Antioxidant Response and Calcium-Dependent Protein Kinases Involvement in Canola (Brassica napus L.) Tolerance to Drought

Canola is an important temperate oil crop that can be severely affected by drought. Understanding the physiological and molecular mechanisms involved in canola tolerance to water deficit is essential to obtain drought-tolerant productive cultivars. To investigate the role of antioxidant response and the possible involvement of calcium-dependent protein kinases (CDPKs) in canola tolerance to drought, we analyzed four genotypes with different sensitivity to water stress. Leaf relative water content, canopy temperature, PSII efficiency, electrolyte leakage index and lipid peroxidation were used as indicators to classify the cultivars as drought-tolerant or drought-sensitive. Antioxidant enzymes superoxide dismutase, guaiacol peroxidase and catalase displayed significantly higher activities in drought-tolerant than in drought-sensitive cultivars subjected to water deficit, suggesting that the efficiency of the antioxidant response is essential in canola drought tolerance. The increased expression of genes BnaCDPK6 and BnaCDPK14 under drought conditions, their differential expression in drought-tolerant and drought-sensitive genotypes, and the presence of multiple cis-acting stress-related elements in their promoter regions suggest that CDPKs are part of the signaling pathways that regulate drought response in canola. We propose the BnaCDPK genes and their regulator elements as potential molecular targets to obtain drought-tolerant productive canola cultivars through breeding or genetic transformation.

Response of morphological and biochemical traits and transcriptomic of CDPKs and P5CS Genes to phytohormones and nutrients in two bread wheat germplasms under salinity conditions

Several enzymes play an important role in the biosynthesis of osmolyte in plants. The main objective of this work is to study the effects of salt stress, kinetin+potassium and giberellic acid+potassium on calcium-dependent protein kinases (CDPKs) and delta-1-pyrroline-5- carboxylate synthase (P5CS) genes expression of two bread wheat varieties. The results shodwe that, salts stress reduced the plant growth of the two wheat germplasms. Moreover, the addition of kinetin+ potassium improved the performance of morphometric parameters while the addition of giberellic acid +potassium has less effect. On the biochemical level, results indicated that salt stress increased the proline contents compared with control plants. Extra increase in proline contents was recorded by kinetin+ potassium, while the treatment of giberellic acid +potassium showed almost similar results as in salt stress only. On the molecular level, salt stress, kinetin and giberellic acid significantly increased the two genes expression of CDPKs and P5CS with more effect in presence of kinetin+ potassium.

Calcium-Dependent Protein Kinase GhCDPK28 Was Dentified and Involved in Verticillium Wilt Resistance in Cotton

Verticillium dahliae is a soil-borne fungus that causes vascular wilt through the roots of plants. Verticillium wilt caused by V. dahliae is one of the main diseases in cotton producing areas of the world, resulting in huge economic losses. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. Calcium-dependent protein kinases (CDPKs) play a pivotal role in plant innate immunity, including regulation of oxidative burst, gene expression as well as hormone signal transduction. However, the function of cotton CDPKs in response to V. dahliae stress remains unexplored. In this study, 96, 44 and 57 CDPKs were identified from Gossypium hirsutum, Gossypium raimondii and Gossypium arboretum, respectively. Phylogenetic analysis showed that these CDPKs could be divided into four branches. All GhCDPKs of the same clade are generally similar in gene structure and conserved domain arrangement. Cis-acting elements related to hormones, stress response, cell cycle and development were predicted in the promoter region. The expression of GhCDPKs could be regulated by various stresses. Gh_D11G188500.1 and Gh_A11G186100.1 was up-regulated under Vd0738 and Vd991 stress. Further phosphoproteomics analysis showed that Gh_A11G186100.1 (named as GhCDPK28-6) was phosphorylated under the stress of V. dahliae. Knockdown of GhCDPK28-6 expression, the content of reactive oxygen species was increased, a series of defense responses were enhanced, and the sensitivity of cotton to V. dahliae was reduced. Moreover, overexpression of GhCDPK28-6 in Arabidopsis thaliana weakened the resistance of plants to this pathogen. Subcellular localization revealed that GhCDPK28-6 was localized in the cell membrane. We also found that GhPBL9 and GhRPL12C may interact with GhCDPK28-6. These results indicate that GhCDPK28-6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.

