Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.)

Carotenoids directly influence citrus fruit color and nutritional value,which is critical to consumer acceptance.Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality.Despite the well-established carotenoid biosynthetic pathways,the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood.Our previous studies have reported that the Myc-type basic helix-loop-helix(bHLH)transcription factor(TF)regulates citrus proanthocyanidin biosynthesis.Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli,but its regulatory mechanism is still unclear.In the present study,we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR,CsHDS,CsHDR,CsPDS,CsLCYE,CsZEP,and CsNCED2,which was accompanied by changes in the contents of abscisic acid and gibberellin.The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR,CsHDS,and CsHDR,the keymetabolic enzymes of the methylerythritol 4-phosphate(MEP)pathway,thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes.In addition,CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes,CsPDS,CsLCYE,CsZEP,and CsNCED2,directly promoting carotenoid biosynthesis.This study reveals a novel network of carotenoid metabolism regulated by CsTT8.Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

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.

Integrated multi-omics analysis of developing‘Newhall’orange and its glossy mutant provide insights into citrus fragrance formation

Sesquiterpene valencene is dominant in flavedo tissues of sweet oranges and imparts a unique woody aroma.However,the interaction between the biosynthetic pathways of valencene and other nutritional compounds is less studied.Sesquiterpenoids were significantly accumulated in a previously reported glossy mutant of orange(MT)than the wild type(WT),especially valencene and caryophyllene.In addition,we identified several other pathways with variations at both the transcriptional and metabolic levels in MT.It’s interesting to found those upregulated metabolites in MT,such as eukaryotic lipids,kaempferol and proline also showed strong positive correlation with valencene along with fruit maturation while those down-regulated metabolites,such as phenylpropanoid coumarins and most of the modified flavonoids exhibited negative correlation.We then categorized these shifted pathways into the‘sesquitepenoid-identical shunt’and the sesquitepenoid-opposite shunt’and confirmed the classification result at transcriptional level.Our results provide important insights into the connections between various fruit quality-related properties.

Lipidomic and transcriptomic analysis reveals reallocation of carbon flux from cuticular wax into plastid membrane lipids in a glossy“Newhall”navel orange mutant

Both cuticle and membrane lipids play essential roles in quality maintenance and disease resistance in fresh fruits.Many reports have indicated the modification of alternative branch pathways in epicuticular wax mutants;however,the specific alterations concerning lipids have not been clarified thus far.Here,we conducted a comprehensive,timeresolved lipidomic,and transcriptomic analysis on the“Newhall”navel orange(WT)and its glossy mutant(MT)“Gannan No.1”.The results revealed severely suppressed wax formation accompanied by significantly elevated production of 36-carbon plastid lipids with increasing fruit maturation in MT.Transcriptomics analysis further identified a series of key functional enzymes and transcription factors putatively involved in the biosynthesis pathways of wax and membrane lipids.Moreover,the high accumulation of jasmonic acid(JA)in MT was possibly due to the need to maintain plastid lipid homeostasis,as the expression levels of two significantly upregulated lipases(CsDAD1 and CsDALL2)were positively correlated with plastid lipids and characterized to hydrolyze plastid lipids to increase the JA content.Our results will provide new insights into the molecular mechanisms underlying the natural variation of plant lipids to lay a foundation for the quality improvement of citrus fruit.

Multiomics-based dissection of citrus flavonoid metabolism using a Citrus reticulata × Poncirus trifoliata population

Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes.Although citrus plants are among the most important fruit crops worldwide,the genetic basis of secondary metabolism in these plants is largely unknown.Here,we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues(young leaf,old leaf,mature pericarp,and mature pulp)of an F_(1) pseudo-testcross citrus population.We detected 80 flavonoids in this population and identified 138 quantitative trait loci(QTLs)for 57 flavonoids in these four tissues.Based on transcriptional profiling and functional annotation,twenty-one candidate genes were identified,and one gene encoding flavanone 3-hydroxylase(F3H)was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions.The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.

QTL analysis reveals reduction of fruit water loss by NAC042 through regulation of cuticular wax synthesis in citrus fruit

Postharvest water loss is a critical factor that determines the quality and shelf life of fresh fruit.Cuticular wax constitutes a key barrier to reduce fruit water loss.Our previous study has shown that HJ(Citrus reticulata)has a significantly higher postharvest water loss rate than ZK(Poncirus trifoliata).Here,we investigated the fruit water loss rate of the HJ×ZK F1pseudo-testcross population in 2016 and 2019.QTL mapping for fruit water loss rate was performed by high-density genetic map and bulk segregant analysis,and QTL9 was identified to be associated with fruit water loss.The expression of NAC042 from QTL9 in ZK was 170-fold that in HJ.Heterologous expression in Arabidopsis showed that NAC042could reduce the water loss of leaves by increasing the cuticular wax content(especially alkanes).Further expression analysis revealed that NAC042 could enhance the expression of many wax-related genes in Arabidopsis leaves,including AtKCS1,AtKCS2,AtKCS9,AtKCS20,At CER1 and At CER3.Therefore,NAC042 might be involved in fruit cuticular wax synthesis to reduce fruit water loss.The findings provide new insights into the regulation of cuticular wax and fruit water loss as well as valuable information for breeding of citrus with better storability.

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.

