Genome-Wide Identification of the F-box Gene Family and Expression Analysis under Drought and Salt Stress in Barley

The F-box protein-encoding gene family plays an essential role in plant stress resistance. In present study, 126 non-redundant F-box genes were identified in barley (Hordeum vulgare L., Hv). The corresponding proteins contained 165– 887 amino acid residues and all were amphiphilic, except 5 proteins. Phylogenetic analysis of F-box protein sequences in barley and stress-related F-box protein sequences in wheat and Arabidopsis thaliana (At) was used to classify barley F-box genes are divided into 9 subfamilies (A–I). A structure-based sequence alignment demonstrated that F-box proteins were highly conserved with a total of 10 conserved motifs. In total, 124 F-box genes were unevenly distributed on 7 chromosomes; another 2 genes have not been anchored yet. The gene structure analysis revealed high variability in the number of exons and introns in F-box genes. Comprehensive analysis of expression profiles and phylogenetic tree analysis, a total of 12 F-box genes that may be related to stress tolerance in barley were screened. Of the 12 detected F-box genes, 8 and 10 were upregulated after drought and salt stress treatments, respectively, using quantitative real-time polymerase chain reaction (qRT-PCR). This study is the first systematic analysis conducted on the F-box gene family in barley, which is of great importance for clarifying this family’s bioinformatic characteristics and elucidating its function in barley stress resistance. These results will serve as a theoretical reference for subsequent research on molecular regulation mechanisms, genetic breeding, and improvement.     

Biological functions of Arabidopsis thaliana MBP-1-like protein encoded by ENO2 in the response to drought and salt stresses

Arabidopsis thaliana ENO2 (AtENO2) plays an important role in plant growth and development. It encodes two proteins, a full-length AtENO2 and a truncated version, AtMBP-1, alternatively translated from the second start codon of the mRNA. The AtENO2 mutant (eno2⁻) exhibited reduced leaf size, shortened siliques, a dwarf phenotype and higher sensitivity to abiotic stress. The objectives of this study were to analyze the regulatory network of the ENO2 gene in plant growth development and understand the function of AtENO2/AtMBP-1 to abiotic stresses. An eno2⁻/35S:AtENO2-GFP line and an eno2⁻/35S:AtMBP-1-GFP line of Arabidopsis were obtained. Results of sequencing by 454 GS FLX identified 578 upregulated and 720 downregulated differential expressed genes (DEGs) in a pairwise comparison (WT-VS-eno2⁻). All the high-quality reads were annotated using the Gene Ontology (GO) terms. The DEGs with KEGG pathway annotations occurred in 110 pathways. The metabolic pathways and biosynthesis of secondary metabolites contained more DEGs. Moreover, the eno2⁻/35S:AtENO2-GFP line returned to the wild-type (WT) phenotype and was tolerant to drought and salt stresses. However, the eno2⁻/35S:AtMBP-1-GFP line was not able to recover the WT phenotype but it has a higher tolerance to drought and salt stresses. Results from this study demonstrate that AtENO2 is critical for the growth and development, and the AtMBP-1 coded by AtENO2 is important in tolerance of Arabidopsis to abiotic stresses.     

Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.     

RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.