Planta (Bed.) 111,279--296 (1973) 9 by Springer-Verlag 1973
Polar Auxin Transport and Auxin-induced Elongation in the Absence of Cytoplasmic Streaming W. Z. Can@, Mary Helen M. Goldsmith, and P. M. t~ay Department of Biological Sciences, Stanford University, Stanford, California 94305, USA Received December 18, 1972 Summary. When cytoplasmic streaming in oat and maize coleoptile ceils is completely inhibited by cytoehalasin B (CB), polar transport of auxin (indole-3acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40 % by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament
material. Introduction The suggestion that cytoplasmic streaming is responsible for the intracellular aspect of polar transport of auxin (indole-3-acetic acid, IAA) dates back to the earliest investigations of polar transport (Went, 1928) and remains almost axiomatic in current thinking about this phenomenon (Arisz, 1969; Leopold and de la Fuente, 1968; Newman, 1970). However, experimental support for the involvement of streaming in auxin transport is very indirect (DuBuy and Olson, 1940), and it is already clear t h a t streaming cannot be the rate limiting process in polar transport (Goldsmith, 1969 ; tIertel and Flory, t968). Streaming has also been invoked to explain the action of auxin on cell elongation (Galston and Purves, 1960 ; Thimann, 1948), based on the observation that streaming in eoleoptiles is promoted almost immediately upon exposure to auxin (Sweeney and Thimann, 1937, 1942).
The fungal metabolite cytochMasin B (CB) (Turner and Carter, 1972), inhibits certain types of cell movements (Carter, 1972) and m a y affect cytoplasmic microfilament systems of animal cells (Wessels et al., 1971). I n plant cells CB reversibly inhibits cytoplasmic streaming (Bradley, 1973; tterth et al., 1972; Mascarenhas and La Fountain, 1972) and certain 19 1Blanta(Berl.),Bd, iii
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o t h e r m o v e m e n t s ( W a g n e r et al., 1972; P a l e v i t z , 1972) in w h i c h involv e m e n t of m i c r o f i l a m e n t s is i m p l i c a t e d . U s i n g C B we h a v e t e s t e d w h e t h e r s t r e a m i n g is i n v o l v e d in p o l a r t r a n s p o r t a n d in t h e a c t i o n of a u x i n on e l o n g a t i o n . Materials and Methods Plant Material. Coleoptile segments 5 mm long were cut under dim red light from dark-grown seedlings of Avena sativa L. (cv. Victory) 3 days old, orZea mays L. (cv. Golden Cross Bantam T51) usually 6 days old. Stem segments 5 mm long were similarly cut from the third internode of etiolated seedlings of Pisum sativum L. (cv. Alaska) grown 7 days in the dark with occasional exposure to dim red light. Maize and pea seeds were from Ferry Morse Seed Co., Mountain View, Calif., and oats from U.S. Department of Agriculture, Branch Experiment Station, Aberdeen, Idaho, U.S.A. Chemicals. Cytochalasin B was provided by Dr. S. B. Carter, Imperial Chemical Industries, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K. The material was dissolved in dimethyl sulfoxide (DMSO) as an 0.25 % stock solution. The stock solution was added to aqueous media to give solutions of 2.5-50 ~zg CB/ml; the media therefore contained 0.1-2.0% DMSO. For all experiments, the control treatments contained the same concentration of DMSO but no CB. DMSO at the concentrations used had no effect, either on coleoptile and pea stem elongation, or on polar transport of auxin. Pretreatments. Prior to the elongation or transport experiments the segments were pretreated in control or CB media on a reciprocating shaker at 25~ in the case of coleoptile segments the hollow center of each segment was filled with pretreatment medium using a syringe. At the end of the pretreatment period and at approximately 1-h intervals during the subsequent transport or elongation experiments the cytoplasmic streaming in epidermal and parenehyma cells was observed under a bright field or a Nomarski differential interference contrast microscope at ca. 500 • magnification. For this purpose, paradermal sections were cut freehand with a razor blade from segments randomly selected from those being carried through the experiment. Light micrographs were taken on a Reichert Zetopan light microscope equipped with Nomarski optics. Electron Microscopy. Stem or coleoptile segments were cut longitudinally into quarters and fixed in 0.1 M veronal buffered 2 % glutaraldehyde, pH 7 at 0 ~for 1-2 h followed by 0.1 M phosphate-buffered 0.5% Os0~, pH 7, overnight a t 0 ~ The material was embedded in Epon, and silver sections obtained with a diamond knife were mounted on copper grids, stained with lead citrate, and examined with a Hitachi HU-11E electron microscope. Elongation Measurements. Segments were incubated in i mM potassium phosphate, pH 6.5, containing 2% DMSO and, where used, CB and IAA and, in the case of oat eoleoptiles only, 50 mM sucrose. The total length of each set of 20 coleoptile or pea stem segments was determined periodically by placing the segments end-toend in a transparent plastic trough mounted over a millimeter scale. Isotope Incorporation Experiments (Table 1.) Tissue segments (50 segments per sample with peas, f0 segments per sample with coleoptiles) were pretreated for 2 h (3 h in case of oats) in media containing 1 mM potassium phosphate, pH 6.5, and f.5 % DMSO without or with CB at the indicated concentrations. Then 0.5 ~Ci of glucose [1-3H] and 0.5 izCi of [14C]lencine, or in the case of the oat coleoptile experiment 0.25 ~zCi of [l~C]sucrose (50 mM) only, was added, along with IAA (17 tzM). Incubation was continued on a reciprocating shaker for 4 h with coleoptile segments
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and 2 h with pea segments, at 25 ~ Tissue was washed with ice water, ground in cold water, and centrifuged for 2 rain at 1000 • g to remove cell wall material; the latter was re-ground and washed with cold water. Soluble radioactivity was determined on an aliquot of the combined supernatants; after this, protein was precipitated with 9% trichloroacetic acid and washed. Radioactivity was determined for all fractions by liquid scintillation spectrometry. Uptake denotes the sum of soluble and cell-wallbound radioactivity. Auxin Transport. Transport was determined by placing coleoptile segments between 1.5 % agar donor and receiver blocks in the conventional manner (Goldsmith, 1969), after draining the pretreatment medium from the central cavity. Donor blocks contained 5 • 10_6 M IAA[5-3H] (Schwarz Bioreseareh, Orangeburg, N. Y., U.S.A. spec. act. 25 Ci/mmol). Donor and receiver blocks contained DMSO and CB at concentrations used in the pretreatment of the segments. Blocks were added directly to a dioxane-based scintillation solvent several hours prior to determination of radioactivity by liquid scintillation spectrometry. Chromatographic Identi/ication o/Transport Material. Donor and receiver blocks from a typical transport experiment were extracted 3 times with acetonitrile on ice overnight on a reciprocating shaker. Unlabeled IAA (10 ~g) was added to each extract as a carrier. The extracts were reduced to a small volume using a rotary vacuum evaporator, then spotted on Eastman Kodak silica gel thin-layer chromatography sheets No. 6061. The following solvent mixtures were used: A) methyl acetate:isopropanol:aqueous ammonia:water (22.5:17.5:9:1,u/v); B) butanol:chloroform (3:2) saturated with 0.05 N aqueous formic acid (organic phase used); C) isopropanol: aqueous ammonia: water (8:1 : 1, u/v). The chromatograms were scanned on a Packard Model 7 200 Radiochromatogram Scanner, then cut up and assayed by liquid scintillation spectrometry.
Results
E//ects o/ Cytochalasin B on Cytoplasmic Streaming I n t h e e p i d e r m a l a n d p a r e n c h y m a cells of maize a n d o a t coleoptiles a n d of p e a stem tissue, t h e n o r m a l p a t t e r n of vigorous l o n g i t u d i n a l bidirectional s t r e a m i n g is d i s s u p t e d w i t h i n 30 m i a after t r e a t m e n t with CB. C h a r a c t e r i s t i c a l l y t h e c y t o p l a s m begins to pile up in one to several conspicuous masses (Figs. 1-4). A t lower CB concentrations or prior to c o m p l e t e i n h i b i t i o n a t higher concentrations t h e c y t o p l a s m i c masses slowly m i g r a t e a n d r o t a t e . U n d e r complete i n h i b i t i o n b y CB t h e cytop l a s m w i t h i n t h e masses is s t a t i o n a r y e x c e p t for some B r o w n i a n m o t i o n of particles. R e c o v e r y of t h e n o r m a l s t r e a m i n g p a t t e r n occurs w i t h i n 30 rain after C B - i n h i b i t e d o a t or maize coleoptfle tissue is r e t u r n e d to water, even after long exposure to CB a t high concentrations (50 Ezg/m], 10 h). D u r i n g t h e r e c o v e r y process, t h e c y t o p l a s m i c masses slowly change in shape. T h e y m a y r o t a t e in place or m i g r a t e along t h e p e r i p h e r y of t h e cell. E v e n t u a l l y the masses f l a t t e n as s t r e a m s of c y t o p l a s m m o v e from t h e m along t h e cell surface. R e c o v e r y of s t r e a m i n g from CB i n h i b i t i o n is n o t r a p i d in p e a s t e m cells, a n d occurs o n l y after exposure to r e l a t i v e l y low c o n c e n t r a t i o n s 19"
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Figs. 1--4
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(5-10 txg/ml) of the drug. Irreversible effects begin to appear after 4-5h exposure even to 10~g/ml CB. The cytoplasmic masses begin to become vaeuolated and lose their compact shape, often forming biconcave bridges of cytoplasm across the cell lumen. The cytoplasm gradually acquires a watery, highly vesieulated appearance. During 24 h exposure to CB, the medium becomes milky due to bacterial growth, indicating leakage of solutes from the CB-treated segments into the medium (Christiansen, 1950). This is not observed in the control medium, nor with CB-treated oat or maize coleoptile segments. Peas also differ from oat and maize cells in sensitivity to CB. Oat and maize eoleoptile cells still display some cytoplasmic movement at 20 ~zg/ ml CB while streaming in pea stem cells is completely stopped b y 10 ~xg/ml CB (Table i). This difference m a y be due in part to differences in penetration of CB through the cuticle. With both pea and coleoptile segments, cells nearest to the cut surfaces in partially inhibited tissues are completely inhibited while cells in the center of the tissue segment m a y display nearly normal streaming. Poor uptake of CB by colcoptile tissue was also indicated b y the finding that the CB effect on streaming in the mid-part of segments could be enhanced by injecting CB solution into the central cavity of the coleoptile cylinder. For example, in oat coleoptile segments simply incubated in 20 Izg/ml CB, streaming was inhibited near the cut ends but not in the middle of the segments. If this solution was also injected into the central cavity, partial inhibition of streaming was seen throughout the segment as noted in Table 1. The addition of auxin had no apparent effect on the inhibition by CB of cytoplasmic streaming. Paradermal sections cut from CB-treated oat and pea segments were examined before and after the addition of 3 mg/1 IAA. Not even a transient burst of streaming occurred after addition of IAA.
Figs. 1--4. Nomarski interference contrast photomicrographs of living epidermal tissue in freehand paradcrmM sections. • 750 Fig. 1. Maize eoleoptile outer epidermis, untreated, showing streaming peripheral cytoplasm (PC) and vigorously streaming transvaeuolar cytoplasmic strands (VS). N nucleus Fig. 2. CB-treated maize eoleoptile, showing cytoplasmic masses (CM) formed in response to CB Fig. 3. Pea stem epidermis, untreated, showing nuclei (N) and streaming peripheral cytoplasm (PC) Fig. 4. CB-treated pea stem, showing cytoplasmic masses (CM), usually 2 or more per cell, of which 1 contains the nucleus
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Figs. 5--7. Electron micrographs of portions of cytoplasmic masses within CBtreated maize coleoptile epidermal cells Fig. 5. Grazing section through an end wall (CW) and adjacent cytoplasm showing microtubules (MT) and micro~ilament bundles (MF) lying near and parallel to the plasma membrane. • 24000
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Table 1. CB effects on streaming, elongation, and metabolism Elongation, and isotope uptake and incorporation, were measured after 4 h (coleoptile segments) or 2 h (peas) in 17 ~M IAA, as described in Methods. Data are expressed as percent of control treated with IAA and DMSO only, a n d each figure is the m e a n for duplicate samples of CB-treated and control segments except for the experiment with maize which involved single samples (..indicates measurements t h a t were omitted). CB (~g/ml)
Streaming a
Elongation (% of control)
Uptake (% of control)
Incorporation (% of control)
Sugar aa
Cell wall Protein
Leucine
Pea stem segments 1.25 2.5 5.0
~b
10
--
20 40
---
92 73 62 55 59 58
20 50
• c --
63 61
50
--
51
~ ~ c
. . 95 . . 82 83 63
.
. 102 . . 83 94 68
.
.
.
. 85
.
.
.
95 .
79 89 34
93 92 44
68 48
.. ..
38
107
Oat coleoptile segments 88 63
.. ..
Maize coleoptile segments 68
70
~- = normal; ~ ~ partial inhibition, cells contain slowly moving cytoplasmic masses; -- ~ completely inhibited, motionless cytoplasmic masses in all cells. b Normal except in cells near cut ends, which are ~ . c Moving cytoplasmic masses in most cells in interior of segments; complete inhibition in cells near cut ends. a~ Glucose was used with pea and maize segments, sucrose with oat segments.
Ultrastructural Observations B u n d l e s of m i e r o f i l a m e n t s a d j a c e n t t o a n d p a r a l l e l t o t h e cell s u r f a c e (Figs. 5 - 7 ) , a n d also j u s t o u t s i d e t h e n u c l e a r e n v e l o p e (Figs, 8, 9), w e r e frequently found within the cytoplasmic masses that form in CB-treated m a i z e e o l e o p t i l e e p i d e r m a l cells. P r o m i n e n t f i l a m e n t b u n d l e s w e r e also c o m m o n l y f o u n d within t h e n u c l e u s of b o t h e p i d e r m a l a n d p a r e n c h y m a
Fig. 6. Longitudinal section of outer epidermal wall (CW) and adjacent cytoplasm showing microtubules (Mr#) in cross section, adjacent microfilament bundles (MF) in oblique section, and mitochondrion (MC) with dense core. • 28000 Fig. 7. Enlarged view of single microfilament bundle in oblique section, showing indications of regular substructure or cross links. • 49000
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Figs. 8 and 9. Electronmicrographs of portions of cytoplasmic masses within CB-treated maize colcoptile epidermal cells Fig. 8. Microfilament bundles (MF) in oblique section, and rnitoehondrion with dense core (MC), adjacent to nucleus (N) containing intranuclear microfi]ament bundles (NF). • 23000
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cells of CB-treated maize coleoptiles (Fig. 8); no such bundles were seen in any of the nuclei observed in control (not CB-treated) cells. Cytoplasmic microfilament bundles were much more difficult to find in control than in CB-treated maize eoleoptile cells although a few microfilament bundles comparable to those shown in Figs. 5-7 were seen in control epidermal cells, as have been reported for oat and ungerminated rice coleoptile cells (O'Brien and Thimann, 1966; 0pik, 1972). Another peculiarity of CB-treatcd maize coleoptiles was the presence of a dense core within m a n y of the mitochondria of epidermal cells (Figs. 6, 8). These cores were not seen in control cells. No microfilament bundles could be clearly identified in our observations of either CB-treated or control epidermal or parenehyma cells from pea stem tissue.
E//ect o / C B on Elongation and Auxin Action Auxin promoted elongation in oat coleoptile and pea stem segments in which streaming was completely inhibited by CB (Figs. i0, i i). CB inhibited the overall elongation of oat coleoptile segments about 40 % during the first 4 h, but segments treated with IAA in the presence of CB nevertheless showed a doubling of elongation over the CB control (Fig. 10). A similar inhibition of overall elongation by CB was obtained with peas, but again in the presence of CB a substantial stimulation of elongation by I A A over the CB control was obtained dm~ the first 4 h (Fig. 11). With oat coleoptiles inhibition of elongation by CB increased to about 60 % in the period after 4 h, but promotion of elongation b y IAA above the CB control was nevertheless apparent throughout at least 28 h (Fig. 10). With peas, however, although the elongation rate in IAA @ CB was initially high the rate declined rapidly and elongation ceased altogether after 6 h (Fig. 11), due probably to degenerative secondary effects of CB on the cells noted above. With pea stem tissue the inhibition of early elongation b y CB increased with concentration up to a m a x i m u m of about 40 % inhibition, this plateau being reached at the same concentration of CB as caused complete inhibition of streaming (Table 1). More limited information on concentration dependence was obtained for coleoptile tissue (Table 1), but here also some inhibition of coleoptile segment elongation b y CB was observed under partial inhibition of streaming (20 ~g/ml CB) and about 40 % inhibition of early elongation was obtained when streaming was corn-
Fig. 9. Longitudinal section of microfilament bundle (MF) adjacent to nucleus (N), exhibiting substructure similar to that in Fig. 7 (arrow). • 29500
W. Z. Cande et aI. :
288 I00
Fig.I0
~
IAA
Fig. II
80
60
o
/
40 X 20
0
4
8
12
16 20 24 TIME(hours) 10
28
32
4
8
,2
TIME (hours) 11
,~
8~
Figs. 10 and 11. Effect of IAA on elongation of normal and CB-treated oat coleoptile (Fig. 10, left) and pea stem (Fig. ll, right) segments. Segments were pretreated 2 h at 25~ in buffered aqueous D~ISO without or with CB (50 ~g/ml in Fig. 10, 20 ~zg/ml in Fig. 11), then (zero time on graphs) incubation was continued in the same media with addition of IAA (3 ~xg/ml) to samples so indicated. Each point is mean elongation of 20 segments that were initially 8 mm (Fig. 10) or 5 mm (Fig. 11) long. Microscopic observations showed that streaming had been completely inhibited by CB prior to addition of IAA in both experiments
pletely stopped (50 Fg/ml CB). Bradley (1973) also found about 50% inhibition of elongation of internodal cells of Nitella by a concentration of CB (30 ~xg/ml) t h a t completely inhibited streaming, but he did not s t u d y the concentration dependence of inhibition of elongation.
