Gene expression and activity of antioxidant enzymes in barley (Hordeum vulgare L.) under controlled severe drought

The objective of the present study was to determine the activity of antioxidant enzymes: superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) and the expression of their genes in two barley genotypes under controlled severe drought. To fulfill this objective, 21-day-old barley plants of two genotypes: Rum and Yarmouk were exposed to controlled severe drought (25% field capacity) for 2, 9, and 16 days. The activity of SOD was significantly high in Rum genotype after 2 days of drought treatment. In Yarmouk genotype, the activity of APX was significantly high after 2 and 9 days of drought treatment. In Rum genotype, CAT2 was upregulated after 9 days of drought treatment and SOD and APX were upregulated after 16 days of drought treatment, whereas CAT2, SOD, and APX were upregulated in Yarmouk genotype after 2 days of drought treatment. The results indicate a unique pattern of activity and gene expression of the antioxidant enzymes in the two barley genotypes under controlled severe drought. Moreover, the data suggest that each genotype utilizes different molecular and biochemical responses under the same drought conditions.     

Studies of soil moisture stress in barley (Hordeum vulgare L.)

Summary The objective was to find the optimum range of water contents for inducing better growth, physiological efficiency and yield potential of barley plants (Hordeum vulgare L. var. K18). A pot culture experiment was conducted in the Division of Crop Physiology and Biochemistry Kanpur-2. The plants were subjected to various soil moisture stresses,i.e., 0.15, 0.30, 0.45, 0.60 and 0.75 atm tension throughout the crop growth period measured by irrometers.Plants maintained at 0.45 soil moisture tension required 19.07 litre of water and had the best water use efficiency (1765 mg dm/litre of water) which favourably influenced the leaf water balance (85.9%), plant growth as measured by plant height (85.4 cm) and tiller production (35.6) per hill, photosynthetic efficiency (2.185 mg CO₂/g dm/h), grain number (722) and grain yield (33.7 g) per hill while plants irrigated at a tension greater than 0.45 SMT did not develop as well. However, protein and gluten percentage increased gradually with the subsequent increase in soil moisture tension. On the other hand respiration rate (2.090 mg CO₂/g dm/hr) and leaf area (4375 cm²) were recorded to be the highest at 0.60 and 0.30 atm SMT respectively.Thus it is suggested that for reaping high harvest of barley crop, the physiological need of water (19.07 litre) in total of plant life should be made available through scheduled irrigation based on maintenance of plant at 0.45 SMT from seeding to maturity.     

Indole acetic acid (IAA) induced changes in growth, relative water contents and gas exchange attributes of barley (Hordeum vulgare L.) grown under water stress conditions

Effect of different concentrations of indole acetic acid (IAA) under varying soil water deficit conditions on two barley cultivars viz. B-99094 and Jau-87 was investigated in soil filled earthen pots. There were six treatments including control each with four replicates. Three concentrations of IAA (0, 15 and 30 mg l⁻¹) were applied as foliar spray 30 days after germination. After hormone application, half of the pots were subjected to one cycle of water stress (withholding of water till incipient wilting), followed by regular watering. Plant height, photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency relative water content, dry biomass, and grain yield/plant were significantly reduced by water stress. However, IAA treatments alleviated the adverse effect of water stress and successful in enhancing the plant growth and yield of barley cultivars. Barley cultivar Jau-87 performed better than B-99094. IAA application␣was effective in enhancing growth and photosynthetic efficiency of barley both under normal and water stress conditions.     

Relationship between gibberellins, height, and stress tolerance in barley (Hordeum vulgare L.) seedlings

The relationship between gibberellin (GA) levels, height, and stress tolerance was investigated using barley (Hordeum vulgare L.) seedlings, which were exposed to heat stress (50 °C for 3 h) and a free radical generator (Paraquat). Barley cv. Perth seedlings were treated with GA-inhibitors, either a triazole or one of two acylcyclohexanediones. They were tested along with four mutants that were either responsive (MC96 and dwf1) or non-responsive (MC90 and Dwf2) to applied growth-active GAs. Analyses of seedlings, whether mutants, or treated with GA-inhibitors, gave a negative correlation between height and tolerance. We also observed that the shorter seedlings with the least amount of growth active GAs were the most tolerant to stress. The overall results of this study suggest that reduced GA levels or sensitivity to GA, with a concomitant reduction in height are important for induction of stress tolerance.     

