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.

Effects of Storage Temperature at the Early Postharvest Stage on the Firmness, Bioactive Substances, and Amino Acid Compositions of Chili Pepper (Capsicum annuum L.).

The commercial and nutritional quality of chili peppers deteriorates rapidly after harvest. So far, little is known about the effect of temperature on postharvest chili pepper quality. This study elucidated the effects of two temperatures (20 °C and 30 °C) on chili peppers' postharvest firmness, flavor, and nutritional attributes. We found that compared to 20 °C, 30 °C escalated the decline in fruit firmness, capsaicin content, and dihydrocapsaicin content, while enhancing the increment in water loss and electrical conductivity, as well as total carotenoids and ascorbic acid content. The contents of most amino acids (AAs) decreased significantly during postharvest storage compared to their initial values, whether stored at 20 °C or 30 °C; however, 30 °C had a more substantial impact than 20 °C. Meanwhile, as for soluble protein and amino acid compositions, the effect of storage temperature was genotype-dependent, as reflected by differential changes in total AA contents, single AA contents, essential AA ratio, delicious AA ratio, etc., under the 20 °C or 30 °C treatments. In conclusion, our findings reveal the influence of temperature on pepper quality, showing that the storage temperature of 20 °C was better for maintaining chili quality than 30 °C from the perspective of overall commercial attributes.

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.

Post-Harvest LED Light Irradiation Affects Firmness, Bioactive Substances, and Amino Acid Compositions in Chili Pepper (Capsicum annum L.).

Chili pepper is an important vegetable and spice crop with high post-harvest deteriorations in terms of commercial and nutritional quality. Light-emitting diodes (LEDs) are eco-friendly light sources with various light spectra that have been demonstrated to improve the shelf-life of various vegetables by manipulating light quality; however, little is known about their effects on the post-harvest nutritional quality of chili peppers. This study investigated the effects of different LED lightings on the post-harvest firmness and nutritional quality of chili peppers. We found that red and blue light could increase the content of capsaicinoids, whereas white and red light could increase the essential and aromatic amino acid (AA) content in pepper. Nonetheless, the influence of light treatments on AA contents and compositions depends strongly on the pepper genotype, which was reflected by total AA content, single AA content, essential AA ratio, delicious AA ratio, etc., that change under different light treatments. Additionally, light affected fruit firmness and the content of nutrients such as chlorophyll, vitamin C, and total carotenoids, to varying degrees, depending on pepper genotypes. Thus, our findings indicate that LED-light irradiation is an efficient and promising strategy for preserving or improving the post-harvest commercial and nutritional quality of pepper fruit.

Systemic H2O2 signaling mediates epigallocatechin-3-gallate-induced cadmium tolerance in tomato.

Toxic heavy metal cadmium (Cd) reduces crop yield and threatens human health via the food chain. The bioactive flavonoid 'Epigallocatechin-3-gallate' (EGCG) affects plant stress response; however, the function of EGCG in Cd tolerance and the molecular pathways remain largely unknown. Here, we revealed that root application of EGCG alleviated Cd stress in tomato plants. While Cd stress decreased Fv/Fm, ФPSII, photosynthetic rate, root growth, root vitality and biomass accumulation by increasing reactive oxygen species (ROS) accumulation and lipid peroxidation, exogenous EGCG minimized excessive ROS accumulation and oxidative stress by promoting the activity of antioxidant enzymes and redox poise in roots and leaves. Moreover, EGCG induced the transcript of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and decreased Cd content and photoinhibition in leaves. Interestingly, similar to EGCG, exogenous H2O2 application also enhanced Cd tolerance; however, the application of an NADPH oxidase inhibitor, diphenyleneiodonium (DPI), aggravated Cd phytotoxicity and attenuated the beneficial effects of EGCG on plant tolerance to Cd stress, suggesting that root applied EGCG-induced expression of RBOH1 and associated H2O2 signaling mediate the EGCG-induced enhanced Cd tolerance. This work elucidates a fundamental mechanism behind EGCG-mediated Cd tolerance and contributes to our existing knowledge of stress resistance properties of EGCG in plants.

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.

[Ultrasound-guided electroacupuncture at suprahyoid muscle group for pharyngeal dysphagia after stroke: a randomized controlled trial].

