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Crop plants experience various abiotic stresses that reduce yield and quality. Although several adaptative physiological and defense responses to single stress have been identified, the behavior and mechanisms of plant response to multiple stresses remain underexamined. Herein, we determined that the leaf and vascular changes in Cucumis sativus Irregular Vasculature Patterning (CsIVP)-RNAi cucumber plants can enhance resistance to nitrogen deficiency and high-temperature stress. CsIVP negatively regulated high nitrate affinity transporters (NRT2.1, NRT2.5) and reallocation transporters (NRT1.7, NRT1.9, NRT1.12) under low nitrogen stress. Furthermore, CsIVP-RNAi plants have high survival rate with low heat injury level under high-temperature condition. Several key high-temperature regulators, including Hsfs, Hsps, DREB2C, MBF1b and WRKY33 have significant expression in CsIVP-RNAi plants. CsIVP negatively mediated high-temperature responses by physically interacting with CsDREB2C. Altogether, these results indicated that CsIVP integrates innate programming of plant development, nutrient transport and high-temperature resistance, providing a potentially valuable target for breeding nutrient-efficient and heat-resistant crops.

The eggplant (Solanum melongena L.) is rich in vitamin P (also known as rutin), which is beneficial for humans. In this study, the vitamin P contents of 53 eggplant lines were measured via UV spectrophotometry. The results showed that the vitamin P content differed significantly among the eggplant lines and the actual contents of vitamin P in fresh fruit pulp and fresh peel were 0.18–0.82 mg · g−1 and 0.42–2.32 mg · g−1, respectively. Furthermore, the mixed major gene and polygene inheritance model method was used for the inheritance analysis of the vitamin P content in F2 populations of three hybridization combinations. Inheritance analysis demonstrated that the vitamin P content of these eggplant lines was a quantitative trait. One or two major genes were the contributor, and the genetic effect was an additive-dominance effect. The high vitamin P content trait was regulated by the main gene and followed an additive effect. The heritability of the major genes controlling the high vitamin P content was low, while the heritability of the major gene that controls the low vitamin P content trait was high. These results provide a theoretical basis for the breeding of eggplants with high vitamin P content.

To improve the understanding of molecular mechanisms of anther and/or pollen development in Chili pepper, in the present study, full-length cDNA and DNA sequences of the pollen development-related gene CaMF5 were obtained from the anthers of a Capsicum annuum nuclear male-fertile line. Sequence analysis indicated that the full length of CaMF5 was 747 bp, containing a maximum opening reading frame of 447 bp. Amino acid sequence alignment and phylogenetic analysis revealed that CaMF5 shared approximately 37%–77% homology with a series of uncharacterized or hypothetical proteins and late embryogenesis abundant (LEA) proteins from other plants. However, no LEA structural domain was detected in CaMF5, which indicated that it might be a new type of LEA gene. CaMF5 was only expressed in flower buds at stages 7 and 8 and in open flowers of the male-fertile line, whereas it exhibited no expression in any examined organs of the male-sterile line. In addition, CaMF5 showed the highest transcript abundance in the anthers of the male-fertile line, with no expression being detected in any other examined organs, such as the sepals, petals, pistils, roots, stems, or leaves. Taken together, our results suggest that CaMF5 is an anther-specific gene that might encode a new type of LEA protein related to anther and/or pollen development in C. annuum. Keywords: Capsicum annuum, CaMF5, Anther-specific gene, Late embryogenesis abundant, Expression analysis

Bacterial wilt (BW) is a soil-borne disease that severely impacts plant growth and productivity globally. Ubiquitination plays a crucial role in disease resistance. Our previous research indicated that NAC transcription factor SmNAC negatively regulates BW resistance in eggplant (Solanum melongena). However, whether the ubiquitin/26S proteasome system (UPS) participates in this regulation is unknown.This study used SmNAC as a bait to screen eggplant cDNA library and obtained SmDDA1b, an E3 ubiquitin ligase. Subcellular location and bimolecular fluorescence complementation assays revealed that SmDDA1b could interact with SmNAC in the nucleus. The in vivo and in vitro ubiquitination experiments indicated that SmDDA1b can degrade SmNAC through UPS. However, the discovery of negative regulation of SmDDA1b expression by SmNAC showed that there was a negative feedback loop between SmNAC and SmDDA1b in eggplant.The SmDDA1b-overexpressed lines showed a higher BW resistance associated with high expression levels of salicylic acid (SA)-related genes and SA content than the wild-type lines. However, SmDDA1b-silencing lines showed the opposite results, indicating that SmDDA1b is a positive regulatory gene for BW resistance.This study provides a candidate gene that can enhance BW resistance in eggplants. In addition, it provides insight into a mechanism that promotes plant disease resistance via the SmDDA1b-SmNAC-SA pathway.

Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious disease affecting the production of Solanaceae species, including eggplant (Solanum melongena). However, few resistance genes have been identified in eggplant, and therefore the underlying mechanism of BW resistance remains unclear. Hence, we investigated a spermidine synthase (SPDS) gene from eggplant and created knock-down lines with virus-induced gene silencing. After eggplant was infected with R. solanacearum, the SmSPDS gene was induced, concurrent with increased spermidine (Spd) content, especially in the resistant line. We speculated that Spd plays a significant role in the defense response of eggplant to BW. Moreover, using the yeast one-hybrid approach and dual luciferase-based transactivation assay, an R2R3-MYB transcription factor, SmMYB44, was identified as directly binding to the SmSPDS promoter, activating its expression. Overexpression of SmMYB44 in eggplant induced the expression of SmSPDS and Spd content, increasing the resistance to BW. In contrast, the SmMYB44-RNAi transgenic plants showed more susceptibility to BW compared with the control plants. Our results provide insight into the SmMYB44-SmSPDS-Spd module involved in the regulation of resistance to R. solanacearum. This research also provides candidates to enhance resistance to BW in eggplant.

Plants produce countless specialized metabolites crucial for their development and fitness, and many are useful bioactive compounds. Capsaicinoids are intriguing genus-specialized metabolites that confer a pungent flavor to Capsicum fruits, and they are widely applied in different areas. Among the five domesticated Capsicum species, Capsicum chinense has a high content of capsaicinoids, which results in an extremely hot flavor. However, the species-specific upregulation of capsaicinoid-biosynthetic genes (CBGs) and the evolution of extremely pungent peppers are not well understood. We conducted genetic and functional analyses demonstrating that the quantitative trait locus Capsaicinoid1 (Cap1), which is identical to Pun3 contributes to the level of pungency. The Cap1/Pun3 locus encodes the Solanaceae-specific MYB transcription factor MYB31. Capsicum species have evolved placenta-specific expression of MYB31, which directly activates expression of CBGs and results in genus-specialized metabolite production. The capsaicinoid content depends on MYB31 expression. Natural variations in the MYB31 promoter increase MYB31 expression in C. chinense via the binding of the placenta-specific expression of transcriptional activator WRKY9 and augmentation of CBG expression, which promotes capsaicinoid biosynthesis. Our findings provide insights into the evolution of extremely pungent C. chinense, which is due to natural variations in the master regulator, and offers targets for engineering or selecting flavor in Capsicum.

Background The basic helix–loop–helix (bHLH) transcription factors (TFs) serve crucial roles in regulating plant growth and development and typically participate in biological processes by interacting with other TFs. Capsorubin and capsaicinoids are found only in Capsicum, which has high nutritional and economic value. However, whether bHLH family genes regulate capsorubin and capsaicinoid biosynthesis and participate in these processes by interacting with other TFs remains unknown. Results In this study, a total of 107 CabHLHs were identified from the Capsicum annuum genome. Phylogenetic tree analysis revealed that these CabHLH proteins were classified into 15 groups by comparing the CabHLH proteins with Arabidopsis thaliana bHLH proteins. The analysis showed that the expression profiles of CabHLH009, CabHLH032, CabHLH048, CabHLH095 and CabHLH100 found in clusters C1, C2, and C3 were similar to the profile of carotenoid biosynthesis in pericarp, including zeaxanthin, lutein and capsorubin, whereas the expression profiles of CabHLH007, CabHLH009, CabHLH026, CabHLH063 and CabHLH086 found in clusters L5, L6 and L9 were consistent with the profile of capsaicinoid accumulation in the placenta. Moreover, CabHLH007, CabHLH009, CabHLH026 and CabHLH086 also might be involved in temperature-mediated capsaicinoid biosynthesis. Yeast two-hybrid (Y2H) assays demonstrated that CabHLH007, CabHLH009, CabHLH026, CabHLH063 and CabHLH086 could interact with MYB31, a master regulator of capsaicinoid biosynthesis. Conclusions The comprehensive and systematic analysis of CabHLH TFs provides useful information that contributes to further investigation of CabHLHs in carotenoid and capsaicinoid biosynthesis.

