Profiling Of Cadmium Translocation In Italian Ryegrass-rice Rotation And Full-length Transcriptome Survey Of Italian Ryegrass(Lolium Multiflorum Lam.) Under Cadmium Stress

Cadmium(Cd)as a hazardous heavy metal element,is one of the main inorganic contaminants of farmland in China.Cd-contaminated paddy field has been concerned by public because of Cd accumulation in rice grain resulting human Cd exposure by food chain.Italian ryegrass(Lolium multiflorum Lam.),belonging to Lolium genus Poaceae family,is a valuable forage grass because of their desirable characteristics,such as high yields,rapid establishment,and high palatability and digestibility for ruminant animals.It can be cultivated in the agricultural offseason after harvesting other major crops such as corn and rice,and has been planted in the middle-lower reaches of Yangtze River area widely.Recent studies also showed that Italian ryegrass could be used for lignocellulosic ethanol conversion programs and phytoremediation,but there was little known in biomass production and phytoremediation planted in field.In this study,an Italian ryegrass-rice rotation experiment was conducted in Cd-polluted paddy field of three consecutive years in Chenzhou city,Hunan province.In the experiment,the yield of biomass and amount of Cd removal of three cropping systems(winter fallow as control,rotation cropping system included non-cutting and cutting)were evaluated.And Cd translocation factors(TF)in the different prat of Italian ryegrass and rice were analysed.Field experiment showed that it would contribute to develop Italian ryegrass-rice rotation in Cd containmated paddy field to increase the Cd TF of Italian ryegrass from root to culm.Then we optimized PacBio ISO-seq integrated with RNA-seq to construct a de novo Italian ryegrass full-length transcriptomic database for an un-sequenced autotetraploid species under Cd stress.With the reference transcriptome,we unveiled the molecular regulatory pathways of Italian ryegrass under Cd stress.And it is promising to be used for breeding high-Cd-accumulated Italian ryegrass cultivars.The main results are as follows:1.The structural carbohydrates in rice and Italian ryegrass straw were determined with HPLC.Acid hydrolysis of rice and Italian ryegrass straw resulted in mainly three monosaccharides(glucose,xylose,and arabinose);the content of glucose was much greater than that of the others.The glucose content in Italian ryegrass straw was significantly higher than that in rice straw(p<0.01).The bioethanol conversion potential of Italian ryegrass is higher than that of rice straw.The biomass and Cd concentration of rice grain showed no significant differences among the different cropping systems.And the Italian ryegrass-rice rotation could supply about 16.6 t ha-1 lignocellulosic feedstocks used for bioethanol production every year,as well removed Cd 98.6 g ha-1 from paddy fields.Italian ryegrassrice rotation system can achieve the purpose of "production while phytoremediation".2.The Cd concentration and TFs in the different part of Italina ryegrass and rice were showed that cutting treatment significantly increased Cd concentration of next sampled Italina ryegrass root,but did not affect overground Cd concentration;the TF of Italina ryegrass from root to culm was higher than that of rice,while TF of rice from culm to leaf was lower than Italina ryegrass.The TF of Cd from root to shoot in Italian ryegrass restricted the application of Italian ryegrass-rice rotation for phytoremediation,to develop new varieties whit high Cd TF from root to culm in Italian ryegrass will facilitate the application of Italian ryegrass-rice rotation system.3.Using PacBio ISO-seq and RNA-seq,we constructed a de novo database of Italina reygrass full-length transcriptome under Cd stress.With the database,we analysed the transcriptome of Italina ryegrass root treated with 50 μM Cd for 6 hours.In total,2367 differentially expressed genes(DEGs)were identified,1944 DEGs were significantly upregulated,and 423 DEGs were significantly downregulated.Gene Ontology(GO)analysis showed that the DEGs mainly enriched in oxidation reduction processes(GO:0055114),response to heat(GO:0009408),and protein folding(GO:0006457)in BP category;in MF category,the DEGs were mainly enriched in unfolded protein binding(GO:0051082),heme binding(GO:0020037),and GTP binding(GO:0005525);extracellular regions(GO:0005576),vacuoles(GO:0005773),and cell wall(GO:0005618)terms were the mainly harbours in the CC category.The Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of DEGs showed that the there were 334 DEGs involed in protein processing in the endoplasmic reticulum pathway(ko04141)in response to Cd stress.4.With the full-length reference transcriptome database,we reconstructed all transcripts as UniTransModels by cogent software,and analysed the complexity of alternative splicing(AS)with a genome-free strategy.In summary,a total of 29.76%of the UniTransModels had more than one isoform,and almost half of the UniTransModels(14.33%)had two isoforms;there were still some UniTransModels with more than 10 isoforms(797,1.12%).The different types of AS events were assessed based on the UniTransModels as the references instead of canonical genome-based AS events.Retained introns(RIs)were the majority of AS events,contributed 51.88%,and together with alternative 5’ or 3’ AS events,these three types of AS events constituted more than 90%of detected events.5.Based on DEGs resulted from RNA-seq and integrating the Cd tolerance studies of Arabidopsis thaliana auxin associated mutants,a DEG annotated as auxin transporter was selected for functional characterization.A blast searched in rice and Arabidopsis thaliana genome database indicated that it coded an auxin transporter,was the homologous gene of A UX1,named as LmA UX1.The relative expression level of this gene in root was significantly decreased in response to Cd stress validated by qRT-PCR.Overexpressed LmA UXl into the aux1-7 mutant with CaMV35S promoter,and the tropic movement of transgenosis mutant roots was regained,the IAA concent of transgenosis mutant shoot was significant lower than that of aux1-7 mutant shoot,the results showed that the function of LmAUX1 was similar with AtAUX1,contributed to auxin polar transport.In previous studies,the loss-of-function mutants of auxin transporter(aux1-7 and axr4-2)enhanced plant Cd tolerance and the IAA concent of root was reduced under Cd stress.The results indicated that Cd disturbed the plant auxin homeostasis,and auxin transport participated in regulating plant Cd tolerance.6.Ectopic overexpression of LmAUXl in Col-0 wild type and aux1-7 mutant.Transgenic lines,aux1-7 mutant,and Col-0 were treated with and without 50 μM CdCl2 in 1/2 MS medium,and the results showed that the relative length of root elongation in the transgenic lines was significant lower than that of the aux1-7 mutant and Col-0.Pot experiments showed that shoot Cd and Fe concentrations of transgenic lines were significant increased compared to aux1-7 mutant and Col-0,and the MDA content,ROS concentrations,and antioxidase activity of shoot were all significantly increased.Taken together,LmAUX1 is involved in regulating plant Cd translocation,then affects plant Cd tolerance;it is suggested that Italian ryegrass enhanced Cd self-tolerance by decreasing the expression level of LmA UX1.In conclusion,with the aim of biomass production in heavy metal containmated field,we set up the Italian ryegrass-rice rotaiont cropping system in Cd containmated paddy field.The yield of biomass and amount of Cd removal of Italian ryegrass-rice rotaiont were evaluated.Simultaneously,the Cd TFs of the different parts in Italian ryegrass and rice indicated that the key to develop Italian ryegrass-rice rotation in Cd containmated paddy field was to breed Cd-phytoremediation Italian ryegrass varieties.Then with PacBio IS O-seq and RNA-seq,we constructed a de novo Italian ryegrass full-length transcriptomic database for an unsequenced autotetraploid species under Cd stress.The complexity and diversity of Italian ryegrass transcriptome was profiled,DEGs of Italian ryegrass uder Cd stress were identified.Then LmAUX1 was confirmed with the function of Cd translocation in plants,and this gene is promising to be used to breed high-Cd-accumulation Italian ryegrass for bioenergy.