| Plant life cycles alternate between the growth of a diploid sporophytic organism and a highly reduced haploid gametophytic phase. For the gametophytes, angiosperms have both female and male gametophytes. Female gametophyte (embryo sac) is critical to many steps of the angiosperm reproductive processes, including pollen tube guidance, fertilization, the induction of seed development upon fertilization, and maternal control of seed development after fertilization. Female gametophyte development, as one of the most important developmental events, is always a hot area of plant reproductive developmental biology and is significant for both fundamental research and agricultural breeding. Two-component system (TCS) mediated phosphorylation is one of the primary mechanisms to regulate singal transduction in plant cells. TCS in angiosperms is a multi-step phosphorelay that employs a hybrid bistidine kinase, a histidine-containing phosphotransfer protein, and a response regulator. So far, functional research of plant two-component system mainly focuses on stress adaption and vegetative growth, research on reproductive development especially female gametophyte development and cell fate specification is still limited.To further study the relationship between plant two-component system and female gametophyte development and cell fate specification, we firstly detected the two-component signals in the embryo sac of model plant,Arabidopsis, and studied the expression patterns of histidine kinase genes involved in TCS signal transduction. Specifically, cytokinin dependent specification of functional megaspore and relationship between CK11 (cytokinin-indepenndent 1) and female gametophytic cell fate specification has been studied. Besides, to study TCS in Brassica rapa, we identified IIPTs (isopentenyl transferases) and CKXs (cytokinin oxidase/dehydrogenases) gene family, TCS genes and CRFs (cytokinin response factors) gene family in B. rapa genome based on whole genome sequencing project and also the genome information, protein characteristics, phylogenetic relationships, evolutionary patterns and expression patterns were analyzed, which provieda good foundation for further functional research. The main results are summarized in the following:(1) Dynamic distribution of TCS signals in Arabidopsis embryo sac development was carefully observed utilizing TCSnpro::NLS-3XeGFP marker line, results showed TCS signals exist in embryo sacs from functional megaspore stage to mature embryo sac stage, suggesting TCS may have some functions in female gametophyte development. Then the promoter activities of 7 histidine kinase genes involved in two-component signal transduction during embryo sac development have been studied, results showed AHK3, AHK4, AHK1, AHK5, CKI1 and ETR1 express in embryo sacs. Studies on the loss of function mutant of cytokinin receptor genes (AHK2, AHK3 and AHK4) showed ahk2 ahk3 ahk4 triple mutants redundantly regulate the specification of functional megaspore.(2) Arabidopsis ckil-9/+ mutant shows female gametophytic abortions. DIC observation of cleared ovules showed two polar nuclei were unable to fuse and three undegenerated antipodal cell nuclei in the chalazal end move towards the micropylar end. We constructed female gametophytic cell specific single marker and double markers and transformed these markers into ckil-9/+ mutant by floral dipping method, results showed antipodal cell fate and central cell fate were completely lost and three antipodal cells and two unfused polar nuclei were transformed into egg cell fate or synergid cell fate. Studies on CKIl protein localization in embryo sacs indicate CKIl protein exhibited polarized localization in the chalazal end starting from FG4 stage, post transcription regulation may account for these polarized localization phenomenon. The ESI promoter to specifically overexpress CKI1 in embryo sacs has been employed, results showed ectopic expression of CKI1 in egg cell and synergid cells could transform egg cell fate and synergid cell fate into central cell fate. Based on these results, we proposed two models of female gametophytic cell fate specification.(3) Promoter activities of AHP1?AHP5 genes in mature embryo sacs have been investigated, results showed AHP1 expresses in central cell and synergid cells, AHP3 expresses specifically in central cell, AHP2 and AHP5 express in central cell, egg cell, synergid cells and antipodal cells, while AHP4 do not express in embryo sac. Afterwards, we constructed ahp multiple mutants and seed development and embryo sac development were studied, results showed ahp2-2 ahp3 ahp5-2/+exhibits similar phenotypes with ckil-9/+ mutant, therefore AHP2, AHP3 and AHP5 together act downstream of CKI1 to regulate female gametophyte development.