Planta 137, 61 64 (1977) Planta �9 by Springer-Verlag 1977

Phytochrome and Potassium Uptake by Mung Bean Hypocotyl Sections

Colin Brownlee and Richard E. Kendrick Department of Plant Biology, The University, Newcastle upon Tyne NE1 7RU, U.K.

Abstract. Uptake of potassium (K) and 86rubidium- labelled potassium (86Rb) by sub-hypocotyl hook sec- tions of Phaseolus aureus L. was inhibited by red light. The effect was reversible with far red light. Using short exposures of high irradiance the effect on 86Rb-labelled K uptake was observed after 5 rain. The response showed no specificity for a particular anion. Uptake of 86Rb-labelled K by sections cut immediately below the cotyledons was enhanced by red light after 10 rain incubation and was also far red reversible. These results are interpreted as a rapid phytochrome-induced change in membrane properties resulting in modified K uptake.

Key words: Phaseolus Phytochrome -- Potassium uptake.

Introduction

Hendricks and Borthwick (1967) proposed that the initial action of phytochrome involved an alteration of membrane properties and much evidence now sup- ports this view (Jaffe, 1968; Newman and Briggs, 1972; Racusen, 1976; Tanada, 1968). Several reports have indicated phytochrome control of K transport. Phytochrome-controlled fluxes of K in the pulvini of Albizz ia ju l ibr iss in and Samanea saman have been shown to be involved in their leaf movements (Gal- ston and Satter, 1976). K6hler and co-workers (K6hler etal., 1968; K6hler, 1969), using 86Rb- labelled K solutions showed changes in the uptake and distribution of 86Rb in etiolated peas after R and F. However, from the results it was not clear whether these effects were consequences of growth. Kendrick and Hillman (1972) failed to show phyto-

Abbreviations." P = P h y t o c h r o m e ; P r - r e d absorbing form of P; P f r = f a r red absorbing form of P; R = r e d light; F = f a r red light

chrome-related changes in K uptake by etiolated pea stem segments after an uptake period of 3 h. Tezuka and Yamamoto (1975) found a R/F reversible inhibi- tion of K uptake by segments of etiolated mung bean hypocotyls after an uptake period of 1 h. This paper reports the influence of light on uptake of K and 86Rb over short time periods in different regions of the mung bean hypocotyl.

Materials and Methods

Mung bean (Phaseolus aureus L.) seeds were soaked for 4 h in tap water and then sown in trays containing vermiculite moistened with distilled water. After 3 days in darkness at 25_+0.5 ~ C, seed- lings were selected, sections of the hypocotyl excised (see Figs. 1 and 3) and pretreated in an aerated solution containing 1 m mol 1-1 CaCI2 and l m tool 1 1 MgC12 for 3h . Experiments were performed at 2 5 + 0 . 5 ~ and all manipulat ions were carried out under dim green safelight (Kendrick et al., 1969).

In the experiments on K uptake (Fig. 1), samples containing 40 sections were either left in darkness, given 5 rain of R, irradiance 0.5 W m 2 or given 5 rain of R followed by 7 rain of F, irradiance 3 W m -2 (for details of these broad band light sources, see Kendrick and Russell, 1975). After irradiation, samples were placed in 10 ml beakers with 1 ml of K a H P O 4 - K H 2 P O 4 buffer pH 6.5, 50 m tool 1 - a with respect to K, and shaken for 15 and 30 min, after which they were removed, washed with distilled water for 15 s, blotted dry and weighed. K was extracted with a mixture of nitric, hydro- chloric and perchloric acids using a method modified from Chapman and Pratt (1961), and measured using a Pye Unicam SP90 atomic absorption spectrophotometer.

For the experiments on 86Rb uptake (Figs. 2 and 3), 40 sections of either A or B were immersed in l m l 5 0 m tool 1 1 KC1 (pH adjusted to 6.5 with 5 0 m tool 1 -a KOH) labelled with 0.1 ~tCi m l - a 86RbC1" Light treatments were given immediately following immersion and both irradiation and uptake were carried out in 1 cm path length spectrophotometric cuvettes. Samples were shaken during incubation and were either left in the dark or irra- diated with 30 s R or 30 s R followed by 30 s F. R and F was obtained from slide projectors in combination with interference filters (R, 661 nm; band width at 50% transmission 10.6 nm, irra- diance 22 W i n - z ; F, 730 mn; 10.2 nm band width at 50 % transmis- sion, irradiance 29 Win-2) . Used in this way both sources bring about complete photoconversion of phytochrome in the hypocotyl

62 C. Brownlee and R.E. Kendrick: Phytochrome and Potassium Uptake

sections as measured spectrophotometrically. Segments were collected after 5, l0 and 15 min incubation and treated as described for K experiments. After drying, segments were arranged on plan- chettes and the radioactivity counted using an end window GM counter with an automatic sample changer.

All treatments consisted of at least 4 replicates. Results are expressed as K uptake g fresh wt - z + SE or 86Rb-labelled K uptake g fresh wt 1 • SE. Differences between treatments were tested for significance using Student's t test (Fisher and Yates, 1963).

