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.

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.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

A Calcium-Dependent Protein Kinase Regulates the Defense Response in Citrus sinensis.

Citrus Huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CLas), is one of the most destructive citrus diseases worldwide, and defense-related Citrus sinensis gene resources remain largely unexplored. Calcium signaling plays an important role in diverse biological processes. In plants, a few calcium-dependent protein kinases (CDPKs/CPKs) have been shown to contribute to defense against pathogenic microbes. The genome of C. sinensis encodes dozens of CPKs. In this study, the role of C. sinensis calcium-dependent protein kinases (CsCPKs) in C. sinensis defense was investigated. Silencing of CsCPK6 compromised the induction of defense-related genes in C. sinensis. Expression of a constitutively active form of CsCPK6 (CsCPK6CA) triggered the activation of defense-related genes in C. sinensis. Complementation of CsCPK6 rescued the defense-related gene induction in an Arabidopsis thaliana cpk4/11 mutant, indicating that CsCPK6 carries CPK activity and is capable of functioning as a CPK in Arabidopsis. Moreover, an effector derived from CLas inhibits defense induced by the expression of CsCPK6CA and autophosphorylation of CsCPK6, which suggests the involvement of CsCPK6 and calcium signaling in defense. These results support a positive role for CsCPK6 in C. sinensis defense against CLas, and the autoinhibitory regulation of CsCPK6 provides a potential genome-editing target for improving C. sinensis defense. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Transcriptome and weighted gene co-expression network analyses reveal key genes and pathways involved in early fruit ripening in Citrus sinensis.

BACKGROUND: The fruit ripening period is an important target trait in fruit tree crop breeding programs. Thus, citrus tree breeders seek to develop extreme early ripening cultivars that allow optimization of citrus maturation periods. In this study, we explored the regulatory network involved in fruit ripening in Citrus sinensis using the 'Newhall' navel orange variety and its early-ripening mutant, 'Gannanzao'. This research will provide a basis for further research on important signaling pathways, gene functions and variety breeding of Citrus sinensis related to fruit ripening period. RESULTS: Physiological analyses suggested that early fruit ripening in 'Gannanzao' is regulated by early accumulation of abscisic acid (ABA), persistently high levels of jasmonic acid (JA), and higher sucrose content in the pericarp. Pericarp samples from 'Gannanzao' and 'Newhall' navel oranges were sampled for RNA sequencing analysis at 180, 200, and 220 days after flowering; 1430 differentially expressed genes (DEGs) were identified. Functional enrichment analysis indicated that these DEGs were mainly enriched in the plant hormone signal transduction and sugar metabolism pathways, as well as other pathways related to fruit ripening. Important DEGs associated with fruit ripening in 'Gannanzao' included genes involved in ABA and JA metabolism and signal transduction, as well as sugar metabolism. Weighted gene co-expression network analysis showed that the deep pink module had the strongest correlations with ABA content, JA content, and early ripening. Based on gene functionality and gene expression analyses of 37 genes in this module, two candidate hub genes and two ethylene response factor 13 (ERF13) genes (Cs_ont_5g000690 and Cs_ont_5g000700) were identified as key genes regulated by ABA and JA signaling. These findings will help to clarify the mechanisms that underlie early citrus fruit ripening and will lead to the development of excellent genetic resources for further breeding of extreme early-ripening varieties. CONCLUSIONS: Through analyses of the 'Newhall' navel orange cultivar and its early-ripening mutant 'Gannanzao', we identified genes involved in ABA and JA metabolism, signal transduction, and sugar metabolism that were related to fruit ripening. Among these, two ERF13 genes were inferred to be key genes in the regulation of fruit ripening. These findings provide insights into the genetic architecture related to early fruit ripening in C. sinensis.

Physiological and molecular responses of 'Hamlin' sweet orange trees expressing the VvmybA1 gene under cold stress conditions.

MAIN CONCLUSION: Overexpression of VvmybA1 transcription factor in 'Hamlin' citrus enhances cold tolerance by increasing anthocyanin accumulation. This results in improved ROS scavenging, altered gene expression, and stomatal regulation, highlighting anthocyanins' essential role in citrus cold acclimation. Cold stress is a significant threat to citrus cultivation, impacting tree health and productivity. Anthocyanins are known for their role as pigments and have emerged as key mediators of plant defense mechanisms against environmental stressors. This study investigated the potential of anthocyanin overexpression regulated by grape (Vitis vinifera) VvmybA1 transcription factor to enhance cold stress tolerance in citrus trees. Transgenic 'Hamlin' citrus trees overexpressing VvmybA1 were exposed to a 30-day cold stress period at 4 °C along with the control wild-type trees. Our findings reveal that anthocyanin accumulation significantly influences chlorophyll content and their fluorescence parameters, affecting leaf responses to cold stress. Additionally, we recorded enhanced ROS scavenging capacity and distinct expression patterns of key transcription factors and antioxidant-related genes in the transgenic leaves. Furthermore, VvmybA1 overexpression affected stomatal aperture regulation by moderating ABA biosynthesis, resulting in differential responses in a stomatal opening between transgenic and wild-type trees under cold stress. Transgenic trees exhibited reduced hydrogen peroxide levels, enhanced flavonoids, radical scavenging activity, and altered phytohormonal profiles. These findings highlighted the role of VvmybA1-mediated anthocyanin accumulation in enhancing cold tolerance. The current study also underlines the potential of anthocyanin overexpression as a critical regulator of the cold acclimation process by scavenging ROS in plant tissues.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus 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.

Weighted gene coexpression correlation network analysis reveals the potential molecular regulatory mechanism of citrate and anthocyanin accumulation between postharvest 'Bingtangcheng' and 'Tarocco' blood orange fruit.

BACKGROUND: Organic acids and anthocyanins are the most important compounds for the flavor and nutritional quality of citrus fruit. However, there are few reports on the involvement of co-regulation of citrate and anthocyanin metabolism. Here, we performed a comparative transcriptome analysis to elucidate the genes and pathways involved in both citrate and anthocyanin accumulation in postharvest citrus fruit with 'Tarocco' blood orange (TBO; high accumulation) and 'Bingtangcheng' sweet orange (BTSO; low accumulation). RESULTS: A robust core set of 825 DEGs were found to be temporally associated with citrate and anthocyanin accumulation throughout the storage period through transcriptome analysis. Further according to the results of weighted gene coexpression correlation network analysis (WGCNA), the turquoise and brown module was highly positively correlated with both of the content of citrate and anthocyanin, and p-type ATPase (PH8), phosphoenolpyruvate carboxylase kinase (PEPCK), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H) and glutathione S transferase (GST) were considered key structural genes. Moreover, MYB family transcription factor (PH4), Zinc finger PHD-type transcription factor (CHR4, HAC12), Zinc finger SWIM-type transcription factor (FAR1) and Zinc finger C3H1-type transcription factor (ATC3H64) were considered hub genes related to these structural genes. Further qRT-PCR analysis verified that these transcription factors were highly expressed in TBO fruit and their expression profiles were significantly positively correlated with the structural genes of citrate and anthocyanin metabolism as well as the content of citrate and anthocyanin content. CONCLUSIONS: The findings suggest that the CHR4, FAR1, ATC3H64 and HAC12 may be the new transcription regulators participate in controlling the level of citrate and anthocyanin in postharvest TBO fruit in addition to PH4. These results may providing new insight into the regulation mechanism of citrate and anthocyanin accumulation in citrus fruit.

Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4 months, with transformation efficiency of 30% and 22% for the respective species.

Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity.

BACKGROUND: Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression. METHODS AND RESULTS: Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura × Pisifera (D × P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21 years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279. CONCLUSION: The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

Metabolite Signature and Differential Expression of Genes in Washington Navel Oranges (Citrus sinensis) Infected by Spiroplasma citri.

Spiroplasma citri is the pathogen that causes citrus stubborn disease (CSD). Infection of citrus with S. citri has been shown to cause leaf mottling, reduce fruit yield, and stunt tree growth. Fruit from trees exhibiting symptoms of CSD are misshapen and discolored. The symptoms of CSD are easily confused with nutrient deficiencies or symptoms of citrus greening disease. In this study, young Washington navel oranges (Citrus sinensis) were graft-inoculated with budwood originating from trees confirmed to be infected with S. citri. Leaf samples were collected monthly for 10 months for metabolomics and differential gene expression analyses. Significant differences in the concentration of metabolites and expressed genes were observed between control and S. citri-infected trees throughout the experiment. Metabolites and genes associated with important defense and stress pathways, including jasmonic acid signaling, cell wall modification, amino acid biosynthesis, and the production of antioxidant and antimicrobial secondary metabolites, were impacted by S. citri throughout the study, and even prior to symptom development. This work fills a current gap in knowledge surrounding the pathogenicity of S. citri and provides an updated mechanistic explanation for the development of CSD symptoms in S. citri-infected plants.

Intra-Specific Variation in Desiccation Tolerance of Citrus sinensis 'bingtangcheng' (L.) Seeds under Different Environmental Conditions in China.

Intra-specific variation in seed storage behaviour observed in several species has been related to different maternal environments. However, the particular environmental conditions and molecular processes involved in intra-specific variation of desiccation tolerance remain unclear. We chose Citrus sinensis 'bingtangcheng' for the present study due to its known variability in desiccation tolerance amongst seed lots. Six seed lots of mature fruits were harvested across China and systematically compared for drying sensitivity. Annual sunshine hours and average temperature from December to May showed positive correlations with the level of seed survival of dehydration. Transcriptional analysis indicated significant variation in gene expression between relatively desiccation-tolerant (DT) and -sensitive (DS) seed lots after harvest. The major genes involved in late seed maturation, such as heat shock proteins, showed higher expression in the DT seed lot. Following the imposition of drying, 80% of stress-responsive genes in the DS seed lot changed to the stable levels seen in the DT seed lot prior to and post-desiccation. However, the changes in expression of stress-responsive genes in DS seeds did not improve their tolerance to desiccation. Thus, higher desiccation tolerance of Citrus sinensis 'bingtangcheng' seeds is modulated by the maternal environment (e.g., higher annual sunshine hours and seasonal temperature) during seed development and involves stable expression levels of stress-responsive genes.

Unleashing the Potential of EIL Transcription Factors in Enhancing Sweet Orange Resistance to Bacterial Pathologies: Genome-Wide Identification and Expression Profiling.

The ETHYLENE INSENSITIVE3-LIKE (EIL) family is one of the most important transcription factor (TF) families in plants and is involved in diverse plant physiological and biochemical processes. In this study, ten EIL transcription factors (CsEILs) in sweet orange were systematically characterized via whole-genome analysis. The CsEIL genes were unevenly distributed across the four sweet orange chromosomes. Putative cis-acting regulatory elements (CREs) associated with CsEIL were found to be involved in plant development, as well as responses to biotic and abiotic stress. Notably, quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that CsEIL genes were widely expressed in different organs of sweet orange and responded to both high and low temperature, NaCl treatment, and to ethylene-dependent induction of transcription, while eight additionally responded to Xanthomonas citri pv. Citri (Xcc) infection, which causes citrus canker. Among these, CsEIL2, CsEIL5 and CsEIL10 showed pronounced upregulation. Moreover, nine genes exhibited differential expression in response to Candidatus Liberibacter asiaticus (CLas) infection, which causes Citrus Huanglongbing (HLB). The genome-wide characterization and expression profile analysis of CsEIL genes provide insights into the potential functions of the CsEIL family in disease resistance.

CsLOB1 regulates susceptibility to citrus canker through promoting cell proliferation in citrus.

Citrus sinensis lateral organ boundary 1 (CsLOB1) was previously identified as a critical disease susceptibility gene for citrus bacterial canker, which is caused by Xanthomonas citri subsp. citri (Xcc). However, the molecular mechanisms of CsLOB1 in citrus response to Xcc are still elusive. Here, we constructed transgenic plants overexpressing and RNAi-silencing of CsLOB1 using the canker-disease susceptible 'wanjincheng' orange (C. sinensis Osbeck) as explants. CsLOB1-overexpressing plants exhibited dwarf phenotypes with smaller and thicker leaf, increased branches and adventitious buds clustered on stems. These phenotypes were followed by a process of pustule- and canker-like development that exhibited enhanced cell proliferation. Pectin depolymerization and expansin accumulation were enhanced by CsLOB1 overexpression, while cellulose and hemicellulose synthesis were increased by CsLOB1 silence. Whilst overexpression of CsLOB1 increased susceptibility, RNAi-silencing of CsLOB1 enhanced resistance to canker disease without impairing pathogen entry. Transcriptome analysis revealed that CsLOB1 positively regulated cell wall degradation and modification processes, cytokinin metabolism, and cell division. Additionally, 565 CsLOB1-targeted genes were identified in chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. Motif discovery analysis revealed that the most highly overrepresented binding sites had a conserved 6-bp 'GCGGCG' consensus DNA motif. RNA-seq and ChIP-seq data suggested that CsLOB1 directly activates the expression of four genes involved in cell wall remodeling, and three genes that participate in cytokinin and brassinosteroid hormone pathways. Our findings indicate that CsLOB1 promotes cell proliferation by mechanisms depending on cell wall remodeling and phytohormone signaling, which may be critical to citrus canker development and bacterial growth in citrus.

Characterization of pectin methylesterase gene family and its possible role in juice sac granulation in navel orange (Citrus sinensis Osbeck).

BACKGROUND: Citrus is one of the most important fresh fruit crops worldwide. Juice sac granulation is a physiological disorder, which leads to a reduction in soluble solid concentration, total sugar, and titratable acidity of citrus fruits. Pectin methylesterase (PME) catalyzes the de-methylesterification of homogalacturonans and plays crucial roles in cell wall modification during plant development and fruit ripening. Although PME family has been well investigated in various model plants, little is known regarding the evolutionary property and biological function of PME family genes in citrus. RESULTS: In this study, 53 non-redundant PME genes were identified from Citrus sinensis genome, and these PME genes were divided into four clades based on the phylogenetic relationship. Subsequently, bioinformatics analyses of gene structure, conserved domain, chromosome localization, gene duplication, and collinearity were performed on CsPME genes, providing important clues for further research on the functions of CsPME genes. The expression profiles of CsPME genes in response to juice sac granulation and low-temperature stress revealed that CsPME genes were involved in the low temperature-induced juice sac granulation in navel orange fruits. Subcellular localization analysis suggested that CsPME genes were localized on the apoplast, endoplasmic reticulum, plasma membrane, and vacuole membrane. Moreover, yeast one-hybrid screening and dual luciferase activity assay revealed that the transcription factor CsRVE1 directly bound to the promoter of CsPME3 and activated its activity. CONCLUSION: In summary, this study conducts a comprehensive analysis of the PME gene family in citrus, and provides a novel insight into the biological functions and regulation patterns of CsPME genes during juice sac granulation of citrus.

Citrus sinensis CBF1 Functions in Cold Tolerance by Modulating Putrescine Biosynthesis through Regulation of Arginine Decarboxylase.

C-repeat (CRT) binding factors (CBFs) are well known to act as crucial transcription factors that function in cold stress response. Arginine decarboxylase (ADC)- mediated putrescine (Put) biosynthesis has been reported to be activated in plants exposed to cold conditions, but it remains elusive whether CBFs can regulate ADC expression and Put accumulation. In this study, we show that cold upregulated ADC gene (Citrus sinensis ADC;CsADC) and elevated endogenous Put content in sweet orange (C.sinensis). The promoter of CsADC contains two CRT sequences that are canonical elements recognized by CBFs. Sweet orange genome contains four CBFs (CsCBF1-4), in which CsCBF1 was significantly induced by cold. CsCBF1, located in the nucleus, was demonstrated to bind directly and specifically to the promoter of CsADC and acted as a transcriptional activator. Overexpression of CsCBF1 led to notable elevation of CsADC and Put levels in sweet orange transgenic plants, along with remarkably enhanced cold tolerance, relative to the wild type. However, pretreatment with D-arginine, an ADC inhibitor, caused a prominent reduction of endogenous Put levels in the overexpressing lines, accompanied by greatly compromised cold tolerance. Taken together, these results demonstrate that the CBF1 of sweet orange directly regulates ADC expression and modulates Put synthesis for orchestrating the cold tolerance. Our findings shed light on the transcriptional regulation of Put accumulation through targeting the ADC gene in the presence of cold stress. Meanwhile, this study illustrates a new mechanism underlying the CBF-mediated cold stress response.

Comparative transcriptomic analyses of citrus cold-resistant vs. sensitive rootstocks might suggest a relevant role of ABA signaling in triggering cold scion adaption.

BACKGROUND: The citrus genus comprises a number of sensitive tropical and subtropical species to cold stress, which limits global citrus distribution to certain latitudes and causes major economic loss. We used RNA-Seq technology to analyze changes in the transcriptome of Valencia delta seedless orange in response to long-term cold stress grafted on two frequently used citrus rootstocks: Carrizo citrange (CAR), considered one of the most cold-tolerant accessions; C. macrophylla (MAC), a very sensitive one. Our objectives were to identify the genetic mechanism that produce the tolerant or sensitive phenotypes in citrus, as well as to gain insights of the rootstock-scion interactions that induce the cold tolerance or sensitivity in the scion. RESULTS: Plants were kept at 1 ºC for 30 days. Samples were taken at 0, 15 and 30 days. The metabolomic analysis showed a significant increase in the concentration of free sugars and proline, which was higher for the CAR plants. Hormone quantification in roots showed a substantially increased ABA concentration during cold exposure in the CAR roots, which was not observed in MAC. Different approaches were followed to analyze gene expression. During the stress treatment, the 0-15-day comparison yielded the most DEGs. The functional characterization of DEGs showed enrichment in GO terms and KEGG pathways related to abiotic stress responses previously described in plant cold adaption. The DEGs analysis revealed that several key genes promoting cold adaption were up-regulated in the CAR plants, and those repressing it had higher expression levels in the MAC samples. CONCLUSIONS: The metabolomic and transcriptomic study herein performed indicates that the mechanisms activated in plants shortly after cold exposure remain active in the long term. Both the hormone quantification and differential expression analysis suggest that ABA signaling might play a relevant role in promoting the cold hardiness or sensitiveness of Valencia sweet orange grafted onto Carrizo citrange or Macrophylla rootstocks, respectively. Our work provides new insights into the mechanisms by which rootstocks modulate resistance to abiotic stress in the production variety grafted onto them.