The dirigent (DIR and DIR-like) proteins involved in lignification, play a pivotal role against biotic and abiotic stresses in plants. However, no information is available about DIR gene family in pepper (Capsicum annuum L.). In this study, 24 putative dirigent genes (CaDIRs) were identified, their gene structure, genome location, gene duplication and phylogenetic relationship were elucidated. Tissue-specific expression analysis displayed the highest transcription levels in flower, stem and leaf. Some CaDIRs were up-regulated by virulent (CaDIR2, 3, 6, 7, 11, 14, 16, 22 and 23) and avirulent (CaDIR3, 5, 7, 16, 20, 22, 23 and 24) Phytophthora capsici strains, as well as by Methyl jasmonate, salicylic acid, NaCl and mannitol stresses. Acid-soluble lignin content increased (103.21%) after P. capsici inoculation (48-hour). Silencing of CaDIR7 weakened plant defense by reducing (~50%) root activity and made plants more susceptible (35.7%) to P. capsici and NaCl (300 mM). Leaf discs of the CaDIR7:silenced plants exposed to NaCl and mannitol (300 mM each), exhibited a significant decrease (56.25% and 48% respectively) in the chlorophyll content. These results suggested that CaDIR7 is involved in pepper defense response against pathogen and abiotic stresses and the study will provide basic insights for future research regarding CaDIRs.

SQUAMOSA promoter binding protein (SBP)-box genes encode plant-specific transcription factors that are extensively involved in many physiological and biochemical processes, including growth, development, and signal transduction. However, pepper (Capsicum annuum L.) SBP-box family genes have not been well characterized. We investigated SBP-box family genes in the pepper genome and characterized these genes across both compatible and incompatible strain of Phytophthora capsici, and also under different hormone treatments. The results indicated that total 15 members were identified and distributed on seven chromosomes of pepper. Phylogenetic analysis showed that SBP-box genes of pepper can be classified into six groups. In addition, duplication analysis within pepper genome, as well as between pepper and Arabidopsis genomes demonstrated that there are four pairs of homology of SBP-box genes in the pepper genome and 10 pairs between pepper and Arabidopsis genomes. Tissue-specific expression analysis of the CaSBP genes demonstrated their diverse spatiotemporal expression patterns. The expression profiles were similarly analyzed following exposure to P. capsici inoculation and hormone treatments. It was shown that nine of the CaSBP genes (CaSBP01, 02, 03, 04, 05, 06, 11, 12, and 13) exhibited a dramatic up-regulation after compatible HX-9 strain (P. capsici) inoculation, while CaSBP09 and CaSBP15 were down-regulated. In case of PC strain (P. capsici) infection six of the CaSBP genes (CaSBP02, 05, 06, 11, 12, and 13) were arose while CaSBP14 was down regulated. Furthermore, Salicylic acid, Methyl jasmonate and their biosynthesis inhibitors treatment indicated that some of the CaSBP genes are potentially involved in these hormone regulation pathways. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles of the pepper CaSBP genes, will help to improve pepper stress tolerance in the future.

The AP2/ERF family is one of the largest transcription factor families in the plant kingdom. AP2/ERF genes contributing to various processes including plant growth, development, and response to various stresses have been identified. In this study, 175 putative AP2/ERF genes were identified in the latest pepper genome database and classified into AP2, RAV, ERF, and Soloist subfamilies. Their chromosomal localization, gene structure, conserved motif, cis-acting elements within the promoter region, and subcellular locations were analyzed. Transient expression of CaAP2/ERF proteins in tobacco revealed that CaAP2/ERF064, CaAP2/ERF109, and CaAP2/ERF127 were located in the nucleus, while CaAP2/ERF171 was located in the nucleus and cytoplasm. Most of the CaAP2/ERF genes contained cis-elements within their promoter regions that responded to various stresses (HSE, LTR, MBS, Box-W1/W-box, and TC-rich repeats) and phytohormones (ABRE, CGTCA-motif, and TCA-element). Furthermore, RNA-seq analysis revealed that CaAP2/ERF genes showed differential expression profiles in various tissues as well as under biotic stresses. Moreover, qRT-PCR analysis of eight selected CaAP2/ERF genes also showed differential expression patterns in response to infection with Phytophthora capsici (HX-9) and in response to phytohormones (SA, MeJA, and ETH). This study will provide basic insights for further studies of the CaAP2/ERF genes involved in the interaction between pepper and P. capsici.

Ethylene-responsive factors (ERF) are usually considered to play diverse roles in plant response to biotic and abiotic stresses. In this study, an ERF gene CaPTI1 was isolated from pepper transcriptome database. CaPTI1 contains an open reading frame (ORF) of 543 bp, which encodes a putative polypeptide of 180 amino acids with a theoretical molecular weight of 20.30 kDa. Results of expression profile showed that CaPTI1 had a highest expression level in roots and this gene could not only response to the infection of Phytophthora capsici and the stresses of cold and drought, but also be induced by the signaling molecule (salicylic acid, Methyl Jasmonate, Ethephon, and hydogen peroxide). Furthermore, virus-induce gene silencing (VIGS) of CaPTI1 in pepper weakened the defense response significantly by reducing the expression of defense related genes CaPR1, CaDEF1 and CaSAR82 and also the root activity. These results suggested that CaPTI1 is involved in the regulation of defense response to P. capsici in pepper.

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.