The CBL-interacting protein kinase CaCIPK13 positively regulates defence mechanisms against cold stress in pepper.

Cold stress is one of the main factors limiting growth and development in pepper. Calcineurin B-like proteins (CBLs) are specific calcium sensors with non-canonical EF-hands to capture calcium signals, and interact with CBL-interacting protein kinases (CIPKs) in the regulation of various stresses. In this study, we isolated a cold-induced CIPK gene from pepper named CaCIPK13, which encodes a protein of 487 amino acids. In silico analyses indicated that CaCIPK13 is a typical CIPK family member with a conserved NAF motif, which consists of the amino acids asparagine, alanine, and phenylalanine. The CaCIPK13 protein was located in the nucleus and plasma membrane. Knock down of CaCIPK13 resulted in enhanced sensitivity to cold stress in pepper, with increased malondialdehyde content, H2O2 accumulation, and electrolyte leakage, while the catalase, peroxidase, superoxide dismutase activities and anthocyanin content were decreased. The transcript level of cold and anthocyanin-related genes was substantially decreased in CaCIPK13-silenced pepper leaves relative to the empty vector control. On the contrary, overexpression of CaCIPK13 in tomato improved cold tolerance via increasing anthocyanin content and activities of reactive oxygen species scavenging enzymes. Furthermore, the interaction of CaCIPK13 with CaCBL1/6/7/8 was Ca2+-dependent. These results indicate that CaCIPK13 plays a positive role in cold tolerance mechanism via CBL-CIPK signalling.

The CaCIPK3 gene positively regulates drought tolerance in pepper.

Drought stress is a major agricultural problem restricting the growth, development, and productivity of plants. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) significantly influence the plant response to different stresses. However, the molecular mechanisms of CBL-CIPK in the drought stress response of pepper are still unknown. Here, the function of CaCIPK3 in the regulation of drought stress in pepper (Capsicum annuum L.) was explored. Transcriptomic data and quantitative real-time PCR (qRT-PCR) analysis revealed that CaCIPK3 participates in the response to multiple stresses. Knockdown of CaCIPK3 in pepper increased the sensitivity to mannitol and methyl jasmonate (MeJA). Transient overexpression of CaCIPK3 improved drought tolerance by enhancing the activities of the antioxidant system and positively regulating jasmonate (JA)-related genes. Ectopic expression of CaCIPK3 in tomato also improved drought and MeJA resistance. As the CaCIPK3-interacting partner, CaCBL2 positively influenced drought resistance. Additionally, CaWRKY1 and CaWRKY41 directly bound the CaCIPK3 promoter to influence its expression. This study shows that CaCIPK3 acts as a positive regulator in drought stress resistance via the CBL-CIPK network to regulate MeJA signaling and the antioxidant defense system.

CaFtsH06, A Novel Filamentous Thermosensitive Protease Gene, Is Involved in Heat, Salt, and Drought Stress Tolerance of Pepper (Capsicum annuum L.).

Harsh environmental factors have continuous negative effects on plant growth and development, leading to metabolic disruption and reduced plant productivity and quality. However, filamentation temperature-sensitive H protease (FtsH) plays a prominent role in helping plants to cope with these negative impacts. In the current study, we examined the transcriptional regulation of the CaFtsH06 gene in the R9 thermo-tolerant pepper (Capsicum annuum L.) line. The results of qRT-PCR revealed that CaFtsH06 expression was rapidly induced by abiotic stress treatments, including heat, salt, and drought. The CaFtsH06 protein was localized to the mitochondria and cell membrane. Additionally, silencing CaFtsH06 increased the accumulation of malonaldehyde content, conductivity, hydrogen peroxide (H2O2) content, and the activity levels of superoxide dismutase and superoxide (·O2-), while total chlorophyll content decreased under these abiotic stresses. Furthermore, CaFtsH06 ectopic expression enhanced tolerance to heat, salt, and drought stresses, thus decreasing malondialdehyde, proline, H2O2, and ·O2- contents while superoxide dismutase activity and total chlorophyll content were increased in transgenic Arabidopsis. Similarly, the expression levels of other defense-related genes were much higher in the transgenic ectopic expression lines than WT plants. These results suggest that CaFtsH06 confers abiotic stress tolerance in peppers by interfering with the physiological indices through reducing the accumulation of reactive oxygen species, inducing the activities of stress-related enzymes and regulating the transcription of defense-related genes, among other mechanisms. The results of this study suggest that CaFtsH06 plays a very crucial role in the defense mechanisms of pepper plants to unfavorable environmental conditions and its regulatory network with other CaFtsH genes should be examined across variable environments.

