Preliminary Study On The Mechanism Of Tillering Regulated By Phytochrome Genes In Rice

Rice is one of the main food crops that nearly half of the world’s population lives on.Rice tillering is one of the important agronomic traits.It is not only one of the key factors closely related to rice yield,but also is an ideal model for studying shoot branching growth and development of mono-cotyledons.Formation and elongation of shoot branch is influenced not only by environmental factors(such as nutrients,light and water),but also by endogenous factors(MOC1/TB1/IPA1)and kinds of plant hormones.Cytokinins,as a positive regulator in tiller growth and development,transports from root through xylem to promote tillering.Strigolactones inhibit tiller growth through SLs synthesis or signal transduction pathway.Both hormones can be used as second messengers to co-regulate the formation of shoot branching.As an important environmental factor,light regulates plant growth and development.Light not only provides energy and substances for plant growth,but also regulates light morphogenesis as a signal molecule.Plants receive light signals mainly through photo receptors such as phytochrome.In rice plants,there are three phytochrome family genes like OsPHYA/OsPHYB/OsPHYC.Phytochrome plays a key role to modulate rice growth and development,including seedling de-yellowing,internode elongation,fertility,flower formation and circadian rhythm biological clock under short-and long-day conditions.However,few have been reported on the agronomic trait of rice tillering affected by photosensitive pigments.It is obscure whether and how phytochrome plays a role in regulating tillering growth.Doplant hormones such as cytokinins and strigolactones participate in phytochrome-regulating rice tiller growth?Above mentioned are the main contents of this paper.We focus on rice tillering regulated by three phytochrome genes.First,bioinformation was analyzed and relative epression during plant growth duration was conducted by qRT-PCR;consequently,osphyA/osphyAB/osphyABC mutants were constructed by Cas9 technology and agronomic traits of the mutants were studied under field conditions;furthermore,hydroponics cultures were employed to verify mutants’ phenotype and possible mechanism.The main results are as follows:1.Bioinformation analysis showed that OsPHYA/OsPHYB/OsPHYC are located on the third chromosome,encoding 1128,1171 and 1137 amino acids,respectively.The homology of three amino acids was 60.83%.And evolutionary tree analysis showed that three Phytochrome genes are located in different branches.The results from gene chip analysis and qRT-PCR showed that are constitutivly expressed in rice plants:OsPHYA was mainly expressed in shoot-root junction from seedling to blooming stage;OsPHYB was highly expressed in leaves;and OsPHYC was richly expressed in leaves and sheathes.Besides,realtive expression of OsPHYA and OsPHYB was induced by low nitrogen relative to control treatments.2.Homozygous mutants of osphyA/osphyAB/osphyABC were obtained by rice transgenic technology.Specific mutation sites were identified by RT-PCR and sequencing analysis.And several mutant lines with stable inheritance were obtained through breeding.3.Compared with wt plants under field experiments,tiller numbers were significantly increased in osphyA/osphyAB/osphyBC/osphyABC mutants,with increment of 30%,50%,75%and 35%respectively.Tiller numbers in osphyA and osphyAB began to exceed that in wild type at the peak tillering stage,and the differences maintained after the tillering stage.In addition,PHYA and PHYB was found to participate in panicle development,due to a significant reduction in panicle length and panicle weight of osphyA and osphyAB in comparison with wt plants.4.The results from seed germination and hydroponics experiments showed that,compared with wt plants,mutation of OsPHYA did not affect seed germination,and no differences were observed in root length and aboveground length.During rice seedling,the number of tiller of osphyA had increment tendency relative to wt plants,but did not reach a significant level.When cultured in rice nutrient solution for about one month,tiller numbers of osphyA increased significantly in comparison with wt plants;and length of seminal roots and number of adventitious roots increased significantly.Quantitative RT-PCR was used to detect the expression of strigolactones and cytokinin-related genes at the junction of mutants and wt plants.Compared with wt plants,relative expression of strigolactone synthetic and signaling genes(OsD3/OsD14/OsD10)and that of cytokinin signaling genes(OsRR6/OsRR9)were significantly down regulated in mutant plants,while the expression of OsD53 tended to increase.In conclusion,rice phytochrome genes affect tillering probably by regulating the synthesis and signal transduction pathways of strigolactones and cytokinins.