Planta

Planta 149, 91-96 (I980)

9 by Springer-Verlag 1980

The Phytoehrome System in Light-grown Zea mays L. Merten Jabben Institut ffir Biologic II, Universitfit Freiburg, D-7800 Freiburg, Federal Republic of Germany

Abstract. The p h y t o c h r o m e system is a n a l y z e d in light-grown m a i z e (Zea mays L.) plants, which were p r e v e n t e d f r o m greening b y a p p l i c a t i o n o f the herbicide S A N 9789. T h e d a r k kinetics o f p h y t o c h r o m e are n o t different in the first, s e c o n d o r t h i r d leaf. It is c o n c l u d e d t h a t in l i g h t - g r o w n m a i z e p l a n t s phyt o c h r o m e levels are r e g u l a t e d b y Pr f o r m a t i o n a n d Pfr a n d Pr destruction, r a t h e r t h a n by Pfr--'Pr d a r k reversion. Pr u n d e r g o e s d e s t r u c t i o n after it has been cycled t h r o u g h Pfr- The consequences o f this Pr destruction on the p h y t o c h r o m e system are discussed.

Key words: L i g h t - g r o w n p l a n t s - P h y t o c h r o m e S A N 9789 -

Zea.

the herbicide S A N 9789, which inhibits c h l o r o p h y l l a c c u m u l a t i o n in white light, b u t leaves the p h y t o c h r o m e system unaffected ( J a b b e n a n d D e i t z e r 1979; F r o s c h et al. 1979; H u n t a n d P r a t t 1979; G o r t o n a n d Briggs 1979). In o r d e r to m o r e closely a p p r o x i m a t e the situation of a m a t u r e plant, m a i z e (Zea mays L.) was used which c o n t a i n s m o r e seed s t o r a g e material. In the presence o f S A N 9789 m a i z e fully d e v e l o p s at least 3 leaves. In this p u b l i c a t i o n e x p e r i m e n t s a r e d e s c r i b e d w h i c h a l l o w for a c o m p a r i s o n o f the p h y t o c h r o m e system in these 3 leaves a n d f r o m which conclusions on the in situ p h y t o c h r o m e system p r e v a l e n t in m a i z e p l a n t s c a n be drawn.

Introduction

Materials and Methods

The p h y t o c h r o m e system in l i g h t - g r o w n p l a n t s has been s t u d i e d in o a t seedlings which were t r e a t e d with the h e r b i c i d e S A N 9789 ( J a b b e n a n d D e i t z e r 1978b). It has been c o n c l u d e d f r o m these e x p e r i m e n t s t h a t in c o n t i n u o u s white light a c o n s t a n t p o o l o f p h y t o c h r o m e (Plot) is e s t a b l i s h e d t h r o u g h an e q u i l i b r i u m between Pr f o r m a t i o n and Pfr d e s t r u c t i o n . Since these o a t seedlings were never e x p o s e d to d a r k n e s s the phyt o c h r o m e system is the p h y t o c h r o m e system o f a truly light-grown seedling. H o w e v e r , the o a t seedlings were o n l y 6 d a y s old a n d the e x p e r i m e n t s were p e r f o r m e d on the t i p - r e g i o n o f the p r i m a r y leaf; one m i g h t suspect the situation to be different in the leaves o f m a ture plants. Since the s p e c t r o p h o t o m e t r i c assay for the q u a n t i t a t i v e d e t e c t i o n o f p h y t o c h r o m e is restricted to n o n - g r e e n p l a n t tissues, p l a n t s were t r e a t e d with

Plant Material. Maize (Zea mays L., Hybrid Inrakorn l, Nungesser, Bad Krozingen) seeds were presoaked for 8 h in water containing 5.10 -s M SAN 9789. Seedlings were grown on moist vermiculite containing 5.10- 5 M SAN 9789 for 9 d under continuous white light or, in one experiment (Fig. 2), for various days under light/ dark (12/12) cycles.

