Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members) using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

SUMMARYMicroRNAs (miRNAs) are important for the regulation of gene expression, and are involved in many developmental processes. A set of miRNAs which were differentially expressed between cells of totipotent (C1) and non-totipotent (C2) Arabidopsis thaliana calli was identified, some of which were affected during callus formation or shoot regeneration. One of those down-regulated after 10 days' incubation in shoot induction medium (SIM) was MIR160a, for which transcript abundance was lower in C1 than in C2. Over-expression of MIR160 compromised shoot regeneration from in vitro cultured A. thaliana cells, while the transgenic expression of a miR160-resistant form of ARF10 was associated with a high level of shoot regeneration. The latter transgenic line also showed an elevated expression level of shoot meristem-specific genes CLAVATA3, CUP-SHAPEDCOTYLEDON1 and -2, and WUSCHEL. ARF10 expression was concentrated at the initiation sites of shoots or leaves, while during the early phase of shoot regeneration, the accumulation of the ARF10 mRNA was lower in the wild type than in the mARF10 transgenics, in contrast to the pattern of miR160 expression. Thus, miR160 and ARF10 both appear to be components of the regulation of shoot regeneration in vitro.

MYB transcription factors are important regulators of the plant response to abiotic stress. Their participation in the salinity stress of the key forage legume species alfalfa (Medicago sativa) was investigated here by comparing the transcriptomes of the two cultivars Dryland (DL) and Sundory (SD), which differed with respect to their ability to tolerate salinity stress. When challenged by the stress, DL plants were better able than SD ones to scavenge reactive oxygen species. A large number of genes encoding transcription regulators, signal transducers and proteins involved in both primary and secondary metabolism were differentially transcribed in the two cultivars, especially when plants were subjected to salinity stress. The set of induced genes included 17 MYB family of transcription factors, all of which were subsequently isolated. The effect of constitutively expressing these genes on the salinity tolerance expressed by Arabidopsis thaliana was investigated. The introduction of MsMYB4 significantly increased the plants’ salinity tolerance in an abscisic acid-dependent manner. A sub-cellular localization experiment and a transactivation assay indicated that MsMYB4 was deposited in the nucleus and was able to activate transcription in yeast. Based on this information, we propose that the MsMYB4 products is likely directly involved in alfalfa’s response to salinity stress.

Cotton is an important economic crop affected by different abiotic stresses at different developmental stages. Salinity limits the growth and productivity of crops worldwide. Na+/H+ antiporters play a key role during the plant development and in its tolerance to salt stress. The aim of the present study was a genome-wide characterization and expression pattern analysis under the salinity stress of the sodium-proton antiporter (NHX) of Gossypium barbadense in comparison with Gossypium hirsutum. In G. barbadense, 25 NHX genes were identified on the basis of the Na+_H+ exchanger domain. All except one of the G. barbadense NHX transporters have an Amiloride motif that is a known inhibitor of Na+ ions in plants. A phylogenetic analysis inferred three classes of GbNHX genes—viz., Vac (GbNHX1, 2 and 4), Endo (GbNHX6), and PM (GbNHX7). A high number of the stress-related cis-acting elements observed in promoters show their role in tolerance against abiotic stresses. The Ka/Ks values show that the majority of GbNHX genes are subjected to strong purifying selection under the course of evolution. To study the functional divergence of G. barbadense NHX transporters, the real-time gene expression was analyzed under salt stress in the root, stem, and leaf tissues. In G. barbadense, the expression was higher in the stem, while in G. hirsutum the leaf and root showed a high expression. Moreover, our results revealed that NHX2 homologues in both species have a high expression under salinity stress at higher time intervals, followed by NHX7. The protein-protein prediction study revealed that GbNHX7 is involved in the CBL-CIPK protein interaction pathway. Our study also provided valuable information explaining the molecular mechanism of Na+ transport for the further functional study of Gossypium NHX genes.