Phytochrome does not associate with ribonucleoprotein particles from Cucurbita pepo

Department of Developmental Biology, Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2601, Australia

Phytochrome does not associate with Ribonucleoprotein Particles from Cucurbita pepo RICHARD

Yu

With 6 figures Received February 27, 1980 . Accepted May 28, 1980

Summary Following red light irradiation of excised zucchini hypocotyl hooks and tissue homogenization in a buffer containing EDTA about 50 Ofo of the cellular phytochrome became sedimentable into a crude mitochondrial fraction. Upon further analysis by sucrose gradient centrifugation, phytochrome in the crude mitochondrial fraction was distributed near the mitochondrial region and the gradient/sample interface of the gradient. Neither of these two areas of phytochrome activity exactly coincided with the RNA-containing material. Treatment of the crude mitochondrial fraction with Triton X 100 solubilized the phytochrome, RNA-containing material, and the NADPH cytochrome c reductase activity but not the cytochrome c oxidase activity. Under this condition phytochrome and RNA-containing material still appeared to sediment separately. When homogenized in the presence of EDTA, phytochrome and RNA-containing material remaining in the post-mitochondrial supernatant were eluted as separate zones from Sepharose 4B/CL columns, phytochrome being the slower eluting material. When red light irradiated hooks were homogenized in a buffer containing Mg++, phytochrome in the post-mitochondrial supernatant was found to elute faster than the RNA-containing material. But in neither case were phytochrome and RNA-containing material eluted together. Pretreatment of red lightirradiated hooks with glutaraldehyde resulted in most of the phytochrome in the crude mitochondrial fraction to sediment apparently with the cytochrome oxidase activity and RNA containing material. Triton X 100 treatment of the crude mitochondrial fraction did not appear to solubilize the phytochrome and RNA-containing material but caused them to sediment separately. They banded with the cytochrome c oxidase activity on prolonged centrifugation. In post-mitochondrial supernatant from glutaraldehyde-treated tissues, phytochrome and RNA-containing material were eluted from Sepharose columns as separate zones, phytochrome being the slower component. Key words: zucchini, phytochrome, RNP, binding, receptor.

Introduction The interaction of phytochrome with subcellular material in Cucurbita has been extensively studied (see QUAIL'S review, 1975 a). It has been reported that when a 20,000 xg sediment from homogenates of zucchini hypocotyl hooks, previously irZ. P/lanzenphysiol. Bd. 99. S. 449-460. 1980.

450

RlCHARD Yu

radiated with red light in vivo and extracted in the presence of either Mg++ or EDTA, was subfractionated on a sucrose density gradient containing EDTA, phytochrome was seen to be associated with RNP (31S) particles and with «heavy» membrane particles (QUAIL, 1975 b, c). Subfractionation of a homologous 20,000 sediment, extracted in the presence or absence of Mg++, on a sucrose density gradient containing no EDTA, did not show the presence of 31S RNP particles and hence showed no evidence of association of phytochrome with 31S RNP. It was concluded that the RNP fraction is probably of ribosomal origin and that the data were accounted for by an electrostatic adsorption of phytochrome onto ribosomal material, either ER-associated in the «heavy» fraction or «free» in the 31S fraction (QUAIL, 1975 c). Further work however seems to contradict such conclusions and tends to suggest that phytochrome in the «heavy» fraction is neither bound directly to 31Slike RNP nor to the ribosomal RNP found in that fraction but to a protein constituent (GRESSEL and QUAIL, 1976). Hence, and also by considering the centrifugation operations involved in earlier experiments (QUAIL, 1975 b, c) it can be inferred that the «31S-RNP and phytochrome association» is generated in vitro due to the presence of EDTA, and quite unrelated to the association of phytochrome with the «heavy» membrane material which may actually occur in situ and have a certain physiological significance. However PRATT (1978) concluded after reviewing the Cucurbita work that: «Unless one is interested in the apparent artifactual in vitro induced association with 31S particles, there is no justification for continued use of Cucurbita» in phytochrome binding studies. This would be true if it could be shown that phytochrome binding to RNP is inevitably the only reaction possible. This point at the present time is already certainly not as definitive as Pratt has stated. It is clear from the data of GRESSEL and QUAIL (1976) that this is not so. Our own work on Cucurbita shows no evidence at all for a direct association of phytochrome and ribonucleoprotein material. This communication reports our findings.

