Water and salt stress-induced alterations in proline metabolism of Triticum durum seedlings

Many plants accumulate proline as a non-toxic and protective osmolyte under saline or dry conditions. Its accumulation is caused by both the activation of its biosynthesis and inactivation of its degradation. We report here on the alterations induced by water and salt stress in the proline metabolism and amino acid content of 5-day-old seedlings of Triticum durum cv. Simeto. Most of the amino acids showed an increase with the induction of either stress, but proline increased more markedly than did other amino acids. We also measured the activities of two enzymes, Δ¹-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) and proline dehydrogenase (EC 1.5.1.2), which are involved in proline biosynthesis and catabolism, respectively. The activity of P5C reductase was enhanced during both water and salt stress, while proline dehydrogenase was inhibited only during salt stress. The results indicate that synthesis de novo is the predominant mechanism in proline accumulation in durum wheat. Use of a cDNA clone that encodes P5C-reductase from Arabidopsis thaliana , showed no differences in the gene expression between controls and stressed plants, implying that the increase in enzyme activity is unrelated to the expression of this gene.     

Genetic manipulation of proline levels affects antioxidants in soybean subjected to simultaneous drought and heat stresses

It was assumed that the genetic manipulation of the proline (Pro) level would also affect the (homo)glutathione content as both compounds have a common precursor, glutamate. To test this hypothesis, the levels of Pro, reduced and oxidized (homo)glutathione [(h)GSH and (h)GSSG] and other antioxidants were compared under simultaneous drought and heat stress conditions and in a control treatment in a time course experiment on wild-type soybean ( Glycine max cv. Ibis) and on transgenic plants containing the cDNA coding for l -Δ¹-pyrroline-5-carboxylate reductase (EC 1.5.1.2), the last enzyme involved in Pro synthesis, in the sense and antisense directions. At the end of the recovery period, the highest H₂O₂ and lipid hydroperoxide concentrations were observed in the antisense transformants, which exhibited the greatest injury, while the lowest H₂O₂ content was detected in the sense transformants, which exhibited the lowest injury percentage. During stress treatment, the highest Pro and ascorbate (AA) levels were detected in the sense transformants, while the highest GSH and hGSH contents, AA/dehydroascorbate (DHA) and (h)GSH/(h)GSSG ratios and ascorbate peroxidase (APX) activity were found in the antisense transformants. The greatest APX (EC 1.11.1.11) activity was observed in the first part of the stress treatment in the antisense transformants, and the greatest glutathione reductase (EC 1.6.4.2) activity was observed in the second part of the treatment in the same genotype. The present experiments indicate that the manipulation of Pro synthesis affects not only the (h)GSH concentrations, but also the levels of other antioxidants.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

Expression of Δ1-pyrroline-5-carboxylate dehydrogenase and proline/arginine homeostasis in Solanum tuberosum

Polyclonal antibodies raised in mouse against purified potato Δ ¹-pyrroline-5-carboxylate dehydrogenase (P5C-DH, EC 1.5.1.12), which catalyses the last step in the catabolism of both proline and arginine, were used to investigate the expression of this enzyme. Distribution of P5C-DH in potato ( Solanum tuberosum L. cv. Desiree) organs was studied at different stages during plant development. Variations in enzyme level were determined in axenically grown plantlets following the addition of exogenous proline, and in cell suspension cultures under hyperosmotic stress and after its relief. Free proline and arginine levels were also quantified, and compared to those of the enzyme. Results were consistent with a developmental, but not with an environmental, control of P5C-DH expression. The possible involvement of specific isozymes in proline and arginine oxidation is discussed.     

NaCl effects on proline metabolism in rice (Oryza sativa) seedlings

Salt-stress effects on osmotic adjustment, ion and proline concentrations as well as proline metabolizing enzyme activities were studied in two rice ( Oryza sativa L.) cultivars differing in salinity resistance: I Kong Pao (IKP; salt-sensitive) and Nona Bokra (salt-resistant). The salt-sensitive cultivar exposed to 50 and 100 m M NaCl in nutritive solution for 3 and 10 days accumulated higher levels of sodium and proline than the salt-resistant cultivar and displayed lower levels of osmotic adjustment. Proline accumulation was not related to proteolysis and could not be explained by stress-induced modifications in Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) or proline dehydrogenase (PDH; EC 1.5.1.2) activities recorded in vitro. The extracted ornithine Δ -aminotransferase (OAT; EC 2.6.1.13) activity was increased by salt stress in the salt-sensitive cultivar only. In both genotypes, salt stress induced an increase in the aminating activity of root glutamate dehydrogenase (GDH; EC 1.4.1.2) while deaminating activity was reduced in the leaves of the salt-sensitive cultivar. The total extracted glutamine synthetase activity (GS; EC 6.3.1.2) was reduced in response to salinity but NaCl had contrasting effects on GS1 and GS2 isoforms in salt-sensitive IKP. Salinity increased the activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) extracted from leaves of both genotypes and increased the activity of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the salt-sensitive cultivar. It is suggested that proline accumulation is a symptom of salt-stress injury in rice and that its accumulation in salt-sensitive plants results from an increase in OAT activity and an increase in the endogenous pool of its precursor glutamate. The physiological significance of the recorded changes are analyzed in relation to the functions of these enzymes in plant metabolism.     

