Overexpression of wheat transcription factor (TaHsfA6b) provides thermotolerance in barley.

MAIN CONCLUSION: Overexpressing a heat shock factor gene (TaHsfA6bT) from wheat provides thermotolerance in barley by constitutive expression of heat and other abiotic stress-response genes. Temperature is one of the most crucial abiotic factors defining the yield potential of temperate cereal crops, such as barley. The regulators of heat shock response (HSR), heat stress transcription factors (Hsfs), modulate the transcription level of heat-responsive genes to protect the plants from heat stress. In this study, an Hsf from wheat (TaHsfA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHsfA6b showed improvement in thermotolerance. The constitutive overexpression of a TaHsfA6b gene upregulated the expression of major heat shock proteins and other abiotic stress-responsive genes. RNA-seq and qRT-PCR analysis confirmed the upregulation of Hsps, chaperonins, DNAJ, LEA protein genes, and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of reactive oxygen species (ROS) occurred in wild-type (WT) plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms, showing lesser ROS accumulation under high temperature. No negative phenotypic changes were observed in overexpression lines. These results suggest that TaHsfA6b is a regulator of HSR and its overexpression altered the expression patterns of some main stress-related genes and enhanced the thermotolerance of this cereal crop.

DREB/CBF expression in wheat and barley using the stress-inducible promoters of HD-Zip I genes: impact on plant development, stress tolerance and yield.

Networks of transcription factors regulate diverse physiological processes in plants to ensure that plants respond to abiotic stresses rapidly and efficiently. In this study, expression of two DREB/CBF genes, TaDREB3 and TaCBF5L, was modulated in transgenic wheat and barley, by using stress-responsive promoters HDZI-3 and HDZI-4. The promoters were derived from the durum wheat genes encoding the γ-clade TFs of the HD-Zip class I subfamily. The activities of tested promoters were induced by drought and cold in leaves of both transgenic species. Differences in sensitivity of promoters to drought strength were dependent on drought tolerance levels of cultivars used for generation of transgenic lines. Expression of the DREB/CBF genes under both promoters improved drought and frost tolerance of transgenic barley, and frost tolerance of transgenic wheat seedlings. Expression levels of the putative TaCBF5L downstream genes in leaves of transgenic wheat seedlings were up-regulated under severe drought, and up- or down-regulated under frost, compared to those of control seedlings. The application of TaCBF5L driven by the HDZI-4 promoter led to the significant increase of the grain yield of transgenic wheat, compared to that of the control wild-type plants, when severe drought was applied during flowering; although no yield improvements were observed when plants grew under well-watered conditions or moderate drought. Our findings suggest that the studied HDZI promoters combined with the DREB/CBF factors could be used in transgenic cereal plants for improvement of abiotic stress tolerance, and the reduction of negative influence of transgenes on plant development and grain yields.

Overexpression of HvAKT1 improves drought tolerance in barley by regulating root ion homeostasis and ROS and NO signaling.

Potassium (K+) is the major cationic inorganic nutrient utilized for osmotic regulation, cell growth, and enzyme activation in plants. Inwardly rectifying K+ channel 1 (AKT1) is the primary channel for root K+ uptake in plants, but the function of HvAKT1 in barley plants under drought stress has not been fully elucidated. In this study, we conducted evolutionary bioinformatics, biotechnological, electrophysiological, and biochemical assays to explore molecular mechanisms of HvAKT1 in response to drought in barley. The expression of HvAKT1 was significantly up-regulated by drought stress in the roots of XZ5-a drought-tolerant wild barley genotype. We isolated and functionally characterized the plasma membrane-localized HvAKT1 using Agrobacterium-mediated plant transformation and Barley stripe mosaic virus-induced gene silencing of HvAKT1 in barley. Evolutionary bioinformatics indicated that the K+ selective filter in AKT1 originated from streptophyte algae and is evolutionarily conserved in land plants. Silencing of HvAKT1 resulted in significantly decreased biomass and suppressed K+ uptake in root epidermal cells under drought treatment. Disruption of HvAKT1 decreased root H+ efflux, H+-ATPase activity, and nitric oxide (NO) synthesis, but increased hydrogen peroxide (H2O2) production in the roots under drought stress. Furthermore, we observed that overexpression of HvAKT1 improves K+ uptake and increases drought resistance in barley. Our results highlight the importance of HvAKT1 for root K+ uptake and its pleiotropic effects on root H+-ATPase, and H2O2 and NO in response to drought stress, providing new insights into the genetic basis of drought tolerance and K+ nutrition in barley.

