Genomics of the origin and evolution of Citrus.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.

Seasonal Transcriptome Profiling of Susceptible and Tolerant Citrus Cultivars to Citrus Huanglongbing.

Citrus huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. Most commercial citrus cultivars are susceptible to HLB, with a few more tolerant exceptions such as 'LB8-9' Sugar Belle mandarin. Transcriptomic analyses have been widely used to investigate the potential mechanisms for disease susceptibility, resistance, or tolerance. Previous transcriptomic studies related to HLB mostly focused on single time point data collection. We hypothesize that changes in day length and temperature throughout the seasons have profound effects on citrus-CLas interactions. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant mandarin 'LB8-9' in winter, spring, summer, and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both cultivars, the highest number of DEGs were found in the spring. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, reactive oxygen species (ROS), and PR genes in both cultivars, consistent with the model that HLB is a pathogen-triggered immune disease. HLB-positive mandarin 'LB8-9' trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, mandarin 'LB8-9' showed higher expression of genes involved in phloem regeneration, which might contribute to its HLB tolerance. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.

Comparative Transcriptomic Analysis of Inarching Invigorating Rootstock onto Incompatible Grafts in Citrus.

Grafting is a technique that is widely used in citrus production. Graft incompatibility often occurs in the orchard. Inarching can effectively improve the vigor of incompatible grafts, but its mechanism remains poorly understood. Our previous studies investigated the scion-rootstock interaction of citrus and highlighted the role of hormonal balance and genes in abscisic acid biosynthesis regulation. To further elucidate the mechanism of inarched grafts rejuvenation, Hm/Pt combination (Citrus maxima (Burm.) Merrill cv. 'Hongmian miyou' grafted onto Poncirus trifoliata) were inarched with 'Pujiang Xiangcheng' (a novel citrus rootstock cultivar recently selected from wild Citrus junos populations), and comprehensive analysis was performed to compare the inarched grafts and controls. Compared with incompatible grafts, the results revealed that inarching could recover the leaf metabolism balance, including reducing starch content, increasing chlorophyll content and restoring the cell structure. Additionally, our results corroborated that hormonal balance and hormone-related genes played a central role in inarching compatibility. Furthermore, the roles of Hsf4, ERF1, NCED3 and PYL were highlighted, and a model for explaining inarched grafts recovery invigoration was proposed. This study shed light on the mechanism of inarching regulation tree vigor and offered deep insights into the scion-rootstock interaction in citrus.

A chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) provides insights into disease resistance, cold tolerance and genome evolution in Citrus.

Trifoliate orange (Poncirus trifoliata), a deciduous close relative of evergreen Citrus, has important traits for citrus production, including tolerance/resistance to citrus greening disease (Huanglongbing, HLB) and other major diseases, and cold tolerance. It has been one of the most important rootstocks, and one of the most valuable sources of resistance and tolerance genes for citrus. Here we present a high-quality, chromosome-scale genome assembly of P. trifoliata. The 264.9-Mb assembly contains nine chromosomal pseudomolecules with 25 538 protein-coding genes, covering 97.2% of the estimated gene space. Comparative analyses of P. trifoliata and nine Citrus genomes revealed 605 species-specific genes and six rapidly evolving gene families in the P. trifoliata genome. Poncirus trifoliata has evolved specific adaptation in the C-repeat/DREB binding factor (CBF)-dependent and CBF-independent cold signaling pathways to tolerate cold. We identified candidate genes within quantitative trait loci for HLB tolerance, and at the loci for resistance to citrus tristeza virus and citrus nematode. Genetic diversity analysis of Poncirus accessions and Poncirus/Citrus hybrids shows a narrow genetic base in the US germplasm collection, and points to the importance of collecting and preserving more natural genetic variation. Two phenotypically divergent Poncirus accessions are found to be clonally related, supporting a previous conjecture that dwarf Flying Dragon originated as a mutant of a non-dwarfing type. The high-quality genome reveals features and evolutionary insights of Poncirus, and it will serve as a valuable resource for genetic, genomic and molecular research and manipulation in citrus.

De Novo Transcriptome Sequencing of Rough Lemon Leaves (Citrus jambhiri Lush.) in Response to Plenodomus tracheiphilus Infection.