Abbreviations : SAN 9789-4-chloro-5-(methylamino)-2-(c~,c~,c~-tri fluoro-m-tolyl)-3(2H) pyridazinone; Pf,-far-red absorbing form of phytochrome; Pr= red absorbing form of phytochrome; P,ot Pfr + P~

Light Sources. White light was obtained from HQIL-lamps (3.6 kW). The light was filtered through a 6 mm Thermophane glass. The spectral distribution of the light in the visible range was very similar to sunlight; the illuminance was 16,000 Ix. Far-red light was provided by a far-red source (emission maximum at 740 rim, bandwidth approximately 120 nm, fluence rate 3.5 Wm- 2) and red light was obtained from a standard red source (emission maximum at 656 nm, bandwidth 15 nm, fluence rate 0.67 Wm -2 or, if indicated, 6.7 Win-2). For faster photoconversions (Table 4) high-intensity red or far-red light was obtained by filtering the light from a modified Zeiss Ikon Xenosol III 2.5 kW Xenon arc source (described in Beggs et al., in press) through an AL interference filter (2max=660nm, bandwidth 17 nm, fluence rate 12 Wm 2) or through a DAL interference filter ()o~,~=729 nm, bandwidth 16 nm, flueace rate 20 Wm 2). Phytochrome Measurements. Maize leaves were harvested and placed vertically in a metal cuvette (1.2.1.2.4 cm aluminum block 0032-0935/80/0149/0091 / $01.20

92

M. Jabben: Phytochrome in Light-grown maize

with glass windows at the front and the rear and a horizontal pathlength of 9 mm). The measuring beam passed through the apical 2 cm of 14 primary leaves or (Table 2) through different regions of 14 secondary leaves. In all other experiments seven secondary and tertiary leaves were cut in the middle and the two halves were layered so that the measuring beam passed through the central area (upper and lower central area) of the leaf. Phytochrome contents were measured at 1~ C with a custom-built spectrophotometer (Pratt and Marm6 1976). A (AA) values were determined between 728 and 793 nm with 662 and 739 nm as the actinic lights (Schott IL interference filters). The difference spectrum was obtained by processing multiple spectra in a digital signal averager (Tracor-Northern Model NS-570).

1. Spectral properties of phytochrome in vivo (from difference spectra)

Table

Sample

maize (light-grown) maize (etiolated) cauliflower curd

Rma x

Isosbestic

FRma x

R/F

(nm)

point (nm)

(nm)

ratio

657 660 665

687 690 688

730 730 725

1.05 1.1 i 0.97

have been reported for etiolated maize coleoptiles (Quail 1974) or for cauliflower curds (Johnson and Hilton 1978). Spectral data are summarized in Table 1.

Results

Spectral Characteristics. The most serious problem involved in the spectrophotometric measurements of phytochrome in light-grown plants is the presence of chlorophyll (Butler 1962). Although the SAN 9789treated maize leaves appear completely white, conclusive evidence that the measured A (AA) signals are due to phytochrome can only be obtained from a difference spectrum. The in vivo difference spectrum of secondary leaves (upper and lower central region) is depicted in Fig. 1. It clearly shows that only phytochrome is responsible for the measured red/far-red reversible absorbance changes. The peak positions (Rma x and FRmax), the isosbestic point, and the ratio of red to far-red absorbance maxima in the difference spectrum (R/F-ratio) are very similar to those which

Phytochrome Levels in Maize Leaves. Nothing is known about the distribution of phytochrome within the leaves of a light-grown plant. Since detailed immunochemical studies on the issue are not yet available, a very rough estimate of the phytochrome distribution within a secondary leaf was made spectrophotometrically. Table 2 shows that phytochrome is distributed relatively homogeneously over the maize leaf and that this distribution does not change drastically during 24 h of darkness. This indicates that during kinetical studies, when phytochrome is measured only in a certain part of a leaf, no spatially differential changes occur.

