Barley HOMOCYSTEINE METHYLTRANSFERASE 2 confers drought tolerance by improving polyamine metabolism.

Drought stress poses a serious threat to crop production worldwide. Genes encoding homocysteine methyltransferase (HMT) have been identified in some plant species in response to abiotic stress, but its molecular mechanism in plant drought tolerance remains unclear. Here, transcriptional profiling, evolutionary bioinformatics, and population genetics were conducted to obtain insight into the involvement of HvHMT2 from Tibetan wild barley (Hordeum vulgare ssp. agriocrithon) in drought tolerance. We then performed genetic transformation coupled with physio-biochemical dissection and comparative multiomics approaches to determine the function of this protein and the underlying mechanism of HvHMT2-mediated drought tolerance. HvHMT2 expression was strongly induced by drought stress in tolerant genotypes in a natural Tibetan wild barley population and contributed to drought tolerance through S-adenosylmethionine (SAM) metabolism. Overexpression of HvHMT2 promoted HMT synthesis and efficiency of the SAM cycle, leading to enhanced drought tolerance in barley through increased endogenous spermine and less oxidative damage and growth inhibition, thus improving water status and final yield. Disruption of HvHMT2 expression led to hypersensitivity under drought treatment. Application of exogenous spermine reduced accumulation of reactive oxygen species (ROS), which was increased by exogenous mitoguazone (inhibitor of spermine biosynthesis), consistent with the association of HvHMT2-mediated spermine metabolism and ROS scavenging in drought adaptation. Our findings reveal the positive role and key molecular mechanism of HvHMT2 in drought tolerance in plants, providing a valuable gene not only for breeding drought-tolerant barley cultivars but also for facilitating breeding schemes in other crops in a changing global climate.

Genome-wide identification of expansin gene family in barley and drought-related expansins identification based on RNA-seq.

Expansins are cell wall loosening proteins and involved in various developmental processes and abiotic stress. No systematic research, however, has been conducted on expansin genes family in barley. A total of 46 expansins were identified and could be classified into three subfamilies in Hordeum vulgare: HvEXPA, HvEXPB, and HvEXLA. All expansin proteins contained two conserved domains: DPBB_1 and Pollen_allerg_1. Expansins, in the same subfamily, share similar motifs composition and exon-intron organization; but greater differences were found among different subfamilies. Expansins are distributed unevenly on 7 barley chromosomes; tandem duplicates, including the collinear tandem array, contribute to the forming of the expansin genes family in barley with few whole-genome duplication events. Most HvEXPAs mainly expressed in embryonic and root tissues. HvEXPBs and HvEXLAs showed different expression patterns in 16 tissues during different developmental stages. In response to water deficit, expansins in wild barley were more sensitive than that in cultivated barley; the expressions of HvEXPB5 and HvEXPB6 were significantly induced in wild barley under drought stress. Our study provides a comprehensive and systematic analysis of the barley expansin genes in genome-wide level. This information will lay a solid foundation for further functional exploration of expansin genes in plant development and drought stress tolerance.

Response of Tibetan Wild Barley Genotypes to Drought Stress and Identification of Quantitative Trait Loci by Genome-Wide Association Analysis.

Tibetan wild barley has been identified to show large genetic variation and stress tolerance. A genome-wide association (GWA) analysis was performed to detect quantitative trait loci (QTLs) for drought tolerance using 777 Diversity Array Technology (DArT) markers and morphological and physiological traits of 166 Tibetan wild barley accessions in both hydroponic and pot experiments. Large genotypic variation for these traits was found; and population structure and kinship analysis identified three subpopulations among these barley genotypes. The average LD (linkage disequilibrium) decay distance was 5.16 cM, with the minimum on 6H (0.03 cM) and the maximum on 4H (23.48 cM). A total of 91 DArT markers were identified to be associated with drought tolerance-related traits, with 33, 26, 16, 1, 3, and 12 associations for morphological traits, H⁺K⁺-ATPase activity, antioxidant enzyme activities, malondialdehyde (MDA) content, soluble protein content, and potassium concentration, respectively. Furthermore, 7 and 24 putative candidate genes were identified based on the reference Meta-QTL map and by searching the Barleymap. The present study implicated that Tibetan annual wild barley from Qinghai⁻Tibet Plateau is rich in genetic variation for drought stress. The QTLs detected by genome-wide association analysis could be used in marker-assisting breeding for drought-tolerant barley genotypes and provide useful information for discovery and functional analysis of key genes in the future.