Mal secco is one of the most severe diseases of citrus, caused by the necrotrophic fungus Plenodomus tracheiphilus. With the main aim of identifying candidate genes involved in the response of citrus plants to "Mal secco", we performed a de novo transcriptome analysis of rough lemon seedlings subjected to inoculation of P. tracheiphilus. The analysis of differential expressed genes (DEGs) highlighted a sharp response triggered by the pathogen as a total of 4986 significant DEGs (2865 genes up-regulated and 2121 down-regulated) have been revealed. The analysis of the most significantly enriched KEGG pathways indicated that a crucial role is played by genes involved in "Plant hormone signal transduction", "Phenylpropanoid biosynthesis", and "Carbon metabolism". The main findings of this work are that under fungus challenge, the rough lemon genes involved both in the light harvesting and the photosynthetic electron flow were significantly down-regulated, thus probably inducing a shortage of energy for cellular functions. Moreover, the systemic acquired resistance (SAR) was activated through the induced salicylic acid cascade. Interestingly, RPM1 interacting protein 4, an essential positive regulator of plant defense, and BIR2, which is a negative regulator of basal level of immunity, have been identified thus representing useful targets for molecular breeding.

Deficiency of valencene in mandarin hybrids is associated with a deletion in the promoter region of the valencene synthase gene.

BACKGROUND: Valencene is a major sesquiterpene in citrus oil and biosynthesized by valencene synthase (Cstps1; EC: 4.2.3.73) from the 15-carbon substrate farnesyl diphosphate. It is abundant in juice of some mandarins (e.g. Citrus reticulata Blanco cv. Fortune), however, it is undetectable in others (e.g. C. reticulata Blanco cv. Murcott), We have discovered that the Murcott mandarin Cstps1 gene expression is severely reduced. A previous genetic mapping study using an F1 population of Fortune × Murcott found that the segregation of valencene production in fruit exhibited a Mendelian inheritance ratio of 1:1. There was only one dominant locus associated with valencene content detected on the mandarin genetic map. The goal of this study was to understand the molecular mechanism underlying the valencene deficiency observed in some citrus hybrids. RESULTS: There was a clear relationship between presence or absence of the valencene synthase gene (Cstps1) expression, and presence or absence of valencene among randomly selected mandarin hybrids. Cloning the coding regions of Cstps1 from Fortune and Murcott mandarin, and aligning with previous reported Valencia orange Cstps1 sequence, showed that they both exhibited extremely high similarity with the known Cstps1. By further cloning and analyzing the promoter region of Cstps1 from Valencia, Fortune and Murcott, a 12-nucleotide deletion at approximately - 270 bp from the Cstps1 coding region was only found in Murcott. Three binary vectors, designated as p1380-FortP-GUSin, p1380-MurcP-GUSin and p1380-MurcP(+ 12)-GUSin, were developed for promoter activity analysis. Transient over-expression of Fortune Cstps1 promoter in sweet orange showed notable GUS activity, but the Murcott Cstps1 promoter did not. In addition, by re-inserting the 12-nucleotide fragment, the activity of the Murcott Cstps1 promoter was mostly recovered. CONCLUSION: The deficiency of valencene production in some mandarins is probably due to a 12-nucleotide deletion in the promoter region of the Cstps1, which could be a crucial switch of Cstps1 transcription. Our results further enhanced the understanding of valencene biosynthesis in citrus.

Comparative analysis of juice volatiles in selected mandarins, mandarin relatives and other citrus genotypes.

BACKGROUND: Citrus fruit flavor is an important attribute prioritized in variety improvement. The present study compared juice volatiles compositions from 13 selected citrus genotypes, including six mandarins (Citrus reticulata), three sour oranges (Citrus aurantium), one blood orange (Citrus sinensis), one lime (Citrus limonia), one Clementine (Citrus clementina) and one satsuma (Citrus unshiu). RESULTS: Large differences were observed with respect to volatile compositions among the citrus genotypes. 'Goutou' sour orange contained the greatest number of volatile compounds and the largest volatile production level. 'Ponkan' mandarin had the smallest number of volatiles and 'Owari' satsuma yielded the lowest volatile production level. 'Goutou' sour orange and 'Moro' blood orange were clearly distinguished from other citrus genotypes based on the analysis of volatile compositions, even though they were assigned into one single group with two other sour oranges by the molecular marker profiles. CONCLUSIONS: The clustering analysis based on the aroma volatile compositions was able to differentiate mandarin varieties and natural sub-groups, and was also supported by the molecular marker study. The gas chromatography-mass spectrometry analysis of citrus juice aroma volatiles can be used as a tool to distinguish citrus genotypes and assist in the assessment of future citrus breeding programs. The aroma volatile profiles of the different citrus genotypes and inter-relationships detected among volatile compounds and among citrus genotypes will provide fundamental information on the development of marker-assisted selection in citrus breeding. © 2017 Society of Chemical Industry.