2. A (AA) for various regions of secondary leaves of 9 d old light-grown maize. All A (AA) values were multiplied by 10 3 Table

Region assayed

9 d WL

9 d W L + 5 min far-red +48h D

Apical 2 cm Upper central Lower central Basal

1.4 1.9 1.6 1.3

3.5 4.5 4.7 4.7

(Pr- Pfr )

; O:,v;

ID 0 e~ 0

I ~

0

i--0.00?.

I

I

M',,,.

650

wavelength ( n m) Fig. 1. The difference spectrum in vivo between far-red and red irradiated maize (secondary leaves). The difference spectrum was recorded 5 times and normalized electronically

In order to determine the age dependency of the phytochrome levels in the plants, maize was grown under light/dark (12/12) cycles and the phytochrome content was measured every day (Fig. 2). In each leaf the phytochrome content slowly diminishes with age and apparently levels off in all 3 leaves after about 11 days at about the same level. Similarly, a low but apparently constant phytochrome level in the light has been observed in light-grown oat seedlings (Jabben and Deitzer 1978b) or in green oat seedlings (Hunt and Pratt 1979). After the 7th day the first maize leaf is fully developed and its size remains constant. The decrease of the A (AA) signal in the second

M. Jabben: Phytochrome in Light-grown maize

93

Maize ?

2

-

9~

x

9 ~

~

9 1 st O

2 nd

x

3 rd

Fig. 2. Age dependency of phytochrome levels in primary (o), secondary (o) and tertiary (x) leaves of maize. Determinations were made at the middle of the light period of the light/dark (12/12) cycle

te0f

I

t

r

r

l

7

8

9

10

11

12

13

DAYS AFTER SOWING

LIGHT

Maize

DARK

2.0

Fig. 3. Light- and dark kinetics of Ptot, Pfr and P, in maize grown for 9 d under continuous WL. At the end of the light period plants received either a saturating red light (R) or far-red light (FR) pulse. Closed symbols (m, o, *) represent Ptot levels after a R-, X-ed symbols ( [], | ~ ) after a FR light pulse. Open symbols represent Pfr (on, o) or Pr (zx) levels. Pr is estimated (=Pto~ Per). Phytochrome levels in secondary leaves (| o, o) are compared to levels in primary ([], m, []) and tertiary ( . , *) leaves. Ptot levels are normalized to Pto, levels after 9 d of WL (time 0 ~ l . 0 ~ l . 5 x 10-3 A (AA))

c~ II

o

n

1.5

1.0

t o w

9

0.5

I 0

6

12 0

HOURS LIGHT

5

12

2~

36

HOURS DARKNESS

leaf between d a y 7 and 9 m a y be partially due to a " d i l u t i o n - e f f e c t " because of leaf growth, although leaf growth is too extensive to completely explain the decrease in A (AA). The A (AA) signal in the third leaf, although it grows rapidly f r o m day 9 to 12, decreases very slowly. This obviously m e a n s that there is net synthesis of p h y t o c h r o m e in these leaves.

Analysis of the Phytochrome System. After 9 d of continuous white light the kinetics of Ptot, P f , and P~ were determined in subsequent darkness in the seco n d a r y leaf and c o m p a r e d with the p h y t o c h r o m e levels in the first and third leaves (Fig. 3). The level of Ptot remains constant for some time during the d a r k period as it did during the light period. Pfr disappears slowly with a half-life of 7-9 h and even after 24 h there is still some Pf~ detectable. P~ (calculated f r o m m e a s u r e d Ptot and Pfr) increases linearly with

6.10 -5 A (AA) h -1. Conversion of Pe, by 5 min of far-red light at the end of the light period results (after a small undershoot) in a linear increase of Ptot - which is P~ - with a rate constant of again 6.10 -5 A (AA) h - I . It becomes obvious that the rates of Pr f o r m a t i o n are not different, either in the beginning of darkness when only part of the p h y t o c h r o m e is present as Pr, or when all the p h y t o c h r o m e is present as P~. It can be seen f r o m Fig. 3 that the dark-kinetics of P~o,, Pfr, and Pr are not different in the first, second, or third leaf. Also, there is no difference in the kinetics of Ptot, Pfr, and P~ between seedlings grown under continuous white light and light/dark (12/12) cycles (data not published).

