Due to the present scenario of climate change, plants have to evolve strategies to survive and perform under a plethora of biotic and abiotic stresses, which restrict plant productivity. Maintenance of plant protein functional conformation and preventing non-native proteins from aggregation, which leads to metabolic disruption, are of prime importance. Plant heat shock proteins (HSPs), as chaperones, play a pivotal role in conferring biotic and abiotic stress tolerance. Moreover, HSP also enhances membrane stability and detoxifies the reactive oxygen species (ROS) by positively regulating the antioxidant enzymes system. Additionally, it uses ROS as a signal to molecules to induce HSP production. HSP also enhances plant immunity by the accumulation and stability of pathogenesis-related (PR) proteins under various biotic stresses. Thus, to unravel the entire plant defense system, the role of HSPs are discussed with a special focus on plant response to biotic and abiotic stresses, which will be helpful in the development of stress tolerance in plant crops.

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 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.

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.

Environmental stress affects growth and development of crops, and reduces yield and quality of crops. To cope with environmental stressors, plants have sophisticated defense mechanisms, including the HSF/HSP pathway. Here, we identify the expression pattern of CaHSP16.4 in thermo-tolerant and thermo-sensitive pepper (Capsicum annuum L.) lines. Under heat stress, R9 thermo-tolerant line had higher CaHSP16.4 expression level than the B6 thermo-sensitive line. Under drought stress, expression pattern of CaHSP16.4 was dynamic. Initially, CaHSP16.4 was downregulated then CaHSP16.4 significantly increased. Subcellular localization assay showed that CaHSP16.4 localizes in cytoplasm and nucleus. In the R9 line, silencing of CaHSP16.4 resulted in a significant increase in malonaldehyde content and a significant reduction in total chlorophyll content, suggesting that silencing of CaHSP16.4 reduces heat and drought stresses tolerance. Overexpression of CaHSP16.4 enhances tolerance to heat stress in Arabidopsis. Under heat stress, the survival rate of CaHSP16.4 overexpression lines was significantly higher than wild type. Furthermore, under heat, drought, and combined stress conditions, the CaHSP16.4-overexpression lines had lower relative electrolytic leakage and malonaldehyde content, higher total chlorophyll content, and higher activity levels of superoxide dismutase, catalase, ascorbic acid peroxidase, and glutathione peroxidase compared to wild type. Furthermore, the expression levels of the stress response genes in the overexpression lines were higher than the wild type. These results indicate that the overexpression of CaHSP16.4 enhances the ability of reactive oxygen species scavenging under heat and drought stress.

Chitin-binding proteins are pathogenesis-related gene family, which play a key role in the defense response of plants. However, thus far, little is known about the chitin-binding family genes in pepper (Capsicum annuum L.). In current study, 16 putative chitin genes (CaChi) were retrieved from the latest pepper genome database, and were classified into four distinct classes (I, III, IV and VI) based on their sequence structure and domain architectures. Furthermore, the structure of gene, genome location, gene duplication and phylogenetic relationship were examined to clarify a comprehensive background of the CaChi genes in pepper. The tissue-specific expression analysis of the CaChi showed the highest transcript levels in seed followed by stem, flower, leaf and root, whereas the lowest transcript levels were noted in red-fruit. Phytophthora capsici post inoculation, most of the CaChi (CaChiI3, CaChiIII1, CaChiIII2, CaChiIII4, CaChiIII6, CaChiIII7, CaChiIV1, CaChiVI1 and CaChiVI2) were induced by both strains (PC and HX-9). Under abiotic and exogenous hormonal treatments, the CaChiIII2, CaChiIII7, CaChiVI1 and CaChiVI2 were upregulated by abiotic stress, while CaChiI1, CaChiIII7, CaChiIV1 and CaChiIV2 responded to hormonal treatments. Furthermore, CaChiIV1-silenced plants display weakened defense by reducing (60%) root activity and increase susceptibility to NaCl stress. Gene ontology (GO) enrichment analysis revealed that CaChi genes primarily contribute in response to biotic, abiotic stresses and metabolic/catabolic process within the biological process category. These results exposed that CaChi genes are involved in defense response and signal transduction, suggesting their vital roles in growth regulation as well as response to stresses in pepper plant. In conclusion, these finding provide basic insights for functional validation of the CaChi genes in different biotic and abiotic stresses.

Dehydrins (DHNs) play a crucial role in enhancing abiotic stress tolerance in plants. Although DHNs have been identified and characterized in many plants, there is little known about Capsicum annuum L., one of the economically important vegetable crops. In this study, seven CaDHNs in the pepper genome were identified, which could be divided into two classes: YnSKn- and SKn-type, based on their highly conserved domains. Quantitative real-time PCR (qRT-PCR) results showed that the seven DHN genes were expressed in all tissues and might be involved in the growth and development of pepper. The gene expression profiles analysis suggested that most of the CaDHN genes were induced by various stresses (low temperature, salt and mannitol) and signaling molecules (ABA, SA and MeJA). Furthermore, the CaDHN3 (YSK2)-silenced pepper plants showed obvious lower resistance to abiotic stresses (cold, salt and mannitol) than the control plants (TRV2:00). So the CaDHN3 might act as a positive role in resisting abiotic stresses. This study lays the foundation for further studies into the regulation of their expression under various conditions.