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Summary: Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA‐mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism‐related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid‐driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5‐CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5‐CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5‐CsbZIP44 precisely modulates ABA signal‐mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops. [ABSTRACT FROM AUTHOR]

Summary: Tetraploids have been reported to exhibit increased stress tolerance, but the underlying molecular and physiological mechanisms remain poorly understood. In this study, autotetraploid plants were identified by screening natural seedlings of trifoliate orange (Poncirus trifoliata). The tetraploids exhibited different morphology and displayed significantly enhanced drought and dehydration tolerance in comparison with the diploid progenitor. Transcriptome analysis indicated that a number of stress‐responsive genes and pathways were differentially influenced and enriched in the tetraploids, in particular those coding for enzymes related to antioxidant process and sugar metabolism. Transcript levels and activities of antioxidant enzymes (peroxidase and superoxide dismutase) and sucrose‐hydrolysing enzyme (vacuolar invertase) were increased in the tetraploids upon exposure to the drought, concomitant with greater levels of glucose but lower level of reactive oxygen species (ROS). These data indicate that the tetraploids might undergo extensive transcriptome reprogramming of genes involved in ROS scavenging and sugar metabolism, which contributes, synergistically or independently, to the enhanced stress tolerance of the tetraploid. Our results reveal that the tetraploids take priority over the diploid for stress tolerance by maintaining a more robust system of ROS detoxification and osmotic adjustment via elevating antioxidant capacity and sugar accumulation in comparison with the diploid counterpart. [ABSTRACT FROM AUTHOR]

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid β-citraurin is responsible for the peel color in "Newhall" orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on β-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and β-citraurin production in "Newhall" orange. A MeJA treatment induced the expression of CsMYC2 , which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for β-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene β-cyclase (CsLCYb), and β-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce β-citraurin biosynthesis in citrus by activating a CsMPK6–CsMYC2 cascade, thereby affecting fruit coloration. [ABSTRACT FROM AUTHOR]

Summary: Arginine decarboxylase (ADC)‐mediated putrescine (Put) biosynthesis plays an important role in plant abiotic stress response. SNF1‐related protein kinases 2s (SnRK2s) and abscisic acid (ABA)‐response element (ABRE)‐binding factors (ABFs), are core components of the ABA signaling pathway involved in drought stress response. We previously reported that ADC of Poncirus trifoliata (PtrADC) functions in drought tolerance. However, whether and how SnRK2 and ABF regulate PtrADC to modulate putrescine accumulation under drought stress remains largely unclear.Herein, we employed a set of physiological, biochemical, and molecular approaches to reveal that a protein complex composed of PtrSnRK2.4 and PtrABF2 modulates putrescine biosynthesis and drought tolerance by directly regulating PtrADC.PtrABF2 was upregulated by dehydration in an ABA‐dependent manner. PtrABF2 activated PtrADC expression by directly and specifically binding to the ABRE core sequence within its promoter and positively regulated drought tolerance via modulating putrescine accumulation. PtrSnRK2.4 interacts with and phosphorylates PtrABF2 at Ser93. PtrSnRK2.4‐mediated PtrABF2 phosphorylation is essential for the transcriptional regulation of PtrADC. Besides, PtrSnRK2.4 was shown to play a positive role in drought tolerance by facilitating putrescine synthesis.Taken together, this study sheds new light on the regulatory module SnRK2.4‐ABF2‐ADC responsible for fine‐tuning putrescine accumulation under drought stress, which advances our understanding on transcriptional regulation of putrescine synthesis. [ABSTRACT FROM AUTHOR]

Background: Previous reports have mainly focused on the volatiles in citrus fruits, and there have been few reports about the volatiles in citrus leaves and flowers. However, citrus leaves and flowers are also rich in volatile compounds with unique aromas. Here, to investigate the volatiles in citrus leaves and flowers, volatile profiling was performed on leaves from 62 germplasms and flowers from 25 germplasms. Results: In total, 196 and 82 volatile compounds were identified from leaves of 62 citrus germplasms and flowers of 25 citrus germplasms, respectively. The dominant volatile terpenoids were more diverse in citrus leaves than in peels. A total of 34 volatile terpenoids were commonly detected in the leaves of at least 20 germplasms, among which 31 were overaccumulated in the leaves of wild or semiwild germplasms. This result was consistent with the high expression levels of five genes and one key gene of the mevalonate and 2-C-methyl-D-erythritol-4-phosphate (MEP) biosynthetic pathways, respectively, as well as the low expression levels of geranylgeranyl diphosphate synthase of the MEP pathway, relative to the levels in cultivars. Fully open flowers showed increased levels of four terpene alcohols and a decrease in sabinene content compared with balloon-stage flowers, especially in sweet orange. A monoterpene synthase gene was identified and functionally characterized as a sabinene synthase in vitro. Conclusions: Collectively, our results suggest that 31 important terpenoids are abundant in wild or semiwild citrus germplasms, possibly because of a negative effect of domestication on the volatiles in citrus leaves. The sweet smell of fully open flowers may be attributed to increased levels of four terpene alcohols. In addition, a sabinene synthase gene was identified by combined transcriptomic and metabolomic analyses. [ABSTRACT FROM AUTHOR]

Summary: Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as 'Mini‐Citrus') for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research. [ABSTRACT FROM AUTHOR]

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′ cis -regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C 30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing β-citraurin, CCD4b is also responsible for the production of another important C 30 apocarotenoid pigment, β-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′ cis -regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C 30 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. Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments. This paper reports that a 5′ cis -regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C 30 apocarotenoids responsible for red coloration of citrus peel. Phylogenetic evidence suggests that functional diversification of CCD4b and naturally occurring variation in the CCD4b promoter resulted in the stepwise evolution of red-peel trait in mandarins and their hybrids. [ABSTRACT FROM AUTHOR]

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7- O - di -glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcT s resulted in flavonoid-7- O -glucosides being catalysed into bitterless flavonoid-7- O - di -glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7- O -neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcT s showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7- O - di -glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation. [ABSTRACT FROM AUTHOR]

Mandarin ( Citrus reticulata ) is one of the most important citrus crops worldwide. Its domestication is believed to have occurred in South China, which has been one of the centers of mandarin cultivation for four millennia. We collected natural wild populations of mandarin around the Nanling region and cultivated landraces in the vicinity. We found that the citric acid level was dramatically reduced in cultivated mandarins. To understand genetic basis of mandarin domestication, we de novo assembled a draft genome of wild mandarin and analyzed a set of 104 citrus genomes. We found that the Mangshan mandarin is a primitive type and that two independent domestication events have occurred, resulting in two groups of cultivated mandarins (MD1 and MD2) in the North and South Nanling Mountains, respectively. Two bottlenecks and two expansions of effective population size were identified for the MD1 group of cultivated mandarins. However, in the MD2 group there was a long and continuous decrease in the population size. MD1 and MD2 mandarins showed different patterns of interspecific introgression from cultivated pummelo species. We identified a region of high divergence in an aconitate hydratase ( ACO ) gene involved in the regulation of citrate content, which was possibly under selection during the domestication of mandarin. This study provides concrete genetic evidence for the geographical origin of extant wild mandarin populations and sheds light on the domestication and evolutionary history of mandarin. [ABSTRACT FROM AUTHOR]