Genome-Wide Identification, Characterization and Expression Analysis of Xyloglucan Endotransglucosylase/Hydrolase Genes Family in Barley (Hordeum vulgare).

Xyloglucan endotransglucosylase/hydrolases (XTHs)-a family of xyloglucan modifying enzymes-play an essential role in the construction and restructuring of xyloglucan cross-links. However, no comprehensive study has been performed on this gene family in barley. A total of 24 HvXTH genes (named HvXTH1-24) and an EG16 member were identified using the recently completed genomic database of barley (Hordeum vulgare). Phylogenetic analysis showed that 24 HvXTH genes could be classified into three phylogenetic groups: (I/II, III-A and III-B) and HvXTH15 was in the ancestral group. All HvXTH protein members-except HvXTH15-had a conserved N-glycosylation site. The genomic location of HvXTHs on barley chromosomes showed that the 24 genes are unevenly distributed on the 7 chromosomes, with 10 of them specifically located on chromosome 7H. A structure-based sequence alignment demonstrates that each XTH possesses a highly conserved domain (ExDxE) responsible for catalytic activity. Expression profiles based on the barley genome database showed that HvXTH family members display different expression patterns in different tissues and at different stages. This study is the first systematic genomic analysis of the barley HvXTH gene family. Our results provide valuable information that will help to elucidate the roles of HvXTH genes in the growth and development of barley.

Genotypic-dependent effects of N fertilizer, glutathione, silicon, zinc, and selenium on proteomic profiles, amino acid contents, and quality of rice genotypes with contrasting grain Cd accumulation.

Soil heavy metal (HM) contamination has posed a serious problem for safe food production. For restricting the translocation of HM into grain, many proteins were regulated to involve in the process. To identify these proteins, 2D-based proteomic analysis was carried out using different rice genotypes with distinct Cd accumulation in grains and as affected by an alleviating regulator (AR) in field experiments. AR application improved grain quality, with increased contents in Glu, Cys, His, Pro, and protein. Twenty-six low-grain HM accumulation-associated protein species were identified and categorized as physiological functions via two-dimensional gel electrophoresis (2DE) and mass spectrometry. Among these proteins, 8, 9, and 9 proteins exhibited higher accumulation, lower accumulation, and unchanged accumulation, respectively, in Xiushui817 (low accumulator) vs R8097 (high accumulator) under control conditions but showed differential accumulation patterns after AR application. These proteins included sucrose synthase 3, alanine aminotransferase, glutelin, cupin family protein, and zinc finger CCCH domain-containing protein 32. The differential expression of these protein species might contribute to decreased HM accumulation in grain via decreasing the protein accumulation which had high affinity to HM or regulating energy metabolism and signal transduction. Our findings provide valuable insights into the mechanisms of low-grain HM accumulation in rice and possible utilization of candidate protein species in developing low-grain HM accumulation genotypes.

Genome-Wide Identification and Expression Analysis of CsCaM/CML Gene Family in Response to Low-Temperature and Salt Stresses in Chrysanthemum seticuspe.

Calmodulin (CaM) and calmodulin-like proteins (CML) act as significant Ca2+ sensors binding Ca2+ with EF-hand motifs and have been reported to be involved in various environmental stresses in plants. In this study, calmodulin CsCaM/CML gene family members were identified based on the genome of Chrysanthemum seticuspe published recently; a phylogenetic tree was constructed; gene structures and chromosomal locations of CsCaM/CML were depicted; cis-acting regulatory elements were predicted; collinearity and duplicate events of CaM/CML were analyzed using MCScanX software; and the expression levels of CsCaM/CML in response to abiotic stress were analyzed, based on the published RNA-seq data. We identified 86 CsCaM/CML (4 CsCaMs and 82 CsCMLs) genes in total. Promoter sequences of CsCaM/CML contained elements related to abiotic stresses (including low-temperature and anaerobic stresses) and plant hormones (including abscisic acid (ABA), MeJA, and salicylic acid). CsCaM/CML genes were distributed on nine chromosomes unevenly. Collinearity analysis indicated that recent segmental duplications significantly enlarged the scale of the CML family in C. seticuspe. Four CsCMLs (CsCML14, CsCML50, CsCML65, and CsCML79) were statistically differentially regulated under low-temperature and salt stress compared with those in the normal condition. These results indicate diverse roles of CsCaM/CML in plant development and in response to environmental stimuli in C. seticuspe.