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Immunofluorescence and electron microscopic investigations of DCPA-treated oat roots
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
lmmunofluorescence
4,
47-57 (191)
and Electron Microscopic DCPA-Treated Oat Roots
Investigations
of
LARRY P. LEHNEN, JR.‘,’ AND KEVIN C. VAUGHN'*~ United States Department of Agriculture, Agricultural Research Service, Southern Weed Science Laboratory, P.O. Box 350, Stoneville, Mississippi 38776 Received September 14, 1990; accepted December 29, 1990 Immunofluorescence and electron microscopy were used to investigate the effects of DCPA (dimethyl tetrachloroterephthalate) on oat roots. Phragmoplast microtubule arrays were the most consistently affected. Multiple phragmoplasts, undulated phragmoplasts, branched phragmoplasts, and phragmoplasts arising at places other than that predicted by the preprophase band were all observed. Cell wall formations resulting from these abnormal phragmoplast configurations are also abnormal and consist of reticulate cell plates, walls formed at abnormal angles, and wall material in net-like arrays containing pockets of cytoplasm. Cortical and preprophase band microtubule arrays were unaffected, and were similar in both treated and control cells. Minispindle and multipolar spindles were observed in some meristematic cells, although many spindle formations were normal or nearly so. Some multiple and highly lobed nuclei were also found as a consequence of these abnormal divisions. Cell plates were abnormal even when there were no nuclear abnormalities that would cause physical disruption of the division plane and even though preprophase bands (which are predictive of the subsequent plane of division) were normal. These data indicate that DCPA has a direct effect on phragmoplast microtubule organizing centers. 8 1991AC&I& Pm, Inc.
INTRODUCTION
tematic survey of these compounds to monitor the steps of mitosis that are altered after treatment. DCPA has been investigated previously by a number of workers but the effects have been mainly described at the light microscopic level. The most extensive studies are those of Holmsen and Hess (4, 5). In a comparison of DCPA with colchicine and CIPC, Holmsen and Hess (5) found that DCPA was the most effective of the three compounds in inducing cell plate abnormalities. Likewise, using light and electron microscopy, Vaughan and Vaughn (6) found that DCPA-induced abnormal cell plates or, in some tissues, the complete absence of cell plates in wheat root tips. These authors (4-6) speculated that DCPA probably causes these effects by disrupting phragmoplast microtubule arrays. In this report, we utilize immunofluorescence microscopy, which more readily detects changes in microtubule profiles, and electron microscopy to determine the effects of DCPA on phragmoplast microtubule and cell plate formation.
Compounds that disrupt cell division are useful in elucidating the processes and proteins that are required to complete mitosis as well as the consequences of deviation from a normal cell division. Many of the compounds utilized to disrupt plant mitosis were originally selected for their ability to disrupt mitosis in animal cells (1). However, compounds that disrupt animal mitosis are much less effective at disrupting mitosis in plant cells (1). For example, almost lOOO-fold more colchicine is required to disrupt mitosis in plant cells than animal cells. A number of herbicides disrupt mitosis and are much more effective at disrupting plant mitosis than the compounds that disrupt animal mitosis (2, 3). We have begun a sys’ Southern Weed Science Laboratory, USDA-ARS, P.O. Box 350, Stoneville, MS 38776. ’ Present address: Plant Cell Biology Group, Australian National University, Canberra, ACT, Australia. ’ To whom correspondence should be addressed. 47
0048-3575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All lights of reproduction in any form reserved.