Background ERF transcription factors (TFs) belong to the Apetala2/Ethylene responsive Factor (AP2/ERF) TF family and play a vital role in plant growth and development processes. Capsorubin and capsaicinoids have relatively high economic and nutritional value, and they are specifically found in Capsicum. However, there is little understanding of how ERFs participate in the regulatory networks of capsorubin and capsaicinoids biosynthesis. Results In this study, a total of 142 ERFs were identified in the Capsicum annuum genome. Subsequent phylogenetic analysis allowed us to divide ERFs into DREB (dehydration responsive element binding proteins) and ERF subfamilies, and further classify them into 11 groups with several subgroups. Expression analysis of biosynthetic pathway genes and CaERFs facilitated the identification of candidate genes related to the regulation of capsorubin and capsaicinoids biosynthesis; the candidates were focused in cluster C9 and cluster C10, as well as cluster L3 and cluster L4, respectively. The expression patterns of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101, which were found in cluster C9 and cluster C10, were consistent with those of accumulating of carotenoids (β-carotene, zeaxanthin and capsorubin) in the pericarp. In cluster L3 and cluster L4, the expression patterns of CaERF102, CaERF53, CaERF111 and CaERF92 were similar to those of the accumulating capsaicinoids. Furthermore, CaERF92, CaERF102 and CaERF111 were found to be potentially involved in temperature-mediated capsaicinoids biosynthesis. Conclusion This study will provide an extremely useful foundation for the study of candidate ERFs in the regulation of carotenoids and capsaicinoids biosynthesis in peppers.

Background: ERF transcription factors (TFs) belong to the Apetala2/Ethylene responsive Factor (AP2/ERF) TF family and play a vital role in plant growth and development processes. Capsorubin and capsaicinoids have relatively high economic and nutritional value, and they are specifically found in Capsicum. However, there is little understanding of how ERFs participate in the regulatory networks of capsorubin and capsaicinoids biosynthesis. Results: In this study, a total of 142 ERFs were identified in the Capsicum annuum genome. Subsequent phylogenetic analysis allowed us to divide ERFs into DREB (dehydration responsive element binding proteins) and ERF subfamilies, and further classify them into 11 groups with several subgroups. Expression analysis of biosynthetic pathway genes and CaERFs facilitated the identification of candidate genes related to the regulation of capsorubin and capsaicinoids biosynthesis; the candidates were focused in cluster C9 and cluster C10, as well as cluster L3 and cluster L4, respectively. The expression patterns of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101, which were found in cluster C9 and cluster C10, were consistent with those of accumulating of carotenoids (β-carotene, zeaxanthin and capsorubin) in the pericarp. In cluster L3 and cluster L4, the expression patterns of CaERF102, CaERF53, CaERF111 and CaERF92 were similar to those of the accumulating capsaicinoids. Furthermore, CaERF92, CaERF102 and CaERF111 were found to be potentially involved in temperature-mediated capsaicinoids biosynthesis. Conclusion: This study will provide an extremely useful foundation for the study of candidate ERFs in the regulation of carotenoids and capsaicinoids biosynthesis in peppers.

Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar ‘Indigo Rose.’ Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar ‘Indigo Rose’ using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. ‘Indigo Rose,’ while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. ‘Indigo Rose.’ Our findings further our understanding of anthocyanin production in tomato and other plant species.

Additional file 2: Table S1. ERF family genes in Arabidopsis, tomato and rice. Table S2. List of primers used in real-time quantitative PCR (qPCR). Table S3. CaERF genes identified and characterized in the pepper. Table S4. Biological functions of characterized ERF proteins that potentially exist in Arabidopsis and tomato. Table S5. Putative CaERF homologs (version 2.0) of pepper ERF proteins with known biological functions. Table S6. Multilevel consensus sequence identified in 144 CaERF genes.

The 2-oxoglutarate and Fe(II)-dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation and hydroxylation reactions. Due to their important biochemical significance in metabolism, a systematic analysis of the plant 2OGD genes family is necessary. Here, we identified 160, 179, and 337 putative 2OGDs from Brassica rapa, Brassica oleracea, and Brassica napus. According to their gene structure, domain, phylogenetic features, function, and previous studies, we also divided 676 2OGDs into three subfamilies: DOXA, DOXB, and DOXC. Additionally, homologous and phylogenetic comparisons of three subfamily genes provided valuable insight into the evolutionary characteristics of the 2OGD genes from Brassica plants. Expression profiles derived from the transcriptome and Genevestigator database exhibited distinct expression patterns of the At2OGD, Br2OGD, and Bo2OGD genes in different developmental stages, tissues, or anatomical parts. Some 2OGD genes showed high expression levels in various tissues, such as callus, seed, silique, and root tissues, while other 2OGD genes were expressed at very low levels in other tissues. Analysis of six Bo2OGD genes in different tissues by qRT-PCR indicated that these genes are involved in the metabolism of gibberellin, which in turn regulates plant growth and development. Our working system analysed 2OGD gene families of three Brassica plants and laid the foundation for further study of their functional characterization.

The isopentytransperase (IPT) gene, coupled to the SAG12 promoter, was successfully transferred into the eggplant (Solanum melongena L.) mediated by Agrobacterium tumefaciens. Four PSAG12 IPT overexpressing transgenic plants were identified by Southern blotting. Reverse transcription polymerase chain reaction revealed that IPT was expressed in senescent leaves under the control of SAG12 promoter. Compared with wild type eggplants, the leaf senescence was significantly delayed and tolerance to drought and cold stress was enhanced in the T1 generation of PSAG12-IPT transgenic eggplants. The contents of chlorophyll, indoleacetic acid, zeatin riboside, and gibberellic acid in the transgenic eggplants were higher than those in the wild type eggplants. The activities of reactive oxygen species-scavenging enzymes in the transgenic eggplants were also higher than those in the wild type eggplants. Abscisic acid and malondialdehyde levels were lower in the transgenic plants than in the wild type plants. Als...

Chinese kale (Brassica oleracea var. chinensis Lei) is an important vegetable crop in South China, valued for its nutritional content and taste. Nonetheless, the thermal tolerance of Chinese kale still needs improvement. Molecular characterization of Chinese kale&rsquo, s heat stress response could provide a timely solution for developing a thermally tolerant Chinese kale variety. Here, we report the cloning of multi-protein bridging factor (MBF) 1c from Chinese kale (BocMBF1c), an ortholog to the key heat stress responsive gene MBF1c. Phylogenetic analysis showed that BocMBF1c is highly similar to the stress-response transcriptional coactivator MBF1c from Arabidopsis thaliana (AtMBF1c), and the BocMBF1c coding region conserves MBF1 and helix-turn-helix (HTH) domains. Moreover, the promoter region of BocMBF1c contains three heat shock elements (HSEs) and, thus, is highly responsive to heat treatment. This was verified in Nicotiana benthamiana leaf tissue using a green fluorescent protein (GFP) reporter. In addition, the expression of BocMBF1c can be induced by various abiotic stresses in Chinese kale which indicates the involvement of stress responses. The BocMBF1c-eGFP (enhanced green fluorescent protein) chimeric protein quickly translocated into the nucleus under high temperature treatment in Nicotiana benthamiana leaf tissue. Overexpression of BocMBF1c in Arabidopsis thaliana results in a larger size and enhanced thermal tolerance compared with the wild type. Our results provide valuable insight for the role of BocMBF1c during heat stress in Chinese kale.

Jasmonates (JAs) play an important role in plant developmental processes and regulate the biosynthesis of various specialized metabolites, and transcription factors are crucial in mediating JA signaling to regulate these processes. Capsaicinoids (Caps) are intriguing specialized metabolites produced uniquely by Capsicum species that give their fruits a pungent flavor to defend against herbivory and pathogens. In this study, we identify a R2R3-MYB transcription factor CaMYB108 and demonstrate its roles in regulating the biosynthesis of Caps and stamen development. Transcriptional analysis indicated that CaMYB108 was preferentially expressed in the flower and fruit, while the subcellular localization of CaMYB108 was shown to be the nucleus. Virus-induced gene silencing of CaMYB108 led to the expression of capsaicinoid biosynthetic genes (CBGs), and the contents of Caps dramatically reduce. Moreover, the CaMYB108-silenced plants showed delayed anther dehiscence and reduced pollen viability. Transient overexpression of CaMYB108 caused the expression of CBGs to be upregulated, and the Caps content significantly increased. The results of dual-luciferase reporter assays showed that CaMYB108 targeted CBG promoters. In addition, the expression of CaMYB108 and CBGs was inducible by methyl jasmonate and was consistent with the increased content of Caps. Overall, our results indicate that CaMYB108 is involved in the regulation of Caps biosynthesis and stamen development.

