Expression Of Calcium-dependent Protein Kinase Research Articles

Combined Transcriptomic and Metabolomic Analyses of Defense Mechanisms against Phytoplasma Infection in Camptotheca acuminata Decne

Camptotheca acuminata Witches’-broom disease (CaWB) is the most destructive disease affecting C. acuminata in China. Previous studies on CaWB have failed to clarify the incidence pattern in C. acuminata after infection with phytoplasma. The time interval between phytoplasma infection of C. acuminata and the onset of Witches’-broom symptoms in C. acuminata was very long. C. acuminata inoculated with CaWB showed leaf margin scorching symptoms at 4 weeks in inoculated leaves. At 16 weeks after infection (WAI), old leaves were shed, while new leaves showed a mild leaf margin scorch; at 28 WAI, typical symptoms appeared. Transcriptomic and metabolomic analyses of the three sampling periods revealed 194 differentially expressed genes, mainly enriched in MAPK signaling, plant–pathogen interaction, phenylpropanoid biosynthesis, starch and phenylpropanoid biosynthesis, and phenylpropanoid biosynthesis pathways. The expression of calcium-dependent protein kinase (CDPK), β Ketoacyl-CoA Synthase1/10 (KCS1/10), and WRKY22/29 genes in the plant–pathogen interaction pathway significantly increased, indicating that they may be key genes in the CaWB phytoplasma-mediated maintenance of ROS homeostasis. Moreover, isochlorogenic acid B, atractylenolide II, and 3-methoxybenzoic acid were found, which might serve as signaling or functional substances in the defense response. Our results provide novel insights into the pathogenesis of CaWB and the defense response of C. acuminata under the influence of phytoplasma. Additionally, we identified potential candidate genes related to the defense response of C. acuminata, laying the foundation for further research.

Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages

BackgroundBabesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies.ResultsIn this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts.ConclusionsSimilar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis.Graphical abstract

Characterization of Glutamate-Mediated Hormonal Regulatory Pathway of the Drought Responses in Relation to Proline Metabolism in Brassica napus L.

Proline metabolism influences the metabolic and/or signaling pathway in regulating plant stress responses. This study aimed to characterize the physiological significance of glutamate (Glu)-mediated proline metabolism in the drought stress responses, focusing on the hormonal regulatory pathway. The responses of cytosolic Ca2+ signaling, proline metabolism, and redox components to the exogenous application of Glu in well-watered or drought-stressed plants were interpreted in relation to endogenous hormone status and their signaling genes. Drought-enhanced level of abscisic acid (ABA) was concomitant with the accumulation of ROS and proline, as well as loss of reducing potential, which was assessed by measuring NAD(P)H/NAD(P)+ and GSH/GSSG ratios. Glu application to drought-stressed plants increased both salicylic acid (SA) and cytosolic Ca2+ levels, with the highest expression of calcium-dependent protein kinase (CPK5) and salicylic acid synthesis-related ICS1. The SA-enhanced CPK5 expression was closely associated with further enhancement of proline synthesis-related genes (P5CS1, P5CS2, and P5CR) expression and a reset of reducing potential with enhanced expression of redox regulating genes (TRXh5 and GRXC9) in a SA-mediated NPR1- and/or PR1-dependent manner. These results clearly indicate that Glu-activated interplay between SA- and CPK5-signaling as well as Glu-enhanced proline synthesis are crucial in the amelioration of drought stress in Brassica napus.

Reactive oxygen species induce cyanide-resistant respiration in potato infected by Erwinia carotovora subsp. Carotovora

Studies have shown that pathogenic bacteria infections induce the overproduction of reactive oxygen species (ROS) in plants. Cyanide-resistant respiration, an energy-dissipating pathway in plants, has also been induced by a pathogenic bacteria infection. However, it is unknown whether the induction of cyanide-resistant respiration under the pathogenic bacteria infection was caused by ROS. In this study, two pathogenic Erwinia strains were used to infect potato tuber, and membrane lipid peroxidation levels and the cyanide-resistant respiration capacity were determined. In addition, StAOX expression and regulation by ROS in potato tuber were analyzed. Moreover, the role of the Ca2+ pathway in regulating cyanide-resistant respiration was determined. The results showed that ROS induced cyanide-resistant respiration in potato tuber infected by Erwinia. Cyanide-resistant respiration inhibited the production of H2O2. Intracellular Ca2+ regulated the expression of calcium-dependent protein kinase (StCDPK1, StCDPK4, and StCDPK5) in potato, which indirectly controlled intracellular ROS levels. These results indicate that Ca2+ metabolism is involved in ROS-induced cyanide-resistant respiration.

Inhibition of nitric oxide synthesis delayed mature-green tomato fruits ripening induced by inhibition of ethylene

Ethylene is a key molecule to quality preservation during fruit ripening. The ethylene biosynthesis is regulated partly by nitric oxide (NO), a signal molecule involved in various physiological reactions. In order to investigate the effect of NO on key factors of ethylene biosynthesis, mature green tomatoes (Solanum lycopersicum Mill. cv. Lichun) were treated with the NO synthesis inhibitor L-Nitro-arginine methylester (L-NAME). The results showed that L-NAME decreased endogenous ethylene release and delayed the breaker stage of fruits. In the process, the activities of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxide (ACO) were inhibited in accordance with the down-regulation of SlACS2/4 and SlACO1/3 genes. In addition, the expression of calcium-dependent protein kinase (CDPK) and mitogen-activated protein kinase (MAPK) genes, SlCDPK1/2 and SlMAPK1/2/3, was delayed or reduced. These results suggest that protein phosphorylation is involved in the ethylene biosynthesis reduction induced by L-NAME and that this lead to a delay in tomato fruit ripening.

Calcium triggers protein kinases-induced signal transduction for augmenting the thermotolerance of developing wheat (Triticum aestivum) grain under the heat stress

Calcium acts as a signaling molecule and plays very important role in plants-tolerance to the abiotic stresses including the heat stress. We studied the effect of exogenous Ca2+ (10 mM) prior to the heat stress (42 °C, 2 h) on different molecular and biochemical parameters associated with thermotolerance in wheat (Triticum aestivum). We could observe 64 and 52 unique protein spots in HD2967 (thermotolerant) and HD2329 (thermosusceptible) cultivars under normal and heat stress condition. Similarly, 13 and 9 (HD2967) and 19 and 9 proteins (HD2329) were observed to be up-regulated and down-regulated in response to HS. MALDI-TOF-TOF/MS characterization identified the differentially expressed protein (DEP) spots as calcium dependent protein kinase (CDPK), oxygen evolving enhancer protein, HSP17, HSP70, Rubisco activase etc. Transcript profiling of identified stress-associated genes (SAGs) showed very high expression of CDPK, HSFA4a, HSP17, SOD and APX in response to Ca2+ + HS in HD2967 compared to HD2329 cultivars of wheat. Similarly accumulation of signaling molecules (H2O2 and CDPK) as well as osmolyte was observed maximum in response to Ca2+ + HS in HD2967 compared to HD2329 cultivars. Very high activities of guaiacol-peroxidase and ascorbate peroxidase were observed in Ca-treated HD2967 compared to HD2329 when exposed to HS. It is the abundance of these chaperones and antioxidant enzymes in thermotolerant cultivar which limit the accumulation of H2O2 in response to Ca2+ and HS. Exogenous Ca2+ application showed negative correlation with lipid peroxidation and positive correlation with total antioxidant capacity of the cell system under the elevated temperature. Alteration in Ca2+ efflux triggers the activities of kinases and peroxidases more in tolerant compared to susceptible cultivars which regulate the accumulation of ROS inside the cell and attenuate the lipid peroxidation process. Ultimately, it enhances the total antioxidant capacity and thermotolerance of the plants under the heat stress.

Expression of calcium-dependent protein kinase (CDPK) genes under abiotic stress conditions in wild-growing grapevine Vitis amurensis

Calcium-dependent protein kinases (CDPKs), which are important sensors of Ca2+ flux in plants, are known to play essential roles in plant development and adaptation to abiotic stresses. In the present work, we studied expression of CDPK genes under osmotic and temperature stress treatments in wild-growing grapevine Vitis amurensis Rupr., which is known to possess high adaptive potential and a high level of resistance against adverse environmental conditions. In this study, using RT-PCR with degenerate primers, DNA sequencing and frequency analysis of RT-PCR products, we identified 13 CDPK genes that are actively expressed in healthy V. amurensis cuttings under high salt, high mannitol, desiccation, and temperature stress conditions. 12 CDPKs, namely VaCPK1, VaCPK2, VaCPK3, VaCPK9, VaCPK13, VaCPK16, VaCPK20, VaCPK21, VaCPK25, VaCPK26, VaCPK29 and VaCPK30, were novel for Vitaceae, and their full cDNAs were obtained and described. Quantitative real-time RT-PCR demonstrated that mRNA levels of 10 VaCPK genes were differentially up-regulated under the osmotic and temperature stress treatments, while the abundance of 3 VaCPK transcript variants, VaCPK3a, VaCPK25, and VaCPK30, was not markedly changed. Expression profiling of the VaCPK genes in leaves, leaf petioles, stems, inflorescences, berries, and seeds of V. amurensis revealed that the genes exhibit different organ-specific expression patterns. The stimulatory effect of abiotic stress on the expression of the VaCPK1, 2, 3, 9, 13, 16, 20, 21, 26, and VaCPK29 genes is suggestive of their implication in the grapevine response to osmotic and temperature stresses, while the variability in their organ-specific expression patterns indicates that the enzymes perform distinct biological functions.

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of cal- cium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02 % dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1-0.8 % DW). Treatment with the CI at a 1 lM con- centration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10 lM significantly decreased resveratrol production by 2-4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1 lM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expres- sion of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concen- trations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.

Calcium dependent protein kinase (CDPK) expression during fruit development in cultivated peanut ( Arachis hypogaea) under Ca 2+-sufficient and -deficient growth regimens

Adequate soil calcium (Ca 2+) levels are crucial for sustained reproductive development of peanut ( Arachis hypogaea). A role for calcium dependent protein kinase was evaluated during peanut fruit development under sufficient and deficient soil Ca 2+ conditions. Quantitative RT-PCR and protein gel blot analyses confirmed transcriptional upregulation of CDPK in seeds developing under inadequate soil Ca 2+ regimen, as well as spatiotemporal regulation of CDPK expression during early mitotic growth and later during the storage phase of seed development. However, a consistent basal level of CDPK was present during similar developmental stages of pod tissue, irrespective of the soil Ca 2+ status. Immunolocalization data showed CDPK decoration primarily in the outer most cell layers of the pericarp and around vascular bundles linked by lateral connections in developing pods, as well as the single vascular trace supplying nutrients to the developing seed. Finally, carbohydrate analyses and qRT-PCR data are provided for peanut genes encoding enzymes involved in sucrose cleavage (orthologs of Vicia faba, VfCWI1 and VfCWI2) and utilization ( AhSuSy and AhSpS), and oleosin gene transcripts ( AhOleo17.8 and AhOleo18.5) validating a role for CDPK in the establishment and maintenance of sink strength, and subsequent onset of storage product biosynthetic phase during seed maturation.

AFLP-based transcript profiling for cassava genome-wide expression analysis in the onset of storage root formation

Cassava (Manihot esculenta Crantz) is a root crop that accumulates large quantities of starch, and it is an important source of carbohydrate. Study on gene expressions during storage root development provides important information on storage root formation and starch accumulation as well as unlock new traits for improving of starch yield. cDNA-Amplified Fragment Length Polymorphism (AFLP) was used to compare gene expression profiles in fibrous and storage roots of cassava cultivar Kasetsart 50. Total of 155 differentially expressed transcript-derived fragments with undetectable or low expression in leaves were characterized and classified into 11 groups regarding to their functions. The four major groups were no similarity (20%), hypothetical or unknown proteins (17%), cellular metabolism and biosynthesis (17%) and cellular communication and signaling (14%). Interestingly, sulfite reductase (MeKD82), calcium-dependent protein kinase (CDPK) (MeKD83), ent-kaurene synthase (KS) (MeKD106) and hexose transporter (HT) (MeKD154) showed root-specific expression patterns. This finding is consistent with previously reported genes involved in the initiation of potato tuber. Semi-quantitative reverse transcription polymerase chain reaction of early-developed root samples confirmed that those four genes exhibited significant expression with similar pattern in the storage root initiation and early developmental stages. We proposed that KS and HT may involve in transient induction of CDPK expression, which may play an important role in the signaling pathway of storage root initiation. Sulfite reductase, on the other hand, may involve in storage root development by facilitating sulfur-containing protein biosynthesis or detoxifying the cyanogenic glucoside content through aspartate biosynthesis.

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration.

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.

Gene expression in cultured strawberry cells subjected to hydrodynamic stress

We investigated gene expression responding to hydrodynamic stress in an agitation tank using RT-PCR. A novel fragment, tuf, was found to be expressed under turbulent flow conditions using a differential display method. The deduced amino acid sequence of the fragment was homologous to those of NBS (nucleotide binding site) domains in plant disease-resistant genes, but differed with respect to some of the functional motifs of those sequences. We also demonstrated that calcium-dependent protein kinase (CDPK) expression increased by agitation. A calcium channel blocker, verapamil, suppressed the expression of tuf, but did not affect the expression of CDPK.