Reduced tolerance to abiotic stress in transgenic Arabidopsis overexpressing a Capsicum annuum multiprotein bridging factor 1.

BACKGROUND: The pepper fruit is the second most consumed vegetable worldwide. However, low temperature affects the vegetative development and reproduction of the pepper, resulting in economic losses. To identify cold-related genes regulated by abscisic acid (ABA) in pepper seedlings, cDNA representational difference analysis was previously performed using a suppression subtractive hybridization method. One of the genes cloned from the subtraction was homologous to Solanum tuberosum MBF1 (StMBF1) encoding the coactivator multiprotein bridging factor 1. Here, we have characterized this StMBF1 homolog (named CaMBF1) from Capsicum annuum and investigated its role in abiotic stress tolerance. RESULTS: Tissue expression profile analysis using quantitative RT-PCR showed that CaMBF1 was expressed in all tested tissues, and high-level expression was detected in the flowers and seeds. The expression of CaMBF1 in pepper seedlings was dramatically suppressed by exogenously supplied salicylic acid, high salt, osmotic and heavy metal stresses. Constitutive overexpression of CaMBF1 in Arabidopsis aggravated the visible symptoms of leaf damage and the electrolyte leakage of cell damage caused by cold stress in seedlings. Furthermore, the expression of RD29A, ERD15, KIN1, and RD22 in the transgenic plants was lower than that in the wild-type plants. On the other hand, seed germination, cotyledon greening and lateral root formation were more severely influenced by salt stress in transgenic lines compared with wild-type plants, indicating that CaMBF1-overexpressing Arabidopsis plants were hypersensitive to salt stress. CONCLUSIONS: Overexpression of CaMBF1 in Arabidopsis displayed reduced tolerance to cold and high salt stress during seed germination and post-germination stages. CaMBF1 transgenic Arabidopsis may reduce stress tolerance by downregulating stress-responsive genes to aggravate the leaf damage caused by cold stress. CaMBF1 may be useful for genetic engineering of novel pepper cultivars in the future.

Silencing of the CaCP gene delays salt- and osmotic-induced leaf senescence in Capsicum annuum L.

Cysteine proteinases have been known to participate in developmental processes and in response to stress in plants. Our present research reported that a novel CP gene, CaCP, was involved in leaf senescence in pepper (Capsicum annuum L.). The full-length CaCP cDNA is comprised of 1316 bp, contains 1044 nucleotides in open reading frame (ORF), and encodes a 347 amino acid protein. The deduced protein belongs to the papain-like cysteine proteases (CPs) superfamily, containing a highly conserved ERFNIN motif, a GCNGG motif and a conserved catalytic triad. This protein localized to the vacuole of plant cells. Real-time quantitative PCR analysis revealed that the expression level of CaCP gene was dramatically higher in leaves and flowers than that in roots, stems and fruits. Moreover, CaCP transcripts were induced upon during leaf senescence. CaCP expression was upregulated by plant hormones, especially salicylic acid. CaCP was also significantly induced by abiotic and biotic stress treatments, including high salinity, mannitol and Phytophthora capsici. Loss of function of CaCP using the virus-induced gene-silencing technique in pepper plants led to enhanced tolerance to salt- and osmotic-induced stress. Taken together, these results suggest that CaCP is a senescence-associated gene, which is involved in developmental senescence and regulates salt- and osmotic-induced leaf senescence in pepper.

Overexpression of the CaTIP1-1 pepper gene in tobacco enhances resistance to osmotic stresses.

Both the gene expression and activity of water channel protein can control transmembrane water movement. We have reported the overexpression of CaTIP1-1, which caused a decrease in chilling tolerance in transgenic plants by increasing the size of the stomatal pore. CaTIP1-1 expression was strongly induced by salt and mannitol stresses in pepper (Capsicum annuum). However, its biochemical and physiological functions are still unknown in transgenic tobacco. In this study, transient expression of CaTIP1-1-GFP in tobacco suspension cells revealed that the protein was localized in the tonoplast. CaTIP1-1 overexpressed in radicle exhibited vigorous growth under high salt and mannitol treatments more than wild-type plants. The overexpression of CaTIP1-1 pepper gene in tobacco enhanced the antioxidant enzyme activities and increased transcription levels of reactive oxygen species-related gene expression under osmotic stresses. Moreover, the viability of transgenic tobacco cells was higher than the wild-type after exposure to stress. The pepper plants with silenced CaTIP1-1 in P70 decreased tolerance to salt and osmotic stresses using the detached leaf method. We concluded that the CaTIP1-1 gene plays an important role in response to osmotic stresses in tobacco.

Characteristic of the pepper CaRGA2 gene in defense responses against Phytophthora capsici Leonian.

