MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes.

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.

Genome-Wide Identification, Classification, and Expression Analysis of the HD-Zip Transcription Factor Family in Apple (Malus domestica Borkh.).

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.

Insertion of a TRIM-like sequence in MdFLS2-1 promoter is associated with its allele-specific expression in response to Alternaria alternata in apple.

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the major fungal diseases in apple. Early field observations revealed, the anther-derived homozygote Hanfu line (HFTH1) was highly susceptible to AAAP, whereas Hanfu (HF) exhibited resistance to AAAP. To understand the molecular mechanisms underlying the difference in sensitivity of HF and HFTH1 to AAAP, we performed allele-specific expression (ASE) analysis and comparative transcriptomic analysis before and after AAAP inoculation. We reported an important immune gene, namely, MdFLS2, which displayed strong ASE in HF with much lower expression levels of HFTH1-derived alleles. Transient overexpression of the dominant allele of MdFLS2-1 from HF in GL-3 apple leaves could enhance resistance to AAAP and induce expression of genes related to salicylic acid pathway. In addition, MdFLS2-1 was identified with an insertion of an 85-bp terminal-repeat retrotransposon in miniature (TRIM) element-like sequence in the upstream region of the nonreference allele. In contrast, only one terminal direct repeat (TDR) from TRIM-like sequence was present in the upstream region of the HFTH1-derived allele MdFLS2-2. Furthermore, the results of luciferase and ß-glucuronidase reporter assays demonstrated that the intact TRIM-like sequence has enhancer activity. This suggested that insertion of the TRIM-like sequence regulates the expression level of the allele of MdFLS2, in turn, affecting the sensitivity of HF and HFTH1 to AAAP.