JXB Advance Access originally published online on November 29, 2004
Journal of Experimental Botany 2005 56(412):557-565; doi:10.1093/jxb/eri034
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RESEARCH PAPER |
Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays
1Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62250, México
2Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, Saskatchewan, Canada S7N 5E5
* To whom correspondence should be addressed. Fax: +52 777 313 9988. E-mail: marco{at}ibt.unam.mx
Received 30 March 2004; Accepted 20 September 2004
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Abstract |
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Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist. Actin is present in all the seed components, but it is mainly concentrated in the embryo axis. The expression of maize actin was induced during post-imbibition at both the protein and mRNA levels. Sharp increases in actin appeared from 24–48 h and again from 72–96 h. A more modest and steady actin mRNA increase in expression was observed; however, it did not appear as dramatic as in the case of common bean due to the presence of readily detectable amounts of message in the dry maize seed. The isoform distribution in the dry seed showed a pattern of at least three isovariants of pIs
5.0, 5.1, and 5.2, which were differentially expressed at the various post-imbibition times analysed. Two of the actin isoforms at 48 h post-imbibition cross-reacted with a phosphotyrosine-specific antibody and they are the product of three expressed genes as shown by in vitro translation assays. These data indicate that maize actin protein and mRNA expression is induced upon the trigger of germination, and the isoform expression kinetics and patterns resemble those from bean, suggesting that, in both species, actin expression at these early germination/growth stages is a highly regulated event. Key words: Actin, germination, isoforms, phosphorylation, seeds, Z. mays
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Introduction |
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Germination in plants could be described as the origination of a new organism from a pre-existing embryo in the seed. This transformation involves a number of dynamic processes triggered by hydration signals, which then relay to other signal-transduction mechanisms to drive elongation, active cell division, and copious vesicle transport to the sites of newly synthesized cell walls (McCurdy and Williamson, 1991
; Wasteneys and Galway, 2003). Cytoskeletal elements are known to be involved in vesicle movement via molecular motors on filamentous cables (Reddy, 2001; Grebe et al., 2003). One fundamental component of these cytoskeletal assemblies is actin, a 42–45 kDa cytoplasmic protein that is capable of self-assembly into dynamic filamentous structures. Actin filaments are fundamental for eukaryotic function and, owing to their importance, are tightly regulated within cells. In plants, actin filaments are presumed to play essential roles in many important processes including cell division, cell elongation, establishment of cytoplasmic organization, cytoplasmic streaming, tropisms, pollen tube growth (reviewed in Wasteneys and Galway, 2003), and changes in response to bacterial signalling molecules arising from pathogens (Dantán-González et al., 2001) or nodulation factors (Cárdenas et al., 1998).
The presence of cytoplasmic actin is now known to be ubiquitous in plants (Villanueva et al., 1990; Zhang et al., 1993; Cleary, 1995; Kim et al., 1995; Kost et al., 1998). Actins are encoded by multigene families with high divergence (Meagher et al., 1999). For example, single plant species have more divergent actin genes than do individual animal species (Meagher et al., 1999). Therefore, different isovariants are likely to be expressed at particular times depending on the cell physiological status. It is now generally accepted that plants have individual isovariants of actin that are specialized for particular functions and/or expression in certain cell types (Meagher et al., 1999; Kandasamy et al., 2001). Furthermore, different myosin isoforms may interact differentially with the various actin isovariants and additional regulation may occur by direct modification of actin or its accessory proteins through kinases and phosphatases (Guillén et al., 1999; 
amaj et al., 2002). Therefore, highly regulated spatio-temporal expression of actin genes and subsequent post-translational modifications are probably required during the germination process.
Most studies on active dynamic elongation processes have been carried out on growing pollen tubes and although they are substantially different from a germinating seed, they probably possess similar signal-transduction mechanisms and cytoskeletal functions. For example, the microfilament cytoskeletal organization in fine networks is a common feature of both pollen tubes and root hairs. The apical region of both cell types is rich in vesicles (Wasteneys and Galway, 2003), and a close co-ordination between cytoskeletal dynamics and signal-transduction events are needed to modulate growth direction (reviewed in Wasteneys and Galway, 2003). Moreover, GTPases of the Rop family have been localized on the tip of growing pollen tubes (Li et al., 1999); these GTPases are known to be key regulators of actin cytoskeletal organization (reviewed in Yang, 2002; Wasteneys and Galway, 2003). Therefore, similarities that parallel germination events and tip-growth mechanisms are likely to be encountered.
