Chromosome movement in lysed mitotic cells is inhibited by vanadate.

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10--100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.

Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline.

The distribution of membrane-associated calcium has been determined at various stages of mitosis in Haemanthus endosperm cells with the fluorescent chelate probe chlorotetracycline (CTC). CTC fluorescence in Haemanthus has two components: punctate, because of mitochondrial and plastid membrane-Ca++; and diffuse, primarily because of Ca++ associated with endoplasmic reticulum membranes. Punctate fluorescence assumes a polar distribution throughout mitosis. Cones of diffuse fluorescence in the chromosomse-to-pole regions of the metaphase spindle appear to coincide with the kinetochore fibers; during anaphase, the cones of fluorescence coalesce and this region of the spindle exhibits uniform diffuse fluorescence. Perturbation of the cellular Ca++ distribution by treatment with lanthanum, procaine, or EGTA results in a loss of diffuse fluorescence with no accompanying change in the intensity of punctate fluorescence. Detergent extraction of cellular membranes causes a total elimination of CTC fluorescence. CTC fluorescence of freshly teased crayfish claw muscle sarcoplasmic reticulum coincides with the A bands and is reduced by perfusion with lanthanum, procaine, and EGTA in a manner similar to that for diffuse fluorescence in the endosperm cells. These results are consistent with the hypothesis that a membrane system in the chromosome-to-pole region of the mitotic apparatus functions in the localized release of sequestered Ca++, thereby regulating the mechanochemical events of mitosis.

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition.

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane-associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.

Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea.

During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while beta-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from the Marsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea beta-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither beta-tubulin- nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita.

Lithium alters mitotic progression in stamen hair cells of Tradescantia in a time-dependent and reversible fashion.

Stamen hair cells of Tradescantia exhibit remarkable precision in the timing of their mitotic events. This precision is altered dramatically with treatment in 50 microM to 1 mM LiCl, an inhibitor of the polyphosphoinositide cycle. Mitotic progression is altered as a function of the time of treatment with LiCl. If cells are treated during late prophase, greater than 80% fail to enter metaphase. Most of the cells that undergo nuclear envelope breakdown become arrested in metaphase. Treatment with LiCl earlier in prophase also results in metaphase arrest. Metaphase arrest can be reversed by the addition of 10 microM myo-inositol or 100 microM CaCl2 to the extracellular medium. The timing of reversal by myo-inositol takes 10 to 14 min while CaCl2 promotes anaphase onset in 2 to 5 min. The difference in kinetics for reversal between these two treatments suggests that myo-inositol addition overrides a biochemical pathway while Ca2+ addition supplants a phosphoinositide-mediated rise in the cation that may be necessary for anaphase onset. Buffer without myo-inositol or CaCl2 is insufficient for reversal. If the cells are treated with LiCl in mid-late-metaphase, at least 5 min prior to the expected time of anaphase onset, sister chromatids split at the normal time, 33 +/- 4 min after nuclear envelope breakdown, but further chromosome separation is arrested. Anaphase chromosome movement can be restored by treatment with either 10 microM myo-inositol or 100 microM CaCl2 in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644.

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.

The buffering of calcium with quin2 reversibly forestalls anaphase onset in stamen hair cells of Tradescantia.

Cells from immature stamen hairs of the spiderwort plant Tradescantia virginiana cv. Zwanenburg Blue exhibit remarkable precision in the timing of their mitotic events: anaphase onset occurs 33 +/- 4 min after nuclear envelope breakdown, and cell plate vesicle aggregation occurs approximately 19 +/- 4 min after anaphase onset. To test the hypothesis that altered calcium levels might affect mitotic events, we incubated stamen hairs with the acetoxymethyl ester of the Ca-chelator, quin2 (quin2AM), or the K+-salt of quin2 free acid, and found that mitotic progression was blocked in metaphase and late anaphase. The inhibition of mitotic progression was dependent upon the quin2AM or quin2 concentration and the duration of incubation with the probe. Metaphase arrest could be reversed within several min by the addition of 100 microM CaCl2 to the extracellular medium, while lower concentrations of Ca2+ or the presence of Mg2+ in the buffer were insufficient to reverse the block. A second perfusion of 100 microM CaCl2 was usually necessary to promote cell plate vesicle aggregation. The effective concentration of quin2AM for reversible mitotic arrest was 50 microM (30 min) or 500 microM (12 min); a 30-min incubation in 50 microM quin2 acted similarly. The similarity of dose responses of Ca2+-reversal of quin2AM- and quin2-induced metaphase arrest suggests that the active species is the free acid and that the mode of inhibition is through Ca2+-chelation. Because the free acid is not permeant, and because extracellular esterase activity exists in the cell wall, the site of quin2 activity is probably outside the plasma membrane, in the wall space.(ABSTRACT TRUNCATED AT 250 WORDS)

Nifedipine reversibly arrests mitosis in stamen hair cells of tradescantia.

