Jianbin Zeng

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.

HvEXPB7, a novel β-expansin gene revealed by the root hair transcriptome of Tibetan wild barley, improves root hair growth under drought stress

Highlight A novel root hair development related gene, HvEXPB7, was identified and cloned from the identified drought tolerance-associated genes. BSMV-VIGS of HvEXPB7 confirmed that this gene was involved in root hair growth under drought.

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.

Identification of Mild Freezing Shock Response Pathways in Barley Based on Transcriptome Profiling

Low temperature is a major abiotic stress affecting crop growth and productivity. A better understanding of low temperature tolerance mechanisms is imperative for developing the crop cultivars with improved tolerance. We herein performed an Illumina RNA-sequencing experiment using two barley genotypes differing in freezing tolerance (Nure, tolerant and Tremois, sensitive), to determine the transcriptome profiling and genotypic difference under mild freezing shock treatment after a very short acclimation for gene induction. A total of 6474 differentially expressed genes, almost evenly distributed on the seven chromosomes, were identified. The key DEGs could be classified into six signaling pathways, i.e., Ca2+ signaling, PtdOH signaling, CBFs pathway, ABA pathway, jasmonate pathway, and amylohydrolysis pathway. Expression values of DEGs in multiple signaling pathways were analyzed and a hypothetical model of mild freezing shock tolerance mechanism was proposed. Expression and sequence profile of HvCBFs cluster within Frost resistance-H2, a major quantitative trait locus on 5H being closely related to low temperature tolerance in barley, were further illustrated, considering the crucial role of HvCBFs on freezing tolerance. It may be concluded that multiple signaling pathways are activated in concert when barley is exposed to mild freezing shock. The pathway network we presented may provide a platform for further exploring the functions of genes involved in low temperature tolerance in barley.

Metabolic analysis of two contrasting wild barley genotypes grown hydroponically reveals adaptive strategies in response to low nitrogen stress

Nitrogen (N) is an essential macronutrient for plants. The increasingly severe environmental problems caused by N fertilizer application urge alleviation of N fertilizer dependence in crop production. In previous studies, we identified the Tibetan wild barley accessions with high tolerance to low nitrogen (LN). In this study, metabolic analysis was done on two wild genotypes (XZ149, tolerant and XZ56, sensitive) to understand the mechanism of LN tolerance, using a hydroponic experiment. Leaf and root samples were taken at seven time points within 18 d after LN treatment, respectively. XZ149 was much less affected by low N stress than XZ56 in plant biomass. A total of 51 differentially accumulated metabolites were identified between LN and normal N treated plants. LN stress induced tissue-specific changes in carbon and nitrogen partitioning, and XZ149 had a pattern of energy-saving amino acids accumulation and carbon distribution in favor of root growth that contribute to its higher LN tolerance. Moreover, XZ149 is highly capable of producing energy and maintaining the redox homeostasis under LN stress. The current results revealed the mechanisms underlying the wild barley in high LN tolerance and provided the valuable references for developing barley cultivars with LN tolerance.

Nitrogen (N) metabolism related enzyme activities, cell ultrastructure and nutrient contents as affected by N level and barley genotype

Abstract Development of the new crop cultivars with high yield under low nitrogen (N) input is a fundamental approach to enhance agricultural sustainability, which is dependent on the exploitation of the elite germplasm. In the present study, four barley genotypes (two Tibetan wild and two cultivated), differing in N use efficiency (NUE), were characterized for their physiological and biochemical responses to different N levels. Higher N levels significantly increased the contents of other essential nutrients (P, K, Ca, Fe, Cu and Mn), and the increase was more obvious for the N-efficient genotypes (ZD9 and XZ149). The observation of ultrastructure showed that chloroplast structure was severely damaged under low nitrogen, and the two high N efficient genotypes were relatively less affected. The activities of the five N metabolism related enzymes, i.e., nitrate reductase (NR), glutamine synthetase (GS), nitrite reductase (NiR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) all showed the substantial increase with the increased N level in the culture medium. However the increased extent differed among the four genotypes, with the two N efficient genotypes showing more increase in comparison with the other two genotypes with relative N inefficiency (HXRL and XZ56). The current findings showed that a huge difference exists in low N tolerance among barley genotypes, and improvement of some physiological traits (such as enzymes) could be helpful for increasing N utilization efficiency.

The differences in physiological responses, ultrastructure changes, and Na+ subcellular distribution under salt stress among the barley genotypes differing in salt tolerance

Abstract Plants adopt several strategies to maintain cellular ion homeostasis, including physiological, biochemical, cellular, subcellular, and molecular mechanisms for fighting against salt stress. We investigated the responses of tolerant Tibetan wild barley (XZ16), tolerant (CM72) and sensitive (Gairdner) barley cultivars at physiological, cellular, and molecular levels. The results revealed that salinity induced a significantly greater reduction in total root length, surface area, diameter, and total volume in Gairdner than in CM72 and XZ16. Analysis of gene expression using quantitative RT-PCR showed that transcripts of vacuolar H+-ATPase and inorganic pyrophosphatase (HvHVA/68 and HvHVP1) were more abundant in leaves and roots of XZ16 and CM72 than those of Gairdner. Observation of electron microscopy detected the difference in the damage of leaf and root ultrastructure among the three genotypes under salt stress, with XZ16 and Gairdner being least and most affected, respectively. Subcellular study showed that a primary strategy to protect the cytosol against sodium toxicity was compartmentalization of sodium ions into soluble fraction (vacuoles). Gairdner showed drastically stronger sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than CM72 and XZ16.

Isobaric Tags for Relative and Absolute Quantitation Proteomic Analysis of Germinating Barley under Gibberellin and Abscisic Acid Treatments

The degradation of starch in barley grains is a primary step of beer production. The addition of an appropriate amount of gibberellin (GA) promotes the production of fermentable sugars, beneficial to the brewing industry. However, the response of proteomics in germinating barley to GA and abscisic acid (ABA) treatments is not thoroughly understood. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) proteomics analysis was performed to illustrate the change of proteins in Tibetan wild barley XZ72 and XZ95 under GA and ABA treatments during germination. XZ72 had more proteins upregulated than XZ95 under GA treatment, while under ABA treatments, XZ95 had more proteins upregulated than XZ72. Concerning the proteins involved in energy metabolism under GA treatment, XZ72 had more proteins upregulated than XZ95. Among the 174 proteins related to starch metabolism, 31 proteins related to starch hydrolysis, such as α-amylase, α-glucosidase, and β-fructofuranosidase, showed higher relative abundance in control and GA treatments in XZ72 than in XZ95. Analysis of correlation between proteins and metabolites indicated that higher hydrolase activity is beneficial for the accumulation of fermentable sugars during germination. On the other hand, 26 starch-synthesis-related proteins were upregulated in XZ95 under ABA treatment. It may be suggested that GA-induced proteins act as accelerators of starch degradation, while ABA-induced proteins inhibit starch degradation. The current results showed that XZ72 is highly capable of allocating the starch-hydrolyzing enzymes, which play important roles in starch breakdown.

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.

A Sodium Transporter HvHKT1;1 Confers Salt Tolerance in Barley via Regulating Tissue and Cell Ion Homeostasis

Our previous studies showed that high salt tolerance in Tibetan wild barley accessions was associated with HvHKT1;1, a member of the high-affinity potassium transporter family. However, molecular mechanisms of HvHKT1;1 for salt tolerance and its roles in K+/Na+ homeostasis remain to be elucidated. Functional characterization of HvHKT1;1 was conducted in the present study. NaCl-induced transcripts of HvHKT1;1 were significantly higher in the roots of Tibetan wild barley XZ16 relative to other genotypes, being closely associated with its higher biomass and lower tissue Na+ content under salt stress. Heterologous expression of HvHKT1;1 in Saccharomyces cerevisiae (yeast) and Xenopus laevis oocytes showed that HvHKT1;1 had higher selectivity for Na+ over K+ and other monovalent cations. HvHKT1;1 was found to be localized at the cell plasma membrane of root stele and epidermis. Knock-down of HvHKT1;1 in barley led to higher Na+ accumulation in both roots and leaves, while overexpression of HvHKT1;1 in salt-sensitive Arabidopsis hkt1-4 and sos1-12 loss-of-function lines resulted in significantly less shoot and root Na+ accumulation. Additionally, microelectrode ion flux measurements and root elongation assay revealed that the transgenic Arabidopsis plants exhibited a remarkable capacity for regulation of Na+, K+, Ca2+ and H+ homeostasis under salt stress. These results indicate that HvHKT1;1 is critical in radial root Na+ transport, which eventually reduces shoot Na+ accumulation. Additionally, HvHKT1;1 may be indirectly involved in retention of K+ and Ca2+ in root cells, which also improves plant salt tolerance.