Molecular Mechanism Of Environmental Adaptation Of Heterophylly In Populus Euphratica Oliv.

Heterophylly is the phenomenon that plants produce different forms of leaves in the same plant based on phenotypic plasticity,which is a survival strategy for plants to adapt to the environment and optimize resource utilization.Populus euphratica Oliv.,a typical heterophyllous woody plant,is the only natural tree species and important grouping tree species in the desert areas of Central Asia and Northwest China,playing an important role in resisting wind and sand,maintaining regional ecological balance and biodiversity.Adult P.euphratica developed various forms of leaves,such as lanceolate leaves,ovate leaves,and dentate broad-ovate leaves,which can acclimatize to adverse conditions and becomes an important model tree for researching the environmental adaptation of trees.To date,the research on environmental adaptability of heteromorphic leaves in P.euphratica mainly focused on morphological structure and physiological characteristics,however,research on its molecular mechanisms is limited.The comparative study of the molecular biology of heteromorphic leaves in P.euphratica could reveal the mechanism of environmental adaptation in P.euphratica to a certain degree.Moreover,there is important theoretical and practical significance to the protection and rejuvenation of P.euphratica forest and vegetation restoration in arid and semi-arid areas.In this study,comparative analysis of physiological and biochemical characteristics,protein expression,expression of coding genes and non-coding RNAs(lncRNA,circRNA,miRNA)has been conducted among lanceolate leaves,ovate leaves,and dentate broad-ovate leaves.Based on these results,the competitive endogenous RNA network was further analyzed,and the function of the key differentially expressed gene PePIP2;5 in the competitive endogenous RNA network was investigated.This study could reveal the molecular mechanism of environmental adaptation of heteromorphic leaves in P.euphratica,and provide a reference for the study of the mechanism of environmental adaptation of heterophyllous plants,especially heterophyllous woody plants.The main results are as follows:(1)Investigation of the relationship between physiological and biochemical characteristics of three typical heteromorphic leaves in P.euphratica and adaptability to the heterogeneous environment was performed.The results showed that the light conditions of the upper canopy are superior to that of the lower canopy.The lanceolate leaves in the lower canopy showed shade resistance,with smaller specific leaf weight,dark respiration rate,light compensation point and light saturation point,making them more effective in capturing light energy in low light conditions and reducing Carbon loss to make photosynthetic product accumulated.The ovate leaves and dentate broad-ovate leaves in the upper canopy showed adaptability to high light environments,with larger specific leaf weight,light compensation point,and light saturation point.It is worth noting that the dentate broad-ovate leaves exhibited obvious xerophytic structure and better stress resistance.This type of leaves developed trichomes and sunken stomata with small size and large density,which could effectively regulate transpiration loss.Furthermore,higher content of flavonoids,phenol,carotenoids,and lignin in dentate broad-ovate leaves,suggested that the tolerance to adverse environments in this type of leaves is better than others.(2)Analysis of the relationship between protein expression and environmental adaptability of three typical heteromorphic leaves was conducted.Thus,the protein-protein interaction network of heteromorphic leaves in P.euphratica was established,and metabolic networks of eco-adaptability mechanism in heteromorphic leaves of P.euphratica was proposed.The results showed that proteins involved in the light reaction of photosynthesis,such as Photosystem I reaction center subunit V family protein and Photosystem II oxygen evolving complex,were abundant in ovate leaves and dentate broad-ovate leaves,indicating that the leaves in the upper canopy have advantages in photosynthetic capacity.The ovate leaves showed high capacity in basic metabolism,with proteins related to carbon metabolism,nitrogen metabolism,and other basic metabolisms,such as pyruvate kinase and glutamic acid decarboxylase,that accumulated in this type of leaves.Proteins involved in plant stress response,such as peroxidase,caffeic acid-O-methyltransferase,glutamine synthetase,etc.,were accumulated in dentate broad-ovate leaves,which could assist the dentate broad-ovate leaves to respond to adverse environments such as strong light,high temperature,and drought conditions.