GhMAKR3, A Boll-preferential Gene, Is Involved In The Development Of Cotton Bolls

Cotton is the leading natural fiber crop in the world, and the production of cotton is a mainstay of China’s national economies. The cotton yield is a composite trait, which is influenced by many factors, especially the development of cotton bolls. Thus, to improve the yield of cotton, we need to pay attention to the development of cotton boll where seeds and fibers are produced. However, little is known about genes that are involved in the development of the boll of cotton.MAKRs(Membrane Associated Kinase Regulator), a kind of membrane-associated protein with kinase regulatory elements, may regulate distinct signaling pathways. Overexpression of MAKR1 had a phenotype similar to overexpression of BKI1 in Arabidopsis, suggesting that MAKR1, like BKI1, negatively regulates BRs signaling. MAKR4 is a molecular component that acts downstream of the IBA-to-IAA conversion pathway in the root tip and converts the prebranch sites into lateral organs along the primary root. However, so far, the function of MAKRs proteins in development of plants has been largely unknown.In this paper, to reveal the function of MAKRs in cotton, a gene that preferentially expressed in cotton bolls named GhMAKR3 was cloned. The expression pattern of GhMAKR3 and the GUS pattern of its upstream sequence were studied. Next, the phenotypes of transgenic cottons in which GhMAKR3 was up/down-regulated were observed. The main results are as follows:1. Homology analysis and the expression pattern of GhMAKR3A gene that preferentially expressed in cotton bolls was cloned according to ESTs of Vitis vinifera. NCBI blast showed that the gene belongs to MAKRs protein family, thus nominated GhMAKR3. Sequence analysis showed that the gene is 1715 bp long, containing a complete ORF(Open Reading Frame, 1035 bp) and encoding 344 amino acid residues. The amino acid sequence of GhMAKR3 is similar to that of Citrus sinensis, Vitis vinifera, Malus domestica, Nelumbo nucifera and Prunus mume. Phylogenetic tree analysis indicated that GhMAKR3 has a closer genetic relationship to CsMAKR4-like, PmMAKR4, MdMAKR4 and an unknown protein in Theobroma cacao.Relative expression level of GhMAKR3 detected by qRT-PCR was higher in seeds, fibers andboll shells than other organs. The gene showed relatively high expression level in young bolls(maximizing at 1 DPA, Days Post Anthesis), and early-stage ovules(maximizing at 6 DPA), and in initiating and fast elongating fibers(maximizing at 7 DPA), respectively.2. Regulatory elements and GUS pattern of GhMAKR3 promoterThe upstream promoter sequence of GhMAKR3 contains a variety of cis-elements, including tissue specific expression components related to the development of endosperm, pollen and root, and elements responsing to phytohormones, e.g., auxin, gibberellin and brassinosteroids, and to environmental signaling such as light, heat and drought. GUS staining in the tissues of pGhMAKR3::GUS transgenic cotton plants showed the activity was higher in petals, stamens and cotton bolls, than that in leaves, petioles and pistils, roughly consistent with the results of GhMAKR3 transcript detection in cotton.3. The expression of GhMAKR3 and BRs signaling pathwayThe expression level of GhMAKR3 was enhanced by adding BRZ(Brassinazole, an inhibitor of BR synthesis) to cotton ovule(0 DPA) culture medium. However, the expression was suppressed by the active Brassinolide(BL). In GhMAKR3 up-regulated cotton plants, GhBRI1, an upstream gene in BRs signaling pathway increased slightly, and no obvious change of GhBKI1 was observed.However, expression levels of GhBSU1 and GhBIN2, the downstream genes in BRs signaling pathway, were dramatically increased, while the expression level of GhBZR1 decreased. In the down-regulated(GhMAKR3RNAi) plants, expression of GhBRI1, GhBSU1 and GhBIN2 decreased level, while that of GhBKI1 and GhBZR1 increased. The results suggested that there may be a crosstalking between GhMAKR3 and BRs signaling.4. GhMAKR3 to fiber-development associated MYB transcription factorsSome MYB transcription factors are involved in the fiber development. The expression levels of GhMYB25-like, GhMYB25 and GhMYB109 were down regulated in GhMAKR3-overexpressing plants, contributing to a lower initial density of fiber on 0 DPA ovules than that of wildtype control at the same time. Meanwhile, in GhMAKR3 RNAi plants, the expression of GhMYB109 was up-regulated, and the levels of GhMYB25-like and GhMYB25 had no much changes. The initial density on 0 DPA ovules did not changed significantly. Therefore, changes of GhMAKR3 expression level may affect fiber initiation through influencing expression of MYB transcription factors.5. Down-regulation of GhMAKR3 promoted sugar transporting to seedsThere were no significant changes in total sugar content in mature seeds of overexpressioncottons. However, the content was raised in the RNAi plants, and expression levels of GhSUT1-9increased too, suggesting that down-regulation of GhMAKR3 can promote sugar transportation.6. Up-regulation of GhMAKR3 decreased the size of bolls and seeds, while down-regulation increased the sizeIn GhMAKR3 up-regulated cotton plants, the boll and seed size was smaller than that of the wildtype control, and the number of cotton bolls per plant decreased too, whereas, GhMAKR3down-regulation led to an increase in boll and seed size.The results demonstrated that GhMAKR3 is a boll-preferential gene; up-regulation of this gene could suppress the boll and seed development, while down-regulation promoted it. Based these datas,it is suggested that there may be a negative relationship between GhMAKR3 expression and the level of BRs. That is to say up-regulation of GhMAKR3 is likely as the phenoptype of BRs decreased cotton, while down-regulation of it as BRs enhanced cotton.