A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library, the Taiwan Rice Insertional Mutant (TRIM). This mutant contained a copy of T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which would be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 or TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown colored phenotypes was proportional to the expression levels of transgenes TDC-1 or TDC-3. The level of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC over-expressing transgenic rice were resulted by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.水稻T-DNA插入突變株M47286具有發育遲緩、低稔實與深褐色外表性狀。此突變株帶有單一T-DNA標誌，其插入位置造成兩個色胺酸脫羧酶 (tryptophan decarboxylase) 基因TDC-1與TDC-3的活化。TDC重組蛋白質之酵素活性分析結果顯示，二者皆具有色胺酸脫羧酶活性，可將色胺酸 (tryptophan) 轉化成色胺 (tryptamine) 。色胺進一步被水稻中的色胺酸-5-羥化酶 (tryptamine 5’hydroxylase , T5H) 轉化成血清素 (serotonin)。大量表現TDC-1與TDC-3之轉殖水稻亦呈現與M47286相似之農藝性狀，如發育遲緩、深褐色外表性狀、低稔實等，其影響程度取決於轉殖水稻中TDC-1及TDC-3之表現量。轉殖水稻中之色胺與血清素的累積亦發現與TDC-1及TDC-3之表現量有正相關。質譜分析結果證實，TDC轉殖水稻之深褐色葉片與穀殼是由於血清素二聚體 (serotonin dimer) 之累積所造成的現象；發育遲緩與低稔實是因為血清素與血清素二聚體的累積所引起，而非色胺所導致的結果。本研究結果提供了初步的證據顯示，大量表現TDC之轉殖水稻具有發育遲緩與低稔實之農藝性狀，而其植株內所累積的血清素及血清素二聚體則會導致植株呈現深褐色的外表性狀。中文摘要 ……………………………………………………………………………………i
Abstract ……………………………………………………………………………………ii
List of Tables .…………………………………………………………………………….vi
List of Figures ……………………………………………………………………...……vi
List of Appendices ……………………………………………………………………..viii
Abbreviations ……………………………………………………………...…………….ix
Introduction ………………………………………………………………..…………….1
Materials and Methods
1. Plant materials and growth conditions……………………………...………..…..6
2. Bioinformatics analysis…………………………………………………….…….6
3. DNA extraction………………………………………………………..……..…..7
4. Plasmid Rescue…………………………………………………………...…..….7
5. Genotyping of the progenies of mutant M47286……………………………...…8
6. Southern hybridization………………………………………………..………….9
6.1 Genomic DNA digestion and blotting in nitrocellulose membrane ……9
6.2 Probe preparation……………………………………....................…….9
6.3 Hybridization of membrane and Autoradiography…………….……10
7. RNA extraction and RT-PCR………………………………….………………..10
8. Antibody preparation……………………………………………………………11
9. Western-Blot Analysis………………………..………………...………………12
9.1 Extraction of total rice protein……………………………..………….12
9.2 SDS PAGE………………………………………….……………..….12
9.3 Western blot…………………………………………………………...13
10. Construction of TDC overexpression vectors and plant transformation………14
11. Enzyme activity and an assay for the substrate-specificity of Tryptophan
decarboxylase………………………………………………………………….14
12. Measurement of tryptamine and serotonin…………………………………….14
13. UV catalyzed photo chemical reaction and identification of serotonin
dimer structure….………………….…………………………………...…….15
14. Pigment extraction, Liquid Chromatography and Mass Spectrometry………..16
15. Analysis of the growth effects of the tryptamine, serotonin or serotonin dimer treatments………………………………………………...…...………...16
16. Pollen Viability Assays……………………………………………....………..17
Results
1. Isolation and identification of mutant M47286……………...……..…...………18
2. Activation of TDC genes and the accumulation of tryptamine and serotonin in
various genotypes of M47286…………………………………………………..20
3. TDC-3 has tryptophan decarboxylase activity………………………………….22
4. Expression profile of TDC-1 and TDC-3 genes in rice………….…...…………23
5. Over-expressing TDCs in transgenic rice recapitulate the mutant phenotype….25
6. Polymerization of serotonin dimer caused the dark-brown colored phenotype in rice plants……………………………………….…...……………26
7. Over-expressing TDCs reduced pollen viability and affected fertilization in female gametophyte…………………………………………………….………28
8. Ectopic expression of rice TDC genes in Arabidopsis affected growth and
produce brown and sterile flower buds…….………….………………………..29
9. Exogenous serotonin and serotonin dimer affect plant growth and flower development……………………………………...……………………………..29
Discussions
1. Serotonin accumulation in TDC-overexpressing transgenic rice affects
plant growth……………………………………………..……………………...31
2. High levels of serotonin accumulation in rice plants are necessary but not sufficient for the development of the dark-brown phenotype………….………33
3. Serotonin dimer involved in brown pigmentation in TDC-overexpressing transgenic and mutant rice and at pathogen infection sites…………………….35
Tables……………………………………………………………………………..……….37
Figures……………………………………………………………………………..…...…43
References…………………………………………………………………………...……73
Appendices..........................................................................................................................8
