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The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene β-cyclase (CitLCYb), β-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene ε-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from β,ε-carotenoid (α-car and lutein) accumulation to β,β-carotenoid (β-car, β-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of β,ε-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive β,β-xanthophyll (β-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive β,β-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in β,β-xanthophyll compositions between Satsuma mandarin and Valencia orange was also discussed on the basis of the substrate specificity of β-ring hydroxylase and the balance of expression between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP).

Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

Background Pomelo is one of the three major species of citrus. The fruit accumulates a variety of abundant secondary metabolites that affect the flavor. UDP-glycosyltransferases (UGTs) are involved in the glycosylation of secondary metabolites. Results In the present study, we performed a genome-wide analysis of pomelo UGT family, a total of 145 UGTs was identified based on the conserved plant secondary product glycosyltransferase (PSPG) motif. These UGT genes were clustered into 16 major groups through phylogenetic analysis of these genes with other plant UGTs (A-P). Pomelo UGTs were distributed unevenly among the chromosomes. At least 10 intron insertion events were observed in these UGT genome sequences, and I-5 was identified to be the highest conserved one. The expression profile analysis of pomelo UGT genes in different fruit tissues during development and ripening was carried out by RNA-seq. Conclusions We identified 145 UGTs in pomelo fruit through transcriptome data and citrus genome database. Our research provides available information on UGTs studies in pomelo, and provides an important research foundation for screening and identification of functional UGT genes.

Lignification of juice sacs is a primary contributor to reductions in fruit quality, with impacts on taste and economic value of pomelo (Citrus grandis). To date, information on the regulation of fruit lignification remains fragmentary. In this study, we first analyzed the relationship between lignification and nutrient status of pomelo juice sacs, which revealed a significant positive correlation between nitrate (NO3−) concentration and lignin concentration, with over 60% of lignin accumulation explained by NO3− levels in three models of machine learning-based regression. Results from field trails in 11 pomelo orchards, as well as in pear fruits and soybean roots exposed to low or high NO3− supplies, further demonstrated that nitrate plays an important role in lignification. Transcriptomic analysis further showed that pomelo laccases (CgLACs) were more intensively up-regulated upon addition of NO3− than any of the genes encoding one of the other 12 enzymes involved in lignin biosynthesis. Among the nine identified CgLACs, CgLAC3 was the most significantly up-regulated CgLAC in high nitrate treated plants. Over-expressing CgLAC3 increased lignin concentrations in both pomelo albedo and soybean hairy roots. Taken together, we conclude that nitrate modulates fruit lignification in pomelo through regulation of CgLAC3 expression, which suggests that NO3−-N fertilization may affect fruit lignification, and thereby can be managed to improve fruit quality.

Citrus canker, a devastating disease caused by Xanthomonas citri subsp. citri (Xcc), poses a significant threat to global citrus production due to the high susceptibility of nearly all commercial citrus cultivars to it. Although transcription factor Citrus PH4 (CitPH4) is well known for regulating fruit acidity, its potential role in plant immunity remains elusive. Here, we demonstrate that CitPH4 positively regulates citrus defence against Xcc. Transcriptomic and biochemical analyses indicate that CitPH4 binds to the promoter of the responsive to dehydration 21a (CsRD21a), a gene encoding a papain‐like cysteine protease (PLCP), thus modulating its expression. Functional characterisation further reveals that CsRD21a is essential for CitPH4‐mediated resistance to Xcc. Mechanistically, CsRD21a interacts with pathogenesis‐related protein 5 (CsPR5) to promote salicylic acid (SA) accumulation. In addition, reciprocal activation of PLCP activity and SA signalling enhances immune defences against Xcc. Collectively, our findings reveal a CitPH4‐mediated citrus immunity mechanism in which the CitPH4–CsRD21a–CsPR5 module constitutes a defence loop by integrating reciprocal activation effects of PLCP activity and SA signalling. This study provides a novel insight into the regulatory network of citrus resistance against Xcc, offering potential targets for breeding citrus cultivars with enhanced disease resistance and superior fruit quality.

Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (CrtB). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the β-carotene hydroxylase gene (HYD), which was suppressed in the calli. Co-expression of CrtB and HYD in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.

ICE1 (Inducer of CBF Expression 1) encodes a MYC-like basic helix–loop–helix transcription factor that acts as a central regulator of cold response. In this study, we elucidated the function and underlying mechanisms of PtrICE1 from trifoliate orange [Poncirus trifoliata (L.) Raf.]. PtrICE1 was upregulated by cold, dehydration, and salt, with the greatest induction under cold conditions. PtrICE1 was localized in the nucleus and could bind to a MYC-recognizing sequence. Ectopic expression of PtrICE1 in tobacco and lemon conferred enhanced tolerance to cold stresses at either chilling or freezing temperatures. Yeast two-hybrid screening revealed that 21 proteins belonged to the PtrICE1 interactome, in which PtADC (arginine decarboxylase) was confirmed as a bona fide protein interacting with PtrICE1. Transcript levels of ADC genes in the transgenic lines were slightly elevated under normal growth condition but substantially increased under cold conditions, consistent with changes in free polyamine levels. By contrast, accumulation of the reactive oxygen species, H₂O₂ and O₂⁻, was appreciably alleviated in the transgenic lines under cold stress. Higher activities of antioxidant enzymes, such as superoxide dismutase and catalase, were detected in the transgenic lines under cold conditions. Taken together, these results demonstrated that PtrICE1 plays a positive role in cold tolerance, which may be due to modulation of polyamine levels through interacting with the ADC gene.

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7′,8′ position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.

Carotenoids are the main pigments responsible of the colouration of Citrus fruits. The β-cyclization of lycopene, catalysed by the lycopene β-cyclases (β-LCY), seems to be a key regulatory step of the carotenoid pathway. In the present study, two β-LCYs from orange fruits (Citrus sinensis), named Csβ-LCY1 and Csβ-LCY2 have been isolated and the activity of the encoded proteins was demonstrated by functional analysis. Csβ-LCY1 was expressed at low levels and remained relatively constant during fruit ripening while Csβ-LCY2 showed a chromoplast-specific expression and a marked induction in both peel and pulp of orange fruits in parallel with the accumulation of β,β-xanthophylls. The potential involvement of Csβ-LCY2 in the accumulation of lycopene, characteristic of some Citrus species such as red grapefruits, was investigated. Expression of Csβ-LCY2 and another seven carotenoid biosynthetic genes were studied in the peel and pulp of the high lycopene-accumulating grapefruit, Star Ruby, and compared with those of ordinary Navel orange. In Star Ruby, the accumulation of lycopene during fruit maturation was associated with a substantial reduction in the expression of both β-LCY2 and β-CHX genes with respect to Navel orange. Moreover, two different alleles of β-LCY2: β-LCY2a and β-LCY2b were isolated from both genotypes, and functional assays demonstrated that the lycopene β-cyclase activity of the allele b was almost null. Interestingly, Star Ruby grapefruit predominantly expressed the unfunctional β-LCY2b allele during fruit ripening whereas Navel oranges preferably expressed the functional allele. It is suggested that the presence of diverse alleles of the β-LCY2 gene, encoding enzymes with altered activity, with different transcript accumulation may be an additional regulatory mechanism of carotenoid synthesis involved in the accumulation of lycopene in red grapefruits.