Salt tolerance of barley (Hordeum vulgare L.) cultivars at the germination stage: Analysis of the response functions

Two models, initially proposed by Van Genuchten (1983) for evaluating salinity-yield response curves at the adult stage, were applied to study the salinity response of 24 barley cultivars at the germination stage. According to the calculated salinity threshold, EC_t (the solution electrical conductivity, EC, at which germination starts to decrease), and EC₅₀ (the solution EC at which germination is reduced by 50%) parameters, both models give similar results, although model 2, a sigmoid-form curve, fits the observed data slightly better than model 1, a piecewise response function. Also, the results suggest that, for model 1, EC_t seems to be the most reliable parameter for screening barley germplasm because it clearly discriminates the relative salt-tolerance of the studied cultivars and, furthermore, it basically determines their salinity response for the 100 to 50% germination interval. On the other hand, the model 1 s parameter — percent germination decrease per unit salinity increase bove EC_t—is less relevant because of its smaller variation interval and lack of correlation with EC₅₀, indicating that the salinity response of the studied cultivars for the 50% germination value is independent of this parameter.     

Expression of XET-related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.)

Five cDNA clones were isolated from barley (Hordeum vulgare L.) that encoded mRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated that the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA₃), or homozygosity at the ‘slender’ (sln1) locus, resulted in a 2.5-fold (approximately) stimulation of blade elongation rate. Three of the five clones detected mRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GA₃-treated or slender seedlings. The remaining two clones detected mRNAs that were maximally expressed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA₃ treatment revealed similar maximal REGR values despite a 2.5-fold difference in leaf elongation rate. Segments of GA₃-treated leaves attained their maximal REGR values more rapidly, this being associated with enhanced expression of the three ‘basal’ XET-related mRNAs. Highest XET activities were detected in the base of the elongation zone, and in GA₃-treated seedlings a second activity peak was observed near the distal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes with different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.     

Functional association between malting quality trait components and cDNA array based expression patterns in barley (Hordeum vulgare L.)

We developed an approach for relating differences in gene expression to the phenotypic variation of a trait of interest. This allows the identification of candidate genes for traits that display quantitative variation. To validate the principle, gene expression was monitored on a cDNA array with 1400 ESTs to identify genes involved in the variation of the complex trait malting quality in barley. RNA profiles were monitored during grain germination in a set of 10 barley genotypes that had been characterized for 6 quality-associated trait components. The selection of the candidate genes was achieved via a correlation of dissimilarity matrices that were based on (i) trait variation and (ii) gene expression data. As expected, a comparison based on the complete set of differentially-expressed genes did not reveal any correlation between the matrices, because not all genes that show differential expression between the 10 cultivars are responsible for the observed differences in malting quality. However, by iteratively taking out one gene (with replacement) and re-computing the correlation, those genes that are positively contributing to the correlation could be identified. Using this procedure between 17 and 30 candidate genes were identified for each of the six malting parameters analysed. In addition to genes of unknown function, the list of candidates contains well-known malting-related genes. Five out of eight mapped candidate genes display linkage to known QTLs for malting quality traits. The described functional association strategy may provide an efficient link between functional genomics and plant breeding.     

Photoinhibition of Photosystem I in field-grown barley (Hordeum vulgare L.): Induction, recovery and acclimation

The effects of exposure of a field-grown winter cultivar of barley (Hordeum vulgare L.) to Photosystem I (PS I) photoinhibitory conditions in the form of bright day-light combined with chilling conditions were investigated. PS I photoinhibition was manifested by damage to the Fe-S centers of PS I and to the PS I-A/B polypeptides. Up to 20% of the PS I complexes were photoinactivated. Upon transfer to room temperature, the plants partially recovered from PS I photoinhibition, although damage was still detectable after one week. These results demonstrate that PS I photoinhibition is a physiologically relevant phenomenon in chilling-tolerant plants grown under field conditions. In order to study the induction of cyclic electron transport around PS I by PS I photoinhibitory conditions, antibodies raised against the NDH-I subunit of the NDH complex (a component of cyclic electron transport) were used to measure NDH levels in the exposed plants. A marked increase in the amount of NDH complex and a corresponding increase in NADPH dehydrogenase activity in the thylakoids were observed. The data indicate that the response to PS I-photoinhibitory conditions may involve regulated changes in cyclic electron transport around PS I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Formation of early-light-inducible-protein complexes and status of xanthophyll levels under high light and cold stress in barley (Hordeum vulgare L.)

