Integrated transcriptomic and metabolomic analyses of a wax deficient citrus mutant exhibiting jasmonic acid-mediated defense against fungal pathogens

Naturally,resistant crop germplasms are important resources for managing the issues of agricultural product safety and environment deterioration.We found a spontaneous mutant of‘Newhall’navel orange(Citrus sinensis Osbeck)(MT)with broad-spectrum protections against fungal pathogens in the orchard,postharvest-storage,and artificial inoculation conditions.To understand the defense mechanism of MT fruit,we constructed a genome-scale metabolic network that integrated metabolome and transcriptome datasets.The coordinated transcriptomic and metabolic data were enriched in two sub-networks,showing the decrease in very long chain fatty acid(by 41.53%)and cuticular wax synthesis(by 81.34%),and increase in the synthesis of jasmonic acid(JA)(by 95.23%)and JA-induced metabolites such as 5-dimethylnobietin(by 28.37%)in MT.Furthermore,cytological and biochemical analyses confirmed that the response to fungal infection in MT was independent of wax deficiency and was correlated with the levels of jasmonates,and the expression of plant defensin gene PDF1.2.Results of exogenous application of MeJA and JA inhibitors such as propyl gallate proved that JA-mediated defense contributes to the strong tolerance against pathogens in MT.Our results indicated that jasmonate biosynthesis and signaling are stimulated by the fatty acid redirection of MT,and participate in the tolerance of pathogenic fungi.

Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit

Mitochondria are crucial for the production of primary and secondary metabolites,which largely determine the quality of fruit.However,a method for isolating high-quality mitochondria is currently not available in citrus fruit,preventing high-throughput characterization of mitochondrial functions.Here,based on differential and discontinuous Percoll density gradient centrifugation,we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species,including satsuma mandarin,ponkan mandarin,sweet orange,and pummelo.Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria.By using this protocol coupled with a label-free proteomic approach,a total of 3353 nonredundant proteins were identified.Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways(such as tricarboxylic acid cycle,pyruvate metabolism,and oxidative phosphorylation)and pathways closely related to fruit quality(such asγ-aminobutyric acid(GABA)shunt,ascorbate metabolism,and biosynthesis of secondary metabolites).In addition,differentially abundant proteins(DAPs)between different types of species were also identified;these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis.In summary,the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes,which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

A comprehensive proteomic analysis of elaioplasts from citrus fruits reveals insights into elaioplast biogenesis and function

Elaioplasts of citrus peel are colorless plastids which accumulate significant amounts of terpenes.However,other functions of elaioplasts have not been fully characterized to date.Here,a LC–MS/MS shotgun technology was applied to identify the proteins from elaioplasts that were highly purified from young fruit peel of kumquat.A total of 655 putative plastid proteins were identified from elaioplasts according to sequence homology in silico and manual curation.Based on functional classification via Mapman,~50%of the identified proteins fall into six categories,including protein metabolism,transport,and lipid metabolism.Of note,elaioplasts contained ATP synthase and ADP,ATP carrier proteins at high abundance,indicating important roles for ATP generation and transport in elaioplast biogenesis.Additionally,a comparison of proteins between citrus chromoplast and elaioplast proteomes suggest a high level of functional conservation.However,some distinctive protein profiles were also observed in both types of plastids notably for isoprene biosynthesis in elaioplasts,and carotenoid metabolism in chromoplasts.In conclusion,this comprehensive proteomic study provides new insights into the major metabolic pathways and unique characteristics of elaioplasts and chromoplasts in citrus fruit.

Natural Variation in CCD4 Promoter Underpins Species-Specific Evolution of Red Coloration in Citrus Peel

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference.Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments.In this study,using an integrated genetic approach we revealed that a 5;c/s-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C3 0 apocarotenoids responsible for red coloration of citrus peel.Functional analyses demonstrated that in addition the known role in synthesizing 3-citraurin,CCD4b is also responsible for the production of another important C3 0 apocarotenoid pigment,p-citraurinene.Furthermore,analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5'c/s-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C3 0 apocarotenoid-rich red-peeled accessions.Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids.Taken together,our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants,and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

Integration of Metabolomics and Subcellular Organelle Expression Microarray to Increase Understanding the Organic Acid Changes in Post-harvest Citrus Fruit

Citric acid plays an important role in fresh fruit flavor and its adaptability to post-harvest storage conditions. In order to explore organic acid regulatory mechanisms in post-harvest citrus fruit, systematic biological analyses were conducted on stored Hirado Buntan Pummelo （HBP; Citrus grandis） fruits. High- performance capillary electrophoresis, subcellular organelle expression microarray, real-time quantitative reverse transcription polymerase chain reaction, gas chromatography mass spectrometry （GC-MS）, and conventional physiological and biochemical analyses were undertaken. The results showed that the concentration of organic acids in HBP underwent a regular fluctuation. GC-MS-based metabolic profiling indicated that succinic acid, ~,-aminobutyric acid （GABA）, and glutamine contents increased, but 2- oxoglutaric acid content declined, which further confirmed that the GABA shunt may have some regulatory roles in organic acid catabolism processes. In addition, the concentration of organic acids was significantly correlated with senescence-related physiological processes, such as hydrogen peroxide content as well as superoxide dismutase and peroxidase activities, which showed that organic acids could be regarded as important parameters for measuring citrus fruit post-harvest senescence processes.

NEW INSIGHTS INTO THE PHYLOGENY AND SPECIATION OF KUMQUAT (FORTUNELLA SPP.) BASED ON CHLOROPLAST SNP, NUCLEAR SSR AND WHOLE-GENOME SEQUENCING

Kumquat(Fortunella spp.)is a fruit and ornamental crop worldwide due to the palatable taste and high ornamental value of its fruit.Although Fortunella is classified into the economically important true citrus fruit tree group together with Citrus and Poncirus,few studies have been focused on its evolutionary scenario.In this study,analysis of five chloroplast loci and 47 nuclear microsatellites(nSSR)loci from 38 kumquat and 10 citrus accessions revealed the independent phylogeny of Fortunella among citrus taxa,and that Fortunella mainly comprises two populations:CUL,cultivated Fortunella spp.(F.margarita,F.crassifolia and F.japonica);and HK,wild Hong Kong kumquat(Fortunella hindsii).Genomic analysis based on whole-genome SNPs indicated that the allele frequency of both pupations deviated from the neutral selection model,suggesting directional selection was a force driving their evolutions.CUL exhibited lower genomic diversity and higher linkage strength than HK,suggesting artificial selection involved in its origin.A high level of genetic differentiation(Fst=0.364)was detected and obviously asynchronous demographic changes were observed between CUL and HK.Based on these results,a new hypothesis for the speciation of Fortunella is proposed.