Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

Jasmonate activates a CsMPK6-CsMYC2 module that regulates the expression of ß-citraurin biosynthetic genes and fruit coloration in orange (Citrus sinensis).

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid ß-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 ß-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and ß-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 ß-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene ß-cyclase (CsLCYb), and ß-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 ß-citraurin biosynthesis in citrus by activating a CsMPK6-CsMYC2 cascade, thereby affecting fruit coloration.

Genomic conservation of crop wild relatives: A case study of citrus.

Conservation of crop wild relatives is critical for plant breeding and food security. The lack of clarity on the genetic factors that lead to endangered status or extinction create difficulties when attempting to develop concrete recommendations for conserving a citrus wild relative: the wild relatives of crops. Here, we evaluate the conservation of wild kumquat (Fortunella hindsii) using genomic, geographical, environmental, and phenotypic data, and forward simulations. Genome resequencing data from 73 accessions from the Fortunella genus were combined to investigate population structure, demography, inbreeding, introgression, and genetic load. Population structure was correlated with reproductive type (i.e., sexual and apomictic) and with a significant differentiation within the sexually reproducing population. The effective population size for one of the sexually reproducing subpopulations has recently declined to ~1,000, resulting in high levels of inbreeding. In particular, we found that 58% of the ecological niche overlapped between wild and cultivated populations and that there was extensive introgression into wild samples from cultivated populations. Interestingly, the introgression pattern and accumulation of genetic load may be influenced by the type of reproduction. In wild apomictic samples, the introgressed regions were primarily heterozygous, and genome-wide deleterious variants were hidden in the heterozygous state. In contrast, wild sexually reproducing samples carried a higher recessive deleterious burden. Furthermore, we also found that sexually reproducing samples were self-incompatible, which prevented the reduction of genetic diversity by selfing. Our population genomic analyses provide specific recommendations for distinct reproductive types and monitoring during conservation. This study highlights the genomic landscape of a wild relative of citrus and provides recommendations for the conservation of crop wild relatives.

Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non-photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty-four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG-localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi-feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops.

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.

Adventitious embryonic causal gene FhRWP regulates multiple developmental phenotypes in citrus reproduction.

Citrus is a model plant for studying adventitious embryos, a form of asexual reproduction controlled by a single dominant gene, RWP. This gene has been identified as the causal gene for nucellar embryogenesis, but its function has not yet been fully understood. In this study, we used the fast-growing Fortunella hindsii as a system to explore chromatin accessibility during the nucellar embryony initiation, emphasizing elevated chromatin accessibility in polyembryonic (PO) genotypes compared to monoembryonic ones (MO). Notably, a higher level of accessible chromatin was observed in one allele of the promoter region of FhRWP, consistent with increased expression of the allele carrying the causal structural variant. By independently performing RNAi and gene editing experiments on PO genotypes, we found the downregulation of FhRWP expression could reduce the number of nucellar embryos, while its knockout resulted in abnormal axillary bud development. In overexpression experiments, FhRWP was identified as having the unique capability of inducing the embryogenic callus formation in MO stem segments, possibly through the regulation of the WUS-CLV signaling network and the ABA and cytokinin pathway, marking the inaugural demonstration of FhRWP's potential to reignite somatic cells' embryogenic fate. This study reveals the pleiotropic function of RWP in citrus and constructs a regulatory network during adventitious embryo formation, providing a new tool for bioengineering applications in plant regeneration.

The transcription factor ERF110 promotes cold tolerance by directly regulating sugar and sterol biosynthesis in citrus.

ERFs (ethylene-responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold-induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold-hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress-responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6-like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP-glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110-overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110-silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110-ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress.

A C2H2-type zinc finger protein ZAT12 of Poncirus trifoliata acts downstream of CBF1 to regulate cold tolerance.

The Cys2/His2 (C2H2)-type zinc finger family has been reported to regulate multiple aspects of plant development and abiotic stress response. However, the role of C2H2-type zinc finger proteins in cold tolerance remains largely unclear. Through RNA-sequence analysis, a cold-responsive zinc finger protein, named as PtrZAT12, was identified and isolated from trifoliate orange (Poncirus trifoliata L. Raf.), a cold-hardy plant closely related to citrus. Furthermore, we found that PtrZAT12 was markedly induced by various abiotic stresses, especially cold stress. PtrZAT12 is a nuclear protein, and physiological analysis suggests that overexpression of PtrZAT12 conferred enhanced cold tolerance in transgenic tobacco (Nicotiana tabacum) plants, while knockdown of PtrZAT12 by virus-induced gene silencing (VIGS) increased the cold sensitivity of trifoliate orange and repressed expression of genes involved in stress tolerance. The promoter of PtrZAT12 harbors a DRE/CRT cis-acting element, which was verified to be specifically bound by PtrCBF1 (Poncirus trifoliata C-repeat BINDING FACTOR1). VIGS-mediated silencing of PtrCBF1 reduced the relative expression levels of PtrZAT12 and decreased the cold resistance of trifoliate orange. Based on these results, we propose that PtrZAT12 is a direct target of CBF1 and plays a positive role in modulation of cold stress tolerance. The knowledge gains new insight into a regulatory module composed of CBF1-ZAT12 in response to cold stress and advances our understanding of cold stress response in plants.

The R2R3 MYB Ruby1 is activated by two cold responsive ethylene response factors, via the retrotransposon in its promoter, to positively regulate anthocyanin biosynthesis in citrus.

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold-induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT-induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis-element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co-activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange.

Phylogenomic analysis of 343 Xanthomonas citri pv. citri strains unravels introduction history and dispersal paths.

Xanthomonas citri pv. citri (Xcc) causes the devastating citrus canker disease. Xcc is known to have been introduced into Florida, USA in at least three different events in 1915, 1986 and 1995 with the first two claimed to be eradicated. It was questioned whether the Xcc introduction in 1986 has been successfully eradicated. Furthermore, it is unknown how Xcc has spread throughout the citrus groves in Florida. In this study, we investigated the population structure of Xcc to address these questions. We sequenced the whole genome of 343 Xcc strains collected from Florida groves between 1997 and 2016. Our analysis revealed two distinct clusters of Xcc. Our data strongly indicate that the claimed eradication of the 1986 Xcc introduction was not successful and Xcc strains from 1986 introduction were present in samples from at least 8 counties collected after 1994. Importantly, our data revealed that the Cluster 2 strains, which are present in all 20 citrus-producing counties sampled in Florida, originated from the Xcc introduction event in the Miami area in 1995. Our data suggest that Polk County is the epicenter of the dispersal of Cluster 2 Xcc strains, which is consistent with the fact that three major hurricanes passed through Polk County in 2004. As copper-based products have been extensively used to control citrus canker, we also investigated whether Xcc strains have developed resistance to copper. Notably, none of the 343 strains contained known copper resistance genes. Twenty randomly selected Xcc strains displayed sensitivity to copper. Overall, this study provides valuable insights into the introduction, eradication, spread, and copper resistance of Xcc in Florida.

Transcription factor CsMYB77 negatively regulates fruit ripening and fruit size in citrus.

MYB family transcription factors (TFs) play essential roles in various biological processes, yet their involvement in regulating fruit ripening and fruit size in citrus remains poorly understood. In this study, we have established that the R2R3-MYB TF, CsMYB77, exerts a negative regulatory influence on fruit ripening in both citrus and tomato (Solanum lycopersicum), while also playing a role in modulating fruit size in citrus. The overexpression of CsMYB77 in tomato and Hongkong kumquat (Fortunella hindsii) led to notably delayed fruit ripening phenotypes. Moreover, the fruit size of Hongkong kumquat transgenic lines was largely reduced. Based on DNA affinity purification sequencing and verified interaction assays, SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA4 (SINAT4) and PIN-FORMED PROTEIN5 (PIN5) were identified as downstream target genes of CsMYB77. CsMYB77 inhibited the expression of SINAT4 to modulate abscisic acid (ABA) signaling, which delayed fruit ripening in transgenic tomato and Hongkong kumquat lines. The expression of PIN5 was activated by CsMYB77, which promoted free indole-3-acetic acid decline and modulated auxin signaling in the fruits of transgenic Hongkong kumquat lines. Taken together, our findings revealed a fruit development and ripening regulation module (MYB77-SINAT4/PIN5-ABA/auxin) in citrus, which enriches the understanding of the molecular regulatory network underlying fruit ripening and size.

Transcriptome and metabolome atlas reveals contributions of sphingosine and chlorogenic acid to cold tolerance in Citrus.

Citrus is one of the most important fruit crop genera in the world, but many Citrus species are vulnerable to cold stress. Ichang papeda (Citrus ichangensis), a cold-hardy citrus species, holds great potential for identifying valuable metabolites that are critical for cold tolerance in Citrus. However, the metabolic changes and underlying mechanisms that regulate Ichang papeda cold tolerance remain largely unknown. In this study, we compared the metabolomes and transcriptomes of Ichang papeda and HB pummelo (Citrus grandis "Hirado Buntan", a cold-sensitive species) to explore the critical metabolites and genes responsible for cold tolerance. Metabolomic analyses led to the identification of common and genotype-specific metabolites, consistent with transcriptomic alterations. Compared to HB pummelo under cold stress, Ichang papeda accumulated more sugars, flavonoids, and unsaturated fatty acids, which are well-characterized metabolites involved in stress responses. Interestingly, sphingosine and chlorogenic acid substantially accumulated only in Ichang papeda. Knockdown of CiSPT (C. ichangensis serine palmitoyltransferase) and CiHCT2 (C. ichangensis hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase2), two genes involved in sphingosine and chlorogenic acid biosynthesis, dramatically decreased endogenous sphingosine and chlorogenic acid levels, respectively. This reduction in sphingosine and chlorogenic acid notably compromised the cold tolerance of Ichang papeda, whereas exogenous application of these metabolites increased plant cold tolerance. Taken together, our findings indicate that greater accumulation of a spectrum of metabolites, particularly sphingosine and chlorogenic acid, promotes cold tolerance in cold-tolerant citrus species. These findings broaden our understanding of plant metabolic alterations in response to cold stress and provide valuable targets that can be manipulated to improve Citrus cold tolerance.

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.

Myo-inositol oxygenase CgMIOX3 alleviates S-RNase-induced inhibition of incompatible pollen tubes in pummelo.

Pummelo (Citrus grandis L. Osbeck) exhibits S-RNase-based self-incompatibility (SI), during which S-RNase cytotoxicity inhibits pollen tubes in an S-haplotype-specific manner. The entry of S-RNase into self-pollen tubes triggers a series of reactions. However, these reactions are still poorly understood in pummelo. In the present study, we used S-RNases as baits to screen a pummelo pollen cDNA library and characterized a myo-inositol oxygenase (CgMIOX3) that physically interacts with S-RNases. CgMIOX3 is highly expressed in pummelo pollen tubes, and its downregulation leads to a reduction in pollen tube growth. Upon entering pollen tubes, S-RNases increase the expression of CgMIOX3 and enhance its activity by directly binding to it in an S-haplotype-independent manner. CgMIOX3 improves pollen tube growth under oxidative stress through ascorbic acid (AsA) accumulation and increases the length of self-pollen tubes. Furthermore, over-expression of CgMIOX3 increases the relative length of self-pollen tubes growing in the style of petunia (Petunia hybrida). This study provides intriguing insights into the pumelo SI system, revealing a regulatory mechanism mediated by CgMIOX3 that plays an important role in the resistance of pollen tubes to S-RNase cytotoxicity.

ACC SYNTHASE4 inhibits gibberellin biosynthesis and FLOWERING LOCUS T expression during citrus flowering.

Flowering is an essential process in fruit trees. Flower number and timing have a substantial impact on the yield and maturity of fruit. Ethylene and gibberellin (GA) play vital roles in flowering, but the mechanism of coordinated regulation of flowering in woody plants by GA and ethylene is still unclear. In this study, a lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylic acid synthase gene (CiACS4) was overexpressed in Nicotiana tabacum and resulted in late flowering and increased flower number. Further transformation of citrus revealed that ethylene and starch content increased, and soluble sugar content decreased in 35S:CiACS4 lemon. Inhibition of CiACS4 in lemon resulted in effects opposite to that of 35S:CiACS4 in transgenic plants. Overexpression of the CiACS4-interacting protein ETHYLENE RESPONSE FACTOR3 (CiERF3) in N. tabacum resulted in delayed flowering and more flowers. Further experiments revealed that the CiACS4-CiERF3 complex can bind the promoters of FLOWERING LOCUS T (CiFT) and GOLDEN2-LIKE (CiFE) and suppress their expression. Moreover, overexpression of CiFE in N. tabacum led to early flowering and decreased flowers, and ethylene, starch, and soluble sugar contents were opposite to those in 35S:CiACS4 transgenic plants. Interestingly, CiFE also bound the promoter of CiFT. Additionally, GA3 and 1-aminocyclopropanecarboxylic acid (ACC) treatments delayed flowering in adult citrus, and treatment with GA and ethylene inhibitors increased flower number. ACC treatment also inhibited the expression of CiFT and CiFE. This study provides a theoretical basis for the application of ethylene to regulate flower number and mitigate the impacts of extreme weather on citrus yield due to delayed flowering.

Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification.

Although interactions between the cytoplasmic and nuclear genomes occurred during diversification of many plants, the evolutionary conflicts due to cytonuclear interactions are poorly understood in crop breeding. Here, we constructed a pan-mitogenome and identified chimeric open reading frames (ORFs) generated by extensive structural variations (SVs). Meanwhile, short reads from 184 accessions of citrus species were combined to construct three variation maps for the nuclear, mitochondrial, and chloroplast genomes. The population genomic data showed discordant topologies between the cytoplasmic and nuclear genomes because of differences in mutation rates and levels of heteroplasmy from paternal leakage. An analysis of species-specific SVs indicated that mitochondrial heteroplasmy was common and that chloroplast heteroplasmy was undetectable. Interestingly, we found a prominent divergence in the mitogenomes and the highest genetic load in the, which may provide the basis for cytoplasmic male sterility (CMS) and thus influence the reshuffling of the cytoplasmic and nuclear genomes during hybridization. Using cytoplasmic replacement experiments, we identified a type of species-specific CMS in mandarin related to two chimeric mitochondrial genes. Our analyses indicate that cytoplasmic genomes from mandarin have rarely been maintained in hybrids and that paternal leakage produced very low levels of mitochondrial heteroplasmy in mandarin. A genome-wide association study (GWAS) provided evidence for three nuclear genes that encode pentatricopeptide repeat (PPR) proteins contributing to the cytonuclear interactions in the Citrus genus. Our study demonstrates the occurrence of evolutionary conflicts between cytoplasmic and nuclear genomes in citrus and has important implications for genetics and breeding.

Laser capture microdissection-based spatiotemporal transcriptomes uncover regulatory networks during seed abortion in seedless Ponkan (Citrus reticulata).

Seed abortion is an important process in the formation of seedless characteristics in citrus fruits. However, the molecular regulatory mechanism underlying citrus seed abortion is poorly understood. Laser capture microdissection-based RNA-seq combined with Pacbio-seq was used to profile seed development in the Ponkan cultivars 'Huagan No. 4' (seedless Ponkan) (Citrus reticulata) and 'E'gan No. 1' (seeded Ponkan) (C. reticulata) in two types of seed tissue across three developmental stages. Through comparative transcriptome and dynamic phytohormone analyses, plant hormone signal, cell division and nutrient metabolism-related processes were revealed to play critical roles in the seed abortion of 'Huagan No. 4'. Moreover, several genes may play indispensable roles in seed abortion of 'Huagan No. 4', such as CrWRKY74, CrWRKY48 and CrMYB3R4. Overexpression of CrWRKY74 in Arabidopsis resulted in severe seed abortion. By analyzing the downstream regulatory network, we further determined that CrWRKY74 participated in seed abortion regulation by inducing abnormal programmed cell death. Of particular importance is that a preliminary model was proposed to depict the regulatory networks underlying seed abortion in citrus. The results of this study provide novel insights into the molecular mechanism across citrus seed development, and reveal the master role of CrWRKY74 in seed abortion of 'Huagan No. 4'.

Small RNAs contribute to citrus Huanglongbing tolerance by manipulating methyl salicylate signaling and exogenous methyl salicylate primes citrus groves from emerging infection.

Citrus production is severely threatened by the devastating Huanglongbing (HLB) disease globally. By studying and analyzing the defensive behaviors of an HLB-tolerant citrus cultivar 'Shatangju', we discovered that citrus can sense Candidatus Liberibacter asiaticus (CLas) infection and induce immune responses against HLB, which can be further strengthened by both endogenously produced and exogenously applied methyl salicylate (MeSA). This immune circuit is turned on by an miR2977-SAMT (encoding a citrus Salicylate [SA] O-methyltransferase) cascade, by which CLas infection leads to more in planta MeSA production and aerial emission. We provided both transgenic and multi-year trail evidences that MeSA is an effective community immune signal. Ambient MeSA accumulation and foliage application can effectively induce defense gene expression and significantly boost citrus performance. We also found that miRNAs are battle fields between citrus and CLas, and about 30% of the differential gene expression upon CLas infection are regulated by miRNAs. Furthermore, CLas hijacks host key processes by manipulating key citrus miRNAs, and citrus employs miRNAs that coordinately regulate defense-related genes. Based on our results, we proposed that miRNAs and associated components are key targets for engineering or breeding resistant citrus varieties. We anticipate that MeSA-based management, either induced expression or external application, would be a promising tool for HLB control.

OrangeExpDB: an integrative gene expression database for Citrus spp.

BACKGROUND: Citrus is a major fruit crop, and RNA-sequencing (RNA-seq) data can be utilized to investigate its gene functions, heredity, evolution, development, and the detection of genes linked to essential traits or resistance to pathogens. However, it is challenging to use the public RNA-seq datasets for researchers without bioinformatics training, and expertise. RESULTS: OrangeExpDB is a web-based database that integrates transcriptome data of various Citrus spp., including C. limon (L.) Burm., C. maxima (Burm.) Merr., C. reticulata Blanco, C. sinensis (L.) Osbeck, and Poncirus trifoliata (L.) Raf., downloaded from the NCBI SRA database. It features a blast tool for browsing and searching, enabling quick download of expression matrices for different transcriptome samples. Expression of genes of interest can be easily generated by searching gene IDs or sequence similarity. Expression data in text format can be downloaded and presented as a heatmap, with additional sample information provided at the bottom of the webpage. CONCLUSIONS: Researchers can utilize OrangeExpDB to facilitate functional genomic analysis and identify key candidate genes, leveraging publicly available citrus RNA-seq datasets. OrangeExpDB can be accessed at http://www.orangeexpdb.com/ .

Genome-wide identification of the GRF family in sweet orange (Citrus sinensis) and functional analysis of the CsGRF04 in response to multiple abiotic stresses.

BACKGROUND: Citrus is one of the most valuable fruits worldwide and an economic pillar industry in southern China. Nevertheless, it frequently suffers from undesirable environmental stresses during the growth cycle, which severely restricts the growth, development and yield of citrus. In plants, the growth-regulating factor (GRF) family of transcription factors (TF) is extensively distributed and plays an vital part in plant growth and development, hormone response, as well as stress adaptation. However, the systematic identification and functional analysis of GRF TFs in citrus have not been reported. RESULTS: Here, a genome-wide identification of GRF TFs was performed in Citrus sinensis, 9 members of CsGRFs were systematically identified and discovered to be scattered throughout 5 chromosomes. Subsequently, physical and chemical properties, phylogenetic relationships, structural characteristics, gene duplication events, collinearity and cis-elements of promoter were elaborately analyzed. In particular, the expression patterns of the CsGRF genes in response to multiple phytohormone and abiotic stress treatments were investigated. Predicated on this result, CsGRF04, which exhibited the most differential expression pattern under multiple phytohormone and abiotic stress treatments was screened out. Virus-induced gene silencing (VIGS) technology was utilized to obtain gene silenced plants for CsGRF04 successfully. After the three stress treatments of high salinity, low temperature and drought, the CsGRF04-VIGS lines showed significantly reduced resistance to high salinity and low temperature stresses, but extremely increased resistance to drought stress. CONCLUSIONS: Taken together, our findings systematically analyzed the genomic characterization of GRF family in Citrus sinensis, and excavated a CsGRF04 with potential functions under multiple abiotic stresses. Our study lay a foundation for further study on the function of CsGRFs in abiotic stress and hormone signaling response.