The most significant threat to pepper production worldwide is the Phytophthora blight, which is caused by the oomycete pathogen, Phytophthora capsici Leonian. In an effort to help control this disease, we isolated and characterized a P. capsici resistance gene, CaRGA2, from a high resistant pepper (C. annuum CM334) and analyzed its function by the method of real-time PCR and virus-induced gene silencing (VIGS). The CaRGA2 has a full-length cDNA of 3,018 bp with 2,874 bp open reading frame (ORF) and encodes a 957-aa protein. The protein has a predicted molecular weight of 108.6 kDa, and the isoelectric point is 8.106. Quantitative real-time PCR indicated that CaRGA2 expression was rapidly induced by P. capsici. The gene expression pattern was different between the resistant and susceptible cultivars. CaRGA2 was quickly expressed in the resistant cultivar, CM334, and reached to a peak at 24 h after inoculation with P. capsici, five-fold higher than that of susceptible cultivar. Our results suggest that CaRGA2 has a distinct pattern of expression and plays a critical role in P. capsici stress tolerance. When the CaRGA2 gene was silenced via VIGS, the resistance level was clearly suppressed, an observation that was supported by semi-quantitative RT-PCR and detached leave inoculation. VIGS analysis revealed their importance in the surveillance to P. capsici in pepper. Our results support the idea that the CaRGA2 gene may show their response in resistance against P. capsici. These analyses will aid in an effort towards breeding for broad and durable resistance in economically important pepper cultivars.

VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves.

The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3'5'H, DFR, ANS, UFGT, ANP, and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H, and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens.

Tapetum-specific expression of a cytoplasmic orf507 gene causes semi-male sterility in transgenic peppers.

Though cytoplasmic male sterility (CMS) in peppers is associated with the orf507 gene, definitive and direct evidence that it directly causes male sterility is still lacking. In this study, differences in histochemical localization of anther cytochrome c oxidase between the pepper CMS line and maintainer line were observed mainly in the tapetal cells and tapetal membrane. Inducible and specific expression of the orf507 gene in the pepper maintainer line found that transformants were morphologically similar to untransformed and transformed control plants, but had shrunken anthers that showed little dehiscence and fewer pollen grains with lower germination rate and higher naturally damaged rate. These characters were different from those of CMS line which does not produce any pollen grains. Meanwhile a pollination test using transformants as the male parent set few fruit and there were few seeds in the limited number of fruits. At the tetrad stage, ablation of the tapetal cell induced by premature programmed cell death (PCD) occurred in the transformants and the microspores were distorted and degraded at the mononuclear stage. Stable transmission of induced semi-male sterility was confirmed by a test cross. In addition, expression of orf507 in the maintainer lines seemed to inhibit expression of atp6-2 to a certain extent, and lead to the increase of the activity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATP synthase. These results introduce the premature PCD caused by orf507 gene in tapetal cells and semi-male sterility, but not complete male sterility.

Characterization and expression profile of CaNAC2 pepper gene.

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A new transcript encoding NAC protein, homologous to nam-like protein 4 from Petunia was identified from an ABA-regulated subtractive cDNA library of Capsicum annuum seedling. Here, this homolog (named CaNAC2) from C. annuum was characterized and investigated its role in abiotic stress tolerance. Our results indicated that a plant-specific and conserved NAC domain was located in the N-terminus domain of CaNAC2 which was predicted to encode a polypeptide of 410 amino acids. Phylogenetic analysis showed that CaNAC2 belonged to the NAC2 subgroup of the orthologous group 4d. The protein CaNAC2 was subcellularly localized in the nucleus and it had transcriptional activity in yeast cell. CaNAC2 was expressed mainly in seed and root. The transcription expression of CaNAC2 was strongly induced by cold, salt and ABA treatment and inhibited by osmotic stress and SA treatment. Silence of CaNAC2 in virus-induced gene silenced pepper seedlings resulted in the increased susceptibility to cold stress and delayed the salt-induced leaf chlorophyll degradation. These results indicated that this novel CaNAC2 gene might be involved in pepper response to abiotic stress tolerance.

Cloning and expression analysis of CaPIP1-1 gene in pepper (Capsicum annuum L.).

Plant aquaporins are responsible for water transmembrane transport, which play an important role on abiotic and biotic stresses. A novel plasma membrane intrinsic protein of CaPIP1-1 was isolated from the pepper P70 according to transcriptome databases of Phytophthora capsici inoculation and chilling stress library. CaPIP1-1, which is 1155 bp in length with an open reading frame of 861 bp, encoded 286 amino acids. Three introns, exhibited CT/AC splice junctions, were observed in CaPIP1-1. The numbers and location of introns in CaPIP1-1 were the same as observed in tomato and potato. CaPIP1-1 was abundantly expressed in pepper fruit. Increased transcription levels of CaPIP1-1 were found in the different stresses, including chilling stress, salt stress, mannitol stress, salicylic acid, ABA treatment and Phytophthora capsici infection. The expression of CaPIP1-1 was downregulated by 50 µM HgCl2 and 100 µM fluridone. The pepper plants silenced CaPIP1-1 in cv. Qiemen showed growth inhibition and decreased tolerance to salt and mannitol stresses using detached leaf method.

Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L.) Leaves under Chilling Stress.

Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.