Regulation of ABA-Non-Activated SNF1-Related Protein Kinase 2 Signaling Pathways by Phosphatidic Acid.

Phosphatidic acid (PA) is involved in the regulation of plant growth and development, as well as responses to various environmental stimuli. Several PA targets in plant cells were identified, including two SNF1-related protein kinases 2 (SnRK2s), SnRK2.10 and SnRK2.4, which are not activated by abscisic acid (ABA). Here, we investigated the effects of PA on various elements of ABA-non-activated SnRK2 signaling. PA 16:0/18:1 was found to modulate the SnRK2 structure and the phosphorylation of some SnRK2 targets. Conversely, phosphorylation by the ABA-non-activated SnRK2s, of one of such targets, dehydrin Early Responsive to Dehydration 14 (ERD14), affects its interaction with PA and subcellular localization. Moreover, PA 16:0/18:1 modulates the activity and/or localization of negative regulators of the ABA-non-activated SnRK2s, not only of the ABA insensitive 1 (ABI1) phosphatase, which was identified earlier, but also of another protein phosphatase 2C, PP2CA. The activity of both phosphatases was inhibited by about 50% in the presence of 50 µM PA. PA 16:0/18:1 also impacts the phosphorylation and subcellular localization of SnRK2-interacting calcium sensor, known to inhibit SnRK2 activity in a calcium-dependent manner. Thus, PA was found to regulate ABA-non-activated SnRK2 signaling at several levels: the activity, phosphorylation status and/or localization of SnRK2 cellular partners.

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.

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 (m(5)C) 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 m(5)C, we evaluate the content of m(5)C 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 h after the treatment followed by a return to the initial level within the next hours. Similar results were obtained using of radio-labeled nucleotides separated by Thin Layer Chromatography (TLC) or using m(5)C-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.

Maize calcium-dependent protein kinase (ZmCPK11): local and systemic response to wounding, regulation by touch and components of jasmonate signaling.

Expression of ZmCPK11, a member of the maize (Zea mays) calcium-dependent protein kinases (CDPKs) family, is induced by mechanical wounding. A rapid increase of the activity of a 56-kDa CDPK has been observed in damaged leaves. In the present work, it is shown that the 56-kDa CDPK, identified as ZmCPK11, is also activated in non-wounded leaves as an element of systemic wound response. Moreover, an increase of the enzyme's activity and induction of ZmCPK11 expression was observed after touching the leaves. To study the role of ZmCPK11 in wound and touch signaling, transgenic Arabidopsis thaliana plants in which c-Myc-ZmCPK11 was expressed under control of the CaMV 35S promoter were generated. Analysis of the transgenic plants showed that c-Myc-ZmCPK11 was activated upon wounding and touching. Furthermore, pre-treatment with acetylsalicylic acid (acSA), an inhibitor of jasmonic acid (JA)-dependent wound signaling, abolished the wound-induced activation of ZmCPK11 in maize and the transgenic A. thaliana plants. Methyl jasmonate (MeJA) and linolenic acid (LA) stimulated the activity of ZmCPK11 as well as induced the expression of ZmCPK11 and other wound-responsive genes, lipoxygenase 1 (ZmLOX1) and proteinase inhibitor 1 (ZmWIP1). These results indicate that ZmCPK11, regulated at the enzymatic and transcriptional level by LA and MeJA, is a component of touch- and wound-induced pathway(s), participating in early stages of local and systemic responses.

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.

Phosphorylation of maize eukaryotic translation initiation factor on Ser2 by catalytic subunit CK2.

Alignment of eukaryotic translation initiation factor 5A (eIF5A) sequences has shown, for plants, in contrast to most other eukaryotes, the presence of N-terminal serine residue (Ser2) which could be phosphorylated by CK2. Using point directed mutagenesis, we demonstrate here that in recombinant maize ZmeIF5Awt Ser2 is exclusively phosphorylated by catalytic subunit of CK2 (CK2α), whereas its mutated variant Ser2Ala is not phosphorylated. To shed light on the physiological significance of this Ser2 phosphorylation, transient expression of fluorescence-labeled proteins was performed in maize protoplast. Wild-type ZmeIF5A was distributed evenly between nucleus and cytoplasm, but the replacement of Ser2 by aspartic acid, which mimics the phosphorylated serine, influences its intracellular localization. We postulate that phosphorylation of Ser2 in maize eIF5A, and most probably in other plant cells, plays a role in specific regulation of nuclear export of eIF5A-bound mRNAs.

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.

Phosphorylation of maize eukaryotic translation initiation factor 5A (eIF5A) by casein kinase 2: identification of phosphorylated residue and influence on intracellular localization of eIF5A.

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the catalytic alpha 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 S2A and S4A were expressed in Escherichia coli and purified. Of these recombinant proteins, only ZmeIF5A-S2A was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A-S2A 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 Ser(2) in ZmeIF5A is indeed phosphorylated. To find a role of the Ser(2) phosphorylation, ZmeIF5A and its variants mutated at Ser(2) (S2A and S2D) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. Although wild-type ZmeIF5A and its S2A variant were distributed evenly between the nucleus and cytoplasm, the variant with Ser(2) replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser(2) plays a role in regulation of nucleocytoplasmic shuttling of eIF5A in plant cells.

Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal.

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1-10 microM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1-20 microM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100-200 microM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.

A novel splicing variant encoding putative catalytic alpha subunit of maize protein kinase CK2.

A cDNA highly homologous to the known catalytic alpha subunit of protein kinase CK2 was cloned from maize (Zea mays). It was designated ZmCK2alpha-4 (accession no. AAF76187). Sequence analysis shows that ZmCK2alpha-4 and the previously identified ZmCK2alpha-1 (accession no. X61387) are transcribed from the same gene, ZmPKCK2AL (accession no. Y11649), but at different levels in various maize organs and at different stages of development. The cDNA encoding ZmCK2alpha-4 has three potential translation initiation sites. The three putative variants of ZmCK2alpha-4 were expressed in Escherichia coli as GST-fusion proteins and purified from bacterial extracts. In contrast to the previously characterized ZmCK2alphas, the obtained GST:ZmCK2alpha-4 proteins were catalytically inactive as monomers or in the presence of equimolar amounts of the human CK2beta. However, GST:ZmCK2alpha-4 did phosphorylate casein in the presence of a large excess of the beta subunit. The activity of ZmCK2alpha-4 toward casein could also be stimulated by increasing ATP concentration. Modeling studies have shown that there is no interaction between the N-terminal segment of ZmCK2alpha-4 and the activation loop responsible for constitutive catalytic activity of CK2alpha. Preliminary results suggest that ZmCK2alpha-4 may function as a negative regulator of other CK2s, and at certain circumstances as a holoenzyme which catalytic activity is stimulated by specific regulatory subunit(s).

[Wound signal transduction pathways in plants]. / Szlaki przekazywania sygnalu w reakcji roslin na zranienie.

A significant advancement in our knowledge and understanding of wound-signaling pathways in plants has been made recently. Essential role in the explanation of these processes came from the genetic screens and analysis of mutants which are defective in either jasmonic acid (JA) biosynthesis, JA perception or systemin function. Plants equally react to wound in the tissues directly damaged (local response) as well as in the non-wounded areas (systemic response). Jasmonides and in particular the most studied JA, produced by the octadecanoid pathway, are responsible for the systemic response. Jasmonides functioning as long-distance signal particles transmit the information about wound to distant, non-wounded tissues where defense response is invoked. Peptyd - systemin, identified in some Solanaceous species, acts locally to the wounded area to elicit the production of JA. Jasmonic acid-dependent and -independent wound signal transduction pathways have been identified and partially characterized. JA-dependent wound signaling pathways are responsible for the activation of systemic responses, whereas JA-independent wound signaling pathways, activated close to wound side, have a role in reparation of damaged tissue and in defense against pathogens.

A wound-responsive and phospholipid-regulated maize calcium-dependent protein kinase.

Using protein sequence data obtained from a calcium- and phospholipid-regulated protein kinase purified from maize (Zea mays), we isolated a cDNA encoding a calcium-dependent protein kinase (CDPK), which we designated ZmCPK11. The deduced amino acid sequence of ZmCPK11 includes the sequences of all the peptides obtained from the native protein. The ZmCPK11 sequence contains the kinase, autoregulatory, and calmodulin-like domains typical of CDPKs. Transcripts for ZmCPK11 were present in every tested organ of the plant, relatively high in seeds and seedlings and lower in stems, roots, and leaves. In leaves, kinase activity and ZmCPK11 mRNA accumulation were stimulated by wounding. The level of ZmCPK11 is also increased in noninjured neighboring leaves. The results suggest that the maize protein kinase is involved in a systemic response to wounding. Bacterially expressed glutathione S-transferase (GST)-ZmCPK11 was catalytically active in a calcium-dependent manner. Like the native enzyme, GST-ZmCPK11 was able to phosphorylate histone III-S and Syntide 2. Phosphorylation of histone was stimulated by phosphatidylserine, phosphatidylinositol, and phosphatidic acid, whereas phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, diolein, and cardiolipin did not increase the enzymatic activity. Autophosphorylation of GST-ZmCPK11 was stimulated by calcium and by phosphatidic acid and, to a lesser extent, by phosphatidylserine. Phosphatidylcholine did not affect autophosphorylation. These data unequivocally identify the maize phospholipid- and calcium-regulated protein kinase, which has protein kinase C-like activity, as a CDPK, and emphasize the potential that other CDPKs are regulated by phospholipids in addition to calcium.