Functional Characterization Of Abscisic Acid8’-Hydroxylases Gene From Upland Cotton (Gossypium Hirsutum L.)

Phytohormones play critical roles in the growth and development of plant. Similarly, they also play important roles in the development of cotton fiber cell, which is one of the longest cells ever characterized in plant kingdom. Many studies have illuminated the functions and regulatory mechanism of phytohormones such as auxin, gibberellin, brassinosteroids, and cytokinins in fiber cell. Compared to these hormones, which can promote plant growth, studies on the function and mechanism of abscisic acid (ABA) was fewer in the cotton fiber development. ABA is a kind of important plant hormone, and plays important roles in plant growth and development as well as in stress responses. Interestingly, there is high ABA content in fiber cell, which is a single cell elongated extremely. This indicates that ABA participates in the regulation of the development of fiber, but the specific function and related mechanism are not clear.The level of ABA is determined by synthesis and catabolism in plants. Previous studies indicated that ABA catabolism is the main way to regulate ABA level. ABA8’-hydroxylases (ABAH) is a key enzyme in ABA catabolism and negatively regulates the ABA level. To reveal the effect and action mechanism of ABA in cotton plant and fiber development, and also to provide more effective genes for gene engineering by which the yield and quality of cotton fiber can be improved, we cloned ABAH homologous genes and their5’-regulated sequence from upland cotton (Gossypium hirsutum L.). The plant expression vectors were constructed and the genetic transformation for tomato and cotton were performed. The gene biological function was verified in transgenic tomato firstly. Transgenic cotton plants in which overexpressing or suppressing GhABAH were obtained by genetic transformation. We have detected the ABA levels and the changes of fiber growth. The relationship among the changes of gene expression, ABA levels, and fiber growth were determined. The effects of GhABAH on regulating fiber growth and development were illuminated. These studies will provide related data for further research on the regulatory mechanism of fiber growth. The following results are obtained:1. GhABAH gene was cloned from upland cotton (Gossypium hirsutum. L)The cloned fragment was1768bp, containing an integral ORF which length was1401bp, a5’-UTR length of58bp and3’-UTR length of309bp. The length of deduced amino acid sequence was466amino acid residues. The protein molecular weight was53KD, and its isoelectric point was9.254. The gene was named GhABAH. The length of genomic sequence of GhABAH was2509bp, containing7exons and6introns. GhABAH protein was a membrane protein, with a transmembrance domain which length was23AA at the N-terminal. GhABAH protein had a high homology to other species’. The identity of GhABAH with the ABAH from Populus trichocarpa, Vitis vinifera, Arabidopsis thaliana and Ricinus communis was85%,85%,79%and85%, the similarity was92%,93%,88%and92%, respectively. In D-subgenome, there were two sequences (GrABAH1and GrABAH2) were existed to encode ABA8’-hydroxylase. One of them, GrABAHl was high similarity with GhABAH. Phylogenetic analysis showed that cloned GhABAH had a very high similarity with Theobroma cacao and Ricinus communis. These results indicated that the cloned GhABAH was ABAH homologous genes in upland cotton.2. The expression pattern of GhABAH gene in upland cotton plantGene expression pattern is an important data to reveal gene function. In order to understand the relationship of GhABAH with the growth and development of cotton plant and fiber cell, we had detected the expression pattern of GhABAH in root, stem, leaf and other different organs and tissues, and also in the fiber cells at different developmental period. Results showed that the GhABAH expression level was much higher in the root than stem and leaf. At various developmental stages of fiber cell, there was a high expression level at the early stage of fiber elongation (6DPA). With the increase of fiber elongation rate, the expression level was reduced gradually and reached the bottom at12DPA, which was a rapid elongation stage of fiber cell. Then with the decrease of fiber elongation rate, the expression level of GhABAH was increased and reached the top at18DPA in which the secondary cell wall started to deposit. These results revealed that the expression pattern of GhABAH is depended on the development of fiber cell. Meanwhile, GhABAH expression was induced by ABA, and inhibited by BRs and IAA. Furthermore, there were expression different between Xuzhou142and its’lintless-fiberless mutant either in-3DPA or0DPA ovules. So the gene was also involved in the fiber cell differentiation and initiation. The expression patterns of GhABAH showed that the gene had a close relationship with fiber cells differentiation, initiation, elongation, and