Functional Characterization Of PvBiP2 Involved In Cadmium Tolerance And Analysis Of PvHsf Gene Family In Switchgrass

Switchgrass(Panicum virgatum L.),originated from north America,is a C4 perennial grass.It has been studied and utilized as one of bioenergy crops in recent years due to high biomass yield and strong adaptability.Molecular chaperone BiPs,members of heat shock protein 70(Hsp70)family,play an important role in plant responses of abiotic stress.In this study,we explored the functions of PvBiP2 on the regulation of Cd tolerance and analyzed PvHsf gene family in order to find Cd tolerant gene resources in switchgrass.Results are summarized as following:1.Analysis of transcriptome changes of root in switchgrass under Cd stressSwitchgrass(cv.Alamo’HR8’)roots treated with 50 μmol·L-1CdCl2 were sampled after Ohr(control,designated as ’A0’),6 hr(A6),and 24 hr(A24)for extracting total root RNA,and furtherly to construct cDNA library.Then RNA-seq analysis was performed using Illumina HiseqTM 2500 platform.Based on the current empirical RNA-Seq data and the latest switchgrass genomic database,a total of 462 novel transcripts were identified and gene structures of 2337 transcripts were refined.A total of254 DEGs were screened out from ’A0vs-A6’,’A0-vs-A24’ and ’A6-vs-A24’ test groups.To validate the RNA-Seq result,relative expression levels of 14 DEGs with different functions were further analyzed using qRT-PCR.The expression levels of 13 DEGs were completely consistent with the RNA-seq results.In both ’A0-vs-A6’ and ’A0-vs-A24’ group,the two top-enriched GO terms were"Oxidation-reduction process" and "metabolic process" in the category of biological processes,"ATP binding" and "metal ion binding" in the category of molecular function,"cytoplasmic membranebounded vesicle" and "integral component of membrane" in the category of cellular compartment.KEGG pathway analysis showed that the "Taurine and hypotaurine metabolism","Vitamin B6 metabolism","Nitrogen metabolism","α-Linolenic acid metabolism","glutathione metabolism" and "Zeatin biosynthesis" were among the top list of enriched pathways.A total of 23 Hsp/chaperone genes were identified as DEGs,and 22 out of them had increased expression after Cd treatment.23 differentially expressed Hsf transcription factors were screened,21 of which were up-regulated in particular.This result indicated that the involvement of Hsfs and their downstream chaperone/Hsp genes might be essential for plant Cd tolerance.The Pavir.Ea01829 with the highest upregulation ratio in Hsp/Chaperon genes was identified as an encoding BiP gene.2.Analysis and functional study of molecular chaperone BiP genes in switchgrassIdentification of PvBiPs was carried out using both BLASTp tool and annotation information(GPR78/BiP/KAR2 conserved protein domain family KOG0100),endoplasmic reticulum(ER)resident signal "HDEL" against the reference genomes of switchgrass.We identified 4 BiP genes,named PvBiP1a and PvBiP1b,PvBiP2 and PvBiP3,locating on chromosome 1 and 5,respectively.Sequence analysis showed that PvBiPs had the typical functional domain of BiP.The evolutionary analysis found that PvBiP1a and PvBiP1b clustered into a clade,and formed a branch with rice OsBiP1,both containing 7 introns.PvBiP2 and PvBiP3 converge to another clade,and neither of them had intron.We further analyzed the 2 Kb promoter sequences of PvBiPs,the results showed that PvBiPs mainly consist of stress defense and hormone response cis-elements.PvBiPs can be expressed in tissues and organs,the expression level of PvBiP1b was significantly higher than that of other BiP genes,and the expression levels of PvBiP2 in seed developmental stages were higher than those in other stages.PvBiPs were induced by ER stress inducer DTT and up-regulated by NaCl,PEG,ABA and CdCl2 treatment.Among them,PvBiP2 had the highest up-regulated peak.PvBiP1a,PvBiP2 and PvBiP3 were then cloned into pGAD426 vector and transformed into yeast to verify their functions under abiotic stresses.The results showed that PvBiPs were tolerant to Cd and salt stress,but not to mannitol stress.3.Study on improvement of Cd tolerance in switchgrass by PvBiP2According to the expression profiles of PvBiPs under abiotic stress and the results of yeast resistance experiment,PvBiP2 gene was furtherly selected to study functions of Cd tolerance.PvBiP2 gene was stably expressed in the roots,stems and leaves of switchgrass,and its expression was continuously up-regulated under Cd stress.We further constructed the plant binary over-expressed vector pCambia1305.2-PvBiP2,and obtained 5 independent transgenic switchgrass lines through agrobacterium-mediated genetic transformation.Under normal conditions,the plant height,stem diameter and leaf width of transgenic switchgrass were significantly higher(P<0.05)than those of wild-type(WT),indicating that overexpressed PvBiP2 could promote the growth of switchgrass.After 30 days treatment of Cd,transgenic switchgrass displayed better performance,physiological and photo synthetic indexes,increased biomass,as well as the ability to maintain higher endogenous Cd content in root and lower in shoot under Cd stress,indicating that PvBiP2 is involved in the regulation of enhancing Cd tolerance in switchgrass.In addition,expression level of downstream molecular chaperone CNX significantly decreased,while increased in upstream heat shock transcription factor HsfA6,suggesting that there may be regulatory relationships among them.4.Analysis of the Hsf gene family in switchgrassSwitchgrass Hsf members were searched from the reference genomes of switchgrass by using HMM profile PF00447.A total of 51 PvHsfs were identified after removing the redundant sequences.PvHsfs were classified into A,B and C classes.There were 28 members in 7 subclasses of class A,3 members in 3 subclasses of class B and 9 members in 2 subclasses of class C.These genes were located on 16 of the 18 switchgrass chromosomes,with no PvHsf on chr08K and chr08N.MCsacnx collinearity search found that there was a pair of tandem duplication gene(PvHsf49 and PvHsf50)and other 46 genes have formed 42 groups of collinear relationships,confirming that the PvHsf genes experienced different gene duplication during evolution.The expression patterns of PvHsf genes in different tissues and organs were analyzed using gene expression atlas obtained from NOBLE database.The result showed that the majority of PvHsfs were constitutive expression with high levels.PvHsf51 was highly expressed in root and seed,indicating important role in seed growth and development of root.PvHsf24,a member of PvHsfA6,was continuously upregulated under Cd stress,which can be used as a candidate gene for Cd response regulation.In our study,we firstly confirmed that PvBiP2 was involved in the positive regulation of switchgrass Cd response,and the PvHsf gene family was analyzed to screen out possible Cd tolerant gene PvHsf24.The results provide theoretical base and valuable resources for molecular genetic improvement of the enhanced Cd tolerant switchgrass plants.