The Molecular Mechanism Of GhHUB2 Involved In Cell Elongation And Secondary Wall Formation In Developing Cotton Fibers

Cotton fiber is the most important natural and renewable resource used in the textile industry.The fibers are highly elongated and thickened single cells derived from epidermal cells on the outer integuments of the ovules.The development of cotton fiber can be divided into four distinct but overlapping stages:initiation,elongation,secondary cell wall(SCW)synthesis and maturation.Fiber development involves active ranscription and metabolism.Each stage contributes to the formation of fiber traits.Identification and characterization of genes involved in fiber development are of great impotance not only for understanding the mechanism underlying fiber development,but also for cotton molecular breeding.In this study,a C3HC4 RING-finger ubiquitin ligase gene,GhHUB2,was cloned from Gossypium hirsutum.GhHUB2 was constitutively expressed in cotton,including within fibers.Transcient expressin of GFP-GhHUB2 in Arabidopsis protoplast demanstrated that GhHUB2 localized to the nucleus.In vitro ubiquitination assay revealed that GhHUB2 had ubiquitin ligase activity.Overexpressing GhHUB2 in Arabidopsis hub2-2 mutant who has lost the function of AtHUB2 rescued its early flowering phenotype,indicating GhHUB2 is a fonctional homologous gene of AtHUB2.To study the role of GhHUB2 in cotton development,both GhHUB2 overexpressing and knockdown cotton lines were obtained via Agrubacterium-mediated transformation.GhHUB2 influenced flowering time as well as fiber development in transgenic cotton.The analysis of cotton fiber in the T3 generation showed that compared with that of wild-type,the mature fiber length of the GhHUB2 overexpression lines increased 7.49%to 8.99%.In contrast,the length of GhHUB2 knockdown fibers was much shorter than that of wild-type fibers,exhibiting a 5.85%to 8.24%reduction.In addition,overexpressing GhHUB2 resulted in elevated content of cellulose and lignin in fibers as well as thicker fiber cell walls.In contrast,the content of cellulose and lignin as well fiber cell wall thickness were significantly reduced in GhHUB2 knockdown fibers.To have an insight into the molecular mechanism that GhHUB2 triggers fiber development,we screened cotton cDNA library using full length GhHUB2 cDNA as bait.GhKNL1,a trasnscriptional repressor predominantly expressed in cotton fiber,as well as components involved in H2B monoubiquitiantion was characterized.GhHUB2 and GhKNL1 co-localized to the nucleus when transient expressed in Arabidopsis protoplast.The interaction between GhHUB2 and GhKNLl was verified by yeast two-hybrid,firefly luciferase complementation and Co-IP assay.GhHUB2 ubiquitinates GhKNLl in vitro and promotes the degradation of GhKNLl through the ubiquitin-26S proteasome pathway as demonstrated by in vivo and semi-in vivo degradation assays.Futher immunoblot assay using specific antibody ageainst GhKNL1 showed that compared with wild-type,the GhHUB2 overexpression lines always exhibited less GhKNL1 protein accumulation throughout cotton fiber development,while the protein levels of GhKNL1 were elevated in the GhHUB2 knockdown lines.RT-qPCR showed that GhHUB2 had no influence on the transcription of GhKNL1 in developing fibers.This indicates that GhHUB2 directs the degradation of GhKNL1 in cotton fibers.The result was also confirmed by cell-free degradation assay.Further study revealed that the expression of genes which are responsible for fiber elongation such as GhXTHl and Gh1,3-?-G,as well as genes that participating in SCW synthesis such as Gh CesA4,Gh4CL1,GhCAD6,GhAGP4,GhCTL1 and GhCOBL4 were significantly upregulated in GhHUB2 overexpressing cotton fibers.Opposite results were found in GhHUB2 knockdown lines.In addition,we identified that GhKNL1 directly binds the promoter and inhibits the expression of GhREV,a homodomain transcription fator involved in SCW synthesis and auxin signaling.The expression of GhREV was elevated in GhHUB2 overexpressing fibers and decresed in GhHUB2 knockdown lines.In conclusion,a novel function of GhHUB2 was uncovered in addition to its role in monoubiquitination of H2B.GhHUB2 ubiquitinates GhKNLl and promotes the degradation of GhKNLl via the ubiquitin-26S proteasome pathway.GhHUB2 negatively regulates GhKNLl and leads to the disinhibition of genes involved in fiber elongation and SCW synthesis.GhHUB2 and GhKNL1 participate in the fine tuning of fiber elongation and SCW synthesis to maintain the homeostasis of fiber development.