Physiological Basis And Molecular Mechanisms Of Kenaf Salt Stress Tolerance Based On Transcriptomic And Proteomic Analyses

The last few decades have witnessed a tremendous influence of anthropogenic climate change which is regarded to be the frontline line contributors of disturbing the natural environments and genuine cause of the increases in different abiotic stress regimes including water scarcity,cold,heat,and soil salinization.Salt stress strongly inhibits the growth,yield,quality,and quantity of crop plants,and this challenging development is fretting over the fate of sustainable agriculture productivity around the globe.Many important crops may reduce up to 50%due to salt stress.Kenaf is a high ranked,valued natural fiber crop,and recognized with great commercial and industrial interests.Although,the kenaf possesses phytoremediation features,however,kenaf production is significantly reduceds under the effect of salt stress.Moreover,the food crops are preferred to be cultivated on fertile soil,and the area under the kenaf cultivation has been shifted to unhealthy,unfertile,and mostly saline soils.Therefore,due to less yield of kenaf industrial demand for kenaf has been compromised greatly.Therefore,the present study was aimed to shed light on physiological and molecular response mechanisms in kenaf exposed to salt stress and further to sort out key genes,target proteins and key molecular regulatory pathways for understanding the salt tolerance mechanism of kenaf and to better use of kenaf for phytoremediation of salt contaminated soils.The core molecular findings in the present study were facilitated through the most recent techniques both at RNA sequencing levels(Illumina Hiseq X^TM),and proteomic levels?iTRAQ-based?,and through reliable bioinformatics analyzing tools.The significant findings during the whole experimental protocols were presented as follows.Initially,a salt-tolerant?P3A?and a salt-sensitive?P3B?kenaf cultivars were selected during preliminary experiments in our Laboratory and the seedlinngs of both the cultivar were treated with salt-stress levels?0 m M and 250 m M Na Cl?,and comparative morphological,physiological and cytological responses were anaysed after 4 days of salt stress treatments.Furthermore,a comparative gene expression analysis through recently advanced RNA sequencing technique(Illumina Hiseq X^TM)was performed and different salt responsive genes and metabolic pathways were analyzed and q RT-PCR was performed to confirm the reliability of RNA sequencing data.After that,iTRAQ-based comparative analyses for protein abundance changes was perfomed and different proteins target and regulatory metabolic pathways were suggested playing significant role in kenaf to normalize salt stress and relative RNA levels for the corresponding differentially abundant proteins were assessed.The results indicated that the seed germination energy?%?,germination rate?%?,germination index?%?and relative rate of salt damage were measured as 73%,73%,33%and 15.14%in P3A and 44%,48%,18%and 70%in P3B,respectively under 250 m M Na Cl.The relative rate of salt damage was almost 5 times greater in P3B that P3A.This indicated that P3A had better adaptability exposed to high salt stress at germination levels.The results of morphological indicators at seedling stage showed that there was a decrease in SPAD value,root fresh weight,root diameter,stem width,plant height,root length,and root volume?cm³?as 52%,61%,32%,23%,34%,55%,and 79%respectively in P3A,and 58%,55%,36%,26,35%,70%and 81%respectively in P3B.This showed that only with exception of the root fresh weight,all other morphological parameters were strongly hampered in P3B compared with P3A.Further,under the influence of salt stress net photosynthetic rate,stomatal conductance to H₂O,transpiration rate with exception of intracellular CO₂concentration,which increased slightly,were recorded significantly higher in P3A than P3B.The chlorophyll?chl a chl b?contents were significantly higher in P3A than P3B,suggesting P3A had better photosynthetic response than P3B.The MDA contents increased in both of the cultivars however,increase was greater in P3B than P3A;at the same time,the SOD activity,soluble proteins and soluble sugar had significant increase in P3A than P3B,indicating that P3A encountered less damage and has a stronger ability in scavenging ROS under salt-stress conditions.Furthermore,cytological studies indicated swelling and vacuolization of chloroplast and thylakoid in cultivar P3A under the influence of alt-stress,however,chloroplast and thylakoid were degraded in cultivar P3B.At the physiological morphological and cytological response,kenaf may be suggested to adapt to salt stress through improved seed germination,maintain photosynthetic efficiency,through better-increased gas exchange parameters and sustained chlorophyll contents and possessing compact chloroplast and thylakoid structures.Furthermore,mitochondrial stability ensures more energy production to address the salt stress regime in kenaf.In the next study to understand the molecular mechanisms of salt stress tolerance both the P3A,and P3B were analyzed for comparative transcriptome response by employing Illumina Hiseq X^TMtechnolog.Results showed that after raw reads elimination,a sum of 178.24 and 161.76 million clean reads from controls,and 172.29 and 170.72 million clean reads from salt-treated samples were gathered from P3A and P3B,respectively.A total of 115045 transcripts and 45724non-redundant unigenes were assembled and the sequence alignment results showed that 42.56%,27.78%,32.28%,48.88%and 62.34%of unigenes and57.08%,36.06%,46.65%,61.18%and 71.68%of transcripts were significantly matched with an E-value of?E<1e-5?by Pfam,string,KEGG,Swissprot and NR databases,respectively.At least 73.49%unigenes and 85.17%transcripts had one significant match by either one of four databases and a sum of 4242 unigenes and17129 transcripts were annotated commonly by all databases.There generated8466 and 9333 differentially expressed unigenes?DEGs??FDR?0.05 and log₂FC?1?between salt-stressed and control treatments in cultivar P3A and cultivar P3B,respectively.Amongst those,3223?38.06%?up and 5243?61.93%?were down-regulated in P3A and similarly 4586?49.13%?up and 4747?50.87%?were down-regulated in P3B.At least,48 Transcription Factors families were also identified and the q RT-PCR result of randomely selected 8 DEGs showed consistency with RNA sequencing results.Several genes encoding transcription factors WRKY,AP2/EREBP,MYB,GRAS and Hsfs families,and genes like LEA,Psb A,PK,GSTs,NPR1,and TGA were induced under salt stress,and expression levels were higher in cultivar P3A compared to cultivar P3B.The GO enrichment and KEGG pathway analysis suggested that these DEGs were related to ionic homeostasis and transport,osmotic adjustment,water deficit response,antioxidants,ROS scavenging,cellular membranes protection,photo-damage repairing cycle of photosystem II?PSII?,thylakoid part,and plant hormone signal transduction pathway and may be related to salt tolerance in kenaf.Furthermore,a comparative iTRAQ-based quantitative proteomic analysis was performed.Results showed that 89?74.78%?and 30?25.2%?of differentially abundant proteins?DAPs?in P3A,and 133?69.27%?and 59?30.7%?in P3B,were up and down-accumulated?FC>1.5 or<0.67;p<0.05?,respectively.Bioinformatics analysis inferred that DAPs were distributed among 160 and 146significantly enriched Gene Ontology categories,and at least 71 and 99 DAPs were mapped to 47 and 61 KEGG pathways in P3A and P3B,respectively.12 KEGG pathways were determined significantly?p<0.05?enriched for each of the two cultivars.Including porphyrin and chlorophyll metabolism,glutathione metabolism,glycine,serine and threonine metabolism,arachidonic acid metabolism,and glyoxylate and dicarboxylate metabolism pathways.q RT-PCR results showed that transcript levels of several genes had consistency with abundance levels of their corresponding proteins.DAPs including ATP,?-D-glucose-1-phosphate adenylyltransferase,aldehyde dehydrogenase?ALDH?,ATP dependent Clp protease?clp B?,glutathione S-transferase?GST?,ATP synthase,and Photosystem II subunit S?Ps BS?were suggested to play essential roles in response to salt.Interestingly,P3A had less DAPs than P3B,however,putative functional role and their distribution amongst the regulatory metabolic pathways suggested that those DAPs were involved in repairing photosystem II?PS II?,maintaining energy as ATP,mitochondrial oxidative phosphorylation,sugar metabolism and starch synthesis,scavenging ROS and aldehydes,cytosolic homeostasis,and ionic balances,de novo synthesis of amino acids,and posttranscriptional and posttranslational changes,and were suggested to play key roles in response to salt stress in kenaf.Inclusively,all these findings could likely shed light on the molecular mechanisms of salt stress tolerance in kenaf.