Integrative Analysis of Metabolome and Transcriptome of Carotenoid Biosynthesis Reveals the Mechanism of Fruit Color Change in Tomato (Solanum lycopersicum).

Tomato fruit ripening is accompanied by carotenoid accumulation and color changes. To elucidate the regulatory mechanisms underlying carotenoid synthesis during fruit ripening, a combined transcriptomic and metabolomic analysis was conducted on red-fruited tomato (WP190) and orange-fruited tomato (ZH108). A total of twenty-nine (29) different carotenoid compounds were identified in tomato fruits at six different stages. The abundance of the majority of the carotenoids was enhanced significantly with fruit ripening, with higher levels of lycopene; (E/Z)-lycopene; and α-, ß- and γ-carotenoids detected in the fruits of WP190 at 50 and 60 days post anthesis (DPA). Transcriptome analysis revealed that the fruits of two varieties exhibited the highest number of differentially expressed genes (DEGs) at 50 DPA, and a module of co-expressed genes related to the fruit carotenoid content was established by WGCNA. qRT-PCR analysis validated the transcriptome result with a significantly elevated transcript level of lycopene biosynthesis genes (including SlPSY2, SlZCIS, SlPDS, SlZDS and SlCRTSO2) observed in WP190 at 50 DPA in comparison to ZH108. In addition, during the ripening process, the expression of ethylene biosynthesis (SlACSs and SlACOs) and signaling (SlEIN3 and SlERF1) genes was also increased, and these mechanisms may regulate carotenoid accumulation and fruit ripening in tomato. Differential expression of several key genes in the fruit of two tomato varieties at different stages regulates the accumulation of carotenoids and leads to differences in color between the two varieties of tomato. The results of this study provide a comprehensive understanding of carotenoid accumulation and ethylene biosynthesis and signal transduction pathway regulatory mechanisms during tomato fruit development.

Comprehensive genomic characterization and expression analysis of calreticulin gene family in tomato.

Calreticulin (CRT) is a calcium-binding endoplasmic reticulum (ER) protein that has been identified for multiple cellular processes, including protein folding, regulation of gene expression, calcium (Ca2+) storage and signaling, regeneration, and stress responses. However, the lack of information about this protein family in tomato species highlights the importance of functional characterization. In the current study, 21 CRTs were identified in four tomato species using the most recent genomic data and performed comprehensive bioinformatics and SlCRT expression in various tissues and treatments. In the bioinformatics analysis, we described the physiochemical properties, phylogeny, subcellular positions, chromosomal location, promoter analysis, gene structure, motif distribution, protein structure and protein interaction. The phylogenetic analysis classified the CRTs into three groups, consensus with the gene architecture and conserved motif analyses. Protein structure analysis revealed that the calreticulin domain is highly conserved among different tomato species and phylogenetic groups. The cis-acting elements and protein interaction analysis indicate that CRTs are involved in various developmental and stress response mechanisms. The cultivated and wild tomato species exhibited similar gene mapping on chromosomes, and synteny analysis proposed that segmental duplication plays an important role in the evolution of the CRTs family with negative selection pressure. RNA-seq data analysis showed that SlCRTs were differentially expressed in different tissues, signifying the role of calreticulin genes in tomato growth and development. qRT-PCR expression profiling showed that all SlCRTs except SlCRT5 were upregulated under PEG (polyethylene glycol) induced drought stress and abscisic acid (ABA) treatment and SlCRT2 and SlCRT3 were upregulated under salt stress. Overall, the results of the study provide information for further investigation of the functional characterization of the CRT genes in tomato.

Functional analysis of fasciclin-like arabinogalactan in carotenoid synthesis during tomato fruit ripening.

Carotenoids are important pigmented nutrients synthesized by tomato fruits during ripening. To reveal the molecular mechanism underlying carotenoid synthesis during tomato fruit ripening, we analyzed carotenoid metabolites and transcriptomes in six development stages of tomato fruits. A total of thirty different carotenoids were detected and quantified in tomato fruits from 10 to 60 DPA. Based on differential gene expression profiles and WGCNA, we explored several genes that were highly significant and negatively correlated with lycopene, all of which encode fasciclin-like arabinogalactan proteins (FLAs). The FLAs are involved in plant signal transduction, however the functional role of these proteins has not been studied in tomato. Genome-wide analysis revealed that cultivated and wild tomato species contained 18 to 22 FLA family members, clustered into four groups, and mainly evolved by means of segmental duplication. The functional characterization of FLAs showed that silencing of SlFLA1, 5, and 13 were found to contribute to the early coloration of tomato fruits, and the expression of carotenoid synthesis-related genes was up-regulated in fruits that changed phenotypically, especially in SlFLA13-silenced plants. Furthermore, the content of multiple carotenoids (including (E/Z)-phytoene, lycopene, γ-carotene, and α-carotene) was significantly increased in SlFLA13-silenced fruits, suggesting that SlFLA13 has a potential inhibitory function in regulating carotenoid synthesis in tomato fruits. The results of the present study broaden the idea of analyzing the biological functions of tomato FLAs and preliminary evidence for the inhibitory role of SlFLA13 in carotenoid synthesis in fruit, providing the theoretical basis and a candidate for improving tomato fruit quality.

Involvement of Alfin-Like Transcription Factors in Plant Development and Stress Response.

Alfin-like (AL) proteins are an important class of transcription factor (TF) widely distributed in eukaryotes and play vital roles in many aspects of plant growth and development. AL proteins contain an Alfin-like domain and a specific PHD-finger structure domain at the N-terminus and C-terminus, respectively. The PHD domain can bind to a specific (C/A) CAC element in the promoter region and affect plant growth and development by regulating the expression of functional genes. This review describes a variety of AL transcription factors that have been isolated and characterized in Arabidopsis thaliana, Brassica rapa, Zea mays, Brassica oleracea, Solanum lycopersicum, Populus trichocarpa, Pyrus bretschenedri, Malus domestica, and other species. These studies have focused mainly on plant growth and development, different abiotic stress responses, different hormonal stress responses, and stress responses after exposure to pathogenic bacteria. However, studies on the molecular functional mechanisms of Alfin-like transcription factors and the interactions between different signaling pathways are rare. In this review, we performed phylogenetic analysis, cluster analysis, and motif analysis based on A. thaliana sequences. We summarize the structural characteristics of AL transcription factors in different plant species and the diverse functions of AL transcription factors in plant development and stress regulation responses. The aim of this study was to provide a reference for further application of the functions and mechanisms of action of the AL protein family in plants.

A mutation in SlCHLH encoding a magnesium chelatase H subunit is involved in the formation of yellow stigma in tomato (Solanum lycopersicum L.).

Chlorophylls are ubiquitous pigments responsible for the green color in plants. Changes in the chlorophyll content have a significant impact on photosynthesis, plant growth and development. In this study, we used a yellow stigma mutant (ys) generated from a green stigma tomato WT by using ethylmethylsulfone (EMS)-induced mutagenesis. Compared with WT, the stigma of ys shows low chlorophyll content and impaired chloroplast ultrastructure. Through map-based cloning, the ys gene is localized to a 100 kb region on chromosome 4 between dCAPS596 and dCAPS606. Gene expression analysis and nonsynonymous SNP determination identified the Solyc04g015750, as the potential candidate gene, which encodes a magnesium chelatase H subunit (CHLH). In ys mutant, a single base C to T substitution in the SlCHLH gene results in the conversion of Serine into Leucine (Ser92Leu) at the N-terminal region. The functional complementation test shows that the SlCHLH from WT can rescue the green stigma phenotype of ys. In contrast, knockdown of SlCHLH in green stigma tomato AC, observed the yellow stigma phenotype at the stigma development stage. Overexpression of the mutant gene Slys in green stigma tomato AC results in the light green stigma. These results indicate that the mutation of the N-terminal S92 to Leu in SlCHLH is the main reason for the formation of the yellow stigma phenotype. Characterization of the ys mutant enriches the current knowledge of the tomato chlorophyll mutant library and provides a novel and effective tool for understanding the function of CHLH in tomato.

Identification of TALE Transcription Factor Family and Expression Patterns Related to Fruit Chloroplast Development in Tomato (Solanum lycopersicum L.).

The TALE gene family is an important transcription factor family that regulates meristem formation, organ morphogenesis, signal transduction, and fruit development. A total of 24 genes of the TALE family were identified and analyzed in tomato. The 24 SlTALE family members could be classified into five BELL subfamilies and four KNOX subfamilies. SlTALE genes were unevenly distributed on every tomato chromosome, lacked syntenic gene pairs, and had conserved structures but diverse regulatory functions. Promoter activity analysis showed that cis-elements responsive to light, phytohormone, developmental regulation, and environmental stress were enriched in the promoter of SlTALE genes, and the light response elements were the most abundant. An abundance of TF binding sites was also enriched in the promoter of SlTALE genes. Phenotype identification revealed that the green shoulder (GS) mutant fruits showed significantly enhanced chloroplast development and chlorophyll accumulation, and a significant increase of chlorophyll fluorescence parameters in the fruit shoulder region. Analysis of gene expression patterns indicated that six SlTALE genes were highly expressed in the GS fruit shoulder region, and four SlTALE genes were highly expressed in the parts with less-developed chloroplasts. The protein-protein interaction networks predicted interaction combinations among these SlTALE genes, especially between the BELL subfamilies and the KNOX subfamilies, indicating a complex regulatory network of these SlTALE genes in chloroplast development and green fruit shoulder formation. In conclusion, our result provides detailed knowledge of the SlTALE gene for functional research and the utilization of the TALE gene family in fruit quality improvement.

Overexpression of a Mitogen-Activated Protein Kinase SlMAPK3 Positively Regulates Tomato Tolerance to Cadmium and Drought Stress.

Mitogen-activated protein kinases (MAPKs) activation is a common defense response of plants to a range of abiotic stressors. SlMPK3, a serine-threonine protein kinase, has been reported as an important member of protein kinase cascade that also functions on plant stress tolerance. In this study, we cloned SlMPK3 from tomato and studied its role in cadmium (Cd2+) and drought tolerance. The results showed that transcripts of SlMAPK3 differentially accumulated in various plant tissues and were remarkably induced by different abiotic stressors and exogenous hormone treatments. Overexpression of SlMAPK3 increased tolerance to Cd2+ and drought as reflected by an increased germination rate and improved seedling growth. Furthermore, transgenic plants overexpressing SlMAPK3 showed an increased leaf chlorophyll content, root biomass accumulation and root activity under Cd2+ stress. Chlorophyll fluorescence analysis revealed that transgenic plants demonstrated an increased photosynthetic activity as well as contents of chlorophyll, proline, and sugar under drought stress. Notably, cadmium- and drought-induced oxidative stress was substantially attenuated in SlMAPK3 overexpressing plants as evidenced by lower malondialdehyde and hydrogen peroxide accumulation, and increased activity and transcript abundance of enzymatic antioxidants under stress conditions compared to that of wild-type. Our findings provide solid evidence that overexpression of SlMAPK3 gene in tomato positively regulates tolerance to Cd2+ and drought stress, which may have strengthen the molecular understanding of SlMAPK3 gene to improve abiotic stress tolerance.

RNA Interference: A Natural Immune System of Plants to Counteract Biotic Stressors.

During plant-pathogen interactions, plants have to defend the living transposable elements from pathogens. In response to such elements, plants activate a variety of defense mechanisms to counteract the aggressiveness of biotic stressors. RNA interference (RNAi) is a key biological process in plants to inhibit gene expression both transcriptionally and post-transcriptionally, using three different groups of proteins to resist the virulence of pathogens. However, pathogens trigger an anti-silencing mechanism through the expression of suppressors to block host RNAi. The disruption of the silencing mechanism is a virulence strategy of pathogens to promote infection in the invaded hosts. In this review, we summarize the RNA silencing pathway, anti-silencing suppressors, and counter-defenses of plants to viral, fungal, and bacterial pathogens.

Genome-Wide Analysis of DCL, AGO, and RDR Gene Families in Pepper (Capsicum Annuum L.).

RNA silencing is an evolutionarily conserved mechanism that regulates variety of cellular processes in plants. Argonaute protein (AGO), Dicer-like protein (DCL) and RNA-dependent RNA polymerase (RDR) are critical components of RNA silencing. These efficient and indispensable components of the RNAi pathway have not been identified and characterized in pepper. In this study, we identified 12 CaAGO, 4 CaDCL and 6 CaRDR genes in pepper and compared them with those of Arabidopsis, tobacco, potato and tomato. Detailed phylogenetic analyses revealed that each CaAGO, CaDCL and CaRDR protein family were classified into four clades. The tissue specific expression and respond to abiotic or biotic stress were studied. The real-time quantitative polymerase chain reaction (PCR) results demonstrated that CaAGO2, CaAGO10b, CaDCL2 and CaDCL4 were upregulated with cucumber mosaic virus (CMV), potato virus Y (PVY) and tobacco mosaic virus (TMV) infections, whereas they showed difference expression patterns in response to abiotic stress. In addition, we found that many of the candidate genes were induced by phytohormones and H2O2 treatment. Our results provide useful information for further elucidation of gene silencing pathways and RNAi-mediated host immunity in pepper.

CaRDR1, an RNA-Dependent RNA Polymerase Plays a Positive Role in Pepper Resistance against TMV.

RNA silencing functions as a major natural antiviral defense mechanism in plants. RNA-dependent RNA polymerases (RDRs) that catalyze the synthesis of double-stranded RNAs, are considered as a fundamental element in RNA silencing pathways. In Arabidopsis thaliana, RDR1, 2 and 6 play important roles in anti-viral RNA silencing. Expression of RDR1 can be elevated following plant treatment with defense hormones and virus infection. RDR1 has been studied in several crop species, but not in pepper (Capsicum annuum L.). Here, a RDR1 gene was isolated from Capsicum annuum L., designated as CaRDR1. The full-length cDNA of CaRDR1 was 3,351 bp, encoding a 1,116-amino acid protein, which contains conserved regions, such as the most remarkable motif DLDGD. The transcripts of CaRDR1 could be induced by salicylic acid (SA), abscisic acid (ABA), H2O2, and tobacco mosaic virus (TMV). Silencing of CaRDR1 in pepper resulted in increased susceptibility to TMV as evident by severe symptom, increased of TMV-CP transcript, higher malondialdehyde (MDA) content and lower antioxidant enzymes activities compared with that of control plants. CaRDR1-overexpressing in Nicotiana benthamiana showed mild disease symptom and reduced TMV-CP transcripts than that of empty vector (EV) following TMV inoculation. The RNA silencing related genes, including NbAGO2, NbDCL2, NbDCL3, and NbDCL4 elevated expression in overexpressed plants. Alternative oxidase (AOX), the terminal oxidase of the cyanide (CN)-resistant alternative respiratory pathway, catalyze oxygen-dependent oxidation of ubiquinol in plants. It has an important function in plant defense against TMV. In addition, CaRDR1 overexpression promoted the expression of NbAOX1a and NbAOX1b. In conclusion, these results suggest that CaRDR1 plays a positive role in TMV resistance by regulating antioxidant enzymes activities and RNA silencing-related genes expression to suppress the replication and movement of TMV.

Transcriptome Profiling of Tomato Uncovers an Involvement of Cytochrome P450s and Peroxidases in Stigma Color Formation.

Stigma is a crucial structure of female reproductive organ in plants. Stigma color is usually regarded as an important trait in variety identification in some species, but the molecular mechanism of stigma color formation remains elusive. Here, we characterized a tomato mutant, yellow stigma (ys), that shows yellow rather than typical green color in the stigma. Analysis of pigment contents revealed that the level of flavonoid naringenin chalcone was increased in the ys stigma, possibly as a result of higher accumulation of p-coumaric acid, suggesting that naringenin chalcone might play a vital role in yellow color control in tomato stigma. To understand the genes and gene networks that regulate tomato stigma color, RNA-sequencing (RNA-Seq) analyses were performed to compare the transcriptomes of stigmas between ys mutant and wild-type (WT). We obtained 507 differentially expressed genes, in which, 84 and 423 genes were significantly up-regulated and down-regulated in the ys mutant, respectively. Two cytochrome P450 genes, SlC3H1 and SlC3H2 which encode p-coumarate 3-hydroxylases, and six peroxidase genes were identified to be dramatically inhibited in the yellow stigma. Further bioinformatic and biochemical analyses implied that the repression of the two SlC3Hs and six PODs may indirectly lead to higher naringenin chalcone level through inhibiting lignin biosynthesis, thereby contributing to yellow coloration in tomato stigma. Thus, our data suggest that two SlC3Hs and six PODs are involved in yellow stigma formation. This study provides valuable information for dissecting the molecular mechanism of stigma color control in tomato. Statement: This study reveals that two cytochrome P450s (SlC3H1 and SlC3H2) and six peroxidases potentially regulate the yellow stigma formation by indirectly enhancing biosynthesis of yellow-colored naringenin chalcone in the stigma of tomato.

SlMAPK3 enhances tolerance to tomato yellow leaf curl virus (TYLCV) by regulating salicylic acid and jasmonic acid signaling in tomato (Solanum lycopersicum).

Several recent studies have reported on the role of mitogen-activated protein kinase (MAPK3) in plant immune responses. However, little is known about how MAPK3 functions in tomato (Solanum lycopersicum L.) infected with tomato yellow leaf curl virus (TYLCV). There is also uncertainty about the connection between plant MAPK3 and the salicylic acid (SA) and jasmonic acid (JA) defense-signaling pathways. The results of this study indicated that SlMAPK3 participates in the antiviral response against TYLCV. Tomato seedlings were inoculated with TYLCV to investigate the possible roles of SlMAPK1, SlMAPK2, and SlMAPK3 against this virus. Inoculation with TYLCV strongly induced the expression and the activity of all three genes. Silencing of SlMAPK1, SlMAPK2, and SlMAPK3 reduced tolerance to TYLCV, increased leaf H2O2 concentrations, and attenuated expression of defense-related genes after TYLCV infection, especially in SlMAPK3-silenced plants. Exogenous SA and methyl jasmonic acid (MeJA) both significantly induced SlMAPK3 expression in tomato leaves. Over-expression of SlMAPK3 increased the transcript levels of SA/JA-mediated defense-related genes (PR1, PR1b/SlLapA, SlPI-I, and SlPI-II) and enhanced tolerance to TYLCV. After TYLCV inoculation, the leaves of SlMAPK3 over-expressed plants compared with wild type plants showed less H2O2 accumulation and greater superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activity. Overall, the results suggested that SlMAPK3 participates in the antiviral response of tomato to TYLCV, and that this process may be through either the SA or JA defense-signaling pathways.