Calcium-dependent Protein Kinases Gene Research Articles

TaCDPK1-5A positively regulates drought response through modulating osmotic stress responsive-associated processes in wheat (Triticum aestivum).

Wheat TaCDPK1-5A plays critical roles in mediating drought tolerance through regulating osmotic stress-associated physiological processes. Calcium (Ca2+) acts as an essential second messenger in plant signaling pathways and impacts plant abiotic stress responses. This study reported the function of TaCDPK1-5A, a calcium-dependent protein kinase (CDPK) gene in T. aestivum, in mediating drought tolerance. TaCDPK1-5A sensitively responded to drought and exogenous abscisic acid (ABA) signaling, displaying induced transcripts in plants under drought and ABA treatments. Yeast two-hybrid and co-immunoprecipitation assays revealed that TaCDPK1-5A interacts with the mitogen-activated protein kinase TaMAPK4-7D whereas the latter with ABF transcription factor TaABF1-3A, suggesting that TaCDPK1-5A constitutes a signaling module with above partners to transduce signals initiated by drought/ABA stressors. Overexpression of TaCDPK1-5A, TaMAPK4-7D and TaABF1-3A enhanced plant drought adaptation by modulating the osmotic stress-related physiological indices, including increased osmolyte contents, enlarged root morphology, and promoted stomata closure. Yeast one-hybrid assays indicated the binding ability of TaABF1-3A with promoters of TaP5CS1-1B, TaPIN3-5A, and TaSLAC1-3-2A, the genes encoding P5CS enzyme, PIN-FORMED protein, and slow anion channel, respectively. ChIP-PCR and transcriptional activation assays confirmed that TaABF1-3A regulates these genes at transcriptional level. Moreover, transgene analysis indicated that these stress-responsive genes positively regulated proline biosynthesis (TaP5CS1-1B), root morphology (TaPIN3-5A), and stomata closing (TaSLAC1-3-2A) upon drought signaling. Positive correlations were observed between yield and the transcripts of TaCDPK1-5A signaling partners in wheat cultivars under drought condition, with haplotype TaCDPK1-5A-Hap1 contributing to improved drought tolerance. Our study concluded that TaCDPK1-5A positively regulates drought adaptation and is a valuable target for molecular breeding the drought-tolerant cultivars in T. aestivum.

Identification of SikCDPK family genes to low-temperature by RNA-seq approaches and functional analysis of SikCDPK1 in Saussurea involucrata (Kar. & Kir.).

Calcium-Dependent Protein Kinases (CDPKs) are a class of serine/threonine protein kinases encoded by several gene families that play key roles in biotic and abiotic stresses response and plant growth and development. However, snow lotus (Saussurea involucrata kar L.) CDPKs has rarely been reported. In this study, 20 CDPK genes in snow lotus were identified based on transcriptome data and classified into four groups (I-IV) based on their structural features and phylogenetic analyses. Among them, the transcript levels of SikCDPK1 were significantly induced by low temperature and multiple hormone treatments, and SikCDPK1 gene was found to have different expression in snow lotus seeds, leaves, stems and roots. The full-length promoter activity of SikCDPK1 gene was higher than that of the 5' end deletion fragment, and the promoter fragment containing the low temperature inducing element had increased activation after low temperature treatment. The promoter activity of SikCDPK1 gene was mainly expressed in roots and rosette leaves. In addition, overexpressing plants of SikCDPK1 were more tolerant compared to the wild type after being subjected to low temperature stress. Physiological analyses indicated that SikCDPK1 improved plant tolerance to low temperature stress by maintaining cell membrane stability and reducing the accumulation of reactive oxygen species (ROS). These findings provided insights into CDPK gene families in snow lotus and broaden our understanding of the biological role of SikCDPK1 and the mechanism of low temperature stress tolerance in snow lotus.

Kinase CPK10 regulates low light-induced tomato flower drop downstream of IDL6 in a calcium-dependent manner.

