Planta (1990)181:269-274
Planta 9 Springer-Verlag1990
Postmitotic 'isodiametric' cell growth in the maize root apex* F. Balu~ka, ~. Kubica, and M. Hauskrecht Institute of Experimental Biologyand Ecology,CBES, SlovakAcademyof Science, Dfibravskfi14, 814 34 Bratislava, Czechoslovakia
Abstract. The onset of rapid cell elongation occurred at different distances from the apex in various tissues of the primary root of maize (Zea mays L.). Furthermore, the comparison of these distances with those determined for the cessation of mitotic divisions revealed a considerable discrepancy. The onset of rapid cell elongation was realized much farther from the root apex than the cessation of cell divisions and therefore a distinct region could be distinguished in every examined maize root tissue. This region was denoted the region of postmitotic 'isodiametric' cell growth. Cells in this region grew in width as well as in length and obtained approximately a square-isodiametric shape. They were also characterized, as are cells in the meristem, by intense nucleic-acid metabolism. This prominent postmitotic 'isodiametric' cell growth was observed in both polyploid and diploid tissues, and indicates that postmitotic 'isodiametric' cell growth, like mitotic division and cell elongation growth, represents an important developmental stage in plant cell ontogeny. Key words: Cell development, g r o w t h - Form factor (cell shape) - Root (cell growth) - Zea (root, cell growth)
Introduction It is widely accepted that plant cells increase their rate of elongation, resulting from extensive vacuolation, immediately after the cessation of mitotic divisions. However several studies indicate that increased cell elongation need not necessarily start simultaneously with the completion of cell division. Cutter and Feldman (1970) observed that non-dividing trichoblasts of Hydroeharis morsus-ranae L. roots continued cytoplasmic growth without conspicuous development of a vacuome. This * The authors dedicate this paper to Dr. M. Luxovfion the occasion of her 65th birthday
growth phase was accompanied by endomitotic synthesis of DNA. Cell elongation, characterized by extensive vacuolar expansion, started only later. Similarly, Cionini et al. (1983) found that chromosome endoreduplication preceded cell elongation growth during the development ,of epidermal cell lines in the first foliage leaf of wheat, and it is clear from results of Demchenko (1984) that the developing metaxylem elements of wheat roots started rapid cell elongation coupled with extensive vacuolation only after two or three endocycles at 8C and 16C DNA contents. That plant cells do not begin accelerated elongation growth simultaneously with the termination of mitotic divisions is supported also by the studies of the influence of several toxic substances on the processes of division and elongation of plant root cells (Ivanov 1987). Ivanov found that the cessation of mitoses and the onset of rapid cell elongation coupled with vacuolation are independent processes. The primary factor which controls growth processes in the distal part of the growth zone in plant roots is the invariable life-span of cells in the meristem. This determines that the onset of rapid cell elongation always occurs after the same time has elapsed from the moment of cell origin, irrespective of the number of mitotic cycles that have occured. The kinetics of cell growth in the maize root were studied by Luxovfi (1980) who found that the transition of meristematic cells to postmitotic cell growth in individual tissues took place at different distances from the root apex. Onset of rapid cell elongation growth did not occur directly after the cessation of cell divisions, and the elongation of cells in this region was slow. Endomitotic DNA synthesis was observed in some tissues of maize primary roots (Balugka 1988). It can be anticipated that, similar to the observations in trichoblasts of Hydrocharis morsus-ranae L., developing epidermal cell lines of the wheat leaf, or the developing metaxylem vessels of the wheat root, an increase in the rate of cell elongation will occur only after completion of the first endomitotic cycles in polyploid tissues of the maize root. Therefore, we have carried out a detailed anatomical analysis of the distal part of the growing zone of maize
270
F. Balu~ka et al. : Postmitotic cell growth in the maize root apex
15.0]
r o o t s w i t h the g o a l o f d e t e r m i n i n g the precise l o c a t i o n , in d i f f e r e n t tissues, o f the o n s e t o f r a p i d cell e l o n g a t i o n a s s o c i a t e d w i t h extensive v a c u o l a t i o n .
125"
Material and methods
10.0-
Primary roots (5 cm long) of Zea mays L. cv. CE-380 seedlings grown at 25 ~ C in darkness were used. Regaud's fixative was used for structural observations (Gray 1954). The suitability of this method of fixation was confirmed by comparing fixed roots with changes in the thickness of living maize roots. Cross-sectional areas of living roots increased in a manner similar to those in paraffin sections, up to the third millimetre from the root-cap junction. During further cell growth, cross-sectional areas of living roots enlarged only slightly (Table 1). Fixation with 5% formaldehyde was used for the detection of starch. After the removal of paraffin, 10-~tm-thick median longitudinal sections from 5-mm-long apical segments were stained with Heidenhain's hematoxylin for structural observations. Starch grains were stained by the periodic acidSchiff (PAS) reaction. After dehydration in a graded ethanol series and xylene, sections were embedded in Canada balsam. The onset of rapid cell elongation was determined in epidermis, hypodermis, cortex (central part), endodermis, pericycle, xylem parenchyma (cells adjoining metaxylem vessels), metaxylem and stelar parenchyma (cells in the central part of the stele). The distances at which cells of these tissues ceased their mitotic activity were as specified earlier (Luxov~ 1980). For determination of the area, the length and the width of cells, together with four cell projections (a, b, a + b , a - b ) an ASBA image analyser (Wild, Heerbrugg, Switzerland) was used. Transverse sections, which characterize the tangential cell dimension, were not observed as, with the exception of the epidermis, the cells of all tissues had an approximately isodiametric shape in transverse sections. Form-factor values were computed by a programme made specially for the ASBA image analyser (Computersysteme Schweizer, Basel, Switzerland). The form factor characterizes the shape of cells (form factor 5.09 for a circular shape, 6 for a square, 7 for a rectangle with a side ratio of 2:1, and 12.4 for a rectangle with a side ratio of 5:1 ; Fig. 1). Cells of individual maize root tissues, from the promeristem up to the region of rapid cell elongation, were measured. The number of measured cells was dependent on the character of the growth process in the individual tissue, which in turn determined the number of cells in a measured section of a given cell file, e.g. in the epidermis the average number of measured cells was greater than 270, in the metaxylem or xylem parenchyma it was less than 150. In all, five roots were examined and the differences in the measured parameters were found to be minimal. The course of curves characterizing lengths and widths of cells as a function of the distance from the root tip was determined by means of a stepwise polynomial regression on a TNS AT (]ZD,
Table 1. Cross-sectional areas of living maize roots at various distances from the apex. Values _-t-SD Distance (mm)
Area ([ttmz)
0.6 1.2 1.8 2.4 3.0 3.6 4.2 4.8 5.4 6.0
514.3 + 34.8 585.9_+53.3 723.9 _+99.5 792.2--+89.2 822.1 -+ 86.0 816.0 _+90.5 819.0-+87.9 816.5-+87.7 824.1 -+ 92.7 830.8+98.4
7.5E u_ 5.0-
2.5-
hl
1:2
1:5
h2 Side ratio
hl
2:1
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Fig. 1. Idealized cell shapes and cell sizes along the growth axis of the maize root apex
Slu~ovice, CSSR) computer with its own software PC-SOFT. Additional calculations were made using the software STATGRAPHICS.