In Phaseolus vulgaris seedlings, the actin content in different parts of the seed has been analysed, as well as the time-course of actin expression and the isoform composition during this period (Villanueva et al., 1999). It was observed that actin is present at very low levels in these seeds and that it was mostly concentrated in the embryo. During the germination process, actin expression increased 5-fold at the protein and mRNA levels. Furthermore, at 48 h post-imbibition, it was observed that certain isoforms increased in quantity but others appeared to be expressed transiently. In this work, our previous analysis was extended to follow the time-course of actin expression in a germinating monocot, maize, and possible modifications of the protein were sought. It was observed that actin protein and mRNA were present at all times in all components of the seed prior to imbibition and at later post-imbibition times. While there was a steady increase in mRNA expression during germination, sharp increases in protein levels were observed. The seedlings displayed a differential actin isoform expression pattern. Expression of seedling mRNA in a reticulocyte lysate translation system and subsequent western blot analysis using 2D gel electrophoresis suggested that the actin isoforms were the products of three different mRNA species, possibly encoded by different genes. At least two of the isoforms isolated from seedling extracts had clearly detected phosphotyrosine modifications. The implications of these data are discussed in terms of the possible role of actin during germination in the monocot maize and how it compares with what has been observed for a similar process in the dicot, common bean.
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Materials and methods |
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Plant material
Maize (Zea mays L. var. Chalqueño) seeds were kindly provided by Dr Jorge Vázquez (National University of Mexico, UNAM, México City, México). Seeds were surface-sterilized in 10% commercial bleach with stirring for 10 min followed by extensive washing in sterile-distilled water. Seeds were air-dried and placed on sterile trays lined with wet paper towels for germination in the dark at 24±1 °C. Maize or seedling tissues were dissected at various stages of germination. This material was used to prepare a fine powder by grinding it with liquid nitrogen in a mortar and pestle. Frozen powders were stored at –80 °C until further analysis.
Antibodies, actin cDNA clone and chemicals
Anti-(calf thymus actin) polyclonal antibodies previously shown to react against soybean (Villanueva et al., 1990) and common bean actin (Díaz-Camino and Villanueva, 1999) were a kind gift from Dr John L Wang (Michigan State University, East Lansing, MI, USA). Anti-actin monoclonal (Clone C4) antibodies were purchased from ICN (Aurora, OH, USA) and anti-phosphotyrosine (PY20) antibodies were purchased from BD Biosciences (Mississauga, ON, Canada). Horseradish peroxidase-conjugated anti-rabbit antibodies were purchased from Zymed (San Francisco, CA, USA), and anti-mouse secondary antibodies, Bradford® protein assay kit, ReadyStrips® IPG Strips, BioLyte® ampholytes, and 2D SDS–PAGE standards were purchased from Bio-Rad (St Louis, MO, USA). Substrates for chemiluminescence (ECL®) were purchased from Amersham (Arlington Heights, IL, USA). Maize actin cDNA from clone 5C01H03 was kindly provided by Theresa A Musket (University of Missouri, CO, USA). TNT® radioactive kit was purchased from Promega (VWR Canlab, Mississauga, ON, Canada), EasyTag® L-[35S]-methionine was purchased from Perkin-Elmer (Boston, MA, USA) and protein A cross-linked to agarose was purchased from Sigma (Oakville, ON, Canada). All other chemicals were reagent grade.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE (2D PAGE) and immunoblotting