48
LEHNENANDVAUGHN
MATERIALS
Plant Material
AND
METHODS
and Treatment
Oat seeds were germinated on moistened Whatman No. 1 paper in petri dishes in the dark at room temperature (approximately 22°C). After 3 days of growth, the seedlings were transferred to new petri dishes supplemented with 10-6-10-4 M DCPA for an additional 24 hr in the dark. The DCPA solutions were prepared from a concentrated acetone stock immediately before use. Acetone concentrations did not exceed 0.1% (v/v). Controls were grown in the dilute acetone solution alone. Microscopic
Techniques
Both immunofluorescence and electron microscopy were performed as described previously (7). In the immunofluorescence preparations, DNA was stained with the fluorescent dye DAPI so that the stage of cell division could be monitored. The experiments were repeated at least four times and several root tips from each treatment were examined by immunofluorescence and electron microscopy. RESULTS
Immunojluorescence
Microscopy
Control oat root tips revealed all of the microtubule arrays typical of higher plant cells. Cortical, preprophase, mitotic, and phragmoplast arrays were unaffected, even with a solution of 0.1% (v/v) acetone (Fig. 1). Treatments with lop4 or 10m5 M DCPA were quite consistent, where as lop6 M treatment often resulted in samples that were either affected or not affected. All of the samples shown below are either from lop5 or 10e4 M treatments. Various microtubule configurations were observed after treatment with DCPA. Some cells, especially in interphase and preprophase configurations appeared unaf-
fected and were virtually identical to the control (Fig. 2A). Prometaphase figures were also similar in treated and control preparations. These are made up of many small microtubule arrays which enmesh the mass of chromosomes, gathered into the center of the cell. Similar profiles have been observed in other grass species at prophase or prometaphase. Occasional profiles of later mitotic stages revealed multipolar and/or abnormal spindle orientations (Fig. 2C), but cells with an apparently normal spindle could also be detected in the same preparation (Fig. 2B). In contrast to the largely normal mitotic and interphase microtubule arrays, phragmoplast microtubule arrays are always abnormal in DCPA-treated cells (Fig. 3). Several different phragmoplast morphologies are observed. In some cells, the two nuclei are separated cleanly at early telophase, the phragmoplast is between the two nuclei but the phragmoplast arrays form in a plane not perpendicular to the side walls (Fig. 3A). The presence of abnormal phragmoplast arrays after DCPA is not due to interference of cell plate formation by portions of the nucleus interrupting the phragmoplast as has been noted for other cell disrupters (e.g., Refs. (7, 9)), although this could be a potential reason for phragmoplast disruption in cells where multipolar divisions are noted (Fig. 3B). Rather, an abnormal phragmoplast appears to be a primary effect. In elongated cells that morphologically resemble epidermal cells, not only are abnormal phragmoplasts found but also those observed are found at places other than the center of the cell (Fig. 3C). Multiple phragmoplast arrays are also noted (Fig. 3D). After the completion of mitosis, cortical arrays of microtubules return even though there are abnormal walls and/or multiple nuclei in the cell (Fig. 2D). To further substantiate that even cells with an apparently normal telophase separation of nuclei produce abnormal phragmoplasts in the presence of DCPA, preparations were stained with DAPI to stain DNA in nuclei and with antitubulin to de-
DCPA
EFFECTS
ON
OAT
ROOTS
49
FIG. 1. Immunojluorescence micrographs of control oat root tips. A. Late preprophase band (arrows) with some microtubule associating with the nucleus. B. Metaphase. C. Early anaphase. D. Late telophase with the phragmoplast arrays clearly separated from the two nuclei (IV). X800.
tect the microtubules. In the cell shown in Fig. 4, the nuclei are well separated and appear to be of the normal spherical morphology associated with a telophase nu-
cleus. However, tubulin profiles in this cell reveal an abnormal curve to the phragmoplast separating the nuclei as well as a second band of microtubules oriented perpen-
50
LEHNEN
AND
VAUGHN
FIG. 2. Immunojluorescence micrographs ofDCPA-treated oat root tips. A. Cells with preprophase band (arrows) and cell in interphase (f) with cortical microtubule arrays. B. Near normal spindle formation. C. Cell with three separate spindle formations in a single cell. D. Return of cortical microtubule arrays after a multipolar division. The three nuclei (N) stand out in negative relief. x800.
DCPA
EFFECTS
ON
OAT
ROOTS
formations in DCPA-treated oat root tips as visualized by FIG. 3. Variations in phragmoplast immunofluorescence microscopy. A. Near normal phragmoplast with slight curving of the phragmoplast, especially on the right side of the cell. B. Branched phragmoplast that separates three nuclei (N). C. Phragmopiast(s) oriented in several directions. One band (arrow) is running across the top third of the cell, with another band running perpendicular to that besides the nucleus. The perpendicular band connects to two other bands at the bottom of the cell. D. Multiple phragmoplasts (arrows) in a multinucleate cell. Phragmoplasts in several directions and orientations are observed. x800.