OBJECTIVE: To compare the effect among ultrasound-guided electroacupuncture (EA) at suprahyoid muscle group, conventional acupuncture and conventional EA at suprahyoid muscle group on pharyngeal dysphagia after stroke, and to explore its biomechanical mechanism. METHODS: A total of 120 patients with pharyngeal dysphagia after stroke were randomly divided into an observation group, a control-1 group and a control-2 group, 40 cases in each group. The patients in the observation group were treated with ultrasound-guided EA at suprahyoid muscle group; the patients in the control-1 group were treated with EA at Lianquan (CV 23), Wangu (GB 12) and Fengchi (GB 20), etc.; the patients in the control-2 group were treated with EA at suprahyoid muscle group according to anatomical location. The EA in the three groups were discontinuous wave, with frequency of 5 Hz and current intensity of 1 mA. The EA was given for 30 minutes, once a day, 6 times were taken as a course of treatment, and 4 courses of treatment were provided. The video floroscopic swallowing study (VFSS) was performed before and after treatment. The Rosenbek penetration-aspiration scale (PAS) score, the forward and upward movement distance of hyoid bone and thyroid cartilage, Ichiro Fujima ingestion-swallowing function score were recorded in the three groups, and the incidences of subcutaneous hematoma were recorded after treatment. RESULTS: Compared before treatment, the PAS scores were reduced and the Ichiro Fujima ingestion-swallowing function scores were increased after treatment in the three groups (P<0.05); the PAS scores in the observation group were lower than those in the control-1 group and the control-2 group, and the Ichiro Fujima ingestion-swallowing function scores in the observation group were higher than those in the control-1 group and the control-2 group (P<0.05). After treatment, the forward and upward movement distance of hyoid bone and thyroid cartilage in the observation group and the control-2 group was increased (P<0.05), and the forward and upward movement distance of hyoid bone was increased in the control-1 group (P<0.05); the forward and upward movement distance of hyoid bone and thyroid cartilage in the observation group was longer than that in the control-1 group and the control-2 group (P<0.05). The incidence of subcutaneous hematoma in the observation group was 0% (0/40), which was lower than 20.0% (8/40) in the control-1 group and 47.5% (19/40) in the control-2 group (P<0.05). CONCLUSION: Ultrasound-guided EA at suprahyoid muscle group could improve the swallowing function in patients with pharyngeal dysphagia after stroke by increasing the motion of hyoid laryngeal complex. Its effect and safety are better than conventional acupuncture and conventional EA at suprahyoid muscle group.

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.

[Effect of electrical stimulation with bilateral scalp acupuncture on time parameters in video fluoroscopic swallowing study and cortical excitability in patients with dysphagia after cortical stroke].

OBJECTIVE: To investigate the effect of electrical stimulation with bilateral scalp acupuncture on time parameters in video fluoroscopic swallowing study and cortical excitability in patients with dysphagia after cortical stroke, as well as its possible mechanism of action. METHODS: A total of 84 patients with dysphagia after cortical stroke were randomly divided into control group with 41 patients and observation group with 43 patients. The patients in the control group were given acupuncture based on acupoint selection for pseudobulbar palsy, and in addition to the treatment in the control group, the patients in the observation group were given electrical stimulation of bilateral scalp acupuncture, with acupuncture and pulse acupuncture at the lower 2/5 of the bilateral anterior oblique parietotemporal lines[on the line connecting Qianshencong (EX-HN1) to Xuanli (GB6)] and the lower 2/5 of the bilateral posterior oblique parietotemporal lines [on the line connecting Baihui (GV20)and Qubin (GB7)], with a needle retaining time of 30 minutes, once a day and 6 times a week for 3 weeks. Before treatment and after 3 weeks of treatment, oral delay time (ODT), oral transit time (OTT), pharyngeal delay time (PDT), and pharyngeal transit time (PTT) were compared between the two group; the Rosenbek Penetration-Aspiration Scale was used to evaluate penetration-aspiration and appro-ximate entropy (ApEn) of EEG nonlinear index. RESULTS: After treatment, both groups had significant reductions in ODT, OTT, PDT, PTT, and Rosenbek Penetration-Aspiration score and a significant increase in ApEn (P<0.05). Compared with the control group after treatment, the observation group had significant reductions in ODT and OTT (P<0.05) and significant increases in the ApEn values of bilateral central, parietal, and posterior temporal regions (P<0.05), while there were no significant differences in PDT, PTT, and Rosenbek Penetration-Aspiration score between the two groups (P>0.05). CONCLUSION: In addition to body acupuncture, electrical stimulation with bilateral scalp acupuncture can improve ODT and OTT in the treatment of patients with dysphagia after cortical stroke, which may be associated with the increased excitability of the swallowing cortex.

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.