Function And Mechanism Of DELLA In Regulating Cotton Fiber Development

Cotton is the most important natural textile fiber crop,and provides about 35% of the world’s total annual fiber demand.Cotton fiber is a single cell originated from ovule epidermis.Cotton fiber’s development is divided into four overlapping stages: initiation,elongation,secondary wall biosynthesis and maturation.Gibberellin(GA)is an important plant hormone that regulates various growth and development processes in plant life cycle.GA has a profound effect on cotton fiber development.For example,increasing GA levels in cotton fibers results in longer mature fibers and thicker fiber cell wall.In recent years,although many researchers have analyzed the mechanism of GA regulating cotton fiber development from different perspectives,the mechanism of GA promoting fiber elongation and secondary wall synthesis in cotton has not been fully elucidated.DELLA protein is a key negative regulator of GA signaling and inhibits almost all known GA-dependent processes.In plants,DELLA protein accumulates in cells and inhibits GA response in the absence of GA;In the presence of GA,GID1 binds with DELLA protein to form GA-GID1-DELLA complex,which promotes ubiquitination and degradation of DELLA protein,and then activates GA response genes inhibited by DELLA protein,resulting in plant response to GA signal.To further explore the function of GA,especially DELLA protein,in cotton fiber development,the effects of DELLA protein on cotton stem development were preliminarily discussed.Next,a DELLA gain-of-function mutant ΔGAI was driven by promoter pro E6 specifically expressed during fiber elongation or promoter pro Fb L2 A specifically expressed during secondary wall synthesis to further investigate the roles and mechanism of DELLA protein in regulating cotton fiber development.The main results are as followed.1.DELLA regulates stem elongation and secondary wall formation in cottonFirst,the internode length and internode diameter of transgenic cotton with overexpression of gibberellin synthase gene GhGA20 ox obtained by our research group were statistically analyzed.It was found that increasing GA level could promote early internode elongation and thickening.Exogenous application of bioactive GA and GA biosynthesis inhibitor paclobutrazole(PAC)promoted and inhibited stem elongation and lignification,respectively.Western blot analysis showed that GA and PAC promoted the degradation and accumulation of GhGAI1,respectively.To further investigate the potential function of the DELLA gene family in stem development,Agrobacteriummediated virus-induced gene silencing(VIGS)was used to down-regulate the expression of GhGAIs in cotton.Compared with control,both GhGAI1&2-and GhGAI3&4-silenced cottons displayed faster plant growth and stem elongation,similar to the effects of exogenous application of GA.Microscopic observation showed that cell length,xylem and phloem of the first internode of GhGAI1&2-silenced plants were significantly increased.These results suggested that GAs regulated stem elongation and lignification in upland cotton via GhGAIs,meanwhile,both Clade Ⅰ and Ⅱ DELLA proteins were involved in this process,although Clade Ⅰ might have dominant effects on stem development.In addition,we carried out RNA-Seq analyses using the first internode to look for genes regulated by GhGAI1&2.There were 97 up-regulated genes and 94 down-regulated genes in GhGAI1&2-silenced plants.KEGG pathway analysis showed that the upregulated genes were mainly involved in phenylpropanoid biosynthesis,starch and sucrose metabolism,galactose metabolism,zeatin biosynthesis.We further employed q RT-PCR method to verify the expression levels of SCW formation related genes.It was showed that SCW-specific cellulose biosynthesis genes(Ces A4,Ces A7 and Ces A8),hemicellulose biosynthesis genes(IRX9 and IRX14),lignin biosynthesis(PAL,4CL,CCo AOMT and F5H)and polymerization genes(LAC4,PER4 and PER21)were upregulated in GhGAI1&2-silenced plants.The expression levels of these genes were also induced by GA treatment,and reduced by PAC treatment.Aforementioned results suggested that DELLA proteins mediated GA signal to regulate SCW formation by regulating the genes related to cellulose biosynthesis,hemicellulose biosynthesis as well as lignin biosynthesis and polymerization.To explore the relationship between GA-DELLA signaling and secondary cell wall formation during cotton stem development,we also examined the interaction between GhGAIs and SCW-related NAC transcription factors in cotton.The results showed that GhGAI1A/D interacted with multiple SCW-related NAC and silencing of these DAELLA-interacting NACs(Gh SND2,Gh FSNs and Gh VND4s)inhibited secondary cell wall formation of cotton stems.2.DELLA protein inhibits the secondary wall deposition of cotton fibersIn order to specifically up-regulate DELLA protein during the secondary wall synthesis of cotton fibers,a specific promoter pro Fb L2 A was selected to drive the expression of ΔGAI.Multiple pro Fb L2A:ΔGAI independent transformants were successfully obtained