APETALA2/Ethylene-Responsive Factor (AP2/ERF) gene family is plant specific transcription factor. It plays critical roles in development process, tolerance to biotic and abiotic stresses, and responses to plant hormones. However, limited data are available on the contributions of AP2/ERF gene family in barley (Hordeum vulgare L.). In the present study, 121 HvAP2/ERF genes in barley were identified by using bioinformatics methods. A total of 118 HvAP2/ERF (97.5%) genes were located on seven chromosomes. According to phylogenetic classification of AP2/ERF family in Arabidopsis, HvAP2/ERF proteins were divided into AP2 (APETALA2), RAV (Related to ABI3/VP), DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and soloist sub families. The analysis of duplication events indicated that tandem repeat and segmental duplication contributed to the expansion of the AP2/ERF family in barley. HvDREB1s/2s genes displayed various expression patterns under abiotic stress and phytohormone. Taken together, the data generated in this study will be useful for genome-wide analysis to determine the precise role of the HvAP2/ERF gene during barley development, abiotic stress and phytohormone responses with the ultimate goal of improving crop production.

Drought and salinity are two major abiotic stresses that severely limit barley production worldwide. Physiological and genetic complexity of these tolerance traits has significantly slowed the progress of developing stress-tolerant cultivars. Marker-assisted selection (MAS) may potentially overcome this problem. In the current research, seventy two double haploid (DH) lines from a cross between TX9425 (a Chinese landrace variety with superior drought and salinity tolerance) and a sensitive variety, Franklin were used to identify quantitative trait loci (QTL) for drought and salinity tolerance, based on a range of developmental and physiological traits. Two QTL for drought tolerance (leaf wilting under drought stress) and one QTL for salinity tolerance (plant survival under salt stress) were identified from this population. The QTL on 2H for drought tolerance determined 42% of phenotypic variation, based on three independent experiments. This QTL was closely linked with a gene controlling ear emergency. The QTL on 5H for drought tolerance was less affected by agronomic traits and can be effectively used in breeding programs. A candidate gene for this QTL on 5H was identified based on the draft barley genome sequence. The QTL for proline accumulation, under both drought and salinity stresses, were located on different positions to those for drought and salinity tolerance, indicating no relationship with plant tolerance to either of these stresses. Using QTL mapping, the relationships between QTL for agronomic and physiological traits and plant drought and salinity tolerance were studied. A new QTL for drought tolerance which was not linked to any of the studied traits was identified. This QTL can be effectively used in breeding programs. It was also shown that proline accumulation under stresses was not necessarily linked with drought or salinity tolerance based on methods of phenotyping used in this experiment. The use of proline content in breeding programs can also be limited by the accuracy of phenotyping.

Waterlogging is one of the major abiotic stresses that affects barley production and yield quality. Proteomics techniques have been widely utilized to explore the mechanisms involved in the responses to abiotic stress. In this study, two barley genotypes with contrasting responses to waterlogging stress were analyzed with proteomic technology. The waterlogging treatment caused a greater reduction in biomass and photosynthetic performance in the waterlogging-sensitive genotype TF57 than that in the waterlogging-tolerant genotype TF58. Under waterlogging stress, 30, 30, 20 and 20 differentially expressed proteins were identified through tandem mass spectrometry analysis in the leaves, adventitious roots, nodal roots and seminal roots, respectively. Among these proteins, photosynthesis-, metabolism- and energy-related proteins were differentially expressed in the leaves, with oxygen-evolving enhancer protein 1, ATP synthase subunit and heat shock protein 70 being up-regulated in TF58. Pyruvate decarboxylase (PDC), 1-amino cyclopropane 1-carboxylic acid oxidase (ACO), glutamine synthetase (GS), glutathione S-transferases (GST) and beta-1, 3-glucanase in adventitious, nodal and seminal roots were more abundant in TF58 than those in TF57 under waterlogging stress. Ten representative genes were selected for validation by qRT-PCR in different genotypes with known waterlogging tolerance, and the expression levels of three candidate genes (PDC, ACO and GST) increased in the roots of all genotypes in response to the waterlogging stress. These three genes might play a significant role in the adaptation process of barley under waterlogging stress. The current results partially determined the mechanisms of waterlogging tolerance and provided valuable information for the breeding of barley with enhanced tolerance to waterlogging.

Salt stress is one of the major environmental factors impairing crop production. In our previous study, we identified a major QTL for salinity tolerance on chromosome 2H on barley (Hordeum vulgare L.). For further investigation of the mechanisms responsible for this QTL, two pairs of near-isogenic lines (NILs) differing in this QTL were developed. Sensitive NILs (N33 and N53) showed more severe damage after exposure to 300 mM NaCl than tolerant ones (T46 and T66). Both tolerant NILs maintained significantly lower Na⁺ content in leaves and much higher K⁺ content in the roots than sensitive lines under salt conditions, thus indicating the presence of a more optimal Na⁺/K⁺ ratio in plant tissues. Salinity stress caused significant accumulation of H₂O₂, MDA, and proline in salinity-sensitive NILs, and a greater enhancement in antioxidant enzymatic activities at one specific time or tissues in tolerant lines. One pair of NILs (N33 and T46) were used for proteomic studies using two-dimensional gel electrophoresis. A total of 53 and 51 differentially expressed proteins were identified through tandem mass spectrometry analysis in the leaves and roots, respectively. Proteins which are associated with photosynthesis, reactive oxygen species (ROS) scavenging, and ATP synthase were found to be specifically upregulated in the tolerant NIL. Proteins identified in this study can serve as a useful resource with which to explore novel candidate genes for salinity tolerance in barley.

Waterlogging stress significantly affects the growth, development, and productivity of crop plants. However, manipulation of gene expression to enhance waterlogging tolerance is very limited. In this study, we identified an ethylene-responsive factor from barley, which was strongly induced by waterlogging stress. This transcription factor named HvERF2.11 was 1158 bp in length and encoded 385 amino acids, and mainly expressed in the adventitious root and seminal root. Overexpression of HvERF2.11 in Arabidopsis led to enhanced tolerance to waterlogging stress. Further analysis of the transgenic plants showed that the expression of AtSOD1, AtPOD1 and AtACO1 increased rapidly, while the same genes did not do so in non-transgenic plants, under waterlogging stress. Activities of antioxidant enzymes and alcohol dehydrogenase (ADH) were also significantly higher in the transgenic plants than in the non-transgenic plants under waterlogging stress. Therefore, these results indicate that HvERF2.11 plays a positive regulatory role in plant waterlogging tolerance through regulation of waterlogging-related genes, improving antioxidant and ADH enzymes activities.