Planta

Planta 150, 371 379 (1980)

9 by Springer-Verlag 1980

The Role of the Epidermis in Auxin-induced and Fusicoccin-induced Elongation of Pisum sativum Stem Segments David A. Brummell and J i . Hall School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.

Abstract. The effects of peeling and wounding on the indole-3-acetic acid (IAA) and fusicoccin (FC) growth response of etiolated Pisum sativum L. cv. Alaska stem tissue were examined. Over a 5 h growth period, peeling was found to virtually eliminate the IAA response, but about 30% of the FC response remained. In contrast, unpeeled segments wounded with six vertical slits exhibited significant responses to both IAA and FC, indicating that peeling does not act by damaging the tissue. Microscopy showed that the epidermis was removed intact and that the underlying tissue was essentially undamaged. Neither the addition of 2% sucrose to the incubation medium nor the use of a range of IAA concentrations down to 1 0 - S M restored IAA-induced growth in peeled segments, suggesting that lack of osmotic solutes and supra-optimal uptake of IAA were not important factors over this time period. It is concluded that, although the possibility remains that peeling merely allows leakage of hydrogen ions into the medium, it seems more likely that peeling off the epidermis removes the auxin responsive tissue. Key words: Auxin - Cell elongation - Epidermis peeling - Fusicoccin - Pisum.

Introduction

The effects of peeling off the epidermis on the auxin response of stem or coleoptile tissue has been in dispute for about fifty years. Both monocotyledons (usually Arena coleoptiles) and dicotyledons (often pea stems) have been studied but with neither tissue have conclusive results been obtained. Bonner (1934) found that peeled Arena coleoptiles showed a distinct Abbreviations. IAA=indole-3-acetic acid, FC=fusicoccin

auxininduced growth for up to 5 h, though it is possible that his coleoptiles were not completely peeled. In contrast, van Overbeek and Went (1937) found that peeling greatly reduced or removed the auxin response. Their explanation, that auxin could not be taken up across a wounded surface, was later disproved (Thimann and Schneider 1938). However, several more recent reports have suggested that peeled Arena coleoptiles respond as well to IAA as do intact ones over periods of up to 7 h (Durand and Rayle 1973 ; Rayle 1973 ; Cleland 1975) although this finding has recently been challenged (Pope, personal communication). With pea stem tissue, the literature is even more contradictory. Van Overbeek and Went (1937) found no effect of several auxin concentrations on peeled etiolated pea stems, whereas Thimann and Schneider (1938) found an auxin-induced growth in peeled pea stem of almost half that of the intact tissue after 5 h. Data in this latter paper, however, also show that while the inner tissue, which was bored out using a very thin-walled glass tube, showed no auxin response at all, the hollow cylinder of outer tissue (including the epidermis) showed a strong response to auxin. They explained this observation by suggesting that the inner core was damaged, though of course the hollow outer cylinder had suffered just as much damage, but on the inside. That hollow cylinders of outer tissue, 10-12 cell layers thick, have a strong auxin response was also shown for light-grown pea stems (Masuda and Yamamoto 1972). Peeling has been reported to have virtually eliminated the auxin response in light-grown pea stem (Tanimoto and Masuda 1971 ; Masuda and Yamamoto 1972; Yamamoto et al. 1974) and Helianthus hypocotyl (Firn and Digby 1977; Mentze et al. 1977). For etiolated pea stem, in contrast to the earlier report of van Overbeek and Went (1937), more recent data indicate that peeling has almost no effect on the auxin

0032-0935/80/0150/0371/$01.80

372 response. Peeled stem showed an auxin-induced g r o w t h o f 8 5 % in 5 h c o m p a r e d w i t h 1 1 0 % f o r i n t a c t s t e m ( T a b l e 2 o f D u r a n d a n d R a y l e 1973). T h e prese n t w o r k was c a r r i e d o u t o n e t i o l a t e d p e a s t e m w i t h the a i m o f r e s o l v i n g this c o n t r o v e r s y , a n d m a k e s use o f f u s i c o c c i n ( F C ) , a f u n g a l t o x i n o f c o m p l e t e l y different s t r u c t u r e to a u x i n b u t h a v i n g s i m i l a r t h o u g h inc r e a s e d effects o n s t e m e l o n g a t i o n a n d h y d r o g e n i o n e x c r e t i o n ( L a d o et al. 1972, 1973; M a r r 6 et al. 1973; C l e l a n d 1976).

Materials and Methods Growth and Preparation of Plant Mater&l. Seeds of Pisum sativum L. cv. Alaska were surface sterilised in approximately 5-fold diluted sodium hypochlorite solution for 15 rain. After thorough rinsing, they were soaked overnight in running water, then grown over water in the dark at 25~ C until the 3rd internode stage was reached (about 7 days). Plants with the 3rd internode between 3.5 and 4.5 cm in length were selected and a 1 cm segment excised from 2-3 mm below the apical hook. These segments were either left intact or wounded with six longitudinal slits using a scalpel blade. For peeled segments (where the epidermis was to be removed), a 1.5 cm segment was excised, the epidermis then being carefully peeled away in two or three strips using fine forceps. Segments were peeled from both ends to ensure complete removal of the epidermis. Peeled segments were then cut to a length of 1 cm. Segments were collected on ice-cold water. All the above procedures were carried out in daylight; this did not appear to have any effect on subsequent results (data not shown). When all the segments of a particular treatment had been collected, the segments were transferred to the dark. All subsequent manipulations were performed under dim green light. Segments were allowed to stabilise for 1 h on distilled water (30 min on ice, 30 rain at room temperature) in the dark before growth measurements began.

Measurement of Growth. Batches of five segments were placed on a glass plate in a photographic enlarger fitted with a green filter, and projected x 10 on to photographic paper. This measurement was time 0, and replicated batches of five segments were then floated on 10 ml of the appropriate solution in the dark at 25 ~ C. IAA (Sigma Chemical Co., St. Louis, Mo., U.S.A.) and FC (gift of Professor E. Marr6, University of Milan, Italy), when present, were at a concentration of 10 -5 M. FC solutions and controls contained ethanol at 5.10-3 M (except Fig. 1b). Ethanol concentrations up to 10- 2 M are not inhibitory to the growth of etiolated peas (Lado et al. 1973). Further shadowgraphs were made at the intervals indicated for each experiment. The lengths of both sides of the segment image were accurately determined by measuring the traced image on to 1 mm squared graph paper. The amount of growth in each treatment was thus found by subtracting the initial mean side lengths from the subsequent means. Results were expressed as mean elongation per segment in ram. Each experiment was repeated several times and mean elongation__+SD was calculated for each treatment. Due to tissue variability, every experiment included replicated intact groups of segments to which direct comparisons of the relative growth of the peeled or wounded segments could be made. Preparation of Tissue for Microscopy. Microscopy was necessary to determine exactly what effects the treatments "peeled" and "wounded" had on the segments. Small pieces of tissue (about 1 mm in length) were excised from the middle of 10 mm stem segments prepared as above, and these pieces fixed in glutaralde-

D.A. Brummell and J.L. Hall: The Epidermis and Stem Elongation hyde followed by OsO4. After dehydration through a graded series of alcohols, the tissue was embedded in resin (TAAB Labs, Reading, U.K.). Thin sections for electron microscopy were post-stained with uranyl acetate/lead citrate. Thick sections for light microscopy were stained with 1% toluidine blue in 1% borax.

Uptake qf[I~C]IAA. The relative IAA uptake of intact and peeled segments was determined by incubating batches of 10 segments for 5 h in 10 -5 M [1-14C]IAA (Radiochemical Centre, Amersham, U.K.) of specific activity 185 GBq tool -1 in unbuffered distilled water containing 0.05% methanol. Segments were allowed a 1 h stabilisation period in the dark before batches of 10 were floated on 3 ml of the IAA solution for 5 h in the dark at 25 ~ C. Uptake of[ 14C]IAA was terminated by removing the segments individually from the uptake medium and immediately rinsing the batches of 10 segments thoroughly in a large volume of distilled water. The segments were blotted briefly before batches of 10 segments were extracted in 2 ml 80% methanol. The segments were thoroughly homogenised with a glass rod and left to extract for 1 h at room temperature. After spinning down the tissue residue in a bench centrifuge (5 min), 1 ml of the methanolic extract was counted in 10 ml aquasol liquid scintillation cocktail (New England Nuclear, Boston, U.S.A.). Determinations of radioactivity were made with a Beckman model LS-233 liquid scintillation counter. Quenching was determined by [14C]toluene standard (171.4 kBq mol 1, Radiochemical Centre, Amersham, U.K.) spike addition and recounting. Counting efficiency was around 92%.

Results Growth Substance - Optimum Concentration. I n t a c t a n d w o u n d e d s e g m e n t s i n c u b a t e d in I A A s h o w e d a n o p t i m u m f o r e l o n g a t i o n at 1 0 - 5 M (Fig, 1 a), w h e r e a s p e e l e d s e g m e n t s h a d a slightly l o w e r o p t i m u m c o n c e n t r a t i o n . A s this d i f f e r e n c e was v e r y small, 1 0 - S M I A A was u s e d in all t h e s u b s e q u e n t g r o w t h e x p e r i ments for consistency. For FC, both intact and peeled s e g m e n t s s h o w e d a n o p t i m u m f o r e l o n g a t i o n at 5 . 1 0 - 5 M (Fig. 1 b), t h o u g h a g a i n it w a s t h o u g h t a d v a n t a g e o u s in s u b s e q u e n t e x p e r i m e n t s to use 1 0 - S M throughout for both IAA and FC. The observations t h a t w o u n d e d s e g m e n t s r e s p o n d e d to F C at 10 - 5 M (Fig. 3), a n d t h a t w i t h b o t h I A A a n d F C p e e l e d a n d w o u n d e d s e g m e n t s h a v e v e r y s i m i l a r o p t i m a to i n t a c t o n e s , i n d i c a t e s t h a t d i f f e r e n c e s in u p t a k e o f g r o w t h substance were not important. Elongation o f Stem Segments in Response to IAA and FC. A f t e r a g r o w t h p e r i o d o f 5 h (a t i m e p e r i o d w h i c h i n c l u d e d all t h e r a p i d e x p a n s i o n p e r i o d , a n d a l l o w e d a c c u r a t e m e a s u r e m e n t s o f e l o n g a t i o n to be m a d e ) , IAA had stimulated the growth of intact segments by 201% above the control, while FC promoted g r o w t h by 3 0 5 % a b o v e t h e c o n t r o l (Fig. 2). T h e e l o n g a t i o n r e s p o n s e t o F C was t h u s a b o u t 5 0 % b e t t e r t h a n t h a t to I A A o v e r this t i m e . I n all cases, w o u n d i n g r e d u c e d b o t h e l o n g a t i o n ( c o m p a r e Figs. 2 a n d 3) a n d I A A / F C - i n d u c e d g r o w t h ( T a b l e 1). N o t e t h a t t h e I A A r e s p o n s e is r e d u c e d r e l a tively m o r e t h a n is t h e F C r e s p o n s e . T h e I A A re-

D.A. Brummell and J.L. Hall: The Epidermis and Stem Elongation

4

a

4

//'/'

3

3

.~- 2 , - ~ " O0 LI..I

373

2

82

1

LU

O

1

~

0.~-8

10-7

166

Conc. of IAA

10:5 (M)

10L4 '~F6

1C~5

Fig. l a and b. Growth of etiolated pea segments in response to varying concentrations of I A A (a) or FC (b). Segments were either intact (e), peeled (A), or wounded with six vertical slits (m). Elongation in the dark at 25 ~ C was determined after 5 h using a photographic technique. Each point is the m e a n of at least 5 segments. All FC solutions contained ethanol at 5.10 2 M (slightly inhibitory, Lado et al. 1973)

+FC

+IAA

82 Hp

0

1

2 3 Time (hi

4

+IAA H20

1Cr4

Conc. of FC (M)

[]

~

',FC

5

Fig. 2. Elongation of intact segments in response to IAA or FC. Segments were allowed to stabilise on distilled water for 1 h (not shown) before the addition of I A A ( e ) or FC (0) at 10 s M, where present. Elongation in the dark at 25 ~ C was followed by a photographic method. Each point is the m e a n of 8 groups of 5 segments from 4 replicated experiments. Bars represent_+ 1 SD

sponse is reduced by wounding to about 30% that of the intact tissue, whereas 75% of the intact FC response remains after wounding (Table 1). Two wounding treatments (with three or six slits) were tried initially (Table 1), but microscopy showed damage to be insufficient with just three slits. Six slits caused a much greater reduction in elongation than three slits (Table 1). The extent of the damage caused by six-slit wounding can be seen in Figs. 4c and d. Damage in vivo is greater than appears in these micrographs, probably due to the wounds becoming compressed by the resin during the process of embedding for microscopy.

Time (h) Fig. 3. Elongation of wounded segments in response to I A A or FC. Segments were wounded with six vertical slits before the 1 h stabilisation period on dist H 2 0 (not shown). I A A (m) or F C (il), where present, were at 10 s M. Elongation in the dark at 25 ~ C was followed by a photographic method. Each point is the m e a n of 8 groups of 5 segments from 4 replicated experiments. Bars represent + 1 SD

Peeling almost always removed two essentially intact cell layers (Fig. 4a). A split occurred along the middle lamella leaving the underlying cortical cells appearing undamaged (Fig. 4b). Electron microscopy showed that the cytoplasm was disrupted in some cells of the outermost layer of the remaining cortex (possibly partially due to the difficulty of fixing such highly vacuolated cells) but that almost all the remaining cells contained intact cytoplasm (Fig. 4e). The epidermal cells did not appear to break along their radial walls as found by Thimann and Schneider (1938) or Burstr6m (1977). Damage caused by peeling may not, therefore, be extensive. As the constraining epidermal layers have been removed, peeled segments expand rapidly due to the release of tissue tension and consequent water uptake (e.g. see Fig. 6). With the usual 1 h stabilisation period, most of this rapid expansion had already occurred before the addition of growth substance (Fig. 5). Peeling almost completely abolished the response to IAA. Standard deviations for the IAA and H 2 0 treatments overlap at every point (Fig. 5) and the IAA response is reduced to about 12% that of the intact tissue (Table 1). On the other hand, the response to FC is much less affected by peeling; there remains a definite FC promotion of elongation (Fig. 5) at 27% that of the intact segments (Table 1).

Effect of Length of Stabilisation Period. Experiments without a stabilisation period (Fig. 6) or with an extended one of 5 h (Fig. 7a and b) were performed to check that this was not a factor affecting the observations described above. With no stabilisation period, the rapid expansion of peeled tissue caused by removing the constraining

Fig. 4a-d. Light micrographs of resin-embedded dark-grown pea stem stained with toluidine blue, showing the peeling (a and b) and wounding (c and d) treatments, a A strip of epidermal peel. Note the essentially intact 2 cell layers (x290). b A segment of stem from which a single strip of epidermis has been removed (x 350). c The extent of the damage caused by 6-slit wounding ( x 72). d A typical wound ( x 275) Fig. 4e. Electron micrograph of cortical surface after peeling, showing most cells contain intact cytoplasm ( x 2,550)

D.A. Brummell and J,L. Hall: The Epidermis and Stem Elongation 4

Table 1. IAA/FC-induced growth of peeled or wounded segments after 5 h relative to the corresponding intact control (100%) run with every experiment. Elongation in the dark at 25~ C, • 10-5 M IAA or FC, was measured photographically. Each figure is the mean _+SD from 4 replicated experiments (5 segments per replicate)

3~

H20

E"

0 Ld

0

1

2 3 4 Time (h)

5

Fig. 5. Elongation of peeled segments in response to IAA or FC. Segments were allowed to stabitise on distilled water for 1 h (not shown) before the addition of IAA (A) or FC (4), where present, at 10 5 M. Elongation in the dark at 25~ C was followed by a photographic method. Each point is the mean of 8 groups of 5 seg ments from 4 replicated experiments. Bars represent • 1 SD

"~2

§

._~ m

1

1

2

3

4

Growth substance l0 5 M

Intact

IAA FC

100 100

Peeled

Wounded 6 slits

11.7_+2.7 27.1_+3.1

(3 slits)

33.3_+14.8 (77.5_+31.7) 73.7_+17.7

epidermal layers is clearly seen (Fig. 6). However, the I A A response in peeled segments here was 11% that of the i n t a c t ; that is, almost identical to the 11.7% o b t a i n e d using a 1 h stabilisation period (Table 1). It is possible that with the u s u a l 1 h stabilisation period the peeled segments were e x p a n d i n g at a rate a p p r o a c h i n g their m a x i m u m , so that I A A or F C could n o t p r o d u c e a further increase. This w o u l d result in a p p a r e n t l y reduced effects of I A A a n d FC. However, with a 5 h stabilisation period a l m o s t all of the e x p a n s i o n in peeled segments due to release of tissue t e n s i o n will have passed, allowing u n i m p e d e d e x a m i n a t i o n of the growth substance response. After a 5 h stabilisation period (all in the dark, c o m p r i s i n g 1 h on ice a n d 4 h at r o o m temperature), the I A A response in peeled segments was 9 % that of the intact tissue (11.7% with 1 h stabilisation, T a b l e 1) a n d F C response 29% that of the intact (27.1% with 1 h stabilisation, T a b l e 1). See Figs. 7a a n d b respectively. It thus appears that the length (or absence) of a stabilisation period does n o t affect I A A / F C - i n d u c e d growth of peeled relative to that of intact segments in this system.

"~+IAA

0

375

5

Time (h)

Fig. 6. Elongation of intact segments in H20 (o) or 10-s M IAA (o), and peeled segments in H20 (zx) or 10 s M IAA (A). No stabilisation period was allowed. Elongation in the dark at 25~ C was followed by a photographic method. Points denote replicate pairs of groups of 5 segments where differences were sufficiently great to be depicted

Presence of 2% Sucrose. D u r i n g the r a p i d cell expansion period observed in stem tissue i m m e d i a t e l y after peeling, the ceils m a y dilute their c o n t e n t s t h r o u g h water u p t a k e to the extent that t u r g o r pressure is reduced sufficiently to prevent further cell e l o n g a t i o n .

b

+IAA

~ 3; v

+FC hi

+IAA

1

2

3

Time (h)

4

b

LLI

I

0

1

2

3

Time (h)

4

5

Fig. 7a and b. Elongation of intact segments in H20 (9 or 10-5 M IAA or FC (o), and peeled segments in H20 (zx) or 10-s M IAA or FC (A). A stabilisation period of 5 h in the dark was allowed (not shown). Elongation in the dark at 25~ C was followed by a photographic method. Points denote replicate pairs of groups of 5 segments where differences were sufficiently great to be depicted

376

D.A. Brummell and J.L. Hall: The Epidermis and Stem Elongation

Discussion

w

~

0

+IAA

1

2

3

4

5

Time (h)

Fig. 8. Elongation of intact segments in 2% sucrose (o) or 10 -5 M I A A + 2 % sucrose (o), and peeled segments in 2% sucrose (zx) or 10-SM I A A + 2 % sucrose (A). Segments were previously allowed to stabilise on 2% sucrose for 1 h in the dark. Elongation in the dark at 25~ was followed by a photographic method. Points denote replicate pairs of groups of 5 segments where differences were sufficiently great to be depicted

This may explain the reduced response of peeled segments to auxin. It has been proposed that the addition of 2% sucrose to the incubation medium will provide a source of osmotic solutes which the cells can take up, and thus maintain a constant osmotic potential despite taking up water during elongation (see Cleland 1977). All segments had a stabilisation period of 1 h on 2% sucrose. Growth of intact segments in the presence of IAA was promoted by sucrose (Fig. 8), these segments exhibiting a stronger growth which appeared less likely to decrease with time (cf. Fig. 2). Peeled segments grew more slowly than usual (cf. Fig. 5), perhaps because the rapid uptake of water after peeling was reduced due to the lower osmotic potential of the surrounding medium. However, auxin-induced growth in peeled segments was 12% that of intact, compared to 11.7% in the absence of sucrose (Table 1).

[14C]IAA Uptake. Another possible explanation of the reduced response of peeled segments to IAA is that it results from a difference in auxin uptake from the medium between intact and peeled segments. The greater absorbing surface of peeled segments may result in the uptake of supra-optimal quantities of IAA, which inhibit growth. The results show a distinct difference in auxin uptake over 5 h, uptake being 2.0+0.2% of that present in the medium by batches of 10 1 cm intact segments, and 3.3 _+0.3% by batches of 10 1 cm peeled segments.

Peeling appears to remove almost all of the auxin response in etiolated pea stem, though a response of peeled stems to FC remains. FC has been shown to cause elongation of peeled stem tissue in lightgrown pea (Yamagata and Masuda 1975) and Helianthus (Mentze et al. 1977) and gives no curvature in the split pea test, suggesting it has a different site of action in the stem to that of IAA (Yamagata and Masuda 1975). Wounding also has a greater effect on the auxin response than on the FC response, but nevertheless does not completely abolish the response of the segment to auxin either in this tissue or in Helianthus hypocotyl (Firn and Digby 1977). Peeling, therefore, does not appear to reduce elongation by damaging the tissue. The observation that the peripheral layers constrain the expansion of the cortical cells has been made for a number of species and goes back to the 19th century (see Firn and Digby 1977). It is easily shown that the epidermal layers are important in stem elongation. Removing them by peeling allows the cortical cells, which are under compression and have a considerable capacity for water uptake, to extend rapidly (see Results and Thimann and Schneider 1938). This release of tension can also be shown by longitudinally splitting a stem for about 3 cm, and watching the outward curvature of the two halves as the inner cortical cells expand. It is also possible that the epidermal layers may contract slightly; observations on isolated epidermal strips show that they do contract by 5-15% on removal from the plant (Heyn 1933; Bonner 1934; Masuda and Yamamoto 1972). Thus, as the outer part of a plant stem is usually under tension and the central core under compression, it can be assumed that the outermost cells of the segments are limiting growth (Thimann and Schneider 1938). For growth to occur, therefore, the walls of these outermost epidermal cells must be loosened, and the rate of stem extension will be determined largely by the rate of expansion of these layers (Firn and Digby 1977). So by regulating the expansion of the epidermis, auxin can control the extension of the whole organ. Loosening of the cell walls may occur by a lowering of their pH in accordance with the acid-growth theory. This theory states that plant cells, upon exposure to auxin, excrete protons outwardly into the cell wall with the result that the pH of the wall solution decreases and the plasticity of the wall increases (for review see Cleland and Rayle 1978). This theory has not been universally accepted (Penny et al. 1975; Pope 1977, 1978; Vanderhoef et al. 1977b) but will be considered here as a working hypothesis.