Metabolic E//ects o / C B Table 1 summarizes tests of the effects of CB on sugar and amino-acid uptake, and on short-term incorporation of sugar into cell wall polymers and of leucine into protein. Higher concentrations of CB caused some inhibition of uptake and incorporation. I n the ease of peas this occurred only at CB concentrations above t h a t required to stop streaming completely and to cause m a x i m u m inhibition of elongation. With eoleoptile segments inhibition of sugar uptake and incorporation was considerable at 50 Fg/ml CB but only slight at 20 Fg/ml CB, even t h o u g h inhibition of elongation was almost m a x i m u m at the latter concentration. Bradley (1973) also found only a slight inhibition of lencine incorporation in Nitdla b y 30 ~zg/ml CB. E//ect o / C B on A u x i n Transport Fig. 12 shows t h a t [aH]IAA was transported in a polar fashion in maize coleoptile segments in which streaming had been completely inhib-
Auxin and Cytoplasmic Streaming
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~-000 Fig.15
Fig.12
6000
~000
4000
~-000
2000
IO00
. /
oB)/
o
E o
/ / / /
Acropelol (• _
~
_/ I
I
2
TIME (hours) 12
0
I
2
5
TIME (hours) 13
Figs. 12 and 13. Effect of CB on polar transport of [3H]IAA in maize (Fig. 12, left) and oat (Fig. 13, right) coleoptile segments. The segments used were 8 or 5 mm long, and the donor blocks contained 25000 or 40000 cpm, respectively; 50 ~g/ml CB was used in pretreatment and in donor and receiver blocks of CB series. Microscopicobservations showed that in both experiments streaming had been completely inhibited by CB prior to supplying donor blocks and remained completely inhibited in the CBtreated segments throughout the transport period. Donor blocks were applied to the apical ends of the segments (basipetal transport) except in the data marked aeropetal in Fig. 12 where donor was applied to the base and receiver to the apex
ited by CB. I n CB-treated segments the arrival of the auxin front (intereept on time axis) was slightly delayed, but the flux (slope of the arrival curve) was similar to that of the controls. I A A was also transported by oat eoleoptile segments in which streaming had been blocked by CB (Fig. 13). I n this ease the arrival of the front was more substantially delayed by CB but the flux was again similar to the control. Fig. 14 shows that most of the radioactivity that was transported into receiver blocks from [sI-I]IAA donors in these experiments chromategraphed like I A A (Rf =0.61 using solvent A listed in Methods). This was true also for two other solvent systems (for both peak radioactivity and known IAA, R f = 0 . 8 3 in solvent B and 0.66 in solvent C). The IAA peak accounted for 85% of the radioactivity in receiver blocks. The [att]IAA stock used to prepare the donors contained a substantial proportion (30 % ) of decomposition products (peaks at origin and near front in Fig. 14 A) that could not be eliminated by reehromatography because of instability
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et al. :
10 5 J o -X
0
A
B 0 L i~
H
r
F
IAA
0
Fig. 14. Radioscans of thin-layer chromatograms of (A)[aH]IAA stock used in donor blocks, and (B) radioactive material that was transported into receiver blocks by maize coleoptiles. 0 origin; F solvent front; bar indicates position of known IAA chromatographed in parallel with radioactive samples. Solvent A was used
Table 2. Effect of CB on auxin transport through segments cut from coleoptiles of different lengths Sections 5 mm long were cut from 4--7-day old eoleoptiles starting 3, 5, or 8 mm below the tip, respectively, in the case of coleoptiles 1.5, 3.5, and 5.5 cm long. Sections were pretreated 2 h in 2% DMSO with or without 50 pg/ml CB. The transport period was 2 h with donors containing 50000 cpm IAA [SH]. Each figure is the mean of triplicate specimens (figures in parentheses are average deviations). Coleoptile length (cm)
Mean length of subepidermal cells a (~m)
Ratio of cell lengths
nil in receivers (cpm) Control
1.5 3.5 5.5
44 127 206
i.O 2.9 4.7
9620 (560) 7780 (550) 16630 (1180) 11520 (810) 17080 (1960) 10750 (850)
CB
Inhibition by CB (%) 19 31 37
a Average length of 80-100 subepidermal parenehyma cells located midway between vascular bundles.
of [aH]IAA. However, very little of these products were f o u n d i n receiver blocks (Fig. 14B) a n d the small a m o u n t s t h a t were seen p r o b a b l y arose b y decomposition of [atI]IAA d u r i n g e x t r a c t i o n a n d c h r o m a t o g r a p h y after the t r a n s p o r t into receiver blocks.
Auxin and Cytoplasmic Streaming
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The effect of CB on auxin transport through segments cut from coleoptiles of different lengths was determined (Table 2) to see if the effect depended on cell length. Inhibition increased with length of the source eoleoptfles. A similar result was obtainedin a comparable second experiment. Discussion
Evidence that streaming was totally stopped in CB-treated tissue was provided not only by microscopic observations but by the finding that after such tissue was centrifuged, the cytoplasm and organelles remained at the centrifugal ends of the cells for hours (Goldsmith and l~ay, 1973). The persistence of polar transport and IAA-induced elongation during complete inhibition of streaming by CB shows not only that streaming is not required for either process but also that promotion of streaming by auxin is not the means by which auxin promotes cell enlargement. The present experiments directly confirm the suggestions advanced previously (Clark, 1938; Showacre and DuBuy, 1947 ; van der Weij, 1932)that auxininduced growth and polar transport are largely independent of streaming. The disputes about the mode of action and specificity of CB (Burnside and Manasck, 1972; Estensen et al., 1971 ; Forer et al., 1972; Krishan and Whitlock, 1972 ; Holtzer and Sanger, 1972) do not affect the strength of the conclusions cb.awn above. The crucial point is that tissues in which streaming has been stopped completely by CB prior to exposure to IAA show substantial auxin transport and promotion of elongation by IAA during the first few hours. The more severe inhibition of elongation by CB in the period after 6 h is not pertinent to the question of primary auxin action and in the case of peas is doubtless due to the degenerative effects of CB on the cells which can be observed within 6 h. Possible Role o/Streaming in Cell Enlargement The partial inhibition of early elongation by CB in the three tissues studied here suggests a beneficial (although not essential) role of streaming in cell enlargement. However, the inhibition could be due to effects of CB other than on streaming; for example, the reported inhibition of sugar uptake by CB (Itaslam, 1972; Estensen and Plagemann, 1972; Kletzien et al., 1972). Since elongation of neither pea nor oat segments is benefited by exogenous sugar during the first 6 h (Christiansen and Thimann, 1950; Ordin et al., 1956) and exogenous sugar was not even provided in the experiments with peas, an effect on sugar uptake could not explain the partial inhibition by CB of early elongation, but it could conceivably explain the stronger inhibition of eoleoptile elongation in the post-6 h period, during which elongation of eoleoptiles becomes dependent on sugar (Schneider, 1938).
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The inhibition of early elongation reaches a m a x i m u m of about 40 % at a CB concentration that just completely inhibits streaming, whereas effects of CB on sugar and amino acid uptake and incorporation increase with CB concentration above this point (Table 1). This suggests that the partial inhibition of early elongation by CB is associated with inhibition of streaming rather than of metabolism. However, since inhibition of streaming is accompanied b y formation of local cytoplasmic masses within the ceils (Figs. 1-4) is could be that withdrawal of much of the cytoplasm into these masses, rather than lack of streaming, is what actually depresses elongation. I n contrast to the present results, elongation of tip-growing cells such as pollen tubes and root hairs is completely inhibited at the same concentrations of CB that inhibit streaming ( g e r t h et al., 1972 ; Masearenhas and La Fountain, 1972). I n tip-growing cells, streaming m a y be essential to supply material synthesized in remote parts of the cell to the region of local growth. Mechanism o / A u x i n Transport I n the absence of streaming, movement of auxin from the apical to the basal and of each cell during polar transport must either be due to diffusion or to a special transport mechanism. Since there is no evidence for the latter (Goldsmith and Ray, 1973) it is of interest to consider whether diffusion could be the mechanism of intraeellular transport. The concentration gradient dc/dx that would be required to transport an amount Q of IAA per see through any cross section of the coleoptile by diffusion m a y be found from Q - - - D A ( d c / d x ) , where A is the crosssectional area of the tissue (0.75 m m 2 for Arena coleoptile; Goldsmith, 1966). From its molecular weight I A A should have a diffusion coefficient (D) of about 7 • 10 -6 cm2/s, a value confirmed by experiment (Larsen, 1944). If in 1 h 1000 dpm of radioactive IAA ( Q ~ 0.3 dpm/s) arc transported from a eoleoptile section into a receiver, dc/dx comes to about 6 • l0 s dpm/cm 3 per em of length. This corresponds to a concentration difference of about 6 • 104 dpm/cm s, between the apex and base of a typical parenchyma ceil about 100 ~m in length, required to sustain by diffusion a delivery of 1000 dpm/h into a receiver. The average concentration of IAA in the transport stream in cells at the basal end of a maize coleoptile segment, next to a receiver, which is the lowest concentration t h a t will exist in the segment since transport stream concentration decreases basipetally (Newman, 1970), will now be estimated. IAA is transported in maize eoleoptiles at a velocity of approximately ] cm/h. In order to deliver 1000 dpm/h to a receiver the basal end of a segment must therefore contain 1000 dpm per em of length or, since its cross sectional area is 0.75 m m 2, the cells at its basal end must contain 13 • 104 dpm/cm a.
Auxin and Cytoplasmic Streaming
293
Therefore, the concentration difference required to transport I A A through each cell by diffusion is less than half of the transport stream concentration. This means t h a t diffusion could account for the intraecllular portion of polar transport. An active mechanism would be needed only to transfer IAA from each cell to the next. The existence of an active cellto-cell transport mechanism for IAA in polar transport has already been inferred from experimental evidence (Christie and Leopold, 1965 ; Hertel and Flory, 1968). The moderate inhibition by CB of polar transport suggests that streaming increases transport somewhat above that due to diffusion. If this is the case, inhibition of polar transport by CB should increase with cell length since the concentration difference required to support diffusion - and thus the increase in IAA concentration that could be achieved at the basal end of each cell by stirring its contents b y streaming - - should increase with cell length. This result was obtained (Table 2). I t is, of course, not excluded that the greater percent inhibition of transport by CB in segments cut from longer eoleoptfles is due to differences other than cell length between coleoptiles of different physiological ages.
Nature o/Cytoohalasin Action It is clear from the observations (Figs. 5-9) that at least in maize eoleoptile cells CB does not cause depolymerization or disorientation of microfilaments or microfilament bundles when it inhibits streaming. A similar observation on NitelIa cells was made by Bradley (1973). If mierofflament bundles are the propulsive organelles for streaming (Nagai and Rebhun, 1966; O'Brien and Thimann, 1966 ; Kamitsubo, 1972 ; Daniel and Jgrlfors, 1972), CB must suppress their action without disorganizing them. That CB induces formation of abnormal intranuclear bundles of mierofflaments (Fig. 8) suggests, on the contrary, that CB m a y cause a hypercondensation of microfilament subunits, like vinblastine does in the case of mierotubule protein (Bryan, 1972). Observations quoted in Table 1 and in the text indicate that CB also has metabolic and membrane effects t h a t do not appear to be related to microfilament function. We thank Margery iVi.Ray for preparation of specimens for Figs. 5-9. This work was supported by grants GB-18499 and 33927X from the 1National Science Foundation, and Grants GM NB 16530 and GM-08886 from the National Institutes of Health.
294
W.Z. Cande et al. : References
Arisz, W. It. : Intercellular polar transport and the role of the plasmodesmata in coleoptiles and Vallisneria leaves. Acta bot. neerl. 18, 14-38 (1969). Bradley, M. O. : Microfilaments and cytoplasmic streaming: inhibition of streaming with cytochalasin. J. Cell Sci. 12, 327-343 (1973). Bryan, J. : Definition of three classes of binding sites in isolated microtubule crystals. Biochemistry 11, 2611-2616 (1972). Burnside, B., Manasek, F. J. : Cytochalasin B : problems in interpreting its effects on cells. Develop. Biol. 27, 443-444 (1972). Carter, S. B. : The cytochalasins as research tools in cytology. Endeavour 31, 77-82 (1972). Christiansen, G. S. : The metabolism of stem tissue during growth and its inhibition. V. ~ature and significance of the exudate. Arch. Biochem. Biophys. 29, 354-368 (1950). Christiansen, G. S., Thimann, K. V. : The metabolism of stem tissue during growth and its inhibition. I. Carbohydrates. Arch. Biochem. Biophys. 26, 230-247 (1959). Christie, A. E., Leopold, A. C. : Entry and exit of indoleacetic acid in corn coleoptiles. Plant Cell Physiol. 6, 453465 (1965). Clark, W. G. : ElectricM polarity and auxin transport. Plant Physiol. 13, 529-552 (1938). Daniel, J. W., J~rlfors, U. : Plasmodial ultrastructure of the myxomycete Physarum polycephalum. Tissue and Cell 4, 15-36 (1972). Du Buy, H. G., 01son, R. A. : The relation between respiration, protoplasmic streaming and auxin transport in the Avena coleoptile, using a polarographic microrespirometer. Amer. J. Bot. 27, 401-413 (1940). Estensen, R. D., Plagemann, P. G. W.: Cytochalasin B: inhibition of glucose and glucosamine transport. Proc. nat. Acad. Sci. (Wash.) 69, 1430-1434 (1972). Estensen, R. D., ]~osenberg, M., Sheridan, J. D. : Cytochalasin B : microfilaments and "contractile" processes. Science 173, 356-357 (1971). Forer, A., Emmersen, J., Behnke, 0.: Cytochalasin B: does it affect actin-like filaments ? Science 175, 774-776 (1972). Galston, A. W., Purves, W. K. : The mechanism of action of auxin. Ann. Rev. Plant Physiol. 11, 239-276 (1960). Goldsmith, )/L It. M. : Movement of indoleaeetic acid in coleoptiles of Avena sativa L. II. Suspension of polarity by total inhibition of the basipetal transport. Plant Physiol. 41, 15-27 (1966). Goldsmith, M. It. M. : Transport of plant growth regulators. In: The physiology of growth and development, M. B. Wilkins, ed., p. 127-162. London: McGraw-Hill Book Co., 1969. Goldmith, M. H. M., l~ay, P. M. : Intracellular loeatization of the active process in polar transport of auxin. Planta (Berl.) 1ll, 297-314 (1973). Goldsmith, M. H.M., Thimann, K. V. : Some characteristics of movement o1 indoleacetic acid in coleoptiles of Arena. I. Uptake, destruction, immobilization and distribution of IAA during basipetal translocation. Plant Physiol. 37, 492505 (1962).
Auxin and Cytoplasmic Streaming
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Haslam, R. J.: Inhibition of blood platelet function by cytochalasins: effects on thrombosthenin and on glucose metabolism. Biochem. J. 127, 34P (1972). Hertel, R., Flory, R.: Auxin movement in corn eoleoptiles. Planta (Berl.) 82, 123-144 (1968). Herth, W., Franke, W. W., Vanderwoude, W. J. : Cytochalasin stops tip growth in plants. Naturwissenschaften 59, 38-39 (1972). Holtzer, H., Sanger, J. W.: Cytochalasin B: mierofilaments, ceil movement and what else ? Develop. Biol..27, 444-446 (1972). Kamitsubo, E. : Mobile protoplasmic fibrils in cells of Characeae. Protoplasma (Wien) 74, 53-70 (1972). Kletzien, R. F., l~erdue, J. F., Springer, A.: Cytochalasin A and B. Inhibition of sugar uptake in cultured cells. J. biol. Chem..247, 2964-2966 (1972). Krishan, A., Whitlock, S. : Cytochalasin B: time-lapse cinematographic studies on its effects on cytokinesis. J. Cell Biol. 54, 657-664 (1972). Larsen, P. : 3-indoleaeetaldehyde as a growth hormone in higher plants. Dansk Botan. Ark. 11, 11-129 (1944). Leopold, A. C., de la Fuente, R. K. : A view of polar auxin transport. In: Transport of plant hormones, p. 24-47, Y. Vardar, ed., Amsterdam: North-Holland Publ. Co.1968. Mascarenhas, J. P., La Fountain, J. : Protoplasmic streaming, cytochalasin B and growth of the pollen tube. Tissue and Cell 4, 11-14 (1972). Nagai, R., P~ebhun, L. I.: Cytoplasmic microfilaracnts in streaming Nitella cells. J. Ultrastruct. Res. 14, 571-589 (1966). Newman, I. A. : Auxin transport in Avena. I. Indoleacetic acid-C14 distributions and speeds. Plant Physiol. 46, 263-272 (1970). O'Brien, T. P., Thimann, K. V. : Intracellular fibers in oat coleoptile cells and their possible significance in cytoplasmic streaming. Proc. nat. Acad. Sci. (Wash.) 56, 888-894 (1966). ()9ik, H. : Some observations on coleoptile cell ultrastructure in ungerminated grains of rice (Oryza sativa L.). Planta (Berl.) 10.2, 61-71 (1972). Ordin, L., Applewhite, T. H., Bonner, J. : Auxin-induced water uptake by Avena coleoptile sections. Plant Physiol. 31, 44-53 (1956). Palevitz, B. A.: Effect of cytoehalasin B on stomatal differentiation in Allium. J. Cell Biol. 55, 198a (1972). Schneider, C. L. : The interdependence of auxin and sugar for growth. Amer. J. Bot. '25, 258 270 (1938). Showaere, J. L., du Buy, K. G. : The relationship of water availability and auxin in the growth of Avena coleoptiles and its meaning for a theory of tropisms. Amer. J. Bot. 84, 175-182 (1947). Sweeney, B. M., Thimann, K. V. : The effect of auxins on protoplasmic streaming, II. J. gen. Physiol. 21, 439-461 (1937). Sweeney, B. M., Thimann, K. V. : The effect of auxins on protoplasmic streaming, III. J. gen. Physiol..25, 841-854 (1942). Thimann, K. V. : Plant growth hormones. In: The hormones, physiology, chemistry and applications, vol. 1, p. 1-76, G. Pineus and K. V. Thimann, eds. New York: Acad. Press 1948. Turner, W. G., Carter, S. B.: The chemistry and some biological properties of the cytochalasins. Biochem. J. 127, 1P (1972). 20 Planta (Berl.), Bd. 111
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Wagner, G., Haupt, W., Laux, A. : Reversible inhibition of chloroplast movement by cytochalasin B in the green alga Mougeotia. Science 176, 808-809 (1972). Weij, H. G., van der: Der Mechanismus des Wuchstofftransportes. Ree. Tray. Bet. N~erl. 29, 379-496 (1932). Went, F. W. : Wuchstoff und Wachstum. Rec. Tray. Bet. N~erl. 25, 1-116 (1928}. Wessels, N. K., Spooner, B. S.,Ash, J. F., Bradley, M. 0., Luduefia, M. A., Taylor, E. L., Wrenn, J. T., Yamada, K. M. : Mierolilaments in cellular and developmental processes. Science 171, 135-143 (1971).
Polar Auxin Transport and Auxin-induced Elongation in the Absence of Cytoplasmic Streaming W. Z. Can@, Mary Helen M. Goldsmith, and P. M. t~ay Department of Biological Sciences, Stanford University, Stanford, California 94305, USA Received December 18, 1972 Summary. When cytoplasmic streaming in oat and maize coleoptile ceils is completely inhibited by cytoehalasin B (CB), polar transport of auxin (indole-3acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40 % by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament
material. Introduction The suggestion that cytoplasmic streaming is responsible for the intracellular aspect of polar transport of auxin (indole-3-acetic acid, IAA) dates back to the earliest investigations of polar transport (Went, 1928) and remains almost axiomatic in current thinking about this phenomenon (Arisz, 1969; Leopold and de la Fuente, 1968; Newman, 1970). However, experimental support for the involvement of streaming in auxin transport is very indirect (DuBuy and Olson, 1940), and it is already clear t h a t streaming cannot be the rate limiting process in polar transport (Goldsmith, 1969 ; tIertel and Flory, t968). Streaming has also been invoked to explain the action of auxin on cell elongation (Galston and Purves, 1960 ; Thimann, 1948), based on the observation that streaming in eoleoptiles is promoted almost immediately upon exposure to auxin (Sweeney and Thimann, 1937, 1942).
The fungal metabolite cytochMasin B (CB) (Turner and Carter, 1972), inhibits certain types of cell movements (Carter, 1972) and m a y affect cytoplasmic microfilament systems of animal cells (Wessels et al., 1971). I n plant cells CB reversibly inhibits cytoplasmic streaming (Bradley, 1973; tterth et al., 1972; Mascarenhas and La Fountain, 1972) and certain 19 1Blanta(Berl.),Bd, iii
280
W . Z . Cande et al. :
o t h e r m o v e m e n t s ( W a g n e r et al., 1972; P a l e v i t z , 1972) in w h i c h involv e m e n t of m i c r o f i l a m e n t s is i m p l i c a t e d . U s i n g C B we h a v e t e s t e d w h e t h e r s t r e a m i n g is i n v o l v e d in p o l a r t r a n s p o r t a n d in t h e a c t i o n of a u x i n on e l o n g a t i o n . Materials and Methods Plant Material. Coleoptile segments 5 mm long were cut under dim red light from dark-grown seedlings of Avena sativa L. (cv. Victory) 3 days old, orZea mays L. (cv. Golden Cross Bantam T51) usually 6 days old. Stem segments 5 mm long were similarly cut from the third internode of etiolated seedlings of Pisum sativum L. (cv. Alaska) grown 7 days in the dark with occasional exposure to dim red light. Maize and pea seeds were from Ferry Morse Seed Co., Mountain View, Calif., and oats from U.S. Department of Agriculture, Branch Experiment Station, Aberdeen, Idaho, U.S.A. Chemicals. Cytochalasin B was provided by Dr. S. B. Carter, Imperial Chemical Industries, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K. The material was dissolved in dimethyl sulfoxide (DMSO) as an 0.25 % stock solution. The stock solution was added to aqueous media to give solutions of 2.5-50 ~zg CB/ml; the media therefore contained 0.1-2.0% DMSO. For all experiments, the control treatments contained the same concentration of DMSO but no CB. DMSO at the concentrations used had no effect, either on coleoptile and pea stem elongation, or on polar transport of auxin. Pretreatments. Prior to the elongation or transport experiments the segments were pretreated in control or CB media on a reciprocating shaker at 25~ in the case of coleoptile segments the hollow center of each segment was filled with pretreatment medium using a syringe. At the end of the pretreatment period and at approximately 1-h intervals during the subsequent transport or elongation experiments the cytoplasmic streaming in epidermal and parenehyma cells was observed under a bright field or a Nomarski differential interference contrast microscope at ca. 500 • magnification. For this purpose, paradermal sections were cut freehand with a razor blade from segments randomly selected from those being carried through the experiment. Light micrographs were taken on a Reichert Zetopan light microscope equipped with Nomarski optics. Electron Microscopy. Stem or coleoptile segments were cut longitudinally into quarters and fixed in 0.1 M veronal buffered 2 % glutaraldehyde, pH 7 at 0 ~for 1-2 h followed by 0.1 M phosphate-buffered 0.5% Os0~, pH 7, overnight a t 0 ~ The material was embedded in Epon, and silver sections obtained with a diamond knife were mounted on copper grids, stained with lead citrate, and examined with a Hitachi HU-11E electron microscope. Elongation Measurements. Segments were incubated in i mM potassium phosphate, pH 6.5, containing 2% DMSO and, where used, CB and IAA and, in the case of oat eoleoptiles only, 50 mM sucrose. The total length of each set of 20 coleoptile or pea stem segments was determined periodically by placing the segments end-toend in a transparent plastic trough mounted over a millimeter scale. Isotope Incorporation Experiments (Table 1.) Tissue segments (50 segments per sample with peas, f0 segments per sample with coleoptiles) were pretreated for 2 h (3 h in case of oats) in media containing 1 mM potassium phosphate, pH 6.5, and f.5 % DMSO without or with CB at the indicated concentrations. Then 0.5 ~Ci of glucose [1-3H] and 0.5 izCi of [14C]lencine, or in the case of the oat coleoptile experiment 0.25 ~zCi of [l~C]sucrose (50 mM) only, was added, along with IAA (17 tzM). Incubation was continued on a reciprocating shaker for 4 h with coleoptile segments
Auxin and Cytoplasmic Streaming
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and 2 h with pea segments, at 25 ~ Tissue was washed with ice water, ground in cold water, and centrifuged for 2 rain at 1000 • g to remove cell wall material; the latter was re-ground and washed with cold water. Soluble radioactivity was determined on an aliquot of the combined supernatants; after this, protein was precipitated with 9% trichloroacetic acid and washed. Radioactivity was determined for all fractions by liquid scintillation spectrometry. Uptake denotes the sum of soluble and cell-wallbound radioactivity. Auxin Transport. Transport was determined by placing coleoptile segments between 1.5 % agar donor and receiver blocks in the conventional manner (Goldsmith, 1969), after draining the pretreatment medium from the central cavity. Donor blocks contained 5 • 10_6 M IAA[5-3H] (Schwarz Bioreseareh, Orangeburg, N. Y., U.S.A. spec. act. 25 Ci/mmol). Donor and receiver blocks contained DMSO and CB at concentrations used in the pretreatment of the segments. Blocks were added directly to a dioxane-based scintillation solvent several hours prior to determination of radioactivity by liquid scintillation spectrometry. Chromatographic Identi/ication o/Transport Material. Donor and receiver blocks from a typical transport experiment were extracted 3 times with acetonitrile on ice overnight on a reciprocating shaker. Unlabeled IAA (10 ~g) was added to each extract as a carrier. The extracts were reduced to a small volume using a rotary vacuum evaporator, then spotted on Eastman Kodak silica gel thin-layer chromatography sheets No. 6061. The following solvent mixtures were used: A) methyl acetate:isopropanol:aqueous ammonia:water (22.5:17.5:9:1,u/v); B) butanol:chloroform (3:2) saturated with 0.05 N aqueous formic acid (organic phase used); C) isopropanol: aqueous ammonia: water (8:1 : 1, u/v). The chromatograms were scanned on a Packard Model 7 200 Radiochromatogram Scanner, then cut up and assayed by liquid scintillation spectrometry.
