Identification,Gene Cloning And Functional Analysis Of Strigolactones In Malus

Strigolactones are a class of new found phytohormone, playing important roles in plants’ development and stress defense. Moreover, as the environmental signals, strigolactones are necessary for the symbiosis of arbuscular mycorrhizal fungi and parasitism of root parasitic plants. Apple(Malus domestica) is a world-wide cultivated fruit crop, which its development and propagation are mostly by grafting. Most apple’s rootstocks belong to the same genus with cultivated apple, and have close genetic relationship. The rootstocks could influence apple’s development and stress defense. There are few reports about strigolactones in Malus. In this study, we determined the contents of strigolactones, explored the functions of strigolactones, and cloned and screened the genes related to strigolactones in Malus. In conclusion, we discovered several significant results shown below:1. The Malus spp. have strigolactones, but their contents vary among different species.The germination bioassay of Orobanche seeds were used for test of strigolactones’ contents in Malus. We chose a total number of 24 strains/cultivars from 9 Malus species for assay. The seedlings were divided into leaf, xylem, phloem and roots. All tested samples could induce the seeds’ germination, indicating that all these species had strigolactones. M. baccata and M. xiaojinensis induced higher germination rates and likely had higher strigolactones’ contents compared with other species. Among four kinds of tissue extracts, those from xylem induced highest seed germination rates, while those from phloem induced lowest germination rates. It indicated that the strigolactones were mainly distributed in xylem.2. Application of exogenous strigolactones(GR24) influenced a series of physiological processes of M. hupehensis and M26(M. pumila).Axillary buds’ growth: GR24 could directly repress the axillary buds’ growth after apical buds’ removal in M. hupehensis. Roots’ development: GR24 could increase the length of primary root and number of root’s hair, and decrease the number of axillary roots. Seed germination: 30 g·L-1 sucrose repressed the seed germination of M. hupehensis and GR24 could improve the seed germination rates, while GR24 had no effect on seed germination under non-sucrose condition. Leaf senescence: when leaves of M. hupehensis developed in the late stage or suffered from pathogen infection, GR24 accelerated their senescence to death. Seedlings in vitro: GR24 increased the height, fresh weight and chlorophyll contents of M26 seedlings in vitro, but decreased the leaves’ stomatal density. Symbiosis of AM fungi: strigolactones could improve the symbiosis rates between M. hupehensis and AM fungi(Glomus mosseae) under drought stress.3. Cloning and analysis of genes related to strigolactones in M. hupehensis.Here we cloned four strigolactones’ synthesis genes and two strigolactones’ signalling genes in M. hupehensis. The four synthesis genes are carotenoid isomerase D27, carotenoid cleavage dioxygenase CCD7, carotenoid cleavage dioxygenases CCD8 and cytochrome P450 monooxygenases MAX1; two signalling genes are F-box family E3 ubiquitin ligases MAX2 and α/β hydrolases D14. Through gene cloning and genomic alignment, we determined that CCD8, MAX2 and D14 had at least two paralogs, and MAX1 had at least three. Tissue-specific expressions presented that CCD7 and CCD8 a were mainly expressed in root, while MAX2 s were expressed ubiquitously. The promoters of CCD7, CCD8 and MAX2 were cloned and many stress-responsive cis-acting elements were in these promoters. The expression level of CCD7 and CCD8 a increased under low phosphorus, but decreased significantly under drought, waterlogging and MeJA treatments. The light oxidative stress induced by methyl viologen decreased MAX2b’s expressions in leaves but increased its expressions in roots.4. Screening of new possible genes and proteins related to strigolactones.Using yeast one-hybrid technology, we determined the transcriptional factor BPC1 could bind to CCD7’s promoter and transient overexpression of BPC1 could decrease the activity of CCD7’s promoter. The transcriptional factor WRKY75 could bind to CCD8’s promoter, which may involve in phosphorus response. Through yeast two-hybrid and bimolecular fluorescence complementation technology, we determined that MAX2 b could interact with CYP38 protein, and CYP38 interacted with some proteins located in plastids. Exogenous GR24 treatments increased the expressions of many plastid proteins.In conclusion, we confirmed that Malus spp. expressed the strigolactones’ synthesis and signalling genes and synthesized the strigolactones. Strigolactones regulated axillary buds and roots’ development, seed germination, stomatal density, leaf senescence and symbiosis of AM fungi in Malus. The expressions of strigolactones’ synthesis genes were repressed by drought, waterlogging and MeJA and improved by low phosphorus. Strigolactones likely regulate some proteins of plastids in root. Our research provided valuable information for future strigolactones’ study in Malus, especially the interaction between strigolactones and stresses.