Planta (Berl.) 129, 229- 233 (1976)
P l a n t a 9 by Springer-Verlag 1976
Rapid-growth Responses of Corn Root Segments: Effect of Citrate-phosphate Buffer on Elongation K a t h r y n L. E d w a r d s * a n d T o m K. Scott Department of Botany, University of North Carolina, Chapel Hill, North Carolina 27514, USA
Summary. T h e effects o f c i t r a t e - p h o s p h a t e buffer on the e l o n g a t i o n rate o f 2 m m r o o t segments o f Z e a m a y s L. was i n v e s t i g a t e d u n d e r 0 2 a n d N2 c o n d i t i o n s by c o m p a r i n g w i t h the effects in n o n - b u f f e r e d solution. T h e buffer at p H 7.0 has an i n h i b i t i n g effect on the e l o n g a t i o n rate o f r o o t segments. A t p H 4.0 the buffer h a s a p r o m o t i n g effect o n the e l o n g a t i o n r a t e twice t h a t o f the H - i o n c o n c e n t r a t i o n . N i t r o g e n e x p e r i m e n t s indicate t h a t g r o w t h in n e u t r a l buffer is O2 d e p e n d e n t . O n the o t h e r h a n d the a c i d - g r o w t h r e s p o n s e o f r o o t segments in c i t r a t e - p h o s p h a t e buffer a p p e a r s to h a v e two c o m p o n e n t s , 1) a H - i o n c o m p o n e n t w h i c h is p a r t i a l l y i n h i b i t e d by N2 a n d fluoride a n d 2) a c i t r a t e c o m p o n e n t w h i c h is i n h i b i t e d in p a r t b y N 2 b u t n o t b y fluoride.
Introduction W e have r e p o r t e d , in a study o f the effect o f p H o n e l o n g a t i o n o f r o o t segments, t h a t the use o f citratep h o s p h a t e buffer m a y l e a d to e r r o n e o u s a s s u m p t i o n s r e g a r d i n g the effect o f H - i o n s ( E d w a r d s a n d Scott, 1974). R o o t segments p l a c e d in c i t r a t e - p h o s p h a t e buffer a t p H 4.0 e l o n g a t e d at a r a t e a p p r o x i m a t e l y twice t h a t o f segments in n o n - b u f f e r e d s o l u t i o n at p H 4.0. T h e p u r p o s e o f this i n v e s t i g a t i o n was to determ i n e s o m e o f the c h a r a c t e r i s t i c s o f the effects o f c i t r a t e - p h o s p h a t e buffer on the e l o n g a t i o n r a t e o f r o o t segments. This was d o n e b y c o m p a r i n g r e s p o n s e s under v a r i o u s c o n d i t i o n s using s o l u t i o n s with a n d witho u t buffer.
Material and Methods The germination and growth of roots of Zea mays L. cv. Burpee Snowcross, the Plexiglas growth apparatus, and the manner of * Present address: Department of Biology, Kline Biology Tower, Yale University, New Haven, Connecticut 06520
growth measurement have already been described in detail in a previous paper (Edwards and Scott, 1974). Roots grown in the dark were harvested after 3 days and averaged 4 cm in length. A segment, 2 mm in length was excised 1 mm behind the apex from ten primary roots. The ten segments were stacked one atop the other with apical ends oriented downwards and topped with a small lead weight. The stacked segments were placed in the specially designed Plexiglas growth apparatus and growth measurements were taken at 5 min intervals under dim green light using a horizontal-travellingmicroscope. All experiments were done at 26~ 1~ Citrate-sodium phosphate (1 mM:2 mM) buffer and non-buffered solution (HC1 in water) were prepared as described previously (Edwards and Scott, 1974). Freshly prepared buffered and nonbuffered solutions were continuously circulated past the experimental tissue without recirculation, and therefore it is assumed that the presence of and the effect of the H-ions on the cell walls was the same in both cases. Sodium fluoride (10 mM) and calcium chloride (10 mM and 20 mM) were prepared in both buffered and non-buffered solutions depending on the experiment. All experiments were repeated three times or more. Generally, the degree of significance of the data presented was assessed by using "Student's" t-test; t values more than 0.05 were considered non-significant. However, due to the great variability of the material, a statistical treatment of this kind often proved to be of no value. Experiments which had similar kinetic profiles or patterns of response (although varying maxima and minima) are considered significant and are reported here when no exceptions occurred (Edwards and Scott, 1974).
