Studies Of The Function Of Genes Involved In Auxin Biosynthesis In Rice

Auxin is one of the most important hormones for plant growth and development.It also plays an important role for plants in responses to environment signals.Rice is one of the main staple foods in the world,but we know very little about its auxin biosynthesis pathways.The study on the auxin biosynthetic pathway in rice not only helps us to understand the basic biological significance of auxin,but enables improvement of rice breeding.The synthesis pathway of auxin is most thoroughly studied in Arabidopsis.So far,only one complete auxin synthesis pathway has been well established in Arabidopsis thaliana.Tryptophan is catalyzed by the tryptophan aminotransferase TAA to produce IPA.Then IPA is catalyzed by flavin-monooxygenase YUC to generate IAA.Pyridoxal-phosphate-dependent aminotransferase VAS1 catalyzes IPA to tryptophan,and also catalyzes methionine to produce KMBA(2-oxo-4-methylthiobutanoic acid),which is involved in the synthesis of ethylene.This auxin synthesis pathway is thought to be conserved in many plants.In order to study the auxin synthesis in rice,we analyzed rice genes homologous to those of Arabidopsis.The main results obtained are the following:1.In previous studies,seven YUC members were identified in rice.Our study extended YUC members to 14 and we confirmed that the YUC functions are conserved between rice and Arabidopsis.Unlike Arabidopsis thaliana where the YUC family genes were functionally redundant and the single mutants had no obvious developmental phenotype,in rice the single mutants of osyuc1 and osyuc8 generated by the CRISPR/Cas9 gene-editing technology had obvious phenotypes.It was demonstrated that the functions of OsYUC1 and OsYUC8 in rice are specific and irreplaceable.The functions of other OsYUCs are also redundant as in Arabidopsis.2.Phylogenetic analysis confirmed the presence of four highly homologous TAA members in rice,and the mutant taa1/fib1 generated by the CRISPR/Cas9 gene editing technique defined the function of OsTAA1.The taa1/fib1 mutants caused severe developmental defects.Specifically,the mutants had extremely short "fishbone" architecture and root developmental defects.3.Overexpression of OsYUC1 produced excessive auxin leading to dramatic phenotypes including ectopic root formation and more root hairs.The same phenotypes appeared in transgenic rice cultivars when other OsYUC genes were overexpressed,supporting that the functions of YUC genes are conserved between rice and Arabidopsis.Overexpressing WOX11 caused phenotypes similar to those caused by overexpression of OsYUCs,allowing us to associate WOX11 with TAA/YUC-mediated auxin synthesis.4.The defects in crown root development in taa1/fib1 and the overproliferliation of crown root in OsYUCs overexpression lines suggested that initiation of rice crown roots is dependent on TAA/YUC-mediated auxin synthesis.On the other hand,the expression level of WOX11 decreased in the taa1/fib1 mutant and increased when OsYUC1 is overexpressed.We show that WOX11 is regulated by the induction of auxin.5.Overexpression of OsYUC1 in the callus of wox11-1 mutant did not result in ectopic crown roots and phenotypes of other excessive roots.Overexpression of OsWOX11 in taa1/fib1 restored the initiation and growth of crown roots,indicating that WOX11 regulates the growth and development of crown roots downstream of the TAA/YUC-mediated auxin synthesis pathway.6.Overexpression of OsVAS1 results in longer primary roots and shorter shoots in seedling stage,and shorter plant heights,larger flag leaf angles,increased number of tillers.Overexpression OsVAS1 also led to fewer primary branches,fewer secondary branches,and longer seeds.The osvas1 mutant generated by CRISPR/Cas9 gene editing technology caused longer primary root,and the plants became taller,with flag leaf angle decreased,the number of tillers decreased,and the number of primary branches increased before harvest stage.Our results demonstrate that OsVAS1 is involved in growth of many tissues and in development of various stages of rice.7.Expression pattern of OsVAS1: GUS determined by GUS staining after three days and five days of seed germination showed that GUS activities mainly took place in shoots.The roots were not stained.The mature stems,leaf sheaths,leaves and stamens of the spikelets,pistil and ovary were not stained,either.RT-PCR showed that OsVAS1 was mainly expressed in leaves,nodes and leaf sheaths.OsVAS1 transcript level was relatively weak in other tissues.8.The results of IAA and ACC treatments of transgenic plants in overexpressing OsVAS1 and in osvas1 mutants demonstrated that OsVAS1 participates in the responses to IAA and ACC and is negatively correlated.At the same time,the direct measurement of IAA content and Edu(5-ethynyl-20-deoxyuridine)staining experiments demonstrated that transgenic plants overexpressing OsVAS1 reduced auxin content but in osvas1 mutant plants it was increased and hence affected root cell activity accordingly.9.vas1taa1 double mutant generated by CRISPR/CAS9 gene-editing technology had increased plant heights,more crown roots,lower leaf curls,and reduced spikelet development defects compared to the taa1/fib1 single mutant indicating that vas1 can rescue the defect phenotype of taa1 and demonstrates that OsVAS1 is in the same pathway as OsTAA1.Our results indicate that the TAA/YUC-mediated auxin biosynthesis pathway is conserved in rice and that the development of rice crown roots depends on the regulation of the YUC-Auxin-WOX11 module.On the other hand,we also genetically demonstrate that OsVAS1 also participates in the synthesis of auxin and ethylene pathways in rice.