(3)Analysis of coding genes and non-coding RNAs(lncRNA,circRNA,miRNA)in heteromorphic leaves by high-throughput sequencing was performed.A total of 586 differentially expressed genes were identified between lanceolate leaves and dentate broad-ovate leaves,including members belonging to 17 important transcription factors families in plants,such as WRKY,NAC,COL,and C2H2.These differentially expressed genes are mainly involved in plant defense responses and secondary metabolism processes such as alkaloids and lignin.The BHLH transcription factor,NAC transcription factor involved in stress response and secondary metabolism of P.euphratica and TCP transcription factor regulating leaf shape were up-regulated in dentate broad-ovate leaves,which could make this type of leaves do better in stress resistance and lead to the emergence of different forms of leaves in P.euphratica.A total of 1725 lncRNAs have been identified in heteromorphic leaves of P.euphratica,of which 54 lncRNAs were differentially expressed between lanceolate leaves and dentate broad-ovate leaves.These differentially expressed lncRNAs involved in regulating the expression of target genes related to light adaptation,protein repair,stress response,and growth and development process,assisting plants to efficiently utilize environmental resources and respond to adverse environmental conditions.Through systematic analysis,a differential expression lncRNAs-target genes interaction network related to environmental adaptation was constructed,and the response mechanism of heteromorphic leaves in P.euphratica to heterogeneous light environments was proposed.The dentate broad-ovate leaves in the upper canopy avoid high-light damage and inhibition by regulating the epidermal wax synthesis and xanthophyll cycle-dependent photoprotection;the lanceolate leaves in the lower canopy can improve light capture in low light environments by the higher expression of antenna protein in photosynthesis.A total of 1702 circRNAs were identified in heteromorphic leaves of P.euphratica,of which 76 circRNAs were differentially expressed between lanceolate leaves and dentate broad-ovate leaves.These differentially expressed circRNAs were mainly involved in oxidation-reduction process,plant defense responses,glyoxylate and dicarboxylate metabolism,nitrogen metabolism pathways.Moreover,circRNA-miRNA targeting diagram of two types of heteromorphic leaves in P.euphratica was constructed,of which 59 circRNAs can be targeted by 72 miRNAs and participate in the post-transcriptional regulation of coding genes.A total of 517 known miRNAs and 127 novel miRNAs were identified in heteromorphic leaves of P.euphratica,of which 22 circRNAs were differentially expressed between lanceolate leaves and dentate broad-ovate leaves.These differentially expressed miRNAs were mainly involved in cellular response to salt stress,RNA degradation,inositol phosphate metabolism,cutin,suberine and wax biosynthesis,base excision repair pathways.The differentially expressed miRNAs can regulate the expression of nuclear transcription factor Y,F-box protein,b HLH transcription factor,NAC transcription factor and Cation/H(+)antiporter that play an important role in drought resistance and salt tolerance of P.euphratica,so that the dentate broad-ovate leaves have advantages in enduring adverse environmental conditions.Competitive endogenous(ce RNA)networks of heteromorphic leaves in P.euphratica were predicted,including 5 circRNAs,9 lncRNAs,17 miRNAs,and 93 m RNAs that were differentially expressed in heteromorphic leaves of P.euphratica.The expression level of 4 ce RNA regulatory groups including aquaporins PePIP2;5 were identified to be consistent with the pattern of ce RNA expression regulation.The regulatory networks of competitive endogenous RNA networks in two types of heteromorphic leaves in P.euphratica were established.(4)The PePIP2;5 gene of P.euphratica was cloned and its expression pattern was analyzed.The results showed that the expression levels of PePIP2;5 were different in tissues and heteromorphic leaves of P.euphratica.High expression of PePIP2;5 in dentate broad-ovate leaves was beneficial to this type of leaves to cope with adverse conditions better,such as high light,high temperature,and water deficit.Using the leaf disc transformation method,the constructed overexpression vector of PePIP2;5 was transformed into wild-type 84 K poplar,and 84 K transgenic plants with PePIP2;5 overexpression were obtained and function study was performed.It was found that PePIP2;5 can enhance the ability of plants to resist the drought stress,enable the transgenic plant to maintain better osmotic adjustment capacity and antioxidant enzyme activity under drought stress conditions,help to retain water and resist oxidative stress.So that the transgenic plant can maintain photosynthetic efficiency under stress conditions,which improves the plant's ability to survive under stress.These results further indicated that the up-regulated expression of PePIP2;5 in dentate broad-ovate leaves can enhance their ability to resist drought stress,alleviate their physiological inhibition under water deficit conditions,and maintain the photosynthetic productivity.Taken together,these above results indicated that the heterophylly of P.euphratica is the mechanism for plants to respond to heterogeneous environments,and is also an adaptation strategy for improving the plant's own survivability and production efficiency.The results of this study will lay a foundation for understanding the molecular mechanisms of environmental adaptability of P.euphratica and other heterophyllous plants.