Comprehensive functional analysis of the PYL-PP2C-SnRK2s family in Bletilla striata reveals that BsPP2C22 and BsPP2C38 interact with BsPYLs and BsSnRK2s in response to multiple abiotic stresses

Abstract

As the core regulation network for the abscisic acid (ABA) signaling pathway, the PYL-PP2C-SnRK2s family commonly exists in many species. For this study, a total of 9 BsPYLs, 66 BsPP2Cs, and 7 BsSnRK2s genes were identified based on the genomic databases of Bletilla striata, which were classified into 3, 10, and 3 subgroups, respectively. Basic bioinformatics analysis completed, including the physicochemical properties of proteins, gene structures, protein motifs and conserved domains. Multiple cis-acting elements related to stress responses and plant growth were found in promoter regions. Further, 73 genes were localized on 16 pseudochromosomes and 29 pairs of paralogous genes were found via intraspecific collinearity analysis. Furthermore, tissue-specific expression was found in different tissues and germination stages. There were two BsPYLs, 10 BsPP2Cs, and four BsSnRK2 genes that exhibited a difference in response to multiple abiotic stresses. Moreover, subcellular localization analysis revealed six important proteins BsPP2C22, BsPP2C38, BsPP2C64, BsPYL2, BsPYL8, and BsSnRK2.4 which were localized in the nucleus and plasma membrane. Finally, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays suggested that BsPP2C22 and BsPP2C38 could interact with multiple BsPYLs and BsSnRK2s proteins. This study systematically reported on the identification and characterization of the PYL-PP2C-SnRK2s family in B. striata, which provided a conceptual basis for deep insights into the functionality of ABA core signal pathways in Orchidaceae.

Comprehensive Functional Analysis of the bZIP Family in Bletilla striata Reveals That BsbZIP13 Could Respond to Multiple Abiotic Stresses

“…Full viable dried pods were selected, and a small number of seeds were extracted for sterilization and then sowed into a solid MS medium with NAA (1.0 mg/L) and 6-BA (2.0 mg/L) and incubated at 30 • C, 55% relative humidity, and 16 h light/8 h dark conditions. The seeds were collected at 0 days (BS0), 5 days (BS1), 10 days (BS2), 20 days (BS3), 28 days (BS4), and 35 days (BS5) of germination for transcriptome sequencing to determine the expression patterns at different germination stages of B. striata [74].…”

“…For hormonal treatments, the surfaces of the aboveground portions of the seedlings were sprayed with 100 µM GA and ABA, respectively. For oxidative stress, the seedlings were sprayed with 100 µM CuSO 4 [74]. The wounding treatment involved an incision (1 cm) that was made along the center of a seedling leaf.…”

“…The recombinant pGADT7-BsSnRK2s vectors were developed based on pDONR207-BsSnRK2s (with the inclusion of the termination codon) using the Gateway system. Here, we used the recombinant vector constructed in the laboratory for the experiment [74]. Following the co-transformation of yeast strain AH109 with pGBKT7-BsbZIP13, yeast cells were cultured on a double-selective medium (SD/−Leu/−Trp) at 28 • C for 3 d. Positive clones were then sequentially screened with X-alpha-gal on SD/−Ade/−His/−Leu/−Trp medium to detect the interactions between BsbZIP13 and BsSnRK2s.…”

“…The primers are shown in Table S5. pEarleyGate201-YN-BsSnRK2s recombinant vectors constructed in the laboratory were used for the experiment [74]. YN-BsSnRK2s and YC-BsbZIP13 recombinant plasmids were transformed into Agrobacterium EHA105, and the onion epidermis pre-grown for one day was put into the Agrobacterium immersion solution containing recombinant plasmid vector and empty plasmid, dark for 16h, observed and photographed under a fluorescence microscope.…”

Genome-Wide Identification and Expression Patterns of the SWEET Gene Family in Bletilla striata and its Responses to Low Temperature and Oxidative Stress

“…It is well-known that metals such as Cu have the potential to generate •OH and induce oxidative stress [ 30 ]. BsPP2C , BsSnPK2.6, and BsPYL6 responded to various stresses, including low temperature and oxidative stress [ 31 ]. The PSII photosynthetic system of B. striata is disrupted by low temperature treatment, which further affects plant growth [ 32 ].…”