Identification and characterisation of a previously unknown drought tolerance-associated microRNA in barley.

Drought is the most serious abiotic stress, and causes crop losses on a worldwide scale. The present study identified a previously unknown microRNA (designated as hvu-miRX) of 21 nucleotides (nt) in length in barley. Its precursor (designated pre-miRX) and primary transcript (designated pri-miRX) were also identified, with lengths of 73 and 559 nt, respectively. The identified upstream sequence of pri-miRX contained both the TATA box and the CAAT box, which are both required for initiation of transcription. Transient promoter activation assays showed that the core promoter region of pri-miRX ranged 500 nt from the transcription start site. In transgenic barley overexpression of the wheat DREB3 transcription factor (TaDREB3) caused hvu-miRX to be highly expressed as compared with the same miRNA in non-transgenic barley. However, the high expression was not directly associated with TaDREB3. Genomic analysis revealed that the hvu-miRX gene was a single copy located on the short arm of chromosome 2 and appeared to be only conserved in Triticeae, but not in other plant species. Notably, transgenic barley that overexpressed hvu-miRX showed drought tolerance. Degradome library analysis and other tests showed that hvu-miRX targeted various genes including transcription factors via the cleavage mode. Our data provides an excellent opportunity to develop drought stress tolerant cereals using hvu-miRX.

Over-expression of (1,3;1,4)-ß-D-glucanase isoenzyme EII gene results in decreased (1,3;1,4)-ß-D-glucan content and increased starch level in barley grains.

BACKGROUND: High content of (1,3;1,4)-ß-d-glucan in barley grains is regarded as an undesirable factor affecting malting potential, brewing yield and feed utilization. Production of thermostable bacterial (1,3;1,4)-ß-glucanase in transgenic barley grain or supplementation of exogenous bacterial (1,3;1,4)-ß-glucanase has been used to improve malt and feed quality. The aim of the present study was to investigate the effect of over-expression of an endogenous (1,3;1,4)-ß-glucanase on ß-glucan content and grain composition in barley. RESULTS: A construct containing full-length HvGlb2 cDNA encoding barley (1,3;1,4)-ß-glucanase isoenzyme EII under the control of a promoter of barley D-Hordein gene Hor3-1 was introduced into barley cultivar Golden Promise via Agrobacterium-mediated transformation, and transgenic plants were regenerated after hygromycin selection. The T2 generation of proHor3:HvGlb2 transgenic lines showed increased activity of (1,3;1,4)-ß-glucanase in grains. Total ß-glucan content was reduced by more than 95.73% in transgenic grains compared with the wild-type control. Meanwhile, over-expression of (1,3;1,4)-ß-glucanase led to an increase in 1000-grain weight, which might be due to elevated amounts of starch in the grain. CONCLUSION: Manipulating the expression of (1,3;1,4)-ß-glucanase EII can control the ß-glucan content in grain with no apparent harmful effects on grain quality of transgenic plants. © 2016 Society of Chemical Industry.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

The wheat Lr34 gene provides resistance against multiple fungal pathogens in barley.

The Lr34 gene encodes an ABC transporter and has provided wheat with durable, broad-spectrum resistance against multiple fungal pathogens for over 100 years. Because barley does not have an Lr34 ortholog, we expressed Lr34 in barley to investigate its potential as a broad-spectrum resistance resource in another grass species. We found that introduction of the genomic Lr34 sequence confers resistance against barley leaf rust and barley powdery mildew, two pathogens specific for barley but not virulent on wheat. In addition, the barley lines showed enhanced resistance against wheat stem rust. Transformation with the Lr34 cDNA or the genomic susceptible Lr34 allele did not result in increased resistance. Unlike wheat, where Lr34-conferred resistance is associated with adult plants, the genomic Lr34 transgenic barley lines exhibited multipathogen resistance in seedlings. These transgenic barley lines also developed leaf tip necrosis (LTN) in young seedlings, which correlated with an up-regulation of senescence marker genes and several pathogenesis-related (PR) genes. In wheat, transcriptional expression of Lr34 is highest in adult plants and correlates with increased resistance and LTN affecting the last emerging leaf. The severe phenotype of transgenic Lr34 barley resulted in reduced plant growth and total grain weight. These results demonstrate that Lr34 provides enhanced multipathogen resistance early in barley plant development and implies the conservation of the substrate and mechanism of the LR34 transporter and its molecular action between wheat and barley. With controlled gene expression, the use of Lr34 may be valuable for many cereal breeding programmes, particularly given its proven durability.

Water uptake in barley grain: Physiology; genetics and industrial applications.

Water uptake by mature barley grains initiates germination and is the first stage in the malting process. Here we have investigated the effects of starchy endosperm cell wall thickness on water uptake, together with the effects of varying amounts of the wall polysaccharide, (1,3;1,4)-ß-glucan. In the latter case, we examined mutant barley lines from a mutant library and transgenic barley lines in which the (1,3;1,4)-ß-glucan synthase gene, HvCslF6, was down-regulated by RNA interference. Neither cell wall thickness nor the levels of grain (1,3;1,4)-ß-glucan were significantly correlated with water uptake but are likely to influence modification during malting. However, when a barley mapping population was phenotyped for rate of water uptake into grain, quantitative trait locus (QTL) analysis identified specific regions of chromosomes 4H, 5H and 7H that accounted for approximately 17%, 18% and 11%, respectively, of the phenotypic variation. These data indicate that variation in water uptake rates by elite malting cultivars of barley is genetically controlled and a number of candidate genes that might control the trait were identified under the QTL. The genomics data raise the possibility that the genetic variation in water uptake rates might be exploited by breeders for the benefit of the malting and brewing industries.

Stress tolerance of transgenic barley accumulating the alfalfa aldose reductase in the cytoplasm and the chloroplast.

Barley represents one of the major crops grown worldwide; its genetic transformation provides an important tool for the improvement of crop quality and tolerance to environmental stress factors. Biotic and abiotic stresses produce reactive oxygen species in the plant cells that can directly oxidize the cellular components including lipid membranes; resulting in lipid peroxidation and subsequently the accumulation of reactive carbonyl compounds. In order to protect barley plants from the effects of stress-produced reactive carbonyls, an Agrobacterium-mediated transformation was carried out using the Medicago sativa aldose reductase (MsALR) gene. In certain transgenic lines the produced MsALR enzyme was targeted to the chloroplasts to evaluate its protective effect in these organelles. The dual fluorescent protein-based method was used for the evaluation of tolerance of young seedlings to diverse stresses; our results demonstrated that this technique could be reliably applied for the detection of cellular stress in a variety of conditions. The chlorophyll and carotenoid content measurements also supported the results of the fluorescent protein-based method and the stress-protective effect of the MsALR enzyme. Targeting of MsALR into the chloroplast has also resulted in increased stress tolerance, similarly to the observed effect of the cytosolic MsALR accumulation. The results of the DsRed/GFP fluorescent protein-based method indicated that both the cytosol and chloroplast accumulation of MsALR can increase the abiotic stress tolerance of transgenic barley lines.