Materials and Methods Plant Material Zucchini seedlings (Cucurbita pepo, L. cv. Greyzini; Arthur Yates & Co. Pty Ltd., New South Wales, Australia) were germinated and cultivated in the dark on moist paper towels, at 25 DC and 85 % relative humidity for 5 days. Hypocotyl hook regions of the seedlings, about 5 mm long, excised immediately below the cotyledons were used as starting material. Harvesting and all subsequent handlings of excised tissues were carried out under dim green safelight (FUAD, 1979). Irradiation Procedures The excised zucchini hooks were irradiated at 21 DC on a petri dish immediately after harvest using a Zeutschel M3 monochromator (Heinz Zeutschel, Tiibingen, Germany) and D.LL. Interference filters (Schott & Gen., Mainz, Germany). Irradiation with red (R) and far red (FR) wavelengths were provided using 661 and 730 nm filters (2000 and 1500 ergs cm- 2 and S-l respectively) for 3 minute duration which was found to be sufficient to estabZ. Pflanzenphysiol. Bd. 99. S. 449-460. 1980.

Phytochrome binding in zucchini

451

lish photoequilibria. Irradiation of cell-free homogenates was performed at OOC and approximately 15 cm from the light source for 5 min with a fluid depth of 5 mm. Glutaraldehyde Pretreatment Zucchini hypocotyl hooks were treated with 2.5 Ofo v/v glutaraldehyde in 0.1 M potassium phosphate buffer at pH 7.0 for 30 minutes at 0 °C in the dark and then washed with water and homogenization buffer as described by Yu (1975 a). Tissue Homogenization and Fractionation Chilled zucchini hooks were finely minced and homogenized in a pre-cooled mortar and pestle in ice-cold SEB which contains 35 mM N-morpholino-3-propansulfonic acid (MOPS), 0.25 M sucrose, 3 mM EDTA, and 14 mM 2-mercaptoethanol at pH 7.6. When prescribed, 10 mM MgCl 2 was included in the homogenization buffer (SEB). Routinely 1.25 gm fresh weight of hooks was homogenized with 4 ml buffer. The brei was filtered through a layer of nylon cloth and centrifuged at 500 xg for 5 min (Sorvall, SS34). The resultant supernatant (0.5 KS) was centrifuged at 30,000 xg for 10 min (Sorvall, SS34) to produce a crude mitochondrial fraction (30 KP) and supernatant (30 KS). Sediments of differential centrifugation were resuspended in SRB (25 mM MOPS, 3 mM EDTA, 0.25 M sucrose, pH 7.0). Sucrose density gradient centrifugation and Sepharose column elution

For sucrose density gradient analysis, resuspended 30 KP or other samples were layered onto 30-75 Ofo w/v linear sucrose gradients containing 25 mM MOPS, 3 mM EDTA and 14 mM 2-mercaptoethanol, pH 7.0. Gradients were centrifuged at 27,000 rpm (Beckman SW27) for the periods specified, and they were fractionated at 1 ml per fraction. Fractions were then assayed for phytochrome (Yu, 1975 b), RNA (FLECK and MUNRO, 1962), cytochrome c oxidase (SMITH, 1955), and NADPH-cytochrome c reductase (COLBEAU, NACHBAUR, and VIGNAIS, 1971). Percentage sucrose was measured refractometrically. For the equilibrium analysis of phytochrome and subcellular components, plant extracts were concentrated by Amicon diaflo equipment (membrane filter PMI0) and chromatographed on Sepharose 4B/CL columns, essentially the same as described by Yu and CARTER (1976). Elution fractions were assayed for phytochrome and RNA.

Results

For the analysis of the association of phytochrome with subcellular components, the cell free homogenate (0.5 KS) was routinely fractionated by differential centrifugation into a 30,000 xg sediment (30 KP) and supernatant (30 KS). Following R light irradiation, phytochrome which would become sedimentable can be quantitatively recovered in a 25,000 Xg sediment (Table 1). Subfractionation of the 30 KP on sucrose density gradient shows that there are roughly two major locations of phytochrome activities in the gradient, one near the mitochondrial fraction identified by cytochrome c oxidase activity and the other near the interface of sample load and gradient (Figure 1 A). There is no obvious correspondence between phytochrome in the mitochondrial area and RNA assayed by absorbance at 260 nm according to the procedure of FLECK and MUNRO (1962). Phytochrome and RNA appear to band together at the load interface. The overall distribution is therefore not very different from that observed by QUAIL (1975 b). The band near the inZ. Pflanzenphysiol. Ed. 99. S. 449-460. 1980.

452

RICHARD

Yu

Table I: Phytochrome pelletability by differential centrifugation. Excised zucchini hypocotyl hooks were irradiated with red light (661 nm) for 3 mi~. They were then homogenized at O°C by chopping and grinding in SEB or SEB containing 10mM MgCl z. The breis were filtered and centrifuged at 500xg for 5 min The resultant supernatants (0.5 KS) were further centrifl,lged at the speed and duration specified in the Table. The sediment of each centrifugation regime was resuspended in SRB or SRB containing lDmM MgCb when it was derived from hooks homogenized in SEB + MgCl z. Phytochrome contents, LI (oLlA) as arbitrary units, in the sediments and supernatants of differential centrifugation were determined and phytochrome pelletability represents Ll(ifA) in sediment 1000;' .LI(4A) in sediment + LI(LlA) in supernatant x ° Differential centrifugation of 0.5KS derived by uuirradiated zucchini hooks homogenized in the presence or absence of MgCb at 100,000 xg for 60 min resulted in about 9 % and 6 % pelletability respectively. Addition to SEB

Differential Centrifugation regIme

10 mM MgCl z

3,000 xg, 5 min 12,000xg, 5min 25,000xg, 10min 41,000xg,25min 100,000xg,60min

NIL

3,000xg, 5min 12,000xg, 5 min 25,000xg, 10min 41,000xg,25min 100,000xg,60min

Percentage phytochrome pelletability 18.1 40.0

58.8 58.6 57.0

8.3 19.5 37.0

42.9 45.0

terface would presumably contain material which may be described as the 31S material reported by QUAIL (1975 b). Upon prolonged centrifugation, the RNA-containing material appears to move further into the gradient and become more incongruent with the phytochrome material in the heavy and light bands (Figure 1 B). Addition of Triton X 100 to the 30,000 xg sediment solubilized the cytochrome c reductase activity in the mitochondrial region of the gradient but not that of cytochrome oxidase. The oxidase activity appears to become more diffuse and heterogenous, banding at slightly higher densities in the gradient. There is no definite banding of phytochrome or RNA-containing material in the heavy band but there is an increase of these materials near the top of the gradient. However there is still no exact correspondence between the RNA and phytochrome material. Phytochrome material remains nonsedimentable while the RNA material migrates further into the gradient upon prolonged centrifugation (Figure 2). About 70 0J0 of the cellular RNA and 55 0J0 of total phytochrome remain in the 30,000 X g supernatant. Further centrifugation to remove microsomal and membranous vesicles at 100,000 X g for 1 hr does not significantly increase further the amount of sedimentable phytochrome but by removing small membrane material it facilitates Z. Pjlanzenphysiol. Bd. 99. S. 449-460. 1980.

Phytochrome binding in zucchini 100

453

1

8+ i

60

40

20

o

I\

•.................... B -..,

....>-

:; >= J

«

1\

.---.~..

80

-R~~

::r

iL:~\~~_'-~~ o

80nOM

10

20

30

TOP

40

FRACTION NUMBER

Fig. 1: Sucrose density gradient centrifugation of the 30,000 xg sediment. Zucchini hooks, irradiated with 661 nm, were homogenized with SEB and fractionated by differential centrifugation to obtain a 30,000 xg sediment (30 KP). The 30 KP was resuspended in SRB and loaded onto a 30-75 Ofo w/v linear sucrose density gradient which contained MOPS and EDTA (see Material and Methods). Gradients were centrifuged in SW27 rotor (Beckman) at 4 DC and 27,000 rpm for 2 hr (A) and 20 hr (B). They were then fractionated at 1 ml per fraction. Fractions were assayed for phytochrome (e---e), RNA (0--0), cytochrome c oxidase (6--6), NADPH cytochrome c reductase (0--0), and refractive index (.---.).

equilibrium studies of a possible association of RNA-containing material and phytochrome, using chromatography on Sepharose columns. When the post-microsomal supernatant of unirradiated hypocotyl hooks was chromatographed on a Sepharose 4B/CL column, a single peak of phytochrome was always eluted behind the predominant RNA-containing protein peak. This is the case irrespective of whether the tissue was homogenized in EDT A or in Mg++ -containing buffer or whether the loaded column was eluted with Mg++ or with EDTA-containing buffers (Figure 3). A similar pattern of elution was obtained when hypocotyl hooks, irradiated with R light was homogenized in EDTA-containing buffer and then the post-microsomal fraction was chromatographed with EDTA buffer. There is no coincidence between the phytochrome and RNA-containing material. The elution pattern of phytochrome Z. Pflanzenphysiol. Ed. 99. S. 449-460. 1980.

