Functional Studies Of GhLDOX&Gh4CL3 Genes In Upland Cotton Gossypium Hirsutum

Anthocyanins are a class of plant flavonoid pigments involved in many biological processes.LDOX is a late gene in the anthocyanin biosynthetic pathway that encodes leucoanthocyanidins and convers it to anthocyanidins.Up to date,there no any functional study has clarified the role of LDOX gene in cotton development.In this study,to functionally characterize Gh LDOX gene in cotton,targeted mutants have been induced in the coding sequence of Gh LDOX gene through CRISPR-Cas9 system,at the same time;the Gh LDOX gene expression has been enhanced through overexpression technology.Results revealed that CRldox mutated plants exhibited a complete absence of anthocyanin pigment whether in the vegetative tissues or in flowers,while OELDOX overexpressing plants showed enhanced accumulation of total anthocyanins.The changes occurred in anthocyanin content is due to the modification in the expression of Gh LDOX gene.Further,to understand the anthocyanin role in cotton trichomes development,cotton leaf-and seed-trichomes have been detected in Gh LDOX transgenic plants.Remarkably,we found a positive correlation between the initiation and elongation of both types of cotton trichomes with anthocyanin production.The absence of anthocyanin in CRldox mutants was associated with a notable reduction in leaf-and seed-trichomes initiation and elongation while OELDOX rich-anthocyanin plants showed improved growth in leaf-and seed-trichomes.To understand the molecular mechanism in Gh LDOX transgenic plants,we have conducted transcriptomic analysis using leaves of the studied lines.Results showed that,by the suppression of Gh LDOX;genes involved in flavonoids biosynthetic pathway have been differently expressed in which most genes along the pathway showed low FPKM values in anthocyaninless samples.However,few genes have been upregulated such as Gh CHS,Gh4 CL,Gh CCR and Gh CCo AOMT in CRldox plants.While the transcript of OELDOX showed no notable differences in flavonoids biosynthesis pathway genes.Only several genes have been significantly upregulated such as Gh4 CL,Gh HCT,Gh C3 H and Gh CCo AOMT.These results indicated that the Gh LDOX suppression impacted the transcriptional levels of most flavonoids biosynthesis genes and has relation in the transcriptional regulation of genes in phenylpropanoid biosynthesis.At the same time,auxin levels in these plants have been altered as well.Anthocyaninless mutants showed great decrease of auxin content in plant leaves,ovules and fibers,this reduction was coupled with a decrease in plant metabolic pathway and trichomes defect.In contrary,when enhanced,anthocyanin overexpressed plants displayed the highest accumulation of IAA at the initiation stage of fibers(0 DPA)and elongation stage at(20 DPA).Comparing these findings with our transcriptomic data,it was affirmed that most of auxin signaling pathway have been positively modulated coupled with anthocyanin content in Gh LDOX plants.Gh ARF2 and Gh ARF18,also known as INDOLEACETIC ACID-INDUCED PROTEINS(IAA14 and IAA16)were differentially modulated in Gh LDOX transgenic plants.In anthocyaninless CRldox plants,these two genes were greatly down regulated and in contrast showed higher transcript level in OELDOX plants.Our findings conclude that the anthocyanin encoded by Gh LDOX gene plays a potential role in cotton trichomes development mediated auxin-signaling pathway.Moreover,anthocyaninless mutations exhibited obvious reduction in several pathways related to photosynthetic activity.Based on(KEGG)functional enrichment analysis,it was observed that porphyrin and chlorophyll metabolism pathways have been suppressed in addition to photosynthesis pathway.These reductions were correlated with significant reduction in plant chlorophyll content and pale plants.These results indicated that anthocyanin pigment can protect plant photosynthetic apparatus by balancing light access to the photosystem.Absence of anthocyanin would increase the oxidative elements in plant tissue that led to photosynthesis inhibition.The reduction in the carbohydrates exhibited in anthocyaninless mutants is due to the low chlorophyll content and anthocyanin deficiency,the available carbon is thought to be redirected to different substrates to compensate the reduction of nitrogen and carbon resources.The notable reduction in sugars content of