The Mechanism Of Formation Of Yellow Characteristic And Re-greening After Shading In Ulmus Pumila ‘Jinye’ Leaves

Ulmus pumila‘Jinye’（hereinafter referred to as‘Jinye’）is a small deciduous tree of Ulmus in Ulmaceae.‘Jinye’,a yellow leaf mutant of Ulmus pumila L.（hereinafter referred to as U.pumila）,was bred by Hebei Academy of Forestry in 2000.‘Jinye’with golden yellow leaves and beautiful tree performance,can be planted as trees and shrubs.The branches of‘Jinye’are easy to prune and shape.‘Jinye’is a common afforestation tree species in arid and saline-alkali areas,as its strong cold and drought resistance,salt and alkali resistance.‘Jinye’has become an important ornamental tree species with great development and application prospects in urban landscape.However,when U.pumila under shading,its yellow leaves turn green,which greatly reduces the ornamental value.The research of leaf color under shading control mechanism is conducive to solving this problem.In this study,the chloroplast genome of‘Jinye’was sequenced and compared with that of U.pumila.Then physiological and transcriptome differences on leaf color between‘Jinye’and U.pumila were studied.On this basis,long-term and short-term gradient shading treatment was used to explore the response mechanism of leaf color variation of‘Jinye’.The phenotypic statistics,physiological indicators determination,subcellular structure observation and transcriptome sequencing and micro RNAs（mi RNAs）analysis was combined to find the mechanism of leaf re-greening.The main findings are as follows:（1）The chloroplast genome of‘Jinye’accords with the quadripartite structure of that of a typical green plant,and the chloroplast genome structure of‘Jinye’is the same as that of U.pumila.The chloroplast genome of‘Jinye’has a total length of 159,113 bp,encoding 131annotated genes,including 8 r RNA genes and 33 t RNA genes.Among the 11 intron-containing genes,except the ycf3 gene with two introns,the other genes have only one intron.The frequency of codons and SSR loci in the chloroplast genome showed that‘Jinye’had a preference for AT base.There were 12 SNP mutations in‘Jinye’,among which Psb gene played an important role in the development of thylakoid membrane structure.The phylogenetic tree cluster analysis of 48 species revealed that‘Jinye’and other 5 Ulmus species were clustered together.Ulmaceae plants has the closest relationship with Moraceae plants,followed by Rosaceae plants.The phylogenetic tree constructed by chloroplast genome sequence is consistent with traditional taxonomy.（2）The comparative analysis of leaf morphology,physiology and transcriptome between‘Jinye’and U.pumila showed significant differences between them.The content of pigments in‘Jinye’leaves was significantly lower than that of U.pumila.The proportion of chlorophyll a between‘Jinye’and U.pumila was similar,but the proportion of chlorophyll b in‘Jinye’was significantly lower than U.pumila.The proportion of carotenoids in‘Jinye’was significantly higher than U.pumila,and the flavonoid biosynthesis in‘Jinye’was more vigorous.The net photosynthetic rate of‘Jinye’was significantly lower than that of U.pumila.Through the ultrastructure of chloroplast observation,‘Jinye’had fewer grains and failed to stack thylakoids compared with U.pumila.There were 1,307 differentially expressed genes（DEGs）between‘Jinye’and U.pumila,and most of them were related to cell formation and transport.Lipopolysaccharide biosynthesis,auxin synthesis and protein transport of‘Jinye’were lower than that of U.pumila.（3）‘Jinye’were treated with different shading intensity.With the increase of shading intensity,the contents of chlorophyll a,b and carotenoids in leaves of‘Jinye’increased gradually,and the leaf area,perimeter length,width and number of serrated teeth showed an increasing trend.At the same time,with the increase of shading intensity,the Lab value of leaf decreased gradually,the chloroplast volume increased,thylakoid stacking gradually formed,and the number of grana lamellae gradually increased.The diurnal variations of photosynthetic rate,transpiration rate,stomatal conductance and intercellular CO₂concentration were correlated.The diurnal variation of F_v/F_m and F_v/F_o of‘Jinye’showed a"single valley"curve,and with the increase of shading intensity,it showed that‘Jinye’had a strong shade-tolerance.After shading treatment,the leaf color of‘Jinye’turned green and its photosynthetic capacity decreased,but the ability to capture light energy is enhanced.The transcriptome results showed that there were differences in chlorophyll biosynthesis and metabolism,which affected the expression of a series of Hem enzymes,so led to the decrease of chlorophyll precursor synthesis in‘Jinye’leaves after shading treatment.Lhcb1 gene played an important role in shading and re-greening.‘Jinye’regulated the synthesis of Lhcb1protein and promoted the accumulation of pigments mainly through IP3/Ca²⁺signal transduction mechanism.（4）Four short-term shading treatment time points（2 h,6 h,26 h,98 h）were selected to determine the pigment content and analyze transcriptome of the treated‘Jinye’leaves.Comparing with the pigment content and transcriptome data with different intensity of long-term shading,that there were significant differences between the two response mechanisms.The shading intensity of short-term shading treatment was 70%,so the result was closer to that of 70%shading treatment in long-term shading.chlorophyll a and chlorophyll b increased with time,but carotenoids did not change significantly.Chlorophyll a and chlorophyll b in‘Jinye’leaves increased with time,but carotenoids did not change significantly.There were 186 differentially expressed genes（DEGs）at 2 h,527 DEGs at 6h,2,132 DEGs at 26 h,and DEGs decreased to 503 at 98 h.Thirteen common DEGs were screened out,of which 3 genes were related to the transport and metabolism of coenzymes,7 genes detected the In Del locus.Two motifs which may be related to leaf color difference were screened out,which provided a theoretical basis for the follow-up study.（5）The expression profiles of micro RNAs in leaves of‘Jinye’at different shading times were analyzed.Total 517 micro RNAs with conservative sequences were annotated,including 59 mi RNA families.There were 16 mi RNAs differentially expressed at different time points.At the early stage of shading,upu-MIR398-2 was overexpressed,and four mi RNAs（upu-MIR398-11,upu-MIR398-1 upu-MIR398-7,and upu-MIR156-11）were overexpressed at the middle stage,while upu-MIR477-6 was overexpressed at the later stage.The target genes of 39 mi RNAs were predicted.Combining with transcriptome data,one gene c60483＿g1,which was inversely expressed with mi RNA（upu-MIR477-6）and belonged to Yellow Stripe-Like（YSL）family,was found.YSL protein was a metal ion transporter,its reduction will reduce Fe²⁺content.