Effects Of Leaf Shape On Photosynthetic Physiological Characteristics And Growth Of Masson Pine Seedling

Heteroblastic foliage,i.e.primary needles and secondary needles appear with the pine seedling development,and there are differences in the photosynthetic capacity of the two types of leaves.However,the mechanism of their effect on seedling growth has not been systematically explained.In the first growth season of Pinus massoniana,primary needle seedling（PNS）and secondary needle seedling（SNS）will appear simultaneously,which are excellent materials for studying the differences of heteroblastic foliage function.Therefore,the PNS and SNS were selected as the research objects,and the morphological and anatomical structure,photosynthetic energy distribution and matter accumulation,transcriptome gene expression and metabolite accumulation of metabolome were studied.The aims are to reveal the molecular mechanism of the effects of heteroblastic foliage on photosynthetic physiological characteristics and growth of P.massoniana seedlings.The main results and conclusions were as follows:（1）There were heterogeneous between the morphology and anatomical structure of primary and secondary needles.The stomata on the surface of primary needles arranged closely,deep concave and not closed.The shape of the cross section was diamond or fan-shaped,with two vascular bundles and relatively high proportion of mesophyll tissue.It is more conducive to the absorption of CO₂and capture of light energy,showing the potential of higher photosynthetic efficiency for primary needles.The secondary needles had smaller stomata and more uniform distribution.The lower stomatal conductance reduced the water loss caused by transpiration,showing obvious xerophytic structure and higher stress resistance.In addition,the cross sections of the base,middle and tip of secondary needles were semi-elliptic,and the central tissue composed of xylem phloem and transport tissue had abundant tracheids and sieve cells,which were more conducive to the efficient transport of water and photosynthate.（2）The light response strategies were significantly different between primary and secondary needles in the first growing season.Primary needles,as juvenile leaves,were sensitive to seasonal climate change,and the reduction of temperature induced the rapid opening of the photoprotection mechanism,non-photochemical quenching（NPQ）increased continuously,enhanced heat dissipation effect,while photochemical efficiency and photochemical activity（φPo and Fv/Fo）decreased gradually.Meanwhile,the proportion of light energy allocated to Y（NO）increased gradually,while the proportion of light energy allocated to Y（NPQ）and Y（II）decreased in primary needles.Especially under high light intensity,primary needles dissipated excess light energy to avoid damage to the photosystem caused by photoinhibition,which reduced the efficiency of light energy utilization and ultimately resulted in the decrease of photosynthate accumulation in seedlings.As mature leaves,secondary needles showed strong resistance.With the decreasing temperature,secondary needles increased the photosynthetic quantum yield and photochemical quantum yield.The ETR and light energy efficiency were increased,and the Y（II）ratio was increased by decreasing the quenching ratio of Y（NPQ）and Y（NO）,and more energy was used for photochemical reaction,which promoted the accumulation of dry matter mass of the whole seedlings,providing a more conservative resource utilization strategy for seedlings.（3）There were significant differences in NSCs allocation and biomass accumulation patterns between PNS and SNS.The results showed that SNS promoted the accumulation of NSCs in leaves and roots,enhanced their ability to obtain light energy,nutrition and water,and facilitated the later seedlings to obtain more photosynthates.Path analysis showed that net photosynthetic rate（Pn）was the key factor determining the biomass accumulation of PNS.Photosynthetic pigments had a direct positive effect,and could promote the biomass accumulation of seedlings by indirectly increasing the synthesis of soluble sugar in stems.However,heat dissipation caused by photoinhibition and water deficit was the main factor limiting carbon accumulation.For SNS,water deficiency caused by transpiration and reduced share of electron transport chain used for photochemical reaction was not be conducive to biomass accumulation of seedlings.（4）we analyzed the differences of primary and secondary needle functional traits at the molecular level.A total of 71 563 full-length Unigenes sequences were obtained.The transcriptome showed that 2037,1920,886 and 515 differentially expressed genes（DEGs）were screened in August,September,October and November,respectively.The enrichment results of KEGG pathway showed that DEGs was mainly enriched in photosynthesis-antenna protein synthesis,polysaccharide metabolism and plant hormone signal transduction,which caused differences in photosynthetic capacity,substance accumulation and stress resistance of heteroblastic foliage of P.massoniana seedlings.With the decrease of seasonal temperature,genes（SWEET,GMPP,CCR and EIN3）related to the biosynthesis of sugar transporter,polysaccharide,lignin,jasmonic acid and ethylene were highly expressed in SNS,which was beneficial to improve the overall utilization efficiency of resources and the resistance of seedlings to adverse environment.The gene co-expression network map was constructed based on weighted gene co-expression network analysis（WGCNA）.And the total of 34 hub genes were identified.Among them,the key genes（CYP86A4S and CYP86B1）regulating cutin and suberin biosynthesis were up-regulated in SNS,which promoted the accumulation of cutin and suberin in secondary needles.The expression levels of genes regulating the synthesis of light harvesting complex protein（LHCB1、LHCB2、LHCB4 and LHCB5）,photosystem II protein（psb S、psb D、psb R、psb O and psb Y）and photosystem I protein（psa D、psa G、psa K and psa E）were continuously up-regulated in SNS,which improved the light-capturing ability and light-using efficiency of secondary needles.Based on time-ordered gene co-expression network（TO-GCN）analysis,transcription factors related to photosynthetic physiology were screened and distributed in 10transcription factor families.The co-expression network mapping revealed that ERF16,ERF38,TCP13 and NAC68 were key factors to improve the stress resistance and growth of SNS,and the transcription factor families AP2/ERF,TCP,NAC and b HLH were closely related to the photosynthetic physiological functions of heteroblastic foliage.（5）The extensive targeted metabonomics analysis of P.massoniana seedlings was performed using UPLC-MS/MS.The results showed that a total of 725metabolites were detected in the two leaf type seedlings,and 58 differential metabolites（DMs）were screened by PCA and OPLS-DA,among which 29metabolites were up-regulated and 29 were down-regulated in SNS.Amino acids derivatives and most flavonols were accumulated in PNS.Secondary needles promoted the synthesis of nucleotides derivatives and flavonoids in seedlings,which was beneficial to enhance the lignification degree of stem,and thus enhanced the radial growth of stem.Through O2PLS model analysis of transcriptome and metabolome data,it was found that DEGs and DMs were mainly enriched in amino acid biosynthesis pathway.In this study,a total of 8 amino acids and derivatives were screened for differential accumulation in two leaf type seedlings,and 12 kinds of enzyme gene that were closely related mainly involved in processes such as glycolysis,plant hormone signal transduction,and response to abiotic stress.This may be an important reason for the different growth strategies of PNS and SNS.