Analyses Of Leaf Membrane Lipid Composition And Chloroplast FAD Gene Function In Ammopiptanthus Mongolicus Under Different Stress Conditions

Membrane lipids are the basic backbone of membrane systems in plant cells.The composition and unsaturation degree of membrane lipids are closely related to the resistance of plants to adverse environments,such as temperature stressors.Ammopiptanthus mongolicus is the only evergreen broadleaf shrub that is endemic to the central Asian desert and has a very high tolerance to cold and drought stresses.In this paper,focusing on the relationship between both the composition and unsaturation degree of membrane lipids in A.mongolicus leaves and stress tolerance of this plant,we carried out three aspects of work:(1)analyzed the changes in unsaturation degree of leaf fatty acids as well as the relative proportion and unsaturation degree of chloroplast membrane lipids under different stress treatments in laboratory and harsh environments in the wild;(2)analyzed expression changes of the genes for key fatty acid desaturases(FADs)located in chloroplast under different stress conditions;(3)cloned the genes for chloroplastic ?-3 FADs,namely AmFAD7 and AmFAD8,and investigated their functionsin stress tolerance.The main results are as follow:(1)In total,eleven classes of fatty acids were detected in A.mongolicus leaves,including linoleic acid(18:2)and linolenic acid(18:3),but without hexadecadienoic acid(16:2)and hexadecatrienoic acid(16:3).Therefore,this species belongs to an 18:3 plant.(2)In the leaves of laboratory-cultured A.mongolicus seedlings,the degree of fatty acid unsaturation increased after the cold and drought treatments but decreased after the heat and salt treatments.In the leaves of A.mongolicus shrubs naturally growing in the wild,the degree of fatty acid unsaturation was quite low in the summer but relatively high during the autumn and winter months.(3)In the leaves of laboratory-cultured A.mongolicus seedlings,the relative proportions of monogalactosyldiacylglycerol(MGDG),phosphatidylglycerol(PG)and sulfoquinovosyldiacylglycerol(SQDG),three major compositions of chloroplast membrane lipids,decreased;while that of digalctosyldiaculglycerol(DGDG),another major composition of chloroplast membrane lipids,increased under cold treatment.Furthermore,the degree of fatty acid unsaturation in all the four lipids increased under the cold treatment.Under heat treatment,the relative proportions of MGDG and DGDG decreased,while those of PG and SQDG increased.Moreover,the degree of fatty acid unsaturation in all the four lipids decreased under the heat stress.Under drought treatment,the relative proportion of MGDG decreased,while those of three other lipids increased.The degree of fatty acid unsaturation increased in DGDG but decreased in three other lipids under the drought stress.Under salt treatment,the relative proportions of MGDG and PG decreased;while those of DGDG and SQDG increased,and the degree of fatty acid unsaturation in all the four lipids also increased.Interestingly,under all the four stress treatments,the relative proportions of MGDG decreased but the ratios of DGDG to MGDG increased,without exception.In the leaves of naturally growing A.mongolicus shrubs in the wild,a high proportion of MGDG and a low proportion of DGDG were detected in the spring and early summer.In other seasons,however,the relative proportions of these two lipids were just the opposite.Moreover,the degree of fatty acid unsaturation was high in PG in the spring.In the summer,both MGDG and DGDG showed a low degree of fatty acid unsaturation.In the autumn,the degree of fatty acid unsaturation was relatively high in both MGDG and SQDG.In the winter,all the four lipids had quite high degree of their fatty acid unsaturation.Compared with laboratory-cultured seedlings,the shrubs growing in the wild had higher values in both the relative proportions of DGDG and the ratios of DGDG to MGDG.Moreover,the degree of fatty acid unsaturation was low in MGDG but high in the other three lipids in the shrubs.(4)In the leaves of A.mongolicus seedlings,the transcription levels of AmFAD7 and AmFAD8 increased but that of AmFAD6 decreased during two weeks of the cold treatment.During two weeks of the heat treatment,the transcription levels of AmFAD6 and AmFAD8 firstly increased and then decreased,while that of AmFAD7 was reduced.The transcription levels of AmFAD6 and AmFAD8(especially the latter)were up-regulated,while that of AmFAD7 was down-regulated under two weeks of the drought treatment.After a week of the salt treatment,all the three AmFADs showed increased transcription levels.In young leaves of naturally growing shrubs in the wild,the transcription levels of AmFAD6 were slightly lower during the winter months than in other seasons;the transcription levels of AmFAD7 were generally higher during the autumn and winter compared with the spring and summer.However,the transcription of AmFAD8 varied irregularly throughout the year.(5)The predicted proteins of AmFAD7 and AmFAD8 consist of 455 and 457 amino acid residues,respectively.Both the proteins contain one chloroplast transit peptide and three conserved histidine clusters.The complete coding region gDNAs of AmFAD7 and AmFAD8 consist of 2247 and 2132 bp,respectively,and both the gDNA sequences contain seven introns and eight exons.(6)Both AmFAD7 and AmFAD8 promoter regions contain the elements for light and hormone responses.The AmFAD8 promoter also contains the elements for the response to low temperature and dehydration.(7)During the stress treatment period of 0-48 h,the transcription level of AmFAD7 in shoots of the seedlings was up-regulated by the cold and heat treatments but down-regulated by the salt treatment.In roots of the seedlings,however,the transcription level of AmFAD7 was increased by the cold and salt treatments but decreased by the heat treatment.The transcription level of AmFAD8 in shoots of the seedlings was induced by the cold and salt treatments but inhibited by the heat treatment.In roots of the seedlings,however,the transcription level of AmFAD8 was up-regulated by the salt treatment but down-regulated by the cold and heat treatments.(8)In different organs of the plants growing in the wild,both AmFAD7 and AmFAD8 were most abundantly expressed in the leaves.In addition,they were most weakly expressed in the twigs and lateral roots,respectively.(9)Both AmFAD7 and AmFAD8 enhanced the tolerance of their transgenic Arabidopsis to freezing and drought stress(AmFAD8 was more tolerant to drought),but decreased the thermostability of the transgenics.In addition,both the membrane stability and oxidative stress in transgenic lines were obviously improved under cold and drought stresses.