Jianjun Lei

De Novo Transcriptome Assembly in Chili Pepper (Capsicum frutescens) to Identify Genes Involved in the Biosynthesis of Capsaicinoids

The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (Capsicum frutescens L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for de novo assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (C. annuum) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in C. frutescens and C. annuum sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of de novo transcriptome assembly is applicable to a wide range of similar studies.

Functional characterization of the translationally controlled tumor protein (TCTP) gene associated with growth and defense response in cabbage

The translationally controlled tumor protein (TCTP) is an important component of the target of rapamycin (TOR) signaling pathway, the major regulator of cell growth in animals and fungi. Despite its relevance, knowledge on plant TCTP homologs is still limited. In the present study, the full-length BoTCTP cDNA was isolated from a cabbage (Brassica oleracea L.) cDNA library. The BoTCTP cDNA encodes a polypeptide of 168 amino acids and shared the highly conserved GTPase binding surface in all of the species analyzed. Northern blotting analysis showed that BoTCTP was specifically expressed in the root and stem. Furthermore, the expression of BoTCTP could be obviously enhanced by stress stimuli, including high temperature and salt stresses, while no significant changes in the BoTCTP expression were observed under ABA stress. Functional analysis of BoTCTP was performed by the silencing of BoTCTP using RNA interference (RNAi) and the BoTCTP RNAi plants exhibited reduced vegetative growth rate and decreased tolerance of the cold, high temperature, and salt stresses. The reported results clearly suggest that the BoTCTP gene is involved in the regulation of both growth and stress response in cabbage.

CaMF2, an anther-specific lipid transfer protein (LTP) gene, affects pollen development in Capsicum annuum L.

Based on the gene differential expression analysis performed by cDNA-amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile-fertile line 114AB of Capsicum annuum L., a variety of differentially expressed cDNA fragments were detected in fertile or sterile lines. A transcript-derived fragment (TDF) specifically accumulated in the flower buds of fertile line was isolated, and the corresponding full-length cDNA and DNA were subsequently amplified. Bioinformatical analyses of this gene named CaMF2 showed that it encodes a lipid transfer protein with 94 amino acids. Spatial and temporal expression patterns analysis indicated that CaMF2 was an anther-specific gene and the expression of CaMF2 was detected only in flower buds at stage 3-7 of male fertile line with a peak expression at stage 4, but not detected in the roots, tender stems, fresh leaves, flower buds, open flowers, sepals, petals, anthers or pistils of male sterile line. Further, inhibition of the CaMF2 by virus-induced gene silencing (VIGS) method resulted in the low pollen germination ability and shriveled pollen grains. All these evidence showed that CaMF2 had a vital role in pollen development of C. annuum.

Purple foliage coloration in tea ( Camellia sinensis L.) arises from activation of the R2R3-MYB transcription factor CsAN1

Purple foliage always appears in Camellia sinensis families; however, the transcriptional regulation of anthocyanin biosynthesis is unknown. The tea bud sport cultivar ‘Zijuan’ confers an abnormal pattern of anthocyanin accumulation, resulting in a mutant phenotype that has a striking purple color in young foliage and in the stem. In this study, we aimed to unravel the underlying molecular mechanism of anthocyanin biosynthetic regulation in C. sinensis. Our results revealed that activation of the R2R3-MYB transcription factor (TF) anthocyanin1 (CsAN1) specifically upregulated the bHLH TF CsGL3 and anthocyanin late biosynthetic genes (LBGs) to confer ectopic accumulation of pigment in purple tea. We found CsAN1 interacts with bHLH TFs (CsGL3 and CsEGL3) and recruits a WD-repeat protein CsTTG1 to form the MYB-bHLH-WDR (MBW) complex that regulates anthocyanin accumulation. We determined that the hypomethylation of a CpG island in the CsAN1 promoter is associated with the purple phenotype. Furthermore, we demonstrated that low temperature and long illumination induced CsAN1 promoter demethylation, resulting in upregulated expression to promote anthocyanin accumulation in the foliage. The successful isolation of CsAN1 provides important information on the regulatory control of anthocyanin biosynthesis in C. sinensis and offers a genetic resource for the development of new varieties with enhanced anthocyanin content.

