Screening Strong Cold-Hardiness And High-Light-Efficiency Triploid Loquat (Eriobotrya Japonica) And Study On The Underlying Mechanisms

Loquat（Eriobotrya japonica）is a subtropical evergreen tree belongs to the Rosaceae family.Loquat blooms in autumn and early winter,and sets fruit in winter,so damage due to below 0℃low temperature and frost conditions often causes dynamic loss of loquat fruit and the industry.Triploid loquat not only produces seedless fruit,but also has greater growth vigor,strong tolerance to abiotic stress.Therefore,in order to improve the limitations in the development of loquat industry,four triploid loquat F₁generation lines produced by crossing of the diploid with tetraploid（B431×GZ23,B431×GZ1,NHB×B431,B431×GZ18）and their parents were used in this study.The triploid F₁ generation lines of B431×GZ23 were screened out by their strong cold hardiness and high photosynthetic efficiency.These triploid F₁ lines and their parents were used for further underlying mechanisms of strong cold-hardiness and high photosynthetic efficiency.The photosynthetic parameter,chlorophyll fluorescence parameters,the cell structure and content of endogenous hormone were measured,and lipidomics and transcriptome analysis were conducted.Two membrane lipid desaturase genes Ej FAD2 and Ej FADC1 were cloned and their functions were studied.The main outcomes from this work are as follows:1 Screening triploid loquat with strong cold-hardiness and high-light-efficiencyFour F₁ generations of triploid loquat（Eriobotrya japonica）and their tetraploid and diploid parents were used as materials to evaluate their growth vigor,photosynthetic efficiency and cold tolerance,respectively.Growth vigor of triploid loquat was stronger than its parents,and the growth rate of triploid loquat leaf length was faster than that of their parents.The order of their cold-hardiness evaluated by conductivity method and chlorophyll fluorescence method is:B431×GZ23>NHB>B431×GZ1>GZ18>GZ1>B431>GZ23>NHB×B431>B431×GZ18.The evaluation model was established by AHP combined with grey correlation analysis.The results showed that triploid loquat F₁ generation line B431×GZ23 had strong cold hardiness and the best photosynthetic efficiency.In order to verify the difference of photosynthetic efficiency among different triploid loquat F₁ under cultivation environment,two F₁ hybrid triploid loquat lines B431×GZ23 and B431×GZ1 and their respective parents were used as materials for the photosynthetic efficiency and photosystem activity analysis.The results showed that the P_n of triploid loquat B431×GZ23 was significantly higher than that of its parents and F₁ triploid from B431×GZ1 under low temperature stress in December,and its photosystem had better stability,which was consistent with its strong cold-hardiness.2 Analysis of ultrastructure of mesophyll cells and physiological and biochemical response under low temperature stressThe B431×GZ23 and its parents were treated at 0℃,-3℃and-5℃for 72 h（25℃as the control）,and the photosynthetic parameters,fluorescence parameters,photosynthetic pigment content,photosynthetic enzyme activities,Endogenous hormone content,cell ultrastructure and lipidomics were measured.The results showed:2.1 Photosynthesis response analysisUnder different low temperature stress,the P_n of triploid loquat was higher than that of its parents,and the difference was significantly at 25℃and 0℃treatment.At-3℃,the P_n of triploid was significantly higher than that of diploids,but no difference with tetraploids.P_n cannot be detected under-5℃treatment.The total chlorophyll content at 25℃was tetraploid>triploid>diploid,and the chlorophyll content of triploid loquat increased at 0℃and-3℃.The electron transfer efficiency and photochemical energy of PSII,photosynthetic enzymes of triploid loquat were higher than that of its parents under different low temperature stress,and the photoprotection ability of triploid loquat was stronger than that of its parents.In conclusion,the photosystem stability,carbon cycling,fixation and recovery ability of these triploid loquat under low temperature stress are stronger than its parents,which might be the important reason for their higher photosynthetic efficiency.2.2 Analysis of contents of endogenous hormoneUnder low temperature stress,the contents of resistance related endogenous hormones SA and ABA in loquat leaves increased,while those related to growth,such as ZT,IAA and GA₃,decreased.The contents of SA and ABA in the leaves of triploid loquat were significantly higher than those of the two parents under-3℃and-5℃temperature stress,and the ABA/GA₃ value increased significantly with the decrease of temperature,and the SA/GA₃ value increased significantly at-3℃and-5℃,which may be related to the inhibition of growth and activation of resistance genes to enhance cold-hardinss under low temperature stress.2.3 Analysis of ultrastructure of mesophyll cells and lipidome responseThere was little damage to mesophyll cells of loquat japonica at 0℃treatment,and the plants could recover quickly.Under-3℃and-5℃stress,the mesophyll cell structure of triploid loquat was more complete and the chloroplast membrane was clearer than that of its parents,and the plant recovery ability was stronger than that of its parents.Further lipidomics analysis showed that a large number of lipids in triploid loquat were up-regulated under-3℃stress.Diploid showed a downward trend,and tetraploid showed a similar degree of upward and downward.Triploid loquat lipids showed the most strong response to low temperature stress,and there were 124 different lipids,which were 3.18 and 4 times of that in tetraploid and diploid,respectively.The main differential lipids were PC（phosphatidyl choline）,TGA（triglyceride）,PE（phosphatidyl