D.A. Brummell and J.L. Hall: The Epidermis and Stem Elongation

377

If there is no effect of length of stabilisation time on subsequent auxin response (see Results), then four explanations for the reduced response of peeled segments to auxin remain. Firstly, it is possible that in peeled segments the osmotic concentration has been sufficiently diluted during the rapid elongation after peeling to prevent further water uptake. A fairly high turgor pressure is required to obtain rapid cell expansion (Cleland 1959 and see Penny et al. 1972). The cells may still be responding to auxin and have loose walls, but no elongation will be seen. It has been shown in long-duration kinetics experiments that this increase in osmotic potential may be important, and that the addition of 2% sucrose can provide a source of osmotic solutes for the cells (Cleland 1977). Sucrose may of course be acting as a substrate, although the same results can be obtained using potassium salts. The presence or absence of 2% sucrose does not appear substantially to affect the growth kinetics during experiments of short duration (see Results). Furthermore, and of great importance, peeled segments can still respond to FC, suggesting that in studies of up to 5 h expansion is not limited by a lack of osmotically active solutes. Secondly, supra-optimal auxin uptake may explain the apparent lack of auxin response in peeled segments. Vesper and Evans (1978) observed that in maize coleoptiles there was a dramatic increase in the magnitude of the response to IAA and dramatic decrease in the latent period of the response with increasing time from excision. This difference could not be explained by a difference in capacity for auxin uptake, as this was not significantly different when measured shortly after excision or 2.5 h after excision. An increase in the sensitivity of the segments to auxin with time from excision was proposed. In contrast, in wheat coleoptile segments the increasing response to auxin with increasing time from excision was shown to be due largely to differential IAA uptake (MacDowall and Sirois 1977). In light-grown pea stems, peeling does enhance auxin uptake slightly by enabling direct entry into more cells, whereas most of the uptake in intact segments is via the cut ends (Davies and Rubery 1978). Although the cuticle of dark-grown pea stems is thinner than in light-grown plants, this is unlikely to affect this observation. Auxin uptake by intact segments would not appear to be a problem as intact segments respond to IAA in less than 15 min (Evans and Ray 1969; Penny 1969; Fig. 2). After the initial rapid uptake lasting 3060 min, IAA uptake continues at a constant rate for about 6 h (Davies 1973). Although uptake is low, the present results show peeled segments do take up more IAA than intact segments, and it is thus possible these segments do contain supraoptimal levels of

IAA. However, peeled segments show virtually no response to IAA at any concentration down to 10 -8 M. Differences in auxin uptake, although evident, do not therefore appear to explain the reduced response of peeled segments to IAA. That the epidermis may be the target tissue for auxin action has been suggested previously (e.g. Tanimoto and Masuda 1971; Masuda and Yamamoto 1972; Firn and Digby 1977), and two lines of evidence support this conclusion. The split pea curvature test (see van Overbeek and Went 1937; Audus 1972) is a measure of auxin activity. When stems of etiolated peas are split lengthways by a medium cut, the split portions bend outwards due to the release of tensions present in normal stem tissues. If such a segment is now placed in a dilute solution of an auxin, the cut portions of the stem will respond by bending in at its tips. This is thought to be due to a differential stimulation of the growth of the inside and the outside of the stem, the cut inside surface growing slower than the intact epidermal region (a slight inhibition of the growth response of the inner tissue due to wounding could be partly involved). The results obtained with this test imply that the epidermal layer(s) control auxin-induced extension growth in stems. Either the epidermis could respond directly to auxin (i.e. the epidermis is the site of action of auxin), or extend in response to hydrogen ions which are produced by the adjacent cells of the cortex and accumulate beneath the cuticle where leakage to the medium is low (Durand and Rayle 1973). This latter explanation is supported by the observation that peeled coleoptile (Cleland 1973; Rayle 1973) and stem tissue (Marr+ et al. 1973) will produce hydrogen ions, though an actual auxin stimulation of this has been questioned in green and etiolated pea stem (Parrish and Davies 1977) and in soybean hypocotyl (Vanderhoef et al. 1977a). It would also appear unlikely that hydrogen ions could accumulate in the epidermal region in sufficient quantities to cause extensive wall loosening whilst the tissue was undergoing an effective free-space wash in pH 6.2 buffer. However, it is possible that the primary control of the rate of cell enlargement in intact stems is by a set of factors different from those controlling floating segments (Cleland 1977), and this should be considered when theories for the control of cell enlargement are assessed. Further evidence that the epidermis could be the direct site of auxin action comes from extensive work on the effects of auxin on the mechanical properties of epidermal and cortical cell walls. Penny et al. (1972) showed using stress-strain analysis that the outermost 2 or 3 cell layers of lupin hypocotyl were more resistant to elongation than the rest of the tissue. Auxin increased the plastic compliance of the outer

378 layers but not in the inner core. A similar conclusion was reached for etiolated pea stem from measurements of Young's modulus (Burstr6m et al. 1967). In light-grown pea stem, auxin caused a conspicuous change in mechanical properties of the epidermal cell wall, as measured by stress-relaxation analysis, but it showed little effect on the inner tissues (Tanimoto and Masuda 1971; Masuda and Y a m a m o t o 1972). Similar results were obtained with A r e n a coleoptiles ( Y a m a m o t o et al. 1970). FC induced an even larger increase than did auxin in the extensibility of the epidermal cell walls of light-grown pea (Yamagata and Masuda 1975). Thus changes in cell wall parameters in response to auxin have been found only in the epidermis. Some evidence that acid can give effects on mechanical properties similar to those of auxin has been presented (Rayle and Cleland 1970), though acid and auxin could act on different linkages in the wall (Rayle and Cleland 1970; Pope 1978). This subject seems worthy of further study. Auxin, but not hydrogen ions, gives an increase in synthesis of some cell wall components, but hydrogen ions could be involved in the initial rapid phase of auxin action (see Masuda 1978). The evidence, therefore, appears to support the idea that auxin has a direct effect on the epidermis. The final explanation for the reduced response of peeled segments to auxin concerns the leakage away of hydrogen ions. It has been suggested that the inner cells may have a larger role to play in stem elongation than merely providing the motive power for it due to their compression (Masuda and Y a m a moto 1972). That differences in p H may occur laterally across a stem was proposed as a mechanism for gravity-induced growth by G a n o t and Reinhold (1970), the resulting curvatures being due to an acid growth response to the higher H § concentration on the lower side of the stem. This might be a consequence rather than a cause of gravity-induced growth (Firn and Digby 1977), but it has also been put forward as an explanation for auxin-induced stem elongation growth. Auxin would cause hydrogen ion secretion mainly in the inner tissue. These ions then accumulate in the region close to the cuticle, probably in the epidermis, resulting in wall loosening of epidermal cells (Durand and Rayle 1973; Y a m a m o t o et al. 1974). The epidermis would still control growth but would not be the direct site of auxin action. In peeled segments, therefore, it is possible auxin acts by inducing hydrogen ion production in the inner cells but that these ions merely leak away into the surrounding medium. As the ions do not accumulate, the cell wall p H does not fall sufficiently to allow wall loosening. With FC, perhaps due to its greater stimulation of hydrogen ion secretion (Marr+ et al. 1973; Cleland

D.A. Brummell and J.L. Hall: The Epidermis and Stem Elongation 1976), H § production exceeds leakage and the wall p H falls sufficiently to allow some stem elongation. One can imagine wounded segments to be intermediate between intact and peeled ones, a low leakage allowing reduced but substantial growth. Although attractive the apparently non-specific H + production in peeled stem (Parrish and Davies 1977; Vanderhoef etal. 1977a; Brummell and Hall, in preparation) makes this theory suspect, at least in dicotyledons. It is, o f course, still possible that auxin-induced H + production is localised exclusively in the epidermis in dicotyledons, when again the epidermis would be the direct site of auxin action. Studies using microelectrodes implanted just beneath the cuticle showed auxin-induced H § production can be observed in the epidermis o f etiolated pea stem (Jacobs and Ray 1976). Although peeling off the epidermis may allow the leakage away of hydrogen ions, it appears more likely that in dicotyledons the auxin responsive tissue is being removed. We wish to thank Drs. D. Pope, A. Browning, J. Allen and J. Thorpe for their help and advice, Professor E. Marr~ for generously providing the fusicoccin, and the Science Research Council for financial support.

