Mechanism In The Inhibition Of Vegetative Branching By High Plant Density And Shading As Well As The Function Of Gene GhNAC4 In Cotton

Cotton has a complex branching pattern,consisting of the distal fruiting branch(FB)and basal vegetative branch(VB).Due to the inhibitory effects arised from vigorous growth,as well as lower boll-setting rate,boll weight and poor fiber quality in VB,manual removal of vegetative branches at early squaring has been widely adopted as an important measure in cotton production for decades in China.However,such labor-consuming practice has confronted severe challenges as a result of society development and fast urbanization.Thus,it is necessary to find an alternative to replace manual plant pruning.Research and practice has indicated that the outgrowth of VB can be considerably inhibited by high plant density;however,the underlying physiological and molecular mechanisms of the inhibition are poorly understood.Various factors are involved to regulate jointly the growth and development of VB.Altered env:ironment factors and the resulting differential expression of genes related to hormone synthesis and metabolism affect the VB growth.As a unique transcription factor in plants,NAC plays a key role in regulating the organogenesis,branching growth and responses to series of stresses.Over-expression of NAC considerably affects the branch number in rice and Arabidopsis.Therefore,revealing the underlying mechanism of how plant density regulates VB growth and development as well as the function of NAC is cruicial for effectively regulating the growth of cotton vegetative branch.Based on that,three experiments were conducted as follows:Firstly,three density gradients,low(3 piant/m2),moderate(6 plant/m2)and high(9 plant/m2)plant densities were arranged in 2015 and 2016.The growth and development as well as fruiting in VB under the three plant densities were comparatively examined.Photosynthesis of VB leaves,hormone-related gene expression and corresponding hormone contents in both the main-stem and VB tips were analysised to reveal the mechanism that high plant density inhibits VB growth and development.Secondly,since light deficiency under high plant density occurs mostly in plant basal parts,its effect on the latter’s leaf and branch growth and development process ought to exceed that incurred in upper parts.Therefore,a VB shading study was conducted at squaring stage during 2017-2018 to further clarify whether high plant density indeed inhibits VB growth due to light deficiency in the lower canopy stratum.Effects of shading on growing status of VB,photosynthesis of VB leaves,hormone-related gene expression as well as corresponding hormone contents were compared with that of control to determine whether or not the inhibition occurs through a similar mechasim as high plant density.Thirdly,the GhNAC4 gene in cotton leaf was cloned by using the specific primers and PCR.GhNAC4 was silenced through VIGS technology in cotton and over-expressed in Arabidopsis and cotton to determine its function in regulation of branching as well as the resistance to salt and drought stress.The main results and conclusions are as follows:1.High plant density inhibits vegetative branching in cotton by decreasing photosynthesis by shading and inhibiting phyB gene expression which alter hormone-related gene expression as well as the corresponding hormone contents1)High plant density significantly inhibited the growth and development of VB.The ratio of VB to total biomass and the number of VB were decreased by high plant density.Compared with low plant density,high plant density reduced the ratio of VB to total biomass and the number of VB by 95.0 and 67.3%at 125 days after seeding(DAS),respectively.2)High plant density significantly inhibited the photosynthesis in VB leaves High plant density decreased the Pn rate by 76.4%at 110 DAS and 83.7%at 125 DAS,RuBP carboxylase activity by 28.1 and 33.2%,chlorophyll contents by 62.8 and 68.6%,soluble sugar content by 25.8 and 26.4%,and starch content in the VB leaves by 44.4 and 40.0%respectively,compared with low plant density.3)High plant density affected the hormone-related gene expression,as well as corresponding hormone contents in the main-stem tips.Meanwhile,the hormone-related gene expression and corresponding hormone contents was hindered through inhibiting phyB.The expression level of IAA biosynthesis gene GhYUC5,IAA transport gene GhPIN1 and CTKs biosynthesis gene Gh1PT3 in the main-stem tips increased with increasing plant density.High plant density increased the expression of GhYUC5 by 62.5%at 110 DAS and 43.7%at 125 DAS,GhPIN1 by 34.1 and 21.5%,and GhIPT3 by 79.7 and 82.9%respectively.High plant density increased the IAA content by 43.9%at 110 DAS and 31.3%at 125 DAS and CTKs content by 29.7 and 38.6%respectively.In contrast,the expression of GhphyB gene in VB tips was by 52.3%at 110 DAS and 62.3%at 125 DAS.The hormone-related gene expression,as well as IAA,CTKs,GA and BR contents in the VB tips decreased with increasing plant density.High plant density decreased the expression of GhYUC5 in the VB tips by 65.8%and 57.0%,GhPIN1 by 61.4 and 60.6%,GhPIN5 by 86.1 and 72.7%,and GhIPT3 by 63.8 and 66.8%,respectively,compared with low plant density.High plant density increased the expression of strigolactone receptor gene GhD14 in VB tips by 57.1%at 110 DAS and 57.8%at 125 DAS.However,it decreased the sucrose biosynthesis gene GhCYFBP in VB leaves by 67.3%at 125 d,compared with low plant density.High plant density reduced the IAA content by 45.3%at 110 DAS and 27.9%at 125 DAS,CTKs content by 38.3 and 25.7%,GA content by 23.1 and 38.2%,and BR contents by 28.8 and 21.5%,respectively.However,the content of SLs in VB tips increased by 