| The slow renewal of natural forest and management of artificial forests have been extensive,resulting in many low-quality forests.Natural secondary forest renewal and nurturing have become important methods and means to improve the ecological and economic benefits of forest land.Understory planting and replanting is one of the important ways to promote forest renewal,and the light environment is an important factor restricting understory renewal.Light is one of the important environmental factors affecting plant growth and development.Therefore,the study of the growth characteristics of main afforestation tree species under different light environments is of great significance.In this study,Juglans mandshurica seedlings were used as the research materials,and different levels of shading treatment(control,80% light,50% light,20% light)and supplemental lighting treatment(control,LED supplemental lighting for 4 hours,LED supplemental lighting for 8 hours)were applied.The growth and physiological indicators of J.mandshurica under different treatments were measured,and further transcriptome and metabolome sequencing and analysis of the leaves were conducted to analyze the effects of different light environments on the morphological and physiological indicators of J.mandshurica,and to explore the molecular response mechanism of J.mandshurica to different light environments.The main research results are as follows:(1)The results of measuring the growth and physiological indicators of J.mandshurica under different levels of shading treatment showed that all indicators differed significantly.Regarding growth indicators,the maximum seedling height and biomass of J.mandshurica were obtained under 50% light treatment.In terms of leaf phenotypes,dry weight ratio was the highest under the 80% light treatment and the lowest under 20% light treatment.As the light intensity decreased,the light compensation point and light saturation point decreased gradually,and the photosynthetic rate and water use efficiency also decreased gradually.The intercellular carbon dioxide concentration increased,and the stomatal conductance and transpiration rate increased first and then decreased.Chlorophyll content increased gradually as the light intensity decreased.Shading treatment increased the content of auxin,gibberellin,abscisic acid,jasmonic acid,and brassinolide in J.mandshurica significantly.The carbon element content was the highest under 80% light,and the nitrogen element content increased gradually as the light intensity decreased.Shading treatment increased the potassium element content significantly.This study shows that shading treatment can improve the growth of J.mandshurica,and the maximum growth was achieved under 50% light treatment.(2)Transcriptome sequencing of 50% shade,20% shade,and control groups revealed that a total of 81.75 Gb of clean reads were obtained from 9 c DNA libraries,and the alignment rates of all samples to the reference genome were over 95%.Compared to the control group,the 50% shade group had the most differentially expressed genes(4298),while the 20% shade group had 4243 differentially expressed genes,with 251 differentially expressed genes found in all three comparison groups.A total of 470 transcription factors were differentially expressed under different shading treatments,with the largest gene families being AP2/ERF,MYB,b HLH,C2C2,and MYB-related.The KEGG analysis of the three comparison groups mainly enriched the following pathways: starch and sucrose metabolism,plant hormone signal transduction,carotenoid biosynthesis,plant-pathogen interaction,phenylpropanoid biosynthesis,flavonoid biosynthesis,galactose metabolism,secondary metabolite biosynthesis,ABC transporters,and tryptophan metabolism,etc.A large number of differentially expressed genes were concentrated in the photosynthesis,plant hormone signal transduction,chlorophyll metabolism,carotenoid biosynthesis,and flavonoid biosynthesis pathways.(3)Extensive targeted metabolomic sequencing results under different levels of shading treatment(50% light,20% light,and control)showed that a total of 1,048 metabolites were detected in this study,including 470 differentially expressed metabolites.Compared with the control,268 and 331 differentially expressed metabolites were identified under 50% and 20%light conditions,respectively,and 46 differentially expressed metabolites were present in all three comparison groups.The 470 differentially expressed metabolites were classified into 11 categories and other groups,with the most abundant being flavonoids,phenolic acids,organic acids,amino acids and their derivatives,and lipids.Flavonoids were further divided into seven categories based on secondary classification,with the most abundant being flavanols,flavones,flavonols,and dihydroflavones.KEGG enrichment analysis showed that flavonoid biosynthesis,arginine and proline metabolism,flavonoid and flavonol biosynthesis,as well as fructose and mannose metabolism pathways were enriched at a high level in all three comparison groups.Nine differentially expressed metabolites were identified in the flavonoid biosynthesis pathway,and the accumulation of naringenin chalcone,naringenin,kaempferol,dihydrokaempferol,quercetin,and dihydroquercetin compounds increased gradually with the increase of shading degree.(4)According to phylogenetic tree analysis,57 Jm HD-Zip genes were identified from the published genome of J.mandshurica,which were divided into four subfamilies: HD-Zip Ⅰ,HD-Zip Ⅱ,HD-Zip Ⅲ and HD-Zip Ⅳ.The genes with similar gene structure were clustered in one branch.Among them,the HD-Zip II subfamily member Jm HDZ43 showed significant differential expression under different shading treatments and had a high expression level.By injecting tobacco cells,Jm HDZ43 was found to be located in the nucleus.Through agrobacterium-mediated transformation,six transgenic tobacco lines were obtained.(5)The results of growth and physiological indicators measurements of J.mandshurica under different supplementary lighting duration showed that all indicators except stomatal conductance had a significant difference between treatments.The plant height of J.mandshurica under LED supplementation for 4 hours was significantly higher than that of other treatments,and the dry weight of J.mandshurica gradually decreased with increasing supplementary lighting duration.Fresh weight of leaf,dry weight of leaf,and leaf area all showed the pattern of 4-hour supplementation > control > 8-hour supplementation.With the extension of lighting time,the light compensation point gradually increased,and the net photosynthetic rate and water use efficiency decreased,while the intercellular carbon dioxide concentration increased,and the transpiration rate showed a trend of decreasing first and then increasing.Regarding plant hormones,supplementary lighting significantly increased the content of gibberellin and abscisic acid,and the concentration of auxin was the lowest under 8-hour supplementary lighting.This study suggests that prolonged lighting time is not conducive to the growth of J.mandshurica.(6)The transcriptome sequencing results showed that a total of 83.77 Gb of clean data were obtained from 9 c DNA libraries under different supplementary lighting duration,and the comparison rate of all samples reached more than 95%.Compared with the control,5607 and4697 differentially expressed genes were identified after 4 and 8 hours of supplementary light,respectively,and 262 differentially expressed genes were found in all the three groups.A total of 501 differentially expressed transcription factors were identified in this study.AP2/ERF,MYB,b HLH and C2C2 were the families with the most differentially expressed genes.Starch and sucrose metabolism,carotenoid biosynthesis,plant MAPK signaling pathway-plant,phenylpropanoid biosynthesis,secondary metabolite biosynthesis,ABC transporter,plantpathogen interaction,and flavonoid biosynthesis pathways had higher KEGG enrichment abundance in the three comparison groups.Plant hormone signal transduction and circadian rhythm-plant pathways were enriched in the three comparison groups,and a large number of differentially expressed genes were found.This study indicated that excessive lighting time broke the original circadian rhythm and disrupted its homeostasis.In summary,shading treatment can significantly improve the growth of J.mandshurica,indicating that it has a certain shade tolerance during the seedling stage and is an optimal tree species for understory regeneration.Transcriptome sequencing analysis revealed a large number of differentially expressed genes responding to changes in light environment,indicating that J.mandshurica responds to changes in light environment by regulating its growth,physiology,and molecular mechanisms,providing an important theoretical basis for the forest management of understory replanting and planting of J.mandshurica. |
