The Barley S-Adenosylmethionine Synthetase 3 Gene HvSAMS3 Positively Regulates the Tolerance to Combined Drought and Salinity Stress in Tibetan Wild Barley.

Drought and salinity are two of the most frequently co-occurring abiotic stresses. Despite recent advances in the elucidation of the effects of these stresses individually during the vegetative stage of plants, significant gaps exist in our understanding of the combined effects of these two frequently co-occurring stresses. Here, Tibetan wild barley XZ5 (drought tolerant), XZ16 (salt tolerant), and cultivated barley cv. CM72 (salt tolerant) were subjected to drought (D), salinity (S), or a combination of both treatments (D+S). Protein synthesis is one of the primary activities of the green part of the plant. Therefore, leaf tissue is an important parameter to evaluate drought and salinity stress conditions. Sixty differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) and classified into 9 biological processes based on Gene Ontology annotation. Among them, 21 proteins were found to be expressed under drought or salinity alone; however, under D+S, 7 proteins, including S-adenosylmethionine synthetase 3 (SAMS3), were exclusively upregulated in drought-tolerant XZ5 but not in CM72. HvSAMS3 carries both N-terminal and central domains compared with Arabidopsis and activates the expression of several ethylene (ET)-responsive transcription factors. HvSAMS3 is mainly expressed in the roots and stems, and HvSAMS3 is a secretory protein located in the cell membrane and cytoplasm. Barley stripe mosaic virus-based virus-induced gene silencing (BSMV-VIGS) of HvSAMS3 in XZ5 severely compromised its tolerance to D+S and significantly reduced plant growth and K+ uptake. The reduced tolerance to the combined stress was associated with the inhibition of polyamines such as spermidine and spermine, polyamine oxidase, ethylene, biotin, and antioxidant enzyme activities. Furthermore, the exogenous application of ethylene and biotin improved the tolerance to D+S in BSMV-VIGS:HvSAMS3-inoculated plants. Our findings highlight the significance of HvSAMS3 in the tolerance to D+S in XZ5.

Tolerance to Drought, Low pH and Al Combined Stress in Tibetan Wild Barley Is Associated with Improvement of ATPase and Modulation of Antioxidant Defense System.

Aluminum (Al) toxicity and drought are two major constraints on plant growth in acidic soils, negatively affecting crop performance and yield. Genotypic differences in the effects of Al/low pH and polyethyleneglycol (PEG) induced drought stress, applied either individually or in combination, were studied in Tibetan wild (XZ5, drought-tolerant; XZ29, Al-tolerant) and cultivated barley (Al-tolerant Dayton; drought-tolerant Tadmor). Tibetan wild barley XZ5 and XZ29 had significantly higher H⁺-ATPase, Ca2+Mg2+-ATPase, and Na⁺K⁺-ATPase activities at pH 4.0+Al+PEG than Dayton and Tadmor. Moreover, XZ5 and XZ29 possessed increased levels in reduced ascorbate and glutathione under these conditions, and antioxidant enzyme activities were largely stimulated by exposure to pH 4.0+PEG, pH 4.0+Al, and pH 4.0+Al+PEG, compared to a control and to Dayton and Tadmor. The activity of methylglyoxal (MG) was negatively correlated with increased levels of glyoxalase (Gly) I and Gly II in wild barley. Microscopic imaging of each genotype revealed DNA damage and obvious ultrastructural alterations in leaf cells treated with drought or Al alone, and combined pH 4.0+Al+PEG stress; however, XZ29 and XZ5 were less affected than Dayton and Tadmor. Collectively, the authors findings indicated that the higher tolerance of the wild barley to combined pH 4.0+Al+PEG stress is associated with improved ATPase activities, increased glyoxalase activities, reduced MG, and lower reactive oxygen species levels (like O2- and H2O2) due to increased antioxidant enzyme activities. These results offer a broad comprehension of the mechanisms implicated in barley's tolerance to the combined stress of Al/low pH and drought, and may provide novel insights into the potential utilization of genetic resources, thereby facilitating the development of barley varieties tolerant to drought and Al/low pH stress.

