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Although some WRKYs have been characterized, regulatory roles of most WRKYs remain poorly understood. Herein, we elucidated function of FcWRKY40 from Fortunella crassifolia in salt tolerance via overexpression and virus-induced gene silencing (VIGS) and unraveled its target genes. Overexpression of FcWRKY40 enhanced salt tolerance in transgenic tobacco and lemon, while silencing of FcWRKY40 increased salt susceptibility. Homolog genes of Salt Overly Sensitive 2 (SOS2) and Δ-1-pyrroline-5-carboxylate synthetase 1 (P5CS1) were dramatically up-regulated in transgenic lemon but down-regulated in VIGS line. Consistently, transgenic lemon displayed lower Na+ and higher proline concentrations, whereas the silenced line accumulated more Na+ but less proline. Treatment of transgenic lemon with 24-epi-brassinolide compromised salt tolerance, while supply of exogenous proline partially restored salt tolerance of the VIGS line. FcWRKY40 specifically binds to and activates promoters of FcSOS2 and FcP5CS1. FcWRKY40 was up-regulated by ABA and salt, and confirmed as a target of ABA-responsive element binding factor 2 (FcABF2). Moreover, salt treatment up-regulated FcABF2 and FcP5CS1, and elevated proline concentrations. Taken together, our findings demonstrate that FcWRKY40 participates in the ABA signaling pathway and as a positive regulator functions in salt tolerance by regulating genes involved in ion homeostasis and proline biosynthesis.

To elucidate different fruit trait mechanisms among Changshou (FOT) ( Fortunella obovata Tanaka), Liuyang (FCSLY) ( Fortunella crassifolia Swingle), and Huapi (FCSHP) ( Fortunella crassifolia Swingle) kumquats, fruit traits, and their metabolite compositions were comprehensively analyzed. The main factors affecting fruit sweetness and bitterness are the sucrose and limonin contents. Differential metabolite analysis showed that FOT peels had the highest redness and more up-regulated anthocyanin and carotenoid compounds. Therefore, anthocyanin and carotenoid metabolites were related to the kumquat peel color. FOT peels had the highest flavonoid content. A total of 1719 metabolites were identified using non-targeted metabolomics, Flavonoid metabolites are more abundant in kumquat peels than in seeds, so the peel’s medicinal value is higher. The total limonin content in seeds is higher than in peels, so the seeds are used as raw materials for extracting limonin compounds. This study analyzed the biochemical mechanisms of fruit trait differences in three kumquat ( Fortunella Swingle) varieties and provided a reference for targeted kumquat development and utilization.

Kumquat and calamondin are two small-size citrus fruits. Owing to their health benefits, they are traditionally used as folk medicine in Asian countries. However, the research on flavonoids and biological activities of kumquat and calamondin have received less attention. This review summarizes the reported quantitative and qualitative data of phenolic compositions in these two fruits. Effects of maturity, harvest time, various solvent extractions and heat treatment of phenolic compositions, and bioactivities were discussed; distributions of the forms of phenolic compounds existing in kumquat and calamondin were also summarized. Furthermore, biological activities, including antioxidant, antityrosinase, antimicrobial, antitumor, and antimetabolic disorder effects, have also been discussed. Effective phenolic components were proposed for a certain bioactivity. It was found that C-glycoside flavonoids are dominant phenolic compounds in kumquat and calamondin, unlike in other citrus fruits. Up to now, biological activities and chemical characteristics of C-glycoside flavonoids in kumquat and calamondin are largely unknown. [Display omitted] •Quantitative and qualitative data of flavonoids and phenolic acids are compared.•C-glycoside flavonoids dominated in kumquat and calamondin.•Biological activities of kumquat and calamondin are summarized.•Phenolic compositions and biological activities by different factors are discussed.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus. Meiwa kumquat (Fortunella crassifolia) has shown a durable resistance against Xcc. Here, we aimed to characterize the mechanisms responsible for such a durable resistance by characterizing the transcriptional and physiological responses of Meiwa kumquat to Xcc. Inoculation of Meiwa kumquat with Xcc promoted immune responses such as upregulation of PR genes, accumulation of salicylic acid, hypersensitive response (HR)-like cell death and early leaf abscission. Hypertrophy and hyperplasia symptoms, which are known to be caused by Xcc-induction of the canker susceptibility gene LOB1 through the transcription activator-like effector (TALE) PthA4, always appear prior to the development of cell death. Mutation of pthA4 in Xcc abolished the induction of LOB1, canker symptoms, cell death, and leaf abscission and reduced the expression of PR genes in inoculated kumquat leaves without reducing Xcc titers in planta. Transcriptome analysis demonstrated that PthA4 promotes plant biotic and abiotic stress responses and the biosynthesis of abscisic acid. Transcriptional induction of LOB1 homologs in Meiwa kumquat by Xcc pthA4 mutant strains carrying a repertoire of designer TALEs promoted the elicitation of HR-like phenotype and leaf abscission, suggesting that kumquat response to Xcc is associated with upregulation of LOB1. Our study suggests a novel mechanism of plant resistance to Xanthomonas via elicitation of immune responses by upregulation of a host susceptibility gene.