454

Yu

RICHARD

A 60

40

20 V)

>-

Z

::>

>-

'"-<

'";:;;>'"-< Z·

80

60

0

;::

U

-<

40

0 >>-

20

'"u..u..

:;;

;::

U

-< 60

40

20

o

80TTOM

TOP

FRACTION NUMBER

Fig. 2: Sucrose density gradient analysis of the 30,000 xg sediment treated with Triton X 100. The 30 KP (derived from 10 gm fresh weight of zucchini hooks) obtained as described in Figure 1 was resuspended in 4 ml of SRB containing 10f0 w/v Triton X 100 at O°c. Resuspended 30 KP was loaded onto 30-75 % w/v linear sucrose gradients and centrifuged at 27,000 rpm and 4°C for 20 min (A), 2 hr (B), and 20 hr (C). Symbols used have the same meaning as those in Figure 1. from R-irradiated tissue was changed when the homogenization and elution buffers contained Mg++. Under this condition one phytochrome peak is eluted ahead of and another behind the RNA peak which continues to elute at about the same position, but there is still no coincidence between them. This change of phytochrome elution property did not appear to result when (1) elution with buffer containing 1 M Kel, (2) irradiating the zucchini hypocotyl hooks with FR light following R, (3) pretreating the red light irradiated hooks with glutaraldehyde before tissue homogenization (Figure 4). This shift in elution did not occur when phytochrome exZ. Pflanzenphysiol. Bd. 99. S. 449--460. 1980.

Phytochrome binding in zucchini

-r--

100 - ----------

C

A

75 -

-lI.OO f75

~

o


U

50 ~

0.50

25-

0.25 ~

z

~

;(


455

o ..: E c

o

'" N

25

20

40

60

FRACTION NUMBER

Fig. 3: Chromatographic analysis of the 100,000 xg supernatant. Unirradiated zucchini hooks were homogenized in SEB or SEB containing 10 mM MgCI 2 • The brei was centrifuged to obtain the 0.5 KS (see Material and Methods), and the 0.5 KS was centrifuged at 100,000 xg for 1 hr at 4°C to obtain the 100,000 xg supernatant (100 KS). The 100 KS (about 16 ml) was concentrated to 2 ml by ultrafiltration and loaded onto a Sepharose 4B/CL (Pharmacia) column (Pharmacia K 15,27 em bed height, Vo = 13 ml). Elution was assisted by a peristaltic pump (LKB Varioperpex) delivering 12 ml per hr. 5 min fractions were collected using a LKB fraction collector. (A) Hypocotyl hooks were homogenized in SEB + 10 mM MgCI 2 , and column eluted with 25 mM MOPS, 3 mM EDTA, 10 mM MgCI 2, pH 7.0; (B) tissue homogenized in SEB and column eluted with 25 mM MOPS and 3 mM EDTA, pH 7.0; (C) tissue homogenized in SEB + 10 mM MgCI 2, 1 M KCI, pH 7.0; (D) unirradiated hypocotyl hooks were pretreated with glutaraldehyde (see Material and Methods) and then homogenized in SEB, column eluted with 25 mM MOPS, 3 mM EDT A, pH 7.0. Elution profiles of phytochrome and RNA containing material were represented by 0--0 and respectively.

e----e

tract from maize was similarly chromatographed (result not shown, see Yu and 1976). When a cell-free homogenate (0.5 KS) of the unirradiated zucchini hooks, extracted in the presence of Mg++, was irradiated with R light at 0 °C, about 40 Ofo of the phytochrome can be sedimented into a crude membrane fraction (40,000 xg, 10 min). This amount represented nearly all the phytochrome which can be sedimented. Little more phytochrome could be pelleted by centrifuging at 100,000 xg for 1 hr. Phytochrome remaining in the supernatant behaved differently depending on whether or not the 0.5 KS has been irradiated with red light. As shown in Figure 5, phytochrome was eluted ahead of the RNA-containing material when the

CARTER,

Z. Pflanzenphysiol. Ed. 99. S. 449-460. 1980.

456

RICHARD

Yu

0.5 KS was irradiated but it eluted after the RNA peak in unirradiated control. This change in phytochrome elution is dependent on the presence of Mg++ in the homogenization buffer and it is eliminated by elution with buffer containing both Mg++ and 1 M KCl. As shown in Figure 4 that pretreatment with glutaraldehyde of R-light irradiated hooks did not affect the elution of phytochrome relative to that of the RNA-containing material. Pretreatment with glutaraldehyde however does have an effect on 100

1.00

o 75

50

25

~

S?