Destruction of Pr. The small u n d e r s h o o t in the Ptot kinetic (Fig. 3) appears to be due to the destruction of Pr which had been previously converted f r o m Prr.

94

M. Jabben: Phytochrome in Light-grown maize

A 1.0

-

-

II -I-, v

g I1.-" ..i.a

0.5

-

o

13..'" Moize

0

I

40

1 45

Fig. 4. Dark kinetics of P,ot (= Pr) in secondary leaves of maize grown for 9 d in WL followed by a FR pulse ([]). After 45 h darkness, plants received either 3 min R (6.7 Wm -2) + 5 rain FR (e) or 5 rain FR (x) or remained in darkness. Ptot levels are normalized to Ptot levels after 45 h darkness (4.3.10 -3 A (AA))

I

50

HOURS D A R K N E S S

Table 3. Phytochrome levels (Ptot) in secondary leaves of maize

Table 4. Phytochrome levels (Pto,) in secondary leaves of maize

(9 d WL+5 min FR) after different light treatments. Treatment with light pulses (R/FR=3 rain R"+5 rain FR) was started after 45 h of darkness

after different light treatments. Light pulses were given after 45 h of darkness (t = 0)

Treatment

A (AA) x 10 3

0 ( : 4 5 h D) R/FR+2 h D R/FR+ 2 h D+R/FR+2 h D R/FR+2hD+R/FR+2hD+R/FR+2hD

4.3_+0.1 3.3_+0.2 2.4_+0.1 2.1_+0.1

a

Ftuence rate: 6.7 Wm 2

In order to investigate this p h e n o m e n o n o f Pr destruction in m o r e detail, maize plants receiving 5 rain of far-red light at the end of the light period were left in darkness until, after about 2 d, the accumulation o f Ptot ceased. After 45 h o f darkness plants received various light treatments (Fig. 4). If a 5 min far-red pulse is given or if plants remain in darkness the Ptot level is not greatly altered. A 3 min red light pulse followed immediately by 5 rain of far-red light, on the other hand, results in a decrease o f Ptot which levels off after 2 h. All 3 treatments in Fig. 4 result in the f o r m a t i o n o f Pr. But the Pr time courses are different. W h e n Pr cycles t h r o u g h Pfr, it undergoes destruction and this destruction ceases after 2 h. One m a y postulate that after 2 h there is no m o r e cycled, destructable Pr left. This in fact seems to be the case: If the plants are irradiated again with red followed by far-red light, there is a further loss o f Pr (Table 3). The table shows that this treatment can be repeated with the same result.

Treatment

A (AA) ( x 10-?)

2h D 5 s R"+5 min D + I min FRb+2 h D 5 s R+ 1 min FR+2 h D

4.4+ 0.2 3.3_+0.2 4.1 _+0.2

b

AL 660 : 12 Win- 2 DAL 729:20 Wm z

In order to estimate the time period required for Pr destruction to begin, the plants were irradiated briefly with 5 s of red light, immediately followed by far-red light, or were left for 5 min in the dark before the far-red light irradiation (Table 4). The results indicate that some time ( > 5 s) is needed for p h y t o c h r o m e in the Pfr f o r m before, after reconversion, Pr destruction can occur.

