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.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Pepper in Shaanxi Province, China.

Pepper (Capsicum annuum L.) is an important solanaceous vegetable crop, with high nutritional and economic value. However, it is susceptible to Colletotrichum spp. infection during its growth and development, which seriously affects production yield and quality. Chili anthracnose, caused by Colletotrichum spp., is one of the most destructive diseases of pepper. In August 2020, chili anthracnose was observed with widespread distribution in the horticulture field of Northwest A&F University (34.16° N, 108.04° E) in Shaanxi Province, China. Approximately 60% of the pepper plants had disease symptoms typical of anthracnose. Lesions on pepper fruits were dark, circular, sunken, and necrotic, with the presence of orange to pink conidial masses (Figure S1A). To perform fungal isolation, the tissue at the lesion margin was cut from eight symptomatic fruits, surface disinfested with 75% ethanol for 30 s, and 2% NaClO for 1 min, then rinsed three times with sterile distilled water and dried on sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 28 ºC in the dark. After 3 days, hyphae growing from tissue of each lesion were recultured on PDA (Liu et al. 2016). A representative single-spore isolate (NWAFU2) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. NWAFU2 colonies had gray-white aerial mycelium, and the reverse side of the colonies was dark gray to light yellow after 10-days growth on PDA (Figure S1B-C). Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, and measured 10.1 to 16.9 (length) × 4.7 to 7.0 (width) µm (n=50) (Figure S1D). Based on morphological features, the isolate was consistent with the description of C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted using a CTAB method and the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial sequences of actin (ACT) genes were amplified and sequenced using primers ITS1F/ITS4, GDF1/GDR1 and ACT-512F/ACT-783R, respectively (Dowling et al. 2020). Using the BLAST, ITS, ACT, GAPDH gene sequences (GenBank accession nos. MW258690, MW258691 and MW258692, respectively) were 100%, 100% and 98.19% identical to ZJL-4 of C. gloeosporioides (GenBank accession nos. MN075757, MN058142 and MN075666, respectively). Phylogenetic analysis was conducted using MEGA-X (Version 10.0) based on the concatenated sequences of published ITS, ACT and GAPDH for Colletotrichum species using Neighbor-Joining algorithm. The identified isolate (NWAFU2) was closely related to C. gloeosporioides (Figure S2). To confirm the pathogenicity, ten healthy pepper fruits were surface-sterilized and 2 µL of conidial suspension (1×106 conidia/mL) was injected the surface of pepper. Five fruits were inoculated with 2µL sterile distilled water as controls. After inoculation, the fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. Anthracnose symptoms similar to those observed in the field, were observed 7 days after inoculation (Figure S1F) and control fruits remained healthy. A similarly inoculated detached leaf assay resulted in water-soaked lesions 3 days after inoculation. C. gloeosporioides was reisolated from the infected pepper fruits, fulfilling Koch's postulates. C. gloeosporioides has been reported to cause chili anthracnose in Sichuan Province, China (de Silva et al. 2019; Liu et al. 2016). However, Shaanxi is one of the main pepper producing areas in china and it is geographically distinct from Sichuan; its climate and environmental conditions are different from Sichuan. Knowledge that C. gloeosporioides causes chili anthracnose of pepper in Shaanxi province, China may aid in the selection of appropriate management tactics for this disease. Reference: de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301-2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180.

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.

Chitinase Gene Positively Regulates Hypersensitive and Defense Responses of Pepper to Colletotrichum acutatum Infection.

Anthracnose caused by Colletotrichum acutatum is one of the most devastating fungal diseases of pepper (Capsicum annuum L.). The utilization of chitin-binding proteins or chitinase genes is the best option to control this disease. A chitin-binding domain (CBD) has been shown to be crucial for the innate immunity of plants and activates the hypersensitive response (HR). The CaChiIII7 chitinase gene has been identified and isolated from pepper plants. CaChiIII7 has repeated CBDs that encode a chitinase enzyme that is transcriptionally stimulated by C. acutatum infection. The knockdown of CaChiIII7 in pepper plants confers increased hypersensitivity to C. acutatum, resulting in its proliferation in infected leaves and an attenuation of the defense response genes CaPR1, CaPR5, and SAR8.2 in the CaChiIII7-silenced pepper plants. Additionally, H2O2 accumulation, conductivity, proline biosynthesis, and root activity were distinctly reduced in CaChiIII7-silenced plants. Subcellular localization analyses indicated that the CaChiIII7 protein is located in the plasma membrane and cytoplasm of plant cells. The transient expression of CaChiIII7 increases the basal resistance to C. acutatum by significantly expressing several defense response genes and the HR in pepper leaves, accompanied by an induction of H2O2 biosynthesis. These findings demonstrate that CaChiIII7 plays a prominent role in plant defense in response to pathogen infection.