by genetic transformation of cotton.Further analysis of pro Fb L2A:ΔGAI transgenic cotton showed that the transcription level and protein level of ΔGAI were highly accumulated during secondary wall biosynthesis stage.Compared with the wild type,the mature fibers length of pro Fb L2A:ΔGAI transgenic cotton were not significantly changed,but the lint percent and lint index were significantly decreased.The fiber quality analysis showed that the micronaire value of mature fibers decreased significantly in four successive generations of multiple transformants.Paraffin section analysis confirmed that the change of fiber quality was caused by the thinning of mature fiber cell wall.These results indicated that the accumulation of DELLA protein during the secondary wall synthesis of cotton fiber cells inhibits the secondary wall biosynthesis and reduces the micronaire value of mature fibers.To clarify how DELLA affects the secondary wall development of cotton fibers,we recorded when wild-type and pro Fb L2A:ΔGAI transgenic cotton fibers entered secondary wall biosynthesis stage.The results showed that there was no difference in the time to enter the secondary wall between the wild-type and transgenic fibers,but the deposition intensity of crystalline cellulose was significantly weaker in pro Fb L2A:ΔGAI transgenic fibers.RNA-seq analysis of 18 DPA fibers revealed 317 differentially expressed genes(174 up-regulated and 143 down-regulated)between transgenic and control.KEGG enrichment analysis showed that starch and sucrose metabolism,pentose and glucuronate interconversions were significantly changed,indicating that the over-expression of ΔGAI affects the sugar metabolism in fiber.Many genes related to secondary wall biosynthesis,such as cellulose synthase Ces A,sucrose synthase SUS and xylan biosynthesis gene IRX,were significantly down-regulated in pro Fb L2A:ΔGAI transgenic cotton.The transcription levels of several SCW-related NAC and MYB TFs showed a downward trend in pro Fb L2A:ΔGAI transgenic cotton.So DELLA affects sugar metabolism and the synthesis of secondary wall components to regulate secondary cell wall biosynthesis.The important function of DELLA protein depends on its ability to establish protein interactions with various regulatory proteins.We detected some transcription factors that were predominanting expressed during secondary biosynthesis stage of fibers,such as Gh NAC56,Gh NAC45,Gh WRKY46,Gh BHLH110,Gh CCCH49,and Gh FNS1,which interacted with GhGAI1A/D.Among them,Gh FSN1 is a known positive regulator of fiber secondary wall development.Combined pro Fb L2A: ΔGAI and FSN1-OE transcriptome data revealed that Gh FSN1-upregulated secondary wall related genes,such as Ces A7,SUS,CTL,KOR,FLA,COBL,BXL,IRX7,and IRX15,were inhibited in pro Fb L2A: ΔGAI fibers.The interaction between GhGAI1 and Gh FSN1 causes GhGAI1 to inhibit the transcriptional activation of Gh FSN1 on its downstream target genes,such as MYBL1,KNL1,DUF231L1 and IRX12.These results indicate that DELLA interacts with positive regulatory factors of fiber secondary wall formation represented by Gh FSN1 during the stage of fiber secondary wall biosynthesis,and inhibits the expression of genes related to secondary wall biosynthesis,thereby inhibiting fiber secondary cell wall biosynthesis.3.DELLA protein inhibits cotton fiber elongationGA promotes fiber elongation.To further explore the mechanism of GA-DELLA signal regulating fiber elongation,a fiber elongation specific promoter pro E6 was uesd to drive the expression of ΔGAI.Several pro E6:ΔGAI transgenic cotton were obtained by genetic transformation of cotton.ΔGAI was specifically expressed during the fiber elongation phase in these transgenic cotton,ΔGAI protein abundance was maintained at a high level during pre-fiber development.The mature fiber length was reduced about5～12mm,and the fiber length was negatively correlated with the expression level of ΔGAI.To further understand the inhibitory effect of ΔGAI on fiber elongation,the detection of endogenous hormone levels showed that the levels of GA and IAA were significantly increased,while the levels of ABA and ethylene precursor ACC were significantly decreased in the fibers of pro E6:ΔGAI.It was also found that the levels of hexose(mainly fructose and glucose)and ROS,which are closely related to fiber elongation,were also significantly decreased in pro E6:ΔGAI fibers.Furthermore,BIFC was used to screen the DELLA interaction proteins which were expressed in cotton fiber elongation phase,and the results showed that seven transcription factors(Ghb ZIP61-1,Ghb ZIP61-2,Gh ILR3,Gh NAC31,Gh MYB66,Gh MYBL,Gh ODO1)dominantly expressed in cotton fiber elongation phase could interact with DELLA protein.These results indicate that DELLA protein interacts with a variety of transcription factors to form a complex multi-level and multi-pathway regulatory network,which suppress the positive regulation of these transcription factors on fiber elongation,and also suppress fiber elongation through hormone levels,VLCFA-ethylene pathway,glucose metabolism,ROS levels,etc.