Functional Characterization Of The AtFEM Gene Involved In Embryogenesis And Rhamnose Synthesis Enzyme AtRHM1 Gene In Arabidopsis

The thesis is constituted of two studies. The first is about an Arabidopsis F-box protein involved in embryogenesis. Precise control of gene expression is critical for embryo and endosperm development in plants. Recent studies revealed that the mutations of over 250 genes resulted in embryoic abnormalities or embryo lethalities, indicating these genes are essential for normal embryo development. The known embryo-defective genes encode proteins with diverse functions, including metabolism, cell growth, transcription, translation, protein fate, protein transport and traffic, and plant defense. In Arabidopsis, there're more than 700 F-box proteins interacting with the rest of the SCF complex for degradation of the substrates. A number of studies have demonstrated that F-box proteins play critical roles in various signaling pathways, while some reports show that F-box proteins are also involved in embryo and endosperm development. The F-box proteins TIR1 and AFB were reported to mediate auxin responses during embryogenesis, and it's found that the embryos of the triple mutant tir1-1 afb2-1 afb3-1 displayed defects during early embryogenesis. Other F-box proteins such as AtSFL61 and AtSFL70 are likely required for the development of embryo or female gametophyte. AtSFL61 mutation led to embryo-lethality with the frequent irregular cell division in atsfl61 embryos. Around 50% of the atsfl70/+ ovules could not survive beyond the zygotic division due to female gametophyte being defective. In this study, we report that F-BOX EMBRYO LETHAL (FEM), an F-box protein, is involved in embryogenesis and endosperm development in Arabidopsis. Loss-of-function mutations of FEM caused recessive embryo-lethal phenotype. Selfed heterozygous plants produced approximately 25% abnormal seeds which were shrunken and aborted. Most of the mutant embryos were arrested at globular stage and some at globular-to-heart transition stage. At 1-cell and 2-cell stages, it was difficult to distinguish the mutant from wild type; when wild-type ovules beared globular embryos, the mutant embryos were at 8-16-cell stage; when wild-type ovules contained heart-shaped embryos, the mutant embryos were at preglobular stage; when wild-type embryos developed mature embryos, the mutant embryos were arrested at globular-like or globular-to-heart transition stages and aborted. None of the fem embryos reached the heart stage. In addition to the embryos being arrested, aberrant cell divisions were observed in the mutant embryos with aberrant positioning of the division planes displayed, which resulted in globular embryos with irregular shape. Moreover, the number of endosperm nuclei of the mutant showed a dramatic reduction compared to wild type. FEM encodes a polypeptide with two LRR domains and two F-Box domains. FEM was ubiquitously expressed in various organs of transgenic Arabidopsis as revealed by FEM promoter-GUS fusion with a consistency with the microarray data (indicate the website). Detailed GUS staining with the transgenic ovules revealed the expression of FEM in the endosperm and developing embryos. Taken together, the data suggest that FEM gene is essential for both normal embryo and endosperm development in Arabidopsis.Second study is about the functional analysis of the Arabidopsis rhamnose biosynthesis gene AtRHM1. Cell wall plays essential roles during plant growth and development. The cell walls determine the boundaries of plant form, represent the main sink for photoassimilated carbon, protect against pathogen invasion, and provide mechanical strength to the plant body. Cell walls comprise primarily cellulose microfibrils, hemicelluloses, pectic polysaccharides, and small amounts of structural proteins. L-Rhamnose (Rha) is an important constituent of pectic polysaccharides, a major component of the cell walls of Arabidopsis, which is synthesized by three rhamnose biosynthesis enzymes (RHM) encoded by AtRHM1, AtRHM2, and AtRHM3, respectively. Mutations in Arabidopsis RHM2 gene, which are allelic to the MUCILAGE-MODIFIED4 (MUM4) locus, reduced the Rha and galacturonic acid content of seed coat mucilage, suggesting that RHM2/MUM4 is involved in RGI biosynthesis. Despite the finding that RHM1 is involved in root hair formation in Arabidopsis, experimental evidence is still lacking for the in vivo enzymatic activity and subcellular compartmentation of the AtRHM1 protein. AtRHM1 displays high similarity to the other members of RHM family in Arabidopsis and in other plant species such as rice and grape. Expression studies with AtRHM1 promoter-GUS (Prhm1) fusion gene showed that AtRHM1 was expressed almost ubiquitously, with more expression in roots and cotyledons of young seedlings and inflorescences, which is consistent with the results of RT-PCR. We made a series of AtRHM1 promoter deletion (Pd1,Pd2,Pd3,Pd4,Pd5). These deletion fragments were fused with GUS reporter gene respectively. The GUS staining showed that the expression pattern of Prhm1 and Pd1 was almost the same, whereas expression levels were different. For Pd1, expression level was a little bit reduced. Compared the expression level of Pd2 to Pd1, no staining was in cotyledon for Pd2, but strong staining was found for Pd1, indicating that -752 bp～-308 bp of RHM1 promoter was related to special expression of root. For Pd3, expression level in cotyledon of young seedling was reduced dramatically compared to Pd2. No staining was in 2 d seedling and a little staining was on top of 4 d and 7 d seedlings for Pd3, indicating that -308 bp～-140 bp was possibly involved in special expression of cotyledon. There were two G-box and a regulatory element of wounding in a range from -752 bp to -308 bp and ACTT and ABRE elements in a range from -308 bp～-140 bp. GUS staining showed that the expression of RHM1 was induced by sugar and wounding. -308 bp～140 bp of RHM1 promoter was possibly a regulatory element of wounding and -931 bp～-752 bp of RHM1 promoter was possibly involved in glucose answering reaction. It has been proposed that many nucleotide sugar interconverting enzymes are localized in cytosol. Reiter and Pai demonstrated that tobacco NbAXS1, a UDP-D-apiose/UDP-D-xylose synthase was mostly localized in the cytosol. GFP::AtRHM1 fusion protein was found to be localized in the cytosol of cotyledon cells, indicating AtRHM1 is a cytosol-localized protein. The overexpression of AtRHM1 gene in Arabidopsis resulted in an increase of rhamnose content as much as 40% in the leaf cell wall compared to the wild type, as well as an alteration in the contents of galactose and glucose. We did not find any significant difference (P < 0.05) in the amounts of the major sugars of cell wall material between wild type and RNAi line including Rha. Fourier-Transform Infrared analyses revealed that surplus rhamnose upon AtRHM1 overexpression contributes to the construction of rhamnogalacturonan.