Studies On Mechanisms Of Low Potassium Tolerance In Tibetan Wild Barley

Potassium (K) is crucial for plant growth and development, as well as the yield and quality formation for crops.In China, most soils show K deficiency, and the case becomes more severe in the recent decades, limiting the agricultural sustainability. Therefore, understanding of physiological and molecular mechanisms of low-K tolerance in crops is imperative for developing the cultivars with high use efficiency of the nutrient element. Tibetan annual wild barley (Hordeum vulgare L. subsp. Spontaneum), recognized as one of the ancestors of cultivated barley, is rich in genetic variation and shows generally wide adaption to poor soil fertility, including K deficiency. In the present study, the physiological and molecular characteristics of Tibetan wild barley were investigated to make sight into the low-K tolerant mechanisms. The main findings were summarized as follows:1. RNA-Seq based comparative transcriptome profiling analysis of different K-tolerant barley genotypes in responses to low potassiumIn this study, Illumina RNA-Sequencing was performed on the two Tibetan wild barley genotypes differing in low K tolerance (XZ153, tolerant and XZ141, sensitive), to determine the genotypic difference in transcriptome profiling. Totally,692 differentially expressed genes (DEGs) were identified 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. Fifteen DEGs were randomly selected for real-time PCR analysis. The results showed that the expression patterns of the most genes were highly consistent with those shown in the RNA-Seq, indicating the accuracy and validity the RNA-Seq data. 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 proposed. 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. Meanwhile, the ethylene response pathway may account for the genotypic difference in low-K tolerance.2. Tandem mass tags (TMT) based quantitative proteomics analysis of the proteins associated with low potassium tolerance in barleyIn this study, ionomic and TMT based high-throughput proteomic methods were performed on the two wild barley genotypes (XZ153, tolerant and XZ141, sensitive), and a low-K tolerance cultivar (Ludaomai), to investigate the genotypic difference in response to low-K stress. At 16 d after low-K treatment, XZ153 was much less affected than the other two genotypes in terms of plant biomass and growth performance. There was a notable genotypic difference in the response of element content changes to low-K stress. XZ153 had the smallest reduction of shoot K content among three genotypes under low K, and XZ141 showed the greatest reduction in P, Zn, Cu and Fe contents in roots. Totally 288 differentially expressed 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 proteomics results showed that XZ153 is higher capable of rearranging ion homeostasis and developing 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 in providing low-K tolerant genetic resource and identified some candidate proteins for use in developing the cultivars with low-K tolerance.3. Metabolites and metabolic pathway responding to low potassium stress in barleyIn order to reveal adaptive metabolic pathways in barley under low potassium (LK) stress, XZ153, XZ141 and one cultivated cultiva (Ludaomai) were used to investigate metabolome changes in response to low potassium (0.01 mM KC1) using gas chromatography-mass spectrometry (GC-MS). A total of 61 key metabolites were identified and their concentrations were significantly affected by low potassium stress. In general, amino acids accumulation was enhanced in both roots and leaves, while TCA cycle was inhibited. In addition, the concentration of the negatively charged amino acids (Asp and Glu) and the negatively charged organic acid (Citric acid) were decreased in leaves and roots, but more positive charged amino acids (Lys) were increased in order to maintain the balance of charges in the cytoplasm. Moreover, the probable physiological basis of barley responding to low potassium was the accumulation of glucose and other sugars by ensuring the energy supply. XZ153 showed increase in the content of active oxygen scavenger (such as proline and ascorbic acid) and the capacity of anti-oxidation stress, resulting in a higher low K tolerance.4. Cloning and identification of HvHKT7 gene in barleyWe isolated one HKT (high-affinity K+ transporter) gene in the RNA-Seq results, which was designated as HvHKT7, being the cDNA length of 1776 bp and containing the complete CDS length of 1749 bp and encoding 582 amino acids, as well as nine transmembrane motifs. Gene structure analysis revealed that the HvHKT7 gene was composed of two introns and three exons. There are a total of five SNPs within the coding region of HvHKT7 between the two genotypes XZ141 and XZ153, leading to three amino acid changed. Meanwhile, the HvHKT7 gene encodes protein with the sequence similarities to OsHKT1;4 and TmHKT1;1. Additionally, HvHKT7 is defined as a class 1 HKT due to the Ser-Gly-Gly-Gly selectivity filter motif. Promoter analysis showed that HvHKT7 not only carry hormone response elements, such as CGTCA-motif and TGACG-motif associated with the JA signal, but also abiotic stress response components, such as 5’UTR Py-rich stretch, HSE and so on. RT-PCR results showed that the HvHKT7 gene expression in XZ153 was higher than that of other genotypes. Moreover, HvHKT7 is a typical membrane protein and located in the plasma and nuclear membranes.