Blanka Szurmak

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 enzymes 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.

Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm

A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.

Isolation of an RNA polymerase II stimulatory protein from wheat germ chromatin

Abstract Resting wheat embryos were found to contain an RNA polymerase II stimulatory factor. It occurred in the embryo cells as a loosely bound constituent of non-histone chromosomal proteins and was isolated as a non-dialysable, heat-labile preparation, containing a polypeptide with MW 37000 as the main component. The isolated preparation showed the stimulatory activity in a simple transcription assay, containing highly purified wheat germ RNA polymerase II as the enzyme source and either denatured or native DNA as template. No activity was found with E. coli RNA polymerase and RNA polymerase III was stimulated to a low extent. Similar stimulatory protein was isolated also from rye germ chromatin and found to be active with both rye and wheat class II RNA polymerases.

A large DNA repeat of the dispersion pattern common to wheat and rye genomes

A Hind III-generated fragment of wheat embryo nuclear DNA has been cloned and sequenced. The cloned fragment corresponds to a 1241 bp long, moderately repeated (60 000 copies/genome) segment of the genomic DNA. The repeat is AT-rich (67%), contains an open reading frame for 151 amino acids and several nucleotide blocks resembling the consensus domain of autonomously replicating sequences. Southern blot hybridization analyses indicate that the repeat is scattered through the wheat genome. A sequence homologous to this repeat occurs also in rye embryo nuclear DNA where it shows the same dispersion pattern as that observed for the wheat repeat.

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.

Preferential stimulation of RNA polymerase IIB by a chromosomal protein from wheat germ

Abstract An RNA polymerase II stimulatory protein was purified from wheat germ chromatin and tested for the ability to stimulate wheat germ RNA polymerases I, II A , II B and III. The form II B of the class II enzyme was stimulated most effectively. The stimulation of[ 14 C]GTP incorporation was accompanied by an increase in the RNA product size, especially if native wheat germ DNA was used as template. The stimulatory protein was also able to reverse inhibition of transcription caused by other chromosomal proteins, and was a particularly preferred substrate for cyclic AMP-independent wheat germ protein kinase. The kinase preparation, however, lost its stimulatory activity. It is concluded that a form II B -specific RNA polymerase II stimulatory protein occurs and may be involved in the regulation of transcription in wheat embryo cell nuclei.