In our previous work we found considerable accumulation of early light-inducible proteins (ELIPs) in barley during adaptation to combined high light and cold stress, an accumulation which occurred preferentially in the apical part of the leaves (M.-H. Montané et al., 1997, Planta 202: 293–302). Here we studied, under the same conditions, the effect of adaptation on the composition of thylakoid membrane proteins and pigments, particularly xanthophylls and chlorophyll, and their distribution within the barley leaf. It was observed that high light fluxes appeared to favour the trimerization of the light-harvesting complex of photosystem II (LHC II) whereas cold appeared to favour the monomers of LHC II. High light, cold or the combination of both factors had only a small effect on the protein composition of the thylakoid membranes except for the proteins of LHC II which were found to decrease under high light to a greater extent at 25 °C than at 5 °C. The total xanthophyll-cycle carotenoid content increased linearly with cellular development, the highest amount being observed in the apical part of the leaf. Cold and high light acted synergistically to induce less than a doubling in the amount of total xanthophylls, while chlorophylls a and b remained nearly constant. The fraction consisting of antheraxanthin plus zeaxanthin was up to 4- to 5-fold higher at 5 °C than at 25 °C. As determined previously (Montané et al. 1997), the same conditions caused a 15-fold increase in the accumulation of ELIPs. Consequently, neither the distribution of total xanthophylls nor that of antheraxanthin plus zeaxanthin along the leaf followed the same pattern as ELIP. Thus, the accumulation of xanthophylls cannot be stoichiometrically correlated with that of ELIPs. Using electrophoresis in the presence of decylmaltoside, we could demonstrate for the first time that ELIPs of 13.5 kDa are contained in high-molecular-mass complexes of >100 kDa, which are located in the unstacked stroma lamellar region of the thylakoid membranes. Received: 6 April 1998 / Accepted: 26 January 1999     

Use of plant growth-promoting bacteria to enhance salinity stress in soybean (Glycine max L.) plants

The effects of three rhizobacterial isolates namely Pseudomonas fluorescens (M1), Pseudomonas putida (M2) and Bacillus subtilis (M3) were examined to enhance growth and chemical components such as chlorophyll and proline of three cultivars of soybean (Glycine max L.) under two levels of salinity stress (S1 = 200 mM and S2 = 400 mM of NaCl salt). Several morphological and physiological parameters were investigated. The highest mean values of final germination percent (FGP) were registered in cultivar Crawford (95%) followed by Giza111 cultivar (93%) in the presence of P. fluorescens, while, FGP of Clark was 85%. Mean germination time was decreased by the application of P. fluorescens or P. putida in both salt stressed and unstressed traits. All growth parameters were significantly decreased by salinity treatments, particularly at S2. A significant increase in stem length and shoot fresh weight was recorded in plants treated with P. fluorescens. This enhancing trend was followed by the application of P. putida then B. subtilis. Chlorophyll contents and plant soluble proteins were decreased, while proline content was increased as compared with control treatment. Results showed that the salt tolerant cultivar, Crawford, may have a better tolerance strategy against oxidative damages by increasing antioxidant enzymes activities under high salinity stress. These results suggest that salt induced oxidative stress in soybean is generally counteracted by enzymatic defense systems stimulated under harsh conditions. Our results showed that inoculation with plant growth-promoting rhizobacterial (PGPR) alleviated the harmful effects of salinity stress on soybean cultivars. The diversity in the phylogenetic relationship and in the level of genetic among cultivars was assessed by SDS-PAGE and RAPD markers. Among the polymorphism bands, only few were found to be useful as positive or negative markers associated with salt stress. The maximum number of bands (17) was recorded in Crawford, while the minimum number of bands (11) was recorded in Clark. Therefore, the ISSR can be used to identify alleles associated with the salt stress in soybean germplasm.     

Exogenous application of gibberellic acid and silicon to promote salinity tolerance in pea (Pisum sativum L.) through Na+ exclusion

Salinity is a worldwide problem limiting the plant growth and risking food security. This study was conducted to examine exogenous application of silicon (Si), gibberellic acid (GA₃) upon the ion transport, growth, yield, and antioxidant enzymes activities of pea plant in saline conditions. Two pea varieties Meteor-FSD and Samrina Zard were pre-treated with GA₃ (10^-4 M) for 12 h. Plants were allowed to grow with or without silicon in washed silica sand. Ten days old seedlings were shifted in pots with 10 kg soil. Twenty-five days old plants were exposed to 0 and 5 dS m^?1 sodium stress. Results showed that exogenous application of GA₃ + Si was the best treatment for increasing plant biomass and yield in the presence and absence of NaCl. Furthermore, application of Si or GA₃ enhanced chlorophyll content in the leaves, thereby increasing the net assimilation rate of pea varieties under NaCl stress by increasing the antioxidant enzyme activity. Treatment of Si alone or in combination with GA₃ significantly reduced Na⁺ movement in both pea varieties. Results showed that Si has more prominent role than GA₃ alone to build-up high plant biomass, yield, soluble protein content and reduction of Na⁺ transport. Samrina Zard variety showed higher yield, shoot and root dry weight as compared to Meteor-FSD variety in presence and absence of salt. It was concluded that Si can be used as a nutrient for pea under saline or non-saline conditions. Moreover, application of GA₃ has a potential role for increasing salinity tolerance, mostly in sensitive pea varieties.     

The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli -glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.NRCC No. 36482     

Foliar application of zinc improves morpho-physiological and antioxidant defense mechanisms,and agronomic grain biofortification of wheat (Triticum aestivum L.) under water stress

Agronomic biofortification with zinc (Zn) may be engaged to improve the nutritious value of food crops along-with tolerance to water deficit conditions. The Zn may increase plant resistance to water stress by boosting physiological and enzymatic antioxidants defense mechanisms. Major objective of this study was to investigate the effect of foliar applied Zn on grain zin biofortification and drought tolerance in wheat. Treatments include application of Zinc at terminal growth phases (BBCH growth stage 49 and BBCH growth stage 65) with five levels: 0 (control-ck), water spray, 5, 10 and 15 mM under two levels of water regimes; well-watered (where 80% water holding capacity (WHC) was maintained in the soil) and water stress, (where 40% WHC was maintained in the soil). Results revealed that water stress significantly reduced relative water contents, gas exchange attributes, plant height, yield and yield related attributes of wheat. In contrast, hydrogen peroxide, free proline levels, activities of malondialdehyde, and concentration of soluble protein were markedly increased under water stress condition. Application of various levels of Zn significantly improved the CAT, SOD, POD and ASP activities at 40% WHC compared with control treatment. Foliarly applied 10 and 15 mM Zn predominantly reduced the damaging impact of water stress by improving the plant status in the form of plant height, RWC and gas exchange attributes. Likewise, wheat plant treated with 10 mM Zn under water stress condition increased the grain yield by improving number of grains per spike, 100 grain weight and biological yield compared with control. Moreover, increasing Zn levels also increased Zn concentration in grains and leaves. Overall, this study suggests that optimum level of Zn (10 mM) might be promising for alleviating the adverse impacts of water stress and enhance the grain biofortification in wheat.     

Expression of cold-inducible genes and frost hardiness in the crown meristem of young barley (Hordeum vulgare L. cv. Igri) plants grown in different environments

The purpose of this work was to examine environmental control of expression, at the mRNA level, of cold-inducible genes and to test the relationship of the expression of the genes to cold acclimation. Barley plants (Hordeum vulgare L. cv. Igri) at the three- to four-leaf stage were (a) grown in different temperature environments between 20/15°C and +4/-4°C or (b) transferred between 20/15°C and 6/2°C or (c) grown under drought or nutrient stress conditions. Frost hardiness (using a regrowth method) and mRNA levels for three cold-induced genes, blt4-9, blt14 and blt101, from meristematic crown tissue (vegetative shoot meristem plus subtending stem and associated root initials) were measured. Hardiness and levels of blt4-9, blt14 and blt101 mRNAs increased with lower growth temperatures, below a maximum inductive temperature. Prior temperature environment and plant age affected the rate of change in mRNA levels of these genes in response to a change of temperature environment. Hardiness was strongly correlated with mRNA levels of these genes in plants grown in different temperature environments. This correlation did not extend to plants exposed to drought or nutrient stresses. Implications are drawn for plant responses to a warmer climate.     

Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting     

Growth, yield and grain quality of barley (Hordeum vulgare L.) in response to nitrogen uptake. II. Plant development and rate of germination

A nutrient culture system supporting stands of barley was used to examine the effect of nitrogen supply on plant development and germination rate. Two contrasting cultivars of barley were grown for two seasons with nitrogen supplied at an optimal rate and at one-third that rate. Mature grain was dissected from ears by grain position; stored until out of dormancy, weighed, and the time from inhibition to germination estimated for different ear positions. A parallel set of samples was analysed for grain nitrogen concentrations. A systematic increase in germination time was observed from the bottom to the top of the ear. This effect was in addition to and of the same order as the effect of grain size on germination time. Although there was significant variation in nitrogen concentration by grain position, these effects were smaller than the variations in grain weight within the ear and were not related to germination rate. These results are discussed in relation to plant development and events at the onset of germination.Keywords: Barley, germination, grain size, ear position, nitrogen.