Identification of QTLs controlling aroma volatiles using a 'Fortune' x 'Murcott' (Citrus reticulata) population.

BACKGROUND: Flavor is an important attribute of mandarin (Citrus reticulata Blanco), but flavor improvement via conventional breeding is very challenging largely due to the complexity of the flavor components and traits. Many aroma associated volatiles of citrus fruit have been identified, which are directly related to flavor, but knowledge of genetic linkages and relevant genes for these volatiles, along with applicable markers potentially for expeditious and economical marker-assisted selection (MAS), is very limited. The objective of this project was to identify single nucleotide polymorphism (SNP) markers associated with these volatile traits. RESULT: Aroma volatiles were investigated in two mandarin parents ('Fortune' and 'Murcott') and their 116 F1 progeny using gas chromatography mass spectrometry in 2012 and 2013. A total of 148 volatiles were detected, including one acid, 12 alcohols, 20 aldehydes, 14 esters, one furan, three aromatic hydrocarbons, 16 ketones, one phenol, 27 sesquiterpenes, 15 monoterpenes, and 38 unknowns. A total of 206 quantitative trait loci (QTLs) were identified for 94 volatile compounds using genotyping data generated from a 1536-SNP Illumina GoldenGate assay. In detail, 25 of the QTLs were consistent over more than two harvest times. Forty-one QTLs were identified for 17 aroma active compounds that included 18 sesquiterpenes and were mapped onto four genomic regions. Fifty QTLs were for 14 monoterpenes and mapped onto five genomic regions. Candidate genes for some QTLs were also identified. A QTL interval for monoterpenes and sesquiterpenes on linkage group 2 contained four genes: geranyl diphosphate synthase 1, terpene synthase 3, terpene synthase 4, and terpene synthase 14. CONCLUSIONS: Some fruit aroma QTLs were identified and the candidate genes in the terpenoid biosynthetic pathway were found within the QTL intervals. These QTLs could lead to an efficient and feasible MAS approach to mandarin flavor improvement.

Proteomic and metabolomic analyses provide insight into production of volatile and non-volatile flavor components in mandarin hybrid fruit.

BACKGROUND: Although many of the volatile constituents of flavor and aroma in citrus have been identified, the knowledge of molecular mechanisms and regulation of volatile production are very limited. Our aim was to understand mechanisms of flavor volatile production and regulation in mandarin fruit. RESULT: Fruits of two mandarin hybrids, Temple and Murcott with contrasting volatile and non- volatile profiles, were collected at three developmental stages. A combination of methods, including the isobaric tags for relative and absolute quantification (iTRAQ), quantitative real-time polymerase chain reaction, gas chromatography, and high-performance liquid chromatography, was used to identify proteins, measure gene expression levels, volatiles, sugars, organic acids and carotenoids. Two thirds of differentially expressed proteins were identified in the pathways of glycolysis, citric acid cycle, amino acid, sugar and starch metabolism. An enzyme encoding valencene synthase gene (Cstps1) was more abundant in Temple than in Murcott. Valencene accounted for 9.4% of total volatile content in Temple, whereas no valencene was detected in Murcott fruit. Murcott expression of Cstps1 is severely reduced. CONCLUSION: We showed that the diversion of valencene and other sesquiterpenes into the terpenoid pathway together with high production of apocarotenoid volatiles might have resulted in the lower concentration of carotenoids in Temple fruit.

Chemical and genetic basis of orange flavor.