Flower drop is a major cause for yield loss in many crops. Previously, we found that tomato (Solanum lycopersicum) INFLORESCENCE DEFICIENT IN ABSCISSION-Like (SlIDL6) contributes to flower drop induced by low light. However, the molecular mechanisms by which SlIDL6 acts as a signal to regulate low light-induced abscission remain unclear. In this study, SlIDL6 was found to elevate cytosolic Ca2+ concentrations ([Ca2+]cyt) in the abscission zone (AZ), which was required for SlIDL6-induced flower drop under low light. We further identified that one calcium-dependent protein kinase gene (SlCPK10) was highly expressed in the AZ and up-regulated by SlIDL6-triggered [Ca2+]cyt. Over-expression and knockout of SlCPK10 in tomato resulted in accelerated and delayed abscission, respectively. Genetic evidence further indicated that knockout of SlCPK10 significantly impaired the function of SlIDL6 in accelerating abscission. Furthermore, Ser-371 phosphorylation in SlCPK10 dependent on SlIDL6 was necessary and sufficient for its function in regulating flower drop, probably by stabilizing the SlCPK10 proteins. Taken together, our findings reveal that SlCPK10, as a downstream component of the IDL6 signaling pathway, regulates flower drop in tomato under low light stress.

Transcriptome Profiles Reveal Key Regulatory Networks during Single and Multifactorial Stresses Coupled with Melatonin Treatment in Pitaya (Selenicereus undatus L.).

In response to evolving climatic conditions, plants frequently confront multiple abiotic stresses, necessitating robust adaptive mechanisms. This study focuses on the responses of Selenicereus undatus L. to both individual stresses (cadmium; Cd, salt; S, and drought; D) and their combined applications, with an emphasis on evaluating the mitigating effects of (M) melatonin. Through transcriptome analysis, this study identifies significant gene expression changes and regulatory network activations. The results show that stress decreases pitaya growth rates by 30%, reduces stem and cladode development by 40%, and increases Cd uptake under single and combined stresses by 50% and 70%, respectively. Under stress conditions, enhanced activities of H2O2, POD, CAT, APX, and SOD and elevated proline content indicate strong antioxidant defenses. We identified 141 common DEGs related to stress tolerance, most of which were related to AtCBP, ALA, and CBP pathways. Interestingly, the production of genes related to signal transduction and hormones, including abscisic acid and auxin, was also significantly induced. Several calcium-dependent protein kinase genes were regulated during M and stress treatments. Functional enrichment analysis showed that most of the DEGs were enriched during metabolism, MAPK signaling, and photosynthesis. In addition, weighted gene co-expression network analysis (WGCNA) identified critical transcription factors (WRKYs, MYBs, bZIPs, bHLHs, and NACs) associated with antioxidant activities, particularly within the salmon module. This study provides morpho-physiological and transcriptome insights into pitaya's stress responses and suggests molecular breeding techniques with which to enhance plant resistance.

CRISPR–Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes

Calcium-dependent protein kinases (CDPKs) act as key signal transduction enzymes in plants, especially in response to diverse stresses, including herbivory. In this study, a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses. We developed a strategy for screening insect-resistance genes from a CRISPR–Cas9 mutant library of GhCPKs. The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants. The average target-gene coverage was 86.18%, the genome editing rate was 89.49%, and the editing heritability was 82%. An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects. The mutant that showed the clearest insect resistance, cpk33/74 (in which the homologous genes GhCPK33 and GhCPK74 were knocked out), was selected for further study. Oral secretions from Spodoptera litura induced a rapid influx of Ca2+ in cpk33/74 leaves, resulting in a significant increase in jasmonic acid content. S-adenosylmethionine synthase is an important protein involved in plant stress response, and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2. In addition, virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S. litura. This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

Genome-wide identification and stress response analysis of BcaCPK gene family in amphidiploid Brassica carinata.

Calcium-dependent protein kinases (CPKs) are crucial for recognizing and transmitting Ca2+ signals in plant cells, playing a vital role in growth, development, and stress response. This study aimed to identify and detect the potential roles of the CPK gene family in the amphidiploid Brassica carinata (BBCC, 2n = 34) using bioinformatics methods. Based on the published genomic information of B. carinata, a total of 123 CPK genes were identified, comprising 70 CPK genes on the B subgenome and 53 on the C subgenome. To further investigate the homologous evolutionary relationship between B. carinata and other plants, the phylogenetic tree was constructed using CPKs in B. carinata and Arabidopsis thaliana. The phylogenetic analysis classified 123 family members into four subfamilies, where gene members within the same subfamily exhibited similar conserved motifs. Each BcaCPK member possesses a core protein kinase domain and four EF-hand domains. Most of the BcaCPK genes contain 5 to 8 introns, and these 123 BcaCPK genes are unevenly distributed across 17 chromosomes. Among these BcaCPK genes, 120 replicated gene pairs were found, whereas only 8 genes were tandem duplication, suggesting that dispersed duplication mainly drove the family amplification. The results of the Ka/Ks analysis indicated that the CPK gene family of B. carinata was primarily underwent purification selection in evolutionary selection. The promoter region of most BcaCPK genes contained various stress-related cis-acting elements. qRT-PCR analysis of 12 selected CPK genes conducted under cadmium and salt stress at various points revealed distinct expression patterns among different family members in response to different stresses. Specifically, the expression levels of BcaCPK2.B01a, BcaCPK16.B02b, and BcaCPK26.B02 were down-regulated under both stresses, whereas the expression levels of other members were significantly up-regulated under at least one stress. This study systematically identified the BcaCPK gene family in B. carinata, which contributes to a better understanding the CPK genes in this species. The findings also serve as a reference for analyzing stress responses, particularly in relation to cadmium and salt stress in B. carinata.

Identification and expression analysis of calcium-dependent protein kinase family in oat (Avena sativa L.) and their functions in response to saline-alkali stresses.

Calcium-dependent protein kinases (CDPKs) serve as calcium ion sensors and play crucial roles in all aspects of plant life cycle. While CDPK gene family has been extensively studied in various plants, there is limited information available for CDPK members in oat, an important cereal crop worldwide. Totally, 60 AsCDPK genes were identified in oat genome and were classified into four subfamilies based on their phylogenetic relationship. The members within each subfamily shared similar gene structure and conserved motifs. Collinearity analysis revealed that AsCDPK gene amplification was attributed to segmental duplication events and underwent strong purifying selection. AsCDPK promoters were predicted to contain cis-acting elements associated with hormones, biotic and abiotic stresses. AsCDPK gene expressions were induced by different salt stresses, exhibiting stress-specific under different salt treatments. Moreover, overexpression of AsCDPK26 gene enhanced salt resistance in C. reinhardtii, a single-cell photoautotrophic model plants. Further analysis revealed a significant correlation between AsCDPK26 and Na+/H+ antiporter 1 (p<0.05), suggesting that AsCDPK26 may interact with ion transporter to modulate salt resistance. These results not only provide valuable insights into AsCDPK genes in response to different salt stresses, but also lay the foundation to mine novel candidates for improving salt tolerance in oat and other crops.

