Dependent Protein Kinase Gene Research Articles

Caffeine Produced in Rice Plants Provides Tolerance to Water-Deficit Stress.

Exogenous or endogenous caffeine application confers resistance to diverse biotic stresses in plants. In this study, we demonstrate that endogenous caffeine in caffeine-producing rice (CPR) increases tolerance even to abiotic stresses such as water deficit. Caffeine produced by CPR plants influences the cytosolic Ca2+ ion concentration gradient. We focused on examining the expression of Ca2+-dependent protein kinase genes, a subset of the numerous proteins engaged in abiotic stress signaling. Under normal conditions, CPR plants exhibited increased expressions of seven OsCPKs (OsCPK10, OsCPK12, OsCPK21, OsCPK25, OsCPK26, OsCPK30, and OsCPK31) and biochemical modifications, including antioxidant enzyme (superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase) activity and non-enzymatic antioxidant (ascorbic acid) content. CPR plants exhibited more pronounced gene expression changes and biochemical alterations in response to water-deficit stress. CPR plants revealed increased expressions of 16 OsCPKs (OsCPK1, OsCPK2, OsCPK3, OsCPK4, OsCPK5, OsCPK6, OsCPK9, OsCPK10, OsCPK11, OsCPK12, OsCPK14, OsCPK16, OsCPK18, OsCPK22, OsCPK24, and OsCPK25) and 8 genes (OsbZIP72, OsLEA25, OsNHX1, OsRab16d, OsDREB2B, OsNAC45, OsP5CS, and OsRSUS1) encoding factors related to abiotic stress tolerance. The activity of antioxidant enzymes increased, and non-enzymatic antioxidants accumulated. In addition, a decrease in reactive oxygen species, an accumulation of malondialdehyde, and physiological alterations such as the inhibition of chlorophyll degradation and the protection of photosynthetic machinery were observed. Our results suggest that caffeine is a natural chemical that increases the potential ability of rice to cope with water-deficit stress and provides robust resistance by activating a rapid and comprehensive resistance mechanism in the case of water-deficit stress. The discovery, furthermore, presents a new approach for enhancing crop tolerance to abiotic stress, including water deficit, via the utilization of a specific natural agent.

Overexpression of Arabidopsis thaliana cysteine2/histidine2-type transcription factor 6 gene enhances plant resistance to a bacterial pathogen

Transcription factors can be used to engineer plants for enhanced productivity. However, the mechanism(s) by which the C2H2-type zinc finger transcription factor enhances pathogen resistance in cells is not fully understood. Here, Agrobacterium tumefaciens carrying the gene for Arabidopsis thaliana cysteine2/histidine2-type transcription factor 6 (ZAT6) was used to engineer rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.) to generate transgenic cell lines. Transgenic cells were then inoculated with the pathogenic bacterium Pseudomonas syringae. Compared to the control, cell viability of transgenic cells increased 39–47% and growth rate increased 9–15% by 7 days after inoculation in rice, cotton and slash pine. Acid phosphatase activity and alkaline phosphatase activity and transcript levels of Ca2+-dependent protein kinase genes OsCPK1, OsCPK2, OsCPK6, and OsCPK8 and mitogen-activated protein kinase genes OsMAPK1, OsMAPK2, OsMAPK3, and OsMAPK8 increased significantly in transgenic rice cells by 3 day after inoculation, and extracellular pH had decreased by 10–14% by 96 min after inoculation in transgenic rice, cotton and slash pine cells. These results suggest that ZAT6 enhances P. syringae resistance in plant cells by modulating transcription of CPK and MAPK and oxidase activity.

Overexpression of Zinc Finger Transcription Factor ZAT6 Enhances Salt Tolerance.

The purpose of the present investigation is to examine the function of the C2H2-type zinc finger transcription factor of Arabidopsis thaliana 6 (ZAT6) in salt stress tolerance in cells of rice (Oryza sativa L.), cotton (Gossypium hirsutum L.) and slash pine (Pinus elliottii Engelm.). Cells of O. sativa, G. hirsutum, and P. elliottii overexpressing ZAT6 were generated using Agrobacterium-mediated genetic transformation. Molecular and functional analysis of transgenic cell lines demonstrate that overexpression of ZAT6 increased tolerance to salt stress by decreasing lipid peroxidation and increasing the content of abscisic acid (ABA) and GA8, as well as enhancing the activities of antioxidant enzymes such as ascorbate peroxidise (APOX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD). In rice cells, ZAT6 also increased expression of Ca2+-dependent protein kinase genes OsCPK9 and OsCPK25 by 5–7 fold under NaCl stress. Altogether, our results suggest that overexpression of ZAT6 enhanced salt stress tolerance by increasing antioxidant enzyme activity, hormone content and expression of Ca2+-dependent protein kinase in transgenic cell lines of different plant species.

Genome wide identification and characterization of Calcium Dependent Protein Kinase gene family in Cajanus cajan

Genome wide identification and characterization of Calcium Dependent Protein Kinase gene family in Cajanus cajan

Genome-Wide Identification of Calcium Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of Novel D-x-D and D-E-L Motifs in EF-Hand Domain.

Calcium ions are considered ubiquitous second messengers in eukaryotic signal transduction pathways. Intracellular Ca2+ concentration are modulated by various signals such as hormones and biotic and abiotic stresses. Modulation of Ca2+ ion leads to stimulation of calcium dependent protein kinase genes (CPKs), which results in regulation of gene expression and therefore mediates plant growth and development as well as biotic and abiotic stresses. Here, we reported the CPK gene family of 40 different plant species (950 CPK genes) and provided a unified nomenclature system for all of them. In addition, we analyzed their genomic, biochemical and structural conserved features. Multiple sequence alignment revealed that the kinase domain, auto-inhibitory domain and EF-hands regions of regulatory domains are highly conserved in nature. Additionally, the EF-hand domains of higher plants were found to contain four D-x-D and two D-E-L motifs, while lower eukaryotic plants had two D-x-D and one D-x-E motifs in their EF-hands. Phylogenetic analysis showed that CPK genes are clustered into four different groups. By studying the CPK gene family across the plant lineage, we provide the first evidence of the presence of D-x-D motif in the calcium binding EF-hand domain of CPK proteins.

Constitutive expression and silencing of a novel seed specific calcium dependent protein kinase gene in rice reveals its role in grain filling

Ca2+ sensor protein kinases are prevalent in most plant species including rice. They play diverse roles in plant signaling mechanism. Thirty one CDPK genes have been identified in rice and some are functionally characterized. In the present study, the newly identified rice CDPK gene OsCPK31 was functionally validated by overexpression and silencing in Taipei 309 rice cultivar. Spikelets of overexpressing plants showed hard dough stage within 15d after pollination (DAP) with rapid grain filling and early maturation. Scanning electron microscopy of endosperm during starch granule formation confirmed early grain filling. Further, seeds of overexpressing transgenic lines matured early (20–22 DAP) and the average number of maturity days reduced significantly. On the other hand, silencing lines showed more number of unfilled spikelet without any difference in maturity duration. It will be interesting to further decipher the role of OsCPK31 in biological pathways associated with distribution of photosynthetic assimilates during grain filling stage.

Overexpression of the Arabidopsis AtEm6 gene enhances salt tolerance in transgenic rice cell lines

The Arabidopsis thaliana late embryogenesis abundant gene AtEm6 is required for normal seed development and for buffering the rate of dehydration during the latter stages of seed maturation. However, its function in salt stress tolerance is not fully understood. In this investigation, cell suspension cultures of three plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and white pine (Pinus strobes L.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pBI-AtEm6. Integration of the AtEm6 gene into the genome of rice, cotton, and white pine has been confirmed by polymerase chain reaction, Southern blotting, and northern blotting analyses. Three transgenic cell lines from each of O. sativa, G. hirsutum, and P. strobus were used to analyze salt stress tolerance conferred by the overexpression of the AtEm6 gene. Our results demonstrated that expression of the AtEm6 gene enhanced salt tolerance in transgenic cell lines. A decrease in lipid peroxidation and an increment in antioxidant enzymes ascorbate peroxidase, glutathione reductase and superoxide dismutase activities were observed in the transgenic cell lines, compared to the non- transgenic control. In rice, AtEM6 increased expression of Ca2+-dependent protein kinase genes OsCPK6, OsCPK9, OsCPK10, OsCPK19, OsCPK25, and OsCPK26 under treatment of salt. These results suggested that overexpression of the AtEM6 gene in transgenic cell lines improved salt stress tolerance by regulating expression of Ca2+-dependent protein kinase genes. Overexpression of the AtEM6 gene could be an alternative choice for engineering plant abiotic stress tolerance.

Transcription factor AtbZIP60 regulates expression of Ca2+-dependent protein kinase genes in transgenic cells

The Arabidopsis thaliana bZIP60 (AtbZIP60) transcription factor regulates stress signaling. However, its molecular mechanism remains to be elucidated. In this investigation, cell suspension cultures of two different plant species rice (Oryza sativa L.) and white pine (Pinus strobes L.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pBI-AtZIP60. Integration of the AtbZIP60 gene into the genome of rice and white pine has been confirmed by polymerase chain reaction (PCR), southern blotting, and northern blotting analyses. Six transgenic cell lines from O. sativa and three transgenic cell lines from P. strobus were used to analyze the salt, drought, and cold tolerance conferred by the overexpression of the AtbZIP60 gene. Our results demonstrated that expression of the AtbZIP60 gene enhanced salt, drought, and cold tolerance in rice and white pine transgenic cell lines. In rice, transcription factor AtbZIP60 increased expression of Ca(2+)-dependent protein kinase genes OsCPK6, OsCPK9, OsCPK10, OsCPK19, OsCPK25, and OsCPK26 under treatment of salt, drought, and cold. These results demonstrated that overexpression of the AtbZIP60 gene in transgenic cell lines improved salt, drought, and cold stress tolerances by regulating expression of Ca(2+)-dependent protein kinase genes. Overexpression of the AtbZIP60 gene could be an alternative choice for engineering plant abiotic stress tolerance.

Cloning and characterization of a calcium dependent protein kinase gene associated with cotton fiber development

The gene GhCPK1 encoding a calcium dependent protein kinase was identified from cotton. Transcripts of GhCPK1 accumulated primarily in the elongating fiber, and Arabidopsis plants transformed with GhCPK1 promoter-GUS construct exhibited GUS activity mainly in the developing trichomes, roots, young leaves and sepals. In the bombarded onion epidermal cells, GhCPK1-GFP fusion proteins showed a subcellular distribution in the plasma membrane. In vitro assays indicated that GhCPK1 was a functional calcium-dependent kinase able to undergo autophosphorylation and phosphorylation of the known substrate histone III-S. Together, these results suggest that GhCPK1 may play a role in the calcium signaling events associated with fiber elongation.

A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum

A full-length cDNA coding a calmodulin (CaM)-dependent protein kinase gene was cloned from Physarum plasmodia poly(A)-RNA by polymerase chain reaction with the oligonucleotide primers that were designed after the amino acid sequence of highly conserved regions of myosin light-chain kinase. Sequence analysis of the cDNA revealed that this Physarum kinase was a 42,519-Da protein with an ATP-binding domain, Ser/Thr kinase active site signature, and CaM-binding domain. Expression of the cDNA in Escherichia coli demonstrated that the Physarum kinase in the presence of Ca 2+ and CaM phosphorylated the recombinant phosphorylatable light chain (PLc) of Physarum myosin II. The peptide analysis after proteolysis of the phosphorylated PLc indicated that Ser 18 was phosphorylated. The site was confirmed by the failure of phosphorylation of PLc, the Ser 18 of which was replaced by Ala. The physiological role of the kinase will be discussed with special reference to the 55-kDa kinase, which had been previously purified from Physarum plasmodia for phosphorylated PLc.

Distinct light-, cytokinin- and tissue-specific regulation of calcium dependent protein kinase gene expression in cucumber ( Cucumis sativus)

In plants, calcium dependent protein kinases (CDPKs) constitute a unique family of enzymes that is characterized by a C-terminal calmodulin (CaM)-like domain. In this study, we have cloned four partial CDPK cDNAs (CsCDPK1–4) from cucumber by reverse transcription polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers designed based on conserved regions of the other known CDPKs. Transcript levels of one of the CDPK messengers viz. CsCDPK3 were measured in intact, etiolated, excised cotyledons, hypocotyls and roots following treatments with light or phytohormones (cytokinin/auxin) using a recently evolved high-sensitivity quantitative RT-PCR method (TaqMan analysis). The highest transcript levels of CsCDPK3 were detected in hypocotyls followed by roots and cotyledons. Exposure to light was found to have a down-regulatory effect on CsCDPK3 transcript levels in excised hypocotyls and roots unlike in excised cotyledons where light was found to exert an up-regulatory effect. Treatment with benzyladenine (cytokinin) up-regulated CsCDPK3 transcript levels in cotyledons as opposed to a down-regulatory effect in roots and did not seem to have a significant effect on CsCDPK3 transcript levels in hypocotyls. On the other hand, 2,4-dichlorophenoxyacetic acid (2,4-D) (auxin) treatments did not cause any significant changes in CsCDPK3 transcript levels in hypocotyls, cotyledons or roots. Thus, our results show that light and cytokinin differentially regulate CsCDPK3 transcript levels in a tissue-specific manner.