Maize Axes Research Articles

Cyclins D, phytoregulators and cell cycle onset in germinating maize

Several different D-type cyclins can be found in plants and in maize, four of these have been characterized: CycD2;1, CycD4;1, CycD5;1 and CycD5;2. These cyclins appear to form complexes with Cdks, with PCNA and also with KRP proteins and in these kinase activity can be measured. The expression of the corresponding genes during maize germination is highly stimulated by phytohormones like auxin and cytokinin, however this is not followed by an equivalent increase in the amount of the corresponding proteins; nonetheless, auxins do stimulate the associated kinase activity, particularly at early germination times. Thus, auxins appear to stimulate the cell cycle during germination at two levels, transcription and kinase activation. Both auxins and cytokinins appear to shorten the G1 phase during germination and stimulate DNA synthesis, but apparently they do it in different ways as the simultaneous addition of both to germinating maize axes eliminates DNA synthesis stimulation. Therefore, similar actions may be achieved by different paths.

Expression of maize eukaryotic initiation factor (eIF) iso4E is regulated at the translational level

Mechanisms for regulation of gene expression at the translational level have been reported at specific developmental stages in eukaryotes. Control of eukaryotic initiation factor (eIF) 4E availability by insulin/growth factors constitutes a main point of translational regulation. The aim of the present research was to understand the regulatory mechanism(s) behind the differential expression of two main 4E factors present in maize embryonic axes during germination. De novo synthesis of eIFiso4E initiates earlier and is faster than that of eIF4E in maize axes. Insulin addition to maize axes stimulated de novo synthesis of the eIFiso4E protein, but not that of eIF4E. Specific recruitment of the eIFiso4E transcript into polysomes was observed in these axes after insulin stimulation. Inhibitors of the insulin signal-transduction pathway, wortmannin and rapamycin, reversed the insulin effect. In vitro translation of maize poly(A)(+) RNAs by S6 ribosomal protein (rp)-phosphorylated ribosomes demonstrated a strong increase in eIFiso4E synthesis, as compared with its translation by S6 rp-non-phosphorylated ribosomes. Other mRNAs from the poly(A)(+) RNA set, including the eIF4E mRNA, did not show differential translation with regard to the S6-phosphorylated status of the ribosomes. The overall results indicate that eIFiso4E, but not eIF4E, cell content is regulated by de novo synthesis in maize axes during germination, most probably by specific mRNA recruitment into polysomes via a signal-transduction pathway involving S6 rp phosphorylation.

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 mM KCl. Analysis of this fraction by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross‐reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P2). Its activity is inhibited by Ca2+ and Zn2+ and is activated by Mg2+, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.

Characterization of ribosomal protein phosphorylation in maize axes during germination

Ribosomal proteins from maize ( Zea mays L.) axes were in vivo 32P-labeled at different germinating periods. These proteins were analyzed by 1- and 2-D gel electrophoresis and autoradiography. Specific 32P-ribosomal protein patterns were found within the 24 h tested period. The main labeled bands corresponded to 37, 31, 16 and 14.5 kDa. The 31 kDa labeled band appeared on the labeled pattern from 8 h incubation onward, while 32P-incorporation on the 16 and 14.5 kDa bands decreased along the germination period. Early phosphorylation of the 31 kDa protein was achieved by stress (heat or anoxia shock) but not by phytohormones. Hydrolysis of the phosphoproteins and paper electrophoretic analysis of the amino acids showed phosphoserine threonine as the labeled residues. Separation of the ribosomal subunits and analysis of the correspondent proteins indicated that the 31 kDa phosphoprotein is located in the small subunit, while the 14.5, 16 and 37 kDa phosphoproteins are part of the large subunit. Furthermore, the 31 kDa protein appeared labeled in the 2-D gel autoradiography and showed cross reaction with antibodies raised vs. rat S6 ribosomal protein by immunoprecipitation. Acidic ribosomal proteins were extracted from the total ribosomal proteins and analyzed by gel electrophoresis and autoradiography. Results indicated that the extracted proteins corresponded to the 37, 16 and 14.5 kDa phosphoproteins. These proteins gave positive reaction in slot blot analysis using antibodies raised vs. the correspondent yeast acidic ribosomal proteins. The possible significance of the phosphorylation of these proteins in regulating protein synthesis during seed germination is discussed.

DNA ligase activity in deteriorated maize embryo axes during germination: a model relating defects in DNA metabolism in seeds to loss of germinability

AbstractChromosome integrity and DNA polymerase activity are negatively affected by improper storage of maize seeds. We have extended our studies on the effect of improper storage on DNA metabolism by analysing the effect of hot–dry and hot–humid conditions on DNA ligase activity. An assay to measure ligase activity was established by using linearized plasmid DNA as substrate and protein extracts from maize axes. Optimal conditions were established for thein vitroassay. Enzyme activity was importantly reduced under both storage conditions. Activity recovered 6–9 h after imbibition. A model for loss of viability and vigour due to damage of DNA metabolism is proposed.

Nuclear DNA polymerase activities of germinating maize embryo axes

Summary DNA polymerase activities were obtained from 0, 3, 6 and 24-h imbibed maize axes and characterized both from nuclear extracts and after their fractionation through DEAE-cellulose. Typical DNA polymerase inhibitors such as N-ethyl maleimide and ddTTP totally inhibited activities at all times whether from nuclear extracts or DEAE-fractions. Activities in nuclear extracts were more sensitive to aphidicolin than in DEAE-fractions and inhibition was higher at earlier times. KCl at 200 mM stimulated all activities in DEAE-fractions, whereas 250 mM KCl caused inhibition. The 6-h enzyme used more efficiently a synthetic ribohomopolymer than the 24-h enzyme. These results suggest the presence of a nuclear γ-type enzyme in cells of germinating maize axes.

Stimulation of DNA synthesis and DNA polymerase activity by benzyladenine during early germination of maize axes

Benzyladenine, a synthetic cytokinin, stimulates DNA synthesis during early germination of maize. A peak of stimulation is observed at 6 h in the period of 0–9 h of imbibition of embryo axes. Stimulation of DNA synthesis by the phytoregulator is dependent on RNA and (or) protein synthesis, since α-amanitin and cycloheximide inhibit such stimulation. Although DNA polymerase activity is stimulated by incubation of axes in the presence of benzyladenine, enzyme amount is apparently not increased. An enzyme located in the nuclei seems to be preferentially stimulated, and studies with specific inhibitors for polymerases and use of different templates suggest this enzyme could be of type γ. The nature of the mechanism of stimulation of DNA synthesis is discussed. Key words: benzyladenine, DNA synthesis, DNA polymerase, maize, germination.

Effect of benzyladenine on DNA metabolism and cell cycle in maize axes

Effect of benzyladenine on DNA metabolism and cell cycle in maize axes