Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach to their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, the electrophoretic mobility shift assay, and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.

AINTEGUMENTA-LIKE (AIL) genes play a key role in various growth and developmental processes in plants. Thus far, the genome-wide identification of AIL genes has been reported for some plant species. However, genome-wide identification of AIL genes has not been conducted in apple (Malus domestica Borkh.). The current study focused on a comprehensive analysis of the AIL genes in the apple genome (i.e., MdAIL genes). In total, 27 MdAIL genes in the apple genome were identified and then divided into four groups according to phylogenetic analysis. The chromosomal locations, gene and protein structures, and physicochemical characteristics of MdAIL genes were analyzed. Synteny analysis revealed that segmental duplication events played a major role in the expansion of the AIL gene family in apple. The analysis of cis-regulatory elements in MdAIL promoter regions indicated that most of the MdAIL genes are involved in embryo development and seed germination. Moreover, the analysis of tissue-specific expression patterns and transcript levels in adventitious bud regeneration indicated that MdAIL genes play an extensive regulatory role in apple growth and development, especially in the regulation of germination and adventitious bud regeneration from in vitro leaves of apple. In conclusion, this is the first genome-wide analysis of the AIL genes in apple. The current results may help in better understanding the evolution and function of MdAIL genes and thus facilitate further research on plant growth and development.

Homeodomain-leucine zipper (HD-Zip) family genes are considered to play an important role in plant growth and stress tolerance. However, a genome-wide analysis of HD-Zip genes in apples (Malus domestica Borkh.) has not been performed. We detected 48 MdHDZ genes in the apple genome, and categorized them into three subfamilies on the basis of phylogenetic analysis. The chromosomal locations, gene/protein structures, and physiological and biochemical properties of these genes were analyzed. Synteny analysis revealed that segmental duplications were key in the expansion of the apple HD-Zip family. According to an analysis of cis-regulatory elements and tissue-specific expression patterns, MdHDZ genes may be widely involved in the regulation of apple growth and tolerance to environmental stresses. Furthermore, the transcript levels of apple HD-Zip I and II genes were up-regulated in response to fungal treatments. Expression of apple HD-Zip Ⅲ genes was enhanced during adventitious bud regeneration. This suggested possible roles of these genes in regulating the apple response to fungal infection, as well as adventitious bud regeneration. The current results may help us to better understand the evolution and function of apple HD-ZIP genes, and thus facilitate further research on plant resistance to fungal infection and in vitro regeneration.

Protein phosphatase 2Cs (PP2Cs) are members of the serine/threonine phosphatase family that play pivotal roles in regulating plant development and responses to environmental stresses. However, comprehensive genome-wide studies of the PP2C gene family in grape (Vitis vinifera L.) have not yet been conducted. In the present study, 78 VvPP2C genes were identified and classified into 12 clades based on their phylogenetic relationships. Analysis of physicochemical properties and gene/protein architectures revealed that the members within each clade shared conserved structural features. Synteny analysis demonstrated that both tandem and segmental duplications substantially contributed to the expansion of the VvPP2C gene family. Tissue-specific transcriptional profiles and cis-element analyses indicated the potential involvement of these genes in grape development and stress responses. Moreover, expression analysis identified VvPP2C26 and VvPP2C41 as the most abscisic acid (ABA)-responsive genes, with expression patterns highly correlated with grape berry development. Functional validation in transgenic tomato lines demonstrated that the overexpression of either gene markedly delayed fruit ripening. Collectively, this study provides new insights into the evolutionary diversification and regulatory functions of the PP2C gene family in grape and identifies VvPP2C26 and VvPP2C41 as key candidates for elucidating ABA-mediated ripening mechanisms in non-climacteric fruits.

Silique development exerts significant impacts on crop yield. CRPs (Cysteine-rich peptides) can mediate cell–cell communication during plant reproduction and development. However, the functional characterization and regulatory mechanisms of CRPs in silique development remain unclear. In this study, we identified many CRP genes downstream of the CRP gene TPD1 (TAPETUM DETERMINANT1) during silique development using a microarray assay. The novel Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) gene AtPCP-Ba, along with TPD1, are essential for silique development. The AtPCP-Ba was significantly down-regulated in tpd1 flower buds but up-regulated in OE-TPD1 flower buds and siliques. The silencing of AtPCP-Ba compromised the wider silique of OE-TPD1 plants and inhibited the morphology of OE-TPD1 siliques to the size observed in the wild type. A total of 258 CRPs were identified with the bioinformatic analysis in Arabidopsis, Brassica napus, Glycine max, Oryza sativa, Sorghum bicolor, and Zea mays. Based on the evolutionary tree classification, all CRP members can be categorized into five subgroups. Notably, 107 CRP genes were predicted to exhibit abundant expression in flowers and fruits. Most cysteine-rich peptides exhibited high expression levels in Arabidopsis and Brassica napus. These findings suggested the involvement of the CRP AtPCP-Ba in the TPD1 signaling pathway, thereby regulating silique development in Arabidopsis.

Soil salinity severely limits grapevine yield and fruit quality; however, the mechanisms underlying salt tolerance remain poorly understood. Small secreted peptides, particularly rapid alkalinization factor (RALF) peptides, are key regulators of plant responses to environmental stress. However, a systematic analysis of the RALF gene family in grapevine (Vitis vinifera L.) has not been conducted. In this study, we identified 11 VvRALFs in the grapevine genome (PN_T2T) and characterized their physicochemical properties, structural features, and evolutionary relationships. Expression profiling revealed that VvRALF9 was consistently upregulated in the roots of the salt-sensitive rootstock ‘Beta’ but downregulated in the salt-tolerant rootstock ‘3309C’. Silencing VvRALF9 in the roots of grapevine rootstocks significantly enhanced their salt tolerance. Protein interaction assays, including yeast two-hybrid, split-LUC, and pull-down assays, identified VvFER2 as a potential receptor of VvRALF9. Overexpression of VvFER2 in the roots of grapevine rootstocks also improved salt tolerance. Transcriptomic analysis of VvRALF9-RNAi roots revealed significant enrichment of the plant hormone signal transduction pathway, together with extensive changes in the expression of transcription factors and genes associated with multiple hormone signaling pathways. Together, these findings suggest that the VvRALF9–VvFER2 module is involved in the regulation of grapevine salt stress responses.