Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.     

Expression analysis of a stress-modulated transcript in drought tolerant and susceptible cultivars of sorghum (Sorghum bicolor)

The present study reports the cloning of a 581 bp sequence, designated as SbEST8, from the osmotically stressed germinated seeds of a drought tolerant cultivar of sorghum (Sorghum bicolor). The SbEST8, which shows no homology with the reported gene sequences, is present in multiple copies and lacks restriction fragment length polymorphism among different sorghum cultivars. The expression of SbEST8 in the germinating seeds of sorghum was modulated by different abiotic stresses. Kinetic studies revealed that imposition of osmotic stress after 8h resulted in maximum levels of SbEST8 mRNA in the germinating seeds of cv. ICSV-272, with further stress causing a decline to undetectable levels by 16 h. However, relieving the stress after 12h resulted in an enhancement of SbEST8 mRNA levels for at least another 4h following which it declined. The decrease in SbEST8 mRNA levels in the leaves at 30 DAS in response to drought stress was observed only in the drought susceptible cultivar (CSV-216), whereas its expression was either increased substantially or remained unaffected in the tolerant cultivars, thus suggesting its role in water stress tolerance.     

Gas exchange of root hemi-parasite Striga hermonthica and its host Sorghum bicolor under short-term soil water stress

The gas exchange of the upper fully expanded leaf of the root parasite Striga hermonthica and of its host Sorghum bicolor was measured under wet and dry conditions to identify the mechanisms of the devastating effects of the parasite on its hosts under drought. The short-term water stress severely reduced photosynthetic rate in infected sorghum, but less in S. hermonthica. Soil water stress did not affect leaf respiration rate in either S. hermonthica or infected sorghum. This suggests that under dry conditions both infected sorghum and S. hermonthica decreased autotrophic carbon gain. The transpiration rate of S. hermonthica, a major driving force for assimilate uptake from the host, was higher and less affected by water stress than that of infected sorghum. Stomatal density on the abaxial surfaces of the leaves was higher in S. hermonthica than in sorghum. Both S. hermonthica infection and water stress decreased stomatal conductance of the sorghum leaves. S. hermonthica, irrespective of soil water status, had greater stomatal aperture on the adaxial and abaxial surfaces of its leaves than infected sorghum. These results indicate that the higher transpiration rate of S. hermonthica even under water stress, achieved through higher stomatal density on the abaxial surfaces of the leaves and greater stomatal aperture on both surfaces of the leaves, may induce the maintenance of water and solute transfers from the host to the parasite leading to severe damage to the host under drought.     

Stress-Induced Changes in Peptidyl-Prolyl cis-trans Isomerase Activity of Sorghum bicolor Seedlings

Developmental changes and effects of various abiotic stresses on peptidyl prolyl cis-trans isomerase (PPIase) activity were studied in the seedlings of sorghum [Sorghum bicolor (L.) Moench cv. CSH-6]. The PPIase activity of sorghum seedlings markedly decreased after two days of germination. Up to 90 % of the PPIase activity was inhibited by cyclosporin-A. Maximal increase in specific PPIase activity in the 3-d-old seedlings was observed in response to osmotic stress and it was transient in nature. The stress-induced enhancement in PPIase activity, depending upon tissue and stress treatment, was due to induction of cyclophilins as well as other PPIases. Osmotic stress-induced enhancement in PPIase activity in the drought susceptible cv. SPRU-94008B was maximal in roots, as compared to shoots in the drought tolerant cv. ICSV-272. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water use efficiency in the drought-stressed sorghum and maize in relation to expression of aquaporin genes

Zea mays L. is less tolerant to drought than Sorghum bicolor L. In the present study, we investigated the response of both plants to drought stress applied under field conditions by withholding water for 10 d. The plant growth in terms of shoot fresh and dry masses was more severely reduced in maize than in sorghum, consistently with reduction of leaf relative water content. Gas exchange was also more inhibited by drought in maize than in sorghum. The water use efficiency (WUE) of maize fluctuated during the day and in response to the drought stress. In contrast, sorghum was able to maintain a largely constant WUE during the day in the well-watered plants as well as in the stressed ones. Studying the expression of four aquaporin genes (PIP1;5, PIP1;6, PIP2;3, and TIP1;2) revealed that PIP1;5 in leaves and PIP2;3 in roots were highly responsive to drought in sorghum but not in maize, where they might have supported a greater water transport. The expression pattern of PIP1;6 suggests its possible role in CO₂ transport in control but not droughty leaves of both the plants. TIP1;2 seemed to contribute to water transport in leaves of the control but not droughty plants. We conclude that PIP1;5 and PIP2;3 may have a prominent role in drought tolerance and maintenance of WUE in sorghum plants.     

