| Drought is one of the major stress factors that restrict agricultural and forestry products in the world.All land plants are prone to short-term/long-term water deficiency,which leads to water loss and water potential decline,as well as decrease of cell turgor pressure,thus affecting plant growth and development.Pinus massoniana Lamb.,a native tree species to China,is widely distributed in subtropical region of south China and has become one of the most important tree species for afforestation in this region.However,seasonal drought occurs all year long in this region,which severely restricts the survival rate of forestation and forest productivity.Therefore,it is of great significance to study the effects of drought on Pinus massoniana and its mechanism of adaptation to drought,and to find ways to improve its drought resistance to overcome the constraints of drought environment on masson's pine forestation and forest productivity.As a typical ectomycorrhiza(ECM)tree species,the formation of symbiosis is conducive to improving the drought resistance of masson pine.The effect of ECM on the drought resistance of masson pine seedlings is the result of a series of physiological and biochemical reactions and molecular biological changes.It has complex and changeable characteristics.To date,the molecular mechanism of regulating drought resistance of Masson's pine by ectomycorrhizal fungi(ECMF)inoculation has not been reported.In this study,we used masson pine,Suillus placidus(Sp)and Suillus luteus(Sl)and Suillus placidus as materials to study the growth,physiological and biochemical changes of different masson pine mycorrhizal seedlings and non-mycorrhizal seedlings under different drought levels,and the absorption and transformation of non-structural carbohydrates(NSC),meanwhile,the characteristics of composition and content of root exudates were preliminarily explored.Moreover,the morphological and structural changes of masson pine mycorrhizal seedlings and non-mycorrhizal seedlings were observed,and the regulation and response mechanisms of mycorrhizal and non-mycorrhizal seedlings to drought stress were discussed from the transcriptome and proteome levels,aiming to analyze the physiological and molecular mechanism of ECMF improving drought resistance of Pinus massoniana.The main results and conclusions are as follows:1.Growth,physiological and biochemical responses of mycorrhizal and non-mycorrhizal seedlings of Pinus massoniana under different drought conditionsThe seedlings of masson pine inoculated with Suillus luteus(Sl),Suillus placidus(Sp)and uninoculated(NM)were used as materials.The potted planting method simulates drought in the greenhouse,and the weighing method is used to maintain the drought.To study the growth,osmotic adjustment,antioxidant system,photosynthetic physiology,and non-structural carbohydrate(NSC)content and distribution of inoculated and uninoculated masson pine seedlings under different drought levels.The results showed:(1)The growth of NM seedlings was inhibited by drought stress,and the inhibition degree increased with the increase of stress,while the growth of the two kinds of inoculated seedlings was inhibited only under moderate and severe drought stress.Both inoculated and uninoculated seedlings increased root-shoot ratio to resist drought stress.Compared with NM,inoculation of two kinds of mycorrhizal fungi promoted the growth of masson pine seedlings under different water conditions.Under watering and mild stress,the promotion effect of Sp was more obvious,while under moderate and severe stress,the promotion effect of Sl was more obvious.Those results showed that both ectomycorrhizal fungi could effectively alleviate the inhibition of drought stress on masson pine seedling growth,but the effects of different bacteria were slightly different.(2)With the increase of drought degree,MDA content in masson pine seedlings continued to increase,and reached the maximum under severe stress.Both inoculated and uninoculated seedlings responded to drought stress by regulating osmotic substances and antioxidant enzyme activities.Among them,with the exception of individual osmotic adjustment substances or enzyme activities in NM and Sp that have decreased relative to watering treatments under different drought conditions,the two inoculated seedlings and NM seedlings can generally increase proline,soluble sugar and soluble protein.Content and activity of ascorbate peroxidase(APX),phenylalanine ammonia lyase(PAL),SOD,POD and CAT respond to varying degrees of drought stress to maintain cell turgor,remove excessive accumulated reactive oxygen species,and reduce membrane lipids The degree of damage.Compared with NM,the two kinds of inoculant seedlings had lower MDA content,higher soluble sugar content and higher SOD,PAL and POD activities.In addition,CAT activity and APX activity in Sl were also higher under different drought degrees.The results showed that inoculation of two kinds of mycorrhizal fungi could alleviate the oxidative damage caused by drought stress on Masson pine by increasing the content of osmotic regulation substances and the activities of various antioxidant enzymes,and enhance its drought resistance.It suggested that the inoculation of two mycorrhizal fungi can alleviate the oxidative damage caused by drought stress to masson pine by increasing the content of osmotic adjustment substances and the activities of various antioxidant enzymes,and enhance its drought resistance ability.(3)Drought stress decreased relative water content,gas exchange capacity(net photosynthetic rate Pn,stomatal conductance Gs,intercellular carbon dioxide concentration Ci and transpiration rate Tr),maximum photochemical efficiency(Fv/Fm)and chlorophyll content,but increased water use efficiency(WUE).Under the same stress,Sp and Sl could alleviate the inhibition of water and photosynthesis under drought stress to a certain extent,so that the two kinds of inoculated seedlings had higher gas exchange capacity and water content.What,s more,S1 seedlings could also protect the photosystem from drought stress by increasing the carotenoid content.(4)Photosynthesis is one of the important physiological processes for plants to obtain carbon.The inhibition of drought on the growth and photosynthesis of Masson pine seedlings changed the content and distribution of NSC in their bodies.Inoculated and uninoculated seedlings responded to different degrees of drought stress by increasing the total NSC content of the plant,but the accumulations of NSC contents in different tissues were inconsistent,and different drought stress levels had different effects on the NSC contents in different seedlings and different tissues,and the content of accumulated NSC was the lowest in stem.Drought stress also changed the distribution of pinus massoniana seedling NSC,with the increase of stress level,the NM NSC content in leaf and stem growth rates continued to rise,and increased first then decreased in root,and negative growth in severe stress with a lower content than watering treatment,which illustreated the NM accumulated the NSC to response to drought stress,but under the severe stress,carbon in root unbalanced,It is not good for seedling growth and drought resistance under drought stress.Under drought stress,the two kinds of inoculated seedlings accumulated more NSC in response to drought stress.Sp mainly increased NSC by increasing starch content in roots,stems and leaves,while Sl mainly increased soluble sugar content.Under moderate and severe drought stress,NSC content in roots,stems and leaves increased steadily,and the growth rate was higher in roots,which was conducive to promoting root growth and enhancing water absorption capacity of seedlings to resist drought stress,which may also be one of the reasons that seedlings under severe drought stress had stronger drought resistance.2.Effect of drought stress on root characters of inoculated and uninoculated masson pine seedlings(1)Under drought stress,compared with NM,Sl and Sp inoculation effectively improved root growth parameters such as root biomass,root surface area,root volume,root tip number and branch number,also root morphology and root vitality,thus enhanced the ability of water absorption and utilization by the roots of the inoculated seedlings and alleviating the damage caused by drought stress.Under watering,mild and moderate stress,Sp had better promoting effect,and Sl was better under severe stress.(2)The exudates of the rhizosphere soil of all treated masson's pine were determined by GC-MS.A total of 38 compounds including acids,aldehydes,terpenoids,esters,ketones,aromatics and alcohols were identified,among which acid organic compounds accounted for the largest proportion of 13-16 species in the exudates.The composition and relative content of exudates were regulated by water content and inoculation.At different stress levels,the two kinds of mycorrhizal fungi had different effects on the species of exudates from masson's pine roots.Under normal water supply,moderate and severe stress,Sl increased the species of exudates,while Sp only promoted the species under mild stress.Regardless of whether it was inoculated or not,the types of secretions increased under drought stress.The relative contents of all kinds of secretions had obvious and different responses to drought and inoculation,and the range and direction of change were related to the types of secretions.Mycorrhizal fungi inoculation changed the secretion of various relative content changes under drought stress,such as the stress degree increased,the relative contents of terpenoids organic matter decreased first and then increased in NM,but a downward trend in the Sp,and a increase-decrease-increase trend in S;,besides,under the stress of moderate and severe,acids relative contents of organic matter decreased more in the mycorrhizal seedlings.In addition,Sl and Sp inoculation promoted the absorption and transformation of nutrients in rhizosphere soil via increasing urease,thus promoting plant growth and resistance to drought stress.To sum up,inoculation of mycorrhizal fungi could promote the growth of masson pine and alleviate drought stress by improving root morphology,increasing root activity and maintaining higher soil enzyme activity,Sl has a better effect on alleviating drought stress under severe stress.3.Effects of drought stress on cell structure of Pinus massoniana seedlings under different treatmentsSl was selected as the test strain to explore the effects of ECMF on the morphology and cell structure of pinus masson needles under different degrees of drought stress.The results indicate that with the increase of the degree of drought stress,the whole needles of the two seedlings shrivelled,and the mesophyll cells,transmission tissue and phloem cells were seriously dehydrated and deformed.Especially under the severe stress,the mesophyll cells in the uninoculated seedlings were seriously damaged and disordered,and the resin canal cells were not visible.Compared with NM,Sl alleviated the damage degree of drought stress on needles,which was mainly manifested as a lower degree of shrinkage.Under severe stress,mesophyll cells were relatively complete in shape,arranged more closely and orderly,resin canal cells were visible,and epidermis was relatively intact.As the increase of stress degree,the ultrastructure of pinus needles,mainly chloroplast and mitochondria,changed significantly,which mainly showed the increase of starch and osmophilic granules,the damage of chloroplast and mitochondria membrane,the expansion of chloroplast to a large number of holes and the dissolution of mitochondrial crest.Under the same stress,the change range of the inoculated seedlings was small,the chloroplast expansion degree was low,the degree of cavitation was low,and the