Identification of the CDPK gene family in patchouli and functional analysis in response to continuous cropping stress

To further reveal the molecular mechanisms underlying the formation of continuous cropping disorders in patchouli, this study analyzed the function of calcium dependent protein kinase (CDPK) genes at the molecular level in patchouli continuous cropping disorders. The findings unveiled the presence of 45 PcCDPK genes within the complete patchouli genome dataset. These genes exhibited a range of molecular weights from 50.78 to 78.96 kDa and aliphatic index values spanning from 74.42 to 88.49, and are shown to be hydrophilic proteins. The evolution of 45 PcCDPK members was divided into 4 subfamilies, with a total of 65 pairs of collinear genes. Each PcCDPK contains a STKc-CAMK domain and four EF-hand structures exhibiting a certain degree of conservatism during evolution. Transcriptome data further supported the significance of PcCDPK25 and PcCDPK38 genes, showing substantial upregulation, which was corroborated by qRT-PCR results. The 1629 bp and 1716 bp CDS sequences were obtained by cloning the PcCDPK25 and PcCDPK38 genes, respectively, and subcellular localization showed that both proteins were localized on the plasma membrane. This comprehensive study provides molecular-level confirmation of the pivotal roles played by CDPK genes in the emergence of continuous cropping challenges in patchouli plants, establishing a crucial foundation for a deeper comprehension of the molecular mechanisms underpinning these obstacles.

Genome-Wide Identification and Characterization of CDPK Gene Family in Cultivated Peanut (Arachis hypogaea L.) Reveal Their Potential Roles in Response to Ca Deficiency.

This study identified 45 calcium-dependent protein kinase (CDPK) genes in cultivated peanut (Arachis hypogaea L.), which are integral in plant growth, development, and stress responses. These genes, classified into four subgroups based on phylogenetic relationships, are unevenly distributed across all twenty peanut chromosomes. The analysis of the genetic structure of AhCDPKs revealed significant similarity within subgroups, with their expansion primarily driven by whole-genome duplications. The upstream promoter sequences of AhCDPK genes contained 46 cis-acting regulatory elements, associated with various plant responses. Additionally, 13 microRNAs were identified that target 21 AhCDPK genes, suggesting potential post-transcriptional regulation. AhCDPK proteins interacted with respiratory burst oxidase homologs, suggesting their involvement in redox signaling. Gene ontology and KEGG enrichment analyses affirmed AhCDPK genes' roles in calcium ion binding, protein kinase activity, and environmental adaptation. RNA-seq data revealed diverse expression patterns under different stress conditions. Importantly, 26 AhCDPK genes were significantly induced when exposed to Ca deficiency during the pod stage. During the seedling stage, four AhCDPKs (AhCDPK2/-25/-28/-45) in roots peaked after three hours, suggesting early signaling roles in pod Ca nutrition. These findings provide insights into the roles of CDPK genes in plant development and stress responses, offering potential candidates for predicting calcium levels in peanut seeds.

Genome-Wide Identification of the CDPK Gene Family and Their Involvement in Taproot Cracking in Radish.

Taproot cracking, a severe and common physiological disorder, markedly reduces radish yield and commercial value. Calcium-dependent protein kinase (CDPK) plays a pivotal role in various plant developmental processes; however, its function in radish taproot cracking remains largely unknown. Here, 37 RsCDPK gene members were identified from the long-read radish genome "QZ-16". Phylogenetic analysis revealed that the CDPK members in radish, tomato, and Arabidopsis were clustered into four groups. Additionally, synteny analysis identified 13 segmental duplication events in the RsCDPK genes. Analysis of paraffin-embedded sections showed that the density and arrangement of fleshy taproot cortex cells are important factors that affect radish cracking. Transcriptome sequencing of the fleshy taproot cortex revealed 5755 differentially expressed genes (DEGs) (3252 upregulated and 2503 downregulated) between non-cracking radish "HongYun" and cracking radish "505". These DEGs were significantly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interaction KEGG pathways. Furthermore, when comparing the 37 RsCDPK gene family members and RNA-seq DEGs, we identified six RsCDPK genes related to taproot cracking in radish. Soybean hairy root transformation experiments showed that RsCDPK21 significantly and positively regulates root length development. These findings provide valuable insights into the relationship between radish taproot cracking and RsCDPK gene function.

Identification of Dioscorea opposite Thunb. CDPK gene family reveals that DoCDPK20 is related to heat resistance.