Total protein extracts were prepared according to Hurkman and Tanaka (1996). Pellets were resuspended in Laemmli's sample buffer and boiled immediately for 8 min. The supernatants were recovered by centrifugation at 14 000 g and the protein concentration from the obtained fractions was determined using the modified Bradford® protein assay with IgG as standard. A protein load of 50 µg was used for standard one-dimensional SDS-PAGE gels (Laemmli, 1970). The protein was then transferred to a nitrocellulose membrane by electroblotting (Towbin et al., 1979). Equal protein loading was confirmed by staining replicate gels with Coomasie Brilliant Blue. For analysis by 2D PAGE, final pellets were resuspended in the appropriate volume of rehydration solution (8 M urea, 2% [w/v] CHAPS, 100 mM DTT, 0.2% [v/v] pH 4–7 BioLyte® ampholytes, 0.001% bromophenol blue). The first dimension isoelectric focusing was performed in immobilized pH gradient 4–7 gel strips of 7 cm. Re-swelling of gels was performed including the protein sample in the rehydration solution. A protein load of 150 µg was used for each condition. Re-swelling of IPG gels was performed in the electrophoresis unit, which is equipped with a gradient power supply (PROTEAN IEF® focusing unit from Bio-Rad), for 12 h at 20 °C and 50 V. Re-swelled gels were focused at 20 °C by using a three-stage linear ramped voltage program (15 min in a gradient from 0–250 V, 1 h from 250–8000 V, 2.5 h at 8000 V). For the second dimension SDS-PAGE, IPG gels were equilibrated according to the manufacturer's instructions (ReadyStrip® IPG Strip Instruction Manual from Bio-Rad). SDS–PAGE was performed according to Laemmli (1970) with a 10% polyacrylamide resolving gel and a 4% polyacrylamide stacking gel. After running, gels were transferred to nitrocellulose (Towbin et al., 1979). For immunoblotting, the nitrocellulose membranes were blocked for 1 h at 50 °C in blocking solution (either 5% BSA or in 5% non-fat dry milk, 0.1% Triton X-100 in PBS [10 mM Na-phosphate, 150 mM NaCl, pH 7.4]) followed by an overnight incubation at 4 °C with primary (anti-[calf thymus actin], anti-actin monoclonal [Clone C4] or anti-phosphotyrosine [PY20]) antibodies diluted 1:2000 in blocking solution. Blots were then washed three times with PBST (0.05% Triton X-100 in 10 mM Na-phosphate, 150 mM NaCl, pH 7.4) and incubated with secondary (horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG) antibodies diluted 1:5000 in blocking solution for 3 h at room temperature. The antigen–antibody complex was detected by chemiluminescence using the ECL® kit. Both, the isoelectric points and the molecular weights of the detected actin isoforms were estimated by comparison with commercial standards.
Northern blot analysis
Part of the original embryo and seedling powder was used for total RNA extraction according to the method of de Vries et al. (1991). An 8 µg sample of total RNA was electrophoresed on a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred onto nylon membranes (Sambrook et al., 1989). The blot was then hybridized with Z. mays actin cDNA from clone 5C01H03. Hybridization and washes were done under light stringency conditions (Church and Gilbert, 1984) and subjected to autoradiography. An equal load of total RNA was standardized and monitored by pre-staining the ribosomal RNA on the membranes with ethidium bromide.
Total polysomal RNA, in vitro translation and immunoblotting
Total RNA was isolated from seedlings at various stages of development according to Wang and Vodkin (1994). TNT® radioactive kit was used following the manufacturer's standard assay instructions. Twenty µg of total RNA and 50 µCi of L-[35S]methionine were added to 50 µl lysate in a final volume of 50 µl. Mixtures were incubated at 30 °C for 60 min. In vitro translation products were loaded in pH gradient 4–7 gel strips of 7 cm. First dimension isoelectric focusing, second dimension SDS-PAGE, protein transfer to nitrocellulose membranes, western blot analysis, and autoradiography were performed as described above.
Densitometric analysis of immunoblots
The films corresponding to the immunoblotted spots of the actin isoforms were scanned using the UMAX vistascan 3.5.2 (UmMAX Data Systems, Inc) program, and analysed with NIH Image 1.60. The integrated density values of the three isoforms for the same developmental time were added to obtain the total density, and the individual integrated density value of each spot divided by this number. This yielded a measurement of individual isoforms relative to the particular blot and time and assured uniformity of the results. Measurements were carried out for each time-course with two independent experimental blots. Statistical uncertainty values ranged from 0.01–0.04. Since they were negligible; and they are not true standard error values, they were omitted from the plotted results.
