| Abies beshanzuensis M.H.Wu belongs to the genus Abies of Pinaceae.Only 3 wild adult plants exist in the field,which is listed as one of the 12 critically endangered species.Despite efforts by the staff of the protected area to preserve the species through near-situ and ex-situ protection,the growth of seedlings is generally poor and the survival rate is low.Furthermore,with the intensification of global warming,frequent and extreme high temperature weather will pose a great threat to the survival of A.beshanzuensis,which will also face high-temperature stress if moved to a lower altitude for near-situ and ex-situ protection.To address these challenges,an urgent research is needed to study the physiological and molecular mechanisms of A.beshanzuensis seedlings in response to altitude and high temperature stress,which can provide a theoretical foundation for climate change and near-situ and ex-situ protection strategies.In this study,we conducted physiological index determinations and transcriptome sequencing to analyze the physiological and molecular mechanisms of A.beshanzuensis seedlings in response to altitude gradient and high temperature stress.The main results of this study are as follows:1.There was no significant difference in leaf morphology of A.beshanzuensis seedlings transplanted at 1550 m,1300m and 1000 m altitudes,while the leaves of A.beshanzuensis seedlings transplanted at 750 m altitude were slightly yellowish.Most of the leaves of A.beshanzuensis seedlings transplanted at 450 m altitude were yellowish brown and burned.The values of Yield,ETR,q P and Fv/Fm of A.beshanzuensis seedlings showed a decreasing trend with decreasing altitude,respectively.The NPQ values increased with decreasing altitude from 1550 m to 700 m,but reached its lowest value at an altitude of 450 m.The chlorophyll a,chlorophyll b,total chlorophyll content and chlorophyll a/b value also decreased significantly with decreasing altitude,respectively.Conversely,the contents of proline,soluble sugar and soluble protein increased significantly with decreasing altitude,respectively.The content of MDA increased significantly with decreasing altitude from 1550 m to 700 m,but was the lowest at an altitude of 450 m.The activities of superoxide dismutase,catalase,and peroxidase initially increased and then decreased with decreasing altitude,with the highest activities observed at an altitude of 1000 m,respectively.Therefore,a decrease in altitude is detrimental to the growth of A.beshanzuensis seedlings,and the altitude of1000 m may be the lower limit for the survival of A.beshanzuensis seedlings.2.Mild high temperature stress(30 ℃/25 ℃)had no significant impact on the leaf morphology,chlorophyll fluorescence parameters,and chlorophyll content of A.beshanzuensis seedlings,respectively.However,under moderate high temperature stress(35 ℃/ 30 ℃)and severe high temperature stress(40 ℃/35 ℃),the leaves of A.beshanzuensis seedlings were damaged on the 9th and 3rd day,respectively.Furthermore,on the 12 th day of severe high temperature stress(40 ℃/35 ℃),almost all the leaves turned yellowish brown.Along with this,the values of Yield,ETR,q P,Fv/Fm and chlorophyll content in the leaves of A.beshanzuensis seedlings decreased significantly with the prolongation of stress time under moderate and severe high temperature stresses,respectively.On the other hand,the NPQ values increased significantly under moderate high temperature stress(35 ℃/30 ℃)and decreased significantly under severe high temperature stress(40 ℃/35 ℃).Mild high temperature stress(30 ℃/25 ℃)did not significantly affect MDA content in leaves of A.beshanzuensis seedlings.However,MDA content increased significantly with the prolongation of stress time under moderate high temperature stress(35 ℃/30 ℃).MDA content initially increased but then decreased,reaching its peak on the 6th day under severe high temperature stress(40 ℃/35 ℃).Osmotic regulating substance content and antioxidant enzyme activities significantly increased in leaves of A.beshanzuensis seedlings under mild high temperature stress(30 ℃/25 ℃)and moderate high temperature stress(35 ℃/30 ℃),respectively.Under severe high temperature stress(40 ℃/35 ℃),proline content significantly increased with the prolongation of stress time,while soluble sugar and soluble protein contents initially increased but then decreased,peaking on the 9th day.Superoxide dismutase and peroxidase activities initially increased and then decreased,while catalase activity initially decreased,then increased,and finally decreased.The activities of the three enzymes peaked on the 6th day.Therefore,A.beshanzuensis seedlings are capable of withstanding high temperatures up to 30 ℃ for brief periods.However,they cannot withstand sustained high temperatures of 35 ℃,and exposure to 40 ℃ temperatures can cause irreversible damage in a short period of time.3.Non-parametric transcriptome sequencing was used to study the leaves of A.beshanzuensis seedlings transplanted to different elevations,and a total of 41260 Unigene were obtained.The results of Unigene protein database annotations showed that NR database annotated the most Unigene(24029)and KEGG database annotated the least Unigene(16436).Pairwise comparisons among different altitude treatments identified 3,819,5,207,and 1,010 differentially-expressed genes(DEGs)in the 450 m vs 1000 m,450 m vs 1550 m,and 1000 m vs 1550 m groups,respectively.KEGG metabolic pathway analysis of DEGs revealed that plant-pathogen interaction,protein processing in the endoplasmic reticulum,plant hormone signal transduction and MAPK signal pathway-plant played a major role in A.beshanzuensis seedlings responding to low altitude stress,while A.beshanzuensis seedlings transplanted to middle altitude could respond to environmental changes through energy metabolism and the production and accumulation of secondary products.Expression of photosynthesis-related DEGs was suppressed in all three comparison groups while DEGs encoding heat shock proteins were mostly up-regulated,over half of which were part of the s HSPs family.It is showed that s HSPs plays an influential role in the response of A.beshanzuensis seedlings to low-altitude environmental stress.Furthermore,most of the DEGs encoding heat shock proteins found in the NOG annotation were induced by high temperature,indicating that high temperature was the primary factor contributing to environmental stress caused by decreasing altitude.4.Non-parametric transcriptome sequencing was used to study the leaves of A.beshanzuensis seedlings treated at different temperatures,and a total of 45758 Unigene were obtained.The results of Unigene protein database annotations showed that NR database annotated the most Unigene(27927)and KEGG database annotated the least Unigene(19360).Compared with the control group(25 ℃/20 ℃),1,868,3,580,and9,344 DEGs were identified in the leaves of A.beshanzuensis seedlings treated at30 ℃/25 ℃,35 ℃/30 ℃ and 40 ℃/35 ℃ temperatures,respectively.KEGG metabolic pathway analysis revealed that protein processing in endoplasmic reticulum played a major regulatory role under 30 ℃/25 ℃ and 35 ℃/30 ℃ high temperature stress,while plant hormone signal transduction was the most important metabolic regulatory pathway under 40 ℃/35 ℃ high temperature stress.Almost all photosynthesis-related DEGs identified under the three high temperature treatments were down-regulated,while almost all the DEGs encoding heat shock proteins were up-regulated,more than half of which belonged to the HSP20 family.It is showed that s HSPs play an influential role in the response of A.beshanzuensis seedlings to high temperature stress.5.A.beshanzuensis is lack of adaptability to low altitude habitats and tolerance to high temperature,which may be a contributing factor to its endangered status. |
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