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Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar 'Fuji', which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis , indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin. [ABSTRACT FROM AUTHOR]

Shoot branching is regulated by phytohormones, including cytokinin (CK), strigolactone (SL), and auxin in axillary buds. The correlative importance of these phytohormones in the outgrowth of apple axillary buds remains unclear. In this study, the outgrowth dynamics of axillary buds of a more-branching mutant (MB) and its wild-type (WT) of Malus spectabilis were assessed using exogenous chemical treatments, transcriptome analysis, paraffin section, and reverse transcription-quantitative PCR analysis (RT-qPCR). High contents of CK and abscisic acid coincided in MB axillary buds. Exogenous CK promoted axillary bud outgrowth in the WT but not in MB, whereas exogenous gibberellic had no significant effect on bud outgrowth in the WT. Functional analysis of transcriptome data and RT-qPCR analysis of gene transcripts revealed that MB branching were associated with CK signaling, auxin transport, and SL signaling. Transcription of the SL-related genes MsMAX1, MsD14 , and MsMAX2 in the axillary buds of MB was generally upregulated during bud outgrowth, whereas MsBRC1 / 2 were generally downregulated both in WT and MB. Exogenous SL inhibited outgrowth of axillary buds in the WT and the apple varieties T337, M26, and Nagafu 2, whereas axillary buds of the MB were insensitive to SL treatment. Treatment with N -1-naphthylphalamic acid (NPA; an auxin transport inhibitor) inhibited bud outgrowth in plants of the WT and MB. The transcript abundance of MsPIN1 was generally decreased in response to NPA and SL treatments, and increased in CK and decapitation treatments, whereas no consistent pattern was observed for MsD14 and MsMAX2. Collectively, the present results suggest that in apple auxin transport from the axillary bud to the stem may be essential for the outgrowth of axillary buds, and at least, is involved in the process of bud outgrowth. [ABSTRACT FROM AUTHOR]

Cytokinins (CKs) play a crucial role in promoting axillary bud outgrowth and targeting the control of CK metabolism can be used to enhance branching in plants. CK levels are maintained mainly by CK biosynthesis (isopentenyl transferase, IPT) and degradation (dehydrogenase, CKX) genes in plants. A systematic study of the IPT and CKX gene families in apple, however, has not been conducted. In the present study, 12 MdIPTs and 12 MdCKXs were identified in the apple genome. Systematic phylogenetic, structural, and synteny analyses were performed. Expression analysis of these genes in different tissues was also assessed. MdIPT and MdCKX genes exhibit distinct expression patterns in different tissues. The response of MdIPT , MdCKX , and MdPIN1 genes to various treatments (6-BA, decapitation and Lovastatin, an inhibitor of CKs synthesis) that impact branching were also investigated. Results indicated that most of the MdIPT and MdCKX , and MdPIN1 genes were upregulated by 6-BA and decapitation treatment, but inhibited by Lovastatin, a compound that effectively suppresses axillary bud outgrowth induced by decapitation. These findings suggest that cytokinin biosynthesis is required for the activation of bud break and the export of auxin from buds in apple tree with intact primary shoot apex or decapitated apple tree. MdCKX8 and MdCKX10 , however, exhibited little response to decapitation, but were significantly up-regulated by 6-BA and Lovastatin, a finding that warrants further investigation in order to understand their function in bud-outgrowth. [ABSTRACT FROM AUTHOR]

• More-branching phenotype was associated with cytokinin content and signaling. • ABA and GA contents were not correlated with axillary bud outgrowth based on the patterns of gene expression. • Expression changes of auxin signaling genes might be complex in apple branching. Branch number is an important trait in grafted apple breeding and cultivation. To provide new information on molecular mechanisms of apple branching, whole reduced-representation genomes and transcriptome of a wild-type (WT) apple (Malus spectabilis) and its more-branching (MB) mutant at the branching stage were examined in this study. Comparison of WT and MB genomes against the Malus domestica reference genome identified 14,908,939 single nucleotide polymorphisms (SNPs) and 173,315 insertions and deletions (InDels) in WT and 1,483,221 SNPs and 1,725,977 InDels in MB. Analysis of the genetic variation between MB and WT revealed 1,048,575 SNPs and 37,327 InDels. Among them, 24,303 SNPs and 891 InDels mapped to coding regions of 5,072 and 596 genes, respectively. GO and KEGG functional annotation of 3,846 and 944 genes, respectively, identified 32 variant genes related to plant hormone signal transduction that were involved in auxin, cytokinin, gibberellin, abscisic acid, ethylene, and brassinosteroid pathways. The transcriptome pathways of plant hormone signal transduction and zeatin biosynthesis were also significantly enriched during MB branching. Furthermore, transcriptome data suggested the regulatory roles of auxin signaling, increase of cytokinin and genes of cytokinin synthesis and signaling, and the suppressed abscisic acid signaling. Our findings suggest that branching development in apple is regulated by plant hormone signal transduction. [ABSTRACT FROM AUTHOR]