P|anta (Berl.) 90, 236---242 (1970)

Phytochromc Action in Oryza sativa L. V. Effects of D e c a p i t a t i o n a n d R e d a n d F a r - R e d L i g h t on Cell Wall Extensibility

u MASUDA Department of Biology, Faculty of Science~Osaka City University,Osaka CE~-JUN PJoN Biological Institute, Faculty of Science, Nagoya University, Nagoya MASAKI F u ~ u Y A Botany Department, Faculty of Science, University of Tokyo, Hongo, Tokyo Received July 10 / October 15, 1969

Summary. The mechanical properties of the cell wall were measured in eoleoptiles of totally etiolated rice seedlings. Coleoptfles were either decapitated or briefly exposed to red (R) and/or far-red (FR) light. The elastic and plastic extensibilities of the cell wall changed with age (length) of the coleoptiles. Decapitation and exposure to R induced changes in these properties, and the time-courses were similar. Following decapitation or R irradiation, the plastic extensibility of the cell wall decreased more conspicuously than elastic extensibility. Exogenous application of auxin immediately following decapitation alleviated the effect of removal of the tip. FR irradiation reduced both kinds of extensibflities, but its effect was much less than that of R, and it reversed the R-induced effect to the level of tissue treated with FI~ only. In repeated R-FR treatments, the decrease of elastic extensibflity by R and its reversal by FR could be repeated, but the effect of a second irradiation with R after FR on plastic extensibility was not as apparent as that of the first. Reduction of cell-wall extensibility of etiolated rice coleoptiles caused by R fight appeared, at least partly, to be due to a reduced auxin supply in the elongating region from the tip, similar to that caused by decapitation. Introduction The elongation of the intact rice coleoptile is controlled b y the p h y t o e h r o m e system (Pjon and F u r u y a , 1967). The extent of growth inhibition observed 2 days after red (R) irradiation was proportional to the logarithm of the spectrophotometrieally detectable a m o u n t of t)fr (far-red absorbing form of p b y t o e h r o m e ) in the coleoptfle a t the time of initial exposure to either R or blue light (Pjon and F u r u y a , 1968). I n intact rice seedlings, the photostimulus resulting in the phytochrome-dependent inhibition of eoleoptile elongation is perceived mainly b y the tip while the most rapidly growing zone is in the middle third of the coleoptile, and polar auxin t r a n s p o r t from the tip is m a r k e d l y reduced after R irradiation, while far-red (FR) light given immediately

Phyteehrome Action in Oryza. V

237

after R reverses the a m o u n t of diffusible a u x i n to t h a t of F R controls ( F u r u y a et al., 1969). Hence polar a u x i n t r a n s p o r t was suggested to act as one of the factors i n the s y s t e m m e d i a t i n g b e t w e e n photoperceptive site a n d growing zone. The significance of changes i n the mechanical porperties of the cell wall i n regulating cell elongation has r e c e n t l y been confirmed (Cleland, 1967; Masuda, 1968). I n the oat coleoptile, d e c a p i t a t i o n was reported to remove the source of the a u x i n being t r a n s p o r t e d to the growing region, resulting i n a decreased growth rate (Dolk, 1926), a n d to cause a decrease i n elastic a n d plastic extensibilities of the cell wall (Masuda, 1969). I n excised coleoptfle segments, exogenously applied a u x i n increased b o t h ceil-wall cxtensibflities a n d the rate of elongation (Cleland, 1967; Masuda, 1968, 1969). I n the p r e s e n t s t u d y the effect of R a n d F R irradiations on the elastic a n d plastic extensibilities of etiolated rice coleoptfles was comp a r e d with t h a t of decapitation.

Material and Methods Plant Material and Light Sources. Seeds of Oryza stativa L. (cv. Aichi-Asahi, supplied by the Tokai-Kinki Agricultural Experiment Station, Issinden, Mie Prefecture) were used throughout the present study. The procedures for the culture of totally etiolated seedlings and the sources of 1~ and FR as well as those of the dim green safelight were the same as those previously reported (Pjon and Furuya, 1968). Decapitation and I A A Application. Totally etiolated, 4-day-old seedlings grown at 25~ and having ca. 15-ram-long coleoptiles, were selected and exposed to 300 ergs cm-2 sec-1 R light for 3 min. The coleoptile tip, 3 mm, was removed with a pair of scissors, and the initial length of the decapitated coleoptile was measured immediately thereafter. Purified lanolin (Iwaki Seiyaku Co., Ltd., Tokyo) had been previously mixed with an equal volume of either 10-4 M indole-3-aeetic acid (IAA) or distilled water, and ca. 1 mm~ of this lanolin paste was smeared onto the cut ends of the decapitated coleoptiles. All procedures described here were carried out as quickly as possible under a very dim, green safelight; afterwards, the coleoptiles were returned to darkness until the measurements were made. The incubations were also carried out at 25~. Measurement o] Elasti~ and Plasti~ Extensibilities. Cell-wall extensibilities were measured using a "Tensilion', model UTN II tensile tester (Masuda, 1968), and expressed as elastic compliance (DE) and plastic compliance (DP) (Cleland, 1967). In brief, rice eoleoptfles variously treated were killed by immersion for 5 min in boiling methanol and after incubation in a pronase solution (200 ~g/ml) for 18 hr at 37~, to remove proteins, were stored in methanol until the measurements. The initial distance between the clamps was 5 ram, and the bottom clamp was lowered at a rate of 2 mm rain-1 (chart speed: 20 cm rain-l). The mechanical extension was discontinued when the load reached 30 g, the damps were returned to their original positions, and the coleoptfles were extended again. After the extension measurements, the segments were removed from the clamps, dried and weighed to determine the mass per unit length.

238

Y. Masuda, C.-J. Pjon and M. Furuya:

Results E//ect o/ Coleoptile Length on Plastic and Elastic Extensibilities. An a t t e m p t was made to find the relationship between the age of intact colcoptiles and the mechanical properties of the cell wall in the growing zone. For this purpose, coleoptfles of different age (length) were used. I n 10-ram-long, intact colcoptfles of etiolated rice seedlings, the most rapidly growing region was reported to be between 4 and 6 m m from the tip (Furuya et al., 1969). Hence this region was invariably included in the part of the coleoptile for which the compliances were determined. The D E and D P values of the cell wall were measured for coleoptfles with initial lengths of 15, 20 and 23 ram. The results in the Table clearly demonstrate that both D E and D P values decrease as the age of the coleoptile increases. Thus, it is imperative to use coleoptiles of a uniform age. Table. Age-dependent changes in DE and DP Initial length (mm)

DE DP ( X 10-11 cm2 dyne-L) ( • 10-11 cm2 dyne-1)

14--15 20 21 23--24

27.4• 0.2 20.34-0.3 17.6 4- 0.2

30.94- 1.4 24.0• 26.2 4- 0.8

Values are averages of 20 separa~ measurements with standard errors. The phot~reversible inhibition of rice-coleoptile growth becomes observable only after the coleoptile has reached a size of ca. 10 m m (Pjon and Furuya, 1967). But since the growth capacity drops as the coleoptile elongates, 14--15-mm-long coleoptiles were used in all further experiments.

E//ect o/ Decapitation and I A A Application on DE and DP. An experiment was carried out to observe the effect of decapitation on the mechanical properties of the cell wall. Seedlings having ca. 15-mm-long coleoptiles were selected, and 3 m m of the tip were removed under the safelight. The coleoptiles were then kept in the dark for 0, 3, 6 and 9 hr prior to measurement. The results of such a time-course study are presented in Fig. 1. The D P value continued decreasing for at least 9 hr after decapitation, and the DE value decreased for the first 6 hr but at a slower rate. Elongation of the coleoptiles stopped almost immediately after decapitation. I n order to test the effect of auxin on the cell wall and hence on elongation, 15-ram-long etiolated coleoptiles were decapitated, and the cut surface was smeared with lanolin paste containing 10-4 M IAA or water. 3 hr later, cell wall extensibility was determined. The results are

Phy~ochrome Action in Oryza. V

239

x 10-~m~ne

4O

30

~

DE

20

--~ oP lo

I

I

I

t

0

3

6

9

TIMEAFTERDECAP/TATION hr,

Fig. 1. Effect of decapitation on DE and DP of etiolated rice coleoptiles. Values are averages of 20 separate measurements, with standard errors (vertical bars) s u m m a r i z e d in Fig. 2. The D E a n d D P values of t h e cell wall decreased in d e c a p i t a t e d coleoptiles a n d in t h o s e t r e a t e d w i t h lanolin p a s t e conraining w a t e r ; in Z A A - t r e a t e d coleoptiles, t h e y were n o t different from t h e values of i n t a c t eoleoptfles. The coleoptiles t r e a t e d with t h e p l a i n p a s t e do n o t grow a t all while those t r e a t e d with I A A p a s t e grow as r a p i d l y as i n t a c t ones ( F u r u y a et al., 1969). These results suggest t h a t cell-wall e x t e n s i b i l i t y as well as e l o n g a t i o n r a t e are r e g u l a t e d b y t h e s u p p l y of a u x i n from t h e tip.

xldlcrn2/dyne i DE DP 30~- -T:-

2o-F

,ol

INITIAL

DEDP -h I I

DECAR DECAP.. § e H~O IAA

Fig. 2. Effects of decapitation and of IAA on DE and DP of etiolated rice coleoptiles. Coleoptiles were decapitated and treated with plain lanolin or lanolin containing 10-4M IAA. The cell-wall properties were measured after 3 hr in the dark. Values are averages of 20 separate measurements, with standard errors (vertical bars)

240

Y. Masuda, C.-J. 1)ion and M. l~uruya: x151~/o ,,e

30

20

~

]O

I

DE

DP rIMEAFTER RED R IRADA ITO Ihr N, 1 3

I 6

I 9

Fig. 3. Effect o~ red light on DE and 91) of e~iolated rice coleop~iles. Values are averages o~ 20 separate measurements, with standard errors (vertical bars) Reversible E / / e c t o/ R e d a n d F a r - r e d L i g h t . Since b a s i p e t a l a u x i n t r a n s p o r t from t h e tips of rice coleoptiles was m a r k e d l y i n h i b i t e d a t least b e t w e e n t h e 2nd a n d 4 t h hour after a brief R i r r a d i a t i o n ( F u r u y a et al., 1969), a question arises as to w h e t h e r t h e D E a n d D P values are affected b y exposure of t h e coleoptfles to 1~ light. Fig. 3 illustrates t h e time-course of changes in t h e D E a n d D P of coleoptfles which were exposed t o 300 ergs em -2 sec - i of R for 3 rain. No significant changes of D E a n d D B were seen in t h e d a r k controls d u r i n g t h i s e x p e r i m e n t .

DP

DE 30-

lo

OFRR R R R FRFR FR RR i

i

i

i

i

fO

DFRR

R R R i

FR

i

i

FRFR

F'R

Fig. 4. Effect of red and far-red light on DE and DP of etiolated rice coleoptiles. D dark control; R red light, 3 min; F R far-red light, 3 rain. The cell wall properties were measured after 3 hr in the dark. Values are averages of 20 separate measurements, with standard errors (vertical bars)

Phy~ochrome Action in Oryza. V

241

It is apparent that 1%has essentially the same effect on cell wall extensibflity of growing 15-ram colcoptfles as has decapitation. An attempt was made to see whether the effect of R on DE and DP could be reversed by F1% light given immediately following the R exposure. The results are shown in Fig. 4. A brief exposure to F1% decreased DE slightly and DP significantly, but the effects were smaller than those of 1%. F1% given immediately after the 1% reversed up to ca. 60 % of the R-induced decrease of DE and ca. 40 % of the decrease of DP. The photocontrol of DE by 1% and F1% was repeatedly reversible when the two kinds of light were given alternately for 3 min each, whereas in the case of DP the effect of 1% and F1% was not as evident in the later exposures as in the first ones. Discussion In a previous study (Furuya et al., 1969), it became evident that phytochrome-mediated inhibition of polar auxin transport from the tip of the rice coleoptilc resulted in a depletion of diffusible auxin in the cells of the growing region, and that cells once starved exhibited a decreased sensitivity to exogenous auxin applied later. The photoreversiblc control of elongation in the rice colcoptile, however, could not be explained simply by the photorcgulation of polar auxin transport. Therefore, further investigations were designed to determine how the cell-wall extensibility of the growing region changes following R irradiation and after auxin depletion.

