Jorge M. Vázquez-Ramos

The cell cycle and seed germination

The cell cycle is the series of molecular events that allows cells to duplicate and segregate their chromosomes to form new cells. The finding that a protein kinase, the product of the yeast cdc2 gene, was fundamental in the regulation of the G 2 /M and G 1 /S transitions, associated with unstable proteins named cyclins, opened a very exciting and dynamic research area. The number of gene products that participate in the development and regulation of the cell cycle may be in the hundreds, and there is a high degree of conservation in protein sequences and regulatory pathways among eukaryotes. Although there are clear differences between plants and animals in cell structure, organization, growth, development and differentiation, the same types of proteins and very similar regulatory pathways seem to exist. Seed germination appears to be an excellent model system for studying the cell cycle in plants. Imbibition will reactivate meristematic cells – most initially with a G 1 DNA content – into the cell cycle in preparation for seedling establishment. Early events include a thorough survey of DNA status, since the drying process and seed storage conditions reduce chromosomal integrity. The initiation of cell cycle events leading to G 1 and S phases, and of the germination process itself, may depend on a G 1 checkpoint control. Most, if not all, cell cycle proteins appear to be already present in unimbibed embryos, although there is evidence of protein turnover in the early hours, suggesting the need for de novo protein synthesis. Regulation also may occur at the level of protein modification, because existing G 1 , S and G 2 cell cycle proteins appear to be activated at precise times during germination. Thus, cell cycle control during seed germination may be exerted at multiple levels; however, knowledge of cell cycle events and their importance for germination is still scarce and fragmentary, and different species may have developed unique control mechanisms, more suited to specific germination characteristics and habitat.

A DNA polymerase from maize axes: its purification and possible role.

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100–120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30–37 °C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28–40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase α. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase α-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme 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

Chromosome 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 the in vitro assay. 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.

Molecular cloning of a maize cDNA clone encoding a putative proliferating cell nuclear antigen.

We report the isolation and sequence of a maize cDNA clone which encodes a protein homologous to proliferating cell nuclear antigen (PCNA). The deduced amino acid sequence predicts a protein of 263 amino acids in length. The amino acid sequence shares 62% identity with the human PCNA and 95% identity with the rice homologue of PCNA.

Natural and artificial seed ageing in maize: germination and DNA synthesis

The effect of natural and artificial seed ageing has been compared in terms of physiological and biochemical responses of several maize genotypes. The physiological parameters were: viability, germinability, emergence in sand and dry matter accumulation. The biochemical parameters were: DNA synthesis and DNA polymerase activity. A close and direct relationship was found between seed deterioration and DNA metabolism in all maize materials which responded to ageing according to their genetic constitution; i.e., vigorous genotypes suffered less severe damage or recovery was faster than in the low-vigour genotypes. Coordination of events at appropriate times would seem a critical factor for proper seed germination.

Maize D4;1 and D5 cyclin proteins in germinating maize. Associated kinase activity and regulation by phytohormones.

We have previously reported the expression of four different maize D cyclins during seed germination and showed that cytokinins and auxins stimulate the expression of every cyclin in a differential way. In this paper we characterize the behavior at the protein level of two of these cyclins, CycD5 and CycD4;1. Antibodies were raised against CycD5;2 (which very likely also recognizes D5;1) and CycD4;1 and Western blot studies demonstrated that neither BA nor indol-3 acetic acid (IAA) stimulate cyclin accumulation during germination, compared with control levels. However, phytohormones, particularly IAA, modify the kinase activity associated to D cyclins preferentially at early hours of germination. The associated kinase moiety to D cyclins appears to be of a Cdk-A type because this protein immunoprecipitates with D cyclins and because kinase activity is strongly inhibited by both olomoucine and also by a peptide corresponding to the carboxy end of a maize kip related protein (KRP) protein. There is thus no correlation between mRNA and protein expression for these maize D cyclins during seed germination, although phytohormones may stimulate a signaling cascade that stimulates activation of protein kinase activity in cyclin-Cdk complexes.

COMPARISON AMONG DNA POLYMERASES 1, 2 AND 3 FROM MAIZE EMBRYO AXES. A DNA PRIMASE ACTIVITY COPURIFIES WITH DNA POLYMERASE 2

Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strenghtening the suggestion that polymerase 2 is a replicative enzyme, of the α-type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature.

The family of maize D-type cyclins: genomic organization, phylogeny and expression patterns

Cyclin proteins, associated to cyclin-dependent kinases (CDKs), play fundamental roles in cell cycle control as they constitute a very important driving force to allow cell cycle progression. D-type cyclins (CycDs) are important both for interpreting external mitogenic signals and in the control of the G1 phase. The maize (Zea mays) genome appears to contain at least 17 different CycD genes, and they fall into the subgroups previously described for other plants. Maize CycDs have been named according to identity percentages of the corresponding orthologs in rice and Arabidopsis. In silico analysis confirmed the presence of characteristic cyclin domains in each maize CycD gene and showed that their genomic organization is similar to their orthologs in rice and Arabidopsis. The expression of maize CycD genes was followed in seeds, during germination in the presence/absence of exogenously added hormones, and also in different plantlet tissues (mesocotyl, root tips and first leaf). Most cyclins were expressed in germinating seeds and at least in one of the plantlet tissues tested; almost all of the detected cyclins show an accumulating pattern of mRNA along germination (0-24 h) and higher levels in root tissue. Interestingly, some cyclins show high levels in non-proliferating tissues as leaf. Addition of auxins or cytokinins does not seem to importantly modify transcript levels; on the other hand, addition of abscisic acid repressed the expression of several cyclins. The role of each CycD during germination and plant growth and its interaction with other cell cycle proteins becomes a topic of the highest interest.

Maize DNA polymerase alpha is phosphorylated by a PCNA-associated cyclin/Cdk complex: effect of benzyladenine

The activity of maize DNA polymerases 1 and 2 (delta and alpha-type enzymes, respectively) is stimulated during germination if embryo axes are imbibed in the presence of benzyladenine. In vivo, DNA pol 2 is a phosphorotein that appears to be maximally phosphorylated previous to the S phase start time (by 12 h of germination, Coello and Vázquez-Ramos 1995a). We find that, in vitro, a PCNA-associated cyclin/kinase activity isolated from maize axes acquires an increasing capacity to phosphorylate DNA pol 2 as germination advances; moreover, the PCNA-associated kinase isolated from BA-treated maize axes germinated at 3 h phosphorylates DNA pol 2 at the same level observed in samples of axes germinated for 13 h in the absence of exogenous BA. PCNA-associated kinase activity from BA-treated axes germinated at 13 h maximal using DNA pol 2 as substrate. However, there is no modification in DNA polymerase activity as a consequence of protein phosphorylation. Results are discussed in terms of their significance for cell cycle regulation during seed germination.

Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings

Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins.