| As an important plant hormone, auxin is closely related to the role of plants due to its biosynthesis and transcription factor regulation in its signal transduction. ARF is a kind of critical auxin response factors, many works have been done in its molecular mechanism. Great progress was made in its function in plant tissues and organs and to explore a variety of physiological activities. However, auxin regulation of plant growth and development is an extremely complex process and there is a strict control mechanism, exploring ARF function mechanism in the plant has important theoretical and practical value. This paper mainly discusses the expression profiles of tomato abscission in ARFs family, followed by the selection of two tomato SlARF genes associated with abscission. Transgenic plants obtained were studied its physiological functions in growth and development of tomato, laying a theoretical foundation in auxin-mediated plant physiological functions. The main results are as follows:1. By investigating abscission rate of tomato flower pedicel explants in different treatment, combined with the abscission defense gene Cystein-type endopeptidase expression signal analysis, we considered 8 h as the key point. Auxin inhibits abscission before 8 h and ethylene promotes after this time point, which is a prerequisite for further study of the gene level.2. We examined the spatial and temporal distribution of IAA during abscission by examining transgenic tomato plants expressing an IAA-inducible promoter fused to the GUS reporter gene (the P5::GUS Chico Ⅲ line). Through observation by transverse and longitudinal scans, we found auxin declined. However, the auxin content is always the highest in abscission zone, the vascular tissue content is higher; while the contents in cortex and pith were quickly dropped. Auxin is completely exhausted at 32 h. Auxin transport, distribution and consumption appeared greater impact in different training direction. Fluorometric GUS analysis indicated that GUS activity in the abscission zone remained stable at 4 h and sharply decreased after 8 h until abscission was complete.3. Flower removal from the explants quickly induced abscission by ethylene, which was inhibited by exogenous auxin or 1-MCP. During early abscission, auxin regulated the expression of various ARFs, including ARF1,2,3,4,5,7,8-1,9,11,12,13,13-1,14 and 17, while ethylene had the opposite effect on most of these genes. Further analysis show that during this stage, auxin may mediate the expression of ARF8-1,9,11,12,13,13-1 and 14, while ethylene may mediate ARF13-1. During the later stage of abscission, ARF5, 6,8,10,11,16 and 19 were upregulated, compared with non-abscissing parts of plants. On the other hand, the absolute amount of SI ARFs gene expression analysis showed that ARF6-1, ARF8-1, ARF13 and ARF19-1 are the highest expression genes, indicating they might have a more important role in the development and separation of abscission zone.4. In order to block the specific gene binding, we designed primer encompassing the B3 DNA-binding domain of ARF proteins. By PCR amplification, we obtained expected 435 bp (SlARF2:512-947 bp) and 422 bp (SIARF 14:562-984 bp) fragment respectively. The Gateway cloning technology was used to build SlARF2 and SlARF14 RNAi vectors, and correctness were validated by sequencing and digestion methods.5. On the basis of established "zhongshu NO.6" tomato efficient regeneration system, we used Agrobacterium-mediated genetic transformation destination vector into plants according to the actual situation. Throughout the process, resistance selection pressure remains 0.5 mg/L PPT. The rooting medium agar content was changed from 0.8% to 0.7 %, in antibacterial premise is more conducive to root growth to increase the transplanted survival rate. PCR amplification was preformed for SlARF2-RNAi PPTR and SlARF14-RNAi PPTR plants, further making sure exogenous gene has been successfully transferred to the tomato genome guides, access to a part of the positive transformed plants, conversion rate is higher than 30%.6. As SlARF14 protein structure and tissue-specific expression analysis showed, S1ARF14 lacks Aux/IAA homodimerization region (CTD), and it expressed in most tissues and organs, especially the pistil, flower buds, leaves and fruit enlargement period. The phylogenetic analysis showed SlARF14 and has been proven with the blade about the growth and development of tomato SlARF10 highly homologous, suggesting that there may be similar to its functionality.12 Aux/IAA genes expression analysis showed no obvious differences between SlARF14-RNAi and control expect IAA7, which suggested Aux/IAAs might not be involved in the effective regulation in SlARF14 down-regulated plants. In another word, SlARF14 may have a relatively independent role in SlARFs family.7. Down-regulation of SlARF14 using RNA interference indicated that this gene plays an important role in leaf morphological development and photosynthesis. The leaves of SlARF14-deficient tomato lines were dark green and thick, and they exhibited reduced lobes, causing a Potato Leaf Tomato-like appearance. Moreover, we observed a lateral development trend in the transformants, as well as significant changes in the expression of the morphology-related genes LeT6 and LeEXPA10. On the other hand, downregulation of SIARF14 stimulated photosynthetic activity at the early stage of development, which appears to be related to increase Rubisco enzyme activity. The altered expression levels of RBCL, RBCS and CAB reflect this notion.8. SlARF14-RNAi floral organ appeared stripped petals and stamens. We found no adverse developmental pistil, stigma, ovary abnormal development, affecting the normal pollination by exposing the ovary. It is also easy to form a deformity fruit. The contents of IAA in pistil and stamen were both low before and after pollination. There was no significant increase in IAA content in pistil after pollination.9. SlARF14-RNAi floral organs appeared abscission at 8 h, indicating inhibition of growth hormone to its pre-weakened. Abscission completed at 36 h, which was earlier than control. The effect of ethylene decreased in ethylene treatment, suggested its performance is not sensitive to ethylene. These results were consistent with the ARF14 gene expression patterns during different periods of abscission.10. As S1ARF2 protein structure and tissue-specific expression analysis showed, S1ARF2 has structural integrity, and it expressed in most tissues and organs, especially the roots, flower organs and seedlings. The phylogenetic analysis showed S1ARF2 has highly homologous with Arabidopsis AtARF2, which affected leaf senescence and flower abscission, suggesting that there may be similar to its functionality.12 Aux/IAA genes expression analysis showed no obvious differences between SlARF2-RNAi and control expect IAA1, IAA3, IAA7 and IAA26, which suggested S1ARF2 functions by interacting with these IAAs.11. We detected the distribution of ARF2 by ProARF2:GUS staining during different developmental stages or different parts of the plant. SIARF2 gene strongly expressed in fresh leaf base, growing point, leaf axils, primary stem parts (especially trichomes), lateral base, fibrous occurrence site and fully open flower stamens, suggested S1ARF2 mainly focused on growth and development of young tissues or organs.12. SlARF2-RNAi plant appeared bright green leaves, reduction in the number of leaflets. Compared with control which rapid aging of mass death, SIARF2-RNAi blades slowed the aging process. The aging gene, SENU1 and SENU5 delay expression. In the same period, its leaves can maintain a relatively high survival rate, and individual leaf photosynthetic rate, maximum photochemical efficiency, chlorophyll remaining amount and Rubisco enzyme activity decreased slowly. At the same time the expression of RBCL and RBCS genes was lagging behind. In addition, antioxidant enzymes decreased more slowly than control significantly. At the level of hormone, leaf senescence pathway was independent of the ZR, and involved IAA regulation.13. The trichomes of SIARF2-RNAi flower stalk and sepals reduced significantly, floral organs (including sepals, petals, stamens and pistils) were shorter than control in lenghth. The contents of IAA in pistil and stamen were both low before and after pollination. The sensitivity to IAA decreased in its stamens.14. SIARF2-RNAi floral organs appeared low abscission rate until 28 h. The critical period was between 16-20 h, because during the time, abscission rate increased very slowly, even delayed, but returned to normal at 20 h later. Abscission rate of SlARF2-RNAi retained at a very low level at the time of 8 h in ethylene treatment, suggesting it would be difficult to be induced by ethylene temporarily. |
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