The CBL-CIPK Pathway in Plant Response to Stress Signals.

Plants need to cope with multitudes of stimuli throughout their lifecycles in their complex environments. Calcium acts as a ubiquitous secondary messenger in response to numerous stresses and developmental processes in plants. The major Ca2+ sensors, calcineurin B-like proteins (CBLs), interact with CBL-interacting protein kinases (CIPKs) to form a CBL-CIPK signaling network, which functions as a key component in the regulation of multiple stimuli or signals in plants. In this review, we describe the conserved structure of CBLs and CIPKs, characterize the features of classification and localization, draw conclusions about the currently known mechanisms, with a focus on novel findings in response to multiple stresses, and summarize the physiological functions of the CBL-CIPK network. Moreover, based on the gradually clarified mechanisms of the CBL-CIPK complex, we discuss the present limitations and potential prospects for future research. These aspects may provide a deeper understanding and functional characterization of the CBL-CIPK pathway and other signaling pathways under different stresses, which could promote crop yield improvement via biotechnological intervention.

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.

CaSBP11 Participates in the Defense Response of Pepper to Phytophthora capsici through Regulating the Expression of Defense-Related Genes.

Squamosa promoter binding protein (SBP)-box genes are plant-specific transcription factors involved in plant growth and development, morphogenesis and biotic and abiotic stress responses. However, these genes have been understudied in pepper, especially with respect to defense responses to Phytophthora capsici infection. CaSBP11 is a SBP-box family gene in pepper that was identified in our previous research. Silencing CaSBP11 enhanced the defense response of pepper plants to Phytophthora capsici. Without treatment, the expression of defense-related genes (CaBPR1, CaPO1, CaSAR8.2 and CaDEF1) increased in CaSBP11-silenced plants. However, the expression levels of these genes were inhibited under transient CaSBP11 expression. CaSBP11 overexpression in transgenic Nicotiana benthamiana decreased defense responses, while in Arabidopsis, it induced or inhibited the expression of genes in the salicylic acid and jasmonic acid signaling pathways. CaSBP11 overexpression in sid2-2 mutants induced AtNPR1, AtNPR3, AtNPR4, AtPAD4, AtEDS1, AtEDS5, AtMPK4 and AtNDR1 expression, while AtSARD1 and AtTGA6 expression was inhibited. CaSBP11 overexpression in coi1-21 and coi1-22 mutants, respectively, inhibited AtPDF1.2 expression and induced AtPR1 expression. These results indicate CaSBP11 has a negative regulatory effect on defense responses to Phytophthora capsici. Moreover, it may participate in the defense response of pepper to Phytophthora capsici by regulating defense-related genes and the salicylic and jasmonic acid-mediated disease resistance signaling pathways.

Transcription Factor CaSBP12 Negatively Regulates Salt Stress Tolerance in Pepper (Capsicum annuum L.).

SBP-box (Squamosa-promoter binding protein) genes are a type of plant-specific transcription factor and play important roles in plant growth, signal transduction, and stress response. However, little is known about the role of pepper SBP-box transcription factor genes in response to abiotic stress. Here, one of the pepper SBP-box gene, CaSBP12, was selected and isolated from pepper genome database in our previous study. The CaSBP12 gene was induced under salt stress. Silencing the CaSBP12 gene enhanced pepper plant tolerance to salt stress. The accumulation of reactive oxygen species (ROS) of the detached leaves of CaSBP12-silenced plants was significantly lower than that of control plants. Besides, the Na+, malondialdehyde content, and conductivity were significantly increased in control plants than that in the CaSBP12-silenced plants. In addition, the CaSBP12 over-expressed Nicotiana benthamiana plants were more susceptible to salt stress with higher damage severity index percentage and accumulation of ROS as compared to the wild-type. These results indicated that CaSBP12 negatively regulates salt stress tolerance in pepper may relate to ROS signaling cascades.

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.

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.

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.

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.