Illuminating the cells:transient transformation of citrus to study gene functions and organelle activities related to fruit quality

Although multiple microscopic techniques have been applied to horticultural research,few studies of individual organelles in living fruit cells have been reported to date.In this paper,we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method.Kumquat(Fortunella crassifolia Swingle)was used;it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window.Fruits were transformed with fluorescent reporters,and confocal microscopy and live-cell imaging were used to study their localization and dynamics.Moreover,various pH sensors targeting different subcellular compartments were expressed,and the local pH environments in cells from different plant tissues were compared.The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits.In summary,our method is effective for studying various membrane trafficking events,protein localization,and cell physiology in fruit and can provide new insight into fruit biology research.

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.

Banana somatic embryogenesis and biotechnological application

As one of the most important economic crops for both staple food and fruit widely cultivated in the tropics and subtropics,banana(Musa spp.)is susceptible to a plethora of abiotic and biotic stresses.Breeding cultivars resistant to abiotic and biotic stressors without adverse effects on yield and fruit quality are the objectives of banana improvement programs.However,conventional breeding approaches are time-consuming and severely hampered by inherent banana problems(polyploidy and sterility).Therefore,genetic transformation is becoming increasingly popular and can provide rapid solutions.Numerous efforts have been made to develop superior banana cultivars with better resistance to abiotic and biotic stresses and optimum yields using genetic modification strategies.Somatic embryogenesis(SE)through embryogenic cell suspension(ECS)cultures is an ideal recipient system for genetic transformation in banana.The purpose of this paper is to review the current status of banana somatic embryo research,clarify the process of banana somatic embryo induction and culture,and summarize the main influencing factors in the process of somatic embryogenesis.At the same time,their applications in breeding technologies such as cryopreservation,protoplast culture,genetic transformation and gene editing were also summarized,in order to provide reference for the research and practical application of banana somatic embryogenesis in the future.

Integrating multiomics data accelerates elucidation of plant primary and secondary metabolic pathways

Plants are the most important sources of food for humans,as well as supplying many ingredients that are of great importance for human health.Developing an understanding of the functional components of plant metabolism has attracted considerable attention.The rapid development of liquid chromatography and gas chromatography,coupled with mass spectrometry,has allowed the detection and characterization of many thousands of metabolites of plant origin.Nowadays,elucidating the detailed biosynthesis and degradation pathways of these metabolites represents a major bottleneck in our understanding.Recently,the decreased cost of genome and transcriptome sequencing rendered it possible to identify the genes involving in metabolic pathways.Here,we review the recent research which integrates metabolomic with different omics methods,to comprehensively identify structural and regulatory genes of the primary and secondary metabolic pathways.Finally,we discuss other novel methods that can accelerate the process of identification of metabolic pathways and,ultimately,identify metabolite function(s).

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.

Prediction of Newhall navel orange internal quality based on digital microscopy

The traditional technology for internal quality detection of citrus fruit is believed to be destructive,inefficient,and error-prone.To address these issues,a novel method has been developed to evaluate nondestructively the sugar-acid ratio(SAR)of soluble solids by using the oil glands density(OGD).In this study,a total of 584 samples with different widths were collected.The sample data were correlated that the SAR and OGD decreased with time during storage.The relevance with time was significant between SAR and OGD of citrus positively correlated in the calibration group(R2=0.82),which indicated that OGD could be used to predict the internal quality of citrus.It indicated that a new generation of digital microscopy could provide an alternative to predict nondestructively the internal quality of citrus fruit,as well as some theoretical insight for further studies of online quality detection in the future.

Transcription factor CsESE3 positively modulates both jasmonic acid and wax biosynthesis in citrus

PLIP lipases can initiate jasmonic acid(JA)biosynthesis.However,little is known about the transcriptional regulation of this process.In this study,an ERF transcription factor(CsESE3)was found to be co-expressed with all necessary genes for JA biosynthesis and several key genes for wax biosynthesis in transcriptomes of‘Newhall’navel orange.CsESE3 shows partial sequence similarity to the well-known wax regulator SHINEs(SHNs),but lacks a complete MM protein domain.Ectopic overexpression of CsESE3 in tomato(OE)resulted in reduction of fruit surface brightness and dwarf phenotype compared to the wild type.The OE tomato lines also showed significant increases in the content of wax and JA and the expression of key genes related to their biosynthesis.Overexpression of CsESE3 in citrus callus and fruit enhanced the JA content and the expression of JA biosynthetic genes.Furthermore,CsESE3 could bind to and activate the promoters of two phospholipases from the PLIP gene family to initiate JA biosynthesis.Overall,this study indicated that CsESE3 could mediate JA biosynthesis by activating PLIP genes and positively modulate wax biosynthesis.The findings provide important insights into the coordinated control of two defense strategies of plants represented by wax and JA biosynthesis.

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.

The metabolic changes that effect fruit quality during tomato fruit ripening

As the most valuable organ of tomato plants,fruit has attracted considerable attention which most focus on its quality formation during the ripening process.A considerable amount of research has reported that fruit quality is affected by metabolic shifts which are under the coordinated regulation of both structural genes and transcriptional regulators.In recent years,with the development of the next generation sequencing,molecular and genetic analysis methods,lots of genes which are involved in the chlorophyll,carotenoid,cell wall,central and secondary metabolism have been identified and confirmed to regulate pigment contents,fruit softening and other aspects of fruit flavor quality.Here,both research concerning the dissection of fruit quality related metabolic changes,the transcriptional and post-translational regulation of these metabolic pathways are reviewed.Furthermore,a weighted gene correlation network analysis of representative genes of fruit quality has been carried out and the potential of the combined application of the gene correlation network analysis,fine-mapping strategies and next generation sequencing to identify novel candidate genes determinants of fruit quality is discussed.