Cotton (Gossypium Hirsutum) GhJAZ3 And GhGAI1 Functions In Jasmonate And Gibberellin Mediated Epidermal Cell Differentiation And Elongation Of Transgenic Arabidopsis

Cotton is the most important fiber crops in the world, is also the important oil crops. Cotton fiber is unicellular hair, differentiated from the ovule epidermis. Cotton fiber length is a significant trait determining cotton fiber quality. Transcription factor and plant hormone play an important role in regulating of cotton fiber cell determination and enlongation. In this study, we isolated GhJAZ3 and GhGAI1 from cotton. We analyzed their expression patterns, subcellular localization and function. The main results are as follows:1. JA and GA3 synergistically induce fiber initiationWe performed ovule culture in vitro, with the flowers were harvested at-3DPA. Different concentrations of JA, GA3, JA+GA3 were added to the BT medium to culture these ovules. After 6 days of culture in the dark, we found that JA and GA3 both promote fiber initiation, respectively. Meanwhile, JA and GA3 synergistically induce fiber initiation.2. Identification and Protein Sequence Analysis of GhJAZ3/GhGAIlGhJAZ3 has an open reading frame of 1116bp, and encodes a protein with 371 amino acid that contains a conserved Jas-domain and a conserved ZIM domain. Phylogenetic analysis showed that GhJAZ3 shares 36%and 35% sequence homology with AtJAZ3 (At3G17860) and AtJAZ4 (At1G48500). GhGAIl has an open reading frame of 1647bp, and encodes a protein wtih 548 amino acid that contains a conserved N-terminal DELL A domain and TVHYNP domain. The GhGAIl protein has 60% and 59.8% identity with Arabidopsis AtRGA (At1G07530) and AtGAIl (At1G14920).3. The expression pattern of GhJAZ3 /GhGAIl and several genes related to fiber developmentThe quantitative RT-PCR results showed that GhJAZ3 was strongly expressed in the stage of 6 to 12 DPA fiber, and at relative high levels in the early stage (3DPA) and secondary cell wall stage (15-20DPA), but no or weak signals were detected in the other tissues. GhGAI1 was widespread expressed in the cotton tissues. The expression of GhGAI1 reached the top at 3 DPA fiber, and gradually reduced from 6DPA fiber. GhDEL65, GhPIF3, GhBZR1 and GhARF6 were also preferentially expressed in cotton fibers. These results suggested that these genes may be involved fiber development.4. The subcellular localization of GhJAZ3 and GhGAI1We constructed the fusion expression vectors of 35S-eGFP-GhJAZ3 and 35S-GhGAI1-eGFP. We transformed the vector to agobacterium and injected agobacterium to tobacco leaf epidermal cells. Green fluorescent signals were detected in cell nuclei of the tobacco leaf epidermal cells by the confocal microscope. These results suggested that GhJAZ3 and GhGAIl protein were localized in the cell nucleus.5. Transcriptional activation analysis of GhJAZ3 and GhGAI1We created the fusion construct of pGBKT7-GhJAZ31 GhGAI1/GhGAI1 △N. The fusion vector was transformed into yeast strains AH 109 and Y187. The full-length GhGAI1 Protein displayed strong transcriptional activation activity, while GhJAZ3 protein lacks the activity of transcriptional activation. GhGAI1 AN, which is a N-domain deletion version of GhGAI1, was cloned into pGBKT7, showed no transcriptional activation.6. GhGAI1 Protein interact with GhJAZ3 and several putative DELLA-bingding proteinsWe constructed the fusion yeast expression vector of pGBKT7-GhGAI1 △N and pGBKT7-GhJAZ3. The fusion vector were transformed into yeast strains Y187. Meanwhile, we also created the fusion expression vector pGADT7-GhGAIl, pGADT7-GhDEL65, pGADT7-GhDEL65NT, pGADT7-GhDEL65CT, pGADT7-GhARF6, pGADT7-BZRl and pGADT7-GhPIF3, which were transformed into yeast strains AH109. According to yeast two-hybrid expeimients, we found that GhGAI1 interact with GhJAZ3, GhPIF3, GhARF6 and GhBZR1. Meanwhile, we found that GhJAZ3/GhGAI1 interact with N-terminal fragment of GhDEL65.7. Phenotypes of Transgenic Arabidopsis Plants Overexpressing GhJAZ3We generated the vector of 35S-GhJAZ3 and 35S-GhJAZ3AC. By agrobacterium mediated transformation of Arabidopsis thaliana, we got GhJAZ3/GhJAZ3AC transgenic plants. Hypocotyls of the GhJAZ3/GhJAZ3AC transgenic plants were longer than those of wild type when these seedlings grown in dark for five days. GhJAZ3/GhJAZ3AC transgenic plants showed longer root length than wild type, produce the early flowering phenotype, and the transgenic plants increased plant height in comparison than the wild type plants. The results of quantitative RT-PCR (qRT-PCR) showed that EXP8, EXPL2 and XTH4 levels were up-regulated in the GhJAZ3/GhJAZ3AC transgenic plants. The GhJAZ3/GhJAZ3AC transgenic plants displayed less trichome number, but showed longer trichome length than the wild type. Eepression of GL2 and MYB23 was down-regulated in GhJAZ3 and GhJAZ3 AC transgenic plants.8. Phenotypes of Transgenic Arabidopsis Plants Overexpressing GhGAI1We generated the vector of 35S-GhGAI1, and obtained the GhGAI1 transgenic plants. GhGAI1 transgenic plants displayed shorter hypocotyls and primary root than wild type. GhGAIl transgenic lines were much more sensitive to JA than the wild type plants. Quantitative RT-PCR results indicated that EXP8, EXPL2 and XTH4 mRNA levels were lower in the GhGAI1 transgenic plants. The GhGAI1 transgenic plants displayed less trichome number and shorter trichome length than the wild type. GL2 and MYB23 mRNA levels were lower in GhGAI1 transgenic plants. Overexpressing GhGAI1 plants showed less root hair than the wild type.9. GhJAZ3 and GhJAR1-8 transgenic cottonWe constructed the overexpression vector of PBI-RDL1-GhJAZ3 and 35S-GhJAR1-8. GhJAR1-8 encodes an enzyme that is invovled in the JA biosynthesis pathway. By agobacterium LBA4404 mediating cotton genetic transformation, we got nine cotton embryonic callus of RDL1-GhJAZ3 and a large number of cotton callus of 35S-GhJAR-8. The results we have got laid a solid foundation to study the function of JA in cotton fiber development.The above results demonstrated that JA and GA synergistically induce fiber initiation, GhJAZ3 and GhGAI1 interact with GhDEL65, GhJAZ3 and GhGAI1 transgenic plants both displayed less trichome number, lower expression of GL2 and MYB23. These results shows that initiation mechanism of cotton fiber similar to that of arabidopsis trichome initiation, GhJAZ3 and GhGAI1 may mediate the synergistical promotion of JA and GA in the period of cotton fiber initiation. In the stage of cotton fiber elongation, GhGAI1 interact with GhJAZ3, and attenuate the repression of GhGAI1 on its downstream transcription factors to promote cell elongation.