Proline accumulation and nitrate reductase activity in contrasting sorghum lines during mid-season drought stress

Six lines of sorghum ( Sorghum bicolor L. Moench) with differing drought resistance (IS 22380, ICSV 213, IS 13441 and SPH 263, resistant and IS 12739 and IS 12744, susceptible) were grown under field conditions in the semi-arid tropics and analysed for proline and nitrate reductase activity (NRA; EC 1.6.6.1) during a mid-season drought. The resistant lines accumulated high levels of proline, while the susceptible lines showed no significant proline accumulation. Most of the proline was accumulated after growth of the plants had ceased. In a separate greenhouse experiment, most of the proline was found in the green rather than the fired portions of leaves. The levels returned to that of irrigated controls within 5 days of rewatering. Proline levels increased as leaf water potential and relative water content fell, and there was no apparent difference among the different sorghum lines with change in plant water status. Susceptible lines accumulated less proline than resistant lines as leaf death occurred at higher water potentials. Proline accumulation may, however, contribute to the immediate recovery of plants from drought. Leaf NRA reached high levels at about 35 days after sowing in both the stressed and irrigated plants, after which it declined. The decline in NRA was more pronounced in the stressed than in the irrigated plants and closely followed changes in the growth rate. Upon rewatering, NRA increased several-fold in all the lines and, in contrast to proline accumulation, genotypic differences in NRA were small, both during stress and upon rewatering. The high sensitivity of NRA to mild drought stress was reflected in the rapid decline of activity with small changes in leaf water potential and relative water content. The results are discussed in the light of a possible role for proline during recovery from drought, and the maintenance of NRA during stress and its recovery upon rewatering.     

Colonization by Arbuscular Mycorrhizal Fungi of Sorghum Leads to Reduced Germination and Subsequent Attachment and Emergence of Striga hermonthica

Two sorghum cultivars: the Striga-tolerant S-35 and the Striga-sensitive CK60-B were grown with or without arbuscular mycorrhizal (AM) fungi, and with or without phosphorus addition. At 24 and 45 days after sowing (DAS) of sorghum, root exudates were collected and tested for effects on germination of preconditioned Striga hermonthica seeds. Root exudates from AM sorghum plants induced lower germination of S. hermonthica seeds than exudates from non-mycorrhizal sorghum. The magnitude of this effect depended on the cultivar and harvest time. A significantly (88–97%) lower germination of S. hermonthica seeds upon exposure to root exudates from AM S-35 plants was observed at both harvest times whereas for AM inoculated CK60-B plants a significantly (41%) lower germination was observed only at 45 DAS. The number of S. hermonthica seedlings attached to and emerged on both sorghum cultivars were also lower in mycorrhizal than in non-mycorrhizal plants. Again, this reduction was more pronounced with S-35 than with CK60-B plants. There was no effect of phosphorus addition on Striga seed germination, attachment or emergence. We hypothesize that the negative effect of mycorrhizal colonization on Striga germination and on subsequent attachment and emergence is mediated through the production of signaling molecules (strigolactones) for AM fungi and parasitic plants.Key Words: arbuscular mycorrhiza, root exudate, sorghum, striga, strigolactones, germination     

Drought- and ABA-induced changes in photosynthesis of barley plants

The changes caused by drought stress and abscisic acid (ABA) on photosynthesis of barley plants (Hordeum vulgare. L. cv. Alfa) have been studied. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. Cycloheximide (CHI), an inhibitor of stress-induced ABA accumulation, was used to distinguish alterations in photosynthetic reactions that are induced after drought stress in response to elevated ABA levels from those that are caused directly by altered water relations. Four hoars after imposition of drought stress or 2 h after application of ABA, Ihe bulk of the leaf's ABA content measured by enzyme-amplified ELISA, increased 14- and 16-fold, respectively. CHI fully blocked the stress-induced ABA accumulation. Gas exchange measurements and analysis of enzyme activities were used to study the reactions of photosynthesis to drought stress and ABA. Leaf dehydration or ABA treatment led to a noticeable decrease in both the initial slope of the curves representing net photosynthetic rate versus intercellular CO₂ concentration and the maximal rate of photosynthesis; dehydration of CHI-treated plants showed much slower inhibition of the latter. The calculated values of the intercellular CO₂ concentration, CO₂ compensation point and maximal carboxylating efficiency of ribulose 1,5-bisphosphate (RuBP) carboxylase support the suggestion that biochemical factors are involved in the response of photosynthesis to ABA and drought stress. RuBP carboxylase activity was almost unaffected in ABA- and CHI-treated, non-stressed plants. A drop in enzyme activity was observed after leaf dehydration of the control and ABA-treated plants. When barley plants were supplied with ABA, the activity of carbonic anhydrase (CA, EC 4.2.2.1) increased more than 2-fold. Subsequent dehydration caused an over 1.5-fold increase in CA activity of the control plants and a more than 2.5-fold increase in ABA-treated plants. Dehydration of CHI-treated plants caused no change in enzyme activity. It is suggested that increased activity of CA is a photosynthetic response to elevated ABA concentration.     