Anthocyanin fruit (Aft) and atroviolacea (atv) were characterized in wild tomato and can enhance anthocyanin content in tomato fruit. However, the gene underlying the Aft locus and the mechanism by which Aft and atv act remain largely unknown. In this study, the Aft locus was fine-mapped to an approximately 145-kb interval on chromosome 10, excluding SlAN2 (Solyc10g086250), SlANT1 (Solyc10g086260) and SlANT1-like (Solyc10g086270), which have previously been suggested as candidates. Thus, the R2R3-MYB transcription factor SlAN2-like (Solyc10g086290) was considered the best candidate gene for Aft. The CRISPR/Cas9-mediated SlAN2-like mutants show a much lower accumulation of anthocyanins associated with the downregulation of multiple anthocyanin-related genes compared to the wild-type tomato, indicating that SlAN2-like is responsible for the Aft phenotype. The repressive function of SlMYBATV also was confirmed through the CRISPR/Cas9 approach. A yeast-two-hybrid assay revealed that SlMYBATV interacts with the bHLH protein SlJAF13. Furthermore, yeast-one-hybrid and dual-luciferase transient expression assays showed that Aft directly binds to the SlMYBATV promoter and activates its expression. The results herein provide candidate genes to enhance anthocyanin content in tomato fruit. This research also provides insight into a mechanism involving the Aft-SlMYBATV pathway that fine-tunes anthocyanin accumulation in tomato fruit.

The yield of pepper plants (Capsicum spp.) is their most important trait and is affected by the flower number and flowering time. Capsicum annuum produces a single flower per node and has an early flowering habit. By contrast, Capsicum chinense yields multiple flowers per node and has a late flowering character. However, the genetic mechanism underlying the control of these floral traits remains largely unknown. In this study, 150 F2 populations from an interspecific cross between the inbred lines 740 (C. chinense) and CA1 (C. annuum) and their parents were used to construct a molecular genetic linkage map using the specific length amplified fragment sequencing (SLAF-seq) technique. This linkage map, spanning 1,586.78 cM in length, contained 9,038 markers on 12 chromosomes, with a mean marker distance of 0.18 cM. Phenotypic data on the flowering time and flower number per node were collected over multiple years, and QTL analysis identified 6 QTLs for the flowering time and flower number per node by composite interval mapping (CIM) and genome-wide composite interval mapping (GCIM) methods at least in two environments. The candidate genes within the major QTL were predicted. In the major flowering time QTL, the candidate gene Capana02g000700, which encodes the homeotic protein APETALA2, was identified. Quantitative reverse-transcription PCR (qRT-PCR) analysis indicated that its expression level in 740 was higher than that in CA1. Gene expression analysis indicated that the expression of Capana02g000700 was significantly upregulated in flowers, and many floral development-related genes were found to be coexpressed with Capana02g000700, supporting the function of this gene in association with flowering time in C. chinense and C. annuum species.

High quantities of anthocyanins in plants confer potential protective benefits against biotic and abiotic stressors. Studies have shown that the bZIP transcription factor HY5 plays a key role in controlling anthocyanin accumulation in response to light. However, in hy5 mutants, residual anthocyanins have been detected, indicating that other regulators exist to regulate anthocyanin biosynthesis in an HY5-independent manner. Here, we employed the CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9) system specifically to induce targeted mutagenesis of SlHY5 in the purple tomato cultivar 'Indigo Rose'. The T2 generation of tomato plants homozygous for the null allele of the SlHY5 frameshift mutated by a 1 bp insertion contained a lower anthocyanin content. Transcriptional analysis showed that most of the anthocyanin biosynthesis structural genes and several regulatory genes were down-regulated in the hy5 mutant lines. With transcriptome analyses of the various tissues from hy5 mutant lines, eight candidate transcription factors were identified that may regulate anthocyanin biosynthesis in an HY5-independent manner. These findings deepen our understanding of how light controls anthocyanin accumulation and facilitate the identification of the regulators of anthocyanin biosynthesis in an HY5-independent manner.

