Physiological And Transcriptomic Responses In Neolitsea Sericea Leaves Under Drought Stress

During the long history of human activity on the Zhoushan archipelago the original oldgrowth broadleaved forests on the islands have been mostly destroyed,including Neolitsea sericea(Lauraceae),which is distributed on a few islands of the Zhoushan archipelago.The rapid degradation and destruction of N.sericea original habitats have resulted in a continuous decline of natural populations.Thus,plenty of studies on N.sericea have been focused on population genetic diversity and genetic structure.But equally important is that N.sericea has well acclimatization,making it survive in the ravines on the islands.It is a fine tree species for afforestation for its strong tolerance to drought,wind,salt and barren soil.The studies of the tolerance on N.sericea were much limited.No research yet has been published that focused on drought tolerance of N.sericea.Plant response to drought stress at molecular level is a complex biological process,involving consideration of the stress effects and regulation physiological events.Thus,it is of great importance to analyze the underlying mechanism systematically at both physiological level and molecular level.To obtain a complete physiological mechanism in N.sericea in responses to drought stress,1-year-old N.sericea seedlings were exposed to normal water supply,light drought,moderate drought,and severe drought for 45 days(d)respectively(relative water content in soil was controlled in 75-80%,55-60%,40-45% and 30-35% of field capacity,respectively).All plants were rehydrated after drought treatments by keeping them under normal water supply for 10 d.The relative water content in soil was controlled by pot-weighing method through the experiment.The dynamic changes of the physiological and photosynthetic traits in leaves were measured every 15 days of stress treatments and at the end of re-watering.The traits include the photosynthetic rate(Pn),stomatal conductance(Gs),water use efficiency(WUE),intercellular CO2 concentration(Ci),transpiration rate(Tr),total content of chlorophyll(Chl),superoxide dismutase(SOD)and peroxidase activity(POD),proline content(Pro),malondialdehyde content(MDA),soluble sugar content(Ss)and relative conductivity(Rc).The results showed that,the Chl,Pn,Tr and Gs was decreased constantly and significantly during severe drought,Ci was decreased in 0-30 d and enhanced in 30-45 d during the severe drought.The results indicated that the decrease of Pn was caused by stomatal limitation during 0-30 d of severe drought,and by non-stomatal limitation during 30-45 d of severe drought.The continuous inhibition of Tr after the rehydration from severe drought,suggesting the afunction of stomatal regulation.The drought stress increased the leaves' overall WUE,although the WUE dropped significantly in 30-45 d of severe drought.The same trend of MDA,SOD and POD content that N.sericea showed during the drought treatments,which was first increased and then decreased,explained the generation and clearing mechanism of MDA.The significantly increase of Rc indicated the gained permeability of cell membrane.The over expressed Ss and Pro in leaves during 30-45 d of drought treatment resulted in the reduction of Rc,which relieved the osmotic pressure in N.sericea.Howerver,Rc staying at high level after rehydration from severe drought indicated the damage on cell membrane.Overall,to reduce the damage from drought,N.sericea aroused the instant stomatal regulation,active oxygen elimination and osmotic adjustment by decreasing the Tr,MDA content,and cell membrane permeability,and by enhancing the WUE in leaves.However,severe and persistent drought brought about shortness and irreversible damage in leaves.To investigate the mechanisms that allow N.sericea seedlings to survive drought,we conducted a transcriptomic analysis of leaves using a pyrosequencing approach after severe drought was induced.Over 17,768,244 reads for each sample were generated,totalling over 4,400 million base pairs.After assembly,129,239 Unigenes were yielded with an average length of 816 base pairs.A total of 51,137 Unigenes(39.6%)were annotated successfully.Among these,11,192 were mapped onto 118 pathways,and the most enriched sequences were ribosome pathways.We identified 61,65,and 67 differentially expressed transcripts after 12,24,and 72 h of drought stress,respectively.Gene Ontology and MapMan functional enrichment analyses revealed that the genes that had accumulated the most were involved in protein transport and the regulation of transcription and miscellaneous enzyme families.These included genes related to osmoregulation,phytohormone,and drought-regulated transcription factors.Genes that encode glycosyl transferase and UDP-glycosyltransferases may modulate the drought-stress responses of N.sericea.Multiple hormones were active in N.sericea leaves during drought stress,including those associated with auxin,abscisic acid,brassinosteroid,and cytokinin.Transcription factors,including AtbHLH112,AtCOL4,AtZFP3,AtCIR1,and AtCCA1,may participate in drought-induced ABA signal transduction in N.sericea.The present study elucidates how N.sericea responds to drought stress at the transcriptome level,which may help reveal the molecular mechanisms associated with drought adaptation.