SUMMARY Propagation through stem cuttings is a popular method worldwide for species such as fruit tree rootstocks and forest trees. Adventitious root (AR) formation from stem cuttings is crucial for effective and successful clonal propagation of apple rootstocks. Strigolactones (SLs) are newly identified hormones involved in AR formation. However, the regulatory mechanisms underpinning this process remain elusive. In the present study, weighted gene co‐expression network analysis, as well as rooting assays using stable transgenic apple materials, revealed that MdBRC1 served as a key gene in the inhibition of AR formation by SLs. We have demonstrated that MdSMXL7 and MdWRKY6 synergistically regulated MdBRC1 expression, depending on the interactions of MdSMXL7 and MdWRKY6 at the protein level downstream of SLs as well as the direct promoter binding on MdBRC1 by MdWRKY6. Furthermore, biochemical studies and genetic analysis revealed that MdBRC1 inhibited AR formation by triggering the expression of MdGH3.1 in a transcriptional activation pathway. Finally, the present study not only proposes a component, MdWRKY6, that enables MdSMXL7 to regulate MdBRC1 during the process of SL‐controlled AR formation in apple, but also provides prospective target genes to enhance AR formation capacity using CRISPR (i.e. clustered regularly interspaced short palindromic repeats) technology, particularly in woody plants.

No abstract

Genetic modifications of floral organs are important in the breeding of Malus species. Flower-specific promoters can be used to improve floral organs specifically, without affecting vegetative organs, and therefore developing such promoters is highly desirable. Here, we characterized two paralogs of the Arabidopsis thaliana gene AGAMOUS (AG) from Malus domestica (apple): MdAG1 and MdAG2. We then isolated the second-intron sequences for both genes, and created four artificial promoters by fusing each intron sequence to a minimal 35S promoter sequence in both the forward and reverse directions. When transferred into tobacco (Nicotiana benthamiana) by Agrobacterium tumefaciens-mediated stable transformation, one promoter, rMdAG2I, exhibited activity specifically in flowers, whereas the other three also showed detectable activity in vegetative organs. A test of the four promoters’ activities in the ornamental species Malus micromalus by Agrobacterium-mediated transient transformation showed that, as in tobacco, only rMdAG2I exhibited a flower-specific expression pattern. Through particle bombardment transformation, we demonstrated that rMdAG2I also had flower-specific activity in the apple cultivar ‘Golden Delicious’. The flower-specific promoter rMdAG2I, derived from M. domestica, thus has great potential for use in improving the floral characteristics of ornamental plants, especially the Malus species.

Apple is one of the most important fruit crops in temperate regions and largely relies on cutting propagation. Adventitious root formation is crucial for the success of cutting propagation. Strigolactones have been reported to function in rooting of woody plants. In this study, we determined that strigolactones have inhibitory effects on adventitious root formation in apple. Transcriptome analysis identified 12 051 differentially expressed genes over the course of adventitious root initiation, with functions related to organogenesis, cell wall biogenesis or plant development. Further analysis indicated that strigolactones might inhibit adventitious root formation through repressing two core hub genes, MdLAC3 and MdORE1. Combining small RNA and degradome sequencing, as well as dual‐luciferase sensor assays, we identified and validated three negatively correlated miRNA–mRNA pairs, including mdm‐miR397–MdLAC3 and mdm‐miR164a/b–MdORE1. Overexpression of mdm‐miR164b and silencing MdORE1 exhibited enhanced adventitious root formation in tobacco and apple, respectively. Finally, we verified the role of mdm‐miR164b–MdORE1 in strigolactone‐mediated repression of rooting ability. Overall, the identified comprehensive regulatory network in apple not only provides insight into strigolactone‐mediated adventitious root formation in other woody plants, but also points to a potential strategy for genetic improvement of rooting capacity in woody plants.

Adventitious root (AR) formation is important for the vegetative propagation. The effects of strigolactones (SLs) on AR formation have been rarely reported, especially in woody plants. In this study, we first verified the inhibitory effects of SLs on AR formation in apple materials. Transcriptome analysis identified 12,051 differentially expressed genes over the course of AR formation, with functions related to organogenesis, cell wall biogenesis or plant senescence. WGCNA suggests SLs might inhibit AR formation through repressing the expression of two core hub genes, MdLAC3 and MdORE1. We further verified that enhanced cell wall formation and accelerated senescence were involved in the AR inhibition caused by SLs. Combining small RNA and degradome sequencing, as well as a dual-luciferase sensor system, we identified and validated three negatively correlated miRNA–mRNA pairs, including mdm-miR397–MdLAC3 involved in secondary cell wall formation, and mdm-miR164a/b–MdORE1 involved in senescence. Finally, we have experimentally demonstrated the role of mdm-miR164b–MdORE1 in SLs-mediated inhibition of AR formation. Overall, our findings not only propose a comprehensive regulatory network for the function of SLs on AR formation, but also provide novel candidate genes for the potential genetic improvement of AR formation in woody plants using transgenic or CRISPR technology.

CRISPR/Cas9-mediated editing of MdSPL6 can be a feasible way to improve adventitious shoot regeneration in apple and other Rosaceous fruit trees.