| Grapevine(Vitis spp.),as one of the most important fruit trees in China,has a long cultivation history and extensive cultivation area.It has been popular with people due to its unique flavor and rich nutrition.Given that the quality and quantity of flower organs are key factors that affect grape fruit yield and quality,the development and sex differentiation of grapevine floral organs have always been important research hotspots in developmental biology.In the current study,the flowers of three grapevine varieties ‘Beichun’(hermaphrodite),‘1613C’(male),and ‘520A’(female)were chosen.The differences in morphology of three kinds of flower organs were compared by morphological observation.The pollen vitality was observed using a scanning electron microscope.Enzyme-linked immunosorbent assay was performed to determine the hormone content of three kinds of grapevine flower at different developmental stages.High-throughput sequencing technology was applied to obtain the transcriptome profiles of flowers with different genders at three developmental stages.We further investigated the mechanisms underlying the grapevine floral organ development and sex differentiation so as to provide theoretical support for molecular research on grapevine flowers.The main results are as follows:(1)At stage 1(S1),there were obvious differences in the size of floral organs with different genders(hermaphrodite> female> male).At stage 2(S2),there were no significant differences in the floral organs of the three flower types,and it was difficult to distinguish them morphologically.At stage 3(S3),hermaphrodite flower showed normal ovary and the anthers were slightly higher than the stigma.Female flower filaments were short,curved,reflexed and lower than the ovary,and the ovary was normal.The filaments of male flower in bloom were upright and higher than the ovary and the ovary was significantly degradated.There were no obvious differences in the pollen grains between hermaphrodite and male.They were oblong-shaped,smooth in surface,having three germination grooves,and the pollen vitality was strong.The pollen grains of female anthers were mostly irregular,smooth in surface,with no germination grooves,and were sterile.(2)Determination of hormone content at different developmental stages of flowers with different genders revealed that the accumulation of various plant hormones in flowers of different genders was different,and so was that in flowers of the same gender at different developmental stages.The trend of hormone content change may be mainly decided by the demand for hormones in the development of specific floral organs.For example,because IAA could promote pistil development,its content was highest in female flower at S1 and was higher than that of male flower.The same was true for other cytokinins such as IPA,ZR,MEJA,etc.There was synergy between hormones(such as IAA / IPA)or antagonism(such as ABA/GA)to jointly regulate the process of flower development and sex differentiation.The ratio between hormones may be an important indicator of fine plant regulation.(3)The transcriptome profiles of ’Beichun’(hermaphrodite),’520A’(female)and ’1613C’(male)flowers at the initiation stage(S1),large bud stage(S2)and full bloom stage(S3)were obtained based on the next-generation sequencing technology.A total of 31965 expressed genes were obtained.1535 genes were directly related to flower development and were mainly involved in phenylpropane synthesis and plant hormone signal transduction pathways.Some genes were continuously up-regulated and down-regulated at different developmental stages.The genes that were continuously up-regulated were mainly involved in phenylpropane synthesis,plant hormone signal transduction and other pathways.The genes that were continuously down-regulated were mainly involved in starch and sugar metabolism,and amino acid synthesis.MADS-box genes,such as AP1,PI,SEP1,AG2,SOC1 and SVP,were significantly differently expressed in different floral organs at developmental stages.During flower sex differentiation,the differential expression of the MADS-box gene,the flower-induced integron gene,and the detoxification and stress-related genes play important roles.q RT-PCR verified the expression characteristics of the genes involved in these pathways,and proved the reliability of transcriptome data.(4)The four MYB genes were all expressed in the genomes of flower organs of different genders,and the expression of VIT＿14s0066g01090 was the highest.The expression levels of VIT＿13s0067g01630and VIT＿12s0059g00700 in ’520A’ were significantly higher than those of ’Beichun’ and ’1613C’ and were not significantly different in ’Beichun’ and ’1613C’.The expression level of VIT＿19s0015g01280 in ‘1613C’was the highest and significantly higher than that of ‘Beichun’,but was not significantly different from that in ‘520A’.The phylogenetic tree indicated that the protein encoded by VIT＿19S0015G01280 and ATMYB80 were clustered together.Promoter element analysis showed that the four MYB genes all contained sex-differentiation-related cis-elements such as multiple light response regulatory elements,cis-regulatory elements involved in endosperm expression,cis-regulatory elements related to specific activation of meristems,and various inductive response elements.(5)A total of 839 new transcripts were identified,among which 697 were annotated.The structure of 10163 annotated genes was optimized. |
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