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Analysis of actin in different parts of the seeds of Zea mays
The presence of actin was detected by western blotting using an antibody to calf thymus actin that is known to cross-react with plant actin (Villanueva et al., 1990
, 1999). When 50 µg of total protein from embryos and storage-tissues from maize and common bean seeds were analysed (Fig. 1A, B), the presence of actin was readily detected preferentially in embryos in both cases (Fig. 1B, lanes 3 and 5, respectively). Actin was still detected in extracts from maize endosperm (Fig. 1B, lane 2) although at lower levels. As found previously, it was not possible to detect the presence of actin in cotyledons of P. vulgaris (Fig. 1B, lane 4) even with the high sensitivity method of chemiluminescence probes. A more detailed analysis of the different parts of the maize seed by western blotting using equal loading of protein in each lane (Fig. 2), confirmed that actin is present mostly in the embryo (Fig. 2, lane 2), followed in quantity by the scutellum (Fig. 2, lane 3) and the least amount in the endosperm (Fig. 2, lane 4). These data indicated that, as observed in common bean, most of the actin present in the seed represents the embryo axis protein. Therefore, an attempt was made to analyse the time-course of actin expression in embryo axes during germination.
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Time-course analysis of maize actin expression at the protein level
Maize seeds were induced to germinate and analysis of their actin content was carried out at various post-imbibition times. The concentration of protein was determined for each extract as described previously (see Materials and methods) and the equal loading of protein on the SDS-gels was confirmed by protein staining with Coomasie Brilliant Blue (Fig. 3A, lanes 3–7). Western blot analysis revealed the presence of actin from low levels at 0 h to maximum levels at 96 h post-imbibition (Fig. 3B, lanes 3–7). Similar to actin expression during germination in common bean (Villanueva et al. 1999
), the actin increases suddenly from 24 h to 48 h and again from 72 h to 96 h. These data indicated that actin is at basal levels in the embryo axis of dry seeds and an increased expression of the polypeptide is triggered at a point between 24 h and 48 h post-imbibition during germination/growth.
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Time-course analysis of actin expression at the RNA level
Analysis of the actin mRNA in embryo axes revealed a steady increase in expression contrary to the burst of expression encountered at the protein level (Fig. 4A, 0–96 h post-imbibition). Also, contrary to common bean mRNA, it was possible to detect maize actin mRNA at 0 h which then increased moderately throughout the germination process. To ensure that the difference in detection of the various RNAs was not due to differential loading of the gel, the RNA amount loaded in all lanes was standardized using the ribosomal RNAs as a reference (Fig. 4B, 0–96 h post-imbibition). These data suggested that the increase in actin detected in embryo axes during germination (Fig. 3B, lanes 3–7) was due, to a very modest extent, to an increase in the expression of the actin mRNA that takes place in the embryo.
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Analysis of actin isoforms expressed at different post-imbibition times
To analyse the characteristics of the actin induction in more detail, the embryo extracts were run on 2D PAGE gels followed by western immunoblotting to detect the isovariants. Similar protein amounts were loaded on the gels. Three distinct actin polypeptides of pIs 5.0, 5.1, and 5.2 were detected initially (Fig. 5, 0 h). In maize, the actin isoforms were generally more acidic (pI 5–5.2; Fig. 5) than those corresponding to common bean (pI 5.5–5.8; Villanueva et al., 1999
). At 24 h post-imbibition, there was little variation in the isoform composition and apparent abundance, except that the pI 5.1 isoform tended to decrease (Fig. 5, 24 h). However, at 48 h post-imbibition, an overall increase of the three isoforms was observed (Fig. 5, 48 h). At 72 h post-imbibition, the increasing trend of all three isoforms continued (Fig. 5, 72 h). Then, by 96 h, a significant and a clear change of expression pattern was detected. The pI 5.0 isoform, previously expressed in increased amounts, decreased significantly and practically disappeared; the pI 5.1 and 5.2 isoforms maintained a constant level of expression (Fig. 5, 96 h). This pattern was observed consistently in different repeats of the time-course analysed. Despite this differential expression pattern, an overall and constant increase in actin, closely matching the one-dimensional SDS-PAGE analysis was observed between 24–96 h in the 2D gel blots (Fig. 5). The change in the maize isoform expression pattern was consistent with the times of significant change in the post-imbibition process; namely, the shift from germination to growth of the hypocotyl (24–48 h), and could be related to the morphological changes in maize seedlings, such as active root expansion, which is known to be concomitant with cell division and morphogenetic processes at later stages of germination (Bewley and Black, 1994). The observed expression behaviour was also similar in most aspects to what had been observed previously for actin isoform expression in P. vulgaris (Villanueva et al., 1999). Densitometric analysis of the protein spots from the two-dimensional western blots allowed the actin isoform expression behaviour to be compared in maize, and to assign a particular trend to each one. It was observed that the most acidic isoform from maize (pI 5.0), was the one with a transient behaviour trend (Fig. 6). By comparison, in common bean, both the most acidic (pI 5.5; and most basic (pI 5.8) isoforms displayed a similar expression trend (Villanueva et al., 1999). There were two main isoforms in both maize (pI 5.1 and 5.2; Fig. 5) and bean (pI 5.6 and 5.7; Villanueva et al., 1999), that contributed to the bulk of the increase in actin protein expression observed throughout the germination process. Thus, except for the expression of one less isoform in maize with respect to common bean, the expression pattern and trends in both species were similar, despite the fact that one is a monocot (maize), and the other one a dicotyledonous (bean) plant.
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The expressed actin isoforms undergo tyrosine phosphorylation
Western immunoblotting analysis of the detected maize actin isoforms at 48 h, which was the time when all three of them were clearly detected (Fig. 7A), was carried out with an anti-phosphotyrosine antibody previously shown to recognize phosphorylated plant actin (Kameyama et al., 2000
). It was observed that at least two of the maize actin isoforms, pI 5.1 and 5.2, were tyrosine phosphorylated at this post-imbibition time (Fig. 7B). A weak spot corresponding to the pI 5.0 isoform was also observed, but it was not significantly present above background (Fig. 7B), and although it may still correspond to a phosphorylated actin, it was not scored as a modified actin isoform. Post-translational modification is another form of regulation and gives plasticity and versatility to a given expressed protein. Therefore, it is possible that these modifications yield more protein isovariants than the directly expressed gene products. To correlate the observed post-translationally modified actin isoforms with the number of expressed genes, in vitro translation assays of the polyA mRNA from this time point were also carried out. The autoradiography of a nitrocellulose membrane containing the 35[S]-methionine labelled in vitro-translated products revealed a well-resolved two-dimensional pattern corresponding to the translated proteins (Fig. 7C). The equivalent location of the maize actin isoforms was at the slightly less acidic positions of pIs 5.1, 5.2, and 5.3, respectively (Fig. 7C, D). Immunoblotting of the same membrane with anti-actin C4 monoclonal antibodies to confirm that the spots indeed corresponded to actin, detected the three same isoforms at slightly less acidic positions (Fig. 7D) than observed originally in total protein extracts (Fig. 7A). In addition, an immunoblot with anti-phosphotyrosine antibodies on a transferred membrane of a 2D gel of in vitro translated RNA from stage 48 h seedlings showed no immunolabelling (data not shown). These data also suggest that the three expressed maize actin isoforms are the products of three distinct genes.
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Discussion |
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The seed has an array of tissues that will provide different functions once germination is initiated. The reserve tissue will be degraded during germination and most of its contents will be used by the embryo for growth. The embryo will use the nutrients coming from this pool, in order to grow and develop the photosynthetic machinery that will allow it to reach autotrophy, and thus, become a new plant. The analysis of the total amount of actin in different seed tissues reflected to some extent the dynamics of the particular tissue during germination. The amount of actin in extracts from different parts of Zea mays and Phaseolus vulgaris (Fig. 1A, B) was different depending on tissue type and species. The reserve tissue showed the most distinctive features (Fig. 1B, lanes 2 and 4). It was not possible to detect the presence of actin in cotyledons of P. vulgaris (Fig. 1B, lane 4) even with the use of more sensitive detection methods than those used previously (Villanueva et al., 1999
). At similar protein amounts loaded on the gels, actin was detectable, although not very abundant, in all the seed reserve tissues of maize (Fig. 1B, lane 2; Fig. 2, lanes 3 and 4). The presence of actin in these maize reserve tissues may be necessary for the assembly of the reserve stores. Clore et al. (1996) showed that there are actin microfilaments that co-localize with eF1
in the endosperm of opaque-2 maize. They proposed that this array allows the assembly of the protein bodies during seed maturation. In addition, it has previously been shown that common bean embryo axes are rich in protein storage vacuoles (Guillén et al., 2001) and recent observations in this tissue have revealed a well-defined actin cytoskeleton that is organized around these vacuoles (MA Villanueva, unpublished observations).
The scutellum contains more actin than the endosperm, but much less than the embryo (Fig. 2, lanes 2–4). This tissue represents a modified cotyledon with specific functions of enzyme secretion and nutrient transport that travels from the reserve tissue to the growing embryo (Bewley and Black, 1994). It is conceivable that cytoskeletal components are necessary for active plasmodesmatal and/or exocytotic transport that might occur during germination (Bewley and Black, 1994; Balu
ka et al., 2001, 2003). When the embryo axis tissue was analysed in P. vulgaris and Z. mays, actin was shown to be a significant protein in both seed species (Fig. 1, lanes 3 and 5). These data are consistent with the view that the embryo will require highly dynamic processes in which actin has fundamental roles once germination is triggered.
An increase of actin expression at the protein and mRNA levels in common bean embryo axes after 48 h post-imbibition has previously been documented (Villanueva et al., 1999). The increase in actin mRNA was very sharp in both whole seeds and embryo axes alone, but the increase in protein was very sharp only in whole seeds and was more moderate in embryo axes (Villanueva et al., 1999). While the time-course analysis of the mRNA and protein expression of actin in the maize embryo axis showed similar increases to those observed in P. vulgaris, a moderate but steady increase in expression at the mRNA level occurs throughout the germination process (Fig. 4A, 0–96 h post-imbibition). However, sharp increases in protein expression were detected at the time points of 24–48 and 72–96 h post-imbibition (Fig. 3B, lanes 3–7). One important observation was that maize actin mRNA was present at easily detectable amounts in the dry seed embryo (Fig. 4, 0 h). For example, in the case of common bean, actin mRNA was not detectable in the dry embryo axis and levels increased conspicuously at 48 h post-imbibition (Villanueva et al., 1999). In common bean, germination stops at some point between 24 and 48 h. In the case of maize, the germination (which concludes with the emergence of the radicle) is one half shorter and it occurs around 12–24 h post-imbibition. To extend the analysis beyond this short germination interval, more points after 24 h, when active elongation of the hypocotyl starts, were also scored, including a further 96 h post-imbibition time point. It is precisely at these time points (24–48 and 72–96 h), when there appears to be an important increase in actin expression level. This could mark important events in germination and later developmental processes. The first increase at 24–48 h might reflect the requirement for newly synthesized actin after most of the initial actin and mRNA have been used during the germination process. Later, at some point around 72 h post-imbibition (Fig. 5), significant morphological changes such as active main root elongation, coleoptile expansion, and development of secondary roots, begin to occur in the maize seedling. Thus, the second increase in actin expression at 72–96 h may reflect the actin requirement in these processes. It is likely that different actin isoforms are required at various developmental stages. This is consistent with the observation of the virtual disappearance of the pI 5.0 isoform and increase of the pI 5.2 and 5.3 isoforms at 96 h (Fig.5, 96 h) post-imbibition. Thus, some of the increase and differential extent of actin expression in the maize seedling may reflect the profound cell changes that accompany