The Regulation Of WRI1 And ABI3 In Oil Accumulation Of Oat Endosperm

Oats(Avena sativa L.)has oil-rich endosperms,which is unique to other cereals with starchy endosperms.Oil of the oat endosperm mainly consists of triacylglycerols.Understanding the special mechanism of oil accumulation in oat endosperm will be of great theoretical significance for the potential oil production in the endosperms of C4 crops in the future.Triacylglycerol biosynthetic pathway has been extensively studied in oil-crop model plant Arabidopsis.However,limited positive regulators in oil accumulation have been reported,such as seed-specific WRINKLED 1(WRI1),FUSCA3(FUS3),LEAFY COTYLEDON 1(LEC1)and LEAFY COTYLEDON 2(LEC2).Based on the characterization of oil accumulation in developing endosperms of various oat cultivars and the transcriptome data from oat seeds,functions of the highly-expressed WRI1 and ABA INSENSITIVE 3(ABI3)have been investigated in this study.Additionally,considering there are scarce molecular biology studies in oat,oat reference genes have also been explored and evaluated.Main results are as follows:(1)The exclusive oat seed transcriptome was compared with the starchy wheat(Triticum aestivum)endosperm transcriptomes and the oily castor(Ricinus communis)endosperm transcriptomes.According to accumulation patterns of various lipid species in developing oat endosperms,combined with gene expression profiles and tissue-specificity,as well as the corresponding phenotypes,WRI1 and ABI3 were selected as the potential regulators participating in oat endosperm oil accumulation.(2)Eleven candidate reference genes(RGs)with one or more copies were selected from the transcriptome of hexaploid oat seeds.The quantitative real-time PCR(qPCR)assays of 18 samples consisting of vegetative and productive organs or tissues were performed with specific primer pairs for candidate RGs above.The expression stabilities were evaluated using four statistical algorithms including the ACt method,geNorm,Normfinder and BestKeeper.EIF4A and HNR showed the highest stability than any other candidate RG sets across all tested samples and in developing endosperms.The combination of EP and EF1A was the best RG set for developing seeds.UBC21,an example of a four-copy duplicated RG,along with HNR were identified as the most stable RG set in shoots and roots of oat seedlings.The expression levels of AsPKPl in different sample sets were normalized to the most and the least stable RGs for verifying the reliability of the evaluation results.(3)Three AsWRI1s(As WRI1a,As WRI1b and As WRI1c)and one Rc WRI1 were identified in the endosperms of oat and castor,respectively.The gene structure and the phylogenetic relationships were analyzed among WRI1 homologous genes from diverse plant species.Expressions of these four endosperm-expressed WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant.Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells,respectively.Moreover,this overexpression also resulted in up-regulations of WRI1 target genes.WRI1 homologs expressed functionally in oil-rich endosperms,and they may be the key regulators that make the difference between oat and other cereals in endosperm oil accumulation.(4)Up to now,the role of ABI3 in plant oil accumulation and its interaction with FUS3 remain unclear.Inducible expression of ABI3 activated oil accumulation in tobacco BY2 cells and Arabidopsis rosette leaves.FUS3 was a concomitant of ABI3 in the mutants and most overexpression lines.Further expressing of ABI3 in rosette leaves of fus3 knock-out mutant still caused up to three folds more triacylglycerol accumulation.Transcriptome analysis and qPCR assays both revealed that lipid droplet protein(LDP)genes including Oleosins and Caleosins were highly up-regulated by ABI3 in the absence of FUS3,while the expression of WRI1 was doubled.Expression levels of some LDP genes and WRI1 decreased in abi3 mutants and increased in ABI3 overexpressed BY2 cells and Arabidopsis leaves.Our results systematically demonstrate characteristics of lipid species and gene expressions during oat endosperm development,identify optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat,and state the similar function of AsWRI1s with different gene structures on oat endosperm oil accumulation.Our results also provides the first genetic evidence that ABI3 activates oil accumulation with or without of FUS3 mainly through up-regulating LDP genes and WRI1.These findings lay a foundation of further understanding the oil accumulation mechanism in oat endosperm and will facilitate molecular breeding and modifications in oil crops.