RNA-sequencing tag profiling of the placenta and pericarp of pungent pepper provides robust candidates contributing to capsaicinoid biosynthesis

Capsaicinoids, metabolites produced in plant placental (seed-bearing) tissue, are responsible for the pungent taste associated with fruits of the genus Capsicum. To identify genes involved in the biosynthesis of capsaicinoids, genome-wide transcriptional profiling of the placenta and pericarp of pungent pepper (C. frutescens L.) was performed using the RNA sequencing (RNA-Seq) approach. The results revealed 4,092 genes with significantly different expression levels between the placenta and pericarp libraries. Among them, 2,049 genes were up-regulated and 2,043 genes were down-regulated in the placenta compared to those of the pericarp. Hundreds of genes potentially regulating capsaicinoid were identified from the up-regulated genes, including genes previously associated with this metabolic process. Subsequent analysis of expression patterns for a subset of these identified genes were verified by semi-quantitative RT-PCR and quantitative RT-PCR. Furthermore, The differential expressed genes were predicted to be involved in microbody, peroxisome, fatty-acid synthase activity, CoA-ligase activity, acyltransferase activity, transaminase activity, phenylalanine metabolism, and other processes through gene ontology and KEGG enrichment analysis. Together, this study provides novel insights into the molecular mechanisms of capsaicinoid biosynthesis.

Overexpression of AtEDT1/HDG11 in Chinese Kale (Brassica oleracea var. alboglabra) Enhances Drought and Osmotic Stress Tolerance.

Plants are constantly challenged by environmental stresses, including drought and high salinity. Improvement of drought and osmotic stress tolerance without yield decrease has been a great challenge in crop improvement. The Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a protein of the class IV HD-Zip family, has been demonstrated to significantly improve drought tolerance in Arabidopsis, rice, and pepper. Here, we report that AtEDT1/HDG11 confers drought and osmotic stress tolerance in the Chinese kale. AtEDT1/HDG11-overexpression lines exhibit auxin-overproduction phenotypes, such as long hypocotyls, tall stems, more root hairs, and a larger root system architecture. Compared with the untransformed control, transgenic lines have significantly reduced stomatal density. In the leaves of transgenic Chinese kale plants, proline (Pro) content and reactive oxygen species-scavenging enzyme activity was significantly increased after drought and osmotic stress, particularly compared to wild kale. More importantly, AtEDT1/HDG11-overexpression leads to abscisic acid (ABA) hypersensitivity, resulting in ABA inhibitor germination and induced stomatal closure. Consistent with observed phenotypes, the expression levels of auxin, ABA, and stress-related genes were also altered under both normal and/or stress conditions. Further analysis showed that AtEDT1/HDG11, as a transcription factor, can target the auxin biosynthesis gene YUCC6 and ABA response genes ABI3 and ABI5. Collectively, our results provide a new insight into the role of AtEDT1/HDG11 in enhancing abiotic stress resistance through auxin- and ABA-mediated signaling response in Chinese kale.

Transcriptional profiling analysis of genic male sterile–fertile Capsicum annuum reveal candidate genes for pollen development and maturation by RNA-Seq technology

Abstract To identify genes involved in the pollen development and maturation, genome-wide transcriptional profiling of the flower buds from fertile and sterile plants of the genic male sterile–fertile line 114AB of Capsicum annuum was performed by the RNA-Seq approach. The results indicated that there were numerous changes in gene expression due to the gene mutation in sterile plants. A total of 668 differential expressed genes were identified. Among them, 562 were up-regulated and 106 were down-regulated genes in the fertile plants compared to those of the sterile plants, including 202 and 2 genes specifically accumulated in fertile and sterile plants, respectively. Hundreds of genes potentially involved in pollen development were identified from the genes specifically expressed in the fertile plants, including pollen coat protein, lipid binding protein, lipid transfer protein, pectin methylesterase, male sterility-related protein, anther-specific protein, pectate lyase gene and so forth. Subsequent analysis of expression patterns for a subset of these genes were verified by semi-quantitative RT-PCR and quantitative RT-PCR. Furthermore, the up-regulated genes were predicted to be associated with intracellular membrane-bounded organelle, cytoplasmic vesicle, oxidoreductase activity, metal ion binding, pollen wall assembly, pollen exine formation, starch and sucrose metabolism, and other processes through Gene ontology and KEGG enrichment analysis. Taken together, this study provides a list of candidate genes and their expression patterns for pollen development and maturation in pepper.

