Improvements in Abiotic Stress Tolerance in Crop Plants: Physiological Approaches

5.3.2.1 Identification of morpho-physiological traits associated with stress tolerance

Identification of wheat genotypes for high temperature. An experiment was conducted in field condition with thirty-six genotypes, selected from all regions of the country, under normal and late sown field conditions. The genotypes, which took longer duration for anthesis, were found to be relatively susceptible while early duration types were found to be more tolerant to high temperature. It is felt that the tolerant genotypes completed their reproductive cycle relatively faster than susceptible types (duration between anthesis and maturity). In tolerant genotypes, heat shock proteins (HSPs) play an important role in acclimation of a genotype. It is suggested that selection on the basis of both yield stability index (YSI) and heat susceptibility index (HSI) could be possible, and could lead to selection of suitable parents for enhancing productivity of wheat under late planting and resource limited conditions.

5.3.2.2 Understanding the mechanism of stress tolerance at cellular and biochemical levels

Differential response of green gram (Vigna radiata (L.) Wilczek) genotypes and wild species to waterlogging and adaptive mechanisms. Waterlogging caused a gradual decrease in relative water content (RWC), chlorophyll content (Chl) in leaves, and membrane stability index (MSI) in leaves and root tissues in all the genotypes. However, wild genotypes Vigna luteola and Vigna sublobata, tolerant genotypes T 44 and MH 96-1 maintained greater levels of RWC, Chl and MSI than those of susceptible genotypes (Pusa Baisakhi and MH 1K- 24). Wild genotype, Vigna luteola performed best with

Heat susceptibility index (S) for dry matter production, grain growth and yield in wheat cultivars grown in control (C) and heated (E) OTCs for the entire period of growth and development, and temperature treatment given only after anthesis

Cultivar S for total S for grain S for 1000

dry matter yield grain

plant^-1 plant^-1 weight High temperature

throughout growing season

HD 2285 1.222*** 1.292*** 0.888**

HD 2329 0.720** 0.648** 2.944***

High temperature after anthesis

HD 2285 0.115* 0.117* 0.326*

HD 2329 1.962*** 1.798*** 1.717***

given after anthesis, the heat susceptibility index showed HD 2285 as highly stress tolerant and HD 2329 as a susceptible type. The greater susceptibility of HD 2285 to moderately high temperature exposure during the entire period of growth and development compared to that of HD 2329 was because of a greater reduction in tillers and consequent reduction in ear number per plant. The present study, therefore, revealed that cultivar HD 2285 which is tolerant to terminal high temperature would become susceptible when high temperature spells come during pre-anthesis period. There is a need, therefore, to develop wheat varieties which are not only tolerant to terminal high temperature stress but also to a warmer climatic condition.

Kinetics of nitrate uptake and expression of transporters. The wheat seedlings were incubated in a range of nitrate solutions (0.01mM-10mM) for various time intervals and time course of nitrate uptake rate was estimated.

Pattern of uptake was similar in seedlings grown under both

reference to physiological, biochemical and molecular characteristics, followed by Vigna sublobata (wild genotype).

However, these were very poor yielder. Tolerant genotypes have higher root carbohydrate levels, while susceptible genotypes have lower root carbohydrate levels. Waterlogging resulted in decline in, non-reducing and reducing sugars in susceptible genotypes, while tolerant genotypes showed increase in reducing sugars, which was parallel to the increase in sucrose synthase (SS) activity. Alcohol dehydrogenase (ADH) gene expression and activity, responsible for fermentation pathway, which allows continuation of glycolytic pathway was greater in tolerant genotypes. Greater SS activity, non-reducing sugars and ADH activity was responsible for better survival of Vigna luteola, T 44, MH 96-1 and Vigna sublobata (tolerant genotypes and wild species).

Deduced protein sequences for sucrose synthase showed 95%, 93% and 98% similarity between Vigna luteola and T 44; T 44 and Pusa Baisakhi; Vigna luteola and Pusa Baisakhi, respectively. Vigna radiata complete DNA sequence (CDS) (Acc. No. D 10266) showed 98%, 94%, and 100% similarity with Vigna luteola, T 44 and Pusa Baisakhi, respectively.

Sucrose synthase (pfam00862) catalyze the synthesis of sucrose from UDP-glucose and fructose. This family includes the bulk of the sucrose synthase protein. However, the carboxyl terminal region of the sucrose synthases belongs to the glycosyl transferase family pfam00534.

RT-PCR amplified alcohol dehydrogenase cDNAs of Vigna luteola, T 44 and Pusa Baisakhi were sequenced and about 650 bp of partial coding sequence were compared with

Phaseolus acutifolius ADH gene (Acc. No. Z23170,complete gene 1143 bp). All the three genotypes showed more than 95% similarity with Phaseolus acutifolius full coding sequence of ADH gene. Conserved domains were identified in all the 3 genotypes using ‘PROSITE’. Partial amino acid sequence of ADH showed two conserved domains: (1) ADH_Zn, Zinc containing alcohol dehydrogenase signature (PS 00059) (amino acid residues 26-40 in Vigna luteola;

24-38 in T 44 and Pusa Baisakhi), where H(Histidine) is the Zinc ligand, which is the 2^nd ligand of the catalytic Zinc atom, and (2) ADH_N, Alcohol dehydrogenase GroES-like domain (amino acid residues 83-96 in Vigna luteola; 80-93 in T 44 and Pusa Baisakhi). Green gram genotype Pusa Baisakhi showed an amino acid replacement at 86^th position by Q(Glutamine) whereas other genotypes had K(Lysine) in that position. This may be one of the reasons for the less expression of ADH in Pusa Baisakhi under waterlogged condition compared to that in other genotypes.

Effect of photosynthetic pigments manipulation effect on photosynthetic activity and yield of green gram under waterlogging. In order to analyze and understand the photosynthetic pigment basis of waterlogging tolerance in green gram, a pot culture experiment using two diverse genotypes, i.e., MH - 96-1(tolerant to waterlogging)

RT-PCR expression analysis of sucrose synthase (A), alcohol dehydrogenase (B), and tubulin (C) genes under waterlogging stress and control conditions (M : ladder; 1 : control - V. luteola; 2 : treated V. luteola; 3 : control T 44; 4 : treated T 44; 5 : control Pusa Baisakhi;

and 6: treated Pusa Baisakhi)

Photosynthetic pigments in two diverse waterlogged genotypes under different treatments. (↑= initiation, ↓ = termination of waterlogging)

MH - 1K - 24 (susceptible to waterlogging) was conducted.

Exposure of blue light and foliar application of urea enhanced the level of photosynthetic pigments and photosynthesis rate under both normal (non-waterlogged) and waterlogged conditions. Foliar spray of urea effectively enhanced the plant height, leaf area and total dry matter accumulation and accelerated the recovery rate. However, dithiothreitol (DTT), SS

Tubulin ADH

an inhibitor of de-epoxidation of violoaxanthin significantly reduced the level of chlorophylls and carotenoids, photosynthesis rate, membrane stability index, and growth parameters under waterlogged conditions in both genotypes.

This in turn indicated the involvement and protective role of carotenoids in waterlogging tolerance. This view is also supported by tolerant genotype, which had higher level of carotenoids compared to that of susceptible genotypes under waterlogging. Further, the level of carotenoids and carotenoids/chlorophyll ratio showed significant relationship with photosynthesis, photosynthetic pigments and waterlogging tolerance during waterlogging and after waterlogging termination.

Photosynthetic pigment profiles developed using the TLC technique revealed various bands, which were very clear particularly in non-waterlogged treatments except non- waterlogged DTT. Poor bands of major photosynthetic pigments and non-traceable minor pigments were noted in waterlogged control, particularly in susceptible genotype and waterlogged DTT treatment. Foliar application of urea and blue light exposed plants exhibited sharp bands of all thirteen pigments under both non-waterlogged and waterlogged conditions.

Cloning of abiotic stress responsive genes encoding transcription factor and signaling proteins (MAPK, PP2C) from wheat (Triticum aestivum L.). A study was undertaken to clone drought stress responsive genes that encode signaling proteins Mitogen Activated Protein Kinase (MAPK) and Protein Phosphatase 2C (PP2C) and a transcription factor (Zinc finger protein). Wheat cultivar C306 (drought and heat stress resistant) and Kharchia 65 (salinity stress resistant) plants were grown in small pots of 6 inches diameter at the National Phytotron Facility, IARI, New Delhi during the rabi season, 2006-07. One-month-old wheat plants were subjected to water-deficit stress by withholding irrigation for 7-8 days.

Another set of plants was regularly irrigated (Control).

RNA was extracted and reverse transcription polymerase chain reactions (RTPCR) were conducted.

Expression analysis showed that wheat TaMAPK, TaPP2C and TaZF genes were induced only under water deficit stress conditions, but not under irrigated conditions. Further, sequence analysis of these genes revealed that the wheat TaMAPK encoded protein differed from a previously reported elicitor induced wheat WCK-1 protein only at a single amino acid indicating that both of these mRNAs are encoded by the same gene. The PP2C gene isolated from wheat cultivars

C306 and Kharchia-65 was found to show 99% identity at the nucleotide level. At the protein level, a single amino acid change was observed (K104 in C306 to E in Kharchia-65).

The ZF gene isolated from wheat was found to encode a PHD type zinc finger protein and showed 66% sequence similarity at the protein level to a previously reported Alfin1 ZF transcription factor from alfalfa. Thus, in this study drought stress responsive genes encoding MAPK, PP2C and ZF have been cloned from abiotic stress resistant wheat cultivars.

Drought induced expression of these wheat genes suggests a potential role of these genes in drought stress signaling and gene regulation. Functional characterization of these genes through transgenic development or genetic approaches will significantly enhance our understanding of drought stress tolerance in wheat.

5.3.2.3 Oxidative stress metabolism in leaves of wheat cultivars differing in senescence under heat stress

Two wheat genotypes, Hindi 62 (heat tolerant) and PBW 343 (heat susceptible) were analyzed for antioxidant defense in the flag leaf under high temperature. The parameters studied include H₂O₂ estimation, lipid peroxidation, antioxidant metabolites and antioxidant enzymes. The first and foremost visible effect of heat induced oxidative stress was to increase the membrane damage. The extent of membrane damage in terms of lipid peroxidation was considerably higher in PBW 343 than that in Hindi 62 under heat stress. The lower level of ROS production, lower lipid peroxidation with less altered and well coordinated antioxidant system during heat stress enabled Hindi 62 to cope with heat stress more efficiently than PBW 343. The ROS scavenging system of heat susceptible genotype PBW 343 was affected more under heat stress and contributed to the faster rate of leaf senescence.

5.3.2.4 Isolation and characterization of chloroplastic Cu/Zn superoxide dismutase gene from Chenopodium murale

The Chl Cu/Zn superoxide dismutase gene (CmCSV) was isolated from Chenopodium murale using RT-PCR and RACE techniques. The 445 bp amplicon obtained by 5´

RACE PCR contained the N-terminus region of the Chl Cu/

Zn SOD. The full length cDNA sequence obtained was 672 bp and had the putative conserved domain of the Cu/Zn SOD with the chloroplast transit peptide. The N-terminal sequence

of the purified SOD protein showed 70% homology with the N-terminal region of the cloned full length cDNA. This is the first report on cloning of chloroplastic Cu/Zn SOD cDNA from C. murale. The expression pattern of the chloroplastic Cu/Zn SOD V (CmCSV) of C. murale was examined under various abiotic stresses like high temperature (45 ^oC) either with or without prior acclimation at 35 ^oC, high light intensitiy (900 ± 50 µmol m^-2 s^-1), and water stress followed by recovery.

The Northern blot analysis clearly indicated that transcript levels of CmCSV were more under high light intensity, high temperature stress after acclimation and water stress recovery in the leaves compared to those of control plants. However, no change in the protein level of chloroplastic Cu/Zn SOD was observed in the western blot using the antibody raised against the thermostable SOD.

5.3.3 Post-harvest Physiology of Fruits,