51
FIG. 4. Fluorescence micrographs of DCPA-treated wheat root cell stained to visualize DNA [A) or tub&in (B and C). In this cell, a telophase separation of the two nuclei is clearly seen in A and the nuclear morphology is regular. B and C are micrographs taken at the same plant of focus as in A (B) or in anotherfocus plant (0. Although the nuclei are well separafed, rhe phragmoplast is curved and contains additional microtubules formed at right angles to this phragmoplast. X800.
DCPA
EFFECTS
dicularly to the major phragmoplast array. This second array is far away from any nucleus. Besides this analysis of double-stained preparations, root tips from the same batches used for immunofluorescence were prepared for Fuelgen-staining to monitor mitotic indices and irregularities. In these preparations, greater than 80% of the telophase preparations were well-separated binucleate cells, with less than 20% of the cells in a multipolar mitosis or with two closely appressed nuclei (Lehnen and Vaughn, unpublished). In preparations from these same batches stained for tubulin, virtually all of the phragmoplast arrays exhibited some type of abnormality. Thus, nuclear interference with phragmoplast arrays could explain less than 20% of the
ON
OAT
ROOTS
53
phragmoplast abnormalities but not the 80% which were normally separated. Electron Microscopy As might be expected from the immunofluorescence microscopy shown herein, and the published reports of wall abnormalities described previously in DCPA-treated cells (34, the major ultrastructural effects observed in DCPA oat roots are wall abnormalities. Several kinds of abnormalities are noted. Some walls contain loops of wall material gathered into mesh-like structures, with pockets of cytoplasm trapped between the loops (Fig. 5). Many cells have wall material originating from the parental cell wall but ending bluntly in the cytoplasm rather than connecting to the opposite parental cell wall (Fig. 6). In other cells, the wall is
FIG. 5. Electron micrograph of a DCPA-treated oat root. Irregularly formed walls (arrows) meander through the cytoplasm, capturing pockets of cytoplasm. An additional reticulate wall(r) runs close to the parental cell wall. N, nuclei. Bar, 5 pm.
54
LEHNEN
AND
VAUGHN
FIG. 6. Electron micrograph of a DCPA-treated oat root. A wall(w), originating along the parental cell wall, grows out into the cytoplasm and ends bluntly without connecting to another cell wall. The nucleus (IV’) is irregular in shape with the larger portion containing an inclusion (*). Bar, 2 pm.
reticulate and appears to undulate between the two nuclei that have resulted from a successful separation of chromosomes during mitosis (Fig. 7A). Many of these walls originate far from the midpoint of the cell and often lay parallel to the parental wall and are far away from any nuclear material. Most other cellular structures appeared normal. Normal-appearing amyloplasts, Golgi, the endoplasmic reticulum, and mitochondria were all observed. As might be expected, there are also some apparently abnormal mitotic figures and the resultant lobed or multiple nuclei typical of cell mitotic disrupters (Fig. 7B). Many apparently normal or near-normal nuclear separations are observed (Fig. 7A) and in these cells nuclear morphology is also normal. DISCUSSION
DCPA is unique among the higher plant mitotic disrupters in its greater specificity in altering phragmoplast microtubule ar-
rays. Many herbicides that disrupt mitosis, such as the dinitroaniline or phosphoric amide herbicides (2, 8), inhibit polymerization of tubulin into microtubules so that no microtubules are formed. All microtubule arrays are affected by these herbicides. Carbamate herbicide mitotic disrupters such as propham (IPC) or chlorpropham (CIPC) do not inhibit microtubule polymerization, but rather cause a fragmentation of the mitotic spindle so that many small spindles are produced (3, 7). Phragmoplast arrays in cells treated with carbamate herbicides are often abnormal. However, the phragmoplast abnormalities in these treated cells may be explained by physical interference of parts of the nuclei in the plane of cell division (7,9). Because of this interference, phragmoplasts grow around the regions of nuclei, resulting in branched or undulating phragmoplast arrays (7, 9). DCPA induces abnormal phragmoplast formation, even when the two telophase nuclei are clearly separated (e.g., Figs. 4 and 7A).