Results
Cell shape. A l l e x a m i n e d tissues were c h a r a c t e r i z e d b y a distinctive course o f c h a n g e s in cell s h a p e w h i c h c o u l d be expressed in terms o f f o r m - f a c t o r values. A n a p p r o x i m a t e l y i s o d i a m e t r i c cell s h a p e was o b s e r v e d at the r o o t tip. C o n s e q u e n t l y , the f o r m f a c t o r was low in this region (Figs. 3b, d; 4 b ; 5 d ; 6b, d). In the case o f the e n d o d e r mis a n d pericycle, this d e v e l o p m e n t a l p h a s e was n o t rec o r d e d as we c o u l d n o t u n a m b i g u o u s l y d i s t i n g u i s h these tissues except at distances g r e a t e r t h a n o f 600 ~tm f r o m the r o o t apex. T h e f o r m f a c t o r i n c r e a s e d d u r i n g the m e r i s t e m a t i c d e v e l o p m e n t a l p h a s e in epidermis, h y p o d e r mis, cortex, e n d o d e r m i s a n d p r o b a b l y also in the pericycle (Figs. 3b, d; 4 b , d; 5b, d; 6b, d). This was caused b y g r o w t h in w i d t h o f the cells a n d by their high r a t e o f m i t o t i c activity. C o n s e q u e n t l y , m e a n cell lengths c h a n g e d o n l y little in spite o f the fact t h a t these cells grew n o t o n l y in w i d t h b u t also in length p r i o r to cell division (Figs. 3a, c; 4 a , c; 5a, c; 6a, c). T h e c e s s a t i o n o f cell division (Luxovfi 1980; Fig. 2) was c o u p l e d with a decrease in the f o r m f a c t o r in these tissues as g r o w t h in cell length was m o r e p r o m i n e n t t h a n g r o w t h in w i d t h a n d , c o n s e q u e n t l y , cells a c q u i r e d a n a p p r o x i m a t e l y s q u a r e ( i s o d i a m e t r i c ) s h a p e (Figs. 3, 4, 5, 6). Values o f the f o r m f a c t o r were very similar, in the r a n g e o f 6.4-6.6, in all e x a m i n e d tissues o f this r o o t region (Figs. 3b, d; 4 b , d; 5b, d; 6 b , d). F a r t h e r f r o m the a p e x the f o r m f a c t o r s t a r t e d to rise a g a i n as a consequence o f intensive cell e l o n g a t i o n (Figs. 3, 4, 5, 6). Like the cessation o f cell division, the increase in the celllength g r a d i e n t p r o c e e d e d centrifugally in the stelar tissues a n d c e n t r i p e t a l l y in the c o r t e x tissues (Fig. 2). Cells o f the x y l e m a n d stelar p a r e n c h y m a , unlike o t h e r tissues,
F. Balugka et al. : Postmitotic cell growth in the maize root apex
271
cortex 100-
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Fig. 4a--d. Dependence on the distance from the maize root tip of changes in length (I) and width (2) (a, e) and form factor (b, d) of cells of the cortex (a, b) and endodermis (c, d). Arrowheads: as for Fig. 3 Fig. 2. The growth pattern in the maize root apex. The crosshatched area represents the meristem (according to Luxovfi 1980), the dotted area represents the zone of postmitotic "isodiametric" cell growth 100.
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hypodermis 100
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Fig. 3a-d. Dependence on the distance from the maize root tip of changes in length (1) and width (2) (a, c) and form factor (b, d) of cells of the epidermis (a, b) and hypodermis (e, d). First arrowhead: termination of mitotic cycle (Luxovfi 1980); second arrowhead: onset of cell elongation growth
were distinguished by small changes in cell width during the period of mitotic and postmitotic 'isodiametric' growth and their form factor started to rise only with the onset of the rapid cell-elongation growth (Figs. 5 c, d; 6a, b). These results clearly show that low form-factor values corresponding to the isodiametric shape of cells were not accompanied by the cessation of mitotic activity. On the contrary, the cessation of cell division was, with the exception of diploid tissues, associated with the first maximum in the curve of form-factor values (Figs. 3b, d; 4b; 6d). The region in which cells ceased to divide while their form factor decreased was denoted as the region of postmitotic 'isodiametric' growth because cells in this region grew in length and width concomitantly, thus becoming approximately square-(isodiametrically) shaped (Figs. 3, 4, 5, 6). Quotation marks are used for the term isodiametric as cells of this region did not grow equally in width and length. A characteristic feature of this region was the intense synthesis of starch in developing metaxylem elements (Fig. 7) and in cortical cells. C e l l w i d t h a n d l e n g t h . The growth in cell width also continued after the cessation of mitotic activity (Luxov~t 1980; Fig. 2) until the point on the curve at which the form factor decreased to its lowest values after the first maximum (Figs. 3, 4, 5, 6). The subsequent increase in the form factor, shown by all cell types, was determined by the high rate of cell elongation while growth in cell width was, compared with the meristematic or the postmitotic 'isodiametric' region, insignificant (Figs. 3, 4, 5,
272
F. Balugka et al. : Postmitotic cell growth in the maize root apex
pert~yc/e
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Fig. 5a-d. Dependence on the distance from the maize root tip of changes in length (1) and width (2) (a, c) and form factor (b, d) of cells of the pericycle (a, b) and stelar parenchyma (c, d). Arrowheads: as for Fig. 3 xylem parenchyma
100a =- 80 60
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/
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bled during their postmitotic 'isodiametric' cell growth (Fig. 6c). By contrast, cells of diploid tissues (endodermis, pericycle, xylem parenchyma, stelar parenchyma) grew in width to a lesser extent (Figs. 4c; 5a, c; 6a). The analysis of cell lengths of individual maize root tissues showed that cells discontinuing mitotic divisions did not immediately embark upon a high rate of cell elongation and at first elongated only slowly. Rapid cell elongation started only at the point of the lowest formfactor values where the growth in cell width ceased (Figs. 3, 4, 5, 6). Cell length diminished in the distal part of the meristem as a consequence of the high rate of cell divisions in individual cell files.