25.3 and 31.2%,respectively.2.VB shading substantially inhibits vegetative branching by altering leaf photosynthesis and affecting the hormone-related gene expression,as well as hormone contents of cotton plants by reducing the phyB gene expression level which was not unlike that of high plant density1)Shading significantly inhibited the growth of VB.Compared with control,the shading treatment decreased the VB number by 54.2%at 55 DAS,53.6%at 62 DAS and 56.2%at 69 DAS,respectively;the corresponding reductions for VB length were 40.8,75.8 and 88.8%and for the ratio of VB to total biomass they were 94.9,89.7 and 91.3%,respectively.By contrast,the accumulated biomass of FB increased by 2.0%at 55 DAS,18.5%at 62 DAS and 30.8%at 69 DAS.2)Shading considerably inhibited photosynthesis in the VB leaves.Compared with control,shading decreased the Pn rate by 42.7%at 55 DAS,86.3%at 62 DAS and 96.4%at 69 DAS;likewise,RuBP carboxylase activity by 23.7,29.1 and 28.0%;the chlorophyll contents by 46.7,44.1 and 82.0%;soluble sugar content by 74.3,48.9 and 51.7%;and starch content of VB leaves by 80.0,58.7 and 86.0%respectively.However,photosynthetic production in the main-stem leaves was considerably increased under shading from 55 to 69 DAS.Compared with control,shading increased the Pn rate by 12,2%at 55 DAS,15.1%at 62 DAS and 12.2%at 69 DAS;likewise,RuBP carboxylase activity by 13.2,5.4 and 9.8%;the chlorophyll contents by 10.0,16.2 and 18.4%;soluble sugar content by 21.1,19.2 and 22.5%and starch content of the main-stem leaves by 12.3,37.4 and 38.1%,respectively.3)Shading also affects hormone-related gene expression and corresponding hormone contents arose from decreasing the accumulation of phyB in VB tips.The expression of the IAA biosynthesis gene GhYUC5,IAA transport genes GhPINl and GhPIN1,CTKs biosynthesis gene GhIPT3,sucrose biosynthesis genes GhCYFBP and GhphyB in the VB tips and leaves all decreased under the shading treatment.Specifically,shading diminished the expression of GhphyB at 55,62 and 69 DAS was lowered by 26.5,32.7 and 56.1%,respectively,GhYUC5 by 79.1%at 55 DAS,86.4%at 62 DAS and 91.2%at 69 DAS,of GhPIN1 by 55.5,76.1 and 81.9%,of GhPIN5 by 23.6,77.1 and 82.2%,GhIPT3 by 41.5,94.2 and 92.9%,respectively.Similarly,the expression level of GhCYFBP likewise reduced by 87.7,88.6 and 96.3%.By contrast,shading correspondingly increased the expression of GhD14 in the VB tips by 11.7,32.7 and 56.1%compared with the control.The auxin(IAA),cytokinins(CTKs)and brassinolides(BRs)contents in the VB tips were significantly decreased by VB shading.At 55,62 and 69 DAS,shading respectively reduced the IAA content by 33.8,19.7 and 40.8%,the CTKs content by 19.3,12.6 and 24.1%and the BR contents by 24.6,26.2 and 27.8%.By contrast,the content of SLs in VB tips correspondingly increased by 21.5,30.5 and 28.6%.3.Although GhNAC4 was not involved in direct regulation of Arapidopsis branching,it delayed plant senescence and positively regulated the resistance of salt and drought stress in cotton1)Different protein sequences from series species were compared through Blastp analysis.The open reading frame of GhNAC4 gene was 1041 bp.2)GhNAC4 was over-expressed in Arabidopsis thaliana.Homozygous transgenic lines were selected and used for detailed analysis.Further phenotypic analysis of homozygous lines showed that there was no difference in the branch number and bolting time,compared with wild type.And the promoter of GhNAC4 was not expressed in the meristematic tissues,which indicated that GhNAC4 cannot regulate directly the branch growth.However,the senescence degree of lotus leaves was significantly different in 3-month plant,compared with that of wild type.Meanwhile,the resistance of transgenic plants to salt and drought stress was enhanced indicating that GhNAC4 gene may be involved in the regulation of plant senescence and stress responses.GUS staining showed that the expression level of GhNAC4 promoter was also induced by salt and drought stress,suggesting that the promoter of GhNAC4 was a stress-inducible promoter.The detailed analysis of the GhNAC4 protein location indicated that the GhNAC4 protein was located in the nucleus of onion.3)The expression pattern of GhNAC4 gene in cotton under different abotic stresses was analyzed.Results showed that the expression level of GhNAC4 was influced by salt and drought stress as well as stress tolerance signals such as hydrogen peroxide and ABA in cotton leaves and roots,indicating that GhNAC4 participate the regulation of response to the stresses.4)VIGS technique was used to silence GhNAC4 gene in cotton.The analysis of stress tolerance of silent lines showed that GhNAC4 gene silencing increased the sensitivity of cotton to salt and drought stress.These results indicated that GhNAC4 gene played an important role in promoting the resistance of stress in cotton.In summary,this study ascertained the inhibition effect of high plant density on VB growth and development,and revealed the underlying physiology and molecular mechanism.Furthermore,we confirmed that VB shading also restrained the VB growth through a similar mechanism as high plant density was in the inhibition of vegetative branching.The results suggested that the inhibited vegetative branching was mainly attributed to light deficiency in lower canopy under high plant density.Although NAC affects tillering or branching growth in rice and Arabidopsis thaliana,NAC4 originated from cotton did not directly involved in the regulation of branching.However,GhNAC4 delayed in leaf senescence and positively regulated the drought resistance and salt tolerance in Arabidopsis thaliana or cotton.Thus,our results provided a theoretical guidance and reference for effectivily controlling the growth of VB.