Regulatory Mechanisms Of Non-photosynthetic Plastids Involved In Starch Degradation In Kiwifruit And Volatile Compounds Accumulation In Citrus Peel

Plastids in plant cells are the sites where a variety of metabolic processes occur,thus play important roles in life activities of most plant organs,such as fruit.Based on the origin of their metabolic precursors,plastids are divided into photosynthetic plastids,which refer to chloroplasts,and non-photosynthetic plastids,which include a variety of heterotrophic plastids such as chromoplasts,amyloplasts,and elaioplast.Climacteric kiwifruit and non-climacteric citrus are important fruit in southern China,which accumulated large amount of starch and volatile organic components（VOCs）,respectively.During the ripening and senescence,starch degradation in kiwifruit pulp and VOCs accumulation in citrus peel are both key physiological processes,which occurred in amyloplasts and elaioplasts,respectively.Starch is the main component of dry matter,and its products soluble sugars are predominant in total soluble solids（TSS）,which determines fruit flavour,texture and storage life.In addition,VOCs are source of fruit aroma,and some components affect the disease resistance.GWD family protein are localized in amyloplasts,starch phosphorylation catalysed by them is the first and key rate-limiting step in starch degradation.Previously,amyloplasts of kiwifruit from different developmental and postharvest stages were isolated in our laboratory for comparative proteomic analysis,results showed that Ac GWD family proteins were closely related to starch metabolism and total soluble solids（TSS）production.However,the biological function of Ac GWD family genes of kiwifruit and its regulation has not been reported yet.Therefore,in this study,we identified Ac GWD family members of kiwifruit,and analyzed the function and regulatory mechanism of them.In addition,elaioplasts of citrus peels are in epithelial cells of the oil glands,while its biological function has not been reported yet.We successfully isolated elaioplasts of high purity from kumquat,and elucidated the function that elaioplasts exerted in the metabolism of volatile terpenes in citrus fruit in combination with plastid proteomics.Moreover,comparison of VOCs,sensory quality and disease resistance were conducted between‘Huapi’kumquat（F.crassifolia Swingle‘HP’）,a mutant with abnormally developed oil glands,and wild type‘Rong’an’kumquat（F.crassifolia Swingle‘RA’）,which provide a new insight in function revelation of elaioplast.The main findings were as follows:1.Amyloplast structure of kiwifruit and its metabolic pattern.To study the changes of amyloplasts in kiwifruit during the development and postharvest ripen stages,we conducted a correlation analysis of the amyloplast morphology,starch content,soluble sugar content and hardness of kiwifruit.The results showed that:1)starch accumulated continuously in the amyloplasts as the fruit developed and peaked at the commercial harvesting stage;2)as the fruit ripened,the membrane of amyloplasts degraded,and the polymeric starch grains aggregated in it became dispersed and start to degrade;starch was gradually degraded into soluble sugars,showing a significant increase in glucose,fructose and sucrose content;3)starch content was closely related to fruit hardness,with a correlation coefficient R of 0.998.2.Plastid-localized Ac GWD family genes were involved in the starch degradation initiation and promote starch degradation in kiwifruit.Previously,people in my lab established a method for amyloplast isolation in kiwifruit.In combination with plastid proteomics techniques,we found that the GWD family proteins（GWD and PWD）with the highest protein abundance were found to be potentially involved in starch degradation.In this study,further analysis using transcriptome data and q RT-PCR showed that GWD family genes showed a‘parabolic’expression pattern with the expression peaking during the harvest window（150-165 days after anthesis）,suggesting that the catalytic product of GWD family proteins,phosphorylated starch,may lay the foundation for adequate starch hydrolysis of postharvest kiwifruit.Subsequently,we constructed a stable genetic transformation system based on‘Hongyang’kiwifruit and transformed Ac GWD1.1 gene using this system.In addition,using CRISPR/Cas9 system,we obtained both single gene and double gene knockout plants of Ac GWD1.1,Ac GWD1.2and Ac PWD1 genes.The results showed that Ac GWD1.1 gene promoted starch degradation,resulting in a significant decrease in starch content and a significant increase in its products fructose,glucose and sucrose.3.Ac BPC6 transcription factor negatively regulates Ac GWD family gene expression and inhibits starch degradation.To investigate the transcriptional regulatory mechanism of Ac GWD genes in promoting ripening and senescence of kiwifruit,a transcription factor library containing 56 members was constructed in this study using transcriptome data,online database prediction.Seven transcription factors that potentially regulate Ac GWD family gene expression were obtained by screening using dual luciferase assay experiment,and further yeast one hybrid（Y1H）and electrophoretic mobility shift assay（EMSA）showed that only Ac BPC6 directly interacted with the Ac GWD family gene promoter and repressed Ac GWD and Ac PWD1gene expression.Overexpression of Ac BPC6 gene in fruit of kiwifruit using transient transformation method demonstrated that Ac BPC6 gene negatively regulated fruit Ac GWD and Ac PWD1 gene expression.Further analysis of the GA/TC tandem repeat region bound by Ac BPC6 was characterized as follows:1)there were two binding regions of（TC）₁₁（-87 to-66）and（TC）₂TT（TC）₂TAGTTTTCT（TC）₃（-29 to-5）in the Ac PWD1gene promoter;2)the（GA/TC）tandem repeat region analysis of Ac GWD family genes in30 different kiwifruit varieties was conducted,results showed that the Ac GWD1.1 gene promoter had 6-18 GA/TC repeats and exhibited significant variety specificity and polymorphism,presumably closely related to starch degradation in kiwifruit of different varieties.4.High-purity kumquat elaioplasts were successfully isolated and confirmed to function in the synthesis of monoterpenes in combination with plastid proteomics.In order to analyze the biological function of elaioplasts,using peel edible kumquat and sucrose density gradient centrifugation method,this study established the method for isolation of highly purified and intact elaioplasts for the first time.Microscopic observation showed that the isolated elaioplasts were intact and sub-circular in structure,with a diameter size of 2-3μm and containing 5-9 plastoglobulus.Western blot using labeling antibodies for cytoplasm,mitochondria and vacuole demonstrated that the isolated elaioplasts were not contaminated by the above three subcellular organelles,indicating that the isolated elaioplasts were of high purity.Subsequently,LC-MS/MS technique was used for the proteomic study of elaioplasts,and a library of 651 plastid-localized proteins was established for the first time.The functional classification showed that these elaioplast localized proteins were mainly involved in the following four metabolic pathways:protein metabolism and processing,protein transport,secondary metabolism,and lipid metabolism,which were similar to other plastids（e.g.chromoplasts and amyloplasts）,indicating the relatively high conserved biological functions of different plastid forms.Meanwhile,the proteomic data of different plastid forms showed that elaioplast are the main sites for monoterpene synthesis,the whole monoterpene synthesis pathway proteins contained in it were of high abundance,showing the specificity of elaioplasts.5.The mutant kumquat with defective oil glands showed reduced volatile compounds（VOCs）content,as well as enhanced sensory quality and disease resistance.In order to analyze the function of elaioplasts in-depth,this study further compared the wild type‘RA’kumquat with the mutant‘HP’kumquat with defective oil glands.Results showed that the number and size of oil glands of‘HP’kumquat were significantly smaller than those of‘RA’kumquat.The results of peel VOCs detection showed that the accumulation of VOCs was significantly inhibited by the defect of oil glands in‘HP’kumquat,among which the most significantly inhibited components like limonene,β-myrcene andα-pinene were all monoterpenes,which was consistent with the results of proteome,indicating that the VOCs accumulated by oil glands were mainly synthesized by elaioplast.In addition,using sensory evaluation and fungi inoculation experiments,this study demonstrated that the sensory quality and disease resistance of‘HP’kumquat were superior to those of‘RA’kumquat,which might be related to the inhibition of elaioplasr function due to oil gland defects.