721
Discussion
8040'i'
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~20-
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around the nucleus in developing metaxylem cells (arrow) during their postmitotic 'isodiametric' growth
metaxylem
160- C
Fig. 7. The intense synthesis of starch grains (arrowhead) arranged
9.2]
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0.4 0'.8 1.2
116 2'.0 2.4
0
ols ~io ~is 21o is
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Distance from roo~ apex Imm)
Fig. 6a--d. Dependence on the distance from the maize root tip of changes in length (1) and width (2) (a, c) and form factor (b, d) of cells of the xylem parenehyma (a, b) and metaxylem (c, d). Arrowheads: as for Fig. 3
6). The most prominent growth in width was observed in polyploid tissues, i.e. epidermis, hypodermis, cortex, metaxylem (Balu~ka 1988; Figs. 3a, c; 4a; 6c). For instance, the width of developing metaxylem elements dou-
Our detailed analysis of the shapes, lengths and widths of cells in individual maize root tissues showed that the cessation of mitotic divisions (Luxovfi 1980) was not accompanied by the immediate onset of rapid cell elongation. Cells continued to grow, as in the meristem, not only in length but also in width. Cell lengths, in contrast to cell widths, did not increase in the meristem because of transverse mitotic divisions. On the contrary, in most tissues they decreased at first. Because of this, the value of the form factor of these cells was high as the cells were much wider than they were long. Together with the cessation of mitotic division, the difference between
F. Balu~ka et al. : Postmitotic cell growth in the maize root apex cell length and width became gradually less as a consequence of the higher rate of cell growth in length. The comparison of increases in cell length and width occurring after the cessation of mitotic divisions supports the results of Luxov~t (1980), according to which maize root cells do not enter the phase of rapid cell elongation growth immediately at the termination of the mitotic cycles but start this process later, when cell lengths correspond to cell widths and values of the form factor are at their lowest level (Figs. 3, 4, 5, 6). We can state that a characteristic feature of maize root cells which have ceased to divide but which have not yet started to elongate rapidly is that they grow both in length and width and acquire an approximately square (isodiametric) shape. These observations have led us to postulate a postmitotic ' isodiametric' cell growth phase in the maize root. The cytophotometric-autoradiographical analysis of the distal part of the maize root growth zone has also confirmed the specific character of cells in the region of postmitotic 'isodiametric' cell growth from a metabolic aspect (Balugka 1988; Kubica et al. 1989). While meristematic cells were characterized the intensive incorporation of [~H]uridine, in the region of postmitotic 'isodiametric' cell growth, [3H]uridine incorporation was observed only in tissues with decondensed chromatin in their nuclei (endodermis, pericycle, stelar parenchyma and metaxylem). Synthesis of D N A continued in all polyploid tissues (epidermis, hypodermis, cortex, metaxylem) in the region of postmitotic 'isodiametric' cell growth with the same intensity as in the meristem. The onset of rapid cell elongation was associated with a slowing-down of endomitotic D N A synthesis and the termination of [3H]uridine incorporation in all tissues of the maize root. A specific growth phase occurring between the periods of meristematic and cell-elongation development was also described by Broekaert and Van Parijs (1978) who distinguished three main growth phases during the embryonic development of cotyledons of several members of the Leguminosae. Fertilization was followed by a phase of mitotic divisions. Then a "preparatory" phase for cell elongation occurred during which the D N A content increased through several endocycles. The phase of rapid cell elongation started when the last endocycle began. The first days of postmitotic cell growth, which corresponded to the "preparatory" phase for cell elongation, were, like the mitotic developmental phase, characterized by a high rate of R N A synthesis. The onset of rapid cell elongation was coupled by a decrease of R N A synthesis and a slowing down of D N A synthesis. This resembles the situation in the maize primary root. We suggest that the period of postmitotic "isodiametric" cell growth in maize roots and the "preparatory" phase for cell elongation described in developing cotyledons of Leguminosae represent a common developmental period for all plant cells. In agreement with these observations, which indicate differences between the initial stage of postmitotic growth and genuine cell elongation growth, several authors have recorded a characteristic of the early phases
273 of postmitotic growth that is not found in its later course. Peeters et al. (1987) compared the growth of fibers in three different species and found that the initial stage of cotton fiber development was characterized by the enlargement and vacuolation of nucleoli, processes which are critical in the production of ribosomes (Deltour and DeBarsy 1985). During subsequent cell elongation, nucleolar vacuolation ceased and the nucleoli became smaller. Chaly and Setterfield (1975) observed the incorporation or [3H]cytidine into the rRNA of cells of growing Pisum sativum roots and noted that the large size of the nucleoli, the high rate of R N A synthesis, and the increase in cell width were distinct not only in the meristem but also in the early stages of postmitotic cell growth. The later course of cell elongation growth in pea roots was associated with the termination of R N A synthesis, the reduction of nucleolar size, and the extensive vacuolation of cells which grew in width only slightly. Our results, together with data from the literature, indicate a close correlation between the synthesis of nucleic acids by plant cells and the phase of postmitotic 'isodiametric' growth. In accordance with this, we observed that the most prominent postmitotic 'isodiametric' cell growth occurred in developing metaxylem elements, which intensively synthesized R N A and reached a 32C D N A content at the time of entering the phase of rapid cell elongation (Balugka 1988; Kubica et al. 1989). Other polyploid tissues were also characterized by a more conspicuous phase of postmitotic 'isodiametric' cell growth compared with diploid tissues of the maize root. It seems that a characteristic cell metabolism in the region of postmitotic 'isodiametric' cell growth