Background: Peanut (Arachis hypogaea L.), an important oilseed and food legume, is widely cultivated in the semi-arid tropics. Drought is the major stress in this region which limits productivity. Microbial communities in the rhizosphere are of special importance to stress tolerance. However, relatively little is known about the relationship between drought and microbial communities in peanuts. Method: In this study, deep sequencing of the V3-V4 region of the 16S rRNA gene was performed to characterize the microbial community structure of drought-treated and untreated peanuts. Results: Taxonomic analysis showed that Actinobacteria, Proteobacteria, Saccharibacteria, Chloroflexi, Acidobacteria and Cyanobacteria were the dominant phyla in the peanut rhizosphere. Comparisons of microbial community structure of peanuts revealed that the relative abundance of Actinobacteria and Acidobacteria dramatically increased in the seedling and podding stages in drought-treated soil, while that of Cyanobacteria and Gemmatimonadetes increased in the flowering stage in drought-treated rhizospheres. Metagenomic profiling indicated that sequences related to metabolism, signaling transduction, defense mechanism and basic vital activity were enriched in the drought-treated rhizosphere, which may have implications for plant survival and drought tolerance. Conclusion: This microbial communities study will form the foundation for future improvement of drought tolerance of peanuts via modification of the soil microbes.

Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na⁺ levels. Salt-responsive genes, including Na⁺/H⁺ exchangers (NHX1) and high-affinity K⁺ transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na⁺. Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.

Pyrabactin resistance 1 (PYR1)/PYR1-like (PYL) abscisic acid (ABA) receptors have been proved to be recruited in the plasma membrane (PM). In order to explain the roles of PYR/PYLs in the PM, PYL4 was used as bait to screen the PM-localized leucine-rich repeat receptor-like kinase family, and five members were found directly interacting with PYL4. Loss of function by T-DNA insertion in C-terminally encoded peptide receptor 2 (CEPR2) together with phloem intercalated with xylem (PXY) and PXY-Like 2 (PXL2) led to ABA hypersensitivity, while CEPR2 overexpression led to ABA insensitivity compared with the wild type, indicating the redundant and negative roles of CEPR2, PXY, and PXL2 in ABA signaling. The PYL4 proteins were strongly accumulated in cepr2/pxy/pxl2 compared with the wild type. Furthermore, higher phosphorylation levels accompanied by lower protein levels of PYL4 in CEPR2 overexpression lines were observed, indicating the requirement of phosphorylation of PYLs for degradation. Subsequently, MS and in vitro kinase assays demonstrated that CEPR2 phosphorylated PYL4 at Ser54, while this phosphorylation was diminished or even eliminated in the presence of ABA. Taken together, CEPR2 promotes the phosphorylation and degradation of PYLs in unstressed conditions, whereas ABA represses this process to initiate ABA response during times of stress.

The cysteine-rich transmembrane module (CYSTM) is comprised of a small molecular protein family that is found in a diversity of tail-anchored membrane proteins across eukaryotes. This protein family belongs to a novel uncharacteristic non-secreted cysteine-rich peptides (NCRPs) according to their conserved domain and small molecular weight, and genome-wide analysis of this family has not yet been undertaken in plants. In this study, thirteen CYSTM genes were identified and located on 5 chromosomes with diverse densities in Arabidopsis thaliana. The CYSTM proteins could be classified into 4 subgroups based on domain similarity and phylogenetic topology. Encouragingly, the CYSTM members were expressed in at least one of the tested tissues and dramatically responded to various abiotic stresses, indicating that they played vital roles in diverse developmental processes, especially in stress responses. CYSTM peptides displayed a complex subcellular localization, and most were detected at the plasma membrane and cytoplasm. Of particular interest, CYSTM members could dimerize with themselves or others through the C-terminal domain, and we built a protein-protein interaction map between CYSTM members in Arabidopsis for the first time. In addition, an analysis of CYSTM3 overexpression lines revealed negative regulation for this gene in salt stress responses. We demonstrate that the CYSTM family, as a novel and ubiquitous non-secreted cysteine-rich peptide family, plays a vital role in resistance to abiotic stress. Collectively, our comprehensive analysis of CYSTM members will facilitate future functional studies of the small peptides.