Mitotic stamen hair cells of Tradescantia virginiana (cv. Zwanenburg Blue) become arrested in metaphase following a 30-min treatment with 10 to 100 microM nifedipine, a Ca2+-channel entry blocker. The time interval between nuclear envelope breakdown and anaphase onset in untreated cells is approximately 33 min +/- 4 min; nifedipine extends this "metaphase transit time" beyond 70 min. Nifedipine can be photoreversed in situ by exposure to 365 nm light. UV illumination inactivates the drug, its inhibitory effect on Ca2+ is abolished, and cells arrested in metaphase enter anaphase within 3 to 18 min of UV exposure if CaCl2 is present in the medium. The interval between UV illumination and anaphase onset is inversely related to the extracellular concentration of CaCl2. If CaCl2 is not added to the medium, the interval between UV exposure and anaphase onset is usually longer than 18 min. The sole addition of 100 microM CaCl2 to the medium is insufficient to reverse nifedipine inhibition; unless the cells are exposed to UV light, anaphase will not commence. The threshold concentration of free Ca2+ for rapid anaphase onset (less than 10 min after UV photoreversal) is between 1 and 10 microM. These results suggest that an influx of Ca2+ from the extracellular medium to the cytosolic compartment is necessary for normal progression from metaphase to anaphase and that this influx may serve as a trigger for chromosome separation.

The coincident distribution of calcium-rich membranes and kinetochore fibers at metaphase in living endosperm cells of Haemanthus.

We have coupled chlorotetracycline (CTC) fluorescence with polarized light microscopic observations of living mitotic endosperm cells of Haemanthus in order to compare the spatial distributions of Ca2+-rich endomembranes in the spindle with that of the kinetochore fibers during metaphase. In either control cells or in those treated with the microtubule-stabilizing drug, taxol, the metaphase distribution of Ca2+-rich membranes (bright spindle fluorescence) coincides exactly with the distribution of kinetochore-fiber birefringence.

Quin2-induced metaphase arrest in stamen hair cells can be reversed by 1,2-dioctanoylglycerol but not by 1,3-dioctanoylglycerol.

Elicitor molecules of the polyphosphoinositide cycle, inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (1,2-DAG) play roles in the entry of calcium into the cytosol and in the elevation of protein kinase C activity, respectively. We have treated stamen hair cells of the spiderwort plant, Tradescantia virginiana, with a solution of quin2 (50 microM) or its acetoxymethyl ester, quin2-AM (50 microM) and have retarded the normally predictable rate of progression through metaphase. Metaphase arrest persists for longer than 80 min after treatment with this Ca2+-chelator, and, shortly thereafter, the cells revert to interphase without dividing. Reversal of metaphase arrest results from treatments with calcium chloride (100 microM) after 5 to 8 min or with 1,2-DAG (i.e., 60 micrograms/ml 1,2-dioctanoylglycerol) after 7 to 11 min. In control experiments, metaphase arrest could not be reversed by treatment with either magnesium sulfate or 1,3-dioctanoylglycerol. Anaphase onset was observed in these control cells after post-treatment with calcium chloride (after 4-9 min) or with 1,2-dioctanoylglycerol (after 7-13 min). The treatment of stamen hair cells in very early prophase with H-7, (i.e., 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a potent protein kinase C inhibitor, extends the duration of metaphase significantly. Neither H-8 nor HA-1004, less active protein kinase C inhibitors in this class of molecules, alter the duration of metaphase to a significant extent. These results suggest that in cells arrested in metaphyase by quin2, calcium translocation plays a role in the sequence of events which culminate in anaphase.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of microtubules in stimulating cytokinesis in animal cells.

The initiation of furrow formation is disrupted when microtubule elongation to the cell surface is inhibited either by promoting microtubule disassembly with hydrostatic pressure or by stabilizing the mitotic astral microtubules with taxol. The pressure studies confirmed Rappaport's earlier observation that stimulation of furrow formation is produced by a pair of asters and does not require chromosomes or a central spindle in the large dividing cells of echinoderm embryos. The taxol studies showed that furrow formation occurs between two stable asters when the asters are within 20 microns of the lateral cell surface. Furrow formation at the lateral edge of the cell does not appear to require microtubule dynamics, microtubule elongation, or contact of microtubule ends with the lateral cell surface. Microtubules may function to increase the concentrations of the active forms of diffusible stimulatory factors that interact with receptors at the inner cell surface to initiate the formation of the furrow apparatus and activate contraction.

Control of development and motility in the spermatozoids of lower plants.

