Functional Studies Of Auxin Biosynthesis In Fruit Development And LAM In Runner Formation In Strawberry

Cultivated strawberry（Fragaria x ananassa）is octoploid,which is popular among consumers because of its fragrant,delicious and nutritious fruit.The fleshy and juicy fruit of strawberry is an accessory fruit,which enlarges in response to auxin accumulation after fertilization.The botanical fruit of strawberry is the dry achene that dots the surface of the receptacle.Clonal propagation through runner is the main propagation method for cultivated strawberries during production.So,receptacle and runner are important organs in strawberry.The critical role of auxin in strawberry fruit set and receptacle enlargement was demonstrated previously.Although fertilization is known to trigger auxin biosynthesis,the specific tissue source of fertilization-induced auxin is not well understood.Moreover,the genetic control of runnering is largely unknown.In order to better utilize the advantages of runners in production,it is necessary to study the formation mechanism of runners.Our study analyzed the spatial and temporal biosynthesis of auxin required for receptacle enlargement and the genetic control of runner formation in diploid woodland strawberry.The main results are shown as followes:Here,the auxin reporter DR5_ver2::GUS was introduced into wild strawberry（F.vesca）to reveal auxin distribution in the seed and fruit receptacle pre-and post-fertilization as well as in the root.The accumulation of auxin in seeds and receptacles before fertilization is undetectable,while auxin accumulation occurred mainly in the chalazal seed coat,embryo,vasculature strands and the surfaces where achenes dot in receptacle after fertilization.In addition,Tryptophan Aminotransferase of Arabidopsis（TAA）and YUCCA genes code for enzymes catalysing the two-step auxin biosynthesis pathway.The transcript level of each of the Fve TAAs and Fve YUCs was analyzed by mining the comprehensive RNA-seq data representing 41 different tissues,mostly floral organs and fruit tissues of F.vesca.Different members of Fve TAAs and Fve YUCs exhibit distinct expression patterns.We generated transgenic F.vesca plants containing the promoter::GUS reporters of two Fve TAR and four Fve YUC genes as they are likely more relevant to auxin-mediated fruit set due to their abundant and specific expression in the seed.Fve YUC4 was shown to be expressed primarily in the embryo inside the achenes after fertilization.Fve TAr1,Fve YUC5,Fve YUC10,and Fve YUC11 were expressed mainly in the endosperm.The two Fve TAAs and four Fve YUCs were specifically expressed in root tips and lateral root primordia.Moreover,we generated knock-out mutants of Fve YUC10,the most abundant YUC in seeds,by CRISPR/Cas9.The fveyuc10 mutants had a lower free auxin level in young fruit,whereas displayed no obvious morphological phenotypes.Overall,the study revealed auxin accumulation in the chalazal seed coat,embryo,receptacle vasculature,root tip,and lateral root primordia and highlighted the endosperm as the main auxin biosynthesis site for receptacle enlargement in strawberry.An EMS mutant that had no stamens in flowers in diploid strawberry F.vesca,loss of axillary meristems（lam）,was found in our lab previously.The causative mutation of lam is located in Fv H4＿3g41310/gene01184,encoding the homologous gene of MOC1 in rice,LAS in Arabidopsis,LS in tomato.LAM is belonging to GRAS family.The function of homologous genes of LAM suggested that LAM maybe influence the runner formation in strawberry.As expected,the branch crowns and runners of lam were decreased as a resulte of the absence of axillary buds.Expression of LAM in the lam mutant could rescue the growth of runners.Consistently,the lam^CR mutants generated by CRISPR/Cas9 produced and fewer runners.LAM is broadly expressed in the meristematic tissues,young leaves,and axillary buds.Gibberellic acid（GA）could promote the development of runners,and GA-induced runnering requires the presence of LAM.Runners were inhibited by the GA biosysthesis inhibitor PBZ,which became branch crowns.Previous study reported that the srl mutant,owing to mutation of DELLA gene Fve RGA1,caused runner formation in the background of YW,a runnerless wild type.Different to srl,the srl lam plants failed to development runners,indicating that lam is epistatic to srl with respect to runnering.We found 125 proteins that could interact with LAM by screening yeast-two hybrid library.Fve RGA1,one of the interacting proteins and the causative gene of srl,was further confirmed to interact with LAM both in vitro and in vivo.Overall,our results demonstrate that LAM is necessary for runner formation by regulating axillary bud initiation,and Fve RGA1 influence the outgrowth of axillary bud subsequently.