Physiological And Molecular Mechanisms Of Pinus Koraiensis Seedlings In Response To Different Light Conditions Research

With the implementation of the regulation prohibiting commercial logging in natural forests,the focus of forest management has shifted towards strengthening the cultivation of reserve resources and restoring natural forests.The construction of mixed forests in the understory is an important measure for the management of both natural and artificial forests at this stage,and the selection and breeding of shade-tolerant and high-value-added tree species is imperative.Pinus koraiensis,an important indigenous species in northeast China,is a second-class protected species in the country with extremely high economic and ecological value.It is one of the important tree species for the restoration of understory vegetation in the northeastern forest region.Therefore,exploring the optimal light conditions during the seedling stage and studying the growth and development of P.koraiensis,under different light conditions are of great significance for its understory regeneration.In this study,four-year-old(S_2-2)P.koraiensis,seedlings were used as the research material,and normal light was used as the control.The changes in growth indicators,photosynthetic parameters,pigment content,hormone content,and element content of red pine under different light conditions were analyzed.Transcriptome and metabolome analyses were also conducted to explore the physiological and molecular mechanisms of red pine seedlings in response to different light conditions.The main research results are as follows:（1）The results of growth and physiological indicators analysis of P.koraiensis seedlings under different shading treatments showed significant differences in all measured indicators under different treatment conditions.The biomass of P.koraiensis,seedlings were the maximum under 80%light intensity and the minimum under 20%light intensity.The instantaneous net photosynthetic rate and water use efficiency decreased gradually with increasing shading intensity.The content of chlorophyll a,chlorophyll b,and carotenoids increased gradually with increasing shading intensity.Shading treatment increased the content of auxin,zeatin,gibberellin,abscisic acid,and brassinosteroids.The carbon and phosphorus element content showed an increasing trend followed by a decreasing trend with increasing shading intensity,while the nitrogen and potassium element content gradually increased with increasing shading intensity.（2）The transcriptome sequencing results of red pine seedlings under control,80%light intensity,and 20%light intensity conditions showed that a total of 86.32 Gb clean data was obtained from 9 c DNA libraries,and the assembly quality evaluation BUSCO coverage rate was 91.90%.A total of 11916 differentially expressed genes were identified,with the least genes（3751）identified under 80%light intensity compared to the control,and the most genes（8556）identified under 20%light intensity,with 522 common differentially expressed genes.A total of 331 differentially expressed transcription factors were identified,mainly including MYB-related,AP2/ERF,b HLH,HB,MYB,C2H2,and NAC families.Many differentially expressed genes were enriched in plant hormone signal transduction,photosynthesis,porphyrin and chlorophyll metabolism,carotenoid biosynthesis,and flavonoid biosynthesis pathways.（3）Under different photoperiod conditions,significant differences were observed in various measured indices of P.koraiensis seedlings.In terms of growth indices,biomass of P.koraiensis significantly decreased under long photoperiod conditions.In terms of plant hormones,under long photoperiod conditions,levels of auxin and gibberellin in P.koraiensis significantly decreased,while levels of abscisic acid,ethylene,jasmonic acid,brassinolide,and strigolactone significantly increased.Transcriptome sequencing results showed that a total of76.70 Gb of clean data were obtained from 9 c DNA libraries.A total of 10614 differentially expressed genes were identified,with 6428 differentially expressed genes identified under LED prolonged illumination for 4 hours and 5206 differentially expressed genes identified under incandescent lamp prolonged illumination for 4 hours compared to the control.A total of 361differentially expressed transcription factors were identified,mainly including MYB-related,AP2/ERF,MYB,Tify,NAC,WRKY,and b HLH families.A large number of differentially expressed genes were mainly enriched in plant hormone signal transduction,circadian rhythm-plant,and flavonoid biosynthesis pathways.（4）The results of broad-targeted metabolomic profiling using high-throughput sequencing of P.koraiensis seedlings under control,80%light,and 20%light conditions showed a total of911 metabolites detected across 9 samples.Among them,243 differential metabolites were identified and classified into 10 categories,including flavonoids（33.74%）,phenolic acids（16.05%）,lignans and coumarins（11.52%）,among others.Compared to the control,the least differential metabolites（62）were identified under 80%light treatment,while 158 differential metabolites were identified under 20%light treatment.The number of down-regulated metabolites was greater than the number of up-regulated metabolites in all comparison groups,with 13 common differential metabolites identified.Flavonoids were the most abundant category of differential metabolites.KEGG pathway analysis showed that the phenylpropanoid biosynthesis,vitamin B6 metabolism,stilbenoid,diarylheptanoid and gingerol biosynthesis were highly enriched in all comparison groups.Correlation analysis of differential genes and differential metabolites revealed a significant positive correlation between differential genes and differential metabolites in all comparison groups.（5）Based on the conserved domain of the HD-Zip family,62 P.koraiensis HD-Zip genes were identified by filtering out sequences lacking typical HD-Zip domains.These genes were classified into four subfamilies:HD-Zip I,HD-Zip II,HD-Zip III,and HD-Zip IV,containing20,13,13,and 16 genes,respectively.Among them,the expression level of Pk HDZ37,a gene in the HD-Zip II subfamily,gradually increased with increasing shading degree,and it had the highest expression level.Pk HDZ37 may be a key gene involved in the response of P.koraiensis seedlings to light conditions.Subcellular localization experiments showed that Pk HDZ37 is located in the nucleus.A plant expression vector of the Pk HDZ37 gene was constructed and used for genetic transformation of tobacco,resulting in the successful acquisition of 5 transgenic lines.In conclusion,slight shading promotes the growth of P.koraiensis seedlings,indicating that P.koraiensis has a high shade tolerance during the seedling stage and can be used as a preferred tree species for undergrowth renewal.Transcriptome analysis revealed a large number of differentially expressed genes closely related to photosynthesis and other pathways,indicating that the physiological and gene expression levels of P.koraiensis have undergone significant changes in response to changing light environment.This provides an important basis for forest management strategies for underplanting and replanting P.koraiensis.