Sweet orange (Citrus sinensis) exhibits limited genetic diversity and high susceptibility to Huanglongbing (HLB). Breeding HLB-tolerant orange-like hybrids is in dire need. However, our understanding of the key compounds responsible for orange flavor and their genetic regulation remains elusive. Evaluating 179 juice samples, including oranges, mandarins, Poncirus trifoliata, and hybrids, distinct volatile compositions were found. A random forest model predicted untrained samples with 78% accuracy and identified 26 compounds crucial for orange flavor. Notably, seven esters differentiated orange from mandarin flavor. Cluster analysis showed six esters with shared genetic control. Differential gene expression analysis identified C. sinensis alcohol acyltransferase 1 (CsAAT1) responsible for ester production in orange. Its activity was validated through overexpression assays. Phylogeny revealed the functional allele was inherited from pummelo. A SNP-based DNA marker in the coding region accurately predicted phenotypes. This study enhances our understanding of orange flavor compounds and their biosynthetic pathways and expands breeding options for orange-like cultivars.

Conserving Citrus Diversity: From Vavilov's Early Explorations to Genebanks around the World.

Citrus is among the most economically important fruit crops. Its vast species diversity and global production was observed by N.I. Vavilov during his international plant explorations from the early to mid-1900s. Currently, ex situ citrus collections located around the world conserve and protect citrus genetic resources, as revealed in a survey conducted in 2021. Responses were received from 43 collections in 27 countries, of which 35 provided data regarding collection composition, management practices, and security, as well as other information. The six largest citrus collections have between 1000 and 1735 accessions. The largest accession holdings are mandarins and sweet oranges, although all citrus fruit types are maintained: mandarin, sweet orange, lemon, pummelo, grapefruit, hybrids, lime, sour orange, citron, kumquat, papeda, finger lime, and crop wild relatives. Diseases pose significant threats to collections, though some collections are maintained in a clean-plant state as a result of intensive sanitation efforts. National and regional quarantine regulations often limit the export and import of citrus plants or propagative materials, thus limiting the availability of materials at an international level. Resources, both financial and human, are necessary to ensure the long-term safety and security of citrus collections on a global scale. Future efforts to develop citrus genebanking communities will provide opportunities for improved conservation, as well as collaborations and training.

Construction of a high density genetic linkage map to define the locus conferring seedlessness from Mukaku Kishu mandarin.

Mukaku Kishu ('MK'), a small sized mandarin, is an important source of seedlessness in citrus breeding. Identification and mapping the gene(s) governing 'MK' seedlessness will expedite seedless cultivar development. In this study, two 'MK'-derived mapping populations- LB8-9 Sugar Belle® ('SB') × 'MK' (N=97) and Daisy ('D') × 'MK' (N=68) were genotyped using an Axiom_Citrus56 Array encompassing 58,433 SNP probe sets, and population specific male and female parent linkage maps were constructed. The parental maps of each population were integrated to produce sub-composite maps, which were further merged to develop a consensus linkage map. All the parental maps (except 'MK_D') had nine major linkage groups, and contained 930 ('SB'), 810 ('MK_SB'), 776 ('D') and 707 ('MK_D') SNPs. The linkage maps displayed 96.9 ('MK_D') to 98.5% ('SB') chromosomal synteny with the reference Clementine genome. The consensus map was comprised of 2588 markers including a phenotypic seedless (Fs)-locus and spanned a genetic distance of 1406.84 cM, with an average marker distance of 0.54 cM, which is substantially lower than the reference Clementine map. For the phenotypic Fs-locus, the distribution of seedy and seedless progenies in both 'SB' × 'MK' (55:42, χ2 = 1.74) and 'D' × 'MK' populations (33:35, χ2 = 0.06) followed a test cross pattern. The Fs-locus mapped on chromosome 5 with SNP marker 'AX-160417325' at 7.4 cM in 'MK_SB' map and between two SNP markers 'AX-160536283' and 'AX-160906995' at a distance of 2.4 and 4.9 cM, respectively in 'MK_D' map. The SNPs 'AX-160417325' and 'AX-160536283' correctly predicted seedlessness of 25-91.9% progenies in this study. Based on the alignment of flanking SNP markers to the Clementine reference genome, the candidate gene for seedlessness hovered in a ~ 6.0 Mb region between 3.97 Mb (AX-160906995) to 10.00 Mb (AX-160536283). This region has 131 genes of which 13 genes (belonging to seven gene families) reportedly express in seed coat or developing embryo. The findings of the study will prove helpful in directing future research for fine mapping this region and eventually underpinning the exact causative gene governing seedlessness in 'MK'.

Effects of Different Rootstocks on the Metabolites of Huanglongbing-Affected Sweet Orange Juices Using a Novel Combined Strategy of Untargeted Metabolomics and Machine Learning.

Huanglongbing (HLB) is one of the most destructive citrus diseases, mainly caused by the Gram-negative bacteria Candidatus Liberibacter asiaticus. Aiming at unraveling the mechanisms of different scion/rootstock combinations on improving HLB-affected orange juice quality, the effects of rootstocks on the metabolites of HLB-affected sweet orange juices were investigated using a combined strategy of untargeted metabolomics and machine learning. A total of 2531 ion features were detected using UHPLC-Q-Orbitrap high-resolution electrospray ionization mass spectrometry, and 54 metabolites including amino acids, amines, flavonoids, coumarins, fatty acids, and glycosides were definitely or tentatively identified as the differential markers based on the random forest algorithm. Furthermore, 24 metabolites were verified and semi-quantified using authentic standards. Notably, the presence of specific amino acids and amines, especially polyamines, indicated that different rootstocks might affect glutamate, aspartate, proline, and arginine metabolism to regulate the physiological response against HLB. Meanwhile, the production of flavonoids and prenylated coumarins suggested that rootstocks influenced phenylalanine and phenylpropanoid metabolism. The possible metabolic pathways were proposed, and the important intermediates were verified by authentic standards. These results provide new insights on the effects of rootstocks on the metabolites of HLB-affected sweet orange juices.

Effects of rootstocks on the flavor quality of huanglongbing-affected sweet orange juices using targeted flavoromics strategy.

Citrus greening disease or Huanglongbing (HLB) is one of the most destructive diseases affecting all varieties of citrus worldwide. Aimed at optimizing the scion/rootstock combination to improve HLB-affected orange juice quality, a flavoromics strategy was used to investigate the effects of six different rootstocks (CH, blue, 1804, FG, SW, and Volk) on flavor quality of HLB affected orange juices. A sensory quality test was conducted by a panel to evaluate the sensory attributes of different orange juices. The orange juice from rootstock CH had the best flavor quality with highest sweetness, low sourness and bitterness, while rootstocks Volk and FG produced the poorest quality orange juices. Chemical profile analysis resulted in semi-quantification of 89 metabolites including 57 nonvolatile compounds and 32 volatile compounds using UHPLC-MS and GC-MS, respectively. Canonical correlation analysis indicated that some specific sugar and sugar alcohols including raffinose, xylose, rhamnose, glucose, sorbitol, and myo-inositol made a strong positive contribution to sweetness. Meanwhile, several amino acids including alanine, glutamic acid, proline, arginine, serine, asparagine, as well as aspartic acid were responsible for positive flavor quality. On the other hand, some nucleotides and limonin increased bitterness. In addition, KEGG pathway enrichment analysis demonstrated different rootstocks could affect aminoacyl-tRNA biosynthesis, ABC transporters, and monoterpenoid biosynthesis. These results indicated different rootstocks can change specific metabolites and thus affect the flavor quality of orange juices. This study also provides reference for optimizing the scion/rootstock combination to improve HLB-affected orange juice quality.

Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to 'Candidatus Liberibacter asiaticus' infection.

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by 'Candidatus Liberibacter asiaticus'. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to 'Ca. L. asiaticus' infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to 'Ca. L. asiaticus'. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping.

BACKGROUND: Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a 'Mediterranean' mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. RESULTS: Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. CONCLUSIONS: A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the 'Mediterranean' mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Mechanism-based inhibition of human cytochrome P450-3A activity by grapefruit hybrids having low furanocoumarin content.

A citrus breeding program aimed at developing low furanocoumarin (FC) grapefruit cultivars provided 40 grapefruit juice (GFJ) samples containing variable concentrations of FC derivatives, established as being mechanism-based (irreversible) inhibitors of human CYP3A isoforms. The principal inhibitory FCs were identified as 6',7'-dihydroxybergamottin, along with a series of dimeric compounds (spiroesters) having high inhibitory potency. A random subset of the GFJ samples (n = 25) were tested as CYP3A inhibitors using an in vitro model based on human liver microsomal metabolism of the index substrate triazolam. The reciprocal values of in vitro 50% inhibitory concentrations (IC(50)) were highly correlated with concentrations of inhibitory FCs in the GFJ samples (r(2) = 0.96). However the correlations were driven mainly by a few samples having high FC content and high reciprocal IC(50) (corresponding to low IC(50)). Among the rest of the samples, the relationship was less robust. Further study is needed to determine how low the FC content needs to be (or how high the IC(50) needs to be) to assure minimal risk of clinical interactions involving GFJ and CYP3A substrate drugs.

Natural Sweeteners and Sweetness-Enhancing Compounds Identified in Citrus Using an Efficient Metabolomics-Based Screening Strategy.

With the high demand for a healthy diet, it is necessary and important to explore natural sweeteners used in food that enhance palatability but minimize calories. Citrus is considered a good potential source of noncaloric sweeteners, but to date, only one sweetness modulator has been found in this most common fruit crop. Herein, an efficient strategy based on an in-house database and the untargeted and targeted metabolomics analyses was proposed to screen sweeteners or sweet-enhancing compounds from citrus. Eight sweeteners or sweetness-enhancing compounds were screened out, seven of which were newly identified from the genus Citrus. Surprisingly, we identified naturally occurring oxime V, which previously was only known as a synthetic compound. The contents of five compounds, in 11 citrus cultivars or unreleased selections across two production years, were compared. Successful identification of these natural sweeteners and sweetness-enhancing compounds in citrus fruit indicated the potential to identify the relevant biosynthetic pathways and to breed new citrus cultivars containing these compounds that provided both palatability and lower sugar consumption. This study also demonstrated that the proposed metabolomics-based screening strategy could greatly boost the identification of taste modulators with low contents in natural resources.

Transcriptome Analysis of Plenodomus tracheiphilus Infecting Rough Lemon (Citrus jambhiri Lush.) Indicates a Multifaceted Strategy during Host Pathogenesis.

The causal agent of mal secco disease is the fungus Plenodomus tracheiphilus, mainly affecting lemon tree survival in the Mediterranean area. Using a fully compatible host-pathogen interaction, the aim of our work was to retrieve the fungus transcriptome by an RNA seq approach during infection of rough lemon (Citrus jambhiri Lush.) to identify crucial transcripts for pathogenesis establishment and progression. A total of 2438 clusters belonging to P. tracheiphilus were retrieved and classified into the GO and KEGG categories. Transcripts were categorized mainly within the "membrane", "catalytic activity", and "primary metabolic process" GO terms. Moreover, most of the transcripts are included in the "ribosome", "carbon metabolism", and "oxidative phosphorylation" KEGG categories. By focusing our attention on transcripts with FPKM values higher than the median, we were able to identify four main transcript groups functioning in (a) fungus cell wall remodeling and protection, (b) destroying plant defensive secondary metabolites, (c) optimizing fungus development and pathogenesis, and (d) toxin biosynthesis, thus indicating that a multifaceted strategy to subdue the host was executed.

An Integrated Metabolome and Transcriptome Analysis Reveal the Regulation Mechanisms of Flavonoid Biosynthesis in a Purple Tea Plant Cultivar.

Purple tea plant cultivars, enrich with flavonoids and anthocyanins, are valuable materials for manufacturing tea with unique color and flavor. Researchers found that 'Zijuan' leaves changed from purple to green mainly caused by the decreased flavonoids and anthocyanins concentrations. The mechanism of flavonoids and anthocyanin biosynthesis has been studied in many purple tea plant cultivars and the key genes which regulated the biosynthesis of flavonoid and anthocyanins in different purple tea plant cultivars were quite different. Also, the molecular regulation mechanism underlying the flavonoids and anthocyanins biosynthesis during leaves development and color changes is less-thoroughly understood. In this study, an integrative analysis of transcriptome and metabolome was performed on the purple leaves and green leaves of 'Zijuan' tea plant to reveal the regulatory networks correlated to flavonoid biosynthesis and to identify key regulatory genes. Our results indicated that the 'Zijuan' new shoots leaves were purple might be due to the copigmentation of quercetin and kaempferol derivatives. In 'Zijuan' tea plant cultivar, flavonoids metabolites concentrations in purple leaves and green leaves were significantly influenced by the genes involved in flavonoid biosynthesis, transcriptional regulation, transport, and hormone response. Transcription factors including NAC008, MYB23, and bHLH96 and transporters such as ABC transporter I might be responsible for the flavonoid and anthocyanins accumulation in purple leaves. This study provides a new insight into the metabolism and molecular mechanisms underlying flavonoid and anthocyanin biosynthesis in tea plant.