secondary wall formation.3. Functional characterization of GhABAH promoter by transgenic tobaccoTo understand accurately the temporal and spatial expression pattern and the regulatory factors to affect GhABAH, the5’-upstream regulatory sequence of2013bp of the gene was cloned. Sequence analysis showed that the sequence contained a number of light responsive elements and enhancer sequences, anaerobic response element, fungi response element, endosperm expression sequence, defense and stress response element, ABA response element, ethylene response element and so on. These indicated that the expression of GhABAH was affected by various factors. Then we have constructed a plant expression vector in which the GUS gene was controlled by the target promoter. Through detecting the GUS activity in transgenic tobacco, the results showed that1811bp and1094bp promoters have no obvious differences in expression patterns. It illustrated the core region of GhABAH promoter was located in1094bp. In seedling of transgenic tobacco, the GUS activity was detected in the root. While in mature plants of transgenic tobacco plant, the GUS signal was detected in the root, stem, leaves, flower and epidermal hair. The GUS signal was focused on young root fragment. No signal was detected in old root and stem medulla. These indicated that GhABAH expression was depended on the development and tissues.4. Analysis the function of GhABAH gene by transgenic tomatoSince the genetic transformation of cotton is time-consuming, we firstly identified the biology function of GhABAH through transgenic tomato. The phenotypic variation of transgenic tomato plants was observed and the content of ABA was detected either in transgenic or wild type plants. Ectopically expressing GhABAH led to endogenous ABA levels decreased by57.89%in transgenic tomato. When compared with wild type, the plant height of the transgenic tomato decreased by29%, leaves were wilting and easy to dry up, stomata can’t close normally, leaves color was dark green, chlorophyll content increased by72%～124%, pollen sterility was reduced, no seeds in the fruit or seeds number was decreased, the fruit coloring and plant senescence were advanced, lateral root number increased by102%. These results suggested that ectopic expression of GhABAH can promote the ABA metabolism, reduced the endogenous ABA content and impacted the growth of tomato, and illuminated the gene was a functional gene.5. Modifying the expression of GhABAH affects the growth of cotton plantsThe phenotypic variation of transgenic cotton plants (TO generation) was characterized and the content of ABA was detected either in transgenic or wild type plants. Compared with wild type, the endogenous ABA content of transgenic cotton was reduced by63.1%, plant were slender, branches were shorter, leaves and bolls were smaller, leaves were wilting, stomata can’t close normally, blooming time was delayed, petals and sepals were smaller, styles elongated, pollen grain not spill out, pollen sterility were reduced. Meanwhile, lateral root number increased by200%under the condition of200mm NaCl in T1generation seedlings. On the contrary, suppressing GhABAH expression level led endogenous ABA level to increase by54.4%, and increased ABA sensitivity in T1generation seedlings. These results indicated that modifying the GhABAH expression level can affect plant endogenous ABA levels and the growth and development of cotton plants. These suggested that ABA plays important regulatory roles in the growth of cotton plants.6. Modifying the expression of GhABAH affects the growth and development of fiber cellOverexpressing GhABAH inhibited fiber cell initiation and elongation in transgenic cotton. Mature fiber length of transgenic cotton was2.64±0.03cm. Compared with wild type, their fiber length was2.97±0.02cm, transgenic fiber was shortened by11.1%, the secondary wall was thicker, the micronaire increased by31%, the fiber strength reduced by20%. At the same time, the transgenic cotton fiber were more resistant to ABA than wild type, including that the degree of transgenic cotton fiber inhibition treated by5μM ABA for10days was lower than wild type, by27%and58%, respectively. Meanwhile, ABA synthesis inhibitors affected transgenic fiber growth less than wild type(23%～25%compared with43%-59%increase, respectively), indicated that the appropriate ABA content was essential for fibre elongation. ABAH specific inhibitors significantly inhibited the growth and development both of fiber and ovule. However, suppressing GhABAH expression didn’t have obvious effect on the mature fiber length, but secondary wall was thicker significantly. Results showed that change of GhABAH expression level not only affected fiber differentiation, initiation and elongation, but also influenced the formation of the secondary wall, and proved that the gene had an important role in regulating the growth and development of cotton fiber.