Molecular Basis Of Rooting Recalcitrance In 'Chistock 1'(Malus Xiaojinensis) Leafy Cuttings

Dwarf and close planting, and intensive mangement is the prospective developing direction of apple production,while dwarf rootstocks are considered as important materials for this aim. Layering is the traditional method used in practice during the propogation of dwarf self-rooted rootstocks.While in compared with layering, several advanced characters are incolved in leafy cutting method,such as high efficiency, short cultivation time and lowcost. However, the lose of juvenility elicits adventitious root recalcitrance in apple rootstocks. Hitherto, one solution to meet the productive demand is to obtain rejuvenated rootstocks and the mature Malus xiaojinensis and M26 rootsocks had already been successfully rejuvenated by series in vitro sublture in previous studies, which could grow roots 85.40% and 81.82%, respectively, after leafy cuttings. Unfortunately, the molecular mechanism of mature cuttings losing adventitious rooting ability is poorly understood.Previous studies indicated that auxin and juvenility are both necessary and important factors for induction of adventitious roots. In the study, juvenile cuttings (Mx-J) and adult cuttings (Mx-A) of Malus xiaojinensis were used as materials and the primary qRT-PCR result showed that the expression level of miR156 was significantly higher in Mx-J than that in Mx-A, and furthermore through the assays of analyzation in 35S:MIR156a6 and 35S:MIM156 transgenic tobacco lines high expression level of miR156 and exogenous auxin application are both necessary factors for the formation of adventitious roots. Based on these foundations, the expression profile of auxin related ARF and PIN genes was analyzed in Mx-A, Mx-J, 35S:MIR156a6 and 35S:MIM156 plant lines during the inducing process of adventitious roots, it demonstrated miR156 regulates the formation of adventitious root independed in the PIN and ARF pathway. However, RTCS (rootless concerning crown and seminal roots)-like gene expressing level was highly upregulated in Mx-J and 35S:MIR156a6 and we speculate that the high induction of RTCS genes may closely related to the induction of adventitious roots.In addition, there are 13 putative miR156-targeted SPL genes in apple genome. Overexpressing the miR 156-resistant MxrSPL genes in tobacco confirmed the involvement of MxSPL20,MxSPL21&22, and MxSPL26 in adventitious root formation. Futher research indicated MxSPL26 could be a key SPL family member involved in adventitious rooting of leafy cuttings. The NbRTCS expression was significantly induced in 35S:MdMIR156a6 transgenic tobacco plants, but no substantial changes were detected in 35S:MIM156 and 35S:rMdSPL26/35S:MdMIR156a6 transfomants, indicating miR156/SPL26 modules was necessary for auxin-induced adventitious root formation via RTCS expression.Furthermore, RNA-seq was conducted by using the samples from the stem barks of Mx-A and Mx-J, respectively, at different time points (0 h, 6 h, 12 h, 24 h, and 48 h) with and without IB A treatments. The data showed the relative expression level of HB13 (homeobox protein 13) and NPRs were significantly down-regulated in Mx-A plants in 6 h -12 h and 12 h -24 h after IB A treatment,rescepitvely. Similarly, the expression level of HB2 (Homeobox protein 2), DOF5.4 (Dof zinc finger protein), NAC78 (NAC domain-containing protein) and lin-54 (Protein lin-54 homolog 4) were significantly down-regulated after 24 h - 48 h after IBA treatment, and then the expression level of auxin transporter genes, auxin induced protein gene, AUX/IAA and also the genes for polygalacturonase, pectate lyase, kinesin-like protein, elongation factor, tubulin and expansin protein,were significantly declined after IBA treatment in Mx-A leafy cuttings. These results indicated adult leafy cuttings actively defended itself against exogenous auxin leading to adventitious rooting recalcitrant. What's more,NAC29 and MYC2,SBP5 and ERF003 and WRKY75, MADS - box,and WRKY29 and C3H67 were significantly upregulated in Mx-J plants in 6 h, 12 h, 24 h, and 48 h after IBA treatment, respectively. In addition, it was found that glutamate decarboxylase (GAD) gene which is the rate-limiting enzyme of GABA biosynthesis, was significantly up-regulated in Mx-A cuttings after IBA treatment. And the application of exgeneous 4 mM GABA could significantly reduced the formation of adventitious roots in apple rootstock. Thus, the upregulation of GAD may lead to adventitious rooting recalcitrant in Malus xiaojinensis.