CaHsfA1d Improves Plant Thermotolerance via Regulating the Expression of Stress- and Antioxidant-Related Genes.

Heat shock transcription factor (Hsf) plays an important role in regulating plant thermotolerance. The function and regulatory mechanism of CaHsfA1d in heat stress tolerance of pepper have not been reported yet. In this study, phylogenetic tree and sequence analyses confirmed that CaHsfA1d is a class A Hsf. CaHsfA1d harbored transcriptional function and predicted the aromatic, hydrophobic, and acidic (AHA) motif mediated function of CaHsfA1d as a transcription activator. Subcellular localization assay showed that CaHsfA1d protein is localized in the nucleus. The CaHsfA1d was transcriptionally up-regulated at high temperatures and its expression in the thermotolerant pepper line R9 was more sensitive than that in thermosensitive pepper line B6. The function of CaHsfA1d under heat stress was characterized in CaHsfA1d-silenced pepper plants and CaHsfA1d-overexpression Arabidopsis plants. Silencing of the CaHsfA1d reduced the thermotolerance of the pepper, while CaHsfA1d-overexpression Arabidopsis plants exhibited an increased insensitivity to high temperatures. Moreover, the CaHsfA1d maintained the H2O2 dynamic balance under heat stress and increased the expression of Hsfs, Hsps (heat shock protein), and antioxidant gene AtGSTU5 (glutathione S-transferase class tau 5) in transgenic lines. Our findings clearly indicate that CaHsfA1d improved the plant thermotolerance via regulating the expression of stress- and antioxidant-related genes.

Characterization of the bZIP Transcription Factor Family in Pepper (Capsicum annuum L.): CabZIP25 Positively Modulates the Salt Tolerance.

The basic leucine zipper (bZIP) proteins compose a family of transcription factors (TFs), which play a crucial role in plant growth, development, and abiotic and biotic stress responses. However, no comprehensive analysis of bZIP family has been reported in pepper (Capsicum annuum L.). In this study, we identified and characterized 60 bZIP TF-encoding genes from two pepper genomes. These genes were divided into 10 groups based on their phylogenetic relationships with bZIP genes from Arabidopsis. Six introns/exons structural patterns within the basic and hinge regions and the conserved motifs were identified among all the pepper bZIP proteins, on the basis of which, we classify them into different subfamilies. Based on the transcriptomic data of Zunla-1 genome, expression analyses of 59 pepper bZIP genes (not including CabZIP25 of CM334 genome), indicated that the pepper bZIP genes were differentially expressed in the pepper tissues and developmental stages, and many of the pepper bZIP genes might be involved in responses to various abiotic stresses and phytohormones. Further, gene expression analysis, using quantitative real-time PCR (qRT-PCR), showed that the CabZIP25 gene was expressed at relatively higher levels in vegetative tissues, and was strongly induced by abiotic stresses and phytohormones. In comparing with wild type Arabidopsis, germination rate, fresh weight, chlorophyll content, and root lengths increased in the CabZIP25-overexpressing Arabidopsis under salt stress. Additionally, CabZIP25-silenced pepper showed lower chlorophyll content than the control plants under salt stress. These results suggested that CabZIP25 improved salt tolerance in plants. Taken together, our results provide new opportunities for the functional characterization of bZIP TFs in pepper.

Heat Shock Proteins: Dynamic Biomolecules to Counter Plant Biotic and Abiotic Stresses.

