Effects Of Transgenic IaaM And ABP1on The Development Of Fibers And Cells Of Cotton And Ramie

Auxin, the most important phytohormone, plays a key role in the regulation of plant growth and development. How the signaling between it and its downstream effectors occurs has attracted widespread attention of researchers. AUXIN BINDING PROTEIN1(ABP1) is the first characterized protein that binds auxin and has been implied as a receptor for a number of auxin responses. Several lines of evidence support a role for ABP1in control of cell expansion and cell division. In this study, we used the fiber plants-cotton and ramie as the model to dissect the roles of auxin and ABP1in cotton fiber elongation, the development of ramie phloem fiber and cell development.Cotton fiber is a highly elongated cell derived from the ovule epidermis. Auxin plays an important role in cotton fiber development. It can promote fiber elongation. A Tryptophan monooxygenase encoded by iaaM gene is the key enzyme in converting tryptophane to indole-3-acetamide which is then hydrolyzed to the indole acetic acid (IAA). So the iaaM gene has been widely used to manipulate the IAA level in various plants. To identify the function of IAA in fiber elongation, we constructed a plant expression vector carrying auxin synthesis gene iaaM coding sequence under the control of the fiber-specific E6promoter from Gossypium hirsutum and it was introduced into the genome of a cotton cultivar,’Xiangzamian14’, by an Agrobacterium-mediated transformation method. Transgenic seedlings were screened out by hygromycin resistance, and then subjected to molecular analysis. PCR analysis showed that iaaM gene was successfully integrated into the cotton genome in five transgenic lines. The result of RT-PCR also verified that iaaM gene was transcripted at mRNA level in transgenic line. The IAA level in fibers at15days post anthesis was increased. However, the length of fibers was not changed significantly. It is clearly shown that excess endogenous auxin can not promote fiber cell elongation.Auxin binding protein1(ABP1) is recently recognized as an essential receptor in auxin signaling. ABP1is especially involved in cell elongation. However, it is lack of evidence on the role of ABP1during postembryonic development due to the embryo lethal in Arabidopsis. To identify the role of ABP1in cotton development, the ABP1gene of cotton was cloned by RT-PCR in Gossypium hirsutum cultivar ’Xiangzamian 14’and its transcription level was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The cDNA of GhABP1was792bp in length and shared99%identity to the cDNA of FJ609678from Gossypium hirsutum cultivar ’Xinluzhong5".It is expressed in leaves, flowers and fibers at15days post anthesis. The transcription level in young leaves is higher than that in other tissues. An over expression vector carrying ABP1gene coding sequence was constructed, and was introduced into the genome of a cotton cultivar’Xiangzamian14’by an Agrobacteriwn-mediated transformation method. Transgenic lines over expressing ABP1were generated. RT-PCR analysis showed that the transcription level of ABP1gene was up-regulated in three transgenic lines which exhibited normal morphological phenotypes. The leaf size and shape were not altered obviously but with leaf trichome density on the leaf margin increased. Moreover, over expression of ABP1did not result in significant alteration in fiber length.Ramie [Boehmeria nivea (L.) Gaud.] is a perennial fiber plant. Its fiber is the longest phloem fiber. The role of auxin signaling in ramie development remains unclear. We were cloned ABP1cDNA (BnABP1) from ramie several years ago. Here, we focused on analyzing the effect of ABP1on ramie development. We constructed an over expression and an antisense expression vectors carrying BnABP1coding sequence under the control of the CaMV35S promoter, and they were transformed into a ramie cultivar,’xiangzhu3’, by an Agrobacterium-mediated transformation method. Transgenic plantlets were screened out by hygromycin resistance and four ABP1over expression plants were generated. The transgenic lines exhibited normal morphological phenotypes. PCR analysis showed that anti-ABP1gene was integrated into the ramie genome in four transgenic lines and the ABP1expression was repressed in ABP1antisense transgenic plantlets. The suppression of ABP1expression led to defects in plant growth including dwarf plant, and decreased apical dominance in the intact plant. In addition, ABP1antisense lines exhibited serious retard of leaf development involving small and twisted leaves, and sparse trichome. Though, the cell type in leaves of ABP1antisense plants was not changed, the cell in epidermis and palisade tissue growed smaller. Moreover, the decreased ABP1expression repressed the development of leaf venation. ABP1antisense lines developed an incomplete leaf venation, which became smaller and lacked in some regions of leaves. Though the cell arrangement was normal in midrib and lateral vein, cell size was decrease and some cell layers were missing in midrib. The alteration of petiole histology was similar to that of leaf histology. ABP1antisense plants had smaller stems, which appeared to be attributable to a decrease in cell size of stems. The decreased ABP1expression repressed the development of fiber. Fiber cells were decreased and smaller. The results showed that ABP1plays an important role in ramie fiber and cell development.In conclusion, the elongation of cotton fiber, as specialized ovule epidermal cell, may be influenced by many factors. It has no significant effect on fiber elongation by increasing either the auxin concentration or ABP1expression level in fiber cell. But the increase of ABP1expression level can has an effect on the development of leaf trichome.For ramie phloem fiber and cell, its development requires a minimum amount of ABP1expression. The over expression of the ABP1have no dose effect on its development. However, the fiber development is significant anomaly when the ABP1expression is below a certain threshold by antisense RNA. The differentiation and elongation of the fibers as well as other cells is affected apparently. It has shown ABP1should keep in an amount for the normal growth and development of ramie. ABP1is a major regulator for the phloem fiber cell differentiation and elongation.All expected cell types in leaves and stems of ABP1antisense lines are present, suggesting that suppression of ABP1gene does not repress primary morphogenesis of the tissues. However, the size and number of cells in the tissues is decreased, which has showed that ABP1can act on cell division and elongation. The mutant phenotypes of ABP1antisense lines are attributed to abnormality of cell division and elongation.