Results and Discussion

Figure 1 shows that K uptake by 5 mm sub-hypocotyl hook sections (section B) is significantly inhibited by R when measured after 15 and 30 min incubation. This inhibition of K uptake by R is reversed by F, demonstrating phytochrome control. The results in Figure 1 are from one experiment, but qualitatively similar results have been repeatedly observed. This is also true for K uptake from 50 m mol 1- ~ KC1, indicating that this response is not specific for a par- ticular anion. Figure 2 demonstrates uptake from 50 m mol 1-t KC1, labelled with the marker 86Rb. By this technique, R/F reversible inhibition of 86Rb

uptake is clearly detectable after 5 min incubation.

"i"

,.E

i... ,,1--

U I |

E ::L

itl

K :3

X:

SECTION B

8- S m m ~

7 -

6- ~- J_

5-

D

I

15 min 30 min

�9 _ _ J

Fig. 1. Uptake of K by hypocotyl section B after 15 and 30 rain incubation in darkness in K-phosphate buffer, pH 6.5, 50 m mol l - z with respect to K. Pretreatments-D: Darkness; R: 5 rain R; R/F: 5 min R followed by 7 min F. Results represent means of at least 4 replicates and brackets represent +SE. D and R/F treaments all significantly different from R, at least at 5% level, but not from each other. Demonstrates inhibition of K uptake by R and its reversal by F

"T t- g~ o

oi

o

: t .

S_4-

~o

SECTION B

T

[

F~/F

t _ _ J

-}

IR JD

i

R :~/F

5min 10 min 15 min

Fig. 2. Uptake of S6Rb-labelled K by hypocotyl section B after 5, 10 and 15 min incubation in 50 m mol 1-1 KC1, pH 6.5. Treat- ments-D: Darkness; R: 30s R; R/F: 30s R followed by 30s F. Results represent means of at least 4 replicates +SE. D and R/F treatments all significantly different from R, at least at 5% level, but not from each other. Demonstrates inhibition of S6Rb-labelled K uptake by R and its reversal by F

The magnitude of the R/F reversible inhibition ap- pears to be constant after 5, 10 and 15 min incubation although total 86Rb uptake increases. Such results indicate a rapid phytochrome-mediated effect on s 6Rb uptake.

In contrast to the sub-hypocotyl hook section, Fig- ure 3 shows that R enhances 86Rb uptake by 3 mm hypocotyl sections cut immediately below the cotyle- dons (section A). In this experiment the response to R was only significant after 10 min incubation, but was readily F reversible. This enhancement of K uptake by R in section A has also been measured directly by atomic absorption after 15 min incubation. Clearly, sections A and B are behaving differently in response to phytochrome photoconversion.

R is known to increase the growth of very young regions of dark-grown stems and to inhibit the growth of older, elongating regions (Thomson, 1954; Klein et al., 1957; Got6 and Esashi, 1976). It is likely that growth responses to R in the mung bean hypocotyl

C. Brownlee and R.E. Kendrick: Phytochrome and Potassium Uptake 63

SECTION A

- - 11 I

oa

::i.

~ i0 J

| 9 .0 ]

5 min 10 rain 15 min

Fig. 3. As for Figure 2, but for hypocotyl section A. D, R, R/F treatments, no significant differences at 5 min. D and R/F treat- ments at 10 and 15 min significantly different from R, at least at 5% level, but not from each other. Demonstrates enhancement of SSRb-labelled K uptake by R and its reversal by F

are in the same direction as phytochrome-mediated changes in K uptake, though no evidence is available concerning such growth responses in mung beans. The possibility thus arises that growth is a causative factor. However, this is unlikely as most growth re- sponses to R are not detectable within the first 15 min after irradiation. Meijer (1968) detected growth inhi- bition of the gherkin hypocotyl by R only after 30 min. K6hler (1969), using 86Rb with intact pea plants, showed that phytochrome-mediated changes in translocation may preceed growth changes. A lag in growth of somewhat more than one hour was recorded. Bertsch and Hillman (1961) demonstrated that sucrose was necessary for a R/F reversible growth response in isolated pea stem segments. We have used isolated segments, preincubated in the absence of su- crose, which would not be expected to show rapid phytochrome-mediated growth responses.

Higinbotham (1974) has reviewed evidence that ion transport across membranes consists of both ac-

tive and passive components. It is possible that phy- tochrome may interact with the cell membrane to modify permeability or an active electrogenic or ex- change pump. Pfr has been found to inhibit auxin- induced proton extrusion by pea stem segments (Ltirssen, 1976). Alternatively, some other process such as extracellular binding or exchange may be modified indirectly by phytochrome photoconversion. Haupt (1976) has speculated that Pfr might alter the binding of Ca 2§ ions to membrane constituents and the concentration of free Ca 2+ in the cytoplasm, which may in turn affect permeability to other sub- stances. Preliminary experiments suggest that Ca 2+ and Mg 2§ enhance the responses described here, and have routinely been included in the preincubation me- dium. At present the exact mechanism of phyto- chrome modulation of K uptake in mung beans is only speculation but appears likely to reflect a change in membrane properties. Clearly R inhibits K uptake in mung bean hypocotyl sections at much shorter time intervals than reported previously (Tezuka and Yamamoto, 1975).

The qualitatively different responses of the two regions of the hypocotyl hook are of great interest. If these modulations are linked to the initial action of phytochrome then they indicate a differential re- sponse to Pfr. Alternatively, changes in K uptake may be secondary consequences of Pfr action depend- ing on the physiological state of the tissue. Currently we are studying K uptake in hypocotyl hook sections at even shorter time intervals after irradiation in order to characterize more exactly the mechanism involved. We are also studying light mediated short term K fluxes in the hypocotyl hooks of intact seedlings in an attempt to relate the K uptake seen in the sections to the physiology of whole seedlings.

Supported by a Science Research Council Studentship (C.B.) and a Scientific Investigations grant from the Royal Society (R.E.K.).

R e f e r e n c e s

Bertsch, W.F., Hillman, W.S.: The photoinhibition of growth in etiolated stem segments. I. Growth caused by sugars in Pisum. Amer. J. Bot. 48, 504-511 (1961)

Chapman, H.D., Pratt, P.F.: Methods of analysis for soils, plants and waters. Univ. of Calif. Div. Agric. Sci., p. 194, 196I

Fisher, R.A., Yates, F. : Statistical tables for biological, agricultural and medical research. Endinburgh: Oliver and Boyd 1963

Galston, A.W., Satter, R.L. : Light, clocks and ion flux: an analysis of leaf movement. In: Light and Plant Development, pp. 159-184, Smith, H., ed. London: Butterworths I976

Got6, N., Esashi, Y.: Aging progression involving dwarfism and its acceleration by red light in bean hypocotyls. Plant Physiol. 57, 547-552 (1976)

64 C. Brownlee and R.E. Kendrick: Phytochrome and Potassium Uptake

Haupt, W., Weisenseel, M.H.: Physiological evidence and some thoughts on localised responses, intracellular loealisation and action of phytochrome. In: Light and Plant Development, pp. 63-74, Smith, H., ed. London: Butterworths 1976

Hendricks, S.B., Borthwick, H.A.: The function of phytochrome in regulation of plant growth. Proc. Natl. Acad. Sci. USA 58, 2125-2130 (1967)

Hinginbotham, N. : Conceptual developments in membrane trans- port, 1924-1974. Plant Physiol. 54, 454462 (1974)

Jaffe, M.J.: Phytochrome-mediated bioelectric potentials in mung bean seedlings, Science, 162, 1016-1017 (1968)

Kendrick, R.E. Hillman, W.S. : Ion relations, chlorophyll synthesis and the question of ' bulk' phytochrome in Pisum sativum. Phys- iol. Plant. 26, 7-12 (1972)

Kendrick, R.E., Russell, J.H. : Photomanipulation of phytochrome in lettuce seeds. Plant Physiol. 56, 332-334 (1975)

Kendrick, R.E., Spruit, C.J.P., Frankland, B.: Phytochrome in seeds of Amaranthus caudatus. Planta (Berl.) 88, 293-302 (1969)

Klein, W.H., Withrow, R.B., Elstad, V., Price, L.: Photocontrol of growth and pigment synthesis in the bean seedling as related to irradiance and wavelength. Amer. J. Bot. 44, 15 19 (1957)

KShler, D. : Phytochrome-dependent ion transport in pea seedlings. Planta (Berl.) 84, 158-165 (1969)

K6hler, D., Willert, K.V., Lfittge, U.: Phytochrome-dependent variation of growth and ion uptake of leaves and internodes

of etiolated pea (Pisum sativum) seedlings. Planta (BerL) 83, 3548 (1968)

Lfirssen, K.: Counteraction of phytochrome to the IAA-induced hydrogen ion excretion in Arena coleoptile cylinders. Plant Sci. Lett. 6, 389-399 (1976)

Meijer, G.: Rapid growth inhibition of gherkin hypocotyls in blue light. Acta. Bot. Neerl. 17(1), 9-14 (1968)

Newman, I.A., Briggs, W.R.: Phytochrome-mediated electric potential changes in oat seedlings. Plant Physiol. 50, 687-693 (1972)

Racusen, R.H.: Phytochrome control of electric potentials and intercellular coupling in oat-coleoptile tissue. Planta (Berl.) 132, 25-29 (1976)

Tanada, T.: A rapid photoreversible response of barley root tips in presence of 3-indole-acetic acid. Proc. Natl. Acad. Sci. USA 59, 376-380 (1968)

Tezuka, T., Yamamoto, Y.: Control of ion absorbtion by phyto- chrome. Planta (Berl.) 122, 239-244 (1975)

Thomson, B.F. : The effect of light on cell division and cell elonga- tion in seedlings of oat and peas. Amer. J. Bot. 41, 326-332 (1954)

Received 31 May; accepted 11 July 1977