=<

<0


~

Z

0

75

50

U

FRACTION NUMBER

Fig. 4: Chromatographic analysis of 100,000 xg supernatants derived from red light-irradiated zucchini hooks. Zucchini hooks were irradiated with red light, 661 nm and then homogezized in SEB or SEB + 10 mM MgCl 2 to obtain the 100,000 xg supernatant as described in Figure 3. The 100 KS was analysed by Sepharose 4B/CL column elution. (A) tissue homogenized in SEB + 10 mM MgCI 2, pH 7.0; (B) tissue homogenized in SEB, and column eluted with 25 mM MOPS, 3 mM EDTA, pH 7.0; (C) tissue homogenized in SEB + 10 mM MgCl 2 and column eluted with 25 mM MOPS, 3 mM EDTA, 10 m MgCI 2 , 1 M KCI, pH 7.0; (D) irradiated tissue was pretreated with glutaraldehyde, homogenized in SEB, and column eluted with 25 mM MOPS, 3' mM EDTA, pH 7.0; (E) tissue was irradiated with 3 min 661 nm and 3 min 730 nm successively, homogenized in SEB + 10 mM MgCI 2 , and column eluted with 25 mM MOPS, 3 m EDTA, 10 mM MgCI 2 , pH 7.0. • . , elution profile or RNA material; 0--0, elution profile of phytochrome.

z. Pjlanzenphysiol. Bd. 99. S. 449-460.

1980.

Phytochrome binding in zucchini

A 75

~

50

~

457

roo J0.75

<



75

>-'

~

z

o

50

u

FRACTION NUMBER

Fig. 5: Chromatographic anlysis of 100,000 xg supernatant derived from in vitro irradiation of the cell-free homogenate. Unirradiated zucchini hooks were homogenized with SEB (Frame B) or SEB + 10 mM MgCl 2 (Frames A and C). The breis were then filtered and centrifuged to obtain the cell-free homogcnates (0.5 KS). The 0.5 KS was irradiated with red light (661 nm) for 5 min at OOC and the irradiated 0.5 KS was further centrifuged at 100,000 xg for 1 hr. The resultant supernatants (100 KS) were concentrated and loaded onto Sepharose 4B/CL columns for elution. A and B, columns eluted with 25 mM MOPS, 3 mM EDTA, 10 mM MgCI 2, pH 7.0; C, column eluted with 25 mM MOPS, 3 mM EDTA, 10 mM MgCI 2, 1 M KCI, pH 7.0.

the relative sedimentability of phytochrome and RNA-containing material in the particulate membrane fraction. As a result of the glutaraldehyde pretreatment, phytochrome, RNA-containing material and the cytochrome oxidase and some of the cytochrome c reductase activity appear to sediment together and band at roughly the same equilibrium density in the gradient (Figure 6 A). Treatment of the particulate membrane fraction with Triton Xl 00 solubilizes the reductase activity but not the cytochrome oxidase, RNA, and phytochrome activities. The latter three ini-

Z. P/lanzenphysiol. Bd. 99. S. 449-460. 1980.

458

RICHARD

Yu

100

ao 60

.0

20

:

---...<:

100

---. "

"'---

~ .~~

lj/~,:~~ o

BOTTOM

w

~

fRACTION NUMBER

w

~

TOP

Fig. 6: Sucrose density gradient analysis of the crude mitochondrial fraction derived from glutaraldehyde-pretreated zucchini hooks. Excised tissues irradiated with red light, were treated with glutaraldehyde as described in Material and Methods. They were washed and then homogenized in SEB. The brei was centrifuged successively to obtain a crude mitochondrial fraction (30 KP). The 30 KP, containing almost all the sedimentable phytochrome, was resuspended either in SRB or SRB + 1 % Triton X 100. Resuspended 30 KP was then loaded onto gradients for centrifugation. A, 30 KP resuspended in SRB; Band C, 30 KP resuspended in SRB + Triton. A and C gradients were centrifuged at 27,000 rpm for 4 hr, and gradient B for 20 min: symbols used have the same meaning as those in Figure 1. tially sediment separately but band at a similar higher density in the gradient upon prolonged centrifugation (Figure 6 B and C).