Drought stress during soybean seed filling affects storage compounds through regulation of lipid and protein metabolism

Soybean seeds have high lipid and protein contents. Adverse environmental conditions restrict seed yield and quality. We examined the changes in storage compounds caused by drought stress from R5 stage (beginning seed growth stage). Under drought stress, contents of lipid in seed were remarkably low compared to control at 24 and 29 days after treatment. Protein contents in seed were immediately decreased after water deficit treatment. On the other hand, soluble sugar contents in seed were increased by drought stress. Drought stress decreased the expression of genes involved in lipid biosynthesis (PK, BCCP2, and KAS1) and increased the genes expression involved in lipid degradation (ACX2, MS, and PEPCK). These results suggest that the increasing of sugar content in seed under drought stress was complemented by degradation of lipids. The expressions of genes encoding storage protein (Gy4 and β-conglycinin) were also decreased by drought stress. This study showed how drought stress during seed filling affects seed quality, especially lipid and protein contents, that may facilitate further research on seed storage compounds metabolism under environmental stresses.     

Antioxidant Responses of Lentil to Cold and Drought Stress

The effects of cold and drought stress on antioxidant responses and growth parameters in shoots and roots of lentil (Lens culinaris M cv Sultan 1) seedlings were investigated. Ten-day-old hydroponically grown seedlings were subjected to drought and cold (4°C) stress for 5 days. The length and fresh weight of shoots decreased significantly under both stress conditions, contrary to the increase in these growth parameters for roots under the same conditions. The oxidative damage as generation of malondialdehyde and hydrogen peroxide, was markedly higher in shoots under cold. Both stress conditions caused a significant increase in malondialdehyde levels in root tissues. The increase in proline levels was more pronounced under cold stress in shoots and roots. The tested stress conditions had no significant effect on chlorophyll contents. Superoxide dismutase activity was differentially altered in shoot and root tissues under drought and cold stress. The catalase activity was higher in roots under drought stress. On the other hand, ascorbate peroxidase activity increased in root tissues under cold stress. The results indicate that improved tolerance to cold and drought stress in root and shoot tissues of lentil might be correlated to the increased capacity of antioxidative defense system.     

Changes in Soybean (Glycine max [L.] Merr.) Glycerolipids in Response to Water Stress

Soybean (Glycine max [L.] Merr.) plants with the first trifoliate leaf fully expanded were exposed to 4 and 8 days of water stress. Leaf water potentials dropped from −0.6 megapascal to −1.7 megapascals after 4 days of stress; then to −3.1 megapascals after 8 days without water. All of the plants recovered when rewatered. The effects of short-term drought stress on triacylglycerol, diacylglycerol, phospholipid, and galactolipid metabolism in the first trifoliate leaves was determined. Leaf triacylglycerol and diacylglycerol content increased 2-fold during the first 4 days of stress and returned to control levels 3 days after rewatering. The polar lipid fraction, which contained phospholipids and galactolipids, changed little during this time. The linolenic acid (18:3) content of the triacylglycerol and diacylglycerol increased 25% during stress and the polar lipid 18:3 content decreased 15%. The pattern of glycerolipid labeling, after applying [2-¹⁴C]acetate to intact leaves was altered by water stress. After 4 days of water stress the radioactivity of phosphatidic acid + phosphatidylinositol, phosphatidylcholine, triacylglycerol, and diacylglycerol increased between 4 and 9% (compared to control plans) while radioactivity of phosphatidylethanolamine, monogalactosyldiglyceride, and digalactosyldiglyceride decreased 2 to 11%. These data indicated that increased levels of triacylglycerol and diacylglycerol observed during water stress were attributed to de novo synthesis rather than breakdown or reutilization of existing glycerolipids and fatty acids.     

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.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.