Discussion

Until recently, investigations o f p h y t o c h r o m e have been restricted almost exclusively to etiolated seedlings. The presence o f chlorophyll made in vivo spect r o p h o t o m e t r i c measurements of p h y t o c h r o m e in green plants impossible. By using the herbicide S A N 9789, the chlorophyll problem can be circumvented (Jabben and Deitzer 1978a). This herbicide blocks the synthesis o f carotenoids, thereby allowing the photodestruction of chlorophyll and other

M. Jabben: Phytochrome in Light-grown maize

plastid components to occur (under white light at a high fluence rate) since the protection of the chlorophyll normally provided by the carotenoids is lost (Bartels and Hyde 1970; Frosch et al. 1979). Components outside the plastid do not appear to be affected. In particular, the physiological effectivity of phytochrome does not seem to be altered at all in dark-grown (Jabben and Deitzer 1979; Frosch et al. 1979; Gorton and Briggs 1979) and light-grown seedlings (Jabben and Deitzer 1979; Beggs et al. in press). In dark-grown oat seedlings, Pfr destruction and Pr reformation occur at the same rate both in SAN 9789-treated seedlings and in the water controls (Jabben and Deitzer 1979). A radio-immuno-assay (RIA) for phytochrome has been developed recently (Hunt and Pratt 1979) which allows the measurement of the Ptot level in the crude extracts of green tissue. When, using this technique, the Ptot levels in white, SAN 9789-treated oats and green oats of the same age are compared, no difference is found (Hunt and Pratt 1979). This indicates that in the light the phytochrome levels in the herbicide-treated plants reflect the levels in normal green plants. With regard to Pr reformation in the dark there are quantitative differences between the experiments using the herbicide method (Jabben and Deitzer 1978b) and the experiments using the RIAssay (Pratt 1979). Experiments are designed to investigate the reasons for this difference. The main conclusion, that the constant phytochrome level in light-grown oat seedlings is established mainly through Pr destruction and Pr reformation, seems to be confirmed by the experiments using the RIAssay. In this paper the phytochrome system in the first 3 leaves of maize was analyzed spectrophotometrically. There does not seem to be any difference in the dark kinetics of Ptot, Pfr, and P~ in the 3 leaves. Although with the herbicide method it is difficult to study the phytochrome system in the 4th, 5th, etc. leaves, one may assume that the system does not vary greatly from that in the first 3 leaves. Hence, one may conclude that the phytochrome levels in light-grown maize plants, at least in the leaves, are established mainly through Pfr destruction and Pr reformation. The participation of Pf,~ P, dark reversion can be excluded (Borthwick and Hendricks 1960). Part of the phytochrome destruction under white light seems to be due to Pr destruction. Pr destruction which has been observed in etiolated oat seedlings (Chorney and Gordon 1966; Dooskin and Mancinelli 1968; Mackenzie et al. 1978; Stone and Pratt 1979) can also be observed in light-grown maize. It can only be observed if Pf, is formed first and then converted to P~ by a pulse with far-red light. But

95

h.v

~A

.

-

re(a xa t i on k/,

k3 h.v

pB r

destruction/J.,r

S";

pB fi" f r ~ destruction

Fig. 5. A scheme of the phytochrome system which takes in vivo experiments on P, destruction into account

in order for the P, destruction to occur phytochrome must stay in the Pfr form for at least 5 seconds. From the results depicted in Fig. 4 it has to be concluded that there are at least two different states of P,, one of which is due to original P, (pA) present after prolonged darkness and one which is cycled through Pfr first and then undergoes relatively rapid destruction (P~). An interpretation of the results presented in Fig. 4 and Tables 3, 4 is proposed in Fig. 5. This scheme takes into account the fact that P, decays after it has cycled through Pfr and that this decay levels off after a certain time (2 h for maize). It is proposed that this leveling off is due to the "relaxation" of the PrB pool in the dark back to the original pA. In this case a second red light pulse, followed by a far-red light pulse, should again produce PrB which would also undergo destruction. The results presented in Table 3 confirm this assumption. A quantitative analysis of these processes (Fig. 4) yields a half-life time of about 90 min for P~ destruction and about 50min for P,~~PrA relaxation. The experiments from which the scheme proposed in Fig. 5 is deduced are in vivo measurements of PtotThis scheme appears similar, except for the P, destruction, to those which were deduced from phytochrome peltetability experiments (Quail et al. 1973; Sch~ifer 1975). Even the half-life time for the relaxation process TI/2-~50 min) is very similar. As has been concluded for etiolated seedlings (Schiller 1975), a phytochrome system consisting of Pr~Pf, photoconversions, P, synthesis, and Pf, destruction seems to be too simplistic for light-grown maize plants as well.

