- Email: [email protected]
Growth promotion of excised cucumber cotyledons by rubbing or by cuticular abrasion
Plant Science Letters, 29 (1983) 97--105 Elsevier Scientific Publishers Ireland Ltd.
97
GROWTH PROMOTION OF EXCISED CUCUMBER COTYLEDONS BY RUBBING OR BY C U T I C U L A R A B R A S I O N
CLEON W. ROSS, GREGORY L. ORR and ROBERT N. BOWMAN
Dept. of Botany and Plant Physiology, Colorado State University, Fort Collins, CO 80523
(U.S.A.) (Received June 2nd, 1982) (Revision received September 10th, 1982) (Accepted September 13th, 1982)
SUMMARY
Growth of cotyledons excised from 5-day-old dark grown cucumber seedlings was measured after certain wounding treatments. Cotyledons rubbed between wet t h u m b and finger with or without carborundum or cut into eight sections grew faster than non-rubbed, non-cut controls. To determine h o w such treatments p r o m o t e d growth, scanning electron micrographs of c o t y l e d o n surfaces and growth responses in H20, zeatin, KCI and ethylene were evaluated. Treatments enhanced growth without zeatin or KCI, b u t were more effective when KCI was present. Abrasion with carborundum caused tears in cotyledon surfaces and faster absorption of KCI. Nevertheless, growth (H20 absorption) in KCI was so much greater that tissue K ÷ concentrations after growth were lower in abraded than in nonrubbed control cotyledons. Increased permeability of zeatin, KCI, H20 or 02 is unlikely to explain most o f the observed growth enhancement, because such enhancement also occurred when only the adaxial, astomatal surface was abraded and placed either down (in contact with the growth medium) or up (in contact with air). Effects of exogenous ethylene indicate that this gas does not cause growth enhancement. Perhaps an unidentified factor produced in w o u n d e d cells increases growth.
Key words: Zeatin -- Cotyledons -- Cucumber -- Growth -- Wood h o r m o n e
INTRODUCTION
To evaluate the importance of H * efflux for zeatin-induced growth and 0304-4211/83/0000--0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
98 wall loosening of detached cotyledons [ 1,2], it was important to minimize the cuticular barrier [3] to H ÷ fluxes between tissues and external media. We cut cucumber cotyledons into small pieces or rubbed both surfaces between thumb and finger with or without a carborundum slurry. Surprisingly, each treatment promoted growth relative to that of non-rubbed controls [2]. Such growth is only in area, not thickness (unpublished observations}. Our present report investigates the mechanism of growth promotion. We consider the roles of enhanced absorption of zeatin, KCI, H20 and 02, wound-induced ethylene production and production of an unidentified wound-induced factor that increases polysome formation and protein synthesis in dicots and monocots [4]. MATERIALS
AND METHODS
All studies were performed with cotyledons detached in light from 5-dayold cucumber (Cucumis sativus L. cv. Marketer) seedlings grown in darkness over wet paper towels [ 1,2 ]. The average fresh weight of such cotyledons was about 18 mg and the average dry weight (70°C, 24 h) was 7.0 mg. No change in dry weight could be detected during subsequent 3-day growth periods involving low intensity light conditions. To measure growth, 10 cotyledons were placed in each of several 9-cm diameter petri dishes, each dish containing a layer of Whatman No. 1 qualitative filter paper and 3.5 ml of test medium. Test media were distilled H20 or KC1 (10--100 mM), containing or lacking 56 ~M zeatin (Sigma Chemical Co.). Petri dishes, with lids, were placed on wet towels in pyrex baking dishes covered with transparent polyvinyl chloride and growth occurred for either 2 or 3 days at 25--28°C under fluorescent illumination of 20--40 ~E m -2 s -~ or in darkness. Fresh weight increases were determined by blotting and weighing, collectively, all ten cotyledons in each petri dish, then subtracting the initial fresh weights. Special treatments before growth involved rubbing with or without carborundum, cutting and exposure to ethylene. Cotyledons rubbed without carborundum were placed between wetted thumb and longest finger and rubbed back and forth up to fifteen times. Cotyledons rubbed with carborundum were treated similarly, but with thumb and finger coated with wet 300 mesh carborundum. Cut cotyledons were sliced transversely into eight pieces on wet filter papers in petri dishes immediately before growth measurements were begun. Ethylene-treated cotyledons were grown on wet filter papers in petri dishes as described above, but dishes were enclosed in transparent, 8-1 vacuum desiccators instead of pyrex baking dishes. At the start of the growth period, a known amount of ethylene mixed with air was injected through a rubber seal covering the inlet of each desiccator. Scanning electron microscopy of cotyledon surfaces was performed with fresh specimens. Potassium was analyzed by atomic absorption spectrometry. Uptake of K ÷ was determined from decreases in concentration of
99
K + and volumes of KCI solutions in growth media. Tissue concentrations of K + after growth were estimated by adding K + uptake values during growth to the amount of K ÷ present in each cotyledon before growth (approx. 0.7 ~mol, see Ref. 5), then dividing that sum by the tissue water content after growth (fresh wt, minus dry wt.). Statistical analyses of growth comparisons were made using Student's t-test. Growth data summarized in each figure were verified directly in 1 additional experiment; data in each table were verified by 2--4 additional experiments. RESULTS AND DISCUSSION
The influence of rubbing both surfaces various numbers of times with carborundum upon growth in light is illustrated in Fig. 1. Five to ten rubs provided maximal growth stimulation in 20 mM KCI and even 15 such rubs increased growth. When both surfaces were rubbed without carborundum, 5--10 rubs also gave maximum growth in light {data not shown). Rubbing with rubber gloves similarly enhanced growth. Although it was impossible to exert equal pressure when rubbing, these results suggested that subsequent experiments could be restricted to 5 such rubs. Scanning electron micrographs of each cotyledon surface were made after such treatments (Figs. 2--7). Rubbing with carborundum caused numerous tears and pits
t
0
70
~
65
/
8 60
//
,'T"
\\
~\
"t
,oI-T/
\\
,,
-z I
30
I
T 0
I
2
J,
5
10 No. of Rubs
A
15
2~0
Fig. 1. Effects o f rubbing frequency (with carborundum) upon subsequent growth of cotyledons in 20 mM KC1 with (+Z) and without (--Z) zeatin. Growth occurred 3 days i n light. Values a r e m e a n s ± S.D. from 2 petri dishes.
100 in both surfaces (Figs. 5 and 6). We therefore refer to cotyledons so treated as abraded. Such tears and pits were n o t detected after robbing w i t h o u t carborundum, although most of the trichomes on the adaxial surface had been broken off, leaving apparent holes in the surface (Fig. 4). Stomates
Fig. 2--7. Effects of rubbing with or without carborundum on adaxial and abaxial cotyledon surfaces. Fresh S.E.M. specimens unshadowed, observed at 20 KeV, orig. mag., × 175. (2) Adaxial, non-rubbed; (3) abaxial, non-rubbed; (4) adaxial, rubbed; (5) abaxial, robbed; (6) adaxial, carborundum abraded; (7) abaxial, carborundum abraded.
101 were detected only on the abaxial surface of this cultivar, although we have found them on both surfaces of other cultivars. Table I contains data showing effects o f rubbing or abrading five times or cutting cotyledons into eight pieces. Subsequent growth occurred in light with or without zeatin in the presence or absence of 20 mM KCI. Without zeatin and KC1, rubbed cotyledons grew faster than non-rubbed, but abraded or cut cotyledons grew even faster. Similar results occurred without zeatin but with KCI. With zeatin, growth promotion by each of the three treatments was less, at least on a percentage basis, than without zeatin, and this was true with or without KCI. These results indicate that rubbing, abrading, or cutting treatments p r o m o t e growth in distilled H20 by an u n k n o w n mechanism, b u t that faster absorption of zeatin or KCI might enhance the effects of that mechanism. The importance of faster zeatin uptake seems minimal, because each treatment p r o m o t e d growth less (on a percentage basis) with than without hormone (Table I). Furthermore, the optimum concentration o f z e a t i n required for maximum growth (20--60 ~M) was the same for non-rubbed and abraded cotyledons (data not shown). The importance of enhanced KCI absorption for growth promotion by such treatments is less easily ignored. Growth of abraded cotyledons {with or w i t h o u t zeatin) was greater than that of non-rubbed cotyledons over a wide range of KCI concentrations (Fig. 8a). Abrasion simultaneously promoted K ÷ absorption, especially w i t h o u t zeatin (Fig. 8b). Because the pH (initially, 7.4) of the external media changed no more than 0.2 of a unit during growth, we assume that absorption of Cl- matched that of K ÷ in each solution. Nevertheless, tissue concentrations of K + were lower in abraded than in non-rubbed tissue after growth in KCI solutions (Fig. 8c). The simplest explanation for this result is that, on a relative basis, abrasion p r o m o t e d absorption of H20 even more than of KCI. Even so, absorption of KCI is important for growth because it helps maintain turgor as internal solutes are diluted [6,7 ]. These results add to those of Green and Muir [ 5,8 ], w h o deduced the importance o f K ÷ for growth of cucumber cotyledons even w i t h o u t measuring its absorption. To determine if abrasion promotes growth because it allows faster absorption of 02 or H20, we first assumed that tissues in previous experiments absorbed much more O2 from the abaxial {stomatal) surface exposed directly to the air than from the adaxial (astomatal) surface contacting the wet filter paper. We also assumed that stomates were at least partly open in light and closed in darkness. We then abraded with five rubs only the adaxial, astomatal surface using a slender stick covered with c o t t o n dipped in a carborundum slurry. Abraded and non-rubbed cotyledons then grew in light or darkness (with 20 mM KCI but w i t h o u t zeatin) with the adaxial surface either up or down. Growth periods were restricted to 2 days to minimize upward curling of cotyledon margins, because such curling exposes both adaxial and abaxial surfaces to air. Table II contains the results. First, growth was faster in light than in darkness, as noted by others [8,9].
Non-rubbed Rubbed Abraded Cut
Treatment
17 23 28 30
-+ 2.1 ± 2.6 -+ 3.0 ± 4.0
--Zeatin
N o KCI
(100%) (140%) (160%) (180%)
a b c c
40 44 42 52
± ± ± ±
5.2 4.1 5.1 4.5
+ Zeatin (100%) (110%) (105%) (130%)
.
a a a b
29 38 58 58
± 2.4 ± 3.5 -+ 6.6 ± 2.9
-- Z e a t i n (100%) (130%) (200%) (200%)
+ 20 m M KC1
a b c c
65 78 84 75
± ± ± ±
4.8 4.7 4.9 4.6
+ Zeatin (100%) (120%) (129%) (115%)
a b b b
G r o w t h o c c u r r e d 3 d a y s in light. F r e s h wt. i n c r e a s e s ( m g c o t y l e d o n ' ) are m e a n s -+ ave. S.D. for at least 3 e x p e r i m e n t s i n v o l v i n g 2 o r 3 s a m p l e s o f 10 c o t y l e d o n s p e r t r e a t m e n t p e r e x p e r i m e n t . W i t h i n a n y c o l u m n , g r o w t h v a l u e s f o l l o w e d b y a d i f f e r e n t l e t t e r are s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r a t t h e 9 5 % c o n f i d e n c e level (t-test).
G R O W T H W I T H O R W I T H O U T Z E A T I N O R KCI AS A F F E C T E D BY V A R I O U S T R E A T M E N T S
TABLE I
ba Q b~
20
40
I 0
®
I 10
~//
/
/
/
/
I 2.5
mM KCI
1 .50
I 60
I 75
\+Z, Nonrubbed._I~ , J ' ~ k _ _ ~
f
. . " ~ +Z, Abraded
I 100
v
,b
/ I
2~
mM KCI
~
/~/
/
///
20
i
I
7s
i
,oo
/"
~.,
i1~/~//111/ / //
-Z, Abraded ~
+Z. Abraded
3
~,~S" "
\
~,Z. Abraded
+Z, Abraded
.~e
\// mM KCI
-Z. Nonrubbed
I©
Fig 8. Effects o f abrasion u p o n growth, K ÷ absorption and final tissue K ÷ c o n c e n t r a t i o n s at various KC1 concentrations. G r c w t h occurred 3 days in light with o r w i t h o u t zeatin (Z). Values are means -+ S.D. f r o m 2 petri dishes. (a) Effects u p o n g r o w t h ; (b) effects upon K ÷ absorption; (c) effects u p o n tissue K + concentrations.
"~
_c ~J
6o
so
120
140
104
Second, growth of non-rubbed cotyledons with adaxial surfaces up was slightly greater than growth with adaxial surfaces down in light and darkness. If somates were not involved, this difference might be attributed to a cuticle more permeable to 02 or H20 on one surface than on the other. Alternatively, this difference might result from inherent morphological and physiological properties related to in situ growth. Growth of cotyledons abraded only on the adaxial surface was greater than that of non-rubbed controls whether that surface was up or down (Table II). A possible explanation is that abrasion promotes growth when the abraded surface is down because it increases the tissue hydraulic conductivity, i.e., it simply allows faster absorption of H20. However, hydraulic conductivity increases cannot explain growth promotion when the abraded surface was up and exposed to air. Such growth enhancement might be explained by faster absorption of 02, if 02 indeed limits growth of nonrubbed cotyledons. Another possible explanation is that non-rubbed cotyledons are so permeable to both H20 and 02 that abrasion does not directly affect absorption of either molecule. If so, abrasion would enhance growth only indirectly, perhaps as a result of greater production of ethylene. Results from experiments in which growth occurred in light with 20 mM KCI disagree with the hypothesis that wound-induced ethylene promoted growth. First, abraded cotyledons did not affect growth of an equal number of non-rubbed cotyledons incubated in the s~me petri dish (data not shown). Second,. exogenous ethylene did not affect growth of non-rubbed cotyledons at low concentrations and was inhibitory at higher concentrations, especially with zeatin (Fig. 9). We do not know whether another wound-induced factor contributes to growth promotion caused by rubbing, abrading or cutting, but the putative wound hormone recently discovered by Davies and Schuster [4] that enhances polysome formation and protein synthesis seems worth considering. The translocatable thigmomorphogenetic factor apparently present in bean stems is probably not involved, because its T A B L E II E F F E C T S O F A B R A S I O N A N D S U R F A C E O R I E N T A T I O N ON C O T Y L E D O N GROWTH IN L I G H T A N D D A R K N E S S
Fresh wt. increases (rag cotyledon -~ ) are means -* ave. S.D. f r o m 4 e x p e r i m e n t s in light and 3 in darkness, each involving either 2 or 3 s a m p l e s o f 10 c o t y l e d o n s per treatment. G r o w t h p e r i o d was 2 days. O n l y the adaxial surface was abraded. Means f o l l o w e d by different letters are significantly different from each other at the 99% confidence level (t-test).
Treatment
Growth in light
G r o w t h in d a r k n e s s
Non-rubbed, adaxial d o w n Non-rubbed, adaxial up Abraded, adaxial down Abraded, adaxial up
21.1 24.9 34.0 31.4
9.1 11.9 15.9 15.3
± ± ± ±
2.1 2.0 2.9 2.9
a h c c
± ± ± ±
1.3 1.8 1.3 1.2
c e f f
105
--.~ 40
8 3O o
g 2o
10
~1 0
I
.001
I
.01
().1
11.0
I
10
Ethylene Conch. (pl 1-1) Fig. 9. Effects of atmospheric ethylene concentrations upon growth with and without zeatin (Z). Growth occurred 3 days in light. Each value is the mean + S.D. from 3 petri dishes. e f f e c t s in s u c h s t e m s are d u p l i c a t e d b y a d d i t i o n o f t h e e t h y l e n e releasing c o m p o u n d e t h e p h o n [ 10]. R e g a r d l e s s o f h o w w o u n d i n g increases g r o w t h , it is i m p o r t a n t t h a t researchers w h o s t u d y p h y s i o l o g i c a l processes in excised c o t y l e d o n s m i n i m i z e or equalize m e c h a n i c a l injury d u r i n g excision and handling. ACKNOWLEDGEMENTS We t h a n k Ellen D r e w H a g e r f o r c o m p e t e n t t e c h n i c a l assistance and the N a t i o n a l Science F o u n d a t i o n f o r a g r a n t ( P C M 7 9 - 2 2 1 3 9 ) t h a t p r o v i d e d financial s u p p o r t . REFERENCES 1 J. Thomas, C.W. Ross, C.J. Chastain, N. Koomanoff, J.E. Hendrix and E. Van Volkenburgh, Plant Physiol., 68 (1981) 107. 2 C.W. Ross and D.L. Rayle, Plant Physiol., 70 (1982) 1470. 3 S.A. Dreyer, V. Seymour and R:'E~YCleland, Plant Physiol., 68 (1981) 664. 4 E. Davies and A. Schuster, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 2422. 5 J.F. Green and R.M. Muir, Physiol. Plant., 43 (1978) 213. 6 J.J. Oertli, Z. Pflanzenphysiol., 74 (1975) 440. 7 T.T. Stevenson and R.E. Cleland, Plant Physiol., 67 (1981) 749. 8 J.F. Green and R.M. Muir, Physiol. Plant., 46 (1979) 19. 9 A.K. Huff and C.W. Ross, Plant Physiol., 56 (1975) 429. 10 Y. Erner, R. Biro and M.J. Jaffe, Physiol. Plant., 50 (1980) 21.
97
GROWTH PROMOTION OF EXCISED CUCUMBER COTYLEDONS BY RUBBING OR BY C U T I C U L A R A B R A S I O N
CLEON W. ROSS, GREGORY L. ORR and ROBERT N. BOWMAN
Dept. of Botany and Plant Physiology, Colorado State University, Fort Collins, CO 80523
(U.S.A.) (Received June 2nd, 1982) (Revision received September 10th, 1982) (Accepted September 13th, 1982)
SUMMARY
Growth of cotyledons excised from 5-day-old dark grown cucumber seedlings was measured after certain wounding treatments. Cotyledons rubbed between wet t h u m b and finger with or without carborundum or cut into eight sections grew faster than non-rubbed, non-cut controls. To determine h o w such treatments p r o m o t e d growth, scanning electron micrographs of c o t y l e d o n surfaces and growth responses in H20, zeatin, KCI and ethylene were evaluated. Treatments enhanced growth without zeatin or KCI, b u t were more effective when KCI was present. Abrasion with carborundum caused tears in cotyledon surfaces and faster absorption of KCI. Nevertheless, growth (H20 absorption) in KCI was so much greater that tissue K ÷ concentrations after growth were lower in abraded than in nonrubbed control cotyledons. Increased permeability of zeatin, KCI, H20 or 02 is unlikely to explain most o f the observed growth enhancement, because such enhancement also occurred when only the adaxial, astomatal surface was abraded and placed either down (in contact with the growth medium) or up (in contact with air). Effects of exogenous ethylene indicate that this gas does not cause growth enhancement. Perhaps an unidentified factor produced in w o u n d e d cells increases growth.
Key words: Zeatin -- Cotyledons -- Cucumber -- Growth -- Wood h o r m o n e
INTRODUCTION
To evaluate the importance of H * efflux for zeatin-induced growth and 0304-4211/83/0000--0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
98 wall loosening of detached cotyledons [ 1,2], it was important to minimize the cuticular barrier [3] to H ÷ fluxes between tissues and external media. We cut cucumber cotyledons into small pieces or rubbed both surfaces between thumb and finger with or without a carborundum slurry. Surprisingly, each treatment promoted growth relative to that of non-rubbed controls [2]. Such growth is only in area, not thickness (unpublished observations}. Our present report investigates the mechanism of growth promotion. We consider the roles of enhanced absorption of zeatin, KCI, H20 and 02, wound-induced ethylene production and production of an unidentified wound-induced factor that increases polysome formation and protein synthesis in dicots and monocots [4]. MATERIALS
AND METHODS
All studies were performed with cotyledons detached in light from 5-dayold cucumber (Cucumis sativus L. cv. Marketer) seedlings grown in darkness over wet paper towels [ 1,2 ]. The average fresh weight of such cotyledons was about 18 mg and the average dry weight (70°C, 24 h) was 7.0 mg. No change in dry weight could be detected during subsequent 3-day growth periods involving low intensity light conditions. To measure growth, 10 cotyledons were placed in each of several 9-cm diameter petri dishes, each dish containing a layer of Whatman No. 1 qualitative filter paper and 3.5 ml of test medium. Test media were distilled H20 or KC1 (10--100 mM), containing or lacking 56 ~M zeatin (Sigma Chemical Co.). Petri dishes, with lids, were placed on wet towels in pyrex baking dishes covered with transparent polyvinyl chloride and growth occurred for either 2 or 3 days at 25--28°C under fluorescent illumination of 20--40 ~E m -2 s -~ or in darkness. Fresh weight increases were determined by blotting and weighing, collectively, all ten cotyledons in each petri dish, then subtracting the initial fresh weights. Special treatments before growth involved rubbing with or without carborundum, cutting and exposure to ethylene. Cotyledons rubbed without carborundum were placed between wetted thumb and longest finger and rubbed back and forth up to fifteen times. Cotyledons rubbed with carborundum were treated similarly, but with thumb and finger coated with wet 300 mesh carborundum. Cut cotyledons were sliced transversely into eight pieces on wet filter papers in petri dishes immediately before growth measurements were begun. Ethylene-treated cotyledons were grown on wet filter papers in petri dishes as described above, but dishes were enclosed in transparent, 8-1 vacuum desiccators instead of pyrex baking dishes. At the start of the growth period, a known amount of ethylene mixed with air was injected through a rubber seal covering the inlet of each desiccator. Scanning electron microscopy of cotyledon surfaces was performed with fresh specimens. Potassium was analyzed by atomic absorption spectrometry. Uptake of K ÷ was determined from decreases in concentration of
99
K + and volumes of KCI solutions in growth media. Tissue concentrations of K + after growth were estimated by adding K + uptake values during growth to the amount of K ÷ present in each cotyledon before growth (approx. 0.7 ~mol, see Ref. 5), then dividing that sum by the tissue water content after growth (fresh wt, minus dry wt.). Statistical analyses of growth comparisons were made using Student's t-test. Growth data summarized in each figure were verified directly in 1 additional experiment; data in each table were verified by 2--4 additional experiments. RESULTS AND DISCUSSION
The influence of rubbing both surfaces various numbers of times with carborundum upon growth in light is illustrated in Fig. 1. Five to ten rubs provided maximal growth stimulation in 20 mM KCI and even 15 such rubs increased growth. When both surfaces were rubbed without carborundum, 5--10 rubs also gave maximum growth in light {data not shown). Rubbing with rubber gloves similarly enhanced growth. Although it was impossible to exert equal pressure when rubbing, these results suggested that subsequent experiments could be restricted to 5 such rubs. Scanning electron micrographs of each cotyledon surface were made after such treatments (Figs. 2--7). Rubbing with carborundum caused numerous tears and pits
t
0
70
~
65
/
8 60
//
,'T"
\\
~\
"t
,oI-T/
\\
,,
-z I
30
I
T 0
I
2
J,
5
10 No. of Rubs
A
15
2~0
Fig. 1. Effects o f rubbing frequency (with carborundum) upon subsequent growth of cotyledons in 20 mM KC1 with (+Z) and without (--Z) zeatin. Growth occurred 3 days i n light. Values a r e m e a n s ± S.D. from 2 petri dishes.
100 in both surfaces (Figs. 5 and 6). We therefore refer to cotyledons so treated as abraded. Such tears and pits were n o t detected after robbing w i t h o u t carborundum, although most of the trichomes on the adaxial surface had been broken off, leaving apparent holes in the surface (Fig. 4). Stomates
Fig. 2--7. Effects of rubbing with or without carborundum on adaxial and abaxial cotyledon surfaces. Fresh S.E.M. specimens unshadowed, observed at 20 KeV, orig. mag., × 175. (2) Adaxial, non-rubbed; (3) abaxial, non-rubbed; (4) adaxial, rubbed; (5) abaxial, robbed; (6) adaxial, carborundum abraded; (7) abaxial, carborundum abraded.
101 were detected only on the abaxial surface of this cultivar, although we have found them on both surfaces of other cultivars. Table I contains data showing effects o f rubbing or abrading five times or cutting cotyledons into eight pieces. Subsequent growth occurred in light with or without zeatin in the presence or absence of 20 mM KCI. Without zeatin and KC1, rubbed cotyledons grew faster than non-rubbed, but abraded or cut cotyledons grew even faster. Similar results occurred without zeatin but with KCI. With zeatin, growth promotion by each of the three treatments was less, at least on a percentage basis, than without zeatin, and this was true with or without KCI. These results indicate that rubbing, abrading, or cutting treatments p r o m o t e growth in distilled H20 by an u n k n o w n mechanism, b u t that faster absorption of zeatin or KCI might enhance the effects of that mechanism. The importance of faster zeatin uptake seems minimal, because each treatment p r o m o t e d growth less (on a percentage basis) with than without hormone (Table I). Furthermore, the optimum concentration o f z e a t i n required for maximum growth (20--60 ~M) was the same for non-rubbed and abraded cotyledons (data not shown). The importance of enhanced KCI absorption for growth promotion by such treatments is less easily ignored. Growth of abraded cotyledons {with or w i t h o u t zeatin) was greater than that of non-rubbed cotyledons over a wide range of KCI concentrations (Fig. 8a). Abrasion simultaneously promoted K ÷ absorption, especially w i t h o u t zeatin (Fig. 8b). Because the pH (initially, 7.4) of the external media changed no more than 0.2 of a unit during growth, we assume that absorption of Cl- matched that of K ÷ in each solution. Nevertheless, tissue concentrations of K + were lower in abraded than in non-rubbed tissue after growth in KCI solutions (Fig. 8c). The simplest explanation for this result is that, on a relative basis, abrasion p r o m o t e d absorption of H20 even more than of KCI. Even so, absorption of KCI is important for growth because it helps maintain turgor as internal solutes are diluted [6,7 ]. These results add to those of Green and Muir [ 5,8 ], w h o deduced the importance o f K ÷ for growth of cucumber cotyledons even w i t h o u t measuring its absorption. To determine if abrasion promotes growth because it allows faster absorption of 02 or H20, we first assumed that tissues in previous experiments absorbed much more O2 from the abaxial {stomatal) surface exposed directly to the air than from the adaxial (astomatal) surface contacting the wet filter paper. We also assumed that stomates were at least partly open in light and closed in darkness. We then abraded with five rubs only the adaxial, astomatal surface using a slender stick covered with c o t t o n dipped in a carborundum slurry. Abraded and non-rubbed cotyledons then grew in light or darkness (with 20 mM KCI but w i t h o u t zeatin) with the adaxial surface either up or down. Growth periods were restricted to 2 days to minimize upward curling of cotyledon margins, because such curling exposes both adaxial and abaxial surfaces to air. Table II contains the results. First, growth was faster in light than in darkness, as noted by others [8,9].
Non-rubbed Rubbed Abraded Cut
Treatment
17 23 28 30
-+ 2.1 ± 2.6 -+ 3.0 ± 4.0
--Zeatin
N o KCI
(100%) (140%) (160%) (180%)
a b c c
40 44 42 52
± ± ± ±
5.2 4.1 5.1 4.5
+ Zeatin (100%) (110%) (105%) (130%)
.
a a a b
29 38 58 58
± 2.4 ± 3.5 -+ 6.6 ± 2.9
-- Z e a t i n (100%) (130%) (200%) (200%)
+ 20 m M KC1
a b c c
65 78 84 75
± ± ± ±
4.8 4.7 4.9 4.6
+ Zeatin (100%) (120%) (129%) (115%)
a b b b
G r o w t h o c c u r r e d 3 d a y s in light. F r e s h wt. i n c r e a s e s ( m g c o t y l e d o n ' ) are m e a n s -+ ave. S.D. for at least 3 e x p e r i m e n t s i n v o l v i n g 2 o r 3 s a m p l e s o f 10 c o t y l e d o n s p e r t r e a t m e n t p e r e x p e r i m e n t . W i t h i n a n y c o l u m n , g r o w t h v a l u e s f o l l o w e d b y a d i f f e r e n t l e t t e r are s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r a t t h e 9 5 % c o n f i d e n c e level (t-test).
G R O W T H W I T H O R W I T H O U T Z E A T I N O R KCI AS A F F E C T E D BY V A R I O U S T R E A T M E N T S
TABLE I
ba Q b~
20
40
I 0
®
I 10
~//
/
/
/
/
I 2.5
mM KCI
1 .50
I 60
I 75
\+Z, Nonrubbed._I~ , J ' ~ k _ _ ~
f
. . " ~ +Z, Abraded
I 100
v
,b
/ I
2~
mM KCI
~
/~/
/
///
20
i
I
7s
i
,oo
/"
~.,
i1~/~//111/ / //
-Z, Abraded ~
+Z. Abraded
3
~,~S" "
\
~,Z. Abraded
+Z, Abraded
.~e
\// mM KCI
-Z. Nonrubbed
I©
Fig 8. Effects o f abrasion u p o n growth, K ÷ absorption and final tissue K ÷ c o n c e n t r a t i o n s at various KC1 concentrations. G r c w t h occurred 3 days in light with o r w i t h o u t zeatin (Z). Values are means -+ S.D. f r o m 2 petri dishes. (a) Effects u p o n g r o w t h ; (b) effects upon K ÷ absorption; (c) effects u p o n tissue K + concentrations.
"~
_c ~J
6o
so
120
140
104
Second, growth of non-rubbed cotyledons with adaxial surfaces up was slightly greater than growth with adaxial surfaces down in light and darkness. If somates were not involved, this difference might be attributed to a cuticle more permeable to 02 or H20 on one surface than on the other. Alternatively, this difference might result from inherent morphological and physiological properties related to in situ growth. Growth of cotyledons abraded only on the adaxial surface was greater than that of non-rubbed controls whether that surface was up or down (Table II). A possible explanation is that abrasion promotes growth when the abraded surface is down because it increases the tissue hydraulic conductivity, i.e., it simply allows faster absorption of H20. However, hydraulic conductivity increases cannot explain growth promotion when the abraded surface was up and exposed to air. Such growth enhancement might be explained by faster absorption of 02, if 02 indeed limits growth of nonrubbed cotyledons. Another possible explanation is that non-rubbed cotyledons are so permeable to both H20 and 02 that abrasion does not directly affect absorption of either molecule. If so, abrasion would enhance growth only indirectly, perhaps as a result of greater production of ethylene. Results from experiments in which growth occurred in light with 20 mM KCI disagree with the hypothesis that wound-induced ethylene promoted growth. First, abraded cotyledons did not affect growth of an equal number of non-rubbed cotyledons incubated in the s~me petri dish (data not shown). Second,. exogenous ethylene did not affect growth of non-rubbed cotyledons at low concentrations and was inhibitory at higher concentrations, especially with zeatin (Fig. 9). We do not know whether another wound-induced factor contributes to growth promotion caused by rubbing, abrading or cutting, but the putative wound hormone recently discovered by Davies and Schuster [4] that enhances polysome formation and protein synthesis seems worth considering. The translocatable thigmomorphogenetic factor apparently present in bean stems is probably not involved, because its T A B L E II E F F E C T S O F A B R A S I O N A N D S U R F A C E O R I E N T A T I O N ON C O T Y L E D O N GROWTH IN L I G H T A N D D A R K N E S S
Fresh wt. increases (rag cotyledon -~ ) are means -* ave. S.D. f r o m 4 e x p e r i m e n t s in light and 3 in darkness, each involving either 2 or 3 s a m p l e s o f 10 c o t y l e d o n s per treatment. G r o w t h p e r i o d was 2 days. O n l y the adaxial surface was abraded. Means f o l l o w e d by different letters are significantly different from each other at the 99% confidence level (t-test).
Treatment
Growth in light
G r o w t h in d a r k n e s s
Non-rubbed, adaxial d o w n Non-rubbed, adaxial up Abraded, adaxial down Abraded, adaxial up
21.1 24.9 34.0 31.4
9.1 11.9 15.9 15.3
± ± ± ±
2.1 2.0 2.9 2.9
a h c c
± ± ± ±
1.3 1.8 1.3 1.2
c e f f
105
--.~ 40
8 3O o
g 2o
10
~1 0
I
.001
I
.01
().1
11.0
I
10
Ethylene Conch. (pl 1-1) Fig. 9. Effects of atmospheric ethylene concentrations upon growth with and without zeatin (Z). Growth occurred 3 days in light. Each value is the mean + S.D. from 3 petri dishes. e f f e c t s in s u c h s t e m s are d u p l i c a t e d b y a d d i t i o n o f t h e e t h y l e n e releasing c o m p o u n d e t h e p h o n [ 10]. R e g a r d l e s s o f h o w w o u n d i n g increases g r o w t h , it is i m p o r t a n t t h a t researchers w h o s t u d y p h y s i o l o g i c a l processes in excised c o t y l e d o n s m i n i m i z e or equalize m e c h a n i c a l injury d u r i n g excision and handling. ACKNOWLEDGEMENTS We t h a n k Ellen D r e w H a g e r f o r c o m p e t e n t t e c h n i c a l assistance and the N a t i o n a l Science F o u n d a t i o n f o r a g r a n t ( P C M 7 9 - 2 2 1 3 9 ) t h a t p r o v i d e d financial s u p p o r t . REFERENCES 1 J. Thomas, C.W. Ross, C.J. Chastain, N. Koomanoff, J.E. Hendrix and E. Van Volkenburgh, Plant Physiol., 68 (1981) 107. 2 C.W. Ross and D.L. Rayle, Plant Physiol., 70 (1982) 1470. 3 S.A. Dreyer, V. Seymour and R:'E~YCleland, Plant Physiol., 68 (1981) 664. 4 E. Davies and A. Schuster, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 2422. 5 J.F. Green and R.M. Muir, Physiol. Plant., 43 (1978) 213. 6 J.J. Oertli, Z. Pflanzenphysiol., 74 (1975) 440. 7 T.T. Stevenson and R.E. Cleland, Plant Physiol., 67 (1981) 749. 8 J.F. Green and R.M. Muir, Physiol. Plant., 46 (1979) 19. 9 A.K. Huff and C.W. Ross, Plant Physiol., 56 (1975) 429. 10 Y. Erner, R. Biro and M.J. Jaffe, Physiol. Plant., 50 (1980) 21.