Temperature affects the growth and yield of yam (Dioscorea opposite Thunb.), and calcium-dependent protein kinases (CDPKs) play an important role in the plant stress response. However, there has been a lack of system analyses of yam's CDPK gene family. In this study, 29 CDPK transcriptome sequences with complete open reading frames (ORFs) were identified from yam RNA sequencing data. The sequences were classified into four groups (I-VI) using phylogenetic analysis. Two DoCDPK genes were randomly selected from each group and the gene patterns of yam leaves were determined using quantitative real-time PCR (qRT-PCR) under high and low temperature stress in order to show their unique functions in mediating specific responses. Among them, DoCDPK20 was significantly induced in high temperatures. The pPZP221-DoCDPK20 was transformed into tobacco leaves using an agrobacterium-mediated method. Under high temperature stress, DoCDPK20 overexpression reduced photosynthesis and improved heat tolerance in transgenic tobacco. Our research offers meaningful perspectives into CDPK genes and new avenues for the genetic engineering and molecular breeding of yam.

Proteome-Level Investigation of Vitis amurensis Calli Transformed with a Constitutively Active, Ca2+-Independent Form of the Arabidopsis AtCPK1 Gene.

Calcium-dependent protein kinases (CDPKs) are one of the main Ca2+ decoders in plants. Among them, Arabidopsis thaliana AtCPK1 is one of the most studied CDPK genes as a positive regulator of plant responses to biotic and abiotic stress. The mutated form of AtCPK1, in which the autoinhibitory domain is inactivated (AtCPK1-Ca), provides constitutive kinase activity by mimicking a stress-induced increase in the Ca2+ flux. In the present study, we performed a proteomic analysis of Vitis amurensis calli overexpressing the AtCPK1-Ca form using untransformed calli as a control. In our previous studies, we have shown that the overexpression of this mutant form leads to the activation of secondary metabolism in plant cell cultures, including an increase in resveratrol biosynthesis in V. amurensis cell cultures. We analyzed upregulated and downregulated proteins in control and transgenic callus cultures using two-dimensional gel electrophoresis, and Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF). In calli transformed with AtCPK1-Ca, an increased amounts of pathogenesis-related proteins were found. A quantitative real-time PCR analysis confirmed this result.

Foliar Application of Chelated Sugar Alcohol Calcium Improves Photosynthesis and Tuber Quality under Drought Stress in Potatoes (Solanum tuberosum L.).

Drought stress is a major threat to sustainable crop production worldwide. Despite the positive role of calcium (Ca2+) in improving plant drought tolerance in different crops, little attention has been paid to its role in mitigating drought stress in potatoes. In the present study, we studied the effect of foliar chelated sugar alcohol calcium treatments on two potato cultivars with different drought responses applied 15 and 30 days after limiting soil moisture. The results showed that the foliar application of calcium treatments alleviated the SPAD chlorophyll loss of the drought-sensitive cultivar 'Atlantic' (Atl) and reduced the inhibition of photosynthetic parameters, leaf anatomy deformation, and MDA and H2O2 content of both cultivars under drought stress. The Ca2+ treatments changed the expression of several Calcium-Dependent Protein Kinase (StCDPK) genes involved in calcium sensing and signaling and significantly increased antioxidant enzyme activities, average tuber weight per plant, and tuber quality of both cultivars. We conclude that calcium spray treatments improved the drought tolerance of both potato cultivars and were especially effective for the drought-sensitive cultivar. The present work suggests that the foliar application of calcium is a promising strategy to improve commercial potato yields and the economic efficiency of potato production under drought stress conditions.

Whole-Genome Identification of Regulatory Function of CDPK Gene Families in Cold Stress Response for Prunus mume and Prunus mume var. Tortuosa.