Results
E//ects o/ Cytochalasin B on Cytoplasmic Streaming I n t h e e p i d e r m a l a n d p a r e n c h y m a cells of maize a n d o a t coleoptiles a n d of p e a stem tissue, t h e n o r m a l p a t t e r n of vigorous l o n g i t u d i n a l bidirectional s t r e a m i n g is d i s s u p t e d w i t h i n 30 m i a after t r e a t m e n t with CB. C h a r a c t e r i s t i c a l l y t h e c y t o p l a s m begins to pile up in one to several conspicuous masses (Figs. 1-4). A t lower CB concentrations or prior to c o m p l e t e i n h i b i t i o n a t higher concentrations t h e c y t o p l a s m i c masses slowly m i g r a t e a n d r o t a t e . U n d e r complete i n h i b i t i o n b y CB t h e cytop l a s m w i t h i n t h e masses is s t a t i o n a r y e x c e p t for some B r o w n i a n m o t i o n of particles. R e c o v e r y of t h e n o r m a l s t r e a m i n g p a t t e r n occurs w i t h i n 30 rain after C B - i n h i b i t e d o a t or maize coleoptfle tissue is r e t u r n e d to water, even after long exposure to CB a t high concentrations (50 Ezg/m], 10 h). D u r i n g t h e r e c o v e r y process, t h e c y t o p l a s m i c masses slowly change in shape. T h e y m a y r o t a t e in place or m i g r a t e along t h e p e r i p h e r y of t h e cell. E v e n t u a l l y the masses f l a t t e n as s t r e a m s of c y t o p l a s m m o v e from t h e m along t h e cell surface. R e c o v e r y of s t r e a m i n g from CB i n h i b i t i o n is n o t r a p i d in p e a s t e m cells, a n d occurs o n l y after exposure to r e l a t i v e l y low c o n c e n t r a t i o n s 19"
282
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et al. :
Figs. 1--4
Auxin and Cytoplasmic Streaming
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(5-10 txg/ml) of the drug. Irreversible effects begin to appear after 4-5h exposure even to 10~g/ml CB. The cytoplasmic masses begin to become vaeuolated and lose their compact shape, often forming biconcave bridges of cytoplasm across the cell lumen. The cytoplasm gradually acquires a watery, highly vesieulated appearance. During 24 h exposure to CB, the medium becomes milky due to bacterial growth, indicating leakage of solutes from the CB-treated segments into the medium (Christiansen, 1950). This is not observed in the control medium, nor with CB-treated oat or maize coleoptile segments. Peas also differ from oat and maize cells in sensitivity to CB. Oat and maize eoleoptile cells still display some cytoplasmic movement at 20 ~zg/ ml CB while streaming in pea stem cells is completely stopped b y 10 ~xg/ml CB (Table i). This difference m a y be due in part to differences in penetration of CB through the cuticle. With both pea and coleoptile segments, cells nearest to the cut surfaces in partially inhibited tissues are completely inhibited while cells in the center of the tissue segment m a y display nearly normal streaming. Poor uptake of CB by colcoptile tissue was also indicated b y the finding that the CB effect on streaming in the mid-part of segments could be enhanced by injecting CB solution into the central cavity of the coleoptile cylinder. For example, in oat coleoptile segments simply incubated in 20 Izg/ml CB, streaming was inhibited near the cut ends but not in the middle of the segments. If this solution was also injected into the central cavity, partial inhibition of streaming was seen throughout the segment as noted in Table 1. The addition of auxin had no apparent effect on the inhibition by CB of cytoplasmic streaming. Paradermal sections cut from CB-treated oat and pea segments were examined before and after the addition of 3 mg/1 IAA. Not even a transient burst of streaming occurred after addition of IAA.
Figs. 1--4. Nomarski interference contrast photomicrographs of living epidermal tissue in freehand paradcrmM sections. • 750 Fig. 1. Maize eoleoptile outer epidermis, untreated, showing streaming peripheral cytoplasm (PC) and vigorously streaming transvaeuolar cytoplasmic strands (VS). N nucleus Fig. 2. CB-treated maize eoleoptile, showing cytoplasmic masses (CM) formed in response to CB Fig. 3. Pea stem epidermis, untreated, showing nuclei (N) and streaming peripheral cytoplasm (PC) Fig. 4. CB-treated pea stem, showing cytoplasmic masses (CM), usually 2 or more per cell, of which 1 contains the nucleus
284
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Figs. 5--7. Electron micrographs of portions of cytoplasmic masses within CBtreated maize coleoptile epidermal cells Fig. 5. Grazing section through an end wall (CW) and adjacent cytoplasm showing microtubules (MT) and micro~ilament bundles (MF) lying near and parallel to the plasma membrane. • 24000
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Table 1. CB effects on streaming, elongation, and metabolism Elongation, and isotope uptake and incorporation, were measured after 4 h (coleoptile segments) or 2 h (peas) in 17 ~M IAA, as described in Methods. Data are expressed as percent of control treated with IAA and DMSO only, a n d each figure is the m e a n for duplicate samples of CB-treated and control segments except for the experiment with maize which involved single samples (..indicates measurements t h a t were omitted). CB (~g/ml)
Streaming a
Elongation (% of control)
Uptake (% of control)
Incorporation (% of control)
Sugar aa
Cell wall Protein
Leucine
Pea stem segments 1.25 2.5 5.0
~b
10
--
20 40
---
92 73 62 55 59 58
20 50
• c --
63 61
50
--
51
~ ~ c
. . 95 . . 82 83 63
.
. 102 . . 83 94 68
.
.
.
. 85
.
.
.
95 .
79 89 34
93 92 44
68 48
.. ..
38
107
Oat coleoptile segments 88 63
.. ..
Maize coleoptile segments 68
70
~- = normal; ~ ~ partial inhibition, cells contain slowly moving cytoplasmic masses; -- ~ completely inhibited, motionless cytoplasmic masses in all cells. b Normal except in cells near cut ends, which are ~ . c Moving cytoplasmic masses in most cells in interior of segments; complete inhibition in cells near cut ends. a~ Glucose was used with pea and maize segments, sucrose with oat segments.
Ultrastructural Observations B u n d l e s of m i e r o f i l a m e n t s a d j a c e n t t o a n d p a r a l l e l t o t h e cell s u r f a c e (Figs. 5 - 7 ) , a n d also j u s t o u t s i d e t h e n u c l e a r e n v e l o p e (Figs, 8, 9), w e r e frequently found within the cytoplasmic masses that form in CB-treated m a i z e e o l e o p t i l e e p i d e r m a l cells. P r o m i n e n t f i l a m e n t b u n d l e s w e r e also c o m m o n l y f o u n d within t h e n u c l e u s of b o t h e p i d e r m a l a n d p a r e n c h y m a
Fig. 6. Longitudinal section of outer epidermal wall (CW) and adjacent cytoplasm showing microtubules (Mr#) in cross section, adjacent microfilament bundles (MF) in oblique section, and mitochondrion (MC) with dense core. • 28000 Fig. 7. Enlarged view of single microfilament bundle in oblique section, showing indications of regular substructure or cross links. • 49000
286
W . Z . Cande et al. :
Figs. 8 and 9. Electronmicrographs of portions of cytoplasmic masses within CB-treated maize colcoptile epidermal cells Fig. 8. Microfilament bundles (MF) in oblique section, and rnitoehondrion with dense core (MC), adjacent to nucleus (N) containing intranuclear microfi]ament bundles (NF). • 23000
Auxin and Cytoplasmic Streaming
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cells of CB-treated maize coleoptiles (Fig. 8); no such bundles were seen in any of the nuclei observed in control (not CB-treated) cells. Cytoplasmic microfilament bundles were much more difficult to find in control than in CB-treated maize eoleoptile cells although a few microfilament bundles comparable to those shown in Figs. 5-7 were seen in control epidermal cells, as have been reported for oat and ungerminated rice coleoptile cells (O'Brien and Thimann, 1966; 0pik, 1972). Another peculiarity of CB-treatcd maize coleoptiles was the presence of a dense core within m a n y of the mitochondria of epidermal cells (Figs. 6, 8). These cores were not seen in control cells. No microfilament bundles could be clearly identified in our observations of either CB-treated or control epidermal or parenehyma cells from pea stem tissue.