Discussion In explaining his data, QUAIL (1975 c) made the following suggestions: (1) R light enhances the amount of phytochrome bound to RNP (31S ribonucleoprotein particle) Z. Pjlanzenphysiol. Bd. 99. S. 449-460. 1980.

Phytochrome binding in zucchini

459

because P lr has a greater initial affinity than P r for this fraction, (2) the phytochrome-RNP association is itself independent of added Mg++, and Mg++-enhanced phytochrome pelletability at 20,000 xg results indirectly from Mg++-enhanced RNP pelletability. The latter would be most likely the result of a combination of (a) Mg++-mediated preservation of existing RNP-membrane associations, the most likely candidate being rough ER, (b) Mg++ -induced self aggregation of the RNP material into particles sedimentable at 20,000 xg, and (c) the possible cross-agglutination of previously free RNP material with existing membrane-bound RNP. However data presented in this paper show clearly that phytochrome in the P lf form does not associate with RNA-containing material, in either the presence or absence of Mg++. This is the case when phytochrome and RNA interaction was examined in the post-microsomal or crude mitochondrial fraction. The claim that phytochrome associates with ribonucleoprotein particles (RNP) when irradiated with R light and aggregation of the phytochrome-RNP complexes occurs in the presence of Mg++ cannot be substantiated. However in the presence of Mg++, phytochrome appears to have a different elution property, due notably to its larger size upon liquid column chromatography. The nature of this size difference has not yet been characterized though it appears that it does not result from an association with RNA-containing material and the apparent size difference can be eliminated by elution with a buffer of high ionic strength. Such an apparent size difference may result from aggregation of phytochrome with itself or with other subcellular components. If such an aggregation, resulting in the apparent phytochrome size difference, occurs in situ, the aggregates appear not to be susceptible to crosslinking by treatment of R light-irradiated hooks with glutaraldehyde. Because a similar size difference can also be induced by irradiating extracts with R light in the presence of Mg++ at °e, its physiological relevance is uncertain. However it should be noted that in situ far red irradation following R can cancel this apparent size difference. Our data also differ from those reported by QUAIL (1975 a, b, c) in several other respects. Treatment of the crude mitochondrial fraction with Triton Xl 00 did not result in the phytochrome sedimenting to the bottom through a sucrose density gradient. Instead, phytochrome and RNA were solubilized to slower sedimenting materials. Pretreatment of red light-irradiated tissue with glutaraldehyde did not yield {he so-called aggregates of ribosomes (or RNP) and phytochrome which sediment abnormally through a standard sucrose gradient to the bottom as described by QUAIL and GRESSEL (1975). Instead, phytochrome in R light-irradiated tissue is crosslinked by glutaraldehyde to membrane material which bands at equilibrium with the cytochrome oxidase activity and RNA-containing material. It should be emphasized that banding together of materials of assayed parameters, i.e. phytochrome, RNA-containing material, and cytochrome oxidase activity, following in situ glutaraldehyde treatment does not mean however that phytochrome is necessarily crosslinked directly to RNA-containing material or mitochondria. In fact, Triton treat-

°

z.

Pflanzenphysiol. Ed. 99. S. 449-460. 1980.

460

RICHARD Yu

ment of the crude mitochondrial fraction resulted initially in separate sedimentation of these three parameters before they achieved apparent density equilibrium. Furthermore, analysis of the post-microsomal fraction from glutaraldehyde-treatment tissues on Sepharose 4B columns showed no apparent association of RNP and phytochrome. The observation that phytochrome can interact with RNP material has been widely hald to be a serious obstacle to attempts to establish specificity of interaction between phytochrome and particulate fractions. It is believed that this is due to a large molar excess of RNP over phytochrome in various subcellular fractions. The capacity of ribosomal material to bind large amounts of extraneous proteins is well-known and notorious. It would provide adequate potential for the artifactual, electrostatic adsorption of phytochrome to the RNP in any of the subcellular fractions (QUAIL and GRESSEL, 1975). However this fear may be ill-founded as is evident from the complete lack of any apparent association between RNP and phytochrome in our own experiments. Acknowledgