Detection and Analysis of VOCs in Cherry Tomato Based on GC-MS and GC×GC-TOF MS Techniques.

Volatile organic compounds (VOCs) play a significant role in influencing the flavor quality of cherry tomatoes (Solanum lycopersicum var. cerasiforme). The scarcity of systematic analysis of VOCs in cherry tomatoes can be attributed to the constraints imposed by detection technology and other contributing factors. In this study, the cherry tomato cultivar var. 'Zheyingfen1' was chosen due to its abundant fruit flavor. Two detection technology platforms, namely the commonly employed headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and the most advanced headspace solid-phase microextraction-full two-dimensional gas chromatography-time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS), were employed in the analysis. The VOCs of cherry tomato cultivar var. 'Zheyingfen1' fruits at red ripening stage were detected. A combined total of 1544 VOCs were detected using the two aforementioned techniques. Specifically, 663 VOCs were identified by through the HS-SPME-GC-MS method, 1026 VOCs were identified by through the HS-SPME-GC×GC-TOFMS, and 145 VOCs were identified by both techniques. The identification of ß-ionone and (E)-2-nonenal as the principal VOCs was substantiated through the application of the relative odor activity value (rOAV) calculation and subsequent analysis. Based on the varying contribution rates of rOAV, the analysis of sensory flavor characteristics revealed that cherry tomato cultivar var. 'Zheyingfen1' predominantly exhibited green and fatty attributes, accompanied by elements of fresh and floral flavor characteristics. In conclusion, our study conducted a comprehensive comparison of the disparities between these two methodologies in detecting VOCs in cherry tomato fruits. Additionally, we systematically analyzed the VOC composition and sensory flavor attributes of the cherry tomato cultivar var. 'Zheyingfen1'. This research serves as a significant point of reference for investigating the regulatory mechanisms underlying the development of volatile flavor quality in cherry tomatoes.

Divergent Retention of Sucrose Metabolism Genes after Whole Genome Triplication in the Tomato (Solanum lycopersicum).

Sucrose, the primary carbon transport mode and vital carbohydrate for higher plants, significantly impacts plant growth, development, yield, and quality formation. Its metabolism involves three key steps: synthesis, transport, and degradation. Two genome triplication events have occurred in Solanaceae, which have resulted in massive gene loss. In this study, a total of 48 and 65 genes from seven sucrose metabolism gene families in Vitis vinifera and Solanum lycopersicum were identified, respectively. The number of members comprising the different gene families varied widely. And there were significant variations in the pattern of gene duplication and loss in the tomato following two WGD events. Tandem duplication is a major factor in the expansion of the SWEET and Acid INV gene families. All the genes are irregularly distributed on the chromosomes, with the majority of the genes showing collinearity with the grape, particularly the CIN family. And the seven gene families were subjected to a purifying selection. The expression patterns of the different gene families exhibited notable variations. This study presents basic information about the sucrose metabolism genes in the tomato and grape, and paves the way for further investigations into the impact of SCT events on the phylogeny, gene retention duplication, and function of sucrose metabolism gene families in the tomato or Solanaceae, and the adaptive evolution of the tomato.

Comprehensive identification of glutathione peroxidase (GPX) gene family in response to abiotic stress in pepper (Capsicum annuum L.).

Plant glutathione peroxidase (GPX) plays an important role in the maintenance of cell homeostasis and in the antioxidant response in plants. In this study, the peroxidase (GPX) gene family was identified in the whole genome of pepper using bioinformatic method. As a result, a total of 5 CaGPX genes were identified, which were unevenly distributed on 3 of the 12 chromosomes of pepper genome. Based on phylogenetic analysis, 90 GPX genes in 17 species from lower plants to higher plants can be divided into 4 groups (GroupⅠ, Group Ⅱ, Group Ⅲ, Group Ⅳ). The MEME Suite analysis of GPX proteins shows that all these proteins contain four highly conserved motifs, as well as other conserved sequences and amino acid residues. Gene structure analysis revealed the conservative exon-intron organization pattern of these genes. In the promoter region of CaGPX genes, many cis elements of plant hormone and abiotic stress response were identified in each of CaGPX proteins. In addition, expression patterns of CaGPX genes in different tissues, developmental stages and responses to abiotic stress were also performed. The results of qRT-PCR showed that the transcripts of CaGPX genes varied greatly under abiotic stress at different time points. There results suggest that the GPX gene family of pepper may play a role in plant development andstress response. In conclusion, our research provides new insights into the evolution of pepper GPX gene family, and understanding for functional of these genes in response to abiotic stresses.

The OsBDR1-MPK3 module negatively regulates blast resistance by suppressing the jasmonate signaling and terpenoid biosynthesis pathway.

Receptor-like kinases (RLKs) may initiate signaling pathways by perceiving and transmitting environmental signals to cellular machinery and play diverse roles in plant development and stress responses. The rice genome encodes more than one thousand RLKs, but only a small number have been characterized as receptors for phytohormones, polypeptides, elicitors, and effectors. Here, we screened the function of 11 RLKs in rice resistance to the blast fungus Magnaporthe oryzae (M. oryzae) and identified a negative regulator named BDR1 (Blast Disease Resistance 1). The expression of BDR1 was rapidly increased under M. oryzae infection, while silencing or knockout of BDR1 significantly enhanced M. oryzae resistance in two rice varieties. Protein interaction and kinase activity assays indicated that BDR1 directly interacted with and phosphorylated mitogen-activated kinase 3 (MPK3). Knockout of BDR1 compromised M. oryzae-induced MPK3 phosphorylation levels. Moreover, transcriptome analysis revealed that M. oryzae-elicited jasmonate (JA) signaling and terpenoid biosynthesis pathway were negatively regulated by BDR1 and MPK3. Mutation of JA biosynthetic (allene oxide cyclase (AOC)/signaling (MYC2) genes decreased rice resistance to M. oryzae. Besides diterpenoid, the monoterpene linalool and the sesquiterpene caryophyllene were identified as unique defensive compounds against M. oryzae, and their biosynthesis genes (TPS3 and TPS29) were transcriptionally regulated by JA signaling and suppressed by BDR1 and MPK3. These findings demonstrate the existence of a BDR1-MPK3 cascade that negatively mediates rice blast resistance by affecting JA-related defense responses.

Molecular Evolution and Functional Divergence of Stress-Responsive Cu/Zn Superoxide Dismutases in Plants.

Superoxide dismutases (SODs), a family of antioxidant enzymes, are the first line of defense against oxidative damage and are ubiquitous in every cell of all plant types. The Cu/Zn SOD, one of three types of SODs present in plant species, is involved in many of the biological functions of plants in response to abiotic and biotic stresses. Here, we carried out a comprehensive analysis of the Cu/Zn SOD gene family in different plant species, ranging from lower plants to higher plants, and further investigated their organization, sequence features, and expression patterns in response to biotic and abiotic stresses. Our results show that plant Cu/Zn SODs can be divided into two subfamilies (group I and group II). Group II appeared to be conserved only as single- or low-copy genes in all lineages, whereas group I genes underwent at least two duplication events, resulting in multiple gene copies and forming three different subgroups (group Ia, group Ib, and group Ic). We also found that, among these genes, two important events-the loss of introns and the loss of and variation in signal peptides-occurred over the long course of their evolution, indicating that they were involved in shifts in subcellular localization from the chloroplast to cytosol or peroxisome and underwent functional divergence. In addition, expression patterns of Cu/Zn SOD genes from Arabidopsis thaliana and Solanum lycopersicum were tested in different tissues/organs and developmental stages and under different abiotic stresses. The results indicate that the Cu/Zn SOD gene family possesses potential functional divergence and may play vital roles in ROS scavenging in response to various stresses in plants. This study will help establish a foundation for further understanding these genes' function during stress responses.

Effect of Shugan Jianpi Recipe Combined with Cross Moxibustion on Biochemical Examination Indexes and Total Score of TCM Symptoms in Patients with Spleen-Stomach Damp-Heat Diarrhea Irritable Bowel Syndrome.

Objective: To explore the effect of Shugan Jianpi recipe combined with cross moxibustion on biochemical examination indexes and total score of TCM symptoms in patients with spleen-stomach damp-heat diarrhea irritable bowel syndrome (IBS). Methods: Sixty patients with spleen-stomach damp-heat diarrhea (IBS) treated in our hospital from January 2019 to September 2021 were enrolled. The patients were randomly assigned into the control group and study group. The control group was treated with Chang Shugan Jianpi recipe, and the study group was treated with Shugan Jianpi recipe combined with cross moxibustion. The curative effect, single symptom score, total score of TCM clinical symptoms, plasma gastrointestinal hormone level, IBS-QOL score, and recurrence were compared. Results: First of all, we compared the curative effects. The study group was significantly effective in 24 cases, effective in 5 cases, and ineffective in 1 case, and the effective rate was 96.67%. In the control group, 13 cases were markedly effective, 10 cases were effective, and 7 cases were ineffective, and the effective rate was 76.67%. The effective rate of the study group was higher compared to the control group (P < 0.05). Secondly, the individual symptom scores were compared. Compared between the two groups, the scores of diarrhea times, stool characteristics, abdominal pain, and abdominal distension in the study group were lower compared to the control group (P < 0.05). After treatment, the total score of TCM clinical symptoms decreased. Compared between the two groups, the total score of TCM clinical symptoms in the study group was lower compared to the control group at 1 week, 2 weeks, and 4 weeks after treatment (P < 0.05). After treatment, the levels of 5-HT and VIP decreased. The levels of 5-HT and VIP in the study group were lower compared to the control group (P < 0.05). The scores of anxieties, health worry, behavioral disorder, social reaction, somatic intention, interpersonal relationship, dietary concern, and sexual behavior in the study group were lower compared to the control group. The IBS-QOL scores were significantly lower compared to the control group (P < 0.05). Finally, we compared the recurrence. The recurrence rate in the study group was lower compared to the control group (P < 0.05). Conclusion: The prescription combined with cross moxibustion has the effect of soothing the liver and invigorating the spleen, resolving dampness and stomach, and can reduce the main clinical symptoms of patients with diarrhea IBS of spleen-stomach damp-heat type, and the overall effect is significant. It can also enhance the emotional state of anxiety and depression and achieve the overall psychological and physical balance and health state, and the recurrence rate is low, which can be further applied in clinic.

The PAP Gene Family in Tomato: Comprehensive Comparative Analysis, Phylogenetic Relationships and Expression Profiles.

Purple acid phosphatase (PAP) plays a vital role in plant phosphate acquisition and utilization, as well as cell wall synthesis and redox reactions. In this study, comprehensive comparative analyses of PAP genes were carried out using the integration of phylogeny, chromosomal localization, intron/exon structural characteristics, and expression profiling. It was shown that the number of introns of the PAP genes, which were distributed unevenly on 12 chromosomes, ranged from 1 to 12. These findings pointed to the existence of complex structures. Phylogenetic analyses revealed that PAPs from tomato, rice, and Arabidopsis could be divided into three groups (Groups I, II, and III). It was assumed that the diversity of these PAP genes occurred before the monocot-dicot split. RNA-seq analysis revealed that most of the genes were expressed in all of the tissues analyzed, with the exception of SlPAP02, SlPAP11, and SlPAP14, which were not detected. It was also found that expression levels of most of the SlPAP gene family of members were changed under phosphorus stress conditions, suggesting potential functional diversification. The findings of this work will help us to achieve a better insight into the function of SlPAP genes in the future, as well as enhance our understanding of their evolutionary relationships in plants.

Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses.

Plant carotenoid cleavage oxygenase (CCO) is an enzyme that catalyzes the synthesis of carotenoids and participates in many important physiological functions. The plant CCOs exist in two forms, namely carotenoid cleavage dioxygenase (CCD) and nine-cis epoxide carotenoid dioxygenase (NCED). Although studies have shown that this gene family has been identified in many species, such as Arabidopsis, grape, and tomato, the evolutionary origin of the CCO family and the expression pattern of pepper genes in response to H2O2 and other abiotic stresses are still unclear. In this study, we used the bioinformatics method to identify and analyze the members of the CCO gene family from pepper and other 13 plants from lower to higher plant species based on the whole genome sequence. A total of 158 CCO genes were identified in different plant species and further divided into two groups (e.g., groups I and II). The former was subdivided into CCD7 and CCD8 and have independent evolutionary origins, respectively, while the latter was subdivided into CCD1, CCD4, CCD-like, and NCED, which may have come from a common ancestor. In addition, the results of RNA-seq showed that the expression patterns of pepper CaCCO genes were different in the tissues tested, and only few genes were expressed at high levels such as CaCCD1a, CaCCD4a, CaNCED3, and CaCCD1b. For hydrogen peroxide (H2O2) and other abiotic stresses, such as plant hormones, heat, cold, drought, and NaCl treatments, induction of about half of the CaCCO genes was observed. Moreover, the expression patterns of CaCCOs were further investigated under heat, cold, drought, and NaCl treatments using quantitative real-time PCR (qRT-PCR), and most members were responsive to these stresses, especially some CaCCOs with significant expression changes were identified, such as CaCCD4c, CaCCD-like1, CaCCD8, and CaCCD1b, suggesting the important roles of CaCCOs in abiotic stress responses. All these results will provide a valuable analytical basis for understanding the evolution and functions of the CCO family in plants.

Genetic Diversity and Identification of Wilt and Root Rot Pathogens of Tomato in China.

Fungal wilt and root rot diseases affecting tomato have become prevalent in China in recent years and have caused considerable damage. In 2016 to 2018, symptoms of putative wilt and root rot diseases were observed in several locations in tomato cultivars with resistance to Fusarium oxysporum f. sp. lycopersici races 1 and 2. The objective of this study was to identify the causative agents of wilt and root rot of tomato in China and provide a basis for disease prevention and resistance breeding programs. Based on DNA sequence analyses of the internal transcribed spacer (ITS) region, 91 isolates from the roots of tomato plants showing symptoms of wilt and root rot were identified, including F. oxysporum (64 isolates), Fusarium solani (11 isolates), Fusarium proliferatum (2 isolates), Fusarium graminearum (2 isolates), Fusarium equiseti (1 isolate), Pythium aphanidermatum (6 isolates), Ascomycota sp. (2 isolates), and Plectosphaerella cucumerina (3 isolates). F. oxysporum accounted for 70.33% of the isolates obtained. In this case, using PCR-based methods for differentiation of F. oxysporum, we identified several formae speciales and races of F. oxysporum: 7 isolates were identified as F. oxysporum f. sp. lycopersici race 1, 2 isolates as F. oxysporum f. sp. lycopersici race 2, 35 isolates as F. oxysporum f. sp. lycopersici race 3, and 13 isolates as F. oxysporum f. sp. radicis-lycopersici. Pathogenicity tests revealed 55 isolates of tomato wilt and root rot pathogens to be virulent. This study demonstrated that F. oxysporum f. sp. lycopersici race 3 was the most widespread and highly virulent race among these tomato pathogens in China, followed by F. oxysporum f. sp. radicis-lycopersici. Therefore, the development of resistant varieties of tomato against F. oxysporum f. sp. lycopersici race 3 and F. oxysporum f. sp. radicis-lycopersici would aid efforts to develop effective disease management strategies.

Genomic Organization and Comparative Phylogenic Analysis of NBS-LRR Resistance Gene Family in Solanum pimpinellifolium and Arabidopsis thaliana.

NBS-LRR (nucleotide-binding site and leucine-rich repeat) is one of the largest resistance gene families in plants. The completion of the genome sequencing of wild tomato Solanum pimpinellifolium provided an opportunity to conduct a comprehensive analysis of the NBS-LRR gene superfamily at the genome-wide level. In this study, gene identification, chromosome mapping, and phylogenetic analysis of the NBS-LRR gene family were analyzed using the bioinformatics methods. The results revealed 245 NBS-LRRs in total, similar to that in the cultivated tomato. These genes are unevenly distributed on 12 chromosomes, and ~59.6% of them form gene clusters, most of which are tandem duplications. Phylogenetic analysis divided the NBS-LRRs into 2 subfamilies (CNL-coiled-coil NBS-LRR and TNL-TIR NBS-LRR), and the expansion of the CNL subfamily was more extensive than the TNL subfamily. Novel conserved structures were identified through conserved motif analysis between the CNL and TNL subfamilies. Compared with the NBS-LRR sequences from the model plant Arabidopsis thaliana, wide genetic variation occurred after the divergence of S. pimpinellifolium and A thaliana. Species-specific expansion was also found in the CNL subfamily in S. pimpinellifolium. The results of this study provide the basis for the deeper analysis of NBS-LRR resistance genes and contribute to mapping and isolation of candidate resistance genes in S. pimpinellifolium.

Evolutionary Conservation and Expression Patterns of Neutral/Alkaline Invertases in Solanum.

The invertase gene family in plants is composed of two subfamilies of enzymes, namely, acid- and neutral/alkaline invertases (cytosolic invertase, CIN). Both can irreversibly cleave sucrose into fructose and glucose, which are thought to play key roles in carbon metabolism and plant growth. CINs are widely found in plants, but little is reported about this family. In this paper, a comparative genomic approach was used to analyze the CIN gene family in Solanum, including Solanumtuberosum, Solanumlycopersicum, Solanumpennellii, Solanumpimpinellifolium, and Solanummelongena. A total of 40 CINs were identified in five Solanum plants, and sequence features, phylogenetic relationships, motif compositions, gene structure, collinear relationship, and expression profile were further analyzed. Sequence analysis revealed a remarkable conservation of CINs in sequence length, gene number, and molecular weight. The previously verified four amino acid residues (D188, E414, Arg430, and Ser547) were also observed in 39 out of 40 CINs in our study, showing to be deeply conserved. The CIN gene family could be distinguished into groups α and ß, and α is further subdivided into subgroups α1 and α2 in our phylogenetic tree. More remarkably, each species has an average of four CINs in the α and ß groups. Marked interspecies conservation and collinearity of CINs were also further revealed by chromosome mapping. Exon-intron configuration and conserved motifs were consistent in each of these α and ß groups on the basis of in silico analysis. Expression analysis indicated that CINs were constitutively expressed and share similar expression profiles in all tested samples from S. tuberosum and S.lycopersicum. In addition, in CIN genes of the tomato and potato in response to abiotic and biotic stresses, phytohormones also performed. Overall, CINs in Solanum were encoded by a small and highly conserved gene family, possibly reflecting structural and functional conservation in Solanum. These results lay the foundation for further expounding the functional characterization of CIN genes and are also significant for understanding the evolutionary profiling of the CIN gene family in Solanum.

Comparative Genomic Analysis Reveals Extensive Genetic Variations of WRKYs in Solanaceae and Functional Variations of CaWRKYs in Pepper.

As a conserved protein family, WRKY has been shown to be involved in multiple biological processes in plants. However, the mechanism of functional diversity for WRKYs in pepper has not been well elucidated. Here, a total of 223 WRKY members from solanaceae crops including pepper, tomato and potato, were analyzed using comparative genomics. A tremendous genetic variation among WRKY members of different solanaceous plants or groups was demonstrated by the comparison of some WRKY features, including number/size, group constitution, gene structure, and domain composition. The phylogenetic analysis showed that except for the known WRKY groups (I, IIa/b/c/d/e and III), two extra WRKY subgroups specifically existed in solanaceous plants, which were named group IIf and group IIg in this study, and their genetic variations were also revealed by the characteristics of some group IIf and IIg WRKYs. Except for the extensive genetic variations, certain degrees of conservatism for solanaceae WRKYs were also revealed. Moreover, the variant zinc-finger structure (CX4,7CX22-24HXC) in group III of solanaceae WRKYs was identified. Expression profiles of CaWRKY genes suggested their potential roles in pepper development and stress responses, and demonstrated a functional division pattern for pepper CaWRKYs. Furthermore, functional analysis using virus induced gene silencing (VIGS) revealed critical roles of two CaWRKYs (CaWRKY45 and CaWRKY58) in plant responses to disease and drought, respectively. This study provides a solid foundation for further dissection of the evolutionary and functional diversity of solanaceae WRKYs in crop plants.

The Tryptophan Decarboxylase in Solanum lycopersicum.

Melatonin plays an important role in plant growth, development, and environmental stress. In this study, a systematic analysis of tomato tryptophan decarboxylase (SlTrpDC), which is the first enzyme of melatonin biosynthesis, was conducted by integrating structural features, phylogenetic relationships, an exon/intron feature, and a divergent expression profile. The results determined that the tomato genome encoded five members (SlTrpDC1-SlTrpDC5). The phylogenetic relationships indicated that gene expansion was proposed as the major mode of evolution of the TrpDC genes from the different plant algae species to the higher plants species. The analyses of the exon/intron configurations revealed that the intron loss events occurred during the structural evolution of the TrpDCs in plants. Additionally, the RNA-seq and qRT-PCR analysis revealed that the expression of the SlTrpDC3 was high in all of the tested tissues, while the SlTrpDC4 and SlTrpDC5 were not expressed. The expression patterns of the remaining two (SlTrpDC1 and SlTrpDC2) were tissue-specific, which indicated that these genes may play important roles within the different tissues. No expression difference was observed in the tomato plants in response to the biotic stresses. This study will expand the current knowledge of the roles of the TrpDC genes in tomato growth and development.

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development.

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

Identification of Optimal Reference Genes for Normalization of qPCR Analysis during Pepper Fruit Development.

Due to its high sensitivity and reproducibility, quantitative real-time PCR (qPCR) is practiced as a useful research tool for targeted gene expression analysis. For qPCR operations, the normalization with suitable reference genes (RGs) is a crucial step that eventually determines the reliability of the obtained results. Although pepper is considered an ideal model plant for the study of non-climacteric fruit development, at present no specific RG have been developed or validated for the qPCR analyses of pepper fruit. Therefore, this study aimed to identify stably expressed genes for their potential use as RGs in pepper fruit studies. Initially, a total of 35 putative RGs were selected by mining the pepper transcriptome data sets derived from the PGP (Pepper Genome Platform) and PGD (Pepper Genome Database). Their expression stabilities were further measured in a set of pepper (Capsicum annuum L. var. 007e) fruit samples, which represented four different fruit developmental stages (IM: Immature; MG: Mature green; B: Break; MR: Mature red) using the qPCR analysis. Then, based on the qPCR results, three different statistical algorithms, namely geNorm, Normfinder, and boxplot, were chosen to evaluate the expression stabilities of these putative RGs. It should be noted that nine genes were proven to be qualified as RGs during pepper fruit development, namely CaREV05 (CA00g79660); CaREV08 (CA06g02180); CaREV09 (CA06g05650); CaREV16 (Capana12g002666); CaREV21 (Capana10g001439); CaREV23 (Capana05g000680); CaREV26 (Capana01g002973); CaREV27 (Capana11g000123); CaREV31 (Capana04g002411); and CaREV33 (Capana08g001826). Further analysis based on geNorm suggested that the application of the two most stably expressed genes (CaREV05 and CaREV08) would provide optimal transcript normalization in the qPCR experiments. Therefore, a new and comprehensive strategy for the identification of optimal RGs was developed. This strategy allowed for the effective normalization of the qPCR analysis of the pepper fruit development at the whole pepper genome level. This study not only explored the optimal RGs specific for studying pepper fruit development, but also introduced a referable strategy of RG mining which could potentially be implicated in other plant species.

Putative WRKYs associated with regulation of fruit ripening revealed by detailed expression analysis of the WRKY gene family in pepper.

WRKY transcription factors play important roles in plant development and stress responses. Here, global expression patterns of pepper CaWRKYs in various tissues as well as response to environmental stresses and plant hormones were systematically analyzed, with an emphasis on fruit ripening. The results showed that most CaWRKYs were expressed in at least two of the tissues tested. Group I, a subfamily of the entire CaWRKY gene family, had a higher expression level in vegetative tissues, whereas groups IIa and III showed relatively lower expression levels. Comparative analysis showed that the constitutively highly expressed WRKY genes were conserved in tomato and pepper, suggesting potential functional similarities. Among the identified 61 CaWRKYs, almost 60% were expressed during pepper fruit maturation, and the group I genes were in higher proportion during the ripening process, indicating an as-yet unknown function of group I in the fruit maturation process. Further analysis suggested that many CaWRKYs expressed during fruit ripening were also regulated by abiotic stresses or plant hormones, indicating that these CaWRKYs play roles in the stress-related signaling pathways during fruit ripening. This study provides new insights to the current research on CaWRKY and contributes to our knowledge about the global regulatory network in pepper fruit ripening.

The SOD Gene Family in Tomato: Identification, Phylogenetic Relationships, and Expression Patterns.

Superoxide dismutases (SODs) are critical antioxidant enzymes that protect organisms from reactive oxygen species (ROS) caused by adverse conditions, and have been widely found in the cytoplasm, chloroplasts, and mitochondria of eukaryotic and prokaryotic cells. Tomato (Solanum lycopersicum L.) is an important economic crop and is cultivated worldwide. However, abiotic and biotic stresses severely hinder growth and development of the plant, which affects the production and quality of the crop. To reveal the potential roles of SOD genes under various stresses, we performed a systematic analysis of the tomato SOD gene family and analyzed the expression patterns of SlSOD genes in response to abiotic stresses at the whole-genome level. The characteristics of the SlSOD gene family were determined by analyzing gene structure, conserved motifs, chromosomal distribution, phylogenetic relationships, and expression patterns. We determined that there are at least nine SOD genes in tomato, including four Cu/ZnSODs, three FeSODs, and one MnSOD, and they are unevenly distributed on 12 chromosomes. Phylogenetic analyses of SOD genes from tomato and other plant species were separated into two groups with a high bootstrap value, indicating that these SOD genes were present before the monocot-dicot split. Additionally, many cis-elements that respond to different stresses were found in the promoters of nine SlSOD genes. Gene expression analysis based on RNA-seq data showed that most genes were expressed in all tested tissues, with the exception of SlSOD6 and SlSOD8, which were only expressed in young fruits. Microarray data analysis showed that most members of the SlSOD gene family were altered under salt- and drought-stress conditions. This genome-wide analysis of SlSOD genes helps to clarify the function of SlSOD genes under different stress conditions and provides information to aid in further understanding the evolutionary relationships of SOD genes in plants.