determines not only the intensity of this specific developmental phase, but also the later cell differentiation. In metaxylem elements and cortical cells, where postmitotic 'isodiametric' cell growth is most prominent, D N A hydrolysis occurs after the completion of growth processes (Balu~ka 1988). By contrast, diploid cells of the pericycle, endodermis, xylem and stelar parenchyma are typified by their inconspicuous postmitotic 'isodiametric' cell growth and preserve the ability to renew mitotic divisions when they form lateral root primordia (Bell and McCully 1970). It is interesting that postmitotic 'isodiametric' cell growth is not a specific feature of polyploid tissues, but is also a feature of diploid tissues. It can be assumed that this phase of growth is, like mitotic divisions or rapid cell elongation, an important developmental period of plant cells. We propose that postmitotic 'isodiametric' cell growth plays a key role in the transition of meristematic cells to rapid elongation. That cell metabolism changes during postmitotic (isodiametric) cell growth is supported by the intensive synthesis of starch in cortical cells and in developing metaxylem elements, which was not observed in meristematic cells. Also, Smith (1973) reported maximum synthesis of starch during the first endocycles in developing cotyledons of pea. It is interesting that starch granules are localized around the nuclei at the time of their synthesis; this was also
274 o b s e r v e d b y S m a r t a n d O ' B r i e n (1983) in scutellar p a r e n c h y m a cells o f the w h e a t e m b r y o . H e l l e b u s t a n d F o r w a r d (1962) e x a m i n e d the a c t i v i t y o f i n v e r t a s e in m a i z e r o o t s a n d f o u n d t h a t the m a x i m u m increase in its activity occ u r r e d in the 2- to 3 - m m r o o t r e g i o n w h i c h c o r r e s p o n d s with the r e g i o n o f p o s t m i t o t i c ' i s o d i a m e t r i c ' cell g r o w t h in m a i z e r o o t s . The question of proper terminology appears relevant in c o n n e c t i o n w i t h o u r results a n d d a t a f r o m the literature. P o s t m i t o t i c p l a n t cell g r o w t h is g e n e r a l l y d e n o t e d as cell e x p a n s i o n o r cell e l o n g a t i o n . C o n s i d e r i n g o u r results, it seems m o r e a p p r o p r i a t e to use the t e r m cell e x p a n s i o n for the p h a s e o f p o s t m i t o t i c ' i s o d i a m e t r i c ' cell g r o w t h . T h e t e r m cell e l o n g a t i o n is a p p r o p r i a t e for the s u b s e q u e n t r a p i d g r o w t h in cell length a n d the extensive d e v e l o p m e n t o f the v a c u o m e .
References Balugka, F. (1988) Character of chromatin structure in individual tissues of the maize primary root. Ph.D. thesis, Slovak Academy of Sciences, Bratislava Bell, J.K., McCully, M. (1970) A histological study of lateral root initiation and development in Zea mays. Protoplasma 70, 179205 Broekaert, D., Van Parijs, R. (1978) The relationship between the endomitotic cell cycle and the enhanced capacity for protein synthesis in Leguminosae embryology. Z. Pflanzenphysiol. 86, 165-175 Chaly, N.M., Setterfield, G. (1975) Organization of the nucleus, nucleolus, and protein-synthesizing apparatus in relation to cell development in roots of Pisum sativum. Can. J. Bot. 53, 200-218 Cionini, P.G., Cavallini, A., Baroncelli, S., Lercari, B., D'Amato, F. (1983) Diploidy and chromosome endoreduplication in the
F. Balu~ka et al. : Postmitotic cell growth in the maize root apex development of epidermal cell lines in the first foliage leaf of durum wheat (Triticum durum Desf.). Protoplasma 118, 38-43 Cutter, E.G., Feldman, L.J. (1970) Trichoblasts in Hydrocharis. II. Nucleic acids, proteins and a consideration of cell growth in relation to endoploidy. Am. J. Bot. 57, 202-211 Deltour, R., DeBarsy, T. (1985) Nucleolar activation and vacuolation in embryo radicle cells during early germination. J. Cell Sci. 76, 67-83 Demchenko, N.P. (1984) Mitotic and endoreduplication cycles in the development of the wheat root metaxylem cell lines. [In Russ.] Tsitologiya 26, 382-391 Gray, P. (1954) Microtomist's formulary and guide. Constable & Co., London Hellebust, J.A., Forward, D.F. (1962) The invertase of the corn radicle and its activity in successive stages of growth. Can. J. Bot. 40, 113-126 Ivanov, V.B. (1987) Root growth tests as a tool for screening toxic substances and the elucidation of the mode of their action. 3rd Int. Symp. Structure and function of roots, Abstr., Nitra, Czechoslovakia, p. 58 Kubica, S., Balu~ka, F., Ga~parlkov/t, O. (1989) Pattern of nucleic acids synthesis in the root apex of Zea mays L. Biol6gia (Bratislava) 44, 201-207 Luxov~, M. (1980) Kinetics of maize root growth. [In Ukr.] Ukr. Zh. Bot. 37, 68-72 Peeters, M.-C., Voets, S., Dayatilake, G., De Langhe, E. (1987) Nucleolar size at early stages of cotton fiber development in relation to final fiber dimension. Physiol. Plant. 71,436-440 Smart, M.G., O'Brien, T.P. (1983) The development of the wheat embryo in relation to the neighbouring tissues. Protoplasma 114, 1-13 Smith, D.L. (1973) Nucleic acid, protein and starch synthesis in developing cotyledons of Pisum arvense L. Ann. Bot. 37, 7958O4
Received 6 April; accepted 28 August 1989
Planta 9 Springer-Verlag1990
Postmitotic 'isodiametric' cell growth in the maize root apex* F. Balu~ka, ~. Kubica, and M. Hauskrecht Institute of Experimental Biologyand Ecology,CBES, SlovakAcademyof Science, Dfibravskfi14, 814 34 Bratislava, Czechoslovakia
Abstract. The onset of rapid cell elongation occurred at different distances from the apex in various tissues of the primary root of maize (Zea mays L.). Furthermore, the comparison of these distances with those determined for the cessation of mitotic divisions revealed a considerable discrepancy. The onset of rapid cell elongation was realized much farther from the root apex than the cessation of cell divisions and therefore a distinct region could be distinguished in every examined maize root tissue. This region was denoted the region of postmitotic 'isodiametric' cell growth. Cells in this region grew in width as well as in length and obtained approximately a square-isodiametric shape. They were also characterized, as are cells in the meristem, by intense nucleic-acid metabolism. This prominent postmitotic 'isodiametric' cell growth was observed in both polyploid and diploid tissues, and indicates that postmitotic 'isodiametric' cell growth, like mitotic division and cell elongation growth, represents an important developmental stage in plant cell ontogeny. Key words: Cell development, g r o w t h - Form factor (cell shape) - Root (cell growth) - Zea (root, cell growth)
