Plant Cell Reports (1981) 1 : 23-25

Plant Cell Reports © Springer-Verlag 1981

DNA Replication During Differentiation of Cortical Cells in Allium Roots M. J. C a r m o n a and C. G u t b r r e z 1 Instituto de Biologia Celular (C.S.I.C.), Vel~zquez, 144, Madrid-6, Spain Received March 25, 1981

ABSTRACT In the elongation zone of A l l i u m roots a certain f r a c t i o n of cortical cells u n d e r g o DNA r e p l i c a t i o n d u r i n g t r a n s i t i o n from newborn n o n - p r o l i f e r a t i v e cells to m a t u r e cells. The percentage of cortical cells in the mature zone w i t h DNA c o n t e n t 2C (G01) and 4C (G02)is r o u g h l y 60% and 40%, r e s p e c t i v e ly. We p r o p o s e that a replication signal whose concentration d e c r e a s e s the farther you go from the root tip, along the root axis, and which acts on cells after their last p a r t i t i o n i n g , could a c c o u n t for the observed d i s t r i b u t i o n of mature cortical n u c l e i w i t h d i f f e r e n t DNA contents. INTRODUCTION A root g r o w i n g under fixed c o n d i t i o n s can achieve a c o n s t a n t rate of g r o w t h w h e n both g r o w t h components, namely, cell production and cell elongation, reach constant values ( Dfez et al. 1970 ) . Under these c i r c u m stances, cells also reach a c o n s t a n t size at the end of the e l o n g a t i o n z o n e . P r o l i f e r ating cells in the m e r i s t e m b e c o m e mature cells t h r o u g h a transition period, w h i c h implies changes in their metabolic and s t r u c t u r a l p a t t e r n s as well as in size. M o r e over, it has b e e n shown that DNA levels in m a t u r e cells from several plant species are not the same. For instance, m o s t of the mature cells in several species showed a 2C DNA level, w h e r e a s the same cell type in other species exhibited a 4C DNA level (McLeish 1959; M c L e i s h and S u n d e r l a n d 1961; Brunori 1971; Evans and V a n ' t Hof 1974). In the p r e s e n t report, we have o b s e r v e d a high rate of labelled cells after a pulse w i t h ~ 3 ] t h y m i d i n e in the e l o n g a t i o n zone of A l l i u m cepa roots ( w h e r e no cell p r o l i f e r ation occurs), and we have i n v e s t i g a t e d this p h e n o m e n o n by a n a l y z i n g the DNA c o n t e n t in non-replicating cortical cells t h r o u g h o u t the e l o n g a t i o n zone. The results suggest that the site along the root axis w h e r e the last cell partitioning occurs might be important in determining the final DNA c o n t e n t of the m a t u r e c e l l s , l i k e l y , t h r o u g h 1

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a " r e p l i c a t i o n signal" whose c o n c e n t r a t i o n d e c r e a s e s u n i f o r m l y from the root apex along the root axis. MATERIAL

AND ~VIETHODS

Root m e r i s t e m s of A l l i u m cepa L., grown in the dark ( 15 ! 0.5°C T in tap water with constant aeration, were used. All the e x p e r i m e n t s w e r e c a r r i e d out after the growing roots r e a c h e d s t e a d y - s t a t e kinetics. The bulbs were labelled w i t h a 20 ~ C i / m l pulse of ~ 3 ] t h y m i d i n e (21 Ci/mmol) for 30n/n, and then the roots w e r e i m m e d i a t e l y d e t a c h e d from the bulbs and t r a n s f e r r e d to an ethanol/ acetic acid (3:1) fixative solution for 1.5 h. A f t e r F e u l g e n s t a i n i n g of the roots, squashes w e r e p r e p a r e d from the m e r i s t e m and from 1 mm segments located 3, 6, and 9 mm from the root apex in the differentiation zone of each root. They w e r e p r o c e s s e d for autoradiography w i t h Kodak AR-10 s t r i p p i n g film, then e x p o s e d for f~ree weeks, d e v e l o p e d with K o d a k D i0 developer and fixed w i t h Kodak u l t r a r a p i d acid fixer. The Mak m e t h o d (Mak 1965), w h i c h c o m b i n e d a u t o r a d i o g r a p h y and c y t o p h o t o m e t r y of u n l a b e l l e d i n t e r p h a s e nuclei, was used to d e t e r m i n e the n u m b e r of nuclei s h o w i n g d i f f e r e n t D N A c o n t e n t s . Cytop h o t o m e t r i c a l m e a s u r e m e n t s were made with an M85 S c a n n i n g M i c r o d e n s i t o m e t e r (Vickers Ltd). The readings on a w a v e l e n g t h of 550 nm w e r e taken w i t h masks, leaving a v i s u a l field of 10-30 ~ m in diameter, a slit w i d t h of 30 nm (i.e.,--wavelengths from 520-580 n m ) and a r e a d i n g spot 0.4 ~ m in diameter. RESULTS

AND DISCUSSION

The l o n g i t u d i n a l d i m e n s i o n of the e l o n g a t i o n zone at 15°C was d e t e r m i n e d by a n a l y z i n g cell size v a r i a t i o n s in cortical a n d m e t a x i l e m a t i c cells along the root axis. This zone extends from the end of t h e m e r i s t e m (about 2 mm from the root apex) to the point along the root axis w h e r e the cell size reaches a c o n s t a n t value ( 9 m m f r o m t h e root apex) (Carmona 1979)~ In order to estimate the p r o p o r t i o n of cells in GI, S, G 2 and mitosis in each m e r i s t e m p o p u l a t i o n we have i n t e g r a t e d the data o b t a i n e d from c y t o p h o t o m e t r y , the label-

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F r e q u e n c y d i s t r i b u t i o n of cortical nuclei w i t h d i f f e r e n t D N A c o n t e n t a t several d i s t a n c e s from the root tip

ling index and the m i t o t i c index (Mak 1965) in each case from the same s q u a s h c o v e r e d w i t h stripping film. Similirly, t h r o u g h o u t the e l o n g a t i o n zone of the root the same parameters were determined. However, since cells have finished p r o l i f e r a t i o n in this zone and no m i t o s i s occurs at all, 2C and 4C cells have been c o n s i d e r e d as G01 a n d G 0 2 , respectively. Fig. 1 shows the d i s t r i b u t i o n of n u c l e i with d i f f e r e n t DNA content, as d e t e r m i n e d by c y t o p h o t o m e t r y of u n l a b e l l e d interphase cortical nuclei, a t s e l e c t e d d i s t a n c e s from the rot tip. As w o u l d be expected, the m e r i s t e m p o p u l a t i o n of U n l a b e l l e d i n t e r p h a s e nuclei is c l e a r l y s e p a r a t e d into two groups: the 2C (GI) and the 4C (G2) cells, each of t h e m w i t h a n a r r o w range of values. On the other hand, this d i s t r i b u t i o n changes p r o gressively throughout the e l o n g a t i o n zone both in the n u m b e r of DNA content i n t e r v a l s and in the f r e q u e n c y of cells in them, in such a w a y that cells w i t h i n t e r m e d i a t e DNA c o n t e n t b e t w e e n 2C and 4C, are in the more d i s t a n t segments from the root apex. Moreover, the fact that A l l i u m cells possess a high DNA c o n t e n t (Sparrow et ai. 1972) and that no cells w i t h higher DNA levels have been found, seems to indicate that the occurrence of e n d o p o l y p l o i d y is r e s t r i c t e d to species w i t h a low basic n u c l e a r DNA c o n t e n t (Nagl 1976). the

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Cumulative frequency of cortical cells in GI, S, G2 and m i t o s i s in the m e r i s t e m and of G01 , S and G02 at several distances along the e l o n g a t i o n zone

are p r e s e n t e d in Fig. 2 w h e r e we can see a s t r i k i n g r e d u c t i o n in the n u m b e r of labelled cells as they go through the e l o n g a t i o n zone. I n the 3 m m - s e g m e n t from the root tip, w h i c h is already n o n - m e r i s t e m a t i c , t h e i n c r e a s e in the f r e q u e n c y of G01 nuclei is due to cells that finish m i t o s i s in t h e m e r i s t e m . H o w e v e r , it is w o r t h n o t i n g that in t h i s n o n - p r o l i f e r ative zone of the root the f r e q u e n c y of cells e n g a g e d in DNA s y n t h e s i s remains similar to that of the m e r i s t e m zone, i n d i c a t i n g that cortical cells not only go t h r o u g h the S p e r i o d but thatthey must a p p a r e n t l y initiate a n e w D N A r e p l i c a t i o n round as well. 6 m m from the root tip, the increase in the number of G02 nuclei runs c o n c u r r e n t l y with the decrease in the frequency of S cells and is likely p r o d u c e d by cells w h i c h have p a s s e d t h r o u g h the DNA s y n t h e s i s p e r i o d and finished it in the p r e c e d i n g regions of the root. Finally, the i n c r e a s e in G01 o b s e r v e d 9 mm from the root tip is quite s u r p r i s i n g and cannot be regarded as c o m i n g from G02 cells since m i t o s i s does not occur at a l l i n this zone of the root. One p l a u s i b l e r e a s o n for the i n c r e a s e in G01 n u c l e i could be that the DNA r e p l i c a t i o n rate is lower in this d i f f e r e n t i a t e d zone of the root, in such a way that the n u c l e i w o u l d not be l a b e l l e d under our e x p e r i m e n t a l protocol. This w o u l d cause us to score a c e r t a i n number of cortical nuclei, a c t u a l l y i n v o l v e d in DNA synthesis, as u n l a b e l l e d G01 nuclei. In our opinion, a p o s s i b l e e x p l a n a t i o n of the results r e p o r t e d above could have something to do w i t h the rate of DNA synthesis d e t e c t e d d u r i n g cell elongation, as a r e s u l t of w h e r e along the root axis the last cell p a r t i t i o n i n g took place. Therefore, we suggest tha~ some kind of DNA r e p l i c a t i o n signal