Genome-Wide Identification and Expression Profiling of Tomato Hsp20 Gene Family in Response to Biotic and Abiotic Stresses.

The Hsp20 genes are involved in the response of plants to environment stresses including heat shock and also play a vital role in plant growth and development. They represent the most abundant small heat shock proteins (sHsps) in plants, but little is known about this family in tomato (Solanum lycopersicum), an important vegetable crop in the world. Here, we characterized heat shock protein 20 (SlHsp20) gene family in tomato through integration of gene structure, chromosome location, phylogenetic relationship, and expression profile. Using bioinformatics-based methods, we identified at least 42 putative SlHsp20 genes in tomato. Sequence analysis revealed that most of SlHsp20 genes possessed no intron or a relatively short intron in length. Chromosome mapping indicated that inter-arm and intra-chromosome duplication events contributed remarkably to the expansion of SlHsp20 genes. Phylogentic tree of Hsp20 genes from tomato and other plant species revealed that SlHsp20 genes were grouped into 13 subfamilies, indicating that these genes may have a common ancestor that generated diverse subfamilies prior to the mono-dicot split. In addition, expression analysis using RNA-seq in various tissues and developmental stages of cultivated tomato and the wild relative Solanum pimpinellifolium revealed that most of these genes (83%) were expressed in at least one stage from at least one genotype. Out of 42 genes, 4 genes were expressed constitutively in almost all the tissues analyzed, implying that these genes might have specific housekeeping function in tomato cell under normal growth conditions. Two SlHsp20 genes displayed differential expression levels between cultivated tomato and S. pimpinellifolium in vegetative (leaf and root) and reproductive organs (floral bud and flower), suggesting inter-species diversification for functional specialization during the process of domestication. Based on genome-wide microarray analysis, we showed that the transcript levels of SlHsp20 genes could be induced profusely by abiotic and biotic stresses such as heat, drought, salt, Botrytis cinerea, and Tomato Spotted Wilt Virus (TSWV), indicating their potential roles in mediating the response of tomato plants to environment stresses. In conclusion, these results provide valuable information for elucidating the evolutionary relationship of Hsp20 gene family and functional characterization of the SlHsp20 gene family in the future.

[Genome-wide identification and analysis of heat shock protein 90 in tomato].

Heat shock proteins 90 (Hsp90) are a kind of specific proteins in plant which were produced under environmental stresses. By referring to the tomato genome database, we identified and analyzed Hsp90 gene family members using bioinformatics methods. Results indicated that the tomato genome contained at least 7 Hsp90 genes, which were distributed unevenly on 6 chromosomes. Amino acid sequence length of these proteins ranged from 267 to 794aa. Numbers of intron ranged from 2 to 19. Microsynteny analysis showed that two pairs of Hsp90 genes (Hsp90-1and Hsp90-3, Hsp90-5 and Hsp90-7) were identified by segment duplication. In addition, multiple conservation motifs were found in Hsp90 proteins. Phylogenetic analysis revealed that Hsp90 genes from tomato, rice and Arabidopsis can be divided into 5 groups. Three pair of orthologous genes and four pairs of homologous genes were found. Expression analysis based on RNA-seq showed that the expression of three genes (Hsp90-5, Hsp90-6 and Hsp90-7) was high in vegetable and reproductive organs, while the expression of other four genes (Hsp90-1, Hsp90-2, Hsp90-3 and Hsp90-4) was relatively low except for its expression at the breaking stage of fruit. Analysis of promoter regions of Hsp90 genes showed that multiple cis-elements were involved in plant responses to biotic and abiotic stresses. The expression of 7 genes under heat stress was also detected by qRT-PCR. Expression of all Hsp90 genes in tomato leaf was enhanced. The results indicated that these genes could be participated in tomato leaf response to heat stresses. Together, these results will lay a foundation for analyzing Hsp90 gene function and molecular evolution in the future.