Introduction It is widely accepted that plant cells increase their rate of elongation, resulting from extensive vacuolation, immediately after the cessation of mitotic divisions. However several studies indicate that increased cell elongation need not necessarily start simultaneously with the completion of cell division. Cutter and Feldman (1970) observed that non-dividing trichoblasts of Hydroeharis morsus-ranae L. roots continued cytoplasmic growth without conspicuous development of a vacuome. This * The authors dedicate this paper to Dr. M. Luxovfion the occasion of her 65th birthday
growth phase was accompanied by endomitotic synthesis of DNA. Cell elongation, characterized by extensive vacuolar expansion, started only later. Similarly, Cionini et al. (1983) found that chromosome endoreduplication preceded cell elongation growth during the development ,of epidermal cell lines in the first foliage leaf of wheat, and it is clear from results of Demchenko (1984) that the developing metaxylem elements of wheat roots started rapid cell elongation coupled with extensive vacuolation only after two or three endocycles at 8C and 16C DNA contents. That plant cells do not begin accelerated elongation growth simultaneously with the termination of mitotic divisions is supported also by the studies of the influence of several toxic substances on the processes of division and elongation of plant root cells (Ivanov 1987). Ivanov found that the cessation of mitoses and the onset of rapid cell elongation coupled with vacuolation are independent processes. The primary factor which controls growth processes in the distal part of the growth zone in plant roots is the invariable life-span of cells in the meristem. This determines that the onset of rapid cell elongation always occurs after the same time has elapsed from the moment of cell origin, irrespective of the number of mitotic cycles that have occured. The kinetics of cell growth in the maize root were studied by Luxovfi (1980) who found that the transition of meristematic cells to postmitotic cell growth in individual tissues took place at different distances from the root apex. Onset of rapid cell elongation growth did not occur directly after the cessation of cell divisions, and the elongation of cells in this region was slow. Endomitotic DNA synthesis was observed in some tissues of maize primary roots (Balugka 1988). It can be anticipated that, similar to the observations in trichoblasts of Hydrocharis morsus-ranae L., developing epidermal cell lines of the wheat leaf, or the developing metaxylem vessels of the wheat root, an increase in the rate of cell elongation will occur only after completion of the first endomitotic cycles in polyploid tissues of the maize root. Therefore, we have carried out a detailed anatomical analysis of the distal part of the growing zone of maize
270
F. Balu~ka et al. : Postmitotic cell growth in the maize root apex
15.0]
r o o t s w i t h the g o a l o f d e t e r m i n i n g the precise l o c a t i o n , in d i f f e r e n t tissues, o f the o n s e t o f r a p i d cell e l o n g a t i o n a s s o c i a t e d w i t h extensive v a c u o l a t i o n .
125"
Material and methods
10.0-
Primary roots (5 cm long) of Zea mays L. cv. CE-380 seedlings grown at 25 ~ C in darkness were used. Regaud's fixative was used for structural observations (Gray 1954). The suitability of this method of fixation was confirmed by comparing fixed roots with changes in the thickness of living maize roots. Cross-sectional areas of living roots increased in a manner similar to those in paraffin sections, up to the third millimetre from the root-cap junction. During further cell growth, cross-sectional areas of living roots enlarged only slightly (Table 1). Fixation with 5% formaldehyde was used for the detection of starch. After the removal of paraffin, 10-~tm-thick median longitudinal sections from 5-mm-long apical segments were stained with Heidenhain's hematoxylin for structural observations. Starch grains were stained by the periodic acidSchiff (PAS) reaction. After dehydration in a graded ethanol series and xylene, sections were embedded in Canada balsam. The onset of rapid cell elongation was determined in epidermis, hypodermis, cortex (central part), endodermis, pericycle, xylem parenchyma (cells adjoining metaxylem vessels), metaxylem and stelar parenchyma (cells in the central part of the stele). The distances at which cells of these tissues ceased their mitotic activity were as specified earlier (Luxov~ 1980). For determination of the area, the length and the width of cells, together with four cell projections (a, b, a + b , a - b ) an ASBA image analyser (Wild, Heerbrugg, Switzerland) was used. Transverse sections, which characterize the tangential cell dimension, were not observed as, with the exception of the epidermis, the cells of all tissues had an approximately isodiametric shape in transverse sections. Form-factor values were computed by a programme made specially for the ASBA image analyser (Computersysteme Schweizer, Basel, Switzerland). The form factor characterizes the shape of cells (form factor 5.09 for a circular shape, 6 for a square, 7 for a rectangle with a side ratio of 2:1, and 12.4 for a rectangle with a side ratio of 5:1 ; Fig. 1). Cells of individual maize root tissues, from the promeristem up to the region of rapid cell elongation, were measured. The number of measured cells was dependent on the character of the growth process in the individual tissue, which in turn determined the number of cells in a measured section of a given cell file, e.g. in the epidermis the average number of measured cells was greater than 270, in the metaxylem or xylem parenchyma it was less than 150. In all, five roots were examined and the differences in the measured parameters were found to be minimal. The course of curves characterizing lengths and widths of cells as a function of the distance from the root tip was determined by means of a stepwise polynomial regression on a TNS AT (]ZD,
Table 1. Cross-sectional areas of living maize roots at various distances from the apex. Values _-t-SD Distance (mm)
Area ([ttmz)
0.6 1.2 1.8 2.4 3.0 3.6 4.2 4.8 5.4 6.0
514.3 + 34.8 585.9_+53.3 723.9 _+99.5 792.2--+89.2 822.1 -+ 86.0 816.0 _+90.5 819.0-+87.9 816.5-+87.7 824.1 -+ 92.7 830.8+98.4
7.5E u_ 5.0-
2.5-
hl
1:2
1:5
h2 Side ratio
hl
2:1
5:1
Fig. 1. Idealized cell shapes and cell sizes along the growth axis of the maize root apex
Slu~ovice, CSSR) computer with its own software PC-SOFT. Additional calculations were made using the software STATGRAPHICS.