FIG. 7. Electron micrographs of DCPA-treated oat roots. A. A new cell plate separates two fairly well-separated nuclei. The plate is reticulate and undulates between the two nuclei. B. An abnormal lobed nucleus with the two major portions connected by a narrow band of nucleoplasm (arrows). An abnormal thickened cell wall (*) is also noted. p, plastid with starch. Bar, 5 km in A and 2 pm in B.
56
LEHNEN
AND VAUGHN
The data herein also support the results of Holmsen and Hess (4, 5) and Vaughan and Vaughn (6) on the mode of action of DCPA. All of these authors noted that DCPA was most effective at disrupting cell wall formation, although other abnormalities are also noted. It is possible that DCPA affects a cellular component, perhaps a microtubule-associated protein (MAP) that has a critical role in the organization of the phragmoplast and a role of lesser importance in other microtubule arrays. Recently, Cyr and Palevitz (11) have described the isolation of MAPS from higher plants and demonstrated that these proteins are involved in microtubule cross-linking and stability. Not all species are sensitive to DCPA (e.g., onion) and it may be possible to identify MAPS associated with DCPA sensitivity by comparing MAPS from sensitive and resistant species. Preprophase bands are believed to predict the plane of the subsequent cell division (11). In DCPA-treated root tips, preprophase bands are apparently normal, although the plane of the subsequent cell division is not. Thus, it is clear that, although the preprophase band is a general indicator of the division plane in control cells, subsequent phragmoplast formation and location may be subsequently disrupted by DCPA. Clayton and Lloyd (9) found normal preprophase bands, followed by the formation of a branched phragmoplast after multipolar division, as a result of CIPC treatment. However, after CIPC treatment, the multiple nuclei that form after a multipolar mitosis often interrupt the plane of pragmoplast formation. Thus, the abnormal phragmoplasts could be the result of steric hindrance rather than a direct effect on the phragmoplast array. DCPA differs from CIPC in that abnormal walls are produced even though the nuclei often do not interfere with the plane of phragmoplast formation. An extreme effect in wheat root tips results in the production of no cell plates in some tissues even though nuclear
divisions are apparently unaffected in these same tissues. Moreover, the presence of phragmoplasts at places far from the plane of a normal cell division and oriented 90” to the plane indicates strongly that DCPA induces phragmoplast abnormalities by direct action on the phragmoplast array. As such, DCPA may serve as a valuable tool in investigating mechanisms of control of phragmoplast orientation as well as the relationship between the preprophase band and phragmoplast location. ACKNOWLEDGMENTS Ms. Ruth H. Jones and Ms. Lynn Libous-Bailey provided excellent technical assistance during the course of these experiments. This work was supported, in part, by USDA Competitive Grant 86CRCR-1933 to K.C.V. Mention of a trademark or product does not constitute endorsement of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
REFERENCES 1. K. C. Vaughn and M. A. Vaughan, Mitotic disrupters from higher plants, effects on plant cells, in “Biologically Active Natural Products Potential Use in Agriculture” (H. G. Cutler, Ed.), p. 273, Am. Chem. Sot., Washington, 1988. 2. K. C. Vaughn, M. D. Marks, and D. P. Weeks, A dinitroaniline-resistant mutant of Eleusine indim exhibits cross-resistance and supersensitivity to antimicrotubule herbicides and drugs, Plant
Physiol.
83, 956 (1987).
3. F. D. Hess, Herbicide interference with cell division in plants, in “Target Sites of Herbicide Action” (P. Boger and G. Sandmann, Eds.), p. 85, CRC Press, Boca Raton, FL, 1989. 4. J. D. Holmsen and F. D. Hess, Growth inhibition and disruption of mitosis by DCPA in oat (Avena sativa) roots, Weed Sci. 32, 732 (1984). 5. J. D. Holmsen and F. D. Hess, Comparison of the disruption of mitosis and cell plate formation, .I. Exp. ht. 36, 1504 (1985). 6. M. A. Vaughan and K. C. Vaughn, DCPA causes cell plate disruption in wheat roots, Ann. Dot. 65, 379 (1990).
DCPA
EFFECTS
7. L. P. Lehnen, M. A. Vaughan, and K. C. Vaughn, Terbutol affects spindle microtubule organizing centres, J. Exp. Sot. 41, 226 (1990). 8. L. C. Morejohn and D. E. Fosket, Inhibition of plant microtubule polymerization in vitro by the phosphoric amide herbicide amiprophos methyl, Science 224, 874 (1984). 9. L. Clayton and C. W. Lloyd, The relationship between the division plane and spindle geometry in Allium treated with CIPC and griseofulvin:
ON
OAT
ROOTS
57
An anti-tubulin study, Eur. J. Cell Biol. 34, 248 (1984). 10. R. J. Cyr and B. A. Palevitz, Microbutublebinding proteins from carrot I. Initial characterization and microtubule binding, Planta 177, 245 (1989). 11. S. C. Tiwari, S. M. Wick, R. E. Williamson, and B. E. Gunning, Cytoskeleton and integration of cellular function in cells of higher plants, J. Cell Biol. 99s. 63 (1984).
BIOCHEMISTRY
AND
PHYSIOLOGY
lmmunofluorescence
4,
47-57 (191)
and Electron Microscopic DCPA-Treated Oat Roots
Investigations
of
LARRY P. LEHNEN, JR.‘,’ AND KEVIN C. VAUGHN'*~ United States Department of Agriculture, Agricultural Research Service, Southern Weed Science Laboratory, P.O. Box 350, Stoneville, Mississippi 38776 Received September 14, 1990; accepted December 29, 1990 Immunofluorescence and electron microscopy were used to investigate the effects of DCPA (dimethyl tetrachloroterephthalate) on oat roots. Phragmoplast microtubule arrays were the most consistently affected. Multiple phragmoplasts, undulated phragmoplasts, branched phragmoplasts, and phragmoplasts arising at places other than that predicted by the preprophase band were all observed. Cell wall formations resulting from these abnormal phragmoplast configurations are also abnormal and consist of reticulate cell plates, walls formed at abnormal angles, and wall material in net-like arrays containing pockets of cytoplasm. Cortical and preprophase band microtubule arrays were unaffected, and were similar in both treated and control cells. Minispindle and multipolar spindles were observed in some meristematic cells, although many spindle formations were normal or nearly so. Some multiple and highly lobed nuclei were also found as a consequence of these abnormal divisions. Cell plates were abnormal even when there were no nuclear abnormalities that would cause physical disruption of the division plane and even though preprophase bands (which are predictive of the subsequent plane of division) were normal. These data indicate that DCPA has a direct effect on phragmoplast microtubule organizing centers. 8 1991AC&I& Pm, Inc.
INTRODUCTION
tematic survey of these compounds to monitor the steps of mitosis that are altered after treatment. DCPA has been investigated previously by a number of workers but the effects have been mainly described at the light microscopic level. The most extensive studies are those of Holmsen and Hess (4, 5). In a comparison of DCPA with colchicine and CIPC, Holmsen and Hess (5) found that DCPA was the most effective of the three compounds in inducing cell plate abnormalities. Likewise, using light and electron microscopy, Vaughan and Vaughn (6) found that DCPA-induced abnormal cell plates or, in some tissues, the complete absence of cell plates in wheat root tips. These authors (4-6) speculated that DCPA probably causes these effects by disrupting phragmoplast microtubule arrays. In this report, we utilize immunofluorescence microscopy, which more readily detects changes in microtubule profiles, and electron microscopy to determine the effects of DCPA on phragmoplast microtubule and cell plate formation.
