Salinity and drought are two major abiotic stresses that limit grape productivity. Responses to stress in grape are known to be regulated by several families of transcription factors. However, little is known about the role of grape Squamosa promoter binding protein (SBP)-box transcription factor genes in response to abiotic stress. To better understand the functions of the grape SBP-box genes in abiotic stress tolerance, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP16 was amplified from Chinese wild grapevine Vitis pseudoreticulata clone “Baihe-35-1”. We observed that the VpSBP16 protein fused to the green fluorescent protein (GFP) reporter accumulated in the nucleus when transiently expressed in onion epidermal cells. Moreover, VpSBP16 was shown to have transcriptional activation activity using a yeast trans-activation assay. We performed a VpSBP16 functional analysis through the characterization of transgenic Arabidopsis thaliana plants constitutively over-expressing VpSBP16. The transgenic lines had longer roots and the seeds had a higher germination rate than the wild type (WT) under osmotic stress. In addition, the accumulation of reactive oxygen species (ROS) of transgenic seedlings was significantly lower than WT in the transgenic lines, as was electrolyte leakage. VpSBP16 overexpression also elevated expression levels of stress-response genes involved in the salt overly sensitive (SOS) pathway. These results indicate that overexpression VpSBP16 in A. thaliana enhances tolerance of salt and drought stress during seed germination, as well in seedlings and mature plants, by regulating SOS and ROS signaling cascades.

The nuclear factor Y (NF-Y) as a transcription factor plays an important role in plants growth and development, and response to stress. However, few genome-wide analyzes and functional research of the NF-Y family has been undertaken in apple (Malus domestica Borkh.) so far. In this study, we comprehensively identified the 43 MdNF-Y genes in apple, which dispersedly distributed among the three subgroups based on their sequence alignment analysis, including 11 MdNF-YAs, 22 MdNF-YBs and 10 MdNF-YCs. The members in the same subgroups had similar evolution relationships, gene structures, and conserved motifs. The gene duplication analysis suggested that all the genes were dispersed followed by 27 segmental duplication. Moreover, based on synteny analysis of MdNF-Ys with eight plant species results suggested that some ortholog genes were preserved during the evolution of these species. Cis-element analysis showed potential functions of MdNF-Ys in apple growth and development and responded to abiotic stress. Furthermore, the interaction among MdNF-Ys protein were investigated in yeast two-hybrid assays. The expression patterns of MdNF-Ys in tissue-specific response reveled divergence and might play important role in apple growth and development. Subsequently, whole MdNF-Y genes family was carried out for RT-PCR in response to five abiotic stress (ABA, drought, heat, cold, and salinity) to identify their expression patterns. Taken together, our study will provide a foundation for the further study to the molecular mechanism of apple in growing development and response to abiotic stresses.

Drought is the major abiotic stress that limits apple productivity and quality. To date, many important and divergent regulatory functions of miR156/SBP genes in plant growth and development have been well understood. However, little is known about the role of apple miR156 in response to abiotic stress. To better understand the functions of MdmiR156 in abiotic stress tolerance, we constructed the overexpression (OE) and short tandem target mimic (STTM) vector of MdmiR156n and performed its functional analysis through the characterization of transgenic apple calli and Arabidopsis thaliana plants. In this study, MdmiR156n overexpression significantly increased the length of primary roots and the number of lateral roots in transgenic Arabidopsis plants under drought stress. In addition, MdmiR156n transgenic Arabidopsis and apple calli had a lower electrolyte leakage rate and less cell membrane damage than WT and STTM156 after drought stress. Further studies showed that MdmiR156n overexpression promoted the accumulation of flavonoids and scavenging of reactive oxygen species (ROS) under drought conditions in transgenic apple calli and A. thaliana plants. Taken together, overexpression MdmiR156n enhances drought tolerance by regulating flavonoid synthesis and ROS signaling cascades in apple calli and A. thaliana.

Flowering occurs in angiosperms during a major developmental transition from vegetative growth to the reproductive phase. Squamosa promoter binding protein (SBP)-box genes have been found to play critical roles in regulating flower and fruit development, but their roles in grapevine have remained unclear. To better understand the functions of the grape SBP-box genes in both vegetative and reproductive growth phases, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP11, was obtained from Chinese wild grapevine Vitis pseudoreticulata Wen Tsai Wang (W. T. Wang) clone ‘Baihe-35-1’. VpSBP11 encoded a putative polypeptide of 170 amino acids with a highly conserved SBP-domain with two zinc-binding sites of the Cx2C-x3-H-x11-C-x6-H (C2HCH) type and a nuclear localization signal. We confirmed that the VpSBP11 protein was targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans-activation assay. Over-expression of VpSBP11 in Arabidopsis thaliana was shown to activate the FUL gene, and subsequently the AP1 and LFY genes, all of which were floral meristem identity genes, and to cause earlier flowering than in wild type (WT) plants. The pattern of vegetative growth was also different between the transgenic and WT plants. For example, in the VpSBP11 over-expressing transgenic plants, the number of rosette leaves was less than that of WT; the petiole was significantly elongated; and the rosette and cauline leaves curled upwards or downwards. These results were consistent with VpSBP11 acting as a transcription factor during the transition from the vegetative stage to the reproductive stage.

The pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) (known as PYLs for short) have been identified and characterized as the ABA receptors in some plants. However, little is known about the details regarding PYL family genes in the apple (Malusdomestica). In this study, we identified 13 apple PYLs, termed MdPYL1-13, which could be classified into four groups according to structural features of the amino acid sequence. The gene structures and conserved motifs analysis found that the majority of MdPYLs had a similar number of exons and similar conserved motif profile in the same group. In addition, 11 gene pairs were identified to exhibit synteny by synteny analysis between the apple and Arabidopsis. Furthermore, we investigated MdPYLs transcript level in various organs of the red-fleshed apple (Malussieversii f. Neidzwetzkyana (Dieck) Langenf) ‘Xinjiang No.1’. The results suggested all MdPYLs within group I were expressed at relatively higher levels in all of the organs tested. However, the genes of group IV had little or no variation. Additionally, we found various hormone and stress-related cis-elements in the promoters of MdPYLs by analyzing cis-elements. Therefore, the expression levels of all MdPYLs were further detected under ABA, PEG, salt, and cold stresses in ‘Xinjiang No.1’ seedlings. We found that all MdPYLs except for MdPYL11 were upregulated by ABA treatment, 10 genes were upregulated by PEG treatment, 12 genes were upregulated by NaCl treatment, and six genes were upregulated by cold treatment (4 °C) while seven genes were downregulated. Thus, these MdPYLs might be involved in the defense against abiotic stresses. In addition, the interaction between 13 MdPYLs and two 2C protein phosphatases in the apple (MdPP2C65 and MdPP2C72) was investigated in yeast two-hybrid assays. These results suggested that MdPYLs may bind to MdPP2C65 and MdPP2C72 in different manners and with different intensity. Our studies provide useful information for further investigating and researching the regulatory mechanisms of PYL family genes in response to abiotic stresses in the apple.