JXB Advance Access originally published online on November 15, 2004
Journal of Experimental Botany 2005 56(412):515-523; doi:10.1093/jxb/eri029
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RESEARCH PAPER |
Differential response of PCNA and Cdk-A proteins and associated kinase activities to benzyladenine and abscisic acid during maize seed germination
1Departamento de Bioquímica, Facultad de Química, UNAM, México D.F. 04510, México
2Department of Vegetable Crops, One Shields Ave, University of California, Davis, CA 95616-8631, USA
* To whom correspondence should be addressed. Fax: +52 56225284; E-mail: jorman{at}servidor.unam.mx
Received 20 August 2004; Accepted 14 September 2004
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Abstract |
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The proliferating cell nuclear antigen (PCNA) is a protein factor required for processive DNA synthesis that is associated with G1 cell cycle proteins. It has been demonstrated previously that, in germinating maize (Zea mays) embryonic axes, PCNA forms protein complexes with two Cdk-A proteins (32 and 36 kDa) and with a putative D-type cyclin. These complexes exhibit protein kinase activity on histone H1 and on the maize homologue of the pRB (retinoblastoma) protein. Flow cytometry has been used to study the influence of the phytohormones benzyladenine (BA) and abscisic acid (ABA) on cell cycle advancement during maize germination. It was found that, while BA accelerates the passage of cells from G1 to G2, ABA delays cell cycle events so that most cells seem to remain in G1. The amounts of PCNA and Cdk-A proteins also vary according to the hormone treatment. In embryonic axes, PCNA increases rapidly during early germination in BA, compared with a gradual increase in water, while ABA treatment had only a marginal effect. However, of the two Cdk-A proteins, the 32 kDa protein is strongly reduced after 15 h of imbibition in water while this occurs later when axes are imbibed in BA or ABA. The PCNA-associated protein kinase activity in the BA and ABA treatments falls after 3 h of imbibition compared with activity in the control; however, while kinase activity in the BA treatment continues to decline during imbibition, it remains relatively constant until 24 h of imbibition in the ABA treatment. By contrast, a p13Suc1-associated Cdk-A kinase is activated after 15 h of imbibition under all treatments, particularly in ABA. These results suggest that, in maize, ABA delays the germination process by affecting cell cycle advancement, stopping cells mostly in a G1 state.
Key words: Cdks, cell cycle, germination, PCNA, phytohormones, Zea mays L
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Introduction |
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Seed germination is a developmental process of reactivation of metabolism to originate a new plant. This process starts with seed imbibition and ends with the protrusion of the embryonic axis (normally the radicle) through the enclosing tissues (Bewley and Black, 1994
). During this time, cells in a seed repair their DNA, and meristematic cells are committed to start the cell cycle, although cell division generally occurs coincident with or after the completion of germination (radicle emergence) (Vázquez-Ramos and Sánchez, 2003).
In mammals, progress through the cell cycle is regulated by distinct families of cyclin-dependent kinases (Cdks) whose activities are co-ordinated by different types of cyclins. These basic mechanisms of cell cycle control are conserved in plant cells (Mironov et al., 1999). Several genes coding for Cdk proteins from maize, rice, alfalfa, soybean, pea, tobacco, and Arabidopsis have been reported (Colasanti et al., 1991; Ferreira et al., 1991; Hashimoto et al., 1992; Hirt et al., 1993; Miao et al., 1993; Fober et al., 1994; Setiady et al., 1996) and these have been divided into two main families, Cdk-A and Cdk-B. Cdk-B function is important in the G2/M phase, whereas Cdk-A participates in both the G1/S and the G2/M transitions. Cdk-A protein is the homologue of mammalian Cdk1 and contains the characteristic PSTAIRE amino acid motif in the cyclin-binding domain that defines Cdc2-type kinases (Mironov et al., 1999; Joubés et al., 2000). In tobacco and Arabidopsis cells, Cdk-A is activated by cyclin D-type proteins and this protein complex is able to phosphorylate the plant homologue of the retinoblastoma protein (pRB) (Nakagami et al., 1999) and also histone H1 (Healy et al., 2001). These results and the isolation of homologues of the E2F transcription factors from wheat, tobacco, carrot, and Arabidopsis (Ramírez-Parra et al., 1999; Sekine et al., 1999; Albani et al., 2000; Magyar et al., 2000) indicate that the entry of cells into the G1 phase in plants is controlled by cyclin D–Cdk-A-type complexes and that these are able to inactivate the growth-suppressing activity of pRb (Gutierrez et al., 2002).
In maize two different types (32 kDa and 36 kDa) of Cdk-A were found. Both proteins form complexes with the proliferating cell nuclear antigen (PCNA) protein (Sánchez et al., 2002). PCNA protein is the sliding clamp of the replicative DNA polymerase 
(Tan et al., 1986; Prelich et al., 1987; Shivji et al., 1992); however, during the G1 phase in higher eukaryotes, PCNA has been found associated with other cell cycle proteins like Cdk4, cyclin D, and p21 (Xiong et al., 1992). During maize germination, PCNA not only associates with Cdk-A proteins, but also with a putative cyclin D (Herrera et al., 2000). This PCNA-associated protein complex exhibits kinase activity that is able to phosphorylate both histone H1 and the maize pRB-related protein (ZmRBR) (Sánchez et al., 2002). Moreover, PCNA-associated kinase activity is higher during the early hours of germination (0–6 h) than later (15–24 h) when the S phase begins, suggesting that it is a G1 phase kinase. Interestingly, in in vitro assays, the fission yeast Cdk-binding protein p13Suc1, which is active during the G2 phase in yeast cells (Hayles et al., 1986), binds with the maize Cdk-A protein from maize protein extracts. Moreover, the protein complex formed shows kinase activity preferentially with Cdk-A obtained from germinating seeds (by 15 h and onward), in contrast to the PCNA-associated kinase (Sánchez et al., 2002).
Plant hormones play essential roles in plant metabolism and can influence cell cycle proteins. Cytokinins stimulate Cdk-A activity in the G2/M phases (Zhang et al., 1996) and, at the G1/S transition, regulate cell cycle progression partly by inducing CycD3,1 transcription (Meijer and Murray, 2001). During maize germination, benzyladenine (BA), a synthetic cytokinin, accelerates the germination process, affecting the amounts of G1/S cell cycle proteins like PCNA and a putative cyclin D (Cruz-García et al., 1998) and stimulating the activity of replicative DNA polymerases during germination (Vázquez-Ramos and Reyes, 1990; Gómez-Roig and Vázquez-Ramos, 2003). On the other hand, abscisic acid (ABA) inhibits germination, perhaps through inhibition of cell cycle processes in G1 (Swiatek et al., 2002), since this phytohormone can induce the expression of cyclin-dependent kinase inhibitors that bind and inhibit Cdk-A activity (Wang et al., 1998).
Assuming that the G1 phase transition is an important process during germination, and given that PCNA-associated kinase is important in the G1 phase, does this kinase complex participate in the regulation of the germination process by phytohormones? It is shown here that there is a differential cell cycle and kinase response when maize axes are imbibed in the presence of BA or ABA, which may indicate that these phytohormones influence the germination status of seeds, at least partly, by affecting the cell cycle.
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Materials
Protein A-agarose and protease inhibitor cocktail tablets (CompleteTM) were from Roche; hyperfilm ECL, ECL western blotting kit, glutathione–Sepharose 4B, anti-rabbit IgG peroxidase conjugate, and [
-32P]ATP (3000 Ci mmol–1) were from Amersham Pharmacia Biotech; histone H1 and glutathione were from Gibco BRL; diamine-2-phenylindol (DAPI), benzyladenine (BA), and abscisic acid (ABA) were from Sigma-Aldrich Chemicals; immobilon PVDF membranes were from Millipore; anti-PSTAIRE rabbit polyclonal IgG cat No. sc53 was from Santa Cruz Biotech; and lambda protein phosphatase and p13Suc1 sepharose were from Upstate Biotechnology.
Flow cytometry
The DNA content in maize radicle tips was measured by flow cytometry following imbibition in water, 1 µM BA or 20 µM ABA. To obtain nuclei, maize (Zea mays L. cv. Chalqueño) radicle tips were chopped with a razor blade in the presence of 1.5 ml of a buffer containing 50 mM TRIS–HCl pH 7.5, 1 mM MgCl2, 0.1% Triton X-100, and 2 mg DAPI. Samples were filtered through a 30 µm nylon membrane. Nuclei were analysed by flow cytometry in a Partec CA II instrument (Partec GMBH, Munster, Germany); 1500 nuclei were counted for each sample at a speed of 10 nuclei s–1. The resulting data were processed using the Multicycle program, version 2.53 (Phoenix Flow Systems, San Diego, CA).
Protein extraction
Proteins were obtained from maize embryonic axes from dry seeds or after imbibition in buffer for 3, 6, 15, or 24 h with or without 1 µM BA or 20 µM ABA. Protein extraction buffer (1 ml/15 embryonic axes) contained 25 mM TRIS–HCl pH 7.5, 15 mM MgCl2, 75 mM NaCl, 25 mM KCl, 5 mM EDTA pH 8.0, 1 mM DTT, 0.2% Triton X-100, 0.25 M sucrose, 60 mM ß-glycerolphosphate, 50 mM NaF, 200 µM Na3VO4, 1 mM EGTA, and a tablet of protease inhibitor cocktail/50 ml buffer. Protein extracts were centrifuged at 150 000 g for 30 min at 4 °C and protein concentrations were determined by the method of Bradford (1976).
Western blots
Proteins (
25 µg/2–3 µl protein extract) were separated by SDS–PAGE. Gels were blotted onto PVDF membranes and these were incubated either with anti-maize PCNA polyclonal antibody (1:1500 dilution) or with anti-PSTAIRE polyclonal antibody (1:1500 dilution) for 12 h at 4 °C, washed twice in PBS buffer (10 ml each) and once in PBS buffer containing 0.5 M NaCl (10 ml), 15 min each at room temperature, and the membranes incubated for 2 h with peroxidase-conjugated anti-rabbit antibody in a 1:10 000 dilution. The membranes were washed again with 10 ml PBS buffer and the peroxidase reaction was detected by the enhanced chemiluminescence method (ECL). All western blots were repeated a minimum of three times using independent protein extracts.
Immunoprecipitation and pull-down assays
Proteins (100 µg/12–15 µl protein extract) were incubated with anti-maize PCNA antibody conjugated with protein A–agarose (30 µl) or with p13Suc1–Sepharose beads (10 µl) in 200 µl of PBS buffer overnight at 4 °C. After incubation, beads were washed five times with 200 µl buffer A (25 mM TRIS–HCl pH 7.5, 125 mM NaCl, 2.5 mM EDTA pH 8.0, 2.5 mM EGTA, 2.5 mM NaF, and 0.1% Triton X-100) and once with kinase assay buffer (200 µl; see below). The resulting protein precipitates or pulled-down proteins were used as the source of kinase activity.
Phosphatase assay
Proteins (25 µg/2–3 µl protein extract) were incubated with phosphatase buffer (20 µl) that contained 50 mM HEPES pH 7.5, 100 µM EDTA, 2 mM MnCl2, 5 mM DTT, 100 µg ml–1 BSA, and 4 U of lambda protein phosphatase for 30 min at 37 °C. Histone H1 phosphorylated by the PCNA-associated kinase activity was used as a control for lambda protein phosphatase activity.
Protein kinase activity
PCNA immunocomplexes or p13Suc1-associated proteins were resuspended in 15 µl of kinase buffer (70 mM TRIS–HCl pH 7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM DTT, 5 mM EGTA, 20 µM ATP, and 5 µCi [-32P]ATP). As a substrate, 4 µg GST–ZmRBR-C fusion peptide (the C-terminal domain of maize RBR protein) was added per sample. Purification of GST–ZmRBR-C fusion peptide was performed according to Ramirez-Parra et al. (1999). Reactions were performed for 30 min at 30 °C and were stopped by adding SDS loading buffer (15 µl). After boiling for 5 min, the reaction products were separated by SDS–PAGE.
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Results |
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Effect of phytohormones on cell cycle progression during maize germination
Radicle tips of embryonic axes were used to follow the cell cycle status during maize imbibition and germination, since cells in these tissues are the first to enter the cycle (Baíza et al., 1989
). Among nuclei from cells of dry seeds, 82% had a G1 (2C) DNA content and 17% had a G2 (4C) DNA content (Table 1). The cell cycle is activated during the first hours after imbibition, and by 15 h the percentage of G1 nuclei dropped significantly to 63%, whereas the percentage of G2 nuclei increased to 35% (Fig. 1A; Table 1). Growth of water-imbibed axes is first evident at 15–20 h of imbibition. Incubation of maize embryonic axes in the presence of the synthetic mitogenic hormone benzyladenine (BA) advances the initiation of growth by 6–9 h (JM Vázquez-Ramos, unpublished results) and caused an earlier fall in the percentage of nuclei in G1 and an increase in the percentage in G2 within 6 h of imbibition; by 15 h 58% of nuclei were in G1 while 40% were in G2 (Fig. 1; Table 1). Abscisic acid (ABA) is known as an inhibitor of germination (Bewley and Black, 1994), and it reduces maize germination by >75% during the first 48 h of imbibition (MP Sánchez et al., unpublished results). Incubation of maize embryonic axes in the presence of ABA notably reduced the number of cells that moved from G1 to G2 so that by 15 h of germination only 26% of nuclei were in G2 while 68% were in G1 (Fig. 1; Table 1). Nuclei with an apparent S-phase (intermediate) DNA content had increased to 12% after 24 h of imbibition in water or BA, while only 5% of nuclei from axes imbibed in ABA contained S-phase DNA content (Table 1).
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PCNA and Cdk-A content in BA- and ABA-treated maize embryonic axes
The mechanism by which phytohormones influence seed germination is not well known, although it may involve modulating the activity of cell cycle proteins (Liu et al., 1994
; Cruz-García et al., 1998; Riou-Khamlichi et al., 1999; de Castro et al., 2001). To relate the effect of phytohormones on cell cycle advancement during germination with specific G1/S phase proteins, PCNA and Cdk-A proteins were followed by western blotting (Fig. 2). To normalize the amounts of PCNA or Cdks in western blots, these were related to the amount of protein loaded per time per treatment (Fig. 3). During imbibition of embryos in water, PCNA showed that the previously reported gradual increase reached a peak by 15–24 h of imbibition (Figs 2A, 3A; Herrera et al., 2000). A faster increase in PCNA amount was observed in BA-treated embryonic axes during the early hours of imbibition, and similar levels to controls were observed later (Figs 2A, 3A). Only a low, gradual increase in PCNA amount was noticeable during germination when embryonic axes were imbibed in the presence of ABA (Figs 2A, 3A), consistent with the reduced or delayed cell cycle progression (Table 1).
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An antibody against the conserved PSTAIRE sequence, characteristic of Cdc2-related proteins, was used to follow maize Cdk-A proteins. As reported previously (Sánchez et al., 2002
), two proteins of 32 and 36 kDa are detected with this antibody in protein extracts from maize embryonic axes, and, whereas the amount of the 36 kDa protein increased as germination advances, the amount of the 32 kDa protein was reduced at 15 and 24 h of imbibition (Figs 2B, 3B, C). When imbibed in BA, the abundance of the 36 kDa protein also increased by 15–24 h, although amounts remained lower than in control axes. On the other hand, the 32 kDa protein amounts were always higher than in control axes, showing a peak by 6 h of imbibition and declining thereafter (Figs 2B, 3B, C). The behaviour of the 36 kDa Cdk-A protein when axes were incubated in ABA was similar to that in BA-treated axes, being less than in control axes at 15 and 24 h of imbibition (Figs 2B, 3B). The 32 kDa Cdk-A protein, however, was present in similar amounts in ABA-treated and control axes at 3 and 6 h, but then declined more slowly in ABA-treated axes at longer times (Figs 2B, 3C). In addition, a third protein is clearly visible at 15 and 24 h of imbibition in BA or ABA, running between the 36 and 32 kDa bands (Fig. 2B). This 34 kDa protein could be a modified form, perhaps by phosphorylation or dephosphorylation, of either the 36 or the 32 kDa protein.
To determine whether this 34 kDa band appearing at 15 h of germination in BA- and ABA-treated axes is the product of a phosphorylated 36 or 32 kDa protein, protein extracts were treated with lambda protein phosphatase, which has been reported to dephosphorylate plant Cdk-2 proteins (Umeda et al., 2000). First, the ability of this phosphatase to dephosphorylate a previously phosphorylated substrate, histone H1, was tested. Histone H1 was incubated in the presence of the PCNA-associated Cdk-A (Sánchez et al., 2002) and [-32P]ATP, and the labelled histone H1 served as substrate for the lambda phosphatase. An aliquot of radioactively labelled histone H1 (Fig. 4A, lane 1) was divided in half, one-half receiving the lambda phosphatase (Fig. 4A, lane 2) and the other only receiving the corresponding buffer (Fig. 4A, lane 3). Lambda phosphatase almost completely removed the labelled P from histone H1. Next, lambda phosphatase was mixed with protein extracts from 6 h and 24 h BA-treated embryonic axes. However, no variation in band intensity, or in the electrophoretic mobility, of the 32, 34, or 36 kDa proteins was observed (Fig. 4B), suggesting that the different mobilities of the three Cdk-A proteins are not due to differential phosphorylation. Similar results had been obtained previously using alkaline phosphatase (Sánchez et al., 2002).
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-Pase) or with lambda phosphatase buffer only (buffer) and then proteins were processed for western blot assay using the anti-PSTAIRE antibody.Protein kinase activity in anti-PCNA immunoprecipitates and the influence of phytohormones
PCNA-associated Cdk-A kinase activity was measured in anti-PCNA immunoprecipitates from control or phytohormone-treated embryonic axes. Kinase activity was normalized to the amount of PCNA immunoprecipitated at each germination time and under the influence of phytohormones. The carboxy-terminal 120 amino acids of the maize pRB homologue, GST–ZmRBR-C, was used as substrate. As reported previously (Sánchez et al., 2002
), kinase activity in control anti-PCNA immunoprecipitates was similar between 0 and 6 h of imbibition and activity subsequently declined so that at 24 h, activity was only one-third of that present at 0 h (Figs 5A, 6A). In BA-treated axes, protein kinase activity in anti-PCNA immunoprecipitates after 3 and 6 h imbibition dropped to about one-half of that present in control axes at 0 h and then further decreased at 15 and 24 h to levels similar to those shown by control axes at the same time periods (Figs 5A, 6A). In ABA-treated embryonic axes, kinase activity in anti-PCNA immunoprecipitates at 3 h of imbibition was reduced to about one-third of that in controls at 0 h; thereafter, activity remained constant up to 24 h of imbibition (Figs 5A, 6A).
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For comparison purposes, the activity of the Cdk-A protein associated with p13Suc1 was also measured. To normalize this, activity values at different times/treatments were related to western blots of Cdk pulled down by p13Suc1 at every time/treatment. In previous work, it was found that an agarose resin containing the p13Suc1 peptide pulled down only the 36 kDa Cdk-A protein and that the associated kinase activity had a contrasting behaviour to the PCNA-associated Cdk (Sánchez et al., 2002
). Here, the p13Suc1-associated kinase activity from control extracts increased slightly at early imbibition times and then increased about 10-fold as germination advanced (Figs 5B, 6B), similar to previous results (Sánchez et al., 2002). Kinase activity from BA-treated axes was very low at 0 and 3 h of imbibition, and then activity increased 7–8-fold at 15–24 h of imbibition, although these levels were generally lower than in control samples (Figs 5B, 6B). During ABA treatment, p13Suc1-associated kinase activity transiently increased at 3 h of imbibition, decreased at 6 h and then increased to reach a 15–17-fold increase at 24 h of imbibition (Figs 5B, 6B). |
Discussion |
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It has been hypothesized that cell cycle activation (particularly the G1 phase) is required for germination to advance and allow seedling growth (Cruz-García et al., 1998
; de Castro et al., 2000, Sánchez et al., 2002; Vázquez-Ramos and Sánchez, 2003). Although seed metabolism during germination may vary enormously, depending on species, some results substantiate this hypothesis: (i) cells in imbibed dormant seeds, although metabolically active, remain in a G1-like state until dormancy is broken (de Castro et al., 2001); (ii) meristematic cells in osmoprimed seeds tend to remain in a G1-like state for days or even weeks, although in some seed species, a number of cells move into G2, but no further (Lanteri et al., 1994; Cruz-García et al., 1995; Liu et al., 1996; Sánchez-Jiménez et al., 1997; Gurusinghe et al., 1999; de Castro et al., 2000); after removal of the osmotic agent and imbibition in water, a burst of DNA synthesis and cell cycle advancement is rapidly triggered (Cruz-García et al., 1995; de Castro et al., 2000); and (iii) in deteriorated or in low viability seeds, initiation of the S phase in meristematic cells can be extremely delayed compared with the time it takes in cells from high viability seeds, and radicle emergence is similarly delayed (Sen and Osborne, 1974; Elder et al., 1987; Gutiérrez et al., 1993). Thus, reactivation of at least the early stages of the cell cycle in meristematic cells is generally associated with advancement towards germination. On the other hand, radicle protrusion in the absence of cell cycle progression has been reported for cabbage seeds imbibed in the presence of hydroxyurea (an inhibitor of the S phase; Górnik et al., 1997) and in maize embryonic axes in the presence of colchicine (an inhibitor of the M phase; Baíza et al., 1989). Some varieties of tomato seeds also exhibited germination rate advancement due to priming without increases in G2 nuclei (Gurusinghe et al., 1999). While initiation of cell cycle activity normally accompanies germination, entry into the cell cycle may not be absolutely required for the early embryo expansion associated with germination, but cell proliferation is an absolute requirement for continued growth and seedling establishment.
In this paper, it is shown that ABA, a known inhibitor of seed germination, slows down cell cycle advancement during maize germination, compared with control or BA-treated embryonic axes, in which cell cycle activation is evident. This is consistent with reports that seeds deficient in ABA synthesis exhibit more rapid germination and advancement of the cell cycle prior to radicle emergence (Liu et al., 1997; Downie et al., 1999). Accumulation of a G1/S marker like PCNA is stimulated by BA, and this accumulation occurs earlier than in control axes, reflecting an earlier entry into the S phase, as has been reported before (Herrera et al., 2000). On the other hand, the PCNA amount in ABA-treated axes shows only a marginal increase, probably indicating that cells are not being stimulated to enter the S phase, corroborating the flow cytometry results. A differential effect was also observed in the amount of another G1/S marker, the Cdk-A proteins. The amount of the 36 kDa protein increases under all conditions until 15 h of imbibition and, whereas this increasing pattern continues for control and BA treatments, in ABA treatments the 36 kDa protein starts decreasing. This 36 kDa protein, and not the 32 kDa Cdk-A, is preferentially bound by the p13Suc1 protein and, in these complexes, kinase activity is notably enhanced by 15 h of imbibition (Fig. 6B; Sánchez et al., 2002), suggesting that it acts during the G2 phase. It is thus puzzling that at 24 h of imbibition in the presence of ABA, the p13Suc1-associated kinase activity is highest when the amount of the 36 kDa protein is being reduced (see below). The behaviour of the 32 kDa Cdk-A is also contrasting, as the protein seems to be more stable during the hormonal treatments, whereas its levels decrease earlier during germination in water. Of interest here is that, whereas the levels of the 32 kDa Cdk-A are not very different at 3 and 6 h of imbibition in the three treatments, PCNA-associated kinase activity is higher in control and BA treatments than in ABA treatment, perhaps indicating an ABA-induced inhibitory effect. The appearance of a third protein of 34 kDa in BA- or ABA-treated axes could indicate that either the 32 or the 36 kDa protein was phosphorylated as a result of the hormonal treatment. However, this protein band did not disappear after dephosphorylation with lambda phosphatase. Whether this is a new Cdk-A kinase or whether the 32 or 36 kDa proteins are modified in ways other than phosphorylation remain to be determined.
As is well known, the complexes formed by Cdks and D-type cyclins bound to other proteins like PCNA and p21Cip1 are central to the activation of the cell cycle in mammals (Xiong et al., 1992). Similar complexes have been found in germinating maize axes. An antibody raised against maize PCNA co-immunoprecipitates two different Cdk-A proteins and a putative cyclin D protein, and this complex contains a protein kinase activity that is active mainly during the early hours of imbibition, a period corresponding to the G1 phase (Cruz-García et al., 1998; Sánchez et al., 2002). The PCNA-associated kinase is of the Cdk-A type, since proteins of this type are recognized by anti-PSTAIRE antibodies and kinase activity is inhibited by specific Cdk-A inhibitors (Sánchez et al., 2002). Here it is shown that anti-PCNA immunoprecipitates from BA-treated axes also show kinase activity that follows a pattern that differs from that described in control axes, since kinase activity is reduced to about half that shown by controls at 3 h of imbibition. Kinase activity from ABA-treated axes is reduced to about one-third of the control. However, while kinase activity is further reduced in control and BA treatments, in ABA treatment this activity remains at similar levels. Thus, the number of G1 kinase complexes associated with PCNA have a tendency to strongly decrease in control and BA treatments, while in ABA treatments, after an initial reduction, kinase activity remains at relatively constant levels and thus is higher at later germination times than in control and BA treatments. Therefore, ABA could be blocking cell cycle progression beyond the G1 phase via an active residual G1 kinase that, in the absence of ABA, should be inactivated so that the next cell cycle phase is triggered. ABA does not appear to produce an ‘all or nothing’ effect, since the p13Suc1-associated Cdk-A, very likely a G2/M marker, is active during late germination in control or BA- or ABA-treated embryonic axes, although surprisingly, this kinase activity is higher in ABA-treated axes.
The present results suggest that establishment of the G1 phase of the cell cycle is an early component of the germination process in maize embryos. BA may accelerate germination, in part, by stimulating cell cycle progression, while ABA has opposite effects on both cell cycle progression and germination. Conflicting results on the role of, and requirement for, cell cycle activity during germination of diverse seeds may be due, in part, to reliance on flow cytometry, which cannot detect cell cycle activities prior to the S/G2 phase. The proteins and molecular processes associated with entry into and progression through the G1 phase may constitute new and perhaps highly specific molecular markers for seed germination.
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Acknowledgements |
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This work was supported by grants from DGAPA-UNAM (IN-202002) and from the University of California Institute for Mexico and the United States (UC MEXUS) and the Consejo Nacional de Ciencia y Tecnología de México (CONACYT). Additional support was provided by the Western Regional Seed Physiology Research Group.
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