Is DNA methylation modulated by wounding-induced oxidative burst in maize?

Plants respond to environmental changes by modifying gene expression. One of the mechanisms regulating gene expression is methylation of cytosine to 5-methylcytosine (m5C) which modulates gene expression by changing chromatin structure. Methylation/demethylation processes affect genes that are controlled upon environmental stresses. Here, on account of the regulatory role of m5C, we evaluate the content of m5C in DNA from normal and wound-damaged maize leaves. Wounding leads to a transient decrease of the global DNA methylation level ca 20-30% 1 hour after the treatment followed by a return to the initial level within the next hours. Similar results were obtained using of radio-labelled nucleotides separated by Thin Layer Chromatography (TLC) or using m5C-specific Enzyme-Linked Immunosorbent Assay (ELISA). Wounding induced in maize leaves a two-step oxidative stress, an early one just after wounding and the second two hours later. It coincides with the transient changes of the cytosine methylation level. In the stress-inducible maize calcium-dependent protein kinase ZmCPK11 gene wounding transiently reduced methylation of cytosines 100 and 126 in the first exon

Genome-wide analysis and expression profiling of calcium-dependent protein kinases in potato (Solanum tuberosum)

Calcium-dependent protein kinases (CDPKs or CPKs), unique to plants and some protists, are involved in growth and developmental processes as well as in defence against diverse environmental stresses. CDPKs are encoded by multi-gene families. Despite extensive studies of the CDPKs in many species, information about the evolutionary history and expression patterns of the CDPK family in the staple crop potato (Solanum tuberosum) remains poorly known. In this study, we performed bioinformatics analysis of the potato whole genome sequence and identified 23 potential CDPK genes. These genes are located in eleven, of twelve, potato chromosomes. Based on the phylogenetic tree and gene structures, the CDPKs were divided into four subfamilies. To determine their expression, reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis was carried out for the CDPK genes in different organs of potato such as young and mature leaves, stems, young shoots, roots, stolons, swollen stolons, flowers and tubers. The CDPKs were expressed in all the organs analysed, but their expression patterns varied greatly. The expression of some CDPKs was strongly organ specific, for example StCPK13 and StCPK18 was found only/ mostly in flowers. In Solanum genotypes differing in resistance to Phytophthora infestans, the expression and activity of CDPKs increased in response to a P. infestans elicitor with different kinetics and intensity. The expression levels and activity of the CDPKs correlated positively with the level of the resistance. Our results support earlier suggestion that CDPKs are involved in potato organ development and defence against stresses. We provide new information about the CDPK gene family in the potato and a perspective on its evolutionary history and biological roles of the individual kinases

Expression of maize Calcium-Dependent Protein Kinase (ZmCPK11) improves salt tolerance in transgenic Arabidopsis plants by regulating sodium and potassium homeostasis and stabilizing photosystem II

In plants, CALCIUM-DEPENDENT PROTEIN KINASES (CDPKs/CPKs) are involved in calcium signaling in response to endogenous and environmental stimuli. Here, we report that ZmCPK11, one of maize CDPKs, participates in salt stress response and tolerance. Salt stress induced expression and upregulated the activity of ZmCPK11 in maize roots and leaves. Activation of ZmCPK11 upon salt stress was also observed in roots and leaves of transgenic Arabidopsis plants expressing ZmCPK11. The transgenic plants showed a long-root phenotype under control conditions and a short-root phenotype under NaCl, abscisic acid (ABA) or jasmonic acid (JA) treatment. Analysis of ABA and JA content in roots indicated that ZmCPK11 can mediate root growth by regulating the levels of these phytohormones. Moreover, 4-week-old transgenic plants were more tolerant to salinity than the wild-type plants. Their leaves were less chlorotic and showed weaker symptoms of senescence accompanied by higher chlorophyll content and higher quantum efficiency of photosystem II. The expression of Na+/K+ transporters (HKT1, SOS1 and NHX1) and transcription factors (CBF1, CBF2, CBF3, ZAT6 and ZAT10) with known links to salinity tolerance was upregulated in roots of the transgenic plants upon salt stress. Furthermore, the transgenic plants accumulated less Na+ in roots and leaves under salinity, and showed a higher K+/Na+ ratio in leaves. These results show that the improved salt tolerance in ZmCPK11-transgenic plants could be due to an upregulation of genes involved in the maintenance of intracellular Na+ and K+ homeostasis and a protection of photosystem II against damage

Phosphorylation of maize eif5a by ck2: identification of phosphorylated residue and influence on intracellular localization of eif5a.

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with catalytic subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants Ser2Ala and Ser4Ala were expressed in E. coli and purified. Of these recombinant proteins, only ZmeIF5ASer2Ala was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A Ser2Ala mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser2 in ZmeIF5A is indeed phosphorylated. To find a role of the Ser2 phosphorylation, ZmeIF5A and its variants mutated at Ser2 (Ser2Ala and Ser2Asp) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. While wild-type ZmeIF5A and its Ser2Ala variant were distributed evenly between the nucleus and the cytoplasm, the variant with Ser2 replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser2 plays a role in the regulation of nucleocytoplasmic shuttling of eIF5A in plant cells

Regulation of wound-responsive calcium-dependent protein kinase from maize (ZmCPK11) by phosphatidic acid

In plant cells, phospholipids are not only membrane components but also act as second messengers interacting with various proteins and regulating diverse cellular processes, including stress signal transduction. Here, we report studies on the effects of various phospholipids on the activity and expression of maize wound-responsive calcium-dependent protein kinase (ZmCPK11). Our results revealed that in leaves treated with n-butanol, a potent inhibitor of phosphatidic acid (PA) synthesis catalyzed by phospholipase D, a significant decrease of ZmCPK11 activity was observed, indicating contribution of PA in the kinase activation. Using lipid binding assays, we demonstrate that among various phospholipids only saturated acyl species (16 : 0 and 18 : 0) of phosphatidic acid are able to bind to ZmCPK11. Saturated acyl species of PA are also able to stimulate phosphorylation of exogenous substrates by ZmCPK11 and autophosphorylation of the kinase. The level of ZmCPK11 autophosphorylation is correlated with its enzymatic activity. RT-PCR analysis showed that transcript level of ZmCPK11 in maize leaves increased in response to PA treatment. The influence of PA on the activity and transcript level of ZmCPK11 suggests an involvement of this kinase in a PA-mediated wound signal transduction pathway

Using Multiplex-Staining to Study Changes in the Maize Leaf Phosphoproteome in Response to Mechanical Wounding

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress