Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.     

Citrulline and DRIP-1 Protein (ArgE Homologue) in Drought Tolerance of Wild Watermelon

Drought-affected plants experience more than just desiccationof their organs due to water deficit. Plants transpire 1000times more molecules of water than of CO₂ fixed by photosynthesisin full sunlight. One effect of transpiration is to cool theleaves. Accordingly, drought brings about such multi-stressesas high temperatures, excess photoradiation and other factorsthat affect plant viability. Wild watermelon serves as a suitablemodel system to study drought responses of C₃ plants, sincethis plant survives drought by maintaining its water contentwithout any wilting of leaves or desiccation even under severedrought conditions. Under drought conditions in the presenceof strong light, wild watermelon accumulates high concentrationsof citrulline, glutamate and arginine in its leaves. The accumulationof citrulline and arginine may be related to the induction ofDRIP-1, a homologue of ArgE in Escherichia coli, where it functionsto incorporate the carbon skeleton of glutamate into the ureacycle. Immunogold electron microscopy reveals the enzyme tobe confined exclusively to the cytosol. DRIP-1 is also inducedby treating wild watermelon with 150 mM NaCl, but is not inducedfollowing treatment with 100 µM abscisic acid. The salttreatment causes the accumulation of -aminobutyrate, glutamineand alanine, in addition to a smaller amount of citrulline.Citrulline may function as a potent hydroxyl radical scavenger.     

Respiratory complex I deficiency induces drought tolerance by impacting leaf stomatal and hydraulic conductances

To investigate the role of plant mitochondria in drought tolerance, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant, which has low-efficient respiration and photosynthesis, high levels of amino acids and pyridine nucleotides, and increased antioxidant capacity. We show that the delayed decrease in relative water content after water withholding in CMSII, as compared to WT leaves, is due to a lower stomatal conductance. The stomatal index and the abscisic acid (ABA) content were unaffected in well-watered mutant leaves, but the ABA/stomatal conductance relation was altered during drought, indicating that specific factors interact with ABA signalling. Leaf hydraulic conductance was lower in mutant leaves when compared to WT leaves and the role of oxidative aquaporin gating in attaining a maximum stomatal conductance is discussed. In addition, differences in leaf metabolic status between the mutant and the WT might contribute to the low stomatal conductance, as reported for TCA cycle-deficient plants. After withholding watering, TCA cycle derived organic acids declined more in CMSII leaves than in the WT, and ATP content decreased only in the CMSII. Moreover, in contrast to the WT, total free amino acid levels declined whilst soluble protein content increased in CMSII leaves, suggesting an accelerated amino acid remobilisation. We propose that oxidative and metabolic disturbances resulting from remodelled respiration in the absence of Complex I activity could be involved in bringing about the lower stomatal and hydraulic conductances.     

Citrulline, a novel compatible solute in drought-tolerant wild watermelon leaves, is an efficient hydroxyl radical scavenger.

Drought-tolerant wild watermelon accumulates high levels of citrulline in the leaves in response to drought conditions. In this work, the hydroxyl radical-scavenging activity of citrulline was investigated in vitro. The second-order rate constant for the reaction between citrulline and hydroxyl radicals was found to be 3.9x10(9) M(-1) s(-1), demonstrating that citrulline is one of the most efficient scavengers among compatible solutes examined so far. Moreover, citrulline effectively protected DNA and an enzyme from oxidative injuries. Liquid chromatography-mass spectrometry analysis revealed that at least four major products were formed by the reaction between citrulline and hydroxyl radicals. Activities of metabolic enzymes were not inhibited by up to 600 mM citrulline, indicating that citrulline does not interfere with cellular metabolism. We reasoned, from these results, that citrulline contributes to oxidative stress tolerance under drought conditions as a novel hydroxyl radical scavenger.     

Composition and Distribution of Free Amino Acids in Flatpea (Lathyrus sylvestris L.) as Influenced by Water Deficit and Plant Age

Drought-stressed flatpea (Lathyrus sylvestris L.) plants from8 to 22 weeks old were analysed for nitrogen, soluble proteinand free amino acids. An increase in nitrogen and free aminoacid concentrations and a decrease in soluble protein levelwere observed in roots of plants up to 16 weeks old. The cumulativeconcentration of free amino acids increased with drought stress.Tissue concentrations of 2, 4-diaminobutyric acid (1.6–2.6%of the dry weight) were highest in leaves. Levels increasedsteadily, nearly doubling, in leaves and stems between weeks10 and 16. Levels in drought-stressed leaves were, on average,11.9% higher than those of controls. Estimated concentrationsof a mixture of 4-aminobutyric acid and an unknown amino acidwere highest in stems, increased in this tissue with age andtended to increase in stems and leaves and decrease in rootsin response to water deficit. Levels of the mixture of homoserineand another unidentified amino acid were not influenced by ageor water status of the plants. Root concentrations of asparagine,arginine, glutamine, aspartate, and another prominent, unidentifiedamino acid increased with plant age and reached a peak at thetime of flowering (14 to 18 weeks). Only the concentration ofthe unknown compound was elevated following drought stress.Concentrations of valine, isoleucine, leucine, phenylalanine,and methionine also increased during this period and were elevatedin drought-stressed plants. Proline levels increased with plantage and drought stress, but proline accounted for only about10% of the total free amino acids in the drought-stressed plants. Key words: 2, 4-Diaminobutyric acid, drought, flatpea     

Drought and watering-dependent oxidative stress: effect on antioxidant content in Triticum aestivum L. leaves

The purpose of the present work was to evaluate both oxidative stress and the antioxidant response system in leaves from wheat (Triticum aestivum cv. Buck Poncho) subjected sequentially to drought and watering. Drought was imposed by withholding water until soil water potential reached -2.0 MPa and maintained under those conditions for 24 h. DCFDA oxidation by wheat leaves was not significantly affected by drought, but watering led to an approximately 2-fold increase in DCFDA oxidation rate. However, no significant effect either on lipid radical content or on hydroperoxide content was measured after drought and drought followed by watering. Microsomes isolated from leaves exposed to drought, and from leaves exposed to drought followed by watering, generated a significantly higher amount of hydroxyl radical as compared to microsomes isolated for control leaves suggesting a higher production of hydroxyl radical in the cellular water soluble phase, after drought and watering as compared to control values. The content of -tocopherol in wheat leaves was increased 2.4-fold after drought and -carotene content was increased by 2.6-fold after drought. Hydration lowered lipid-soluble antioxidant content to control values. Total thiol content as increased by 70% after drought, and watering did not significantly alter the enhanced values. Drought decreased by 28.5% the content of reduced ascorbic acid. Taken as a whole, active species formed at wheat membranes after exposure to moderate water stress, are efficiently removed upon rehydration by reaction with an increased content of -tocopherol and -carotene. Moreover a co-ordinated response involving glutathione reductase activity, thiols and ascorbic acid is triggered to limit free radical dependent effects.Key words: Antioxidants, lipid radicals, oxygen radicals, water stress, wheat      

The effects of drought stress on free amino acid accumulation and protein synthesis in Brassica napus

Changes in the concentrations of free amino acids and specific organic acids were analysed during the induction of drought stress in Brassica napus . Most of the amino acids showed a characteristic linear increase with the induction of drought stress in Brassica leaves, increasing an average of 5.9-fold over control levels, followed by a reduction in concentration upon rehydration of the plants. Pyruvate concentrations doubled after 4 days of drought stress whereas 2-oxoglutarate concentrations remained relatively constant. The activities of two of the enzymes involved in amino acid biosynthesis, alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1), were also measured. Neither enzyme showed any increase in activity, except when the plants were rehydrated. This suggests that the increase in both alanine and aspartate levels results from the increase in their precursors pyruvate and glutamate and may not require increased enzyme activity. The effect of drought stress upon changes in protein synthesis was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that there was an overall decrease in protein synthesis with the induction of drought stress, followed by a resumption of synthesis upon rehydration. In addition, the synthesis of a number of specific polypeptides was found to decrease upon water loss in the leaves.     

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23^fyp and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.     

Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought

We used proteomic analysis to determine the response of rice plant seedlings to drought-induced stress. The expression of 71 protein spots was significantly altered, and 60 spots were successfully identified. The greatest down-regulated protein functional category was translation. Up-regulated proteins were mainly related to protein folding and assembly. Additionally, many proteins involved in metabolism (e.g. carbohydrate metabolism) also showed differences in expression. cDNA microarray and GC-MS analysis showed 4756 differentially expressed mRNAs and 37 differentially expressed metabolites. Once these data were integrated with the proteomic analysis, we were able to elucidate the metabolic pathways affected by drought-induced stress. These results suggest that increased energy consumption from storage substances occurred during drought. In addition, increased expression of the enzymes involved in anabolic pathways corresponded with an increase in the content of six amino acids. We speculated that energy conversion from carbohydrates and/or fatty acids to amino acids was increased. Analysis of basic metabolism networks allowed us to understand how rice plants adjust to drought conditions.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

Responses of the photosynthetic electron transport system to excess light energy caused by water deficit in wild watermelon