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.

Knockdown of CaHSP60-6 confers enhanced sensitivity to heat stress in pepper (Capsicum annuum L.).

MAIN CONCLUSION: HSP60 gene family in pepper was analyzed through bioinformatics along with transcriptional regulation against multiple abiotic and hormonal stresses. Furthermore, the knockdown of CaHSP60-6 increased sensitivity to heat stress. The 60 kDa heat shock protein (HSP60) also known as chaperonin (cpn60) is encoded by multi-gene family that plays an important role in plant growth, development and in stress response as a molecular chaperone. However, little is known about the HSP60 gene family in pepper (Capsicum annuum L.). In this study, 16 putative pepper HSP60 genes were identified through bioinformatic tools. The phylogenetic tree revealed that eight of the pepper HSP60 genes (50%) clustered into group I, three (19%) into group II, and five (31%) into group III. Twelve (75%) CaHSP60 genes have more than 10 introns, while only a single gene contained no introns. Chromosomal mapping revealed that the tandem and segmental duplication events occurred in the process of evolution. Gene ontology enrichment analysis predicted that CaHSP60 genes were responsible for protein folding and refolding in an ATP-dependent manner in response to various stresses in the biological processes category. Multiple stress-related cis-regulatory elements were found in the promoter region of these CaHSP60 genes, which indicated that these genes were regulated in response to multiple stresses. Tissue-specific expression was studied under normal conditions and induced under 2 h of heat stress measured by RNA-Seq data and qRT-PCR in different tissues (roots, stems, leaves, and flowers). The data implied that HSP60 genes play a crucial role in pepper growth, development, and stress responses. Fifteen (93%) CaHSP60 genes were induced in both, thermo-sensitive B6 and thermo-tolerant R9 lines under heat treatment. The relative expression of nine representative CaHSP60 genes in response to other abiotic stresses (cold, NaCl, and mannitol) and hormonal applications [ABA, methyl jasmonate (MeJA), and salicylic acid (SA)] was also evaluated. Knockdown of CaHSP60-6 increased the sensitivity to heat shock treatment as documented by a higher relative electrolyte leakage, lipid peroxidation, and reactive oxygen species accumulation in silenced pepper plants along with a substantial lower chlorophyll content and antioxidant enzyme activity. These results suggested that HSP60 might act as a positive regulator in pepper defense against heat and other abiotic stresses. Our results provide a basis for further functional analysis of HSP60 genes in pepper.

Identification of CBL and CIPK gene families and functional characterization of CaCIPK1 under Phytophthora capsici in pepper (Capsicum annuum L.).

BACKGROUND: Calcineurin B-like proteins (CBLs) are major Ca2+ sensors that interact with CBL-interacting protein kinases (CIPKs) to regulate growth and development in plants. The CBL-CIPK network is involved in stress response, yet little is understood on how CBL-CIPK function in pepper (Capsicum annuum L.), a staple vegetable crop that is threatened by biotic and abiotic stressors. RESULTS: In the present study, nine CaCBL and 26 CaCIPK genes were identified in pepper and the genes were named based on their chromosomal order. Phylogenetic and structural analysis revealed that CaCBL and CaCIPK genes clustered in four and five groups, respectively. Quantitative real-time PCR (qRT-PCR) assays showed that CaCBL and CaCIPK genes were constitutively expressed in different tissues, and their expression patterns were altered when the plant was exposed to Phytophthora capsici, salt and osmotic stress. CaCIPK1 expression changed in response to stress, including exposure to P. capsici, NaCl, mannitol, salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), ethylene (ETH), cold and heat stress. Knocking down CaCIPK1 expression increased the susceptibility of pepper to P. capsici, reduced root activity, and altered the expression of defense related genes. Transient overexpression of CaCIPK1 enhanced H2O2 accumulation, cell death, and expression of genes involved in defense. CONCLUSIONS: Nine CaCBL and 26 CaCIPK genes were identified in the pepper genome, and the expression of most CaCBL and CaCIPK genes were altered when the plant was exposed to stress. In particular, we found that CaCIPK1 is mediates the pepper plant's defense against P. capsici. These results provide the groundwork for further functional characterization of CaCBL and CaCIPK genes in pepper.

