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.

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.

miR-4709 overexpression facilitates cancer proliferation and invasion via downregulating NR3C2 and is an unfavorable prognosis factor in colon adenocarcinoma.

To date, microRNA-4709 (miR-4709) has not been studied in colon adenocarcinoma (COAD) on the basis of experiments. In our study, we aimed to investigate the biological roles and clinical significance of miR-4709 in COAD. The expression difference between miR-4709 and NR3C2 was measured based on The Cancer Genome Atlas database and cells. Kaplan-Meier and logrank tests were applied to determine the overall survival (OS) differences according to the miR-4709 and NR3C2 expression levels. To measure whether the miR-4709 level was associated with COAD cell growth, migration, and invasion, we respectively conducted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and transwell assays. A luciferase reporter assay was applied to confirm the relationship between miR-4709 and its predicted target. High expression of miR-4709 was found in COAD tissues and cells. The OS rate in the miR-4709 low expression group was significantly higher than that in the miR-4709 high expression group. Univariate and multivariate analyses exhibited that miR-4709 expression was an independent adverse prognostic factor. Exogenous miR-4709 overexpression promoted proliferation, migration, and invasion of LOVO and SW480 cells. Bioinformatics analysis and luciferase assay demonstrated that miR-4709 directly binds to the 3'-untranslated region of NR3C2. NR3C2 was lowly expressed in COAD and high expression of NR3C2 had a better prognosis compared with that in the low expression of NR3C2. Correlation analysis showed that there is a close association between the level of expression of NR3C2 and miR-4709. Accordingly, miR-4709 may function as an oncogene in COAD and provide a preclinical proof for candidate management to target new miR-4709-NR3C2 signaling for COAD therapy.

ANKRD33 is overexpressed in gastric adenocarcinoma and predictive for poor prognosis.

The aim of the current study was to investigate and discuss the function of ANKRD33 gene in the pathogenesis of gastric adenocarcinoma. The marked up-regulated expression of ANKRD33 gene in gastric adenocarcinoma tissues compared to normal tissues was found by bioinformatics analysis. Kaplan-Meier analysis revealed that high expression of ANKRD33 is correlated with lower overall survival of gastric adenocarcinoma patients. The results of qPCR revealed that mRNA expression level of ANKRD33 was dramatically higher in AGS, SGC7901, and BGC823 cell lines than that in the GES1 cells. Knockdown of ANKRD33 remarkably inhibited the viability, invasion, and migration of AGS and BGC823 cells. Furthermore, the ratio of p-AKT/AKT and p-mTOR/mTOR was significantly decreased in AGS cells which transfected with si- ANKRD33. All the above results illustrated that ANKRD33 would act as a tumor forwarder in gastric adenocarcinoma development and have a high potential to be a marker molecule in the diagnosis and treatment of gastric tumors.

Biosynthesis of (S)-naproxen starch ester by Carica papaya lipase in intermittent opening reaction mode.

Inorder to brought S-naproxen into small intestine, an optically pure (S)-naproxen starch ester was produced by lipase through enantio-selective trans-esterification of racemic naproxen methyl ester with pretreatment starch in solvent system. With carefully selection of the reaction medium (isooctane), lipase (Carica Papaya Lipase, CPL) and the reaction mode (intermittent opening), a high conversion rate (48.6%) and enantiomeric excess of product (99.6%) was obtained. The slow release macromolecular (S)-Naproxen had been synthesized to improve the efficacy of racemic naproxen and overcome its side effects. The enanitomeric ratio of CPL (E=52.5) was higher than CRL (E=22) and greatly influenced by the byproduct methyl alcohol. The intermittent opening reaction mode was the effective way to remove the inhibition of methyl alcohol and to improve the enantio-selectivity of CPL. S-naproxen starch was confirmed by HPLC and 1H NMR. This method may also apply to preparation the other optically pure 2-phenylpropionic acid derivatives. S-naproxen starch was a new optically pure derivatives possessing emulsifying and slow release properties would be widely applied to the food, pharmaceutical and biomedical industries.

A novel gene, CaATHB-12, negatively regulates fruit carotenoid content under cold stress in Capsicum annuum.

BACKGROUND: Carotenoids, the secondary metabolites terpenoids, are the largest factors that form the fruit color. Similar to flavonoids, they are not only safe and natural colorants of fruits but also play a role as stress response biomolecules. METHODS: To study the contribution of the key genes in carotenoids biosynthesis, fruit-color formation, and in response to cold stress, we characterized the key regulatory factor CaATHB-12 from the HD-ZIP I sub-gene family members in pepper. RESULTS: Cold stress enhanced carotenoid accumulation as compared with the normal condition. CaATHB-12 silencing through virus-induced gene silencing changed the fruit color by regulating the carotenoid contents. CaATHB-12 silencing increased the antioxidant enzyme activities in the fruits of pepper, exposed to cold stress, whereas CaATHB-12 overexpression decreased the activities of antioxidant enzymes in the transgenic Arabidopsis lines, exposed to cold stress, suggesting that CaATHB-12 is involved in the regulation of cold stress in the pepper fruits. CONCLUSION: Our research will provide insights into the formation of fruit color in pepper and contribution of CaATHB-12 in response to cold stress. Further study should be focused on the interaction between CaATHB-12 and its target gene.

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.

Inhibition of inflammatory cytokines after early decompression may mediate recovery of neurological function in rats with spinal cord injury.

A variety of inflammatory cytokines are involved in spinal cord injury and influence the recovery of neuronal function. In the present study, we established a rat model of acute spinal cord injury by cerclage. The cerclage suture was released 8 or 72 hours later, to simulate decompression surgery. Neurological function was evaluated behaviorally for 3 weeks after surgery, and tumor necrosis factor α immunoreactivity and apoptosis were quantified in the region of injury. Rats that underwent decompression surgery had significantly weaker immunoreactivity of tumor necrosis factor α and significantly fewer apoptotic cells, and showed faster improvement of locomotor function than animals in which decompression surgery was not performed. Decompression at 8 hours resulted in significantly faster recovery than that at 72 hours. These data indicate that early decompression may improve neurological function after spinal cord injury by inhibiting the expression of tumor necrosis factor α.