| Platycladus orientalis,which has drought and barren soil resistance,long-life,disease and insect resistance,strong adaptability and low water consumption,is a good pioneer species for forestation in arid and semi-arid region.The P.orientalis is widely distributed naturally in China,and it has always been the important cultivated species in the ancient temple.And those have the age of more than thousand years can be found in many regions of China.At present,the research on the ancient P.orientalis is still lagging behind.There are some scientific problems that need to be solved,such as the molecular mechanism of P.orientalis growth for more than a thousand years without any degradation,and the mechanism of why some signs of recession showed in recent years.As a longevity tree species,the resistance of P.orientalis to environmental stress may be one of the important factors for its growth for thousands of years,and extreme environmental factors may also contribute to the declination of old trees.This research studied deeply the molecular mechanism of P.orientalis under water stress and carried out molecular mechanism of ancient P.orientalis under low temperature stress.It will lay the foundation of revealing the mechanism of P.orientalis anti-aging and recession.The main results were listed as follows:1.Root transcriptome sequencing and differentially expressed drought-responsive genes in the P.orientalisExperiment was carried out in greenhouse to simulate drought stress of P.orientalis seedlings.After two month of drought stress,c DNA libraries were established by collected P.orientalis roots and 53.1 to 63.4 million clean reads were obtained.After the high quality clean reads de novo assembly and removal of the redundancy,148,392 unigene were eventually got.Finally,116,716 unigenes were retained for the subsequent analysis.Of these 116,716 unigenes,29.9 %(34,845/116,716)were annotated using the NCBI nr database,and 20.2 %(23,573/116,716)shared significant identity with proteins in the Swiss-Prot protein database.In addition,all of the assembled unigenes were annotated via alignment with other public databases,including the Protein family(Pfam)(26,172/116,716),KEGG(13,312/116,716)and String(12,041/116,716)databases.In total,there were 36,900 unigenes(31.62 %)successfully annotated in at least one of the NCBI nr,Swiss-Prot,KEGG,GO and Pfam database,with 7998 unigenes(6.85 %)in all five databases.In total,116,716 unigenes with BLAST matches to known proteins were mapped to GO terms.A total of 17,012 unigenes were assigned to 4441 GO terms and categorized into 60 functional groups.To further analyze the root transcriptome of P.orientalis,all of the unigenes were analyzed using the KEGG pathway database.Of the 116,716 identified unigenes,13,312(11.41 %)were assigned to five main categories that included 98 KEGG pathways.A total of 3930 DEGs were detected between the drought-treated and well-watered plants,and these DEGs included both up-regulated(881 unigenes)and down-regulated(3049 unigenes)genes in the plants exposed to drought.Among them,194 TFs exhibited significant changes in expression during drought stress.To determine the molecular events that occur during the drought process and to identify the biological pathways that may govern the responses of DEGs,we performed GO and KEGG pathway enrichment analysis of the DEGs.The results showed that these DEGs involve in signaling transduction,biosynthesis,reactive oxygen species(ROS)scavenging,ion/energy transporters,proteases,protection factors of macromolecules.This study obtained the mechanisms of drought-stress defense/response of P.orientalis at the transcriptional level,which were based on the protection mechanism of many genes directly involved in the drought stress,including a large number of signal transduction system genes,regulatory genes and functional genes involved.Based on this,we have obtained a complex regulatory mechanism of P.orientalis defense/responses to drought stress: At the first stage of drought stress,the root cells sense water deficit through membrane and/or other cellular changes.Then,stressactivated Ca2+ signaling,reactive oxygen species(ROS)signaling,and hormone signaling that modulate the expression of stress-responsive genes,which include transcription factors,ion/energy transporters,protection factors of macromolecules,detoxification,key enzymes for osmolyte biosynthesis,proteases.In general,gene products can be classified into two groups.The first group contain proteins,involved in further regulation of gene expression that probably function in drought response(regulatory proteins),and the second group contain proteins factors that probably function in stress tolerance(functional proteins)directly.The transcription controls which activate stress-responsive mechanisms to re-establish homeostasis,protect and repair damaged proteins and membranes.2.Changes in protein profile and physiological characteristics of P.orientalis roots and leaves in response to drought stressBased on the sequencing of P.orientalis transcripts,in order to further understand the response and defense mechanism of drought stress,the physiological and proteomics analysis of root and leaf tissue samples of P.orientalis were collected under four water control conditions [(i)well-watered,80 ± 5% of field capacity(control),(ii)70 ± 5% of field capacity(mild water stress),(iii)50 ± 5% of field capacity(moderate water stress),and(iv)35 ± 5% of field capacity(severe water stress)].Given the high drought resistance of P.orientalis,mild water stress did not have a significant impact on soluble protein and chorophyll contents,consistent with the accumulative growth of seedlings.Moderate and severe water stress caused both morphological and physiological changes in plants under stress,resulting in marked reductions in leaf RWC,root WC and accumulative growth.Based on accumulative growth,20 days of severe drought stress and 25 days of moderate water stress produced significant differences in seedling growth compared to the growth observed in the control.Analysis of stress-responsive proteins showed that drought greatly affected the expression of proteins in P.orientalis.Of these,97 and 65 abundant proteins with significant quantitative variations and 1.5-fold differences in root and leaf tissues,respectively,were selected and identified using MALDI TOF/TOF MS/MS.The majority of proteins were related to stress response/defense,the carbohydrate metabolic process and nitrogen metabolism.To survive under the drought environment,plants employed a series of strategies,such as the maintenance of protein stability,activation,and folding;ROS detoxification;and the regulation of cell osmotic conditions and cell wall integrity,energy metabolism,and cell skeleton stabilization.Comprehensive root transcriptome and proteomics results when drought stress occurring the regulation of transcription and protein level have greater similarity.3.Drought stress response genes expression analysis under drought and re-waterIn order to further validation of drought stress defense/response genes screened previous,we investigated the genes expression of transcription factors WRKY53,NAC72 and AP2/ERF;protein kinase CDPK34,CDPK16 and CDPK1;reactive oxygen scavenging genes Fe-SOD,CAT3,POD3,APX3,APX6;osmotic regulation related genes SUS1,SUS3,SUS6,TRE6 PS,P5CS2 and P5CR;protection factors of macromolecules s HSP(HSP18.1,HSP23.5 and HSP25.3),HSP70 and LEA;14-3-3like protein,bark protein-like proteins [bark storage protein(BSP)] after drought and rewatering.The genes expression patterns analysis showed that:(1)Transcription factor NAC72 was involved in the regulation of drought stress in P.orientalis.The NAC72 gene may play an important role in the regulation of downstream genes.The transcription factor AP2/ERF showed an obvious characteristics of rehydration compensation,which may be related to the tissue damage repair and growth of P.orientalis seedlings after drought stress.(2)The expression of APX3,Tre6 PS,LEA,14-3-3 protein genes significantly raised under drought-stress illustrated that these genes may participate in the drought response related regulation of P.orientalis seedlings.(3)Key genes of proline synthesis gene(P5CS2)analysis showed that P5CS2 may play important roles in biosynthesis of osmotic substances.(4)The s HSP and HSP70 genes expression results showed that these two genes were up-regulated at different time points during drought stress,and this may be the two genes involved stress repair or response at different degrees in drought.The study also found that some genes in root and leaf tissue have a synchronicity and also have an asynchronism in response to drought stress.4.Physiology and proteomics research on the leaves of ancient P.orientalis during winterThe physiological and proteomics studies of leaf tissues on 20±5-,500±100-and1200±200-year-old P.orientalis under natural low temperature stress confirmed that the ancient trees were more affected by the low temperature,the significant decrease of the chlorophyll content and soluble protein content,the increase of MDA content and the decrease of SOD and POD activities were a primary characteristic of the leaves of ancient trees under low-temperature stress during winter,and were also indicates that the premature aging of ancient trees leaves and environmental impact may have an important relationship.Leaf proteomes were obtained using two-dimensional electrophoresis gels,and 77 protein spots were identified successfully using MALDI TOF/TOF MS/MS.The majority of the identified protein species were classified into functional categories including defense/stress-related,energy and carbohydrate metabolism,photosynthesis,and hormone-related functions.A general reduction in the abundance of protein species was observed as the age of the studied trees increased;reduction in photosynthesis and defense/stress-related categories were particularly apparent in the leaves of ancient trees.However,the number of protein species with functions in energy and carbohydrate metabolism increased with age.From the proteomics analysis,the ancient trees in the millennium showed more obvious signs of decline at low temperature.However,some high-abundance stress defense,macromolecule protection substances,hormone synthesis and other related proteins were also detected in the leaves of ancient trees,these proteins may be an important factor in anti-aging of ancient P.orientalis.Physiological and proteomics analysis presents a complex framework of physiological and molecular differences in response to low temperature stress of ancient trees. |
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