Glucoraphanin is a plant secondary metabolite that is involved in plant defense and imparts health-promoting properties to cruciferous vegetables. In this study, three genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4 (BCAT4), methylthioalkylmalate synthase 1 (MAM1) and dihomomethionine N-hydroxylase (CYP79F1), were cloned from Chinese kale (Brassica oleracea var. alboglabra Bailey). Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The transcript levels of BCAT4, MAM1 and CYP79F1 were higher in cotyledon, leaf and stem compared with flower and silique. BCAT4, MAM1 and CYP79F1 were expressed throughout leaf development with lower transcript levels during the younger stages. Glucoraphanin content varied extensively among different varieties, which ranged from 0.25 to 2.73 µmol·g(-1) DW (dry weight). Expression levels of BCAT4 and MAM1 were high at vegetative-reproductive transition phase, while CYP79F1 was expressed high at reproductive phase. BCAT4, MAM1 and CYP79F1 were expressed significantly high in genotypes with high glucoraphanin content. All the results provided a better understanding of the roles of BCAT4, MAM1 and CYP79F1 in the glucoraphanin biosynthesis of Chinese kale.

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.

Improvement of biotic and abiotic stress tolerance without yield decrease has been a great challenge in vegetable breeding. Functional stacking of multiple resistant genes could potentially be an effective strategy against more different stress. Here, we report that constitutive co-expression of Arabidopsis thaliana homodomain-leucine zipper transcription factor Enhanced Drought Tolerance/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11) and Bacillus thuringiensis Cry2Aa2 was able to increase pepper drought tolerance and insect resistance. More importantly, fruit yield was improved under both normal and drought stress conditions. The improved tolerance to drought stress was associated with improved root system and reduced stomatal density. Compared to the non-transgenic control, the transgenic pepper plants also had higher levels of chlorophyll content, chlorophyll fluorescence (Fm/Fv), proline, soluble sugar, abscisic acid, and ROS scavenging capacity under dehydration stress conditions. The increased flower number, elevated fruit weight, and improved fruit number all contributed to the increased yield of transgenic pepper. In addition, bioassays showed that the transgenic plants were effective to control the Prodenia litura, producing a mortality rate of 81.3 % after 5 days of infection. Therefore, our study indicates that stacking of AtEDT1/HDG11 and Cry2Aa2 in pepper plants can improve drought tolerance and insect resistance, demonstrating application of AtEDT1/HDG11 and Cry2Aa2 has the potential to enhance drought tolerance and insect resistance.

Ralstonia solanacearum species complex is a devastating family of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the genetic background behind variation remains vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than other reported genomes of R. solanacearum strains, both chromosome and mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, VI strains unveiled substantial sequence rearrangement, insertions and deletions among them. Genome sequences are relatively conserved among the 4 phylotype I strains, but less conserved in the different phylotype strains. Moreover, distinct variance and diversities were found among different strains in the key virulence genes, including those encoding secretion systems and type III effectors. Our results would be useful in further analysis of genetic basis of variation in virulence and host range of the R. solanacearum species.

Ralstonia solanacearum species complex is a devastating family of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the genetic background behind variation remains vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than other reported genomes of R. solanacearum strains, both chromosome and mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, VI strains unveiled substantial sequence rearrangement, insertions and deletions among them. Genome sequences are relatively conserved among the 4 phylotype I strains, but less conserved in the different phylotype strains. Moreover, distinct variance and diversities were found among different strains in the key virulence genes, including those encoding secretion systems and type III effectors. Our results would be useful in further analysis of genetic basis of variation in virulence and host range of the R. solanacearum species.

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.

Previous work on gene expression analysis based on RNA sequencing identified a variety of differentially expressed cDNA fragments in the genic male sterile-fertile line 114AB of Capsicum annuum L. In this work, we examined the accumulation of one of the transcript-derived fragments (TDFs), CaMF3 (male fertile 3), in the flower buds of a fertile line. The full genomic DNA sequence of CaMF3 was 1,951 bp long and contained 6 exons and 5 introns, with the complete sequence encoding a putative 25.89 kDa protein of 234 amino acids. The predicted protein of CaMF3 shared sequence similarity with members of the isoamyl acetate-hydrolyzing esterase (IAH1) protein family. CaMF3 expression was detected only in flower buds at stages 7 and 8 and in open flowers of a male fertile line; no expression was observed in any organs of a male sterile line. Fine expression analysis revealed that CaMF3 was expressed specifically in anthers of the fertile line. These results suggest that CaMF3 is an anther-specific gene that may be essential for anther or pollen development in C. annuum.

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.

The molecular basis of flower bud differentiation in flowering Chinese cabbage (Brassica rapa L. ssp. Chinensis var. utilis Tsen et Lee) was studied in this work. Samples were taken from two varieties, the early-blooming "Youqin 49" and the late-blooming "Youqingtiancaixin 80", at five different developmental stages and studied via cDNA-AFLP. Nineteen expression sequence tags (ESTs) associated with bolting or flowering were isolated and cloned. Blast results indicated that 15 ESTs were involved in the synthesis of anthocayanins, photosynthesis, signal transduction, and phytochrome production. Two ESTs had high similarity to hypothetical proteins with unknown function. Two other ESTs shared no similarity to any sequence in the NCBI database and potentially may be newly identified genes. The deduced amino acid sequences of EST amplified by primer A6T4 or A8T4 had high similarity to both dihydroflavonol reductase (DFR) and UDP-D: -apiose/UDP-D: -xylose synthase (AXS), thus was named BrcuDFR-like/BrcuAXS. Using the cDNA sequence, a putative BrcuDFR-like/BrcuAXS gene was cloned and characterized from flowering Chinese cabbage via rapid amplification of cDNA ends (RACE). The full-length cDNA has 1332 bp with an open frame of 919 bp which codes for a polypeptide of 313 amino acids. The corresponding genome sequence is 2,046 bp. Comparison of cDNA and its corresponding genomic sequence indicates that BrcuDFR-like/BrcuAXS contains 9 exons and 8 introns. The temporal expression patterns indicated the gene is more likely to encode the DFR protein, which catalyzes the synthesis of anthocayanins, than UDP-D: -apiose/UDP-D: -xylose synthase (AXS), which catalyzes the conversion of UDP-D: -glucuronate to a mixture of UDP-D: -apiose and UDP-D: -xylose. Further work is needed to determine what role BrcuDFR-like/BrcuAXS plays during floral organ development.

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.

A pectin methylesterase gene homolog, designated CaPME1, was identified by RNA-sequencing in the genic male sterile–fertile line 114AB of Capsicum annuum L. The full-length transcript of CaPME1 was 1,747 bp, including a complete coding sequence of 1,662 bp (GenBank accession number: JN246474). The derived protein CaPME1 shares high similarity to members of pectin methylesterase protein family, containing a conserved domain of PMEI at the N terminus and a domain of PME at C-terminal end. The expression of CaPME1 was detected only in flower buds at stage 8 and open flowers of male fertile line, with no detection in any examined organ of male sterility line. The fine expression analysis revealed that CaPME1 expressed specifically in anthers of male fertile line, and there was no expression detected in any other examined organ such as sepals, petals, pistils, roots, stems, or leaves. The putative function of CaPME1 involved in pollen development was also discussed in the present paper. These results suggested that CaPME1 was an anther-specific gene and might be essential for pollen maturation and germination in C. annuum.

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/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

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.

In order to understand the function of TuR2, a candidate disease-resistance gene was isolated from cabbage, we transformed it into mustard (Brassica juncea L. Linshicaoyaozi) which was susceptible to TuMV through Agrobacterium iumefacine-mediated method. Transgenic plants were detected by Southern blotting and Northern blotting. Our results confirmed that the TuR2 gene had been integrated into the mustard genome, and it showed different expression levels among primary transplants (T0). The primary transplants (T0) and the first progenies of transgenic plants (T1) were inoculated with TuMV in a greenhouse. The transgenic plants had high TuMV-resistance, whereas the serious virus disease symptom was observed in CK (no transformation plants). The TuR2 gene in the first progenies of transgenic plants (T1) showed dominant monogenic inheritance. Compared with CK, the progenies containing TuR2 gene had stronger resistance to TuMV. The TuR2 gene which was isolated from cabbage had the function of TuMV-resistance.