cell division and differentiation. Two-dimensional analysis of the actin isovariant composition also revealed that, similar to P. vulgaris, the increase in the actin expression was the consequence of two main isoforms. In maize seedlings, two of the three actin isoforms detected at the 48 h post-imbibition time also cross-reacted with an antibody against phosphotyrosine (Fig. 7B), previously shown to recognize tyrosine phosphorylated plant actin (Kameyama et al., 2000). Although no further analysis was carried out to see if this phosphorylation pattern changed or persisted throughout germination and later developmental stages, it is significant that the maize actin is modified in this particular amino acid since this is a protein that is tightly regulated at all levels. Tyrosine phosphorylation in actin has previously been reported in several organisms and it has been proposed that this modification is related to cell-shape changes and spore activity in the unicellular slime mould Dictyostelium (Howard et al., 1993; Kishi et al., 1998), and control of movements in plants (Kameyama et al., 2000). The pI of the corresponding non-phosphorylated maize actin isoforms was only slightly less acidic (Fig. 7D). This suggests that the maize actin isoforms are not hyperphosphorylated and phosphorylation probably occurs in only one tyrosine residue on the actin molecules. In fact, it has been shown that a direct correlation exists between the shift in pI, and the number of modifications by phosphates, on proteins (Kumar et al., 2004). Thus, for a slightly acidic protein such as actin, a modification of 1–2 phosphates would only cause a shift of 0.1–0.15 pI units to more acidic locations as observed in this study. It was also interesting in this respect, that the pI range of the maize actin isoforms was more acidic (5.0–5.2), compared with the pI range previously reported for common bean actin (5.5–5.8; Villanueva et al., 1999) and soybean (McLean et al., 1990). Nontheless, the predicted pI of the only complete reported maize actin sequence (GeneBank accession No. P02582), the ACT1 gene product, is precisely 5.22. Consequently, the protein isoforms intrinsically possess a more acidic nature than those of common bean. This sequence also shows two potential tyrosine phosphorylation sites at amino acids 191–198 and 210–218, which also agrees with the view of a maximum possibility of two phosphates added post-translationally.
At least six copies of actin genes exist in the Z. mays genome (Moniz de Sá and Drouin, 1996; http://www.expasy.org). It is suggested that the three isoforms detected in maize embryos may be expressed from three different genes. Two-dimensional western blot analysis of protein expressed in the reticulocyte lysate from mRNA templates isolated from maize embryos revealed the presence of three distinct proteins. Since that phosphorylation does not occur in vitro in the reticulocyte expression system, it is suggested that the three isoforms detected in maize embryos arise from different genes, rather than from post-translational modifications. However, it cannot be ruled out that differential splicing of a single gene product could also give rise to the three detected actin isoforms.
The reduced number of actin isoforms compared with the number of genes indicate that, in the early growth stages, only a few of these genes are expressed. Later stages of growth and development of this plant where other isoforms may be expressed for specific functions have not been analysed. It is now clear that specific actin isoform expression occurs in different tissues (Huang et al., 1997; Meagher et al., 1999), and under specific conditions (Kandasamy et al., 2001). Furthermore, specific actin expression is required when specific developmental requirements arise as in the case of ACT2 which is required for root hair formation and elongation in Arabidopsis (Nishimura et al., 2003).
These results on the comparison of the expression of actin in both maize, a monocot, and in black bean, a dicot, showing that: (i) both protein and mRNAa expression are similarly induced; (ii) similar qualitative and quantitative changes are seen during the germination process; and (iii) only a subset of genes from the whole maize genome are expressed, suggest a more general and highly regulated expression of actin genes at this stage of germination/growth in plants. These changes might be correlated with morphological changes in seedling development. Future work will focus on the analysis of tissue specificity of the various isoforms and on establishing the significance of these observations at the functional level for root and shoot differentiation during the establishment of the seedling.
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Acknowledgements |
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This study was supported by grants IN-230399 and IN-224103 from DGAPA-UNAM to M Villanueva and an NSERC grant to Nick Ovseneck. Claudia Díaz was partially supported by a scholarship from DGAPA-UNAM and by grant IN-230399. We thank Theresa A Musket (University of Missouri, Columbia, USA) for providing the maize actin clone 5C01H03. We thank Dr Federico Sánchez for providing some of the reagents used in this study. The photographic work of Sergio Trujillo is also acknowledged.
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References |
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