Calcium-dependent protein kinases (CDPK) are known to mediate plant growth and development and respond to various environmental changes. Here, we performed whole-genome identification of CDPK families in cultivated and wild mei (Prunus mume). We identified 14 and 17 CDPK genes in P. mume and P. mume var. Tortuosa genomes, respectively. All 270 CPDK proteins were classified into four clade, displaying frequent homologies between these two genomes and those of other Rosaceae species. Exon/intron structure, motif and synteny blocks were conserved between P. mume and P. mume var. Tortuosa. The interaction network revealed all PmCDPK and PmvCDPK proteins is interacted with respiratory burst oxidase homologs (RBOHs) and mitogen-activated protein kinase (MAPK). RNA-seq data analysis of cold experiments show that cis-acting elements in the PmCDPK genes, especially PmCDPK14, are associated with cold hardiness. Our results provide and broad insights into CDPK gene families in mei and their role in modulating cold stress response in plants.

Identification, characterization, and expression analysis of PpCPK gene family in response to Alternaria-infected peach fruits collected from different geographical regions of China

Peach is a popular and economically important fruit tree highly susceptible to biotic and abiotic stresses, including diseases caused by fungi, bacteria and viruses, pests and insects that feed fruits and leaves. Alteranria is a deadly disease agent, a genus of fungi that can cause significant damage to plants and crops such as peaches. Here, we investigated the role of CPK genes against in Alternaria species. The calcium-dependent protein kinase (CPK) gene family plays a vital role in the biotic stress response of plants. In this study, we aimed to identify, characterize, and analyze the expression pattern of the PpCPK gene family in response to Alternaria-infected peach fruits collected from different geographical regions of China. Based on phylogenetic analysis, 17 PpCPK genes were identified and classified into seven groups. The physicochemical properties, gene structure, conserved domains, and motifs of the PpCPK genes were analyzed to understand their molecular characteristics further. The Ka/Ks ratio analysis revealed the evolutionary facets of duplication events of PpCPKs and predicted the divergence time between 23.5 and 43.6 million years ago. The promoter region analysis confirmed the presence of plant growth, light, stress, and hormone-related cis-elements in PpCPKs. Additionally, the expression pattern of PpCPKs was analyzed using real-time quantitative PCR in Alternaria-infected peach fruits from different regions of China. The results indicated a differential expression pattern of PpCPK genes responding to Alternaria infection, suggesting their potential role in defense against biotic stress. Overall, our study provides insights into the molecular mechanisms of PpCPK genes in response to biotic stress, which could aid in developing strategies for improving peach resistance to biotic stress.

Fine mapping and transcriptome sequencing reveal candidate genes conferring all-stage resistance to stripe rust on chromosome arm 1AL in Chinese wheat landrace AS1676

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), threatens wheat production worldwide, and resistant varieties tend to become susceptible after a period of cultivation owing to the variation of pathogen races. In this study, a new resistance gene against Pst race CYR34 was identified and predicted using the descendants of a cross between AS1676, a highly resistant Chinese landrace, and Avocet S, a susceptible cultivar. From a heterozygous plant from a F7 recombinant inbred line (RIL) population lacking the Yr18 gene, a near-isogenic line (NIL) population was developed to map the resistance gene. An all-stage resistance gene, YrAS1676, was identified on chromosome arm 1AL via bulked-segregant exome-capture sequencing. By analyzing a large NIL population consisting of 6537 plants, the gene was further mapped to the marker interval between KA1A_485.36 and KA1A_490.13, spanning 485.36–490.13 Mb on 1AL. A total of 66 annotated genes have been reported in this region. To characterize and predict the candidate gene(s), an RNA-seq was performed using NIL-R and NIL-S seedlings 3 days after CYR34 inoculation. Compared to NIL-S plants, NIL-R plants showed stronger immune reaction and higher expression levels of genes encoding pathogenesis-associated proteins. These differences may help to explain why NIL-R plants were more resistant to Pst race CYR34 than NIL-S plants. By combining fine-mapping and transcriptome sequencing, a calcium-dependent protein kinase gene was finally predicted as the potential candidate gene of YrAS1676. This gene contained a single-nucleotide polymorphism. The candidate gene was more highly expressed in NIL-R than in NIL-S plants. In field experiments with Pst challenge, the YrAS1676 genotype showed mitigation of disease damage and yield loss without adverse effects on tested agronomic traits. These results suggest that YrAS1676 has potential use in wheat stripe rust resistance breeding.

Genome-wide analysis of the CDPK gene family and their important roles response to cold stress in white clover

ABSTRACT Calcium-dependent protein kinases (CDPKs) are an important class of calcium-sensitive response proteins that play an important regulatory role in response to abiotic stresses. To date, little is known about the CDPK genes in white clover. White clover is a high-quality forage grass with high protein content, but it is susceptible to cold stress. Therefore, we performed a genome-wide analysis of the CDPK gene family in white clover and identified 50 members of the CDPK genes. Phylogenetic analysis using CDPKs from the model plant Arabidopsis divided the TrCDPK genes into four groups based on their sequence similarities. Motif analysis showed that TrCDPKs within the same group had similar motif compositions. Gene duplication analysis revealed the evolution and expansion of TrCDPK genes in white clover. Meanwhile, a genetic regulatory network (GRN) containing TrCDPK genes was reconstructed, and gene ontology (GO) annotation analysis of these functional genes showed that they contribute to signal transduction, cellular response to stimuli, and biological regulation, all of which are important processes in response to abiotic stresses. To determine the function of TrCDPK genes, we analyzed the RNA-seq dataset and found that most TrCDPK genes were highly up-regulated under cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiments, implying that TrCDPK genes are involved in various gene regulatory pathways in response to cold stress. Our study may help to further investigate the function of TrCDPK genes and their role in response to cold stress, which is important for understanding the molecular mechanisms of cold tolerance in white clover and improving its cold tolerance.

Genome-wide Analysis of Drought-related Genes of “Alphonso” and “Tommy Atkins” Mango (Mangifera indica L.) Cultivars

Mango (Mangifera indica L.) is one of the most popular fruit crops grown and traded globally. Mango can withstand seasonal drought periods, especially during flowering. However, prolonged drought stress can cause the weakening of the plant and may encourage injuries and diseases caused by abiotic and biotic factors. With the availability of the mango genome, genomic studies related to drought adaptation of mango can now be extensively explored. In this study, genome- wide drought-related genes (DRGs) of mango were analyzed using the whole genome sequences (WGS) of “Alphonso” and “Tommy Atkins” cultivars (PRJNA487154 and PRJNA450143, respectively). A total of 261 and 257 DRGs were identified in the genome of “Alphonso” and “Tommy Atkins,” respectively, using BLASTP. Approximately 50% of these genes are involved in both molecular and physiological adaptations of mango to drought. Among the drought stress genes, the ABC transporter gene ABCG40 had the highest number of homologs in mango, followed by calcium-dependent protein kinase genes ZmCPK4, CPK21, and CDPK7, as well as the plasma membrane proton ATPase OST2. Gene ontology (GO) analysis of the DRGs revealed that protein binding, ATP binding, and mRNA binding are the most common molecular functions, whereas the predominant biological process of these DRGs is related to their response to water deprivation. Phylogenetic analysis showed that the drought-related proteins in “Alphonso” and “Tommy Atkins” are broadly clustered into seven and six major clades, respectively. This study, to date, offers the most comprehensive information on the genome-wide DRGs of the mango, which can strengthen marker-assisted breeding programs for drought tolerance in mango and other related fruit trees, as well as the future incorporation of favorable alleles toward improving the overall agronomic characteristics of this Philippine important fruit crop.

Comparative transcriptome analysis provides novel insights into molecular response of salt-tolerant and sensitive polyembryonic mango genotypes to salinity stress at seedling stage.

Increased soil salinity in the recent years has adversely affected the productivity of mango globally. Extending the cultivation of mango in salt affected regions warrants the use of salinity tolerant/resistant rootstocks. However, the lack of sufficient genomic and transcriptomic information impedes comprehensive research at the molecular level. We employed RNA sequencing-based transcriptome analysis to gain insight into molecular response to salt stress by using two polyembryonic mango genotypes with contrasting response to salt stress viz., salt tolerant Turpentine and salt susceptible Mylepelian. RNA sequencing by Novaseq6000 resulted in a total of 2795088, 17535948, 7813704 and 5544894 clean reads in Mylepelian treated (MT), Mylepelian control (MC), Turpentine treated (TT) and Turpentine control (TC) respectively. In total, 7169 unigenes annotated against all the five public databases, including NR, NT, PFAM, KOG, Swissport, KEGG and GO. Further, maximum number of differentially expressed genes were found between MT and MC (2106) followed by MT vs TT (1158) and TT and TC (587). The differentially expressed genes under different treatment levels included transcription factors (bZIP, NAC, bHLH), genes involved in Calcium-dependent protein kinases (CDPKs), ABA biosynthesis, Photosynthesis etc. Expression of few of these genes was experimentally validated through quantitative real-time PCR (qRT-PCR) and contrasting expression pattern of Auxin Response Factor 2 (ARF2), Late Embryogenesis Abundant (LEA) and CDPK genes were observed between Turpentine and Mylepelian. The results of this study will be useful in understanding the molecular mechanism underlying salt tolerance in mango which can serve as valuable baseline information to generate new targets in mango breeding for salt tolerance.

Tinospora crispa Ethanolic Extract Downregulates Protein Kinase Genes Expression and Activity during Toxoplasma gondii Infection: A Prospective Drug Target for Lytic Cycle Inhibition

Infection with Toxoplasma gondii remains widespread among humans and animals as water, soil and food continue to serve as the major carriers of the sporulated oocyst. The infection is poorly controlled due to lack of a potent vaccine against the parasite, and the current medication presents severe side effects on the host, less efficacy on the parasite and is accompanied by the potential development of resistance by the parasite. The aim of this study was to evaluate the in vitro activities of ethanolic extract of Tinospora crispa (EETC) on protein kinases involved in the lytic cycle of T. gondii infection in Vero cell line. The EETC was obtained through the maceration of dried stem powder. Vero cells infected with the RH strain of T. gondii were used to evaluate the inhibitory effect of EETC against T. gondii calcium dependent protein kinase (CDPK) genes and microneme proteins (MIC) that are essential for host cell invasion and intracellular replication of the tachyzoite. Gene expression profiling of CDPK genes was determined through quantitative real-time PCR (qPCR) after 24 h of treatment. The expression of microneme protein was determined through western blot technique. The RT-qPCR revealed downregulation of most protein kinase (PK) genes after treatment with EETC. The expression of CDPK1, PKG, CDPK3, CDPK6, CDPK7 genes that participate in the lytic cycle of T. gondii infection was downregulated. The expression of the TgMIC1 and TgMIC2 proteins were observed to have decreased in both 4 and 24 h post-infection treatment models. This study showed that EETC contains promising drug candidates effective against T. gondii that can target the protein kinase genes involved in the lytic cycle of the parasite to prevent disease progression. HIGHLIGHTS This study explored the possible mechanism of action of phytochemicals on Toxoplasma gondii parasite-host cell invasion and intracellular replication The study evaluated the effects of ethanolic extract of Tinospora crispa Miers on protein kinase genes that are involved in host cell invasion The target protein kinase genes were downregulated which prevents the secretion of microneme proteins Inhibition of microneme secretion prevents host cell invasion and intracellular replication by Toxoplasma gondi GRAPHICAL ABSTRACT