Correlation between the induction of a gene for Δ1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress

The isolation and characterization is reported of a cDNA for Δ¹-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana . Southern blot analysis suggested that only one copy of the corresponding gene ( AtP5CS ) is present in A. thaliana . The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia . Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana . A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.     

Metabolic response to cold and freezing of Osteospermum ecklonis overexpressing Osmyb4

The constitutive expression of the rice Osmyb4 gene in Arabidopsis plants gives rise to enhanced abiotic and biotic stress tolerance, probably by activating several stress-inducible pathways. However, the effect of Osmyb4 on stress tolerance likely depends on the genetic background of the transformed species.In this study, we explored the potential of Osmyb4 to enhance the cold and freezing tolerance of Osteospermum ecklonis, an ornamental and perennial plant native to South Africa, because of an increasing interest in growing this species in Europe where winter temperatures are low.Transgenic O. ecklonis plants were obtained through transformation with the Osmyb4 rice gene under the control of the CaMV35S promoter.We examined the phenotypic adaptation of transgenic plants to cold and freezing stress. We also analysed the ability of wild-type and transgenic Osteospermum to accumulate several solutes, such as proline, amino acids and sugars. Using nuclear magnetic resonance, we outlined the metabolic profile of this species under normal growth conditions and under stress for the first time. Indeed, we found that overexpression of Osmyb4 improved the cold and freezing tolerance and produced changes in metabolite accumulation, especially of sugars and proline. Based on our data, it could be of agronomic and economic interest to use this gene to produce Osteospermum plants capable of growing in open field, even during the winter season in climatic zone Z9.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.     

AN INCREASE OF ORNITHINE δ-AMINOTRANSFERASE-MEDIATED PROLINE SYNTHESIS IN RELATION TO HIGH-TEMPERATURE INJURY IN GRACILARIA TENUISTIPITATA (GIGARTINALES, RHODOPHYTA)

Changes of proline biosynthesis in relation to high-temperature (35° C) injury were investigated in Gracilaria tenuistipitata var. liui Zhang et Xia. On exposure to 35° C, the specific growth rate decreased after 5 days while free proline levels increased gradually after 2 days and reached the maximal level on days 4–6 but decreased at day 7. The repair ability of thalli treated at 35° C by measuring the growth rate after transfer to 25° C for another 5 days decreased in thalli that had been grown at 35° C for more than 2 days, and the extent increased as treatment at 35° C was prolonged. After 4 days of treatment at 35° C, the activities of both ornithine δ-aminotransferase (δ-OAT; EC 2.6.11.3) and Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) increased, but that of γ-glutamyl kinase (γ-GK; EC 2.7.2.11) remained unchanged, and that of glutamate-5-semialdehyde dehydrogenase (GSAd; EC 1.4.1.3) decreased. The application of 10 μM gabaculine, an irreversible inhibitor of δ-OAT, at 35° C recovered the growth ability but inhibited the increase of both δ-OAT activity and free proline level; its effects were reversed by 1 mM proline. G. saliconia, which is relatively tolerant to high temperature, showed a decrease of both δ-OAT activity and free proline level at 35° C. It seems that a stimulation of proline synthesis from the ornithine pathway via an increase in both δ-OAT and P5CR activities might be associated with high-temperature injury in G. tenuistipitata.     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

转拟南芥P5CS1基因增强羽衣甘蓝的耐旱性

为提高羽衣甘蓝的耐旱性,本文将拟南芥Δ1-吡咯啉-5-羧酸合成酶（P5CS1）基因经农杆菌介导转入羽衣甘蓝植株中,检测转基因株系与野生型植株在干旱胁迫下P5CS1 mRNA表达量、幼苗脯氨酸含量、株系根系性状、整株干重、鲜重和整株存活率。结果表明,在15%PEG6000渗透胁迫下,转基因植株的P5CS1基因mRNA表达量明显增加,转基因植株脯氨酸含量是野生型的2.4倍;主根长、最长侧根长、侧根数目、整株干重和鲜重均高于野生型,干重/鲜重则低于野生型,转基因植株的平均存活率为78%,极显著高于野生型。数据显示,AtP5CS1基因在羽衣甘蓝中的表达明显改善了转基因植株的耐旱性。     

Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance

Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.     

Enhanced proline accumulation and salt stress tolerance of transgenic <Emphasis Type="Italic">indica</Emphasis> rice by over-expressing <Emphasis Type="Italic">P5CSF129A</Emphasis> gene

Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T₁) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.