This work was supported by the Deutsche Forschungsgemeinschaft (Ja273/2 and SFB 46/E6). I wish to thank Dr. E. Schiller for discussions and Sandoz AG, Basel, Switzerland, for providing the herbicide SAN 9789.

96

References Bartels, P.G., Hyde, A. (1970) Chloroplast development in 4chloro - 5 - (dimethyl - amino) - 2 - (c~,c~,c~-trifluoro-m-tolyl)- 3 (2H)pyridazinone (Sandoz 6706) treated wheat seedlings. Plant Physiol. 45, 807 810 Beggs, C.J., Holmes, M.G., Jabben, M., Sch~ifer, E. (in press) Action spectra for inhibition of hypocotyl growth in light and dark grown Sinapis alba L. seedlings. Plant Physiol. Borthwick, H.A., Hendricks, S.B. (1960) Photoperiodism in plants. Science 132, 1223 1228 Butler, W.L. (1962) Effects of red and far-red light on the fluorescence yield of chlorophyll in vivo. Biochim. Biophys. Acta 64, 309-317 Chorney, W., Gordon, S.A. (1966) Action spectrum and characteristics of the light activated disappearance of phytochrome in oat seedlings. Plant Physiol. 41, 891 896 Dooskin, R.H., Mancinell, A.L. (1968) Phytochrome decay and coleoptile elongation in Arena following various light treatments. Bull. Torrey Bot. Club 95, 474 487 Frosch, S., Jabben M., Bergfeld, R., Kleinig, H., Mohr, H. (1979) Inhibition of carotenoid biosynthesis by the herbicide SAN 9789 and its consequences for the action of phytochrome on plastogenesis. Planta 145, 497-505 Gorton, H.L., Briggs, W.R. (1979) Phytochrome responses of light grown corn seedlings in the presence and absence of Sandoz. Abstr. of Ann. Europ. Symp. Photomorph. 85 Antwerpen Hunt, R.E., Pratt, L.H. (1979) Phytochrome radio-immunoassay. Plant Physiol. 64, 327 331 Jabben, M., Deitzer, G.F. (1978a) A method for measuring phytochrome in plants grown in white light. Photochem. Photobiol. 27, 799 802

M. Jabben: Phytochrome in Light-grown maize Jabben, M., Deitzer, G.F. (1978b) Spectrophotometric phytochrome measurements in light-grown Arena sativa L. Planta 143, 309 313 Jabben, M., Deitzer, G.F. (1979) Effects of the herbicide SAN 9789 on photomorphogenic responses. Plant Physiol. 63, 481-485 Johnson, C.B., Hilton, J. (1978) Effects of light on phytochrome in cauliflower curd. Planta 144, 13-17 MacKenzie, J.M. Jr., Briggs, W.R., Pratt, L.H. (1978) Phytochrome photoreversibility: empirical test of the hypothesis that it varies as a consequence of compartmentalization. Planta 141, 129-134 Pratt, L.H., Marm+, D. (1976) Red light-enhanced phytochrome pelletability: a re-examination and further characterization. Plant Physiol. 58, 686 692 Pratt, L.H. (1979) Phytochrome purification and assay. Abstr. of Ann. Europ. Symp. Photomorph. 16, Antwerpen Quail, P.H., Marm6, D., Schfifer, E. (1973) Particle-bound phytochrome from maize and pumpkin. Nature (London) 245, 189 191 Quail, P.H. (1974) Particle-bound phytochrome : spectral properties of bound and unbound fractions. Planta 118, 345-355 Schiller, E. (1975) A new approach to explain the "High Irradiance Responses" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 Stone, H.J., Pratt, L.H. (1979) Characterization of the destruction of phytochrome in the red-absorbing form. Plant Physiol. 63, 680-682

Received 28 November 1979; accepted 20 January 1980