E//ect o / C B on Elongation and Auxin Action Auxin promoted elongation in oat coleoptile and pea stem segments in which streaming was completely inhibited by CB (Figs. i0, i i). CB inhibited the overall elongation of oat coleoptile segments about 40 % during the first 4 h, but segments treated with IAA in the presence of CB nevertheless showed a doubling of elongation over the CB control (Fig. 10). A similar inhibition of overall elongation by CB was obtained with peas, but again in the presence of CB a substantial stimulation of elongation by I A A over the CB control was obtained dm~ the first 4 h (Fig. 11). With oat coleoptiles inhibition of elongation by CB increased to about 60 % in the period after 4 h, but promotion of elongation b y IAA above the CB control was nevertheless apparent throughout at least 28 h (Fig. 10). With peas, however, although the elongation rate in IAA @ CB was initially high the rate declined rapidly and elongation ceased altogether after 6 h (Fig. 11), due probably to degenerative secondary effects of CB on the cells noted above. With pea stem tissue the inhibition of early elongation b y CB increased with concentration up to a m a x i m u m of about 40 % inhibition, this plateau being reached at the same concentration of CB as caused complete inhibition of streaming (Table 1). More limited information on concentration dependence was obtained for coleoptile tissue (Table 1), but here also some inhibition of coleoptile segment elongation b y CB was observed under partial inhibition of streaming (20 ~g/ml CB) and about 40 % inhibition of early elongation was obtained when streaming was corn-
Fig. 9. Longitudinal section of microfilament bundle (MF) adjacent to nucleus (N), exhibiting substructure similar to that in Fig. 7 (arrow). • 29500
W. Z. Cande et aI. :
288 I00
Fig.I0
~
IAA
Fig. II
80
60
o
/
40 X 20
0
4
8
12
16 20 24 TIME(hours) 10
28
32
4
8
,2
TIME (hours) 11
,~
8~
Figs. 10 and 11. Effect of IAA on elongation of normal and CB-treated oat coleoptile (Fig. 10, left) and pea stem (Fig. ll, right) segments. Segments were pretreated 2 h at 25~ in buffered aqueous D~ISO without or with CB (50 ~g/ml in Fig. 10, 20 ~zg/ml in Fig. 11), then (zero time on graphs) incubation was continued in the same media with addition of IAA (3 ~xg/ml) to samples so indicated. Each point is mean elongation of 20 segments that were initially 8 mm (Fig. 10) or 5 mm (Fig. 11) long. Microscopic observations showed that streaming had been completely inhibited by CB prior to addition of IAA in both experiments
pletely stopped (50 Fg/ml CB). Bradley (1973) also found about 50% inhibition of elongation of internodal cells of Nitella by a concentration of CB (30 ~xg/ml) t h a t completely inhibited streaming, but he did not s t u d y the concentration dependence of inhibition of elongation.
Metabolic E//ects o / C B Table 1 summarizes tests of the effects of CB on sugar and amino-acid uptake, and on short-term incorporation of sugar into cell wall polymers and of leucine into protein. Higher concentrations of CB caused some inhibition of uptake and incorporation. I n the ease of peas this occurred only at CB concentrations above t h a t required to stop streaming completely and to cause m a x i m u m inhibition of elongation. With eoleoptile segments inhibition of sugar uptake and incorporation was considerable at 50 Fg/ml CB but only slight at 20 Fg/ml CB, even t h o u g h inhibition of elongation was almost m a x i m u m at the latter concentration. Bradley (1973) also found only a slight inhibition of lencine incorporation in Nitdla b y 30 ~zg/ml CB. E//ect o / C B on A u x i n Transport Fig. 12 shows t h a t [aH]IAA was transported in a polar fashion in maize coleoptile segments in which streaming had been completely inhib-
Auxin and Cytoplasmic Streaming
8000
289
~-000 Fig.15
Fig.12
6000
~000
4000
~-000
2000
IO00
. /
oB)/
o
E o
/ / / /
Acropelol (• _
~
_/ I
I
2
TIME (hours) 12
0
I
2
5
TIME (hours) 13
Figs. 12 and 13. Effect of CB on polar transport of [3H]IAA in maize (Fig. 12, left) and oat (Fig. 13, right) coleoptile segments. The segments used were 8 or 5 mm long, and the donor blocks contained 25000 or 40000 cpm, respectively; 50 ~g/ml CB was used in pretreatment and in donor and receiver blocks of CB series. Microscopicobservations showed that in both experiments streaming had been completely inhibited by CB prior to supplying donor blocks and remained completely inhibited in the CBtreated segments throughout the transport period. Donor blocks were applied to the apical ends of the segments (basipetal transport) except in the data marked aeropetal in Fig. 12 where donor was applied to the base and receiver to the apex
ited by CB. I n CB-treated segments the arrival of the auxin front (intereept on time axis) was slightly delayed, but the flux (slope of the arrival curve) was similar to that of the controls. I A A was also transported by oat eoleoptile segments in which streaming had been blocked by CB (Fig. 13). I n this ease the arrival of the front was more substantially delayed by CB but the flux was again similar to the control. Fig. 14 shows that most of the radioactivity that was transported into receiver blocks from [sI-I]IAA donors in these experiments chromategraphed like I A A (Rf =0.61 using solvent A listed in Methods). This was true also for two other solvent systems (for both peak radioactivity and known IAA, R f = 0 . 8 3 in solvent B and 0.66 in solvent C). The IAA peak accounted for 85% of the radioactivity in receiver blocks. The [att]IAA stock used to prepare the donors contained a substantial proportion (30 % ) of decomposition products (peaks at origin and near front in Fig. 14 A) that could not be eliminated by reehromatography because of instability
290
W.Z. Cande
et al. :
10 5 J o -X
0
A
B 0 L i~
H
r
F
IAA
0
Fig. 14. Radioscans of thin-layer chromatograms of (A)[aH]IAA stock used in donor blocks, and (B) radioactive material that was transported into receiver blocks by maize coleoptiles. 0 origin; F solvent front; bar indicates position of known IAA chromatographed in parallel with radioactive samples. Solvent A was used
Table 2. Effect of CB on auxin transport through segments cut from coleoptiles of different lengths Sections 5 mm long were cut from 4--7-day old eoleoptiles starting 3, 5, or 8 mm below the tip, respectively, in the case of coleoptiles 1.5, 3.5, and 5.5 cm long. Sections were pretreated 2 h in 2% DMSO with or without 50 pg/ml CB. The transport period was 2 h with donors containing 50000 cpm IAA [SH]. Each figure is the mean of triplicate specimens (figures in parentheses are average deviations). Coleoptile length (cm)
Mean length of subepidermal cells a (~m)
Ratio of cell lengths
nil in receivers (cpm) Control
1.5 3.5 5.5
44 127 206
i.O 2.9 4.7
9620 (560) 7780 (550) 16630 (1180) 11520 (810) 17080 (1960) 10750 (850)
CB
Inhibition by CB (%) 19 31 37
a Average length of 80-100 subepidermal parenehyma cells located midway between vascular bundles.
of [aH]IAA. However, very little of these products were f o u n d i n receiver blocks (Fig. 14B) a n d the small a m o u n t s t h a t were seen p r o b a b l y arose b y decomposition of [atI]IAA d u r i n g e x t r a c t i o n a n d c h r o m a t o g r a p h y after the t r a n s p o r t into receiver blocks.
Auxin and Cytoplasmic Streaming
291
The effect of CB on auxin transport through segments cut from coleoptiles of different lengths was determined (Table 2) to see if the effect depended on cell length. Inhibition increased with length of the source eoleoptfles. A similar result was obtainedin a comparable second experiment. Discussion
Evidence that streaming was totally stopped in CB-treated tissue was provided not only by microscopic observations but by the finding that after such tissue was centrifuged, the cytoplasm and organelles remained at the centrifugal ends of the cells for hours (Goldsmith and l~ay, 1973). The persistence of polar transport and IAA-induced elongation during complete inhibition of streaming by CB shows not only that streaming is not required for either process but also that promotion of streaming by auxin is not the means by which auxin promotes cell enlargement. The present experiments directly confirm the suggestions advanced previously (Clark, 1938; Showacre and DuBuy, 1947 ; van der Weij, 1932)that auxininduced growth and polar transport are largely independent of streaming. The disputes about the mode of action and specificity of CB (Burnside and Manasck, 1972; Estensen et al., 1971 ; Forer et al., 1972; Krishan and Whitlock, 1972 ; Holtzer and Sanger, 1972) do not affect the strength of the conclusions cb.awn above. The crucial point is that tissues in which streaming has been stopped completely by CB prior to exposure to IAA show substantial auxin transport and promotion of elongation by IAA during the first few hours. The more severe inhibition of elongation by CB in the period after 6 h is not pertinent to the question of primary auxin action and in the case of peas is doubtless due to the degenerative effects of CB on the cells which can be observed within 6 h. Possible Role o/Streaming in Cell Enlargement The partial inhibition of early elongation by CB in the three tissues studied here suggests a beneficial (although not essential) role of streaming in cell enlargement. However, the inhibition could be due to effects of CB other than on streaming; for example, the reported inhibition of sugar uptake by CB (Itaslam, 1972; Estensen and Plagemann, 1972; Kletzien et al., 1972). Since elongation of neither pea nor oat segments is benefited by exogenous sugar during the first 6 h (Christiansen and Thimann, 1950; Ordin et al., 1956) and exogenous sugar was not even provided in the experiments with peas, an effect on sugar uptake could not explain the partial inhibition by CB of early elongation, but it could conceivably explain the stronger inhibition of eoleoptile elongation in the post-6 h period, during which elongation of eoleoptiles becomes dependent on sugar (Schneider, 1938).
292
W.Z. Cande et al. :
The inhibition of early elongation reaches a m a x i m u m of about 40 % at a CB concentration that just completely inhibits streaming, whereas effects of CB on sugar and amino acid uptake and incorporation increase with CB concentration above this point (Table 1). This suggests that the partial inhibition of early elongation by CB is associated with inhibition of streaming rather than of metabolism. However, since inhibition of streaming is accompanied b y formation of local cytoplasmic masses within the ceils (Figs. 1-4) is could be that withdrawal of much of the cytoplasm into these masses, rather than lack of streaming, is what actually depresses elongation. I n contrast to the present results, elongation of tip-growing cells such as pollen tubes and root hairs is completely inhibited at the same concentrations of CB that inhibit streaming ( g e r t h et al., 1972 ; Masearenhas and La Fountain, 1972). I n tip-growing cells, streaming m a y be essential to supply material synthesized in remote parts of the cell to the region of local growth. Mechanism o / A u x i n Transport I n the absence of streaming, movement of auxin from the apical to the basal and of each cell during polar transport must either be due to diffusion or to a special transport mechanism. Since there is no evidence for the latter (Goldsmith and Ray, 1973) it is of interest to consider whether diffusion could be the mechanism of intraeellular transport. The concentration gradient dc/dx that would be required to transport an amount Q of IAA per see through any cross section of the coleoptile by diffusion m a y be found from Q - - - D A ( d c / d x ) , where A is the crosssectional area of the tissue (0.75 m m 2 for Arena coleoptile; Goldsmith, 1966). From its molecular weight I A A should have a diffusion coefficient (D) of about 7 • 10 -6 cm2/s, a value confirmed by experiment (Larsen, 1944). If in 1 h 1000 dpm of radioactive IAA ( Q ~ 0.3 dpm/s) arc transported from a eoleoptile section into a receiver, dc/dx comes to about 6 • l0 s dpm/cm 3 per em of length. This corresponds to a concentration difference of about 6 • 104 dpm/cm s, between the apex and base of a typical parenchyma ceil about 100 ~m in length, required to sustain by diffusion a delivery of 1000 dpm/h into a receiver. The average concentration of IAA in the transport stream in cells at the basal end of a maize coleoptile segment, next to a receiver, which is the lowest concentration t h a t will exist in the segment since transport stream concentration decreases basipetally (Newman, 1970), will now be estimated. IAA is transported in maize eoleoptiles at a velocity of approximately ] cm/h. In order to deliver 1000 dpm/h to a receiver the basal end of a segment must therefore contain 1000 dpm per em of length or, since its cross sectional area is 0.75 m m 2, the cells at its basal end must contain 13 • 104 dpm/cm a.
Auxin and Cytoplasmic Streaming
293
Therefore, the concentration difference required to transport I A A through each cell by diffusion is less than half of the transport stream concentration. This means t h a t diffusion could account for the intraecllular portion of polar transport. An active mechanism would be needed only to transfer IAA from each cell to the next. The existence of an active cellto-cell transport mechanism for IAA in polar transport has already been inferred from experimental evidence (Christie and Leopold, 1965 ; Hertel and Flory, 1968). The moderate inhibition by CB of polar transport suggests that streaming increases transport somewhat above that due to diffusion. If this is the case, inhibition of polar transport by CB should increase with cell length since the concentration difference required to support diffusion - and thus the increase in IAA concentration that could be achieved at the basal end of each cell by stirring its contents b y streaming - - should increase with cell length. This result was obtained (Table 2). I t is, of course, not excluded that the greater percent inhibition of transport by CB in segments cut from longer eoleoptfles is due to differences other than cell length between coleoptiles of different physiological ages.
Nature o/Cytoohalasin Action It is clear from the observations (Figs. 5-9) that at least in maize eoleoptile cells CB does not cause depolymerization or disorientation of microfilaments or microfilament bundles when it inhibits streaming. A similar observation on NitelIa cells was made by Bradley (1973). If mierofflament bundles are the propulsive organelles for streaming (Nagai and Rebhun, 1966; O'Brien and Thimann, 1966 ; Kamitsubo, 1972 ; Daniel and Jgrlfors, 1972), CB must suppress their action without disorganizing them. That CB induces formation of abnormal intranuclear bundles of mierofflaments (Fig. 8) suggests, on the contrary, that CB m a y cause a hypercondensation of microfilament subunits, like vinblastine does in the case of mierotubule protein (Bryan, 1972). Observations quoted in Table 1 and in the text indicate that CB also has metabolic and membrane effects t h a t do not appear to be related to microfilament function. We thank Margery iVi.Ray for preparation of specimens for Figs. 5-9. This work was supported by grants GB-18499 and 33927X from the 1National Science Foundation, and Grants GM NB 16530 and GM-08886 from the National Institutes of Health.
294
W.Z. Cande et al. : References
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Haslam, R. J.: Inhibition of blood platelet function by cytochalasins: effects on thrombosthenin and on glucose metabolism. Biochem. J. 127, 34P (1972). Hertel, R., Flory, R.: Auxin movement in corn eoleoptiles. Planta (Berl.) 82, 123-144 (1968). Herth, W., Franke, W. W., Vanderwoude, W. J. : Cytochalasin stops tip growth in plants. Naturwissenschaften 59, 38-39 (1972). Holtzer, H., Sanger, J. W.: Cytochalasin B: mierofilaments, ceil movement and what else ? Develop. Biol..27, 444-446 (1972). Kamitsubo, E. : Mobile protoplasmic fibrils in cells of Characeae. Protoplasma (Wien) 74, 53-70 (1972). Kletzien, R. F., l~erdue, J. F., Springer, A.: Cytochalasin A and B. Inhibition of sugar uptake in cultured cells. J. biol. Chem..247, 2964-2966 (1972). Krishan, A., Whitlock, S. : Cytochalasin B: time-lapse cinematographic studies on its effects on cytokinesis. J. Cell Biol. 54, 657-664 (1972). Larsen, P. : 3-indoleaeetaldehyde as a growth hormone in higher plants. Dansk Botan. Ark. 11, 11-129 (1944). Leopold, A. C., de la Fuente, R. K. : A view of polar auxin transport. In: Transport of plant hormones, p. 24-47, Y. Vardar, ed., Amsterdam: North-Holland Publ. Co.1968. Mascarenhas, J. P., La Fountain, J. : Protoplasmic streaming, cytochalasin B and growth of the pollen tube. Tissue and Cell 4, 11-14 (1972). Nagai, R., P~ebhun, L. I.: Cytoplasmic microfilaracnts in streaming Nitella cells. J. Ultrastruct. Res. 14, 571-589 (1966). Newman, I. A. : Auxin transport in Avena. I. Indoleacetic acid-C14 distributions and speeds. Plant Physiol. 46, 263-272 (1970). O'Brien, T. P., Thimann, K. V. : Intracellular fibers in oat coleoptile cells and their possible significance in cytoplasmic streaming. Proc. nat. Acad. Sci. (Wash.) 56, 888-894 (1966). ()9ik, H. : Some observations on coleoptile cell ultrastructure in ungerminated grains of rice (Oryza sativa L.). Planta (Berl.) 10.2, 61-71 (1972). Ordin, L., Applewhite, T. H., Bonner, J. : Auxin-induced water uptake by Avena coleoptile sections. Plant Physiol. 31, 44-53 (1956). Palevitz, B. A.: Effect of cytoehalasin B on stomatal differentiation in Allium. J. Cell Biol. 55, 198a (1972). Schneider, C. L. : The interdependence of auxin and sugar for growth. Amer. J. Bot. '25, 258 270 (1938). Showaere, J. L., du Buy, K. G. : The relationship of water availability and auxin in the growth of Avena coleoptiles and its meaning for a theory of tropisms. Amer. J. Bot. 84, 175-182 (1947). Sweeney, B. M., Thimann, K. V. : The effect of auxins on protoplasmic streaming, II. J. gen. Physiol. 21, 439-461 (1937). Sweeney, B. M., Thimann, K. V. : The effect of auxins on protoplasmic streaming, III. J. gen. Physiol..25, 841-854 (1942). Thimann, K. V. : Plant growth hormones. In: The hormones, physiology, chemistry and applications, vol. 1, p. 1-76, G. Pineus and K. V. Thimann, eds. New York: Acad. Press 1948. Turner, W. G., Carter, S. B.: The chemistry and some biological properties of the cytochalasins. Biochem. J. 127, 1P (1972). 20 Planta (Berl.), Bd. 111
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