Compounds that disrupt cell division are useful in elucidating the processes and proteins that are required to complete mitosis as well as the consequences of deviation from a normal cell division. Many of the compounds utilized to disrupt plant mitosis were originally selected for their ability to disrupt mitosis in animal cells (1). However, compounds that disrupt animal mitosis are much less effective at disrupting mitosis in plant cells (1). For example, almost lOOO-fold more colchicine is required to disrupt mitosis in plant cells than animal cells. A number of herbicides disrupt mitosis and are much more effective at disrupting plant mitosis than the compounds that disrupt animal mitosis (2, 3). We have begun a sys’ Southern Weed Science Laboratory, USDA-ARS, P.O. Box 350, Stoneville, MS 38776. ’ Present address: Plant Cell Biology Group, Australian National University, Canberra, ACT, Australia. ’ To whom correspondence should be addressed. 47
0048-3575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All lights of reproduction in any form reserved.
48
LEHNENANDVAUGHN
MATERIALS
Plant Material
AND
METHODS
and Treatment
Oat seeds were germinated on moistened Whatman No. 1 paper in petri dishes in the dark at room temperature (approximately 22°C). After 3 days of growth, the seedlings were transferred to new petri dishes supplemented with 10-6-10-4 M DCPA for an additional 24 hr in the dark. The DCPA solutions were prepared from a concentrated acetone stock immediately before use. Acetone concentrations did not exceed 0.1% (v/v). Controls were grown in the dilute acetone solution alone. Microscopic
Techniques
Both immunofluorescence and electron microscopy were performed as described previously (7). In the immunofluorescence preparations, DNA was stained with the fluorescent dye DAPI so that the stage of cell division could be monitored. The experiments were repeated at least four times and several root tips from each treatment were examined by immunofluorescence and electron microscopy. RESULTS
Immunojluorescence
Microscopy
Control oat root tips revealed all of the microtubule arrays typical of higher plant cells. Cortical, preprophase, mitotic, and phragmoplast arrays were unaffected, even with a solution of 0.1% (v/v) acetone (Fig. 1). Treatments with lop4 or 10m5 M DCPA were quite consistent, where as lop6 M treatment often resulted in samples that were either affected or not affected. All of the samples shown below are either from lop5 or 10e4 M treatments. Various microtubule configurations were observed after treatment with DCPA. Some cells, especially in interphase and preprophase configurations appeared unaf-
fected and were virtually identical to the control (Fig. 2A). Prometaphase figures were also similar in treated and control preparations. These are made up of many small microtubule arrays which enmesh the mass of chromosomes, gathered into the center of the cell. Similar profiles have been observed in other grass species at prophase or prometaphase. Occasional profiles of later mitotic stages revealed multipolar and/or abnormal spindle orientations (Fig. 2C), but cells with an apparently normal spindle could also be detected in the same preparation (Fig. 2B). In contrast to the largely normal mitotic and interphase microtubule arrays, phragmoplast microtubule arrays are always abnormal in DCPA-treated cells (Fig. 3). Several different phragmoplast morphologies are observed. In some cells, the two nuclei are separated cleanly at early telophase, the phragmoplast is between the two nuclei but the phragmoplast arrays form in a plane not perpendicular to the side walls (Fig. 3A). The presence of abnormal phragmoplast arrays after DCPA is not due to interference of cell plate formation by portions of the nucleus interrupting the phragmoplast as has been noted for other cell disrupters (e.g., Refs. (7, 9)), although this could be a potential reason for phragmoplast disruption in cells where multipolar divisions are noted (Fig. 3B). Rather, an abnormal phragmoplast appears to be a primary effect. In elongated cells that morphologically resemble epidermal cells, not only are abnormal phragmoplasts found but also those observed are found at places other than the center of the cell (Fig. 3C). Multiple phragmoplast arrays are also noted (Fig. 3D). After the completion of mitosis, cortical arrays of microtubules return even though there are abnormal walls and/or multiple nuclei in the cell (Fig. 2D). To further substantiate that even cells with an apparently normal telophase separation of nuclei produce abnormal phragmoplasts in the presence of DCPA, preparations were stained with DAPI to stain DNA in nuclei and with antitubulin to de-
DCPA
EFFECTS
ON
OAT
ROOTS
49
FIG. 1. Immunojluorescence micrographs of control oat root tips. A. Late preprophase band (arrows) with some microtubule associating with the nucleus. B. Metaphase. C. Early anaphase. D. Late telophase with the phragmoplast arrays clearly separated from the two nuclei (IV). X800.