Results
Cell shape. A l l e x a m i n e d tissues were c h a r a c t e r i z e d b y a distinctive course o f c h a n g e s in cell s h a p e w h i c h c o u l d be expressed in terms o f f o r m - f a c t o r values. A n a p p r o x i m a t e l y i s o d i a m e t r i c cell s h a p e was o b s e r v e d at the r o o t tip. C o n s e q u e n t l y , the f o r m f a c t o r was low in this region (Figs. 3b, d; 4 b ; 5 d ; 6b, d). In the case o f the e n d o d e r mis a n d pericycle, this d e v e l o p m e n t a l p h a s e was n o t rec o r d e d as we c o u l d n o t u n a m b i g u o u s l y d i s t i n g u i s h these tissues except at distances g r e a t e r t h a n o f 600 ~tm f r o m the r o o t apex. T h e f o r m f a c t o r i n c r e a s e d d u r i n g the m e r i s t e m a t i c d e v e l o p m e n t a l p h a s e in epidermis, h y p o d e r mis, cortex, e n d o d e r m i s a n d p r o b a b l y also in the pericycle (Figs. 3b, d; 4 b , d; 5b, d; 6b, d). This was caused b y g r o w t h in w i d t h o f the cells a n d by their high r a t e o f m i t o t i c activity. C o n s e q u e n t l y , m e a n cell lengths c h a n g e d o n l y little in spite o f the fact t h a t these cells grew n o t o n l y in w i d t h b u t also in length p r i o r to cell division (Figs. 3a, c; 4 a , c; 5a, c; 6a, c). T h e c e s s a t i o n o f cell division (Luxovfi 1980; Fig. 2) was c o u p l e d with a decrease in the f o r m f a c t o r in these tissues as g r o w t h in cell length was m o r e p r o m i n e n t t h a n g r o w t h in w i d t h a n d , c o n s e q u e n t l y , cells a c q u i r e d a n a p p r o x i m a t e l y s q u a r e ( i s o d i a m e t r i c ) s h a p e (Figs. 3, 4, 5, 6). Values o f the f o r m f a c t o r were very similar, in the r a n g e o f 6.4-6.6, in all e x a m i n e d tissues o f this r o o t region (Figs. 3b, d; 4 b , d; 5b, d; 6 b , d). F a r t h e r f r o m the a p e x the f o r m f a c t o r s t a r t e d to rise a g a i n as a consequence o f intensive cell e l o n g a t i o n (Figs. 3, 4, 5, 6). Like the cessation o f cell division, the increase in the celllength g r a d i e n t p r o c e e d e d centrifugally in the stelar tissues a n d c e n t r i p e t a l l y in the c o r t e x tissues (Fig. 2). Cells o f the x y l e m a n d stelar p a r e n c h y m a , unlike o t h e r tissues,
F. Balugka et al. : Postmitotic cell growth in the maize root apex
271
cortex 100-
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Fig. 4a--d. Dependence on the distance from the maize root tip of changes in length (I) and width (2) (a, e) and form factor (b, d) of cells of the cortex (a, b) and endodermis (c, d). Arrowheads: as for Fig. 3 Fig. 2. The growth pattern in the maize root apex. The crosshatched area represents the meristem (according to Luxovfi 1980), the dotted area represents the zone of postmitotic "isodiametric" cell growth 100.
:~
epidermis
hypodermis 100
80.
80.
60.
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40"
r
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LL
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Disfance from roof apex (ram}
Fig. 3a-d. Dependence on the distance from the maize root tip of changes in length (1) and width (2) (a, c) and form factor (b, d) of cells of the epidermis (a, b) and hypodermis (e, d). First arrowhead: termination of mitotic cycle (Luxovfi 1980); second arrowhead: onset of cell elongation growth
were distinguished by small changes in cell width during the period of mitotic and postmitotic 'isodiametric' growth and their form factor started to rise only with the onset of the rapid cell-elongation growth (Figs. 5 c, d; 6a, b). These results clearly show that low form-factor values corresponding to the isodiametric shape of cells were not accompanied by the cessation of mitotic activity. On the contrary, the cessation of cell division was, with the exception of diploid tissues, associated with the first maximum in the curve of form-factor values (Figs. 3b, d; 4b; 6d). The region in which cells ceased to divide while their form factor decreased was denoted as the region of postmitotic 'isodiametric' growth because cells in this region grew in length and width concomitantly, thus becoming approximately square-(isodiametrically) shaped (Figs. 3, 4, 5, 6). Quotation marks are used for the term isodiametric as cells of this region did not grow equally in width and length. A characteristic feature of this region was the intense synthesis of starch in developing metaxylem elements (Fig. 7) and in cortical cells. C e l l w i d t h a n d l e n g t h . The growth in cell width also continued after the cessation of mitotic activity (Luxov~t 1980; Fig. 2) until the point on the curve at which the form factor decreased to its lowest values after the first maximum (Figs. 3, 4, 5, 6). The subsequent increase in the form factor, shown by all cell types, was determined by the high rate of cell elongation while growth in cell width was, compared with the meristematic or the postmitotic 'isodiametric' region, insignificant (Figs. 3, 4, 5,
272
F. Balugka et al. : Postmitotic cell growth in the maize root apex
pert~yc/e
1003
stelar parenchyma
100,
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6.5 .! 3 0 0'.5 I.'0 115 2'.0 2'.5 3.0 Oisfance from roof apex (mm)
Fig. 5a-d. Dependence on the distance from the maize root tip of changes in length (1) and width (2) (a, c) and form factor (b, d) of cells of the pericycle (a, b) and stelar parenchyma (c, d). Arrowheads: as for Fig. 3 xylem parenchyma
100a =- 80 60
2O
/
2007
B~4
bled during their postmitotic 'isodiametric' cell growth (Fig. 6c). By contrast, cells of diploid tissues (endodermis, pericycle, xylem parenchyma, stelar parenchyma) grew in width to a lesser extent (Figs. 4c; 5a, c; 6a). The analysis of cell lengths of individual maize root tissues showed that cells discontinuing mitotic divisions did not immediately embark upon a high rate of cell elongation and at first elongated only slowly. Rapid cell elongation started only at the point of the lowest formfactor values where the growth in cell width ceased (Figs. 3, 4, 5, 6). Cell length diminished in the distal part of the meristem as a consequence of the high rate of cell divisions in individual cell files.