The time-course study clearly demonstrated that R brought about a lowering of both DE and DP (Fig. 3); the effect was quite similar to that of decapitation (Fig. 1). It is noteworthy that the effects of R and F R given alternately on DE (Fig. 4) and coleoptile elongation (Pjon and Furuya, 1967) were repeatedly reversible, while a second exposure to R no longer had any effect on DP (Fig. 4) or on polar auxin transport from the tip (Furuya et al., 1969). Hence the D P of the cell wall appears to be correlated with the polar transport of auxin but it appears not to be strongly correlated with the in-vivo state of phytochrome. I t is thus suggested that the photo-induced lowering of DP is not caused by a direct effect of light on the growing region of the coleoptile, but rather is due to the blockage of basipetal auxin transport. Although DE was correlated with auxin transport in the time-course study (Figs. 1, 3), DE was not greatly correlated with auxin in terms of photoreversibility (Fig. 4) where it seems to be under the control of the phytochrome system in the growing region. As reported previously (Furuya et al., 1969, Table), the growth of the coleoptile is greatly inhibited by g applied to only the tip, but it

242 Y. Masuda, C.-J. Pjon and M. Furuya: Phy~oehrome Action in Oryza. V is also inhibited, although much less, if 1~ is applied to the growing region. I t is thus possible t h a t the elongation of the growing region is, at least to some extent, under the control of p h y t o e h r o m e located in the growing region, as reflected b y changes in D E caused by 1~ and/or FR. I n this case, participation of auxin, although possibly not diffusible auxin, cannot necessarily be rejected. I n this connection, it is interesting to note t h a t an increase in D E was supposed to be primarily required for the initiation of cell expansion, and an increase in I ) P needed in order to maintain a high growth rate in oat eoleoptfles (Masuda, 1969). If the same is true in rice eoleoptfles, the phytoohrome system m a y be more closely related to the initiation of cell expansion rather t h a n to the maintenance of the growth rate. The authors are grateful to the Tokai-Kinki Agricultural Experiment Station for providing the rice seeds, and to Professor J. Ashida, Kyoto University, for careful reading of the manuseipt. The investigation was supported in part by a Research Grant (No. 92186) to lg. F. from the Ministry of Education of Japan. C.J.P. is indebted to the Democratic People's Republic of Korea for a scholarship during the present study. References

Clel~nd, R.: Extensibility of isolated cell walls: Measurement and changes during cell elongation. Planta (Berl.) 74, 197--209 (1967). Dolk, H. E. : Concerning the sensibility of decapitated coleoptiles of Avena sativa for light and gravitation. Proc. kon. med. Akad. Wet. 29, 1113--1117 (1929). Furuya, M., Pjon, C. J., Fujii, T., Ito, M. : Phyteehrome action in Oryza sativa L. III. The separation of photoperceptive site and growing zone in coleoptfles, and auxin transport as effector system. Develop. Growth Differentiation 11, 62--76 (1969). Masuda, Y. : Role of cell wall degrading enzymes in the cell wall loosening in oat coleoptile. Planta (Berl.) 88, 171--184 (1968). -Auxin-induced cell expansion in relation to cell wall extensibility. Plant and Cell Physiol. 19, 1--9 (1969). Olson, A. C., Bonner, J., Morre, D. J. : Force extension analysis of Avena coleoptile cell wall. Planta (Berl.) 66, 126--134 (1965). Pjon, C. J., Furuya, M. : Phytochrome action in Oryza sativa L. I. Growth responses of etiolated coleoptiles to red, far-red and blue light. Plant and Cell Physiol. 8, 709--718 (1967). - - - - II. The speetrophotometric versus the physiological status of phytochrome in coleoptiles. Planta (Berl.) 81, 303--313 (1968). Dr. Yoshio Masuda Department of Biology, Faculty of Science Osaka City University Sumiyoshi-ku, Osaka, Japan