Download The Physiology of Gibberellin-Induced Elongation

The Physiology of Gibberellin-Induced Elongation
R.L. JONES
1
IN Plant Growth Substances:
Proceedings of the 10th Intl.
Gonf. on Plant Growth Substances, Madison, Wisconsin,
July 22-26, 1979. F. Skoog, ed.
Springer-Verlag, N.Y. (1980).
Despite the fact that the discovery of the gibberellins (GA s) resulted from the dramat·
ic effect these compounds exert on stem elongation, our understanding of the physiol·
ogy of this process has progressed slowly. Progress has been hampered by both meth·
odological and conceptual limitations, particularly by the lack of an appropriate test
system. Experiments have been conflned largely to studies with whole plants (I, 2)
and to studies with excised sections which show a very limited growth response to GA
(3,4) or some dependence on, or response to, auxins (3,5,6).
Among the conceptuallirnitations to progress in elucidating the mechanism of GA
action, attempts to resolve the roles of cell division and ceil elongation in the process
of GA·induced elongation (7,8) have commanded considerable attention. It is now
clear, however, that only cell elongation, and not cell division, can be involved in the
process of surface growth (11). Thus, GA may affect the process of cell division in in·
tact plants or isolated plant parts (7 -1 0), but elongation growth results only from the
process of cell extension.
The primacy of the role of GA in the stimulation of growth in GA·sensitive plants
has also been disputed (12, 13). Several workers have argued that the role of GA is to
stimulate the synthesis of auxin (14, 15), and that it is auxin which functions to stirn·
ulate elongation growth. Although GA may indeed stimulate the synthesis of indole·
acetic acid in intact plants, the evidence that it is auxin which stimulates elongation
in GA·treated plants is weak.
The precise mechanism of GA·induced elongation has been the object of consider·
able experimentation. Although altered cell·wall plasticity was recognized to playa
central role in auxin-stimulated growth as early as the 1930's (16), the relative contrib·
ution of pressure and osmotic potentials to the water potential of cells of GA·treated
tissue have only recently been resolved. Indeed circumstantial evidence has led most
workers to believe that unlike auxins, the GA s stimulated ceil extension by influenc·
ing the osmotic potential of the cell (17 -19).
The development of suitable excised test systems for studying the physiology of
GA·induced growth has provided answers to many of the questions posed above.
Kaufman and his colleagues (20-25) have explited the response of the excised inter·
node of 45-day-old oat plants, and they have described many aspects of the physiolog:,
of GA·induced elongation in this tissue. The author's laboratory (26-30) has concen·
trated on aspects of the response of the excised lettuce hypocotyl to GA. This review
will emphasize progress in elucidating the physiology of GA action in this system (26).
although reference will be made to the work of others where appropriate.
1 Department of Botany, University of California, Berkeley, California 94720, USA
189
The Physiology of Gibberellin-induced Elongation
Auxins and Growth in GA-Responsive Test Systems
Excised tissue sections which are responsive to GA provide useful models to test the
hypothesis that GA stimulates elongation via an increase in auxin biosynthesis. With an
excised system, the hypothesis can be tested both directly by the addition of auxin
and indirectly by the use of antiauxin (Fig . 1). Silk and Jones (26) reported that IAA
A
140
8
I
!<_ x _ _ .+GA
-~
120
80
80
60
60
x
/
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9-~-o~o
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Log [NAA] (M)
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,I
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!
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1
1
I
-7 -6 -5 -4
log [PC I B] ( M )
Fig. lA and B. The effect of the synthetic auxin naphthaleneacetic acid (NAA) (A) and the antiauxin p-chlorophenoxyisobutyric acid (PCIB) (B) on the elongation of lettuce hypocotyl sections
incubated in 5 I-Ig ml-' GA or H,o
did not stimulate growth of excised lettuce hypocotyls. Neither did the antiauxin triiodobenzoic acid (TIBA) affect the response of the tissue to GA. We have now extended this study to include the application of synthetic auxins, e.g., 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid (NAA), neither of which stimulates elongation
of the lettuce hypocotyl section (Fig. IA). Also the powerful antiauxin p-chlorophenoxyisobutyric acid (PCIB) does not affect GA-induced growth at concentrations up to
10-4 M (Fig . 1B), and ethylene at concentrations up to 5,000 ppm does not affect the
increase in length or weight of hypocotyls treated with GA (Fig. 2). This latter observation is particularly significant since aUxin-responsive tissues, e.g., pea internodes,
soybean hypocotyls, and cereal coleoptiles, exhibit a marked inhibition of elongation
in response to ethylene, and this inhibition of elongation is generally accompanied by
a pronounced swelling of the tissue.
Effects of GA on Cell Division and Cell Elongation
The GA s have been implicated as regulators of both cell division and cell elongation.
Sachs (1) among others (35) has suggested that GA s can promote meristematic activity in whole plants, while Kaufman (20) has presented evidence that in Avena GA can
R.L. Jones
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110
80
90
60
70
40
50
20
~
1000
10000
Lt,
100
1000
10000
[C 2 H4 ] ppm
Fig. 2A and B. The effect of ethylene on fresh weight (A) and elongation (B) of lettuce hypocotyl
sections incubated in GA or H10
inhibit cell division. In the excised hypocotyl of lettuce we have demonstrated that
GA does not affect the process of cell division and that cell division has little if any influence on elongation (27). During the first 12 h of incubation of excised lettuce
hypocotyls in either H 2 0 or GA, there is a 50% increase in the number of cells in the
hypocotyl, but after 12 h few if any mitoses are observed. If hypocotyl sections are
excised from seedlings of 'Y-irradiated seeds, cell division in the section is completely
inhibited, but their GA-induced elongation is not significantly altered (27). Despite
the fact that hypocotyl sections from seedlings of nonirradiated seeds have 50% more
cells than those from irradiated seeds, their growth is essentially the same, indicating
that factors other than cell number influence section length. These results emphasize
the conclusions of Green (II) concerning the distinctions between the processes of
cell elongation and cell division.
Although cell division does not contribute to the process of extension growth, it
is often important to establish the extent to which cross-wall formation occurs after
hormone treatment. If cell wall metabolism is to be studied in elongating tissue, for
example, the contribution which new cross walls might make to the overall metabolism of polysaccharide could be significant.
There are other qualitative aspects of the response of lettuce hypocotyls to GA
which distinguish it from the typical growth response to auxin. The effect of GA in
this tissue is to overcome light-induced inhibition of elongation growth (26); in dark-
The Physiology of Gibberellin-Induced Elongation
191
ness elongation is rapid and unaffected by GA, while in blue and far-red light elongation is inhibited and the inhibition can be overcome by GA. In this respect the response oflettuce to GA is similar to that demonstrated in intact plants, e.g., pea (17),
bean (13), and com (31). The response of tissues to auxin, however, does not exhibit
the same absolute light dependence; indeed, most auxin responses are unaffected by
the presence or absence of light.
A further point of distinction between the GA and auxin response concerns the
effect of a short pulse of hormone. Typically in aUxin-responsive tissues, growth rates
begin to decline to control rates soon after termination of the hormone pulse (32,33),
while in lettuce hypocotyl (34) and Avena internode sections (23) elongation growth
is sustained for many hours after the hormone supply is withdrawn.
We conclude that in excised lettuce hypocotyl sections, elongation in response to
GA is neither mediated through nor influenced by auxin, and that other physiological
characteristics of the GA response of lettuce distinguish it from the auxin response.
Physiology of Cell Elongation in GA-Treated Tissue
From the foregoing it is axiomatic that a study of GA-induced elongation must emphasize changes in the rate of cell elongation. Does GA influence the rate of cell elongation by altering the tensile properties of the cell wall or by affecting the osmotic concentration of the cell sap? Although until recently, circumstantial evidence favored the
latter, experiments with both lettuce hypocotyls (28, 29, 35) and Avena internodes
(25) show that changes in cell wall plasticity accompany GA-induced growth. In lettuce, for example, both direct measurements of cell wall plasticity using Instron techniques (35) and indirect measurements of the properties of the cell walls of living tissue (28) indicate that elongation growth and increased cell wall plasticity are parallel
events. During rapid growth, however, the osmotic concentration of the cell sap of
GA-treated tissues decreases (29).
We have resolved the roles of altered pressure potential (iJt p) and osmotic potential
(iJ; rr) in elongating lettuce hypocotyl tissue by incubating sections in dilute KCI or
NaCl. When sections are incubated in 10 mM KCI in the absence of GA, there is no
change in elongation rate, although the iJt 7T of the tissue decreases (becomes more negative) because of KCI uptake (Fig. 3). Sections incubated in GA alone exhibit an elevated elongation rate with a concomitant increase in iJt . When GA-treated sections are
incubated in KCl, however, the rate of elongation is fu~her enhanced, and the iJt 7T is
maintained at a constant level (Fig. 3). From these data we conclude that elongation
of lettuce hypocotyls is normally regulated by changes in cell wall extensibility, i.e.,
changed iJt p , and that the elongation rate can be further modified by changes in iJt 11'
In the absence of increased cell wall extensibility, an increase in the osmotic concentration of the cell sap has little direct influence on elongation.
Of the numerous hypotheses which have been advanced to account for changes in
cell wall extensibility, the acid growth hypothesis has gained wide acceptance, particularly among those investigating the response of tissue systems to auxin (36, 37). We
have examined both the elongation oflettuce hypocotyl sections at various pHs and the
capacity of the tissue to secrete protons in response to GA treatment (30). Although
192
R.L. Jones
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14
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40
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X
_
36
C
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r
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Time (hr)
24
36
48
. Ti me(hr)
Fig. 3. Analysis of growth (A), K+ uptake (8, D) and osmolarity of expressed scap (C) of lettuce
hypocotyl sections incubated in GA or H10. From Stuart and Jones (28)
hypocotyl sections elongate in response to media of low pH, growth is only 25% over
that of control sections incubated at pH 6.0, and furthermore, preincubation at low
pH does not affect the magnitude of the GA response when sections are subsequently
incubated in GA. Lettuce hypocotyl sections incubated in GA do not secrete protons,
although under certain conditions of fusicoccin treatment sections will acidify the incubation medium. These data provide convincing evidence that for lettuce, elongation
growth in response to GA treatment is not accompanied by proton secretion. These
data contrast with the observations of Hebard et al. (25), who have shown that acid
production accompanies GA treatment of Avena stem segments.
If protons are not responsible for changing the tensile properties of the cell wall,
we must consider other possible agents. Enzymes which cleave specific bonds in the
cell wall have been postulated by many to play an important role in regulating wall
extensibility, but the evidence has been equivocal. Most of the experimental evidence
is based on the extraction of enzymes from whole plants or tissue sections, and few
attempts have been made to localize the extracted enzymes in the cell wall. The fact
that extracted enzymes may use cell wall polysaccharides as substrates does not establish the enzymes as modifiers of the cell wall in intact tissue.
We have used a tissue centrifugation technique in an attempt to localize and characterize changes which might occur in cell wall polymers (40). This centrifugation
technique has been used by Terry and Bonner (41) and Terry et al. (42) to examine
changes in the polysaccharides of pea internode sections. Sections are inf1.ltrated with
water and then centrifuged at 1000 x g. This procedure removes the solution from the
The Physiology of Gibberellin-Induced Elongation
193
free space of the tissue with little detectable cytoplasmic contamination. Using this
technique Terry (40) has shown that following auxin treatment of pea internode sections, there is an increase in the xyloglucan concentration of the free space solution
which can be centrifuged from sections. This observation is in agreement with the experiments of Labavitch and Ray (43), who examined cell wall metabolism using conventional extraction techniques, and it provides evidence that the centrifugation technique does indeed measure changes in the cell wall of this tissue. We have begun to
examine the proteins of the extra-protoplastic fluid from auxin-treated pea stem sections by means of electrophoresis on polyacrylamide, and our preliminary data indicate qualitative differences between control and auxin-treated tissue.
We propose to continue the application of this centrifugation technique to the
problem of cell wall metabolism with emphasis on the role of GA in this process. Thus
far we have examined changes in polysaccharides of GA-treated pea internode sections.
In contrast to auxin, which increases the level of xylose and glucose in the free space
of pea internodes, GA does not significantly affect xylose, glucose, arabinose, galactose, or rhamnose in the solution centrifuged from sections (Table 1). These data also
Table 1. GA and the neutral sugar composition of
centrifugate from pea internodes a
I"g/gm initial weight
+GA
-GA
+GA/- GA
Ara
Xyl
Gal
Glu
8.6
9.5
0.91
10.1
10.6
0.95
21.2
22.2
0.95
19.2
19.9
0.95
Xyl
Gal
Glu
17.7
20.3
0.87
16
18.2
0.88
I"g/gm final weight
Ara
+GA
-GA
+ GA/- GA
a GA growth
7.2
8.7
0.83
8.4
9.7
0.87
= 148% of control
lend support to our observations on changes in water-soluble polysaccharides extracted
by homogenization from GA-treated lettuce hypocotyl sections (Table 2). GA treatment
does not enhance the metabolism of arabinose, xylose, galactose, or glucose in lettuce
hypocotyl sections. We conclude that GA treatment of pea internode or lettuce hypocotyl sections does not result in an enhancement of the metabolism of xyloglucan or a
similar alcohol-insoluble, water-soluble polysaccharide.
194
R.L. Jones
Table 2. The neutral sugar composition of the homogenate + external
medium of lettuce hypocotyls incubated ± GA
,ug!gm initial weight
+GA
-GA
+ GA/- GA
Ara
Xyl
Gal
Glu
128.2
98.2
1.31
73.5
58.7
1.25
257.4
225.1
1.14
73.7
73.8
1.00
Ara
Xyl
Gal
Glu
32.3
34.4
0.94
114.2
133.5
0.85
35.2
42.3
0.83
J.lg/gm final weight
+GA
-GA
+GA/- GA
56.6
69.9
0.81
Conclusions
The evidence that the GA s serve to stimulate cell elongation in plant stems is convincing. The rate of elongation is enhanced by changes in the extensibility of the cell wall,
but acidification of the cell wall does not seem to be involved in the process of cell
wall softening in lettuce as it may be in auxin-responsive tissues. Also by contrast with
auxins, there is no evidence for the turnover or synthesis ofaxyloglucan or similar
water-soluble polysaccharide in the tissues of pea or lettuce after GA treatment. Using
a centrifugation technique to isolate the cell wall free-space solution, we are pursuing
changes in both polysaccharides and proteins in the cell walls of GA-responsive tissues
with the aim of correlating changes in these polymers with altered rates of elongation
of the tissue.
Acknowledgment. I wish to thank Wendy K. Silk, David A. Stuart, Deborah 1. Durnam, and
Maurice E. Terry who completed much of the work cited above. This work was supported by
grants from the National Science Foundation (B~lS 75·18870 and PCM 78·13286).
References
1. Sachs R.M.: Annu. Rev. Plant Physiol. 16, 73-96 (1965)
2. Sachs. R.~1.. Bretz, c., Lang, A.: 1. Exp. Cell Res. 18, 230-244 (1959)
3. Brian, P.W.. Hemming, H.G.: Ann. Bot. 22,1-17 (1958)
4. Penny, D., Penny, P.: Can. J. Bot. 52,959-969 (1974)
5. Shibaoka. H.: Plant Cell Physiol. 13, 461-469 (1972)
6. Galston, A.W., Warburg, H.: Plant Physiol. 34, 16-22 (1959)
7. Nitsan, 1., Lang. A.: Dev. BioI. 12, 358 -3 76 (1965)
8. Arney, S.E., Mancinelli, P.: New Phytol. 65,161-175 (1966)
9. Liu. P.B., Loy,J.B.: Am. J. Bot. 63,325-336 (1976)
The Physiology of Gibberellin-Induced Elongation
195
10. Greulach, V.A., Haesloop. J.G.: Am. J. Bot. 45,566-570 (1958)
Il. Green, P.B.: Bot. Gaz. 137. 187 -202 (1976)
12. Cleland, R.E.: In: The Physiology of Plant Growth and Development. Wilkins, M.B. (ed.),
pp. 49-81. London: McGraw-Hili 1969
13. Bnan, P.W.: Int. Rev. Cyto!. 19, 229-266 (1966)
l~. Kuraishi. S., Muir, R.M.: Naturwissenschaften 50,337-338 (1963)
15. Skytt-Andersen, A., Muir, R.M.: Physio!. Plant.22, 354-363 (1969)
16. Heyn, A.N.J.: Proc. K. Akad. Wet. 33, 1045 -I 058 (1930)
17. Lockhart J.A.: Plant Physio!. 35,129-135 (1960)
18. Pa1eg, L.G.: Annu. Rev. Plant Physiol.16, 319-322 (1965)
19. Cleland, R.E., Thompson, M.L., Rayle, D.L., Purves, W.K.: Nature (Lond.) 214, 510-511
(1968)
20. Kaufman, P.B.: Physio!. Plant. 18,703-724 (1965)
21. Jones, R.A., Kaufman, P.B.: Plant Physio!. 24, 491-497 (1971)
22. Kaufman, P.B., Petering, L.B., Adams, P.B.: Am. 1. Bot. 56,918-927 (1969)
23. Montagne, M.J., Ikuma, H., Kaufman, P.B.: Plant Physio!. 51,1026-1032 (1973)
24. Adams, P.A., Motagne, M.J., Tepfer, M., Rayle, D.L., Ikuma, H.: Plant Physio!. 56,757-760
(1975)
25. Hebard, F.V., Amatangelo, S.J., Dayanandan, S.J., Kaufman, P.B.: Plant Physio!. 58,670-674
(1976)
26. Silk, W.K., Jones, R.L.: Plant Physio!. 56,267-272 (1975)
:7. Stuart, D.A., Durnam, D.L., Jones, R.L.: Planta 135,249-255 (1977)
28. Stuart, D.A., Jones, R.L.: Plant Physio!. 59, 61-68 (1977)
29. Stuart, D.A., Jones, R.L.: Plant Physio!. 61,180-183 (1978)
30. Stuart, D.A .. Jones, R.L.: Planta 142, 135-145 (1978)
31. Phinney, B.O.: Proc. Nat!. Acad. Sci. USA 42, 185-189 (1957)
32. De La Fuente, R.K., Leopold, A.C.: Plant Physio!. 45,19-24 (1969)
33. De La Fuente, R.K., Leopold, A.C.: Plant Physio!. 46,186-189 (1970)
34. Silk, W.K., Jones, R.L., Stoddart, 1.L.: Plant Physio!. 59, 211-216 (1977)
35. Silk, W.K.: Ph.D. Thesis, University of California, Berkeley (1977)
36. Cleland. R.E.: Proc. Nat!. Acad. Sci. USA 70, 3092-3093 (1973)
37. Cleland, R.E.: Plant Physio!. 58,210-213 (1976)
38. Masuda, Y., Yamamoto, Y.: Dev. Growth Differ. 11,287-294 (1970)
39. Fan, D.F., MacLachlan, G.A.: Can. J. Bot. 44,1025 (1966)
40. Terry, M.: Ph.D. Thesis, University of California, Davis (1978)
41. Terry, M.. Bonner, B.A.: Plant Physio!. (in press, 1980)
42. Terry, M.. Rubinstein, B., Bonner, B.A., Jones, R.L.: Abstr. 10th Int. Conf. Plant Growth Substances. p. 40 (1979)
~3. Labavitch, 1.M., Ray, P.M.: Plant Physio!. 53, 669-673 (1974)