tect the microtubules. In the cell shown in Fig. 4, the nuclei are well separated and appear to be of the normal spherical morphology associated with a telophase nu-
cleus. However, tubulin profiles in this cell reveal an abnormal curve to the phragmoplast separating the nuclei as well as a second band of microtubules oriented perpen-
50
LEHNEN
AND
VAUGHN
FIG. 2. Immunojluorescence micrographs ofDCPA-treated oat root tips. A. Cells with preprophase band (arrows) and cell in interphase (f) with cortical microtubule arrays. B. Near normal spindle formation. C. Cell with three separate spindle formations in a single cell. D. Return of cortical microtubule arrays after a multipolar division. The three nuclei (N) stand out in negative relief. x800.
DCPA
EFFECTS
ON
OAT
ROOTS
formations in DCPA-treated oat root tips as visualized by FIG. 3. Variations in phragmoplast immunofluorescence microscopy. A. Near normal phragmoplast with slight curving of the phragmoplast, especially on the right side of the cell. B. Branched phragmoplast that separates three nuclei (N). C. Phragmopiast(s) oriented in several directions. One band (arrow) is running across the top third of the cell, with another band running perpendicular to that besides the nucleus. The perpendicular band connects to two other bands at the bottom of the cell. D. Multiple phragmoplasts (arrows) in a multinucleate cell. Phragmoplasts in several directions and orientations are observed. x800.
51
FIG. 4. Fluorescence micrographs of DCPA-treated wheat root cell stained to visualize DNA [A) or tub&in (B and C). In this cell, a telophase separation of the two nuclei is clearly seen in A and the nuclear morphology is regular. B and C are micrographs taken at the same plant of focus as in A (B) or in anotherfocus plant (0. Although the nuclei are well separafed, rhe phragmoplast is curved and contains additional microtubules formed at right angles to this phragmoplast. X800.
DCPA
EFFECTS
dicularly to the major phragmoplast array. This second array is far away from any nucleus. Besides this analysis of double-stained preparations, root tips from the same batches used for immunofluorescence were prepared for Fuelgen-staining to monitor mitotic indices and irregularities. In these preparations, greater than 80% of the telophase preparations were well-separated binucleate cells, with less than 20% of the cells in a multipolar mitosis or with two closely appressed nuclei (Lehnen and Vaughn, unpublished). In preparations from these same batches stained for tubulin, virtually all of the phragmoplast arrays exhibited some type of abnormality. Thus, nuclear interference with phragmoplast arrays could explain less than 20% of the
ON
OAT
ROOTS
53
phragmoplast abnormalities but not the 80% which were normally separated. Electron Microscopy As might be expected from the immunofluorescence microscopy shown herein, and the published reports of wall abnormalities described previously in DCPA-treated cells (34, the major ultrastructural effects observed in DCPA oat roots are wall abnormalities. Several kinds of abnormalities are noted. Some walls contain loops of wall material gathered into mesh-like structures, with pockets of cytoplasm trapped between the loops (Fig. 5). Many cells have wall material originating from the parental cell wall but ending bluntly in the cytoplasm rather than connecting to the opposite parental cell wall (Fig. 6). In other cells, the wall is
FIG. 5. Electron micrograph of a DCPA-treated oat root. Irregularly formed walls (arrows) meander through the cytoplasm, capturing pockets of cytoplasm. An additional reticulate wall(r) runs close to the parental cell wall. N, nuclei. Bar, 5 pm.
54
LEHNEN
AND
VAUGHN
FIG. 6. Electron micrograph of a DCPA-treated oat root. A wall(w), originating along the parental cell wall, grows out into the cytoplasm and ends bluntly without connecting to another cell wall. The nucleus (IV’) is irregular in shape with the larger portion containing an inclusion (*). Bar, 2 pm.
reticulate and appears to undulate between the two nuclei that have resulted from a successful separation of chromosomes during mitosis (Fig. 7A). Many of these walls originate far from the midpoint of the cell and often lay parallel to the parental wall and are far away from any nuclear material. Most other cellular structures appeared normal. Normal-appearing amyloplasts, Golgi, the endoplasmic reticulum, and mitochondria were all observed. As might be expected, there are also some apparently abnormal mitotic figures and the resultant lobed or multiple nuclei typical of cell mitotic disrupters (Fig. 7B). Many apparently normal or near-normal nuclear separations are observed (Fig. 7A) and in these cells nuclear morphology is also normal. DISCUSSION
DCPA is unique among the higher plant mitotic disrupters in its greater specificity in altering phragmoplast microtubule ar-
rays. Many herbicides that disrupt mitosis, such as the dinitroaniline or phosphoric amide herbicides (2, 8), inhibit polymerization of tubulin into microtubules so that no microtubules are formed. All microtubule arrays are affected by these herbicides. Carbamate herbicide mitotic disrupters such as propham (IPC) or chlorpropham (CIPC) do not inhibit microtubule polymerization, but rather cause a fragmentation of the mitotic spindle so that many small spindles are produced (3, 7). Phragmoplast arrays in cells treated with carbamate herbicides are often abnormal. However, the phragmoplast abnormalities in these treated cells may be explained by physical interference of parts of the nuclei in the plane of cell division (7,9). Because of this interference, phragmoplasts grow around the regions of nuclei, resulting in branched or undulating phragmoplast arrays (7, 9). DCPA induces abnormal phragmoplast formation, even when the two telophase nuclei are clearly separated (e.g., Figs. 4 and 7A).
FIG. 7. Electron micrographs of DCPA-treated oat roots. A. A new cell plate separates two fairly well-separated nuclei. The plate is reticulate and undulates between the two nuclei. B. An abnormal lobed nucleus with the two major portions connected by a narrow band of nucleoplasm (arrows). An abnormal thickened cell wall (*) is also noted. p, plastid with starch. Bar, 5 km in A and 2 pm in B.
56
LEHNEN
AND VAUGHN
The data herein also support the results of Holmsen and Hess (4, 5) and Vaughan and Vaughn (6) on the mode of action of DCPA. All of these authors noted that DCPA was most effective at disrupting cell wall formation, although other abnormalities are also noted. It is possible that DCPA affects a cellular component, perhaps a microtubule-associated protein (MAP) that has a critical role in the organization of the phragmoplast and a role of lesser importance in other microtubule arrays. Recently, Cyr and Palevitz (11) have described the isolation of MAPS from higher plants and demonstrated that these proteins are involved in microtubule cross-linking and stability. Not all species are sensitive to DCPA (e.g., onion) and it may be possible to identify MAPS associated with DCPA sensitivity by comparing MAPS from sensitive and resistant species. Preprophase bands are believed to predict the plane of the subsequent cell division (11). In DCPA-treated root tips, preprophase bands are apparently normal, although the plane of the subsequent cell division is not. Thus, it is clear that, although the preprophase band is a general indicator of the division plane in control cells, subsequent phragmoplast formation and location may be subsequently disrupted by DCPA. Clayton and Lloyd (9) found normal preprophase bands, followed by the formation of a branched phragmoplast after multipolar division, as a result of CIPC treatment. However, after CIPC treatment, the multiple nuclei that form after a multipolar mitosis often interrupt the plane of pragmoplast formation. Thus, the abnormal phragmoplasts could be the result of steric hindrance rather than a direct effect on the phragmoplast array. DCPA differs from CIPC in that abnormal walls are produced even though the nuclei often do not interfere with the plane of phragmoplast formation. An extreme effect in wheat root tips results in the production of no cell plates in some tissues even though nuclear
divisions are apparently unaffected in these same tissues. Moreover, the presence of phragmoplasts at places far from the plane of a normal cell division and oriented 90” to the plane indicates strongly that DCPA induces phragmoplast abnormalities by direct action on the phragmoplast array. As such, DCPA may serve as a valuable tool in investigating mechanisms of control of phragmoplast orientation as well as the relationship between the preprophase band and phragmoplast location. ACKNOWLEDGMENTS Ms. Ruth H. Jones and Ms. Lynn Libous-Bailey provided excellent technical assistance during the course of these experiments. This work was supported, in part, by USDA Competitive Grant 86CRCR-1933 to K.C.V. Mention of a trademark or product does not constitute endorsement of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
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