for investigation as fluorescent probe labels. The treatment of maize seedling roots with QDs showed that the surfactant silwet L-77 aided the efficient transport of QDs into maize roots. Under a concentration ranging from 0.128 to 1.28 microM, QDs caused very low cytotoxicity on maize seed germination and root growth. The addition of mercuric chloride to the Hoagland solution resulted in a decrease of QD content in root tissues, and this decrease was reversed upon the addition of beta-mercaptoethanol, which suggests that mercury-sensitive processes play a significant role in regulating QD flow in the maize root system. We speculate that the apoplastic pathway can contribute substantially to the total quantity of QDs reaching the stele. Therefore, based on this transport approach, MAA-coated QDs can be utilized for live imaging in plant systems to verify known physiological processes.
Light-induced and lumen acidity-dependent quenching (qE) of excited chlorophylls (Chl) in vivo has been amply documented in plants and algae, but not in cyanobacteria, using primarily the saturation pulse method of quenching analysis which is applied to continuously illuminated samples. This method is unsuitable for cyanobacteria because the background illumination elicits in them a very large Chl a fluorescence signal, due to a state 2 to state 1 transition, which masks fluorescence changes due to other causes. We investigated the qE problem in the cyanobacterium Synechococcus sp. PCC 7942 using a kinetic method (Chl a fluorescence induction) with which qE can be examined before the onset of the state 2 to state 1 transition and the attendant rise of Chl a fluorescence. Our results confirm the existence of a qE mechanism that operates on excited Chls a in Photosystem II core complexes of cyanobacteria.
Top-cited authors
Bernard R. Glick
  • University of Waterloo
Andrzej Bajguz
  • University of Bialystok
Shamsul Hayat
  • Aligarh Muslim University
Dr. Vijay Pratap Singh
  • C.M.P. Degree College: A Constituent Post Graduate College of University of Allahabad
Sheo Mohan Prasad
  • University of Allahabad