| Seed germination is the beginning of a new life cycle and is essential for plant growth and crop production.Rice is one of the most important staple food of China.Improving rice germination rates and seedling surviving rates are important preconditions of ensuring high rice yield,and the key of it lies in excellent seed germination and rapid growth after germination.The rice seed germination involves many complex physiological and biochemical reactions,including seed reserve mobilization in the endosperm and nutrient transport and reuse in the embryo.Brassinosteroid(BR)is known as the sixth phytohormone and involved in a wide range of plant growth and development events.Most importantly,BR could regulate a number of key agronomic traits of crops,such as plant height,leaf angle,seed size,and seed germination.Therefore,BR has a great potential in agricultural applications.However,the underlying molecular mechanisms on BR control of rice seed germination are mostly unclear.In this study,we firstly analyzed the effect and mechanism of BR in regulation of rice seed germination.We demonstrated BZR1 transcription factor centered transcriptional regulatory module mediated BR's regulation of rice seed germination and post-germination growth.Gibberellin(GA)is a most important phytohormone with promotion effect on seed germination.GA interacts with BR in various biological processes of many plants.However,it is still obscure whether they coordinate rice seed germination and what is the underlying mechanism.Therefore,we focused on the study of the effect and mechanism of BR and GA coordinated rice seed germination.We confirmed that both the independent and interdependent correlations existed between BR and GA during rice seed germination.Moreover,we revealed that BR and GA co-regulated rice seed germination via activating the mobilization of storage starch and glutelin.In addition,a number of germination-related down-stream target genes were identified and molecular regulatory network of seed germination was established which was mediated by BR signaling and its interaction with GA pathway.The present study not only advanced our understanding of the regulatory mechanisms on rice seed germination and dormancy,but also provided important theoretical support,gene resources,and genetic materials for breeding elite rice varieties with good performance in seed germination.The main results are as follows:1.Blocking either BR biosynthesis or signaling,including BRZ treatment,downregulation of DWARF11 expression,defective BRI1 receptor and overexpression of GSK2 kinase,delayed seed germination and inhibited post-germination embryo growth.In addition,evidences indicated that BZR1 was the key downstream transcription factor that mediated BR regulation of seed germination by binding to the RAmy3D promoter,thus affecting the expression and activity of ?-amylase,as well as the degradation of starch and soluble sugar content in the endosperm.2.The identified BZR1-RAmy3D regulatory module played important roles in controlling storage starch degradation,which was independently from the established GAMYB-RAmy1A module of the GA pathway.Moreover,the BZR1-RAmy3D module also functioned in regulating transient starch degradation in embryo-related tissues.RNA-seq analysis identified more potential BZR1-responsive genes.The above results not only showed the pivotal role of BZR1 in mediating BR's regulation of rice seed germination,but also identified the downstream targets of BZR1 and established the transcription regulatory module of rice seed germination centered by BZR1 transcription factor.The present study will facilitate the isolation of more novel genes that were responsive to BR signal and specifically regulate key agronomic traits such as seed germination.3.Knock-out of OsBZR1 led to the slight decrement of plant height and compact architecture of rice,which meet the requirements of modern crop breeding programs.Although the 1,000-grain weight of the bzr1-il and bzr1-d4 mutants was slightly lower than that of the control,they had the potential to increase rice yield under appropriately dense planting conditions.Most importantly,the bzr1 mutants exhibited a better resistance in pre-harvest sprouting(PHS)than that of the wild-type control,thus ensuring the stable rice yield and minimizing the lose of rice yield and quality caused by PHS.4.The proteomic study of the embryos in the early stage of germination showed that the changes of protein abundance of many starch synthases were strikingly,with the Wx protein as the most obvious one.It is well-known that Wx protein is the key enzyme controlling amylose biosynthesis in rice.Thus,Wx protein is one of the most important regulators of rice eating and cooking quality.Expression analysis showed that BR and GA promoted the accumulation of Wx protein in embryos.The analysis of Wx-related near-isogenic lines and transgenic lines indicated the seeds with Wxa allele germinated faster than the seeds with wx allele.The reason was that the starch in the rice seeds was easier for degradation.In addition,the expression and activity of the ?-amylase in the Wxa rice was higher,thus resulting in the higher conversion efficiency of starch to soluble sugar,and consequently providing more energy and substance for seed germination and post-germination growth.In addition,in natural rice resources,non-waxy rice seeds(Wxa type)germinated faster than waxy rice seeds(wx type),which further indicated that the composition and structure of starch affected rice seed germination.Therefore,the result provided new targets and ideas for breeding new rice varieties with good performance in rice yield,quality and seed germination characteristics.5.The results of both proteomic and qRT-PCR analysis showed that the expression of several glutelin genes in rice embryo was induced by BR and GA.Further expression assay suggested that the increased accumulation of glutelin proteins in the embryo was due to the accelerated degradation of storage glutelin by enhanced expression of cysteine proteinase(REP-1)in the endosperm.The degradation efficiency of glutelin in the endosperm showed a strict positive correlation with the shoot length of rice.The GluA2 mutation led to the reduced degradation rate of glutelin and slower seed germination,and the promotion effect of GA on seed germination was attenuated in the glua2 mutant.In vitro culture assay of rice embryos showed that glutelin mobilization functioned at the downstream of the GA and BR pathways,thus promoting shoot elongation.In conclusion,BR and GA promoted rice seed germination and embryo growth by coordinating glutelin mobilization in the endosperm.6.Exogenous application of GA restored germination defects caused by BR deficiency or insensitivity.A total of 42 confident differentially expressed proteins in response to GA were identified by using proteomic approach,and most of them showed consistent expression change in different groups of GA-treated rice seeds.A potential protein-protein interaction network was constructed by using bioinformatic methods.The expression of five members of the LEA(Late Embryogenesis Abundant)family proteins were notably suppressed by GA in both the transcription and protein levels.These LEA genes were specifically expressed in rice seeds,reaching the expression peak at the latter stages of seed development.Finally,the mutants of LEA33,as one representative member of the LEAs,were generated.The analysis result indicated that LEA33 mutation affected rice grain size and seed germination,possibly realized by reducing BR accumulation but enhancing GA biosynthesis.The above findings improved our knowledge of the mechanisms on the coordinated regulation of seed germination by GA and BR. |
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