Results Equilibration F r e s h l y cut 2 m m r o o t segments s h o w e d a high initial e l o n g a t i o n r a t e w h e n p l a c e d in o x y g e n a t e d citratep h o s p h a t e b u f f e r e d o r n o n - b u f f e r e d s o l u t i o n at p H 7.0. Therefore, segments were a l l o w e d to equilib r a t e in either o f these solutions for 2.25 h b e f o r e e x p e r i m e n t a l t r e a t m e n t s were begun. T h e final equili b r i u m e l o n g a t i o n rate is 2 • higher for r o o t segments in n o n - b u f f e r e d s o l u t i o n at p H 7.0 (averaging 27 lain/ rain/10 segments) (Fig. 1). A n o t h e r c h a r a c t e r i s t i c o f the r o o t segment e q u i l i b r i u m rate in n e u t r a l buffer is o b s e r v e d w h e n s o l u t i o n s a r e gassed with n i t r o g e n d u r i n g e q u i l i b r a t i o n . T h e final e q u i l i b r i u m r a t e in
230
K.L. Edwards and T.K. Scott: Rapid-growth Responses of Corn Root Segments
ROOTS
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P l a n t a 9 by Springer-Verlag 1976
Rapid-growth Responses of Corn Root Segments: Effect of Citrate-phosphate Buffer on Elongation K a t h r y n L. E d w a r d s * a n d T o m K. Scott Department of Botany, University of North Carolina, Chapel Hill, North Carolina 27514, USA
Summary. T h e effects o f c i t r a t e - p h o s p h a t e buffer on the e l o n g a t i o n rate o f 2 m m r o o t segments o f Z e a m a y s L. was i n v e s t i g a t e d u n d e r 0 2 a n d N2 c o n d i t i o n s by c o m p a r i n g w i t h the effects in n o n - b u f f e r e d solution. T h e buffer at p H 7.0 has an i n h i b i t i n g effect on the e l o n g a t i o n rate o f r o o t segments. A t p H 4.0 the buffer h a s a p r o m o t i n g effect o n the e l o n g a t i o n r a t e twice t h a t o f the H - i o n c o n c e n t r a t i o n . N i t r o g e n e x p e r i m e n t s indicate t h a t g r o w t h in n e u t r a l buffer is O2 d e p e n d e n t . O n the o t h e r h a n d the a c i d - g r o w t h r e s p o n s e o f r o o t segments in c i t r a t e - p h o s p h a t e buffer a p p e a r s to h a v e two c o m p o n e n t s , 1) a H - i o n c o m p o n e n t w h i c h is p a r t i a l l y i n h i b i t e d by N2 a n d fluoride a n d 2) a c i t r a t e c o m p o n e n t w h i c h is i n h i b i t e d in p a r t b y N 2 b u t n o t b y fluoride.
Introduction W e have r e p o r t e d , in a study o f the effect o f p H o n e l o n g a t i o n o f r o o t segments, t h a t the use o f citratep h o s p h a t e buffer m a y l e a d to e r r o n e o u s a s s u m p t i o n s r e g a r d i n g the effect o f H - i o n s ( E d w a r d s a n d Scott, 1974). R o o t segments p l a c e d in c i t r a t e - p h o s p h a t e buffer a t p H 4.0 e l o n g a t e d at a r a t e a p p r o x i m a t e l y twice t h a t o f segments in n o n - b u f f e r e d s o l u t i o n at p H 4.0. T h e p u r p o s e o f this i n v e s t i g a t i o n was to determ i n e s o m e o f the c h a r a c t e r i s t i c s o f the effects o f c i t r a t e - p h o s p h a t e buffer on the e l o n g a t i o n r a t e o f r o o t segments. This was d o n e b y c o m p a r i n g r e s p o n s e s under v a r i o u s c o n d i t i o n s using s o l u t i o n s with a n d witho u t buffer.
Material and Methods The germination and growth of roots of Zea mays L. cv. Burpee Snowcross, the Plexiglas growth apparatus, and the manner of * Present address: Department of Biology, Kline Biology Tower, Yale University, New Haven, Connecticut 06520
growth measurement have already been described in detail in a previous paper (Edwards and Scott, 1974). Roots grown in the dark were harvested after 3 days and averaged 4 cm in length. A segment, 2 mm in length was excised 1 mm behind the apex from ten primary roots. The ten segments were stacked one atop the other with apical ends oriented downwards and topped with a small lead weight. The stacked segments were placed in the specially designed Plexiglas growth apparatus and growth measurements were taken at 5 min intervals under dim green light using a horizontal-travellingmicroscope. All experiments were done at 26~ 1~ Citrate-sodium phosphate (1 mM:2 mM) buffer and non-buffered solution (HC1 in water) were prepared as described previously (Edwards and Scott, 1974). Freshly prepared buffered and nonbuffered solutions were continuously circulated past the experimental tissue without recirculation, and therefore it is assumed that the presence of and the effect of the H-ions on the cell walls was the same in both cases. Sodium fluoride (10 mM) and calcium chloride (10 mM and 20 mM) were prepared in both buffered and non-buffered solutions depending on the experiment. All experiments were repeated three times or more. Generally, the degree of significance of the data presented was assessed by using "Student's" t-test; t values more than 0.05 were considered non-significant. However, due to the great variability of the material, a statistical treatment of this kind often proved to be of no value. Experiments which had similar kinetic profiles or patterns of response (although varying maxima and minima) are considered significant and are reported here when no exceptions occurred (Edwards and Scott, 1974).
Results Equilibration F r e s h l y cut 2 m m r o o t segments s h o w e d a high initial e l o n g a t i o n r a t e w h e n p l a c e d in o x y g e n a t e d citratep h o s p h a t e b u f f e r e d o r n o n - b u f f e r e d s o l u t i o n at p H 7.0. Therefore, segments were a l l o w e d to equilib r a t e in either o f these solutions for 2.25 h b e f o r e e x p e r i m e n t a l t r e a t m e n t s were begun. T h e final equili b r i u m e l o n g a t i o n rate is 2 • higher for r o o t segments in n o n - b u f f e r e d s o l u t i o n at p H 7.0 (averaging 27 lain/ rain/10 segments) (Fig. 1). A n o t h e r c h a r a c t e r i s t i c o f the r o o t segment e q u i l i b r i u m rate in n e u t r a l buffer is o b s e r v e d w h e n s o l u t i o n s a r e gassed with n i t r o g e n d u r i n g e q u i l i b r a t i o n . T h e final e q u i l i b r i u m r a t e in
230
K.L. Edwards and T.K. Scott: Rapid-growth Responses of Corn Root Segments
ROOTS
z
tu
pH 4.0 B U F F E R
Z uJ 125
pH 4.0 N O N - B U F F E R
==:= ==:= =:== ,:===:
o
_z
~.
=k
uJ
ul
,oo
re
Z O
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.......:=: =: ...... ,,:==::
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50
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Z 14. Z