structure was relatively intact.All results demonstrated that inoculation of mycorrhizal fungi could alleviate the damage caused by drought stress.4.Transcriptome analysis of Pinus massoniana mycorrhizal seedlings in response to drought stressIn order to explore the mechanism of ECMF helping masson pine seedlings cope with drought stress,we selected needles inoculated with Sl and NM seedlings under four water conditions for RNA-Seq analysis.Results showed,a total of 2388 and 8952 differentially expressed genes(DEGs)(FDR<0.01,|log2FoldChange|?1)were identified in the NM and Sl masson's pine seedlings,respectively,and 60 and 525 differentially expressed genes co-expressed in the three drought degrees.GO and KEGG analysis showed that under different drought stress levels,DEGs in NM were enriched into multiple pathways,among which the most important metabolic pathways in response to drought stress mainly included phenylpropanoid biosynthesis,oxidative phosphorylation and photosynthesis metabolism,while plant pathogenic bacteria and glutathione metabolism were also involved in response to drought stress.DEGs enriched in these pathways were mostly up-regulated,but under severe stress,oxidative phosphorylation and photosynthetic metabolic pathway genes were down-regulated,leading to ROS production and aggravating drought stress to cause serious oxidative damage to plant cells.Compared with NM,Sl regulated the enrichment of more genes into each pathway and changed the response of each metabolic pathway to drought stress,including inhibiting the synthesis of phenylpropanoid biosynthesis and various phenolic substances in the flavonoid metabolic pathway.However,it promoted carotenoid metabolism,plant pathogen interaction,sucrose and starch metabolism,oxidative phosphorylation and glutathione metabolism in response to drought stress,especially under moderate and severe stress,so as to mitigate oxidative damage caused by drought stress.What,s more,photosynthetic pathways in the inoculated seedlings were also involved in response to drought stress,and the differential genes enriched in this pathway were up-regulated under severe stress,while the differential genes in the uninoculated seedlings were down-regulated,suggesting that inoculation of Sl could alleviate the damage to the photosystem caused by drought stress and reduce the production of ROS.In brief,Sl can regulate the enrichment of more differential genes into various drought-resistant pathways,maintain relatively stable energy metabolism and antioxidant capacity,and thus improve the drought-resistant capacity of masson pine.In this study,7 conservative genes in response to drought were excavated in Masson pine seedlings,and they were all up-regulated under different degrees of stress.Among them,?-glucosidase E1.11.1.7 and threonine/serine protein kinase FLS2 participating in glucose synthesis and pathogen signal identification and transmission,respectively,were important metabolic pathways that actively respond to drought stress.5.Proteome analysis of Pinus massoniana mycorrhizal seedlings in response to drought stressIn order to explore the mechanism of ECMF helping masson pine seedlings cope with drought stress,we selected needles inoculated with Sl and NM seedlings under normal water supply and severe drought conditions for TMT technology analysis.Results showed,under water supply and drought conditions,there were 394 and 357 differentially expressed proteins(DEPs)in Sl compared with NM,while there were 665 and 1100 differentially expressed proteins in NM and Sl compared with water supply.GO and KEGG analysis showed that drought stress inhibited carbon fixation in Sl and NM,and NM provided energy for plant metabolic activities by promoting the synthesis and accumulation of reactive oxygen scavengers such as phenylpropanoids and flavonoids,and degrading starch and sucrose.Increasing the contents of soluble sugars such as fructose and glucose can improve the osmotic regulation ability and the activities of antioxidant enzymes such as glutathione transferase(GST),glutathione hydrolase(GGT),glutathione peroxidase(GPX),APX and POD to resist drought stress.However,drought stress also inhibited the synthesis and photorespiration of NM terpenoids(diterpenoids and carotenoids without oxygen),resulting in limited energy supply and photosystem protection,which was not conducive to plant growth under stress.Phenylpropanolates and diterpenoids may not be the key pathways for seedlings to resist stress under drought stress,but they can respond to drought stress by increasing the synthesis and accumulation of flavonoids,increasing the activities of SOD and APX,promoting starch degradation,increasing glucose synthesis and maintaining photorespiration.In addition,the inoculation of Sl can also promote the proline and linolenic acid pathway in C6 green leaf volatiles and jasmonic acid(JA)synthesis,activation of abscisic acid(ABA)and ethylene and brassinolide(BR)signal transduction pathway and MAPK cascade specific ways,the activation and regulation of these pathways may be one of the important reasons for the inoculation of Sl to improve the drought resistance of masson pine.Under drought stress,there were 203 DEPs co-expressed by NM and Sl treatments,99 and 92 jointly up-regulated and down-regulated DEPs,respectively.These DEPs may be conserved proteins of masson's pine in response to drought stress,which can provide data support for the mining and identification of drought tolerant proteins in the later stage.The remaining 12 proteins with inconsistent trends may be important proteins that cause the difference in drought resistance between inoculated and uninoculated seedlings.The functional identification of these proteins will provide important basis for the study on the enhancement of drought resistance of masson pine by ectomycorrhizal fungi. |
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