Study on Agrobacterium-Mediated Transformation of Pepper with Barnase and Cre Gene

Abstract This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper (Capsicum annuum L.). Chili pepper inbred lines (A, D, E, and I) were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9–11-day-old seeding was pre-cultured on media MR [MB (MS mineral +vitamine B5)+BA (6-Benzyladenine) 5.0 mg L−1 + IAA (indoleacetic acid) 1.0 mg L−1 + GA3 (gibberellic acid) 1.0 mg L−1 + sucrose 3% + agar 6.5 g L−1] for 2 d. The explants were infected by Agrobacterium tumefaciens when their OD600 (optical density at 600 nm) reached 0.6–0.9. After co-cultured for 4–5 d on media MC [MB + BA 5.0 mg L−1 + IAA 1.0 mg L−1 + GA3 1.0 mg L−1 + sucrose 3% + agar 6.5 g L−1 + AS (acetosyringone) 200 μmol L−1], these cotyledons with petiole were cultured on selective differentiation medium in the media MT [MB medium supplemented with BA [5.0 mg L−1 + IAA 1.0 mg L−1 + GA3 1.0 mg L−1 + AgNO3 5.0 mg L−1 + CW (coconut water) 5% + Km (kanamycin) 65 mg L−1 + Cb (carbenicillin) 500 mg L−1 + 3% sucrose + agar 6.5 g L−1]. The Kmr (kanamycin resistant) bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen germination experiments.

De novo Transcriptome Assembly of Chinese Kale and Global Expression Analysis of Genes Involved in Glucosinolate Metabolism in Multiple Tissues

Chinese kale, a vegetable of the cruciferous family, is a popular crop in southern China and Southeast Asia due to its high glucosinolate content and nutritional qualities. However, there is little research on the molecular genetics and genes involved in glucosinolate metabolism and its regulation in Chinese kale. In this study, we sequenced and characterized the transcriptomes and expression profiles of genes expressed in 11 tissues of Chinese kale. A total of 216 million 150-bp clean reads were generated using RNA-sequencing technology. From the sequences, 98,180 unigenes were assembled for the whole plant, and 49,582~98,423 unigenes were assembled for each tissue. Blast analysis indicated that a total of 80,688 (82.18%) unigenes exhibited similarity to known proteins. The functional annotation and classification tools used in this study suggested that genes principally expressed in Chinese kale, were mostly involved in fundamental processes, such as cellular and molecular functions, the signal transduction, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in various tissues of Chinese kale. A large number of candidate genes involved in glucosinolate metabolism and its regulation were identified, and the expression patterns of these genes were analyzed. We found that most of the genes involved in glucosinolate biosynthesis were highly expressed in the root, petiole, and in senescent leaves. The expression patterns of ten glucosinolate biosynthetic genes from RNA-seq were validated by quantitative RT-PCR in different tissues. These results provided an initial and global overview of Chinese kale gene functions and expression activities in different tissues.

Molecular Characterization of MYB28 Involved in Aliphatic Glucosinolate Biosynthesis in Chinese Kale (Brassica oleracea var. alboglabra Bailey)

Glucosinolates are Brassicaceae-specific secondary metabolites that act as crop protectants, flavor precursors, and cancer-prevention agents, which shows strong evidences of anticarcinogentic, antioxidant, and antimicrobial activities. MYB28, the R2R3-MYB28 transcription factor, directly activates genes involved in aliphatic glucosinolate biosynthesis. In this study, the MYB28 homology (BoaMYB28) was identified in Chinese kale (Brassica oleracea var. alboglabra Bailey). Analysis of the nucleotide sequence indicated that the cDNA of BoaMYB28 was 1257 bp with an ORF of 1020 bp. The deduced BoaMYB28 protein was a polypeptide of 339 amino acid with a putative molecular mass of 38 kDa and a pI of 6.87. Sequence homology and phylogenetic analysis showed that BoaMYB28 was most closely related to MYB28 homologs from the Brassicaceae family. The expression levels of BoaMYB28 varies across the tissues and developmental stages. BoaMYB28 transcript levels were higher in leaves and stems compared with those in cotyledons, flowers, and siliques. BoaMYB28 was expressed across all developmental leaf stages, with higher transcript accumulation in mature and inflorescence leaves. Over-expression and RNAi studies showed that BoaMYB28 retains the basic MYB28 gene function as a major transcriptional regulator of aliphatic glucosinolate pathway. The results indicated that over-expression and RNAi lines showed no visible difference on plant morphology. The contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes increased in over-expression lines and decreased in RNAi lines. In over-expression lines, aliphatic glucosinolate contents were 1.5- to 3-fold higher than those in the wild-type, while expression levels of aliphatic glucosinolate biosynthesis genes were 1.5- to 4-fold higher than those in the wild-type. In contrast, the contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes in RNAi lines were considerably lower than those in the wild-type. The results suggest that BoaMYB28 has the potential to alter the aliphatic glucosinolates contents in Chinese kale at the genetic level.