(4) Alignment of amino acid sequences of plant HK family proteins was conducted and we found CKIl specific motifs with sequences SHD and GLGLG underling HK domain and HATPase domain, respectively. CKIl domains were swapped by overlap extension PCR to study the roles of CKIl domains on CKIl function, and results showed HK domain and HATPase domain rather than Rec domain affect CKIl function. Besides, by transient expression system with tobacco, we found CKI1 N-terminal region determines the ER localization of CKIl protein.(5) Studies with different lengths of CKI1 promoters indicated that core and minimal functional promoter elements located in the first intron of CKI1. Also yeast one hybrid has been utilized to search for transcription regulation factors upstream of CKI1 and 7 transcription factor genes showing expressions in central cell were found.(6) Thirteen IPT genes and twelve CKX genes were identified in B. rapa genome, genomic information and protein characteristics were analyzed. Analysis with multiple alignment, conserved domains and phylogenetic relationships showed both IPT and CKX genes in B.rapa could be divided into four groups, ????? and ?. IPT and CKX genes from three crucifer plants, B.rapa, A. thaliana and A. lyrata, were chosen for genome level comparation, segmental duplications and tandem duplications were analyzed, and Ks and Ka values of orthologous genes and paralogous genes were determined to investigate their evolutionary patterns. Besides, expression patterns of IPT and CKX genes in B.rapa were analyzed by qRT-PCR and genes with organ-specific expressions were screened. Response patterns of IPT and CKX genes in B. rapa to sress (drought stress and salt stress) and exogenous plant hormones (cytokinin and ABA) were studied and candidate genes involved in abiotic stress and phytohormone regulation networks were screened.(7) Eighty-five TCS gene elements were identified in B. rapa genome, including 20 HK genes,8 HP genes and 57 RR genes, genomic information and protein characteristics were analyzed. Analysis with multiple alignment, conserved domains and phylogenetic relationships showed HK genes could be further divided into cytokinin receptor subfamily, AHK1 subfamily, AHK5 subfamily, CKI1 subfamily, ethylene receptor subfamily and phytochrome subfamily. RR genes could be further divided into Type-A RR, Type-B RR, Type-C RR and pseudo-RR. Segmental duplications and tandem duplications of TCS genes in B. rapa and A. thaliana were analyzed and compared, Ks and Ka values of orthologous genes and paralogous genes were determined to investigate their evolutionary patterns. Besides, expression patterns of TCS genes in B. rapa were analyzed by qRT-PCR and genes with organ-specific expressions were screened. Response patterns of TCS genes in B. rapa to abiotic sress (drought stress and salt stress) and exogenous plant hormones (cytokinin, auxin and ABA) were studied and candidate genes involved in abiotic stress and phytohormone regulation networks were screened.(8) Two hundred eighty one AP2/ERF super gene family members were identified in B.rapa genome. Based on conserved domains and phylogenetic relationships, AP2/ERF super gene family could be divided into three families, AP2 family, RAV family and ERF family. ERF family could be further divided into DREB subfamily and ERF subfamily, with 13 groups I-XI,21 CRF genes located in group VI and VI-L were mainly studied, CRF genes in B. rapa could be divided into 5 clades, I?V, encoding three kinds of proteins, type A, type B and type C. Expression patterns of CRF genes in various organs of B.rapa were analyzed by qRT-PCR, showing that CRF genes were ubiquitously expressed but with different expression levels in different organs. Response patterns of CRF genes in B. rapa to sress (drought stress and salt stress) and exogenous plant hormones (cytokinin and ABA) were studied and candidate genes involved in abiotic stress and phytohormone regulation networks were screened.These results could provide a new perspective to understanding plant female gametophyte development process, also could provide theoretical foundation and technical support for understanding molecular mechanism of sexual reproduction in economic crops, improving research foundation of reproductive biology in Brassica vegetable crops, regulating crops fertility, and achieving good quality, high yield and high-efficiency breeding. |
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