25 exist, but w i t h d e c r e a s i n g c o n c e n t r a t i o n the farther you go from the root tip. It has also been p r o p o s e d that this type of p o s i t i o n a l signal controls the p r o l i f e r a t i v e c a p a c i t y of cells inside the m e r i s t e m and in this way the m e r i s t e m b o u n d a r y too (Barlow 1976; Carmona 1979). M o r e o v e r , it has been demonstrated that the half of the meristem, e x t e n d i n g from the m i d d l e to the end of the meristem, is the zone in w h i c h the last partitioning mostly occurs ( Carmona, unpublished). Thus, cells w h o s e last partition takes place near the m i d d l e of the meristem, are able to d e t e c t e n o u g h signal to initiate DNA synthesis, so that in the mature zone these cells should be o b s e r v e d as 4C cells ( that is, in G02 ) . On the other hand, m e r i s t e m cells p a r t i t i o n i n g near the end of the m e r i s t e m w o u l d only exhibit a 2C DNA c o n t e n t in the m a t u r e zone, w h e r e the p r o p o r t i o n of cells w i t h DNA c o n t e n t 2C and 4C w o u l d be r o u g h l y 60% and 4 0 % , r e s p e c t i v e l y . Our h y p o t h e s i s also e x p l a i n why cells w i t h i n t e r m e d i a t e D N A c o n t e n t appear at increasing d i s t a n c e s from the root tip. In short, a l t h o u g h increases in DNA c o n t e n t should not be c o n s i d e r e d e s s e n t i a l in tissue d i f f e r e n t i a t i o n of h i g h e r plants, at least, as a general rule (Evans and V a n ' t Hof 1975), our results s t r o n g l y suggest that i n i t i a t i o n of DNA r e p l i c a t i o n occurs in the d i f f e r e n t i a t e d zone of the A l l i u m root. Thus, cells l e a v i n g the m e r i s t e m can i n i t i a t e DNA r e p l i c a t i o n a l t h o u g h they never divide.

Acknowledgements The authors are iDdebted to P r o f . J . F . L 6 p e z S~ez for advice and critical reading of the m a n u s c r i p t . W e are grateful to Mrs. M.L. Martfnez for her skilful t e c h n i c a l assistance. This w o r k was p a r t i a l l y s u p p o r t e d by the "Comisidn A s e s o r a para la I n v e s t i g a c i 6 n Cientffica y T6cnica" and by the "Acuerdo de Cooperaci6n Cientffica between CSIC ( Spain ) and U n i v e r s i d a d de Chile (Chile). One of the authors (C.G.) is a r e c i p i e n t of a r e s e a r c h f e l l o w s h i p from the "Caja de A h o r r o s y M o n t e de P i e d a d de Madrid". REFERENCES B a r l o w P (1976) J T h e o r Biol 57: 433-451 B r u n o r i A (1971) C a r y o l o g i a 24: 209-215 C a r m o n a M J (1979) D o c t o r a l Thesis. U n i v e r sity A u t o n o m a of M a d r i d Dfez J L, L 6 p e z - S ~ e z J F, G o n z ~ l e z - F e r n ~ n d e z A (1970) P l a n t a 91: 87-95 Evans L S, V a n ' t H o f J (1974) Am J Bot 61: 1104-1111 Evans L S, V a n ' t Hof J (1975) Am J Bot 62: 1060-1064 Mak S (1965) Exp Cell Res 39: 286-289 M c L e i s h J (1959) Chromos'oma i0: 686-710 M c L e i s h J, S u n d e r l a n d N (1961) Exp Cell Res 24: 527-540 Nagl W (1976) N a t u r e 261: 614-615 S p a r r o w A H, Price H J~ U n d e r b r i n k A G (1972) B r o o k h a v e n Symp Biol 23: 451-494