721
Discussion
8040'i'
10,21
2118-
u_
I
~20-
0
g
around the nucleus in developing metaxylem cells (arrow) during their postmitotic 'isodiametric' growth
metaxylem
160- C
Fig. 7. The intense synthesis of starch grains (arrowhead) arranged
9.2]
I512.
0.4 0'.8 1.2
116 2'.0 2.4
0
ols ~io ~is 21o is
3.0
Distance from roo~ apex Imm)
Fig. 6a--d. Dependence on the distance from the maize root tip of changes in length (1) and width (2) (a, c) and form factor (b, d) of cells of the xylem parenehyma (a, b) and metaxylem (c, d). Arrowheads: as for Fig. 3
6). The most prominent growth in width was observed in polyploid tissues, i.e. epidermis, hypodermis, cortex, metaxylem (Balu~ka 1988; Figs. 3a, c; 4a; 6c). For instance, the width of developing metaxylem elements dou-
Our detailed analysis of the shapes, lengths and widths of cells in individual maize root tissues showed that the cessation of mitotic divisions (Luxovfi 1980) was not accompanied by the immediate onset of rapid cell elongation. Cells continued to grow, as in the meristem, not only in length but also in width. Cell lengths, in contrast to cell widths, did not increase in the meristem because of transverse mitotic divisions. On the contrary, in most tissues they decreased at first. Because of this, the value of the form factor of these cells was high as the cells were much wider than they were long. Together with the cessation of mitotic division, the difference between
F. Balu~ka et al. : Postmitotic cell growth in the maize root apex cell length and width became gradually less as a consequence of the higher rate of cell growth in length. The comparison of increases in cell length and width occurring after the cessation of mitotic divisions supports the results of Luxov~t (1980), according to which maize root cells do not enter the phase of rapid cell elongation growth immediately at the termination of the mitotic cycles but start this process later, when cell lengths correspond to cell widths and values of the form factor are at their lowest level (Figs. 3, 4, 5, 6). We can state that a characteristic feature of maize root cells which have ceased to divide but which have not yet started to elongate rapidly is that they grow both in length and width and acquire an approximately square (isodiametric) shape. These observations have led us to postulate a postmitotic ' isodiametric' cell growth phase in the maize root. The cytophotometric-autoradiographical analysis of the distal part of the maize root growth zone has also confirmed the specific character of cells in the region of postmitotic 'isodiametric' cell growth from a metabolic aspect (Balugka 1988; Kubica et al. 1989). While meristematic cells were characterized the intensive incorporation of [~H]uridine, in the region of postmitotic 'isodiametric' cell growth, [3H]uridine incorporation was observed only in tissues with decondensed chromatin in their nuclei (endodermis, pericycle, stelar parenchyma and metaxylem). Synthesis of D N A continued in all polyploid tissues (epidermis, hypodermis, cortex, metaxylem) in the region of postmitotic 'isodiametric' cell growth with the same intensity as in the meristem. The onset of rapid cell elongation was associated with a slowing-down of endomitotic D N A synthesis and the termination of [3H]uridine incorporation in all tissues of the maize root. A specific growth phase occurring between the periods of meristematic and cell-elongation development was also described by Broekaert and Van Parijs (1978) who distinguished three main growth phases during the embryonic development of cotyledons of several members of the Leguminosae. Fertilization was followed by a phase of mitotic divisions. Then a "preparatory" phase for cell elongation occurred during which the D N A content increased through several endocycles. The phase of rapid cell elongation started when the last endocycle began. The first days of postmitotic cell growth, which corresponded to the "preparatory" phase for cell elongation, were, like the mitotic developmental phase, characterized by a high rate of R N A synthesis. The onset of rapid cell elongation was coupled by a decrease of R N A synthesis and a slowing down of D N A synthesis. This resembles the situation in the maize primary root. We suggest that the period of postmitotic "isodiametric" cell growth in maize roots and the "preparatory" phase for cell elongation described in developing cotyledons of Leguminosae represent a common developmental period for all plant cells. In agreement with these observations, which indicate differences between the initial stage of postmitotic growth and genuine cell elongation growth, several authors have recorded a characteristic of the early phases
273 of postmitotic growth that is not found in its later course. Peeters et al. (1987) compared the growth of fibers in three different species and found that the initial stage of cotton fiber development was characterized by the enlargement and vacuolation of nucleoli, processes which are critical in the production of ribosomes (Deltour and DeBarsy 1985). During subsequent cell elongation, nucleolar vacuolation ceased and the nucleoli became smaller. Chaly and Setterfield (1975) observed the incorporation or [3H]cytidine into the rRNA of cells of growing Pisum sativum roots and noted that the large size of the nucleoli, the high rate of R N A synthesis, and the increase in cell width were distinct not only in the meristem but also in the early stages of postmitotic cell growth. The later course of cell elongation growth in pea roots was associated with the termination of R N A synthesis, the reduction of nucleolar size, and the extensive vacuolation of cells which grew in width only slightly. Our results, together with data from the literature, indicate a close correlation between the synthesis of nucleic acids by plant cells and the phase of postmitotic 