Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana

Dehydroascorbate reductase (DHAR) is a key component of the ascorbate recycling system. Three functional DHAR genes are encoded in the Arabidopsis genome. Ozone exposure increased the expression of the cytosolic DHAR (cytDHAR) gene alone. We characterized an Arabidopsis mutant with a deficient cytDHAR. The mutant completely lacked cytDHAR activity and was highly ozone sensitive. The amounts of total ascorbate and glutathione were similar in both lines, but the amount of apoplastic ascorbate in the mutant was 61.5% lower. These results indicate that the apoplastic ascorbate, which is generated through the reduction of DHA by cytDHAR, is important for ozone tolerance.     

Gluten quality of bread wheat is associated with activity of RabD GTPases

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER‐to‐Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down‐regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT‐PCR to be down‐regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE‐HPLC found that a smaller proportion of HMW‐GS and large proportion of LMW‐GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS‐PAGE was seen in the transgenic grain.     

Characterization of some bread wheat genotypes using molecular markers for drought tolerance

Because of its wide geographical adaptation and importance in human nutrition, wheat is one of the most important crops in the world. However, wheat yield has reduced due to drought stress posing threat to sustainability and world food security in agricultural production. The first stage of drought tolerant variety breeding occurs on the molecular and biochemical characterization and classification of wheat genotypes. The aim of the present study is characterization of widely grown bread wheat cultivars and breeding lines for drought tolerance so as to be adapted to different regions in Turkey. The genotypes were screened with molecular markers for the presence of QTLs mapped to different chromosomes. Results of the molecular studies identified and detected 15 polymorphic SSR markers which gave the clearest PCR bands among the control genotypes. At the end of the research, bread wheat genotypes which were classified for tolerance or sensitivity to drought and the genetic similarity within control varieties were determined by molecular markers. According to SSR based dendrogram, two main groups were obtained for drought tolerance. At end of the molecular screening with SSR primers, genetic similarity coefficients were obtained that ranged from 0.14 to 0.71. The ones numbered 8 and 11 were the closest genotypes to drought tolerant cultivar Gerek 79 and the furthest genotypes from this cultivar were number 16 and to drought sensitive cultivar Sultan 95. The genotypes as drought tolerance due to their SSR markers scores are expected to provide useful information for drought related molecular breeding studies.     

Evaluation of some physiological traits as indicators of drought tolerance in bread wheat genotypes

The objective of this work was to evaluate the ability of some physiological traits to identify drought-tolerant bread wheat genotypes. To this end, twenty bread wheat genotypes were assessed under post anthesis drought stress (rain-fed) and irrigated conditions. The Stress Tolerance Index (STI) was used as a measure of drought tolerance. Relationships between STI and Cell Membrane Stability (CMS), Proline Concentration (PC), Relative Water Content (RWC), Chlorophyll a/b Ratio (Chl a/b), Relative Chlorophyll Content (RCC), Excised Leaf Water Retention (ELWR), and Relative Water Loss (RWL) were determined in order to find out whether these physiological traits could be used as the indicators of drought tolerance. The results showed that ELWR, RWL, and CMS could be considered as reliable indicators in screening wheat genotypes for drought tolerance.     

Physiological traits associated with heat tolerance in bread wheat (Triticum aestivum L.)

Field experiments for evaluating heat tolerance-related physiological traits were conducted for two consecutive years using a mapping population of recombinant inbred lines (RILs) from the cross RAJ4014/WH730. Chlorophyll content (Chl) and chlorophyll fluorescence (CFL) were recorded under timely sown (TS) and late sown (LS) conditions. Late sowing exposes the terminal stage of plants to high temperature stress. Pooled analysis showed that CFL and Chl differed significantly under TS and LS conditions. The mean value of CFL (Fv/Fm) and Chl under both timely and late sown conditions were used as physiological traits for association with markers. Regression analysis revealed significant association of microsatellite markers viz., Xpsp3094 and Xgwm131 with coefficients of determination (R²) values for CFL (Fv/Fm) and Chl as 12 and 8 %, respectively. The correlation between thousand grain weight (TGW) with Chl and CFL were 14 and 7 % and correlation between grain wt./spike with Chl and CFL were 15 and 8 %, respectively. The genotypes showing tolerance to terminal heat stress as manifested by low heat susceptibility index (HSI = 0.43) for thousand grain weight, were also found having very low Chl, HSI (−0.52). These results suggest that these physiological traits may be used as a secondary character for screening heat-tolerant genotypes.     

Expansin-regulated cell elongation is involved in the drought tolerance in wheat

Water stress restrains plant growth. Expansin is a cell wall protein that is generally accepted to be the key regulator of cell wall extension during plant growth. In this study, we used two different wheat cultivars to study the involvement of expansin in drought tolerance. Wheat coleoptile was used as the material in experiment. Our results indicated that water stress induced an increase in acidic pH-dependant cell wall extension, which is related to expansin activity; however, water stress inhibited coleoptile elongation growth. The increased expansin activity was mainly due to increased expression of expansin protein that was upregulated by water stress, but water stress also resulted in a decrease in cell wall acidity, a negative factor for cell wall extension. Decreased plasma membrane H⁺-ATPase activity was involved in the alkalinization of the cell wall under water stress. The activity of expansin in HF9703 (a drought-tolerant wheat cultivar) was always higher than that in 921842 (a drought-sensitive wheat cultivar) under both normal and water stress conditions, which may be correlated with the higher expansin protein expression and plasma membrane H⁺-ATPase activity observed in HF9703 versus 921842. However, water stress did not change the susceptibility of the wheat cell wall to expansin, and no difference in this susceptibility was observed between the drought-tolerant and drought-sensitive wheat cultivars. These results suggest the involvement of expansin in cell elongation and the drought resistance of wheat.     

普通小麦中双脱氢抗坏血酸还原酶(TaDHAR)基因的克隆与生化特性分析

利用同源克隆技术从六倍体普通小麦中获得了两个不同的双脱氢抗坏血酸还原酶(TaDHAR)基因的cDNA克隆。器官表达模式分析表明,这两个TaDHAR基因(暂时命名为TaDHAR1和TaDHAR2)在小麦根、茎、叶、幼穗以及开花后10d、20d和30d的种子中均有表达,为组成型表达基因。原生质体表达实验表明,两个基因的产物均可能定位在细胞质中。在细菌中表达并提纯了两个基因的重组蛋白。体外生化测定表明两个重组蛋白均具有将双脱氢抗坏血酸还原成抗坏血酸的能力,其最适pH为7.5,在37oC时的活性比25oC高,但25oC条件下pH6.0和7.0时,两个DHAR蛋白的活性显著不同。本研究的结果为进一步揭示TaDHAR基因在小麦抗坏血酸代谢中的生理作用奠定了基础。     

Terminal drought and seed priming improves drought tolerance in wheat

Plants retain the preceding abiotic stress memory that may aid in attainment of tolerance to subsequent stresses. This study was conducted to evaluate the influence of terminal drought memory (drought priming) and seed priming in improving drought tolerance in wheat (Triticum aestivum L.). During first growing season, wheat was planted in field under optimal (well-watered) and drought stress imposed at reproductive stage (BBCH growth stage 49) until maturity (BBCH growth stage 83). Seeds collected from both sources were subjected to hydropriming or osmopriming (with 1.5% CaCl₂ solution); while, dry seed was taken as control. Treated and control seeds, from both sources, were sown in soil filled pots. After the completion of seedling emergence, pots were maintained at 50% water holding capacity (drought) or 100% water holding capacity (well-watered). Drought stress suppressed the plant growth (2–44%), perturbed water relations (1–18%) and reduced yield (192%); however, osmolytes accumulation (3–14%) and malondialdehyde contents (26–29%) were increased under drought. The crop raised from the seeds collected from terminal drought stressed plants had better growth (5–63%), improved osmolyte accumulation (13–45%), and lower lipid peroxidation (3%) than the progeny of well-watered crop. Seed priming significantly improved the crop performance under drought stress as compared to control. However, osmopriming was more effective than hydropriming in this regard as it improved leaf area (9–43%), tissue water status (2–47%), osmolytes accumulation (6–48%) and grain yield (14–79%). In conclusion, terminal drought induced modifications in seed composition and seed priming improved transgenerational drought tolerance through improvement in tissue water status and osmolytes accumulation, and decrease in lipid peroxidation.     

Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance

Maize (Zea mays) production, which is of global agro‐economic importance, is largely limited by herbivore pests, pathogens and environmental conditions, such as drought. Zealexins and kauralexins belong to two recently identified families of acidic terpenoid phytoalexins in maize that mediate defence against both pathogen and insect attacks in aboveground tissues. However, little is known about their function in belowground organs and their potential to counter abiotic stress. In this study, we show that zealexins and kauralexins accumulate in roots in response to both biotic and abiotic stress including, Diabrotica balteata herbivory, Fusarium verticillioides infection, drought and high salinity. We find that the quantity of drought‐induced phytoalexins is positively correlated with the root‐to‐shoot ratio of different maize varieties, and further demonstrate that mutant an2 plants deficient in kauralexin production are more sensitive to drought. The induction of phytoalexins in response to drought is root specific and does not influence phytoalexin levels aboveground; however, the accumulation of phytoalexins in one tissue may influence the induction capacity of other tissues.     

Improvement in drought tolerance in bread wheat is related to an improvement in osmolyte production,antioxidant enzyme activities,and gaseous exchange

Water deficit stress negatively affects wheat growth, physiology, and yield. In lab and hydroponic experiments, osmotic stress levels (control, −2, −4, −6 and −8 Bars) created by PEG-6000, caused a significant decline in germination, mean germination time, root, shoot, and coleoptile length in both wheat genotypes examined. Germination was inhibited more in Wafaq-2001 than in Chakwal-50. Wafaq-2001 showed a higher susceptibility index based on root and shoot dry weight than did Chakwal-50. Wheat plants exhibited osmotic adjustment through the accumulation of proline, soluble sugars, soluble proteins, and free amino acids, and increased antioxidation activities of superoxide dismutase, peroxidase, catalase, and malondialdehyde. Increasing water deficit stress caused a linear decline in chlorophyll contents, leaf membrane stability, and relative water content in all wheat plants, with Wafaq-2001 showing a more severe negative impact on these parameters with increasing stress levels. The results suggest the possibility of utilizing some of these parameters as quantitative indicators of water stress tolerance in plants. Gas exchange measurements (photosynthesis, transpiration, stomatal conductance), leaf osmotic potential, water potential, and yield attributes decreased more abruptly with increasing water deficit, whereas leaf cuticular wax content increased in both genotypes, with more severe impacts on Wagaq-2001. More reduction in biochemical, physiological, and yield attributes was observed in Wafaq-2001 than was observed in Chakwal-50. Based on these results, we can conclude that Chakwal-50 is a more drought-tolerant genotype, and has excellent potential for future use in breeding programs to improve wheat drought tolerance.     

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.     

Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.     

Chlorophyll fluorescence parameters and drought tolerance in a mapping population of winter bread wheat in the highlands of Iran

The usefulness of fluorescence parameters as drought tolerance selection criteria for winter bread wheat in the highlands of Iran was studied. A population of 142 recombinant inbred lines, derived from a cross between two common wheat varieties, Azar2 (winter type) and 87Zhong291 (facultative type), was used to analyze the correlation between grain yield and chlorophyll fluorescence parameters at the grain-filling stage under drought stress and supplementary irrigation conditions during 2006–2007 and 2007–2008 seasons at Maragheh experiment station of the Dryland Agricultural Research Institute (DARI) using a RCBD with three replications. The results showed significant differences among the lines in the grain yield and all fluorescence parameters under rainfed and irrigation conditions. The values of chlorophyll content, F ₀, F _m, F _v, F _v/F _m, LWP, YPEC, NPQ, and PI in the drought-tolerant genotypes were significantly higher than those in drought-sensitive genotypes under drought stress. Significant differences were observed between slope coefficients under drought, but not under supplementary irrigation conditions except NPQ (P = 5%). It was concluded that chlorophyll content, F ₀, F _m, F _v, F _v/F _m, LWP, YPEC, NPQ, and PI could be used as additional indicators in screening wheat germplasm for drought tolerance.     

Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester to aldehyde conversion in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis and its biological function, a cDNA encoding CCR was identified from wheat (Triticum aestivum L.), and designated as Ta-CCR1. Phylogenetic analysis indicated that Ta-CCR1 grouped together with other monocot CCR sequences while it diverged from Ta-CCR2. DNA gel-blot and mapping analyses demonstrated that Ta-CCR1 is present as a single copy gene in the wheat genome. Recombinant Ta-CCR1 protein converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA, and caffeoyl CoA, but feruloyl-CoA was the best substrate, suggesting the preferential biosynthesis of G-type lignin. RNA gel-blot analysis indicated that Ta-CCR1 was highly expressed in stem, with lower expression in leaves, and undetectable expression in roots. CCR enzyme activity was increased progressively along with the lignin biosynthesis and stem maturity. During stem development, Ta-CCR1 mRNA levels remained high at elongation, heading, and milky stages in the wheat H4564 cultivar, while they declined dramatically at the heading and milky stages in stems of the C6001 cultivar. Ta-CCR1 mRNA expression paralleled extractable CCR enzyme activity in these two cultivars. Furthermore, high Ta-CCR1 mRNA levels and high CCR enzyme activity in wheat stem were correlated with a higher Klason lignin content and greater stem mechanical strength in the H4564 cultivar. This suggests that Ta-CCR1 and its related CCR enzyme may be involved in the regulation of lignin biosynthesis during stem maturity and then contributes to stem strength support in wheat.     

NADH-ferric reductase activity associated with dihydropteridine reductase

In mammals dietary ferric iron is reduced to ferrous iron for more efficient absorption by the intestine. Analysis of a pig duodenal membrane fraction revealed two NADH-dependent ferric reductase activities, one associated with a b-type cytochrome and the other not. Purification and characterization of the non-cytochrome ferric reductase identified a 31 kDa protein. MALDI-MS analysis and amino acid sequencing identified the ferric reductase as being related to the 26 kDa liver NADH-dependent quinoid dihydropteridine reductase (DHPR). The NADH-dependent DHPR ferric reductase activity was found to be pteridine-independent since exhaustive dialysis did not reduce activity and heat-inactivation destroyed activity. In intestinal Caco-2 cells, DHPR mRNA levels were found to be regulated by iron. Thus, DHPR appears to be a dual function enzyme, a NADH-dependent dihydopteridine reductase and an iron-regulated, NADH-dependent, pteridine-independent ferric reductase.     

Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco

Aluminum (Al) inhibits plant growth partly by causing oxidative damage that is promoted by reactive oxygen species and can be prevented by improving antioxidant capacity. Ascorbic acid (AsA), the most abundant antioxidant in plants, is regenerated by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We investigated the role of MDAR and DHAR in AsA regeneration during Al stress using transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis cytosolic MDAR (MDAR-OX) or DHAR (DHAR-OX). DHAR-OX plants showed better root growth than wild-type (SR-1) plants after exposure to Al for 2 weeks, but MDAR-OX plants did not. There was no difference in Al distribution and accumulation in the root tips among SR-1, DHAR-OX, and MDAR-OX plants after Al treatment for 24 h. However, DHAR-OX plants showed lower hydrogen peroxide content, less lipid peroxidation and lower level of oxidative DNA damage than SR-1 plants, whereas MDAR-OX plants showed the same extent of damage as SR-1 plants. Compared with SR-1 plants, DHAR-OX plants consistently maintained a higher AsA level both with and without Al exposure, while MDAR-OX plants maintained a higher AsA level only without Al exposure. Also, DHAR-OX plants maintained higher APX activity under Al stress. The higher AsA level and APX activity in DHAR-OX plants contributed to their higher antioxidant capacity and higher tolerance to Al stress. These findings show that the overexpression of DHAR, but not of MDAR, confers Al tolerance, and that maintenance of a high AsA level is important to Al tolerance.