References Audus, L.J. (1972) Plant growth substances Vol. 1, 3rd edn., pp. 33-35. Leonard Hill: London Bonner, J. (1934) The relation of hydrogen ions to the growth rate of the Arena coleoptile. Protoplasma 21,406-423 Burstr6m, H.G. (1977) Tissue structure and hormone responses. Plant Sci. Lett. 10, 341-345 Burstr6m, H.G., Uhrstr6m, I., Wurscher, R. (1967) Growth, turgor, water potential, and Young's modulus in pea internodes. Physiol. Plant. 20, 213-231 Cleland, R.E. (1959) Effects of osmotic concentration on auxin action and on irreversibleand reversible expansion of the Arena coleoptile. Physiol. Plant. 12, 809-825 Cleland, R.E. (1973) Auxin-induced hydrogen ion excretion from Arena coleoptiles. Proc. Natl. Acad. Sci. USA 70, 3092-3093 Cleland, R.E. (1975) Auxin-induced hydrogen ion excretion: correlation with growth, and control by external pH and water stress. Planta 127, 233-242 Cleland, R.E. ('t976) Fusicoccin-induced growth and hydrogen ion excretion of Arena coleoptiles: relation to auxin responses. Planta 128, 201-206 Cleland, R.E. (1977) The control of cell enlargement. In: Integration of activity in the higher plant. Society for Experimental Biology Symposium 31, pp. 101-115, Jennings, D.H., ed. University Press~ Cambridge Cleland, R.E., Rayle, D.L. (1978) Auxin, H+-excretion and cell elongation. Bot. Mag. Tokyo, Spec. Issue 1, 125-139 Davies, P.J. (1973) The uptake and fractional distribution of differentially labelled indoleacetic acid in light grown stems. Physiol. Plant. 2& 95-100 Davies, P.J., Rubery, P.H. (1978) Components of auxin transport in stem segments of Pisum sativum L. Planta 142, 211-219

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379 Penny, P. (1969) The rate of response of excised stem segments to auxins. N.Z.J. Bot. 7, 290-301 Penny, D., Miller, K.F., Penny, P. (1972) Studies on the mechanism of cell elongation of lupin hypocotyl segments. N . Z . J . Bot. 10, 97-111 Penny, P., Dunlop~ J., Perley, J.E., Penny, D. (1975) pH and auxin-induced growth: a causal relationship? Plant Sci. Lett. 4, 3 5 4 0 Pope, D.G. (1977) Separation of indol-3-ylacetic acid-induced growth from acid-induced growth in Arena coleoptiles. Ann. Bot. 41, 1069-1071 Pope, D.G. (1978) Does indoleacetic acid promote growth via cell wall acidification? Planta 140, 137-142 Rayle, D.L. (1973) Auxin-induced hydrogen-ion secretion in Arena coleoptiles and its implications. Planta 114, 63-73 Rayle, D.L., Cleland, R.E. (1970) Enhancement of wall loosening and elongation by acid solutions. Plant Physiol, 46, 250-253 Tanimoto, E., Masuda, Y. (1971) Role of the epidermis in auxininduced elongation of light-grown pea stem segments. Plant Cell Physiol. 12, 663-673 Thimann, K.V., Schneider, C.L. (1938) Differential growth in plant tissues. Am. J. Bot, 25, 627-64I van Overbeek, J., Went, F.W. (1937) Mechanism and quantitative application of the pea test. Bot. Gaz. Chicago 99, 22-41 Vanderhoef, L.N., Findlay, J.S., Burke, J.J., Blizzard, W.E. (1977a) Auxin has no effect on modification of external pH by soybean hypocotyl cells. Plant Physiol. 59, 1000-1003 Vanderhoef, L.N., Lu, T-Y.S., Williams, C.A. (1977b) Comparison of auxin-induced and acid-induced elongation in soybean hypocotyls. Plant Physiol. 59, 1004-1007 Vesper, M.J., Evans, M.L. (1978) Time-dependent changes in the auxin sensitivity of coleoptile segments. Apparent sensory adaptation. Plant Physiol. 61,204-208 Yamagata, Y., Masuda, Y. (1975) Comparative studies on auxin and fusicoccin actions on plant growth. Plant Cell Physiol. 16, 41-52 Yamamoto, R., Shinozaki, K., Masuda, Y. (1970) Stress-relaxation properties of plant cell walls with special reference to auxin action. Plant Cell Physiol. 11,947-956 Yamamoto, R., Maki, K., Yamagata, Y., Masuda, Y. (1974) Auxin and hydrogen ion actions on light-grown pea epicotyl segments. I. Tissue specificity of auxin and hydrogen ion actions. Plant Cell Physiol. 15, 823-831 Received 25 May; accepted 11 July 1980