Analysis Of Transcriptome And Expression Profiles In Response To Desiccation Stress In Pyropia Haitanensis

Pyropia haitanensis is an economically important marine crop, which has beencultivated widely along the coasts of South China, accounting for75%of the totaloutput of cultivated Pyropia in China. P. haitanensis is also considered to be researchmodels for studying physiological ecology and the molecular mechanisms for stresstolerance of intertidal seaweed communities. However, for P. haitanensis, limitedstudies have been conducted. So far, the genomic information on Pyropiaremains incomplete, and systematic molecular investigations of the mechanisms foracclimation to intertidal stress environment are very lacking at present.Using Illumna high-throughout sequencing technology, the transcriptome andexpression profiles of P. haitanensis were sequenced and analyzed. And using454and Sanger sequencing technology, the complete plastid genomes of P. hatanensis andP. yezoensis were fully sequenced. Comprehensive and systematic study oftranscriptomic characteristics and deep exploration of molecular mechanism forresponse to the dehydration/rehydration stress environment will not only provide newunderstanding of the mechanisms for response to intertidal desiccation stressenvironment, but also provide new reference for stress-resistance breeding in future.The major findings are as follows:The global transcriptome of P. haitanensis was sequenced using Illuminahigh-throughput sequencing technology. A total of47,764,168clean reads (4.5Gbclean bases) were generated from the cDNA library constructed for a RNA mixture ofP. hatanensis at different development stages and under different stress treatments.These clean reads were assembled into28,536unigenes with a mean length of584bp.Based on sequence similarity searches with known proteins, a large number of genesrelated to stress response were found, including genes related to desiccation stress,temperature stress, light stress, starvation, antioxidant system and other genes relatedto stress processes, and of which group1type LEA and vanadium-dependent haloperoxidase (vHPO) homologous sequences were firstly reported. And Manygenes related to developmental process were also found in P. hatanensis. Searchingagainst the KEGG database indicated that there existed284KEGG pathways intranscriptome of P. hatanensis, including apoptosis pathway and MAPK signalingpathways related to stress environment. The MAPK signaling pathways contain manygenes involved in mammalian JNK and p38type MAPK pathway and yeast MAPKpathway. After annotation, there are still10,378unigenes unannotated (36.4%),indicating that there may be a lot of novel genes with unknown function.In order to study the expression profile characteristics in response to desiccationstress environment in P. hatanensis, the cDNA library was constructed for eightsamples from the different dehydration and rehydration stress treatments and thensequenced by Illumina sequencing technology (two biological replicates for eachtreatment). A total of11.83M-15.04M clean reads were abtained for each sample.Moreover, the number of reads mapped to transcriptome is96.78%-97.74%of thetotal reads number. Within each treatment, correlation of gene expression levelsbetween two biological replicates was high (R∧2=0.916-0.941). By analysis of theoverall pattern of gene expression during moderate dehydration (compared with thecontrol group), a total of1681differentially expressed genes were identified. Avariety of new motif type MAPKs were firstly found, and of which the new TQAmotif type MAPK was involved in signal transduction pathway in response to thedehydration stress. A total of10molecular chaperones genes (including the membersof HSP90superfamily, HSP70superfamily and cochaperone proteins of HSP70) weresignificantly up-regulated in response to the moderate dehydration stress，whereas themembers of HSP60superfamily and the Hsp33were expressed constitutively.Additionally, peroxiredoxin and Fe-SOD were expressed constitutively to removereactive oxygen species, the protein disulfide-isomerase genes in antioxidant systemwere up-regulated and involved in repair of oxidative damage during moderatedehydration stress. And the induction of apoptosis was involved in tolerance tomoderate dehydration stress, and in which the mitochondria played a centralregulatory role. Compared with moderate dehydration stress treatment, only18differentially expressed genes were identified during the severe dehydration stresstreatment, indicating that the gene expression patterns were quite similar between thetwo stress treatment groups. Compared with severe dehydration stress treatment,1199 differentially expressed genes were identified during the rehydration stress treatment,and of which one activator of caspases involved in induction of apoptosis andcochaperone proteins of HSP70significantly downregulated, the thioredoxin inantioxidant system upregulated and was involved in repair of oxidative damage duringrehydration stress.The complete plastid genomes of P. hatanensis (195,597bp) and P. yezoensis(191,975bp) were fully sequenced using454high-throughout sequencing togetherwith Sanger sequencing, the largest of all the plastid genomes of the red lineagesequenced to date. Organization and gene contents of the two plastids were similar,with211-213protein-coding genes,37tRNA genes, and6ribosomal RNA genes,suggesting a largest coding capacity in the red lineage. In each genome,14proteingenes overlapped and no interrupted genes were found, indicating a high degree ofgenomic condensation. Pyropia maintains an ancient gene content and conserved geneclusters in their plastid genomes, containing nearly complete repertoires of the plastidgenes known in photosynthetic eukaryotes. Similarity analysis based on the wholeplastid genome sequences showed the distance between P. haitanensis and P.yezoensis (0.146) was much smaller than that of Porphyra purpurea and P.haitanensis (0.250), and P. yezoensis (0.251); this supports re-grouping the twospecies in a resurrected genus Pyropia while maintaining P. purpurea in genusPorphyra. Phylogenetic analysis supports a sister relationship betweenBangiophyceae and Florideophyceae, though precise phylogenetic relationshipsbetween multicellular red alage and chromists were not fully resolved. Combiningwith transcriptome and expression profiling results, coordination of plastid andnuclear gene expression was found to be mainly mediated by nuclear-encoded sigmafactors and activity of Mg-chelatase in response to dehydration stress.