Identification of the differentially accumulated proteins associated with low phosphorus tolerance in a Tibetan wild barley accession.

Low phosphorus (LP) in soil is a widely-occurred limiting factor for crop production in the world. In a previous study we identified a highly LP-tolerant Tibetan wild barley accession (XZ99). Here, a comparatively proteomic analysis was conducted using three barley genotypes differing in LP tolerance to reveal the mechanisms underlying the LP tolerance of XZ99. Totally, 31 differentially accumulated proteins were identified in the roots and leaves of the three genotypes using 2-dimensional gel electrophoresis coupled with mass spectrometry. They were involved in the various biological processes, including carbon and energy metabolism, signal transduction, cell growth and division, secondary metabolism, and stress defense. In comparison with XZ100 (LP sensitive) and ZD9 (LP moderately-tolerant), XZ99 had a more developed root system, which is mainly attributed to enhanced carbohydrate metabolizing proteins under LP conditions. The current results showed that Tibetan wild barley XZ99 and cultivated barley cultivar ZD9 differ in the mechanism of LP tolerance. The changes of the proteins associated with carbohydrate metabolism could account for the difference between the LP-tolerant and LP-sensitive genotypes. In addition, the mRNA expression levels of 9 LP responsive proteins were verified by qRT-PCR. The current results may open a new avenue of understanding the LP tolerance in plants on the proteomic basis.

Physiological and molecular analysis on root growth associated with the tolerance to aluminum and drought individual and combined in Tibetan wild and cultivated barley.

MAIN CONCLUSION: The drought-stimulated gene expression of NCED, SUS, and KS - DHN and ABA signal cross-talk with other phytohormones maintains barley root growth under drought stress at pH 4.0 plus polyethylene glycol plus aluminum. Aluminum (Al) toxicity and drought are two major factors that limit barley production. In this work, the individual and combined effects of Al/acid and polyethylene glycol (PEG 6000) induced drought stress that suppressed root growth and caused oxidative damage as characterized by increased H2O2 and O2(.-) accumulation. The wild-barley genotypes, XZ5 and XZ29, exhibited a higher tolerance than the two cultivars Dayton (Al tolerant) and Tadmor (drought tolerant) under combined stress (pH 4.0 + PEG + Al). The oxidative damage induced by PEG was more severe at pH 4.0 than at pH 6.0. In XZ29, the highest root secretion of malate and citrate was recorded, and the least Al uptake in the four genotypes. In XZ5, a peak accumulation of ABA and minor synthesis of zeatin riboside and ethylene were found being essential in maintaining primary root elongation and root hair development. PEG-induced drought stress repressed Al uptake in root tips, with a lower increase in callose formation and HvMATE (Hordeum vulgare multidrug and toxic compound exudation) expression compared to Al-induced callose production. Stress by pH 4.0 + PEG + Al up-regulated 9-cis-epoxycarotenoid dioxygenase (NCED) which is involved in ABA biosynthesis. Such treatment stimulated the regulation of ABA-dependent genes sucrose synthase (SUS) and KS-type dehydrin (KS-DHN) in root tips. Our results suggest that the tolerance ranking to pH 4.0 + PEG + Al stress in Tibetan wild barley by gene expression is closely correlated to physiological indices. The results show that acclimatisation to pH 4.0 + PEG + Al stress involves specific responses in XZ5 and XZ29. The present study provides insights into the effects of Al/acid and drought combined stress on the abundance of physiological indices in the roots of barley varieties.

Identification of the proteins associated with low potassium tolerance in cultivated and Tibetan wild barley.