In plants, drought stress coupled with high levels of illumination causes not only dehydration of tissues, but also oxidative damage resulting from excess absorbed light energy. In this study, we analyzed the regulation of electron transport under drought/high-light stress conditions in wild watermelon, a xerophyte that shows strong resistance to this type of stress. Under drought/high-light conditions that completely suppressed CO(2) fixation, the linear electron flow was diminished between photosystem (PS) II and PS I, there was no photoinhibitory damage to PS II and PS I and no decrease in the abundance of the two PSs. Proteome analyses revealed changes in the abundance of protein spots representing the Rieske-type iron-sulfur protein (ISP) and I and K subunits of NAD(P)H dehydrogenase in response to drought stress. Two-dimensional electrophoresis and immunoblot analyses revealed new ISP protein spots with more acidic isoelectric points in plants under drought stress. Our findings suggest that the modified ISPs depress the linear electron transport activity under stress conditions to protect PS I from photoinhibition. The qualitative changes in photosynthetic proteins may switch the photosynthetic electron transport from normal photosynthesis mode to stress-tolerance mode.     

Alterations in photosynthetic pigments, protein and osmotic components in cotton genotypes subjected to short-term drought stress followed by recovery

In order to assess drought tolerance mechanism in cotton, short-term drought-induced biochemical responses were monitored in two cotton (Gossypium hirsutum L.) genotypes contrasting their tolerance to water deficit. The seeds of two genotypes, namely GM 090304 (moderately drought tolerant) and Ca/H 631 (drought sensitive), were sown in pots containing soil, sand and peat in the ratio of 1:1:1, and irrigated every alternate day up to 45 days after sowing when each genotype was subjected to a cycle of water stress by withholding irrigation for 7 days. The stress cycle was terminated by re-watering the stressed plants for 7 days. The leaf of the drought tolerant genotype (GM 090304) maintained higher relative water content under water stress than that of the drought sensitive genotype (Ca/H 631). The levels of biochemical components, such as chlorophylls, carotenoids, total protein, free proline, total free amino acids, sugars, starch and polyphenols, were measured during the stress as well as the recovery periods. The chlorophylls, carotenoids, protein and starch contents decreased in drought stressed plants as compared to control and tended to increase when the plants were recovered from stress. The degree of decrease in chlorophylls, carotenoids and protein contents under drought was higher in the sensitive genotype (Ca/H 631) as compared to the moderately tolerant genotype (GM 090304). However, proline, total free amino acids, total sugars, reducing sugars and polyphenol contents were increased in drought stressed plants and tended to decrease during the period of recovery. Drought-induced increases in total free amino acids, proline, sugars and polyphenols were significantly higher in the moderately tolerant genotype (GM 090304) than in the sensitive genotype (Ca/H 631). These results suggest that proline, sugars and polyphenols act as main compatible solutes in cotton in order to maintain osmotic balance, to protect cellular macromolecules, to detoxify the cells, and to scavenge free radicals under water stress condition.     

Overexpression of wheat <Emphasis Type="Italic">TaNCED</Emphasis> gene in <Emphasis Type="Italic">Arabidopsis</Emphasis> enhances tolerance to drought stress and delays seed germination

Abscisic acid (ABA) regulates various plant physiological processes, especially participates in the plant responses to harsh environments. The 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis pathway. Here, a TaNCED with an 1 887-bp open reading frame was cloned from wheat, which encodes a peptide of 628 amino acids. A chloroplast transit peptide sequence was found at the N-terminus of the TaNCED protein. Multiple sequence alignments indicate that the TaNCED protein shared high similarities with other NCEDs from different species. Real-time quantitative PCR analysis shows that expression of TaNCED was strongly up-regulated by treatments with ABA, polyethylene glycol, and drought stress, and it was down-regulated during germination of the wheat seeds. Ectopic overexpression of the TaNCED gene in Arabidopsis resulted in an increase of endogenous ABA and free proline content. A lower water loss rate and stomatal conductance of leaves were found in the transgenic plants in comparison with the wild type. Subsequently, the transgenic plants displayed an enhanced tolerance to drought stress but delayed seed germination. These data provide evidence that the TaNCED might play a primary role in regulation of ABA content during water stress and seed dormancy.     

Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C

A protein phosphatase 2C (PP2C)-homologous cDNA was isolated from Nicotiana tabacum (NtPP2C1). The deduced protein sequence of 416 amino acids showed the highest degree of similarity to the PP2C of Arabidopsis thaliana (AtPP2CA) implicated in abscisic acid signalling. The expression of NtPP2C1 was strongly induced by drought, but repressed by oxidative stress and heat shock. It is suggested that NtPP2C1 operates at the junction of drought, heat shock and oxidative stress.