Genome-wide identification and in silico expression analysis of CCO gene family in Citrus sinensis (orange) in response to citrus greening

Mildew resistance Locus O (MLO), a gene family specific to plants, plays significant roles in the resistance to powdery mildew (PM) and response to a variety of abiotic stresses, plant growth and development. Despite their importance as barley, rice, wheat, few studies are reported in dicots except Arabidopsis; no global analysis has been performed in the burgeoning model fruit plant sweet orange (Citrus sinensis). The recent release of the genome sequences of C. sinensis provides an opportunity to conduct a comprehensive overview the evolution and features of the MLO gene family in sweet orange. In this study, amount to 14 members of the Citrus sinensis MLO gene (CisMLO) family according to their gene structures, conserved motifs, and similitude among their presumptive Arabidopsis and rice orthologs were identified in silico. Based on these analyses, all CisMLOs were grouped into six clades and expanded partly due to one tandem duplication and two segmental duplication events. Survey of their chromosomal distributions uncovered that 14 CisMLOs are localized across 6 chromosomes. Multiple-sequence alignments showed that 11 of them shared seven highly conserved transmembrane domains (TMs), while all of the sweet orange MLO proteins except CisMLO4/14 had a calmodulin-binding domain for MLO function. Expression analysis demonstrated that the MLO gene family has a diverse tissue-specific expression profiles in the sweet orange development and plays potential critical roles in stress responses. These findings will facilitate further studies of evolutionary pattern and biological functions of MLO genes in sweet orange.

Lectins are sugar-binding proteins found abundantly in plants. Lectin superfamily members have diverse roles, including plant growth, development, cellular processes, stress responses, and defense against microbes. However, the genome-wide identification and functional analysis of lectin genes in sweet orange (Citrus sinensis L.) remain unexplored. Therefore, we used integrated bioinformatics approaches (IBA) for in-depth genome-wide identification, characterization, and regulatory factor analysis of sweet orange lectin genes. Through genome-wide comparative analysis, we identified a total of 141 lectin genes distributed across 10 distinct gene families such as 68 CsB-Lectin, 13 CsLysin Motif (LysM), 4 CsChitin-Bind1, 1 CsLec-C, 3 CsGal-B, 1 CsCalreticulin, 3 CsJacalin, 13 CsPhloem, 11 CsGal-Lec, and 24 CsLectinlegB.This classification relied on characteristic domain and phylogenetic analysis, showing significant homology with Arabidopsis thaliana's lectin gene families. A thorough analysis unveiled common similarities within specific groups and notable variations across different protein groups. Gene Ontology (GO) enrichment analysis highlighted the predicted genes' roles in diverse cellular components, metabolic processes, and stress-related regulation. Additionally, network analysis of lectin genes with transcription factors (TFs) identified pivotal regulators like ERF, MYB, NAC, WRKY, bHLH, bZIP, and TCP. The cis-acting regulatory elements (CAREs) found in sweet orange lectin genes showed their roles in crucial pathways, including light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and more. These findings will aid in the in-depth molecular examination of these potential genes and their regulatory elements, contributing to targeted enhancements of sweet orange species in breeding programs.

GRAS genes are suggested to be grouped into plant-specific transcriptional regulatory families that have been reported to participate in multiple processes, including plant development, phytohormone signaling, the formation of symbiotic relationships, and response to environmental signals. GRAS genes have been characterized in a number of plant species, but little is known about this gene family in Citrus sinensis. In this study, we identified a total of 50 GRAS genes and characterized the gene structures, conserved motifs, genome localizations and cis-elements within their promoter regions. According to their structural and phylogenetic features, the identified sweet orange GRAS members were divided into 11 subgroups, of which subfamily CsGRAS34 was sweet orange-specific. Based on publicly available RNA-seq data generated from callus, flower, leaf and fruit in sweet orange, we found that some sweet orange GRAS genes exhibited tissue-specific expression patterning. Three of the six members of subfamily AtSHR, particularly CsGRAS9, and two of the six members of subfamily AtPAT1 were preferentially expressed in leaf. Moreover, protein-protein interactions with CsGRAS were predicted. Gene expression analysis was performed under conditions of phosphate deficiency, and GA3 and NaCl treatment to identify the potential functions of GRAS members in regulating stress and hormone responses. This study provides the first comprehensive understanding of the GRAS gene family in the sweet orange genome. As such, the study generates valuable information for further gene function analysis and identifying candidate genes to improve abiotic stress tolerance in citrus plants.

Genome-wide identification and characterization of laccase gene family in Citrus sinensis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that plays important roles in multiple cellular processes including phytohormone signaling, plant development, and transcriptional regulation. Although GAPDH genes have been well characterized in various plant species such as Arabidopsis, tobacco, wheat, rice, and watermelon, comprehensive analysis has yet to be completed at the whole genome level in sweet orange (Citrus sinensis). In this study, six GAPDH genes distributed across four chromosomes were identified within the sweet orange genome. Their gene structures, conserved subunits, and subcellular localization were also characterized. Cis-element analysis of CsGAPDHs’ promoter regions and the results of dark treatments indicate that CsGAPDH may be involved in photosynthesis. CsGAPDH genes expressed either in a tissue-specific manner or constitutively were ultimately identified along with their expression response to phosphorus deficiency treatments. In addition, a dual-luciferase transient assay was performed to reveal the transcriptional activation of CsGAPDH proteins. Gene Ontology (GO) analysis for proteins interacting with CsGAPDHs helped to uncover the roles these CsGAPDHs play in other plant processes such as citrus seed germination. This study provides a systematic analysis of the CsGAPDH gene family in the sweet orange genome, which can serve as a strong foundation for further research into the biochemical properties and physiological functions of CsGAPDHs.

BackgroundLeucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs. The functions of most LRR-RLKs have remained undiscovered, and a few that have been experimentally characterized have been shown to have important roles in growth and development as well as in defense responses. Although RLK subfamilies have been previously studied in many plants, no comprehensive study has been performed on this gene family in Citrus species, which have high economic importance and are frequent targets for emerging pathogens. In this study, we performed in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of Citrus clementina (clementine) and Citrus sinensis (sweet orange). In addition, we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and further narrowed the analysis to the LRR-XII group, which contains several previously described cell surface immune receptors.ResultsWe built integrative protein signature databases for Citrus clementina and Citrus sinensis using all predicted protein sequences obtained from whole genomes. A total of 300 and 297 proteins were identified as LRR-RLKs in C. clementina and C. sinensis, respectively. Maximum-likelihood phylogenetic trees were estimated using Arabidopsis LRR-RLK as a template and they allowed us to classify Citrus LRR-RLKs into 16 groups. The LRR-XII group showed a remarkable expansion, containing approximately 150 paralogs encoded in each Citrus genome. Phylogenetic analysis also demonstrated the existence of two distinct LRR-XII clades, each one constituted mainly by RD and non-RD kinases. We identified 68 orthologous pairs from the C. clementina and C. sinensis LRR-XII genes. In addition, among the paralogs, we identified a subset of 78 and 62 clustered genes probably derived from tandem duplication events in the genomes of C. clementina and C. sinensis, respectively.ConclusionsThis work provided the first comprehensive evolutionary analysis of the LRR-RLKs in Citrus. A large expansion of LRR-XII in Citrus genomes suggests that it might play a key role in adaptive responses in host-pathogen co-evolution, related to the perennial life cycle and domestication of the citrus crop species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2930-9) contains supplementary material, which is available to authorized users.

Thaumatin-like proteins (TLPs), a subfamily of pathogenesis-related (PR) proteins, play a vital role in plant defense against pathogens. In this study, 23 CsTLP genes were identified in the Citrus sinensis genome. These genes encode proteins ranging from 203 to 512 amino acids, with molecular weights between 21.88 and 53.75 kDa, classifying them as small molecular weight proteins. The CsTLP genes are unevenly distributed across eight chromosomes, with chromosome 3 containing the highest number (6 genes). Subcellular localization predictions indicate that most CsTLPs are located in the extracellular space. Phylogenetic analysis with Arabidopsis thaliana TLPs classified the CsTLPs into 10 clades, with clade 5 being the largest. Three segmentally duplicated gene pairs were identified, suggesting a mechanism for the expansion of this gene family. Expression profiling revealed tissue-specific patterns, with the highest expression levels observed in roots and leaves. Under biotic stress, qRT-PCR analysis of 12 selected CsTLPs demonstrated pathogen-specific responses: CsTLP9 and CsTLP22 were strongly upregulated during Huanglongbing (HLB, bacterial) infection, by 21.70-fold and 9.47-fold, respectively. Multiple genes, including CsTLP5/13/18/21/23, exhibited over 10-fold upregulation following Citrus Anthracnose (CA, fungal) infection; however, most genes showed only weak responses to Citrus tristeza virus (CTV, viral). These findings underscore the regulatory significance of CsTLPs in pathogen responses and provide an important theoretical foundation for enhancing molecular disease-resistance breeding in Citrus sinensis.

Sweet orange (Citrus sinensis) is a sub-tropical fruit crop with important economic value that is popular worldwide; however, various pathogens significantly affect citrus cultivation and distribution. AlkB homolog (ALKBH) proteins play crucial roles in RNA metabolism and translation in plants; however, no systematic investigations have been performed on ALKBH in sweet oranges. In this study, ten ALKBH gene family members were identified in Citrus sinensis genome. Standardized analyses, including physical properties, phylogenetic analysis, gene structure, motif composition, cis-acting element prediction, chromosome distribution, and synteny analysis, were conducted. The phylogenetic analysis suggested that the ten proteins were clustered into three groups, each of which had similar motifs and gene structures. Gene expression profiling revealed that almost all CsALKBH proteins were highly expressed in callus, and ALKBH9/10-like group members responded positively to biotic stress. Overall, this study is the first to report a genome-wide assessment of the ALKBH family in sweet oranges and provides valuable insights for candidate gene selection and elucidating the molecular mechanism of sweet orange response to pathogenic infections.

Three-amino-acid-loop-extension (TALE) transcription factors comprise one of the largest gene families in plants, in which they contribute to regulation of a wide variety of biological processes, including plant growth and development, as well as governing stress responses. Although sweet orange (Citrus sinensis) is among the most commercially important fruit crops cultivated worldwide, there have been relatively few functional studies on TALE genes in this species. In this study, we investigated 18 CsTALE gene family members with respect to their phylogeny, physicochemical properties, conserved motif/domain sequences, gene structures, chromosomal location, cis-acting regulatory elements, and protein–protein interactions (PPIs). These CsTALE genes were classified into two subfamilies based on sequence homology and phylogenetic analyses, and the classification was equally strongly supported by the highly conserved gene structures and motif/domain compositions. CsTALEs were found to be unevenly distributed on the chromosomes, and duplication analysis revealed that segmental duplication and purifying selection have been major driving force in the evolution of these genes. Expression profile analysis indicated that CsTALE genes exhibit a discernible spatial expression pattern in different tissues and differing expression patterns in response to different biotic/abiotic stresses. Of the 18 CsTALE genes examined, 10 were found to be responsive to high temperature, four to low temperature, eight to salt, and four to wounding. Moreover, the expression of CsTALE3/8/12/16 was induced in response to infection with the fungal pathogen Diaporthe citri and bacterial pathogen Candidatus Liberibacter asiaticus, whereas the expression of CsTALE15/17 was strongly suppressed. The transcriptional activity of CsTALE proteins was also verified in yeast, with yeast two-hybrid assays indicating that CsTALE3/CsTALE8, CsTALE3/CsTALE11, CsTALE10/CsTALE12, CsTALE14/CsTALE8, CsTALE14/CsTALE11 can form respective heterodimers. The findings of this study could lay the foundations for elucidating the biological functions of the TALE family genes in sweet orange and contribute to the breeding of stress-tolerant plants.

BackgroundSalicylic Acid (SA) is a pivotal phytohormone in plant innate immunity enhancement of triggered by various pathogens, such as Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). WRKY is a plant specific transcription factor (TF) family, which plays crucial roles in plant response to biotic stresses. So far, the evolutionary history, functions, and expression patterns under SA treatment and CLas infection of WRKY family are poorly understood in Citrus, despite the release of the genome of several Citrus species. A comprehensive genomic and expressional analysis is worth to conduct for this family.ResultsHere, a genome-wide identification of WRKY TFs was performed in two Citrus species: Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). In total, 52 CsWRKYs and 51 PtrWRKYs were identified, whose physical and chemical properties, chromosome locations, phylogenetic relationships and structural characteristics were comparatively analyzed. Especially, expression patterns of these WRKY genes before and after SA treatment and CLas infection were compared. Based on this result, seven pairs of orthologous WRKY genes showing opposite expression patterns in two Citrus species were screened out. Moreover, two pairs of orthologous WRKY genes with significant differences in the number or type of stress-responsive cis-elements in the promoter regions were discovered. Subcellular localization and transcriptional activation activity assays revealed that these two pairs of orthologous genes are classic WRKY TFs localize in the nucleus and could function as transcriptional activators.ConclusionIn this study, we systematically analyzed the genomic characterization of WRKY family in two Citrus species, together with the analyses of expression patterns under SA signaling and CLas infection. Our study laid a foundation for further study on the function of WRKY TFs in HLB response and SA signaling of Citrus.

Genome-wide identification and expression analysis of the polyamine oxidase gene family in sweet orange (Citrus sinensis)

In eukaryotic systems, messenger RNA regulations, including splicing, 3′-end formation, editing, localization, and translation, are achieved by different RNA-binding proteins and noncoding RNAs. The YTH domain is a newly identified RNA-binding domain that was identified by comparing its sequence with that of splicing factor YT521-B. Previous study showed that the YTH gene plays an important role in plant resistance to abiotic and biotic stress. In this study, 211 YTH genes were identified in 26 species that represent four major plant lineages. Phylogenetic analysis revealed that these genes could be divided into eight subgroups. All of the YTH genes contain a YT521 domain and have different structures. Ten YTH genes were identified in navel orange ( Citrus sinensis ). The expression profiles of these CitYTH genes were analyzed in different tissues and at different fruit developmental stages, and CitYTH genes displayed distinct expression patterns under heat, cold, salt, and drought stress. Furthermore, expression of the CitYTH genes in response to exogenous hormones was measured. Nuclear localization was also confirmed for five of the proteins encoded by these genes after transient expression in Nicotiana benthamiana cells. This study provides valuable information on the role of CitYTHs in the signaling pathways involved in environmental stress responses in Citrus .

Auxin response factors (ARFs) are an important family of proteins in auxin-mediated response, with key roles in various physiological and biochemical processes. To date, a genome-wide overview of the ARF gene family in citrus was not available. A systematic analysis of this gene family in citrus was begun by carrying out a genome-wide search for the homologs of ARFs. A total of 19 nonredundant ARF genes (CiARF) were found and validated from the sweet orange. A comprehensive overview of the CiARFs was undertaken, including the gene structures, phylogenetic analysis, chromosome locations, conserved motifs of proteins, and cis-elements in promoters of CiARF. Furthermore, expression profiling using real-time PCR revealed many CiARF genes, albeit with different patterns depending on types of tissues and/or developmental stages. Comprehensive expression analysis of these genes was also performed under two hormone treatments using real-time PCR. Indole-3-acetic acid (IAA) and N-1-napthylphthalamic acid (NPA) treatment experiments revealed differential up-regulation and down-regulation, respectively, of the 19 citrus ARF genes in the callus of sweet orange. Our comprehensive analysis of ARF genes further elucidates the roles of CiARF family members during citrus growth and development process.

Genome-wide identification, characterization, and expression profile of NBS-LRR gene family in sweet orange (Citrus sinensis)

Polyamines (PAs) contribute to diverse plant processes, environmental interaction, and stress responses. In citrus, the mechanism underlying the biosynthesis of polyamines is poorly understood. The present study aims to identify the biosynthesis of PA gene family members in satsuma mandarin (Citrus unshiu) and investigate their response against various stresses. The identified biosynthesis of PA genes in C. unshiu showed clustering in six groups, i.e., SPMS, SPDS, ACL5, ADC, ODC, and SAMDC. Syntenic analysis revealed that segmental duplication was prevalent among the biosynthesis of PA genes compared to tandem duplication. Thus, it might be the main reason for diversity in the gene family in C. unshiu. Almost all biosynthesis of PA gene family members in C. unshiu showed syntenic blocks in the genome of Arabidopsis, Citrus sinensis, Poncirus trifoliata, and Citrus reticulata. Analysis of Cis-regulatory elements (CREs) indicated the occurrence of hormones, light, defense, and environmental stress responses as well as the development and other plant mechanisms-related elements in the upstream sequence of the biosynthesis of PA genes. Expression profiling revealed that the biosynthesis of PA gene expression modulates in different organs during various developmental stages and stress in C. unshiu. This information will provide a deep understanding of genomic information and its expression in multiple tissues to better understand its potential application in functional genomics.