In previous studies, we found Tibetan wild barley accessions with high tolerance to low K. In this study, ionomics and proteomics analyses were done on two wild genotypes (XZ153, tolerant and XZ141, sensitive), and a cultivar (B1031, tolerance to low K) to understand the mechanism of low-K tolerance. XZ153 was much less affected by low K stress than the other two genotypes in plant biomass and shoot K content. A total of 288 differentially accumulated proteins were identified between low-K and normal K treated plants. Among them, 129 proteins related to low-K tolerance were mainly involved in defense, transcription, signal transduction, energy, and protein synthesis. The analysis of tandem mass tag (TMT) detected 51 proteins which were increased in relative abundance under low K in XZ153, but unaltered or decreased in XZ141. The proteomics results showed that XZ153 is highly capable of rearranging ion homeostasis and developing an antioxidant defense system under low-K stress. Moreover, ethylene response and phenylpropanoid pathways could determine the genotypic difference in low-K tolerance. The current results confirmed the possibility of Tibetan wild barley providing low-K tolerant germplasm and identified some candidate proteins for use in developing the cultivars with low-K tolerance.

Comparative transcriptome profiling of two Tibetan wild barley genotypes in responses to low potassium.

Potassium (K) deficiency is one of the major factors affecting crop growth and productivity. Development of low-K tolerant crops is an effective approach to solve the nutritional deficiency in agricultural production. Tibetan annual wild barley is rich in genetic diversity and can grow normally under poor soils, including low-K supply. However, the molecular mechanism about low K tolerance is still poorly understood. In this study, Illumina RNA-Sequencing was performed using two Tibetan wild barley genotypes differing in low K tolerance (XZ153, tolerant and XZ141, sensitive), to determine the genotypic difference in transcriptome profiling. We identified a total of 692 differentially expressed genes (DEGs) in two genotypes at 6 h and 48 h after low-K treatment, including transcription factors, transporters and kinases, oxidative stress and hormone signaling related genes. Meanwhile, 294 low-K tolerant associated DEGs were assigned to transporter and antioxidant activities, stimulus response, and other gene ontology (GO), which were mainly involved in starch and sucrose metabolism, lipid metabolism and ethylene biosynthesis. Finally, a hypothetical model of low-K tolerance mechanism in XZ153 was presented. It may be concluded that wild barley accession XZ153 has a higher capability of K absorption and use efficiency than XZ141 under low K stress. A rapid response to low K stress in XZ153 is attributed to its more K uptake and accumulation in plants, resulting in higher low K tolerance. The ethylene response pathway may account for the genotypic difference in low-K tolerance.

Differential changes in grain ultrastructure, amylase, protein and amino acid profiles between Tibetan wild and cultivated barleys under drought and salinity alone and combined stress.

Grain phytochemical profiles were compared in Tibetan wild barley XZ5 (drought-tolerant), XZ16 (salinity/aluminum-tolerant) and cv CM72 (salinity-tolerant) in response to drought and salinity alone and combination (D+S) during anthesis. Total antioxidant capacity assessed by determining ferric-reducing antioxidant potential (FRAP) in grains increased significantly as follows: D+S>drought>salinity, and XZ5>XZ16>CM72. A marked increase in the total phenol (TP) from individual and combined stresses was observed in XZ5, while a decrease occurred in CM72. Moreover, the activity of α-/ß-amylase in the grains under combined stress was 81.8%/16.9% in XZ5 and 48.6%/18.7% in XZ16 higher than that of CM72. Increases in amino acids, protein content and protein fractions of albumin, globulin, hordein and glutelin were maximised under D+S, with larger values in the Tibetan wild genotypes. Observation with a scanning electron microscopy showed a distinct genotypic difference under D+S; for example, XZ5 and XZ16 maintained a relatively integral starch granule with a greater protein deposit/matrix, while CM72 degraded by pitting. This research expands our understanding of barley drought and salt-tolerance mechanisms and provides possibility of Tibetan wild barley in developing barley cultivars with both tolerance to drought and salinity.