'isodiametric' growth. In accordance with this, we observed that the most prominent postmitotic 'isodiametric' cell growth occurred in developing metaxylem elements, which intensively synthesized R N A and reached a 32C D N A content at the time of entering the phase of rapid cell elongation (Balugka 1988; Kubica et al. 1989). Other polyploid tissues were also characterized by a more conspicuous phase of postmitotic 'isodiametric' cell growth compared with diploid tissues of the maize root. It seems that a characteristic cell metabolism in the region of postmitotic 'isodiametric' cell growth determines not only the intensity of this specific developmental phase, but also the later cell differentiation. In metaxylem elements and cortical cells, where postmitotic 'isodiametric' cell growth is most prominent, D N A hydrolysis occurs after the completion of growth processes (Balu~ka 1988). By contrast, diploid cells of the pericycle, endodermis, xylem and stelar parenchyma are typified by their inconspicuous postmitotic 'isodiametric' cell growth and preserve the ability to renew mitotic divisions when they form lateral root primordia (Bell and McCully 1970). It is interesting that postmitotic 'isodiametric' cell growth is not a specific feature of polyploid tissues, but is also a feature of diploid tissues. It can be assumed that this phase of growth is, like mitotic divisions or rapid cell elongation, an important developmental period of plant cells. We propose that postmitotic 'isodiametric' cell growth plays a key role in the transition of meristematic cells to rapid elongation. That cell metabolism changes during postmitotic (isodiametric) cell growth is supported by the intensive synthesis of starch in cortical cells and in developing metaxylem elements, which was not observed in meristematic cells. Also, Smith (1973) reported maximum synthesis of starch during the first endocycles in developing cotyledons of pea. It is interesting that starch granules are localized around the nuclei at the time of their synthesis; this was also
274 o b s e r v e d b y S m a r t a n d O ' B r i e n (1983) in scutellar p a r e n c h y m a cells o f the w h e a t e m b r y o . H e l l e b u s t a n d F o r w a r d (1962) e x a m i n e d the a c t i v i t y o f i n v e r t a s e in m a i z e r o o t s a n d f o u n d t h a t the m a x i m u m increase in its activity occ u r r e d in the 2- to 3 - m m r o o t r e g i o n w h i c h c o r r e s p o n d s with the r e g i o n o f p o s t m i t o t i c ' i s o d i a m e t r i c ' cell g r o w t h in m a i z e r o o t s . The question of proper terminology appears relevant in c o n n e c t i o n w i t h o u r results a n d d a t a f r o m the literature. P o s t m i t o t i c p l a n t cell g r o w t h is g e n e r a l l y d e n o t e d as cell e x p a n s i o n o r cell e l o n g a t i o n . C o n s i d e r i n g o u r results, it seems m o r e a p p r o p r i a t e to use the t e r m cell e x p a n s i o n for the p h a s e o f p o s t m i t o t i c ' i s o d i a m e t r i c ' cell g r o w t h . T h e t e r m cell e l o n g a t i o n is a p p r o p r i a t e for the s u b s e q u e n t r a p i d g r o w t h in cell length a n d the extensive d e v e l o p m e n t o f the v a c u o m e .
References Balugka, F. (1988) Character of chromatin structure in individual tissues of the maize primary root. Ph.D. thesis, Slovak Academy of Sciences, Bratislava Bell, J.K., McCully, M. (1970) A histological study of lateral root initiation and development in Zea mays. Protoplasma 70, 179205 Broekaert, D., Van Parijs, R. (1978) The relationship between the endomitotic cell cycle and the enhanced capacity for protein synthesis in Leguminosae embryology. Z. Pflanzenphysiol. 86, 165-175 Chaly, N.M., Setterfield, G. (1975) Organization of the nucleus, nucleolus, and protein-synthesizing apparatus in relation to cell development in roots of Pisum sativum. Can. J. Bot. 53, 200-218 Cionini, P.G., Cavallini, A., Baroncelli, S., Lercari, B., D'Amato, F. (1983) Diploidy and chromosome endoreduplication in the
F. Balu~ka et al. : Postmitotic cell growth in the maize root apex development of epidermal cell lines in the first foliage leaf of durum wheat (Triticum durum Desf.). Protoplasma 118, 38-43 Cutter, E.G., Feldman, L.J. (1970) Trichoblasts in Hydrocharis. II. Nucleic acids, proteins and a consideration of cell growth in relation to endoploidy. Am. J. Bot. 57, 202-211 Deltour, R., DeBarsy, T. (1985) Nucleolar activation and vacuolation in embryo radicle cells during early germination. J. Cell Sci. 76, 67-83 Demchenko, N.P. (1984) Mitotic and endoreduplication cycles in the development of the wheat root metaxylem cell lines. [In Russ.] Tsitologiya 26, 382-391 Gray, P. (1954) Microtomist's formulary and guide. Constable & Co., London Hellebust, J.A., Forward, D.F. (1962) The invertase of the corn radicle and its activity in successive stages of growth. Can. J. Bot. 40, 113-126 Ivanov, V.B. (1987) Root growth tests as a tool for screening toxic substances and the elucidation of the mode of their action. 3rd Int. Symp. Structure and function of roots, Abstr., Nitra, Czechoslovakia, p. 58 Kubica, S., Balu~ka, F., Ga~parlkov/t, O. (1989) Pattern of nucleic acids synthesis in the root apex of Zea mays L. Biol6gia (Bratislava) 44, 201-207 Luxov~, M. (1980) Kinetics of maize root growth. [In Ukr.] Ukr. Zh. Bot. 37, 68-72 Peeters, M.-C., Voets, S., Dayatilake, G., De Langhe, E. (1987) Nucleolar size at early stages of cotton fiber development in relation to final fiber dimension. Physiol. Plant. 71,436-440 Smart, M.G., O'Brien, T.P. (1983) The development of the wheat embryo in relation to the neighbouring tissues. Protoplasma 114, 1-13 Smith, D.L. (1973) Nucleic acid, protein and starch synthesis in developing cotyledons of Pisum arvense L. Ann. Bot. 37, 7958O4
Received 6 April; accepted 28 August 1989