A small heat shock protein CaHsp25.9 positively regulates heat, salt, and drought stress tolerance in pepper (Capsicum annuum L.).

Extreme environmental conditions seriously affect crop growth and development, resulting in a decrease in crop yield and quality. However, small heat shock proteins (Hsp20s) play an important role in helping plants to avoid these negative impacts. In this study, we identified the expression pattern of the CaHsp25.9 gene in a thermo-tolerance pepper line R9 and thermo-sensitive line B6. The transcription of CaHsp25.9 was strongly induced by heat stress in both R9 and B6. The expression of CaHsp25.9 was induced by salt and drought stress in R9. Additionally, the CaHsp25.9 protein was localized in the cell membrane and cytoplasm. When silencing the CaHsp25.9 gene in the R9 line, the accumulation of malonaldehyde (MDA), relative electrolytic leakage, hydrogen peroxide, superoxide anion were increased, while total chlorophyll decreased under heat, salt, and drought stress. Over-expression of CaHsp25.9 in Arabidopsis resulted in decreased MDA, while proline, superoxide dismutase activity, germination, and root length increased under heat, salt, and drought stress. However, peroxidase activity was higher in drought stress but lower in heat and salt stress in transgenic Arabidopsis compared to the wild type (WT). Furthermore, the transcription of stress related genes was more highly induced in transgenic lines than WT. Our results indicated that CaHsp25.9 confers heat, salt, and drought stress tolerance to plants by reducing the accumulation of reactive oxygen species, enhancing the activity of antioxidant enzymes, and regulating the expression of stress-related genes. Therefore, these results may provide insight into plant adaption mechanisms developed in variable environments.

Knockdown of the chitin-binding protein family gene CaChiIV1 increased sensitivity to Phytophthora capsici and drought stress in pepper plants.

Phytophthora capsici has been the most destructive pathogen of pepper plants (Capsicum annuum L.), possessing the ability to quickly overcome the host defense system. In this context, the chitin-binding protein (CBP) family member CaChiIV1 regulates the response to P. capsici and abiotic stresses. The relevance of functional characterization and regulation of CaChiIV1 has not been explored in horticultural crops, especially pepper plants. The target gene (CaChiIV1) was isolated from pepper plants and cloned; the encoded protein carries a chitin-binding domain (CBD) that is rich in cysteine residues and has a hinge region with an abundance of proline and glycine residues. Additionally, the conserved regions in the promoter have a remarkable motif, "TTGACC". The expression of CaChiIV1 was markedly regulated by methyl-jasmonate (MeJA), hydrogen peroxide (H2O2), melatonin, mannitol and P. capsici (PC and HX-9) infection. Knockdown of CaChiIV1 in pepper plants increased sensitivity to P. capsici (PC strain). Higher malondialdehyde (MDA) content and relative electrolyte leakage (REL) but lower antioxidant enzyme activities, chlorophyll content, root activity, and proline content were observed in CaChiIV1-silenced plants than in control plants. In conclusion, CaChiIV1-silenced pepper plants displayed increased susceptibility to P. capsici infection due to changes in expression of defense-related genes, thus showing its coregulation affect in particular conditions. Furthermore, antioxidant enzymes and proline content were largely diminished in CaChiIV1-silenced plants. Therefore, this evidence suggests that the CaChiIV1 gene plays a prominent role in the defense mechanism of pepper plants against P. capsici infection. In the future, the potential role of the CaChiIV1 gene in defense regulatory pathways and its coregulation with other pathogen-related genes should be identified.

Classification and Genome-Wide Analysis of Chitin-Binding Proteins Gene Family in Pepper (Capsicum annuum L.) and Transcriptional Regulation to Phytophthora capsici, Abiotic Stresses and Hormonal Applications.

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.