Multiple-model GWAS identifies optimal allelic combinations of quantitative trait loci for malic acid in tomato.

Malic acid (MA) is an important flavor acid in fruits and acts as a mediator in a series of metabolic pathways. It is important to understand the factors affecting MA metabolism for fruit flavor improvement and to understand MA-mediated biological processes. However, the metabolic accumulation of MA is controlled by complex heredity and environmental factors, making it difficult to predict and regulate the metabolism of MA. In this study, we carried out a genome-wide association study (GWAS) on MA using eight milestone models with two-environment repeats. A series of associated SNP variations were identified from the GWAS, and 15 high-confidence annotated genes were further predicted based on linkage disequilibrium and lead SNPs. The transcriptome data of candidate genes were explored within different tomato organs as well as various fruit tissues, and suggested specific expression patterns in fruit pericarp. Based on the genetic parameters of population differentiation and SNP distribution, tomato MA content has been more influenced by domestication sweeps and less affected by improvement sweeps in the long-term history of tomato breeding. In addition, genotype × environment interaction might contribute to the difference in domestication phenotypic data under different environments. This study provides new genetic insights into how tomato changed its MA content during breeding and makes available function-based markers for breeding by marker-assisted selection.

Pepper bHLH transcription factor CabHLH035 contributes to salt tolerance by modulating ion homeostasis and proline biosynthesis.

Members of the bHLH family of transcription factors play important roles in multiple aspects of plant biological processes, for instance, abiotic stress responses. Previously, we characterized CaNAC035, a gene that positively regulates stress tolerance and identified CabHLH035, a CaNAC035-interacting protein in pepper (Capsicum annuum L.). In this study, we describe the role of CabHLH035 in the response to salt stress. Our results show that the expression of CabHLH035 increased following salt treatment. Transient expression of CabHLH035 (CabHLH035-To) in pepper enhanced salt tolerance, ectopic expression of CabHLH035 in Arabidopsis increased the salt stress tolerance, whereas knocking down the expression of CabHLH035 in pepper plants resulted in decreased salt tolerance. Homologs of the Salt Overly Sensitive 1 (SOS1) and pyrroline-5-carboxylate acid synthetase (P5CS) genes showed drastically increased expression in transgenic Arabidopsis plants expressing CabHLH035 and CabHLH035-To plants, but expression decreased in CabHLH035-silenced plants. Our results also showed that CabHLH035 can directly bind to the CaSOS1 and CaP5CS gene promoters and positively activate their expression. We found that transgenic Arabidopsis plants, ectopic expression of CabHLH035 and pepper plants transiently overexpressing CabHLH035 (CabHLH035-To) showed lower Na+ and higher proline contents in response to NaCl treatment, while CabHLH035-silenced plants had higher Na+ and lower proline concentrations. Overall, CabHLH035 plays important roles in salt tolerance through its effects on the intracellular Na+ : K+ ratio and proline biosynthesis.

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.

Melatonin Mitigates the Infection of Colletotrichum gloeosporioides via Modulation of the Chitinase Gene and Antioxidant Activity in Capsicum annuum L.

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most damaging pepper (Capsicum annum L.) disease. Melatonin induces transcription of defense-related genes that enhance resistance to pathogens and mediate physiological activities in plants. To study whether the melatonin-mediated pathogen resistance is associated with chitinase gene (CaChiIII2), pepper plants and Arabidopsis seeds were treated with melatonin, then CaChiIII2 activation, hydrogen peroxide (H2O2) levels, and antioxidant enzymes activity during plant-pathogen interactions were investigated. Melatonin pretreatment uncoupled the knockdown of CaChiIII2 and transiently activated its expression level in both control and CaChiIII2-silenced pepper plants and enhanced plant resistance. Suppression of CaChiIII2 in pepper plants showed a significant decreased in the induction of defense-related genes and resistance to pathogens compared with control plants. Moreover, melatonin efficiently enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhanced the activities of antioxidant enzymes, which possibly improved disease resistance. The activation of the chitinase gene CaChiIII2 in transgenic Arabidopsis lines was elevated under C. gloeosporioides infection and exhibited resistance through decreasing H2O2 biosynthesis and maintaining H2O2 at a steady-state level. Whereas melatonin primed CaChiIII2-overexpressed (OE) and wild-type (WT) Arabidopsis seedlings displayed a remarkable increase in root-length compared to the unprimed WT plants. Using an array of CaChiIII2 knockdown and OE, we found that melatonin efficiently induced CaChiIII2 and other pathogenesis-related genes expressions, responsible for the innate immunity response of pepper against anthracnose disease.

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.

The CaChiVI2 Gene of Capsicum annuum L. Confers Resistance Against Heat Stress and Infection of Phytophthora capsici.

Extreme environmental conditions seriously affect crop growth and development, resulting in substantial reduction in yield and quality. However, chitin-binding proteins (CBP) family member CaChiVI2 plays a crucial role in eliminating the impact of adverse environmental conditions, such as cold and salt stress. Here, for the first time it was discovered that CaChiVI2 (Capana08g001237) gene of pepper (Capsicum annuum L.) had a role in resistance to heat stress and physiological processes. The full-length open-reading frame (ORF) of CaChiVI2 (606-bp, encoding 201-amino acids), was cloned into TRV2:CaChiVI2 vector for silencing. The CaChiVI2 gene carries heat shock elements (HSE, AAAAAATTTC) in the upstream region, and thereby shows sensitivity to heat stress at the transcriptional level. The silencing effect of CaChiVI2 in pepper resulted in increased susceptibility to heat and Phytophthora capsici infection. This was evident from the severe symptoms on leaves, the increase in superoxide (O2 -) and hydrogen peroxide (H2O2) accumulation, higher malondialdehyde (MDA), relative electrolyte leakage (REL) and lower proline contents compared with control plants. Furthermore, the transcript level of other resistance responsive genes was also altered. In addition, the CaChiIV2-overexpression in Arabidopsis thaliana showed mild heat and drought stress symptoms and increased transcript level of a defense-related gene (AtHSA32), indicating its role in the co-regulation network of the plant. The CaChiVI2-overexpressed plants also showed a decrease in MDA contents and an increase in antioxidant enzyme activity and proline accumulation. In conclusion, the results suggest that CaChiVI2 gene plays a decisive role in heat and drought stress tolerance, as well as, provides resistance against P. capsici by reducing the accumulation of reactive oxygen species (ROS) and modulating the expression of defense-related genes. The outcomes obtained here suggest that further studies should be conducted on plants adaptation mechanisms in variable environments.

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.