Zhimiao Li

Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L.)

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technology for gene expression and transcriptome analysis. Normalization is a process that is necessary to accurately analyze qRT-PCR data. Stability of reference gene expression is required for this process. Due to the large variation in expression levels of reference genes obtained from different experimental conditions, gene expression stabilities must be evaluated and identified in all experimental systems. In the present paper, the stability of the expression levels of seven potential reference genes in pepper are assessed using qRT-PCR analysis to determine optimal reference genes. These reference genes are evaluated in different pepper tissues, abiotic stress, and hormonal treatment samples. Three common statistical algorithms, geNorm, NormFinder, and BestKeeper, are used to identify expression stability and provide an accurate selection of reference genes. Two reference genes, beta tubulin and ubiquitin-conjugating protein (UBI-3), showed high stability in sample pools with abiotic stress and hormonal treatments. Among the sample pools tested, UBI-3 and glyceraldehyde-3-phosphate dehydrogenase expression levels were the most stable in different tissues. Therefore, these reference genes are selected for qRT-PCR analysis under the experimental conditions tested in pepper. In contrast, ubiquitin-conjugating enzyme and actin genes are identified as the least stable reference genes in all the groups tested, confirming that they are not suitable for normalization. Validation of these candidate genes could provide useful guidelines for reference gene selection in qRT-PCR studies in pepper.

High invertase activity in tomato reproductive organs correlates with enhanced sucrose import into, and heat tolerance of, young fruit

Heat stress can cause severe crop yield losses by impairing reproductive development. However, the underlying mechanisms are poorly understood. We examined patterns of carbon allocation and activities of sucrose cleavage enzymes in heat-tolerant (HT) and -sensitive (HS) tomato (Solanum lycopersicum L.) lines subjected to normal (control) and heat stress temperatures. At the control temperature of 25/20 °C (day/night) the HT line exhibited higher cell wall invertase (CWIN) activity in flowers and young fruits and partitioned more sucrose to fruits but less to vegetative tissues as compared to the HS line, independent of leaf photosynthetic capacity. Upon 2-, 4-, or 24-h exposure to day or night temperatures of 5 °C or more above 25/20 °C, cell wall (CWIN) and vacuolar invertases (VIN), but not sucrose synthase (SuSy), activities in young fruit of the HT line were significantly higher than those of the HS line. The HT line had a higher level of transcript of a CWIN gene, Lin7, in 5-day fruit than the HS line under control and heat stress temperatures. Interestingly, heat induced transcription of an invertase inhibitor gene, INVINH1, but reduced its protein abundance. Transcript levels of LePLDa1, encoding phospholipase D, which degrades cell membranes, was less in the HT line than in the HS line after exposure to heat stress. The data indicate that high invertase activity of, and increased sucrose import into, young tomato fruit could contribute to their heat tolerance through increasing sink strength and sugar signalling activities, possibly regulating a programmed cell death pathway.

Analysis of TIR- and non-TIR-NBS-LRR disease resistance gene analogous in pepper: characterization, genetic variation, functional divergence and expression patterns

BackgroundPepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. However, its yield and fruit quality can be severely threatened by several pathogens. The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens. Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs.ResultsA total of 78 R gene analogues (CaRGAs) were identified in pepper by degenerate PCR amplification and database mining. Phylogenetic tree analysis of the deduced amino acid sequences for 51 of these CaRGAs with typically conserved motifs ( P-loop, kinase-2 and GLPL) along with some known R genes from Arabidopsis and tomato grouped these CaRGAs into the non-Toll interleukin-1 receptor (TIR)-NBS-LRR (CaRGAs I to IV) and TIR-NBS-LRR (CaRGAs V to VII) subfamilies. The presence of consensus motifs (i.e. P-loop, kinase-2 and hydrophobic domain) is typical of the non-TIR- and TIR-NBS-LRR gene subfamilies. This finding further supports the view that both subfamilies are widely distributed in dicot species. Functional divergence analysis provided strong statistical evidence of altered selective constraints during protein evolution between the two subfamilies. Thirteen critical amino acid sites involved in this divergence were also identified using DIVERGE version 2 software. Analyses of non-synonymous and synonymous substitutions per site showed that purifying selection can play a critical role in the evolutionary processes of non-TIR- and TIR-NBS-LRR RGAs in pepper. In addition, four specificity-determining positions were predicted to be responsible for functional specificity. qRT-PCR analysis showed that both salicylic and abscisic acids induce the expression of CaRGA genes, suggesting that they may primarily be involved in defence responses by activating signaling pathways.ConclusionThe identified CaRGAs are a valuable resource for discovering R genes and developing RGA molecular markers for genetic map construction. They will also be useful for improving disease resistance in pepper. The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.

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.

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.

A Comprehensive Analysis of Carotenoid Cleavage Dioxygenases Genes in Solanum Lycopersicum

Carotenoid cleavage dioxygenases (CCDs) in plant species is one of the most important enzymes in the carotenoid metabolism. In this study, we performed a comprehensive analysis for the CCDs family in Solanum lycopersicum based on the whole tomato genome sequences and explored their expression pattern. At least seven CCD genes were discovered in the tomato genome sequence. Two pairs of them were arranged in tandem. The tandem duplication events could be dating to approximately 14 and 21 Mya, and the tandem duplication genes experienced a purifying selection during the course of evolution after diversification. Additionally, subcellular localization revealed that four members were predicted to be cytoplasm-localized and the three remaining members plastids-localized. Subsequently, a number of cis-regulatory elements, which were involved in light responsiveness, hormone regulation, and abiotic and biotic stresses, were identified in the promoter sequences of SlCCD genes. Phylogenetic tree revealed that the CCDs from Solanaceae crops have a closer genetic relationship. The difference in abundance and distinct expression patterns during the vegetative and reproductive development suggests different functions for these seven SlCCDs. Our findings suggest that SlCCDs family play important roles throughout the whole life course and will lay the foundation for further elaborating the regulatory mechanism of each member in tomato.

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.

Identification, Phylogeny, and Expression Analysis of Pto-like Genes in Pepper

The Pto gene from the wild tomato (Solanum pimpinellifolium Mill.) encodes a serine/threonine kinase that plays an important role in bacterial speck resistance in the cultivated tomato (Solanum lycopersicum Mill.). In this paper, 10 classes of Pto-like genes are identified using degenerate polymerase chain reaction (PCR) primers and database mining in pepper. Sequences alignment reveals that many features of the gene family, such as subdomains, autophosphorylation sites, and important amino acid residues for tomato Pto, are well conserved in pepper. A phylogenetic tree of pepper Pto-like genes along with those of other plant species, including tomato Pto genes, suggests that these genes share a common evolutionary origin, and they may have evolved prior to the divergence of monocotyledonous and dicotyledonous plants. Expression analysis has revealed that nine selected Pto-like genes can be detected in at least one of the tissues grown under normal growth conditions; however, these genes are differentially expressed. In addition, some of these genes are regulated by at least one of the subjected treatments, including hormones, abiotic stress, and pathogen infection. These findings will contribute to expanding our knowledge of the roles of Pto-like genes in growth, development, and stress tolerance in pepper.

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.

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.