The spermatozoids of lower plants have long been recognized as remarkably complex motile gametes. Spermatozoids differ markedly from the other gametophyte cells that surround or give rise to them. Their differentiation process involves the synthesis and assembly of a complex cytoskeleton and a motile apparatus that can be simple or complex, having as few as two to as many as thousands of ciliary axonemes. An important aspect of spermiogenesis involves the de novo synthesis of basal bodies in a cytoplasmic particle known as the blepharoplast: that is, the cells that produce spermatocytes do not contain centrioles. Thus, these cells provide an ideal system in which to study the formation of basal bodies. The cytoskeletons of spermatozoids from different organisms display a common architecture, with a multilayered structure (MLS) at the anterior end of the cell and a dorsally situated planar ribbon of crosslinked microtubules extending the length of the elongated gamete. The function of the MLS is not known, but it could be involved in cell-body elongation during development and in the control of ciliary motility in the mature gamete, particularly during chemotaxis. The application of modern techniques on these cells can shed light on long-standing problems relating to spermiogenesis and motility.

The regulation of mitotic spindle function.

The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.

Physiological requirements for ciliary reactivation of bracken fern spermatozoids.

Physiological parameters affecting reactivated ciliary beat in spermatozoids of braken fern (Pteridium aquilinum) were studied using a Triton/glycerol permeabilized cell model system. Reactivation frequencies of polylysine-tethered cells equalled in vivo rates at neutral pH. Frequency was dependent on ATP and Mg2+ concentration, and reactivation was inhibited by millimolar or greater free calcium. Reactivation was reversibly inhibited by micromolar concentrations of sodium ortho-vanadate, while intact cells were not affected by millimolar levels of the inhibitor. This is the first characterization of in vitro ciliary beat in a non-algal plant cell and demonstrates that the nucleotide and ionic requirements for reactivation of bracken cilia are similar to those of other systems.

Molecular cloning and characterization of maize ZmMEK1, a protein kinase with a catalytic domain homologous to mitogen- and stress-activated protein kinase kinases.

Mitogen- or stress-activated protein kinase kinases (M/SAPKKs) are dual-specificity protein kinases that are components of highly conserved signal transduction pathways. A cDNA clone (ZmMEK1) was isolated from a Zea mays (L.) root tip library. ZmMEK1 contains a complete open reading frame, encoding a 355-amino-acid protein with an estimated molecular mass of 39,874 Da. The predicted protein contains the 11 catalytic sub-domains conserved in all protein kinases, and a version of the sub-domain VIII S/TxxxS/TxVGT motif that is characteristic of M/SAPKK proteins (EC 2.7.1.37). The catalytic domain of ZmMEK1 is most closely related (65% identity) to the tomato M/SAPKK homolog LeMEK1, but exhibits similar identity (39-60%) to M/SAPKKs from other plants, animals and fungi. Northern blotting revealed ZmMEK1 mRNA in maize seedling roots and coleoptiles; in mature plants transcripts were detected in stems and low levels in leaves. Transcription of ZmMEK1 mRNA was also affected by environmental stimuli. The catalytic domain of ZmMEK1 was expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Nanogram quantities of the purified fusion protein reacted with anti-M/SAPKK antibodies on immunoblots. In vitro, the GST-ZmMEK1 fusion protein undergoes autophosphorylation, and will phosphorylate myelin basic protein, but will not phosphorylate histone H1. ZmMEK1 encodes an enzyme that is structurally and functionally similar to other M/SAPKK proteins.

The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase.

Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors.

Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10-20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15-20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15-26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the mitogen-activated protein kinase kinase ZmMEK1 in the primary root of maize.

We have analyzed changes in the distribution and abundance of a mitogen-activated protein kinase kinase (MAPKK) enzyme (EC 2.7.1.37) known as ZmMEK1. in root apices of Zea mays L. under normal growth conditions, and after treatments that alter patterns of proliferation, as a means to assess the potential physiological role of the MAP kinase cascade in growth and development. The ZmMEK1 protein is most abundant within immature tissues such as the roots and leaves of seedlings, and is nearly undetectable in mature leaf tissue. Along the longitudinal axis of growing roots, ZmMEK1 mRNA and protein are abundant throughout the apical 12 mm. Two anti-ZmMEK1 antibody-reactive proteins can be resolved within the apical 4 mm of the root, spatially coinciding with the meristem and distal elongation zone. Phosphatase treatments suggest that both immunoreactive bands are forms of ZmMEK1 that differ in their state of phosphorylation. Expression of ZmMEK1, histone H4 and cyclin-dependent protein kinase (CDK) in roots after 7 days of exposure to low temperature and during a 48-hour recovery period was monitored during the coincident alterations in growth. Levels of ZmMEK1 mRNA and protein within these roots were indistinguishable from those of control roots. However, a slower-migrating form of ZmMEK1 temporally coincided with the observed increase in CDK levels during the transition into proliferative activity. We demonstrate that the ZmMEK1 MAPK activator is expressed and is differentially phosphorylated within the root meristem and distal elongation zone. We suggest that post-translational modifications of the protein regulate the function of ZmMEK1 within the root. Changes in ZmMEK1 phosphorylation state correlate with changes in proliferation in the root apex.