Genome-wide identification of the AP2/ERF transcription factor family in pepper (Capsicum annuum L.).

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.

A Novel Transcription Factor CaSBP12 Gene Negatively Regulates the Defense Response against Phytophthora capsici in Pepper (Capsicum annuum L.).

SBP-box (Squamosa-promoter binding protein) genes are a type of plant-specific transcription factor and play important roles in plant growth, signal transduction and stress response. However, little is known about the SBP-box genes in pepper (CaSBP), especially in the process of Phytophthora capsici infection. In this study, a novel gene (CaSBP12) was selected from the CaSBP gene family, which was isolated from the pepper genome database in our previous study. The CaSBP12 gene was located in the nucleus of the cell and its silencing in the pepper plant enhanced the defense response against Phytophthora capsici infection. After inoculation with Phytophthora capsici, the root activity of the CaSBP12-silenced plants is compared to control plants, while malondialdehyde (MDA) content is compared viceversa. Additionally, the expression of defense related genes (CaPO1, CaSAR8.2, CaBPR1, and CaDEF1) in the silenced plants were induced to different degrees and the peak of CaSAR8.2 and CaBPR1 were higher than that of CaDEF1. The CaSBP12 over-expressed Nicotiana benthamiana plants were more susceptible to Phytophthora capsici infection with higher EC (electrical conductivity) and MDA contents as compared to the wild-type. The relative expression of defense related genes (NbDEF, NbNPR1, NbPR1a, and NbPR1b) in transgenic and wild-type Nicotiana benthamiana plants were induced, especially the NbPR1a and NbPR1b. In conclusion, these results indicate that CaSBP12 gene negative regulates the defense response against Phytophthora capsici infection which suggests their potentially significant role in plant defense. To our knowledge, this is the first report on CaSBP gene which negative regulate defense response.

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.

A mutant in the CsDET2 gene leads to a systemic brassinosteriod deficiency and super compact phenotype in cucumber (Cucumis sativus L.).

KEY MESSAGE: A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase. Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.

Analysis of tandem repeat units of the promoter of capsanthin/capsorubin synthase (Ccs) gene in pepper fruit.

Capsanthin/capsorubin synthase (Ccs) gene is a key gene that regulates the synthesis of capsanthin and the development of red coloration in pepper fruits. There are three tandem repeat units in the promoter region of Ccs, but the potential effects of the number of repetitive units on the transcriptional regulation of Ccs has been unclear. In the present study, expression vectors carrying different numbers of repeat units of the Ccs promoter were constructed, and the transient expression of the ß-glucuronidase (GUS) gene was used to detect differences in expression levels associated with the promoter fragments. These repeat fragments and the plant expression vector PBI121 containing the 35s CaMV promoter were ligated to form recombinant vectors that were transfected into Agrobacterium tumefaciens GV3101. A fluorescence spectrophotometer was used to analyze the expression associated with the various repeat units. It was concluded that the constructs containing at least one repeat were associated with GUS expression, though they did not differ from one another. This repeating unit likely plays a role in transcription and regulation of Ccs expression.

Cloning and characterization of the pepper CaPAO gene for defense responses to salt-induced leaf senescence.

BACKGROUND: Pheophorbide a oxygenase (PAO) is an important enzyme in the chlorophyll catabolism pathway and is involved in leaf senescence. It opens the porphyrin macrocycle of pheophorbide a and finally forms the primary fluorescent chlorophyll catabolite. Previous studies have demonstrated the function of PAO during cell death. However, the characterizaton of PAO during leaf senescence induced by environmental factors is not well understood. METHODS: Homology-based cloning and RACE techniques were used to obtain the full-length cDNA of the CaPAO gene. CaPAO expression was determined by quantitative real-time PCR. Function of CaPAO gene were studied using virus-induced gene silencing and transgenic techniques with tobacco plants (Nicotiana tabacum). RESULTS: A novel PAO gene CaPAO was isolated from pepper (Capsicum annuum L.). The full-length CaPAO cDNA is comprised of 1838 bp, containing an open reading frame of 1614 bp, and encodes a 537 amino acid protein. This deduced protein belongs to the Rieske-type iron-sulfur superfamily, containing a conserved Rieske cluster. CaPAO expression, as determined by quantitative real-time PCR, was higher in leaves than roots, stems and flowers. It was upregulated by abscisic acid, methyl jasmonate and salicylic acid. Moreover, CaPAO was significantly induced by high salinity and osmotic stress treatments and also was regulated by Phytophthora capsici. The virus-induced gene silencing technique was used to silence the CaPAO gene in pepper plants. After 3 days of high salt treatment, the chlorophyll breakdown of CaPAO-silenced pepper plants was retarded. RD29A promoter-inducible expression vector was constructed and transferred into tobacco plant. After 7 days of salt treatment, the leaves of transgenic plants were severely turned into yellow, the lower leaves showed necrotic symptom and chlorophyll content was significantly lower than that in the control plants. CONCLUSIONS: The expression of CaPAO gene was induced in natural senescence and various stresses. The CaPAO gene may be related to defense responses to various stresses and play an important role in salt-induced leaf senescence.

Genome-wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.).

BACKGROUND: Heat shock factors (Hsfs) play crucial roles in plant developmental and defence processes. The production and quality of pepper (Capsicum annuum L.), an economically important vegetable crop, are severely reduced by adverse environmental stress conditions, such as heat, salt and osmotic stress. Although the pepper genome has been fully sequenced, the characterization of the Hsf gene family under abiotic stress conditions remains incomplete. RESULTS: A total of 25 CaHsf members were identified in the pepper genome by bioinformatics analysis and PCR assays. They were grouped into three classes, CaHsfA, B and C, based on highly conserved Hsf domains, were distributed over 11 of 12 chromosomes, with none found on chromosome 11, and all of them, except CaHsfA5, formed a protein-protein interaction network. According to the RNA-seq data of pepper cultivar CM334, most CaHsf members were expressed in at least one tissue among root, stem, leaf, pericarp and placenta. Quantitative real-time PCR assays showed that all of the CaHsfs responded to heat stress (40 °C for 2 h), except CaHsfC1 in thermotolerant line R9 leaves, and that the expression patterns were different from those in thermosensitive line B6. Many CaHsfs were also regulated by salt and osmotic stresses, as well as exogenous Ca(2+), putrescine, abscisic acid and methyl jasmonate. Additionally, CaHsfA2 was located in the nucleus and had transcriptional activity, consistent with the typical features of Hsfs. Time-course expression profiling of CaHsfA2 in response to heat stress revealed differences in its expression level and pattern between the pepper thermosensitive line B6 and thermotolerant line R9. CONCLUSIONS: Twenty-five Hsf genes were identified in the pepper genome and most of them responded to heat, salt, osmotic stress, and exogenous substances, which provided potential clues for further analyses of CaHsfs functions in various kinds of abiotic stresses and of corresponding signal transduction pathways in pepper.

A novel allele of monoecious (m) locus is responsible for elongated fruit shape and perfect flowers in cucumber (Cucumis sativus L.).

KEY MESSAGE: A 14 bp deletion in CsACS2 gene encoding a truncated loss-of-function protein is responsible for elongated fruit shape and perfect flowers in cucumber. In cucumber (Cucumis sativus L.), sex expression and fruit shape are important components of biological and marketable yield. The association of fruit shape and sex expression is a very interesting phenomenon. The sex determination is controlled primarily by the F (female) and M (monoecy) loci. Homozygous recessive mm plants bear bisexual (perfect) flowers, and the fruits are often round shaped. CsACS2 encoding the 1-aminocyclopropane-1-carboxylic acid synthase has been shown to be the candidate gene for the m locus. We recently identified an andromonoecious cucumber line H38 that has bisexual flowers but elongated fruits. To rapidly clone this monoecious gene in H38, we developed a tri-parent mapping strategy, which took advantage of the high-density Gy14 × 9930 cucumber genetic map and the powder of bulk segregant analysis. Microsatellite markers from the Gy14 × 9930 map were used to screen two pairs of unisexual and bisexual bulks constructed from H38 × Gy14 and H38 × 9930 F2 populations. Polymorphic markers were identified and used to quickly develop a framework map and place the monoecious locus of H38 in cucumber chromosome 1. Further fine mapping allowed identification of a novel allele, m-1, at the monoecious locus to control the bisexual flower in H38, which was due to a 14 bp deletion in the third exon of the CsACS2 gene encoding a truncated loss-of-function protein of the cucumber 1-aminocyclopropane-1-carboxylic acid synthase. This new allele provides a valuable tool in understanding the molecular mechanisms of CsACS2 in the relationships of sex determination, fruit shape, and CsACS activities in cucumber.