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.

The volatiles of different aerial parts of three kumquat species (Fortunella margarita Swingle–Nagami, Fortunella japonica Swingle–Marumi, and Fortunella crassifolia Swingle–Meiwa) growing in Greece were analyzed via GC-MS and evaluated for their antimicrobial properties against nine human pathogenic microorganisms. A total number of 23 compounds were identified in the peel, 38 in the leaves, and 30 in the flowers of the examined species. Limonene was the dominant metabolite in the peels of all three species, germacrene-D was present in the leaves of Nagami and Marumi kumquats, while limonene was the most abundant in the flower of Marumi and Meiwa kumquat but with significant differences in the composition of the total fracture of the essential oil, since compounds with high antimicrobial activity were only present in the flower of Meiwa kumquat. The essential oils from the leaf and peel of the three kumquat species were either inactive or showed weak antimicrobial activity, respectively, against Gram-positive and Gram-negative bacterial strains and pathogenic fungi. Only the essential oil from the flower of F. crassifolia Swingle (Meiwa) showed a stronger effect (MIC values 3.5–7.48 mg/mL) against all the assayed microorganisms. Furthermore, through multivariate statistical analysis, we studied the relationships between the samples regarding their origin (species and plant part), as well as between the chemical composition of the corresponding essential oils and their antimicrobial activity. Considering its chemical profile and antimicrobial activity, the Greek Meiwa flowers’ essential oil seemed a promising essential oil for further exploitation in the food and/or medicinal industry.

The aim of this study was to determine the main constituents of the essential oil isolated from Fortunella crassifolia Swingle peel by hydro-distillation, and to test the efficacy of the essential oil on antimicrobial activity. Twenty-five components, representing 92.36% of the total oil, were identified by GC-MS analysis. The essential oil showed potent antimicrobial activity against both Gram-negative (E. coli and S. typhimurium) and Gram-positive (S. aureus, B. cereus, B. subtilis, L. bulgaricus, and B. laterosporus) bacteria, together with a remarkable antifungal activity against C. albicans. In a food model of beef extract, the essential oil was observed to possess an effective capacity to control the total counts of viable bacteria. Furthermore, the essential oil showed strongly detrimental effects on the growth and morphological structure of the tested bacteria. It was suggested that the essential oil from Fortunella crassifolia Swingle peel might be used as a natural food preservative against bacteria or fungus in the food industry.

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating biotic stresses affecting the citrus industry. Meiwa kumquat (Fortunella crassifolia) is canker-resistant, while Newhall navel orange (Citrus sinensis Osbeck) is canker-sensitive. To understand the molecular mechanisms underlying the differences in responses to Xcc, transcriptomic profiles of these two genotypes following Xcc attack were compared by using the Affymetrix citrus genome GeneChip. A total of 794 and 1324 differentially expressed genes (DEGs) were identified as canker-responsive genes in Meiwa and Newhall, respectively. Of these, 230 genes were expressed in common between both genotypes, while 564 and 1094 genes were only significantly expressed in either Meiwa or Newhall. Gene ontology (GO) annotation and Singular Enrichment Analysis (SEA) of the DEGs showed that genes related to the cell wall and polysaccharide metabolism were induced for basic defense in both Meiwa and Newhall, such as chitinase, glucanase and thaumatin-like protein. Moreover, apart from inducing basic defense, Meiwa showed specially upregulated expression of several genes involved in the response to biotic stimulus, defense response, and cation binding as comparing with Newhall. And in Newhall, abundant photosynthesis-related genes were significantly down-regulated, which may be in order to ensure the basic defense. This study revealed different molecular responses to canker disease in Meiwa and Newhall, affording insight into the response to canker and providing valuable information for the identification of potential genes for engineering canker tolerance in the future.

In this study, we reported the complete chloroplast genome of Fortunella crassifolia Swingle using the HiSeq-4000 sequencing. The chloroplast genome size is 160,229 bp, which consists of a large single-copy region (87,774 bp), a small single-copy region (18,721 bp), and a pair of IR regions (26,867 bp). The chloroplast genome contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs, and 4 rRNAs. Phylogenetic maximum likelihood analysis showed that F. crassifolia was closest to Hongkong kumquat (F. hindsii). The complete chloroplast genome would be subsequently used for citrus species researches.

We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst; H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E; Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidization processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even number) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.