A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo.

Granulation of juice sacs is a physiological disorder, which affects pomelo fruit quality. Here, the transcriptome and ubiquitinome of the granulated juice sacs were analyzed in Guanxi pomelo. We found that lignin accumulation in the granulated juice sacs was regulated at transcription and protein modification levels. In transcriptome data, we found that the genes in lignin biosynthesis pathway and antioxidant enzyme system of the granulated juice sacs were significantly upregulated. However, in ubiquitinome data, we found that ubiquitinated antioxidant enzymes increased in abundance but the enzyme activities decreased after the modification, which gave rise to reactive oxygen species (ROS) contents in granulated juice sacs. This finding suggests that ubiquitination level of the antioxidant enzymes is negatively correlated with the enzyme activities. Increased H2O2 is considered to be a signaling molecule to activate the key gene expressions in lignin biosynthesis pathway, which leads to the lignification in granulated juice sacs of pomelo. This regulatory mechanism in juice sac granulation of pomelo was further confirmed through the verification experiment using tissue culture by adding H2O2 or dimethylthiourea (DMTU). Our findings suggest that scavenging H2O2 and other ROS are important for reducing lignin accumulation, alleviating juice sac granulation and improving pomelo fruit quality.

An integrated multi-omics approach reveals polymethoxylated flavonoid biosynthesis in Citrus reticulata cv. Chachiensis.

Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, its dried mature peels named "Guangchenpi", has been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. However, the biosynthesis of the crucial natural products polymethoxylated flavonoids (PMFs) in CRC remains unclear. Here, we report a chromosome-scale genome assembly of CRC with the size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, we discover a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3',4'-heptamethoxyflavone (HPMF). Based on transient overexpression and virus-induced gene silencing experiments, we propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. In addition, a potential gene regulatory network associated with PMF biosynthesis is identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.

Transcription-related metabolic regulation in grafted lemon seedlings under magnesium deficiency stress.

Magnesium is one of the essential nutrients for plant growth, and plays a pivotal role in plant development and metabolism. Soil magnesium deficiency is evident in citrus production, which ultimately leads to failure of normal plant growth and development, as well as decreased productivity. Citrus is mainly propagated by grafting, so it is necessary to fully understand the different regulatory mechanisms of rootstock and scion response to magnesium deficiency. Here, we characterized the differences in morphological alterations, physiological metabolism and differential gene expression between trifoliate orange rootstocks and lemon scions under normal and magnesium-deficient conditions, revealing the different responses of rootstocks and scions to magnesium deficiency. The transcriptomic data showed that differentially expressed genes were enriched in 14 and 4 metabolic pathways in leaves and roots, respectively, after magnesium deficiency treatment. And the magnesium transport-related genes MHX and MRS2 may respond to magnesium deficiency stress. In addition, magnesium deficiency may affect plant growth by affecting POD, SOD, and CAT enzyme activity, as well as altering the levels of hormones such as IAA, ABA, GA3, JA, and SA, and the expression of related responsive genes. In conclusion, our research suggests that the leaves of lemon grafted onto trifoliate orange were more significantly affected than the roots under magnesium-deficient conditions, further indicating that the metabolic imbalance of scion lemon leaves was more severe.

The genome of Citrus australasica reveals disease resistance and other species specific genes.

BACKGROUND: The finger lime (Citrus australasica), one of six Australian endemic citrus species shows a high natural phenotypic diversity and novel characteristics. The wide variation and unique horticultural features have made this lime an attractive candidate for domestication. Currently no haplotype resolved genome is available for this species. Here we present a high quality, haplotype-resolved reference genome for this species using PacBio HiFi and Hi-C sequencing. RESULTS: Hifiasm assembly and SALSA scaffolding resulted in a collapsed genome size of 344.2 Mb and 321.1 Mb and 323.2 Mb size for the two haplotypes. The nine pseudochromosomes of the collapsed genome had an N50 of 35.2 Mb, 99.1% genome assembly completeness and 98.9% gene annotation completeness (BUSCO). A total of 41,304 genes were predicted in the nuclear genome. Comparison with C. australis revealed that 13,661 genes in pseudochromosomes were unique in C. australasica. These were mainly involved in plant-pathogen interactions, stress response, cellular metabolic and developmental processes, and signal transduction. The two genomes showed a syntenic arrangement at the chromosome level with large structural rearrangements in some chromosomes. Genetic variation among five C. australasica cultivars was analysed. Genes related to defense, synthesis of volatile compounds and red/yellow coloration were identified in the genome. A major expansion of genes encoding thylakoid curvature proteins was found in the C. australasica genome. CONCLUSIONS: The genome of C. australasica present in this study is of high quality and contiguity. This genome helps deepen our understanding of citrus evolution and reveals disease resistance and quality related genes with potential to accelerate the genetic improvement of citrus.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Transcriptome differential expression analysis of defoliation of two different lemon varieties.

'Allen Eureka' is a bud variety of Eureka lemon with excellent fruiting traits. However, it suffers from severe winter defoliation that leads to a large loss of organic nutrients and seriously affects the tree's growth and development as well as the yield of the following year, and the mechanism of its response to defoliation is still unclear. In order to investigate the molecular regulatory mechanisms of different leaf abscission periods in lemon, two lemon cultivars ('Allen Eureka' and 'Yunning No. 1') with different defoliation traits were used as materials. The petiole abscission zone (AZ) was collected at three different defoliation stages, namely, the pre-defoliation stage (CQ), the mid-defoliation stage (CZ), and the post-defoliation stage (CH). Transcriptome sequencing was performed to analyze the gene expression differences between these two cultivars. A total of 898, 4,856, and 3,126 differentially expressed genes (DEGs) were obtained in CQ, CZ, and CH, respectively, and the number of DEGs in CZ was the largest. GO analysis revealed that the DEGs between the two cultivars were mainly enriched in processes related to oxidoreductase, hydrolase, DNA binding transcription factor, and transcription regulator activity in the defoliation stages. KEGG analysis showed that the DEGs were concentrated in CZ and involved plant hormone signal transduction, phenylpropanoid biosynthesis, glutathione metabolism, and alpha-linolenic acid metabolism. The expression trends of some DEGs suggested their roles in regulating defoliation in lemon. Eight gene families were obtained by combining DEG clustering analysis and weighted gene co-expression network analysis (WGCNA), including ß-glucosidase, AUX/IAA, SAUR, GH3, POD, and WRKY, suggesting that these genes may be involved in the regulation of lemon leaf abscission. The above conclusions enrich the research related to lemon leaf abscission and provide reliable data for the screening of lemon defoliation candidate genes and analysis of defoliation pathways.

Octanal enhances disease resistance in postharvest citrus fruit by the biosynthesis and metabolism of aromatic amino acids.

Octanal was found to be able to reduce green mold incidence in citrus fruit by a defense response mechanism. However, the underlying mechanism remains largely unclear. Herein, the metabolomics, RNA-seq and biochemical analyses were integrated to explore the effect of octanal on disease resistance in harvested citrus fruit. Results showed that octanal fumigation at 40 µL L-1 was effective in controlling citrus green mold. Metabolomics analysis showed that octanal mainly led to the accumulation of some plant hormones including methyl jasmonate, abscisic acid, indole-3-butyric acid, indoleacetic acid (IAA), salicylic acid, and gibberellic acid and many phenylpropanoid metabolites including cinnamyl alcohol, hesperidin, dihydrokaempferol, vanillin, quercetin-3-O-malonylglucoside, curcumin, naringin, chrysin, coniferin, calycosin-7-O-ß-D-glucoside, trans-cinnamaldehyde, and 4',5,7-trihydroxy-3,6-dimethoxyflavone. Particularly, IAA and hesperidin were dramatically accumulated in the peel, which might be the contributors to the resistance response. Additionally, transcriptome analysis showed that octanal greatly activated the biosynthesis and metabolism of aromatic amino acids. This was further verified by the accumulation of some metabolites (shikimic acid, tryptophan, tyrosine, phenylalanine, IAA, total phenolics, flavonoids and lignin), increase in some enzyme activities (phenylalanine ammonia-lyase, tyrosine ammonia-lyase, 4-coumarate CoA ligase, cinnamic acid 4-hydroxylase, polyphenol oxidase, and peroxidase), up-regulation of some genes (tryptophan pyruvate aminotransferase, aldehyde dehydrogenase, shikimate kinase and shikimate dehydrogenase) expressions and molecular docking results. Thus, these results indicate that octanal is an efficient strategy for the control of postharvest green mold by triggering the defense response in citrus fruit.

Three new discovery effector proteins from Candidatus Liberibacter asiaticus psy62 inhibit plant defense through interaction with AtCAT3 and AtGAPA.

KEY MESSAGE: We identify three SDEs that inhibiting host defence from Candidatus Liberibacter asiaticus psy62, which is an important supplement to the pathogenesis of HLB. Candidatus Liberibacter asiaticus (CLas) is the main pathogen of citrus Huanglongbing (HLB). 38 new possible sec-dependent effectors (SDEs) of CLas psy62 were predicted by updated predictor SignalP 5.0, which 12 new SDEs were found using alkaline phosphate assay. Among them, SDE4310, SDE4435 and SDE4955 inhibited hypersensitivity reactions (HR) in Arabidopsis thaliana (Arabidopsis, At) and Nicotiana benthamiana leaves induced by pathogens, which lead to a decrease in cell death and reactive oxygen species (ROS) accumulation. And the expression levels of SDE4310, SDE4435, and SDE4955 genes elevated significantly in mild symptom citrus leaves. When SDE4310, SDE4435 and SDE4955 were overexpressed in Arabidopsis, HR pathway key genes pathogenesis-related 2 (PR2), PR5, nonexpressor of pathogenesis-related 1 (NPR1) and isochorismate synthase 1 (ICS1) expression significantly decreased and the growth of pathogen was greatly increased relative to control with Pst DC3000/AvrRps4 treatment. Our findings also indicated that SDE4310, SDE4435 and SDE4955 interacted with AtCAT3 (catalase 3) and AtGAPA (glyceraldehyde-3-phosphate dehydrogenase A). In conclusion, our results suggest that SDE4310, SDE4435 and SDE4955 are CLas psy62 effector proteins that may have redundant functions. They inhibit ROS burst and cell death by interacting with AtCAT3 and AtGAPA to negatively regulate host defense.

Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri).

Enzymatic browning poses a significant challenge that limits in vitro propagation and genetic transformation of plant tissues. This research focuses on investigating how adding antioxidant substances can suppress browning, leading to improved efficiency in transforming plant tissues using Agrobacterium and subsequent plant regeneration from rough lemon (Citrus × jambhiri). When epicotyl segments of rough lemon were exposed to Agrobacterium, they displayed excessive browning and tissue decay. This was notably different from the 'Hamlin' explants, which did not exhibit the same issue. The regeneration process failed completely in rough lemon explants, and they accumulated high levels of total phenolic compounds (TPC) and polyphenol oxidase (PPO), which contribute to browning. To overcome these challenges, several antioxidant and osmoprotectant compounds, including lipoic acid, melatonin, glycine betaine, and proline were added to the tissue culture medium to reduce the oxidation of phenolic compounds and mitigate browning. Treating epicotyl segments with 100 or 200 µM melatonin led to a significant reduction in browning and phenolic compound accumulation. This resulted in enhanced shoot regeneration, increased transformation efficiency, and reduced tissue decay. Importantly, melatonin supplementation effectively lowered the levels of TPC and PPO in the cultured explants. Molecular and physiological analyses also confirmed the successful overexpression of the CcNHX1 transcription factor, which plays a key role in imparting tolerance to salinity stress. This study emphasizes the noteworthy impact of supplementing antioxidants in achieving successful genetic transformation and plant regeneration in rough lemon. These findings provide valuable insights for developing strategies to address enzymatic browning and enhance the effectiveness of plant tissue culture and genetic engineering methods with potential applications across diverse plant species.

Genome-Wide Identification and Characterization of the Sweet Orange (Citrus sinensis) GATA Family Reveals a Role for CsGATA12 as a Regulator of Citrus Bacterial Canker Resistance.

Citrus bacterial canker (CBC) is a severe bacterial infection caused by Xanthomonas citri subsp. citri (Xcc), which continues to adversely impact citrus production worldwide. Members of the GATA family are important regulators of plant development and regulate plant responses to particular stressors. This report aimed to systematically elucidate the Citrus sinensis genome to identify and annotate genes that encode GATAs and evaluate the functional importance of these CsGATAs as regulators of CBC resistance. In total, 24 CsGATAs were identified and classified into four subfamilies. Furthermore, the phylogenetic relationships, chromosomal locations, collinear relationships, gene structures, and conserved domains for each of these GATA family members were also evaluated. It was observed that Xcc infection induced some CsGATAs, among which CsGATA12 was chosen for further functional validation. CsGATA12 was found to be localized in the nucleus and was differentially upregulated in the CBC-resistant and CBC-sensitive Kumquat and Wanjincheng citrus varieties. When transiently overexpressed, CsGATA12 significantly reduced CBC resistance with a corresponding increase in abscisic acid, jasmonic acid, and antioxidant enzyme levels. These alterations were consistent with lower levels of salicylic acid, ethylene, and reactive oxygen species. Moreover, the bacteria-induced CsGATA12 gene silencing yielded the opposite phenotypic outcomes. This investigation highlights the important role of CsGATA12 in regulating CBC resistance, underscoring its potential utility as a target for breeding citrus varieties with superior phytopathogen resistance.

Seasonal drought promotes citrate accumulation in citrus fruit through the CsABF3-activated CsAN1-CsPH8 pathway.

Plenty of rainfall but unevenly seasonal distribution happens regularly in southern China. Seasonal drought from summer to early autumn leads to citrus fruit acidification, but how seasonal drought regulates citrate accumulation remains unknown. Herein, we employed a set of physiological, biochemical, and molecular approaches to reveal that CsABF3 responds to seasonal drought stress and modulates citrate accumulation in citrus fruits by directly regulating CsAN1 and CsPH8. Here, we demonstrated that irreversible acidification of citrus fruits is caused by drought lasting for > 30 d during the fruit enlargement stage. We investigated the transcriptome characteristics of fruits affected by drought and corroborated the pivotal roles of a bHLH transcription factor (CsAN1) and a P3A-ATPase gene (CsPH8) in regulating citrate accumulation in response to drought. Abscisic acid (ABA)-responsive element binding factor 3 (CsABF3) was upregulated by drought in an ABA-dependent manner. CsABF3 activated CsAN1 and CsPH8 expression by directly and specifically binding to the ABA-responsive elements (ABREs) in the promoters and positively regulated citrate accumulation. Taken together, this study sheds new light on the regulatory module ABA-CsABF3-CsAN1-CsPH8 responsible for citrate accumulation under drought stress, which advances our understanding of quality formation of citrus fruit.

The CrMYB33 transcription factor positively coordinate the regulation of both carotenoid accumulation and chlorophyll degradation in the peel of citrus fruit.

Citrus, cultivated extensively across the globe, possesses considerable economic importance and nutritional value. With the degradation of chlorophyll and accumulation of carotenoids, mature citrus fruits develop an orange-yellow peel, enhancing fruit value and consumer preference. MYB transcription factors (TFs) exert a significant role in diverse plant developmental processes and investigating their involvement in fruit coloration is crucial for developing new cultivars. This work aimed to characterize a citrus TF, CrMYB33, whose expression was found to be positively correlated with carotenoid biosynthesis during fruit ripening. The interference of CrMYB33 expression in citrus fruit resulted in inhibition of carotenoid accumulation, down-regulation of carotenoid biosynthetic genes, and a slower rate of chlorophyll degradation. Conversely, overexpression of CrMYB33 in tomato (Solanum lycopersicum) enhanced chlorophyll degradation and carotenoid biosynthesis, resulting in a deeper red coloration of the fruits. Furthermore, the transcription of associated genes was upregulated in CrMYB33-overexpressing tomato fruits. Additional assays reveal that CrMYB33 exhibits direct links and activation of the promoters of lycopene ß-cyclase 2 (CrLCYb2), and ß-carotene hydroxylases 2 (CrBCH2), both crucial genes in the carotenoid biosynthetic pathway. Additionally, it was found to inhibit chlorophyllase (CrCLH), a gene essential in chlorophyll degradation. These findings provide insight into the observed changes in LCYb2, BCH2, and CLH expression in the transgenic lines under investigation. In conclusion, our study revealed that CrMYB33 modulates carotenoid accumulation and chlorophyll degradation in citrus fruits through transcriptionally activating genes involved in metabolic pathways.

Genome-wide identification and characterization of flowering genes in Citrus sinensis (L.) Osbeck: a comparison among C. Medica L., C. Reticulata Blanco, C. Grandis (L.) Osbeck and C. Clementina.

BACKGROUND: Flowering plays an important role in completing the reproductive cycle of plants and obtaining next generation of plants. In case of citrus, it may take more than a year to achieve progeny. Therefore, in order to fasten the breeding processes, the juvenility period needs to be reduced. The juvenility in plants is regulated by set of various flowering genes. The citrus fruit and leaves possess various medicinal properties and are subjected to intensive breeding programs to produce hybrids with improved quality traits. In order to break juvenility in Citrus, it is important to study the role of flowering genes. The present study involved identification of genes regulating flowering in Citrus sinensis L. Osbeck via homology based approach. The structural and functional characterization of these genes would help in targeting genome editing techniques to induce mutations in these genes for producing desirable results. RESULTS: A total of 43 genes were identified which were located on all the 9 chromosomes of citrus. The in-silico analysis was performed to determine the genetic structure, conserved motifs, cis-regulatory elements (CREs) and phylogenetic relationship of the genes. A total of 10 CREs responsible for flowering were detected in 33 genes and 8 conserved motifs were identified in all the genes. The protein structure, protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to study the functioning of these genes which revealed the involvement of flowering proteins in circadian rhythm pathways. The gene ontology (GO) and gene function analysis was performed to functionally annotate the genes. The structure of the genes and proteins were also compared among other Citrus species to study the evolutionary relationship among them. The expression study revealed the expression of flowering genes in floral buds and ovaries. The qRT-PCR analysis revealed that the flowering genes were highly expressed in bud stage, fully grown flower and early stage of fruit development. CONCLUSIONS: The findings suggested that the flowering genes were highly conserved in citrus species. The qRT-PCR analysis revealed the tissue specific expression of flowering genes (CsFT, CsCO, CsSOC, CsAP, CsSEP and CsLFY) which would help in easy detection and targeting of genes through various forward and reverse genetic approaches.

Transcriptomic and targeted metabolome analyses revealed the regulatory mechanisms of the synthesis of bioactive compounds in Citrus grandis 'tomentosa'.

Exocarpium Citri Grandis is a popular Chinese herbal medicine prepared from Citrus grandis 'tomentosa', and it is rich in several bioactive compounds, including flavonoids, coumarins, and volatile oils. However, studies are yet to elucidate the mechanisms of synthesis and regulation of these active components. Therefore, the present study examined the profiles of flavonoids and volatile oil bioactive compounds in plant petals, fruits, and tender leaves, and then performed RNA sequencing on different tissues to identify putative genes involved in the synthesis of bioactive compounds. The results show that the naringin, naringenin, and coumarin contents of the fruitlets were significantly higher than those of the tender leaves and petals, whereas the tender leaves had significantly higher levels of rhoifolin and apigenin. A total of 49 volatile oils, of which 10 were mainly found in flowers, 15 were mainly found in fruits, and 18 were mainly found in leaves, were identified. RNA sequencing identified 9,942 genes that were differentially expressed in different tissues. Further analysis showed that 20, 15, and 74 differentially expressed genes were involved in regulating flavonoid synthesis, regulating coumarin synthesis, and synthesis and regulation of terpenoids, respectively. CHI1 (Cg7g005600) and 1,2Rhat gene (Cg1g023820) may be involved in the regulation of naringin synthesis in C. grandis fruits. The HDR (Cg8g006150) gene, HMGS gene (Cg5g009630) and GGPS (Cg1g003650) may be involved in the regulation and synthesis of volatile oils in C. grandis petals. Overall, the findings of the present study enhance our understanding of the regulatory mechanisms of secondary metabolites in C. grandis, which could promote the breeding of C. grandis with desired characteristics.

Hub Genes and Pathways Related to Lemon (Citrus limon) Leaf Response to Plenodomus tracheiphilus Infection and Influenced by Pseudomonas mediterranea Biocontrol Activity.

The lemon industry in the Mediterranean basin is strongly threatened by "mal secco" disease (MSD) caused by the fungus Plenodomus tracheiphlilus. Leaf pretreatments with Pseudomonas mediterranea 3C have been proposed as innovative tools for eco-sustainable interventions aimed at controlling the disease. In this study, by exploiting the results of previously performed RNAseq analysis, WCGNA was conducted among gene expression patterns in both inoculated (Pt) and pretreated and fungus-inoculated lemon plants (Citrus limon L.) (3CPt), and two indicators of fungal infection, i.e., the amount of fungus DNA measured in planta and the disease index (DI). The aims of this work were (a) to identify gene modules significantly associated with those traits, (b) to construct co-expression networks related to mal secco disease; (c) to define the effect and action mechanisms of P. mediterranea by comparing the networks. The results led to the identification of nine hub genes in the networks, with three of them belonging to receptor-like kinases (RLK), such as HERK1, CLAVATA1 and LRR, which play crucial roles in plant-pathogen interaction. Moreover, the comparison between networks indicated that the expression of those receptors is not induced in the presence of P. mediterranea, suggesting how powerful WCGNA is in discovering crucial genes that must undergo further investigation and be eventually knocked out.

Transcription factor CrWRKY42 coregulates chlorophyll degradation and carotenoid biosynthesis in citrus.

Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for the coloration and nutritional value of fruits. However, the synergistic regulation of these 2 processes at the transcriptional level remains largely unknown. In this study, we identified a WRKY transcription factor, CrWRKY42, from the transcriptome data of the yellowish bud mutant "Jinlegan" ([Citrus unshiu × C. sinensis] × C. reticulata) tangor and its wild-type "Shiranui" tangor, which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 directly bound to the promoter of ß-carotene hydroxylase 1 (CrBCH1) and activated its expression. The overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene ß-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could bind to the promoters of NONYELLOW COLORING (CrNYC) and STAY-GREEN (CrSGR) and activate their expression, thus promoting chlorophyll degradation. The overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expression of genes involved in both pathways. Our data revealed that CrWRKY42 acts as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening.

Molecular regulation of oil gland development and biosynthesis of essential oils in Citrus spp.

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis-regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

The CsAP2-09-CsWRKY25-CsRBOH2 cascade confers resistance against citrus bacterial canker by regulating ROS homeostasis.

Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc-inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2-09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2-09 targeted the promoter of the Xcc-inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2-09 knockdown. Moreover, CsWRKY25 was found to bind directly to W-box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2-09-CsWRKY25 transcriptionally reprograms CsRBOH2-mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2-09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC-resistant citrus varieties.

Diversity in storage age enables discrepancy in quality attributes and metabolic profile of Citrus grandis "Tomentosa" in China.

The folk proverb "the older, the better" is usually used to describe the quality of Citrus grandis "Tomentosa" (CGT) in China. In this study, CGT aged for 6-, 12-, 16-, and 19-years were collected for the investigation of infusion color, main bioactive components, antioxidant activity, metabolic composition, and pathway. The results found that infusion color, the total phenolic and flavonoid, and antioxidant activity of CGT were obviously changed by aging process. Through untargeted metabolomics, 55 critical metabolites were identified to in discrimination of CGT with different storage ages, mainly including phenylpropanoids, lipids, and organic oxygen compounds. Twenty compounds that showed good linear relationships with storage ages could be used for year prediction of CGT. Kyoto encyclopedia of genes and genomes enrichment pathway analysis uncovered important metabolic pathways related to the accumulation of naringin, kaempferol, and choline as well as the degradation of benzenoids, thus supporting that aged CGT might be more beneficial to health. Correlation analysis provided that some key metabolites with bitter taste and biological activity were involved in the darkening and reddening of CGT infusion during aging, and total phenolic and flavonoid were more strongly associated with the antioxidant activity of CGT. This study systematically revealed the quality changes and key metabolic pathways during CGT aging at first time. PRACTICAL APPLICATION: This study reveals the differences in quality attributes and metabolic profile between CGT with different storage ages, providing guidance for consumers' consumption, and also providing more scientific basis for the quality evaluation and improvement of CGT.