Molecular Mechanisms Of PvHSFA4a In Regulating Cadmium-Tolerance In Seashore Paspalum

China has rich heavy metal mineral resources,but a large number of surrounding lands,water bodies and vegetation suffer from heavy metal pollution with the great achievements of mineral resources exploitation in recent years.Now,the heavy metal cadmium(Cd)is one of the most severe and biologically toxic elements contaminated in the soil of the mining area as well as the surrounding urban and rural areas.To repair the ecosystem and follow the national policy of "Beautiful countryside construction",environmental governance,vegetation restoration,and landscape construction are becoming more and more important in these areas.Turfgrasses have been utilized by human to enhance environment for more than 10 centuries.They play a vital role in landscape construction,maintenance and environment protection,especially in heavy metal contaminated area.Researches showed that they had the ability to uptake the heavy metal by roots and translocate to above-ground organs,which were cut away by pruning.Seashore paspalum(Paspalum vaginatum)is one of the most cadmium-tolerant turfgrasses among warm-season turfgrasses,so it is an ideal material for investigating cadmium-tolerance mechanism of turfgrasses.Exploring the molecular mechanism of cadmium tolerance in seashore paspalum can provide a theoretical basis for the development of new turfgrass germplasm,which are the key measures for turf establishment and vegetation phytoremediation in cadmium-contaminated areas.Previous studies have shown that a heat shock transcription factor,HSFA4a,positively regulates cadmium tolerance in wheat and rice,but the regulatory mechanisms need to be further explored.PvHSFA4a was obtained through screening of expression library of seashore paspalum earlier in our lab.Based on this,the present study focused on three aspects that haven't been clarified in previous researches:direct regulation of downstream pathways in cadmium resistance,amino acid functional sites verification and protein interaction mechanisms.The main results are as follows:1.The analyses of amino acid sequence,expression pattern,subcellular localization and transcriptional activation activity of PvHSFA4a:First,the sequence of PvHSFA4a was cloned from seashore paspalum,and it includes a 1305-bp open reading frame(ORF)predicted to encode 435 amino acids that showed high sequence identity with TaHSFA4a and TaHSFA4b,followed by OsHSFA4a.Additionally,analysis of PvHsfA4a polypeptide structure showed that PvHSFA4a has a highly conserved N-terminal DNA binding domain(DBD),oligomerization domain(OD),nuclear localization signal(NLS),C-terminal activation domain(CTAD)and nuclear export signal(NES).qRT-PCR analysis showed that the transcription level of PvHSFA4a was significantly induced by cadmium stress in seashore paspalum.To verify its subcellular localization,a GFP-fused PvHSFA4a was constructed and transiently transformed into tobacco leaves.The aligned fluorescence of GFP indicated the nuclear localization of PvHSFA4a.Transcriptional activation analysis by yeast system showed that PvHSFA4a has self-activation activity.2.In order to confirm the key role of PvHSFA4a in regulating cadmium tolerance,the integrated plasmids were heterologously transformed into yeast and homologously transformed into seashore paspalum.Overexpression of PvHSFA4a improved the cadmiumtolerance of yeast under cadmium stress,but failed to enhance the tolerance of yeast under other heavy metal stresses.Overexpression of PvHSFA4a in transgenic plants enhanced the cadmium tolerance of seashore paspalum.Physiological parameters,such as plant survival rate,leaf unfolding rate and relative water content(RWC)in transgenic seashore paspalum plants were significantly higher than WT under cadmium stress.In addition,overexpression of PvHSFA4a resulted in increased accumulation of cadmium in roots and stems of transgenic seashore paspalum plants.To further explore the downstream molecular mechanism of PvHSFA4a in regulating cadmium tolerance,PvHSFA4a were heterologously transgened to rice and also CRISPR/Cas9-induced mutation of OsHSFA4a was obtained.Overexpression of PvHSFA4a in transgenic rice plants enhanced the cadmium tolerance than WT plants,while CRISPR/Cas9 knockout mutant showed cadmium sensitive phenotype.These results suggested that PvHSFA4a uniquely regulates cadmium tolerance,like HSFA4a in rice and wheat.3.Analysis of the downstream molecular mechanism of PvHSFA4a:Through analyzing the promoter sequence of cadmium-tolerant genes in rice,we found several potential heat shock elements(HSE).Among them,NRAMP5,ABCG43,CLT1,HMA2 and IRT1 promoter sequences were proved to interact with PvHSFA4a through yeast one-hybrid system.Also,expression levels of MT-I-1a,HMA2 and CLT1 were up-regulated in transgenic rice plants overexpressing PvHSFA4a under cadmium stress.Electrophoresis mobility shift assay(EMSA)verified the authenticity of interaction that PvHSFA4a interacted directly with HSE1/2(MT-I-1a),HSE7(CLT1)and HSE17(HMA2)respectively.These results suggested that Expression levels of MT-I-1a,HMA2 and CLT1 were up-regulated in transgenic rice plants overexpressing PvHSFA4a under cadmium stress,thus we suppose that PvHSFA4a may regulate cadmium tolerance through promoting the detoxification and transport of cadmium in plants.4.Identification of specific functional sites of PvHSFA4a:previous studies suggested that the presence of N-terminal DBD in HSFA4a was necessary in its function.Through amino acid alignment,we found a K76 sites conserved with the K80 acetylation site in the human HSF1,and a functionally different C49 site.To determine whether K76 was the key site of PvHSFA4a function,we mutated the lysine residue into arginine R(K76R),glutamine(K76Q),alanine A(K76A),and threonine T(K76T).The K76 mutated PvHSFA4a lost the function in inducing cadmium tolerance of yeast.Overexpression of PvHSFA4a enhances cadmium tolerance in transgenic rice while K76R and K76Q mutated plants appeared less survival rate.Similarly,we mutated the cysteine residue into serine S(C49S)and tryptophan W(C49W).The C49S mutated PvHSFA4a failed to induce cadmium tolerance of yeast and rice,while overexpression of PvHSFA4a and the C49W mutated PvHSFA4a still maintained the same cadmium resistance.These results indicate that K76 and C49 are important amino acid sites affecting the function of PvHSFA4a in cadmium-tolerance.5.Screening and identification of PvHSFA4a interacting proteins:Based on the known acetylation modification pathway of HSF1 in humans and the proved function of K76,13 histone deacetylases were cloned and constructed to related plasmids.Through yeast twohybrid and luciferase complementation assay(LCA),three histone deacetylases(PvHDAC15,PvHDAC6,PvHDAC9)were proved to interact with PvHSFA4a.In addition,108 effective clones,including 34 proteins,were screened from yeast two-hybrid library of seashore paspalum,and twelve of them were confirmed to interact with PvHSFA4a by yeast twohybrid.Thereafter,the interaction of PvHSFA4a with three nuclear localization proteins(PvHSFA4a,PvTATl and PvCHYR1)were detected through subcellular localization and LCA.RT-PCR results showed that the expression level of these five interacting proteins were all up-regulated under cadmium stress.The discovery of histone deacetylases and interacting proteins could provide a basis for further studies exploring regulatory mechanisms of PvHSFA4a induced cadmium-resistance.