anthocyaninless mutants led to defect in cotton trichomes development by which cotton trichomes are the major sinks for carbon partitioning.Further,OELDOX plants have activated the porphyrin and chlorophyll metabolism,photosynthesis,carbon fixation and metabolism pathways and nitrogen metabolism.From the above statement,we can firmly propose cotton Gh LDOX as an elite regulatory gene that involved in cotton trichomes development.Flavonoids are biochemical compounds that play important role in plant defense against biological stresses.Hence,the behavior of Gh LDOX transgenic plants has been tested in response to cotton bollworm infection.Anthocyaninless mutants were more favorable to cotton bollworm larva than wild type while overexpression lines faced less damaged by cotton bollworm larva.Altering Gh LDOX expression modulated the metabolic flux in the flavonoids pathways.It is thought that the increased contents of flavonoids and gossypol in OELDOX plants likely played as a feeding deterrent to cotton bollworm.Plants have evolved a complex network of preexisting and inducible defense mechanisms to resist pathogens.The plant cell wall is the first barrier that comes across pathogens which needs to be overcome for successful invasion.Regarding the traditional concept for being a passive barrier,plant cell wall is a dynamic structure acting as signaling molecules to trigger the plant immune responses.The plant cell wall also interacts with pathogens and evolves inducible defense mechanisms including physical and chemical mechanisms.On the other hand,plant secondary cell wall gives the cell wall its rigidity and reinforce its structure.Thus,enhanced secondary cell wall would increase plant defense ability.Lignification is a dynamic,flexible process reinforcing cell wall and enhancing the host defensive system against pathogen invasion.The lignin biosynthesis pathway is relatively well described in many plant species,however studies in cotton plants are limited.As pathogens and pests are the major constraints to cotton production,engineering plants with enhanced defense system is of great value.Adenosine monophosphate(AMP)binding domain is widely spread domain in many plant species,however;its molecular basis in plant development and plantpathogen interaction is still unclear.In this study,4-coumarate: coenzyme A ligase(4CL);one of the key enzymes in the monolignol biosynthesis pathway that shares AMP binding protein domain,has been characterized and verified its role in cotton plant defense system through lignin and phenylpropanoid pathways under pathogens and pests infection.Here;based on phylogenetic analysis,35 putative 4CL gene members were identified in the uoland cotton genome.One of which(Gh4CL3)gene,that corresponding to the 4CL3 from Arabidopsis,was selected for further study due to its distinctiveness than the other two 4CLs.Via subcellular localization analysis using GFP as a reporter gene,it was found that Gh4CL3 was localized in nucleus and plasma membrane.Next,for functional characterization,we have generated specific targeted mutations in Gh4CL3 gene using the CRISPR/Cas9 system.Three independent mutants were successfully generated and termed as CR4CL-1,CR4CL-2 and CR4CL-3.The role of the Gh4CL3 generated plants in cotton defense response to Verticillium dahliae(V991)and Helicoverpa armigera treatments was studied.Results indicated that all CR4CL3 lines were hypersensitive to pathogen and insects treatments.Suppression of Gh4CL3 led to downregulate the expression level of lignin biosynthesis genes,thereby a great reduction in the lignin content of plants stem and roots.This reduction was coupled with an increased content of salicylic acid,high expression of PR genes and low expression of flavonoids biosynthetic genes.At the same time,for mutation results confirmation,we have developed Gh4CL3 overexpressed cotton plants as well via Agrobacterium-mediated transformation.As a result;three independent lines were produced and designated as OE4CL-1,OE4CL-2 and OE4CL-3.Interestingly,Gh4CL3 overexpressed lines showed enhanced resistance against fungi treatment,high lignin accumulation in stem and roots and higher expression in flavonoids related genes.Taken all together,Gh4CL3 acts as a positive regulator in cotton resistance against biotic stresses through lignin-mediated enhanced-cell wall rigidity that strengthen plants defense mechanism.