Improvement of the Biosynthesis of Resveratrol in Endophytic Fungus (Alternaria sp. MG1) by the Synergistic Effect of UV Light and Oligomeric Proanthocyanidins

Resveratrol, a natural polyphenol compound with multiple bioactivities, is widely used in food and pharmaceutical industry. Endophytic fungus Alternaria sp. MG1, as a native producer of resveratrol, shows increasing potential application. However, strategies for improvement of the biosynthesis of resveratrol in this species are still scarce. In this study, different elicitors were used to investigate their effect on the biosynthesis of resveratrol in MG1 and the induction mechanism. Ultrasound and sodium butyrate had no effect and slight inhibition on the resveratrol production and related gene expression, respectively. UV radiation and co-culture with Phomopsis sp. XP-8 significantly promoted the biosynthesis of resveratrol with the highest production (240.57μg/l) coming from UV 20min. Co-culture altered the profiles of secondary metabolites in MG1 by promoting and inhibiting the synthesis of stilbene and lignin compounds, respectively, and generating new flavonoids ((+/−)-taxifolin, naringin, and (+)-catechin). Oligomeric proanthocyanidins (OPC) also showed an obviously positive influence, leading to an increase in resveratrol production by 10 to 60%. Two calcium-dependent protein kinases (CDPK) were identified, of which CDPK1 was found to be an important regulatory factor of OPC induction. Synergistic treatment of UV 20min and 100μm OPC increased the production of resveratrol by 70.37% compared to control and finally reached 276.31μg/l.

Cadmium Accumulation Involves Synthesis of Glutathione and Phytochelatins, and Activation of CDPK, CaMK, CBLPK, and MAPK Signaling Pathways in Ulva compressa

In order to analyze the effect of cadmium in Ulva compressa (Chlorophyta), the alga was cultivated with 10, 25, and 50 μM of cadmium for 7 days, and the level of intracellular cadmium was determined. Intracellular cadmium showed an increase on day 1, no change until day 5, and an increase on day 7. Then, the alga was cultivated with 10 μM for 7 days, and the level of hydrogen peroxide, superoxide anions, and lipoperoxides; activities of antioxidant enzymes ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR), and glutathione reductase (GR); the level of glutathione (GSH) and ascorbate (ASC); and the level of phytochelatins (PCs) and transcripts encoding metallothioneins (UcMTs) levels were determined. The level of hydrogen peroxide increased at 2 and 12 h, superoxide anions on day 1, and lipoperoxides on days 3 to 5. The activities of AP and GR were increased, but not the DHAR activity. The level of GSH increased on day 1, decreased on day 3, and increased again on day 5, whereas ASC slightly increased on days 3 and 7, and activities of enzymes involved in GSH and ASC synthesis were increased on days 3 to 7. The level of PC2 and PC4 decreased on day 3 but increased again on day 5. The level of transcripts encoding UcMT1 and UcMT2 increased on days 3 to 5, mainly that of UcMT2. Thus, cadmium accumulation induced an oxidative stress condition that was mitigated by the activation of antioxidant enzymes and synthesis of GSH and ASC. Then, the alga cultivated with inhibitors of calcium-dependent protein kinases (CDPKs), calmodulin-dependent protein kinases (CaMKs), calcineurin B-like protein kinases (CBLPKs), and MAPKs and 10 μM of cadmium for 5 days showed a decrease in intracellular cadmium and in the level of GSH and PCs, with the four inhibitors, and in the level of transcripts encoding UcMTs, with two inhibitors. Thus, CDPKs, CaMK, CBLPKS, and MAPKs are involved in cadmium accumulation and GSH and PC synthesis, and GSH and PCs and/or UcMTs may participate in cadmium accumulation.

The protein kinase CPK28 phosphorylates ascorbate peroxidase and enhances thermotolerance in tomato

High temperatures are a major threat to plant growth and development, leading to yield losses in crops. Calcium-dependent protein kinases (CPKs) act as critical components of Ca2+ sensing in plants that transduce rapid stress-induced responses to multiple environmental stimuli. However, the role of CPKs in plant thermotolerance and their mechanisms of action remain poorly understood. To address this issue, tomato (Solanum lycopersicum) cpk28 mutants were generated using a CRISPR-Cas9 gene-editing approach. The responses of mutant and wild-type plants to normal (25°C) and high temperatures (45°C) were documented. Thermotolerance was significantly decreased in the cpk28 mutants, which showed increased heat stress-induced accumulation of reactive oxygen species (ROS) and levels of protein oxidation, together with decreased activities of ascorbate peroxidase (APX) and other antioxidant enzymes. The redox status of ascorbate and glutathione were also modified. Using a yeast two-hybrid library screen and protein interaction assays, we provide evidence that CPK28 directly interacts with cytosolic APX2. Mutations in APX2 rendered plants more sensitive to high temperatures, whereas the addition of exogenous reduced ascorbate (AsA) rescued the thermotolerance phenotype of the cpk28 mutants. Moreover, protein phosphorylation analysis demonstrated that CPK28 phosphorylates the APX2 protein at Thr-59 and Thr-164. This process is suggested to be responsive to Ca2+ stimuli and may be required for CPK28-mediated thermotolerance. Taken together, these results demonstrate that CPK28 targets APX2, thus improving thermotolerance. This study suggests that CPK28 is an attractive target for the development of improved crop cultivars that are better adapted to heat stress in a changing climate.

The Role of Calcium/Calcium-Dependent Protein Kinases Signal Pathway in Pollen Tube Growth

Pollen tube (PT) growth as a key step for successful fertilization is essential for angiosperm survival and especially vital for grain yield in cereals. The process of PT growth is regulated by many complex and delicate signaling pathways. Among them, the calcium/calcium-dependent protein kinases (Ca2+/CPKs) signal pathway has become one research focus, as Ca2+ ion is a well-known essential signal molecule for PT growth, which can be instantly sensed and transduced by CPKs to control myriad biological processes. In this review, we summarize the recent progress in understanding the Ca2+/CPKs signal pathway governing PT growth. We also discuss how this pathway regulates PT growth and how reactive oxygen species (ROS) and cyclic nucleotide are integrated by Ca2+ signaling networks.

Characterization of Three Calcium-Dependent Protein Kinases of Cryptosporidium parvum

In Cryptosporidium spp., calcium-dependent protein kinases (CDPKs) are considered promising targets for the development of pharmaceutical interventions. Whole-genome sequencing has revealed the presence of 11 CDPKs in Cryptosporidium parvum (CpCDPKs). In this study, we expressed recombinant CpCDPK4, CpCDPK5, and CpCDPK6 in Escherichia coli. The biological characteristics and functions of these CpCDPKs were examined by using quantitative reverse transcription PCR (qRT-PCR), immunofluorescence microscopy, and an in vitro neutralization assay. The expression of the CpCDPK4 gene peaked at 12 h post-infection, the CpCDPK5 gene peaked at 12 and 48 h, and the CpCDPK6 gene peaked at 2–6 h. CpCDPK4 protein was located in the anterior and mid-anterior regions of sporozoites, and CpCDPK5 protein was located over the entire sporozoites, while CpCDPK6 protein was expressed in a spotty pattern. Immune sera of CpCDPK4 and CpCDPK6 exhibited significant inhibitory effects on host cell invasion, while the immune sera of CpCDPK5 had no effects. These differences in protein localization, gene expressions, and neutralizing capacities indicated that the CpCDPK proteins may have different roles during the lifecycle of Cryptosporidium spp.