ethanolamine）,PG（phosphatidylglycerol）,DGDG（digalactosyl diglyceride）,MGDG（monogalactosyl diglyceride）,SQDG（sulfoquinoxyl diacylglycerol）.These lipids play an important role in the stability of plasma membrane,chloroplast membrane and thylakoid membrane,and their relative content and unsaturation in the triploid are significantly increased,and there was no obvious change in diploids.In conclusion,membrane lipid response under low temperature stress has important influence on membrane lipid remodeling,which determines the low temperature tolerance of loquat with different ploidy.3 Transcriptome analysis and key genes miningTranscriptomics and physiological responses were used to analyze the differential expression of genes in membrane lipid,photosynthesis and hormone signal transduction pathways in triploid loquat and its parents.3.1 Analysis DEGs of Lipid synthesis and metabolism pathwayAfter low temperature treatment,the differentially expressed genes were mainly enriched in lipid metabolism pathway in the polyploid loquat plants.There were 48up-regulated genes in the metabolic pathway of glycerophospholipid in triploid loquat plants,including PE,PC and PG synthesis,which were related to the stability of the membrane.PC is an important component of plasma membrane and chloroplast outer envelope,which is closely related to the cold hardiness of plants.The up-regulation of PC synthesis related genes Ej GPAT,Ej PAH1.1,Ej LACS8.2,down-regulation of PC homeostasis related genes Ej NPC,EJPLA1-IIδ,Ej PLDδ1.2,and the dominant expression of FAD gene may play an important role in the formation of cold tolerance in triploids.Under low temperature stress,the expression level of five FAD genes in polyploid loquat plants was higher than that of diploid loquat plants,especially the Ej FAD2.The q RT-PCR results showed that the expression level of Ej FAD2 gene in triploid loquat plants was significantly higher than that of its parens.At the same time,the q RT-PCR showed that Ej FADC1 gene were similar to Ej FAD2 gene,which may also contribute to the cold-hardiness of these triploid loquat.In addition,the ratio of MGDG/DGDG was correlated with the stability of thylakoid membrane.Lipid group analysis showed that the ratio of MGDG/DGDG was the highest in triploid,which may be related to the high expression of Ej MGD1 and low expression of Ej DGD1.3.2 Analysis DEGs of hormone signal transduction pathwayUnder low temperature stress,the expression of growth-related hormone IAA,CTK,GA and BR（Brassinosteroid）signal transduction pathway genes in loquat leaves was significantly down-regulated,resistance-related hormone ETH（Ethylene）,ABA,JA（Jasmonic acid）and SA signal transduction pathway genes was significantly up-regulated.while the expression levels of three ethylene responsive transcription factors ERF were significantly higher than those of their parents after stress,which may be related to the signal transduction of triploid under stress.TIF family of transcription factors of JA metabolism pathway was the most strongly responsive to low temperature and significantly up-regulated after stress,among which 6 transcription factors were significantly up-regulated in triploid than in the two parents.In addition,three SA receptor protein genes NPR were up-regulated in triploids and expressed at higher levels than their parents.These genes may all be associated with triploid cold-hardiness.3.3 Analysis DEGs of photosynthesis related pathwayThe light capture and photosystem-related gene expression levels of triploid loquat plants were higher than its tetraploid parent under normal growth conditions.The expression levels of some genes related carbon assimilation pathway in triploid loquat plant was higher than that of their diploid parents.The dominant expression of key genes of light capture and carbon assimilation in triploid loquat plants during photosynthesis might be the key factors to its higher photosynthetic rate than that of its parents,and also the important reason for its better growth.Low temperature stress resulted in a large number of down-regulated genes in light capture,photosystem and carbon assimilation pathways in triploids and its parents,which was consistent with their physiological phenotypes,suggesting that loquat responded to low temperature stress by down-regulated the whole photosystem to limit energy inflow.In addition,the high expression of Psb Z1.2 and Ej ATPF genes,as well as the key genes Ej GAPDH1.1,Ej RPI3.2 and Ej PRK1.1 in carbon fixing in triploid under stress may be related to maintain the stability of photosynthetic membrane and enhance its strong cold-hardiness.4 Clone and functional analysis of Ej FAD2 and Ej FADC1 genesSubcellular localization showed that Ej FAD2 was localized in endoplasmic reticulum,and Ej FADC1 was localized in chloroplast.Under low temperature stress,Heterologous over-expression of Ej FAD2、Ej FADC1 significantly reduced electrolyte leakage and MDA content in Arabidopsis leaves and increased its cold-hardiness.At the same time,the stability of the photosynthetic membrane was enhanced and significantly improved the PSII photosystem activity under low temperature stress.The research showed that Ej FAD2 and Ej FADC1 genes have the function of enhancing photosystem activity and cold-hardiness in Arabidopsis thaliana under low temperature stress,which suggested that they play an important role in cold-hardiness and high light efficiency in triploid loquat.In conclusion,membrane lipid remodeling of triploid loquat under low temperature stress is a key factor to maintain the stability of plasma membrane and chloroplast membrane,maintain photosystem activity,and obtain cold-hardiness.Especially,Ej FAD2 and Ej FADC1 genes plays an important role in maintaining membrane stability under low temperature stress.