Most citrus species exhibit polyembryony, a mode of sporophytic apomixis that asexual embryos develop in the nucellus. Polyembryony can propagate clonal seedlings but impedes crossbreeding. A 21-nt novel miRNA, CitmiRn23, and target gene CitXS20, encoding a XS domain protein, antagonistically express between polyembryonic and monoembryonic (sexual reproductive) ovules. CitmiRn23 overexpression and RNA interference (RNAi) of CitXS20 was conducted in minicitrus (Fortunella hindsii). The protein interacted with CitXS20 was identified, and exogenous treatments validated the effect of H2O2 on polyembryony. CitmiRn23 overexpression and CitXS20 RNAi both repressed nucellar embryogenesis (NE) and thus increased monoembryonic seed rate and hybrid rate. CitXS20 interacted with CitCAT2 and impaired its catalase activity. In ovaries with downregulated CitXS20, catalase activity was upregulated, while H2O2 level was decreased, indicating that interaction with CitXS20 might retard CitCAT2 activity in H2O2 scavenging. H2O2 preferentially accumulated in polyembryonic ovules. The treatment with H2O2 increased polyembryony level, whereas treatment with diphenyliodonium chloride reduced H2O2 level and polyembryony level, and thus increased hybrid rate. The citrus-specific CitmiRn23-CitXS20 module regulates NE through CitCAT2-mediated modulation of H2O2 level in ovule, which provides insights into apomixis mechanisms and promising approaches to regulate polyembryony, and thus facilitates crossbreeding in citrus.

Somatic embryogenesis (SE) serves as an ideal model for studying plant cell totipotency and embryonic development mechanisms, yet the transcriptional and epigenetic regulation underlying this process remains poorly characterized. We found that citrus nucellar polyembryony (a form of apomixis) gene, RWP-RK domain-containing transcription factor (CitRWP) is highly expressed in embryogenic callus with strong SE capacity, and its expression is constantly upregulated during SE induction. Overexpression of CitRWP induced spontaneous somatic embryo formation in a citrus variety that is resistant to SE bypass exogenous inducer. Suppression of CitRWP expression by RNA interference (RNAi) impairs SE capacity. Integrated RNA-seq and Chromatin immunoprecipitation sequencing (ChIP-seq) analyses combined with molecular assays revealed that CitRWP directly binds to the DUO1-ACTIVATED ZINC FINGER 3 (CsDAZ3) promoter and activates its transcription. Overexpression of CsDAZ3 significantly enhanced SE capacity. Comparative analysis of ChIP-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) datasets demonstrated that CitRWP is capable of binding to regions of low chromatin accessibility. CitRWP binds to a condensed chromatin site in the CsDAZ3 promoter and promotes increased local chromatin accessibility at the CsDAZ3 locus. Our findings delineate a novel regulatory framework for citrus SE, highlighting that CitRWP coordinates transcriptional regulation with chromatin remodeling. This research provides insights into plant cell reprogramming and provides pivotal molecular targets for optimizing breeding protocols by modulating SE.

Citrus, a key medicinal and food crop, produces fruits rich in health-promoting secondary metabolites, among which coumarins represent an important class of bioactive components contributing to its nutritional and medicinal value. However, a systematic understanding of their accumulation and variation across citrus species remains limited. In this study, we performed targeted metabolomic profiling using LC-MS/MS to quantify 21 coumarin compounds in peel and flesh tissues from 121 citrus accessions spanning eight species. Our results revealed markedly higher total coumarin contents in pummelo, grapefruit, lemon and primitive varieties compared to loose-skin mandarin and sweet orange. This interspecific variation was largely driven by furanocoumarins such as oxypeucedanin and isoimperatorin in pummelo, whereas byakangelicol and byakangelicin primarily distinguished lemon from mandarin. Notably, pummelo exhibited profound tissue-specific coumarin accumulation, with the peel-to-flesh ratio of the simple coumarin osthol reaching up to 396-fold, while those of osthenol and aurapten were also considerably high. Wild and medicinal accessions, including Daoxianensis, Jiangyongensis, Mangshanensis, and Chachiensis, consistently showed elevated coumarin levels compared to cultivated counterparts within the same species. Furthermore, analysis of callus cultures from 19 varieties revealed a distinct coumarin distribution pattern, decoupled from that observed in mature fruits. This study clarifies the distribution and variation patterns of coumarins in citrus fruits, offering valuable insights for the targeted development of both dietary and therapeutic citrus resources.

Summary Most citrus species exhibit polyembryony, a mode of sporophytic apomixis that asexual embryos develop in the nucellus. Polyembryony can propagate clonal seedlings but impedes crossbreeding. A 21‐nt novel miRNA, CitmiRn23, and target gene CitXS20 , encoding a XS domain protein, antagonistically express between polyembryonic and monoembryonic (sexual reproductive) ovules. CitmiRn23 overexpression and RNA interference (RNAi) of CitXS20 was conducted in minicitrus ( Fortunella hindsii ). The protein interacted with CitXS20 was identified, and exogenous treatments validated the effect of H 2 O 2 on polyembryony. CitmiRn23 overexpression and CitXS20 RNAi both repressed nucellar embryogenesis (NE) and thus increased monoembryonic seed rate and hybrid rate. CitXS20 interacted with CitCAT2 and impaired its catalase activity. In ovaries with downregulated CitXS20 , catalase activity was upregulated, while H 2 O 2 level was decreased, indicating that interaction with CitXS20 might retard CitCAT2 activity in H 2 O 2 scavenging. H 2 O 2 preferentially accumulated in polyembryonic ovules. The treatment with H 2 O 2 increased polyembryony level, whereas treatment with diphenyliodonium chloride reduced H 2 O 2 level and polyembryony level, and thus increased hybrid rate. The citrus‐specific CitmiRn23‐ CitXS20 module regulates NE through CitCAT2‐mediated modulation of H 2 O 2 level in ovule, which provides insights into apomixis mechanisms and promising approaches to regulate polyembryony, and thus facilitates crossbreeding in citrus.

Plant growth-promoting rhizobacteria (PGPR) interact with host plants through chemical signals. However, the specific signals in citrus-PGPR interactions remain unclear. Here, we show that a predominant and growth-promoting Burkholderia strain (Burk_2H3) isolated from citrus rhizosphere promotes plant growth by secreting N-(3-oxo-octanoyl)-L-homoserine lactone (PGPHL). Metabolomic analysis revealed that PGPHL abundance in Burk_2H3 secretions was 9.7- to 17.2-fold higher than that in three non-promoting Burkholderia strains. Exogenous application of PGPHL, but not other secretory metabolites, increased citrus seedling dry weight by 43.12%. Transcriptomic analysis showed that Burk_2H3, its cell-free supernatant, or PGPHL consistently upregulated key nutrient transporter genes in roots. Consistently, ionomic analysis confirmed higher root concentrations of nitrogen, phosphorus, and potassium. Field trials further demonstrated that PGPHL increased biomass by 21% in pepper, 15% in celery, and 18% in mustard. Together, these findings identify PGPHL as a candidate for developing plant growth stimulants and biofertilizers.

Trichomes develop as outward projections from the epidermal surface. By contrast, as subepidermal secretory cavities, citrus oil glands originate from epidermal cells and develop into specialized hollow structures. During the development of these epidermis-derived structures in vascular plants, transcription factors, such as the homeodomain-leucine zipper and APETALA2/Ethylene responsive factor families, regulate their initiation and morphogenesis. Subsequent biosynthesis of secondary metabolites within these secretory structures is often mediated by jasmonic acid signaling and basic helix-loop-helix proteins, particularly MYC transcription factors. Here, we compare the regulatory mechanisms governing the development of glandular trichomes and secretory cavities, along with the biosynthesis of secondary metabolites. These insights provide the basic knowledge for harnessing these secretory structures as chassis in synthetic biology applications.

Ripening of kiwifruit (Actinidia spp.) is highly sensitive to ethylene, but reliance on exogenous ethylene often results in over-softening, greatly reducing shelf life. Here, we discovered a pathway induced by cool temperature (CT; 5°C-10°C) that directly orchestrates starch-to-sugar conversion in kiwifruit under conditions in which ethylene perception is inhibited by 1-methylcyclopropene. Through transcriptomic and metabolomic profiling, we identified AcBAM3.3, a β-amylase gene that is specifically induced by CT but not by ambient temperature. A CT-inducible ERF transcription factor, AcCTS1 (CT-specific factor 1), was found to directly bind the promoters of AcBAM3.3 and AcBAM3.5 and activate their transcription, as confirmed by dual-luciferase, electrophoretic mobility shift, and yeast one-hybrid assays. We also identified an E3 ubiquitin ligase, AcPUB11, which targets AcCTS1 for 26S proteasomal degradation, repressing starch degradation at room temperature. Under CT, reduced AcPUB11 abundance allows for AcCTS1 accumulation, driving AcBAM3.3 and AcBAM3.5 expression and promoting ripening. Functional characterization via overexpression, RNAi, and CRISPR-Cas9 in both callus and fruit confirmed the AcPUB11-AcCTS1-AcBAM3s module as the central regulator of CT-induced starch metabolism. Our findings define a ubiquitination-controlled transcriptional regulatory module that mediates fruit adaptation to cool environments, providing a mechanistic foundation for temperature-controlled starch degradation during ripening.

Citric acid is the major organic acid affecting citrus fruit taste, which varies widely in the citrus family. An acidless kumquat, a small-fruited citrus species (Citrus crassifolia), has been developed and has become popular on the market. To investigate the molecular basis of the acidless phenotype in Huapi (HP) kumquat, it was crossed with early-flowering, high-acid Hong Kong (HK) kumquat to generate an F1 population, providing a system to dissect the genetic and molecular basis of citric acid accumulation. Organic acid content exhibited a wide and continuous distribution across three consecutive years, which is consistent with quantitative inheritance. Bulked segregant analysis sequencing mapped a major locus, and integration with transcriptomic data identified the GRAS transcription factor SCARECROW-like protein 9 (ChSCL9) as a candidate major-effect gene, showing high expression in HP fruit and low expression in HK fruit. Subcellular localization confirmed that ChSCL9 is a transcription factor. Functional analyses-including CRISPR-Cas9 gene editing, overexpression in kumquat, and RNA interference (RNAi) in citrus juice sacs-demonstrated that ChSCL9 negatively regulates citric acid accumulation. Biochemical experiments showed that ChSCL9 directly represses the expression of the R2R3-MYB gene ChPH4 and the vacuolar P-ATPase gene ChPH5, both of which are key genes for vacuolar acidification, thereby inhibiting citric acid accumulation. These results identify ChSCL9 as a key regulator of citric acid in kumquat, reveal an upstream regulator of PH4, and provide targets for citrus flavor improvement and rational design for citrus breeding.

Rising global temperature threatens fruit quality. As one of the most economically valuable fruits worldwide, citrus suffers from high-temperature-induced chlorophyll retention during ripening, significantly reducing commercial value. Comparative physiological analyses revealed that citrus fruit stored at 30°C exhibited markedly impaired chlorophyll degradation and failed to undergo normal degreening, in stark contrast to those maintained at 20°C. This temperature-dependent suppression of degreening was closely associated with a significant downregulation of key chlorophyll catabolic genes (CCGs). By integrating weighted gene co-expression network analysis (WGCNA) of a stay-green mutant, we identified the transcription factor CsbHLH60, a nuclear-localized transcriptional activator, from a chlorophyll-associated module. CsbHLH60 directly binds E-box motifs in the promoters of CsSGR and CsRCCR to drive their expression, a finding validated by ChIP-qPCR and EMSA assays. Transient overexpression of CsbHLH60 in citrus peel accelerated chlorophyll degradation and, importantly, overcame high-temperature-induced repression to restore degreening, whereas RNAi-mediated silencing at 20°C significantly impeded normal degreening of the peel. Mechanistically, high temperature imposes a dual "double-lock" suppression on CsbHLH60, concurrently repressing its transcription and promoting its protein degradation, thereby depleting functional activator levels. Collectively, our findings establish CsbHLH60 as a pivotal temperature-responsive regulator that directly couples temperature signaling to chlorophyll catabolism and is capable of overriding the high-temperature-induced suppression of chlorophyll degradation, thereby highlighting its potential as a molecular target for mitigating climate-driven declines in citrus fruit appearance quality.

Abstract Citric acid and anthocyanins are key metabolites that determine fruit flavor and coloration. Although the accumulation mechanisms of citric acid and anthocyanins have been known in citrus, little is known about the cross-regulatory mechanism between the two important metabolites. In this study, we identified a purple wampee (Clausena lansium) that exhibits simultaneous accumulation of citric acid and anthocyanins in fruit. Functional analyses confirmed that the ClPH4 positively regulate citric acid accumulation, however, the homologous gene of Ruby1, the activator of anthocyanin biosynthesis, has lost the capacity to induce anthocyanin accumulation in the purple wampee. We subsequently identified a PH4-Like based on gene family and expression analyses. Stable overexpression of ClPH4-like in citrus, along with transient silencing in purple wampee pulp, demonstrated that it can simultaneously induce both anthocyanin and citric acid accumulation. Moreover, PH4-Like is specifically and highly expressed in purple wampee, consistent with the observation that its promoter activity is much higher in purple wampee than in other citrus species. Biochemical assays showed that PH4-Like binds to the promoters of Flavonoid 3’-Hydroxylase (F3’H), Dihydroflavonol 4-reductase (DFR), Anthocyanidin synthase gene (ANS) and the proton pump gene PH5 and VHA-a2, the key genes in the anthocyanin and citric acid accumulation. Together, our findings uncover a key gene that coordinately regulates citric acid and anthocyanins accumulation, providing insight into the genetic events for the diversification of fruit color and tastes in Aurantioideae, and offering valuable targets for simultaneous improvement of fruit quality.

Somatic mutations are widespread in all organisms and cause abundant phenotypic changes, constituting an important source of variation especially for clonally propagated plants. Somatic mutants can serve as a mutation-introducing method for exploring the regulatory mechanism of mutated trait formation in clonally propagated plants. Red-fleshed and orange-fleshed pomelo varieties derive from independent mutations of the wild-type white-fleshed Guanxi honey pomelo (Citrus grandis), offering an ideal system for dissecting core regulatory transcription factors behind the fruit color trait. Here, we performed a meta-analysis comprising genome, transcriptome, DNA methylome and chromatin accessibility assays of the two color mutants and their wild type, resulting in haplotype-scaled pan-genome. The chromatin openness analysis suggested that binding sites for TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors are significantly enriched in the differentially open regions, with different footprint patterns in the mutants and wild type. We identified CgTCP3, CgTCP7, and CgTCP20 as regulators of key carotenogenic genes including ZDS, BCH, and NCED2. Furthermore, CgTCP7 underwent positive selection in pomelo germplasm, with various alleles differentially affecting carotenogenic gene expression and modulating carotenoid accumulation. We therefore identified the core transcription factor genes responsible for color trait formation, providing resources for breeding varieties with diversified phenotypes.

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), severely threatens citrus production. Here, we identify Sec-delivered effector 10 (SDE10), a key effector from CLas, whose ectopic expression in citrus mimics HLB symptoms. Genetic and biochemical analyses demonstrate that SDE10 suppresses salicylic acid (SA)-mediated defense by inhibiting xylem cysteine peptidase 2 (CsXCP2). CsXCP2 positively regulates SA homeostasis through two activity-dependent mechanisms. First, it processes pathogenesis-related 1 (CsPR1) to generate cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP)-derived peptide 9 (CsCAPE9), which activates SA-mediated defense and significantly reduces CLas titers under both greenhouse and field conditions. Second, it degrades the CLas-derived SA hydroxylase (SahA), thereby preventing SA signaling disruption. Notably, engineered CsXCP2 variants that evade SDE10 binding while retaining moderate activity can counteract SDE10-mediated defense suppression. Collectively, our findings delineate a sophisticated molecular arms race between CLas virulence strategies and citrus immune countermeasures centered on SA signaling modulation, providing a mechanistic foundation for breeding HLB-resistant citrus varieties.

Fruit quality is shaped by both crop genetics and cultivation environments, with soil conditions driving rhizosphere microbiome assembly. While rhizosphere microbes are known to enhance nutrient utilization and plant metabolism, their direct contribution to fruit quality regulation remains poorly understood. In this study, we demonstrate that the Satsuma mandarin (Citrus unshiu Marc.) and Navel orange (Citrus sinensis L. Osbeck) rhizosphere microbiome influence fruit sugar concentration, a key determinant of fruit quality. The rhizosphere core microbiota and soil mineral nutrients were positively correlated with fruit quality indices. Fruit quality-correlated bacterial operational taxonomic units (OTUs) explained an average of 32.6% of the observed variation in quality parameters. Inoculation with three bacterial strains (affiliated with Burkholderia, Pseudomonas, Rhizobium) and two bacterial consortia significantly increased fruit sugar concentrations. Metagenomic analysis linked sugar-associated microbes to iron (Fe) utilization, revealing genomic enrichment of siderophore biosynthesis gene clusters. Consistently, the selected bacterial strains exhibited siderophore secretion capabilities, increased leaf Fe content by 23.3-47.8% in citrus rootstock. Further field application of chelated-Fe fertilizer also increased fruit sugar concentration. Collectively, our results revealed an influence of the rhizosphere microbiome on fruit quality that is related to Fe acquisition optimization and subsequent sugar accumulation in citrus.

Very-long-chain fatty acids (VLCFAs) are essential building blocks for sphingolipids, phospholipids, triacylglycerols, suberin, and cuticular waxes, all of which play crucial roles in plant development and environmental adaptation. The elongation of VLCFAs is governed by plant-specific β-ketoacyl-CoA synthase (KCS) enzymes and ECERIFERUM2 (CER2) family proteins. However, the structural catalytic mechanisms underlying VLCFA elongation remain poorly understood. Here, we present structural snapshots of the KCS6-CER2 complex from the evolutionarily representative land vascular plant Selaginella moellendorffii in multiple elongation reaction states. These structures delineate how the acyl chain of the substrate C22:0 acyl-CoA (behenoyl-CoA), the two-carbon donor malonyl-CoA, and the product C24:0 β-ketoacyl-CoA (3-oxo-lignoceroyl-CoA) sequentially engage the subtrate binding channel of the KCS6-CER2 complex on the cytosolic side of the ER membrane. We further identify a previously unrecognized side channel that accommodates an additional acyl-CoA molecule, indicating an unexpected structural feature for VLCFA biosynthesis. In addition, structural and biochemical analyses of the KCS6-CER2 complex reveal that CER2 acts as a structural cofactor, extending the hydrophobic subtrate binding channel of KCS6.Together, these findings uncover the molecular mechanism by which KCS6 and CER2 collaborate to drive chain-length-specific VLCFA elongation and provide a structural framework for understanding and engineering VLCFA elongation across the plant kingdom.

Postharvest wax coating reduces water loss and enhances visual appeal but impairs gas exchange in citrus fruit, inducing anaerobic fermentation and off-flavor development. This study demonstrates that exogenous gamma-aminobutyric acid (GABA) mitigates these physiological disorders. GABA enhanced peel gas permeance by 1.7- to 2.2-fold through suppression of C25-C31 cuticular alkane biosynthesis and enlargement of stomatal apertures, thereby alleviating internal hypoxia. This metabolic shift from anaerobic fermentation toward aerobic respiration was mediated by GABA shunt reprogramming and amino acid metabolic remodeling, which reduced ethanol accumulation by 57% while preserving key flavor compounds. Transcriptomic analysis revealed coordinated regulation of genes associated with wax synthesis, fermentation, and hypoxia responses. This simple amino acid treatment preserves citrus fruit flavor and nutritional quality under waxing-induced hypoxia by mitigating physiological disorders.

The extreme phenotypic diversity in fruit coloration across the Citrinae subtribe poses a fascinating evolutionary puzzle. While carotenoids are essential for plant ecological interactions and human nutrition, the genomic basis underlying their vast metabolic variation in the context of complex species hybridization remains largely elusive. Here, we integrated whole-genome resequencing of 80 representative Citrinae accessions with high-resolution metabolic and transcriptional profiling to elucidate the evolutionary and genetic architectures of citrus fruit coloration. Population genomic modeling demonstrated that pervasive interspecific hybridization and multi-stage introgression from primary progenitors (C. reticulata, C. maxima, and C. medica) profoundly reshaped the mosaic metabolic landscapes of modern hybrid taxa (C. sinensis, C. aurantium, and C. limon). We uncovered a remarkable tissue-specific decoupling of metabolic flux: CitLCYB2 acts as the primary bottleneck limiting β-branch flux in the flavedo (peel), coupled with massive CitNCED2-mediated degradation in pummelos, whereas CitPSY1 serves as the core rate-limiting enzyme dictating source-driven accumulation in the pulp. Transgenic validation in vivo using a woody plant callus system directly confirmed the pivotal role of CitPSY1, yielding a > 10-fold surge in carotenoid content. Furthermore, by integrating high-resolution local ancestry inference with demography-robust genome-wide scans (CLR, iHS, and XP-CLR), we elucidated the divergent evolutionary trajectories of CitPSY1. We revealed that the ancestral mandarin lineage (C. reticulata) experienced a strong, targeted regulatory selective sweep to rapidly fix a highly active promoter haplotype for intense pigmentation, whereas pummelos and citrons retained basal metabolic capacities shaped predominantly by natural demographic history. Our findings provide a comprehensive paradigm for how historical gene flow and divergent selection orchestrate secondary metabolism in woody perennials, offering premier genetic targets for the nutritional biofortification of commercial fruits.

Salt stress severely limits global crop productivity. Poncirus trifoliata, a widely used citrus rootstock valued for its disease resistance, exhibits sensitivity to salt stress, presenting a major constraint for sustainable citriculture. A stepwise screening strategy identified PtWRKY20 as a key negative regulator of salt tolerance in P. trifoliata. Transcript analysis revealed that PtWRKY20 expression is rapidly induced within 1 h of exposure to salt stress, peaks at 3 h, and then gradually declines from 6 to 24 h, before dropping below the control level by 48 h. Subcellular localization confirmed that PtWRKY20 is a nuclear-localized transcription factor. Functional validation via CRISPR/Cas9-mediated knockout in hairy roots demonstrated enhanced salt tolerance in Ptwrky20-KO plants, evidenced by reduced chlorosis, higher chlorophyll content, lower electrolyte leakage, and decreased malondialdehyde accumulation under salt stress. Conversely, PtWRKY20 overexpression increased sensitivity, showing opposite physiological trends. Integrated DNA affinity purification sequencing and RNA-seq analyses, validated by yeast 1-hybrid and dual-luciferase assays, identified 10 direct target genes bound and transcriptionally regulated by PtWRKY20, including 6 transcription factor genes (eg PtMYB78), a sugar metabolism gene (PtHXK3), an ion homeostasis mediator (PtAMT2), and 2 unannotated genes. Our findings establish PtWRKY20 as a negative regulator of the salt stress response and suggest that its targeted knockout via rootstock-adapted CRISPR is a promising strategy for developing salt-tolerant citrus rootstocks.

Citrus huanglongbing (HLB), caused by "Candidatus Liberibacter" spp., is one of the most disastrous citrus diseases worldwide. HLB-affected citrus fruits are significantly more acidic than healthy fruits. However, the molecular mechanism behind this phenomenon remains to be elucidated. Here, we report that HLB-affected fruits have higher levels of citric acid (CA) than healthy fruits. Moreover, Citrus PH4 (CitPH4), which encodes a MYB transcription factor that functions as a key regulator of CA accumulation, was upregulated in HLB-affected fruits relative to healthy fruits. Heterologous overexpression of CitPH4 in tobacco (Nicotiana tabacum) plants enhanced tolerance to HLB. Subsequently, overexpression and gene-editing experiments indicated that CitPH4 can affect the salicylic acid (SA) pathway, which directly binds to and activates the promoter of CsPBS3, a key gene of SA biosynthesis. HLB-affected fruits had higher SA levels than healthy fruits. Furthermore, application of SA activated CA biosynthesis and application of CA activated SA biosynthesis and signalling in citrus fruits and decreased "Candidatus Liberibacter asiaticus" (CLas) titres in infected leaves. This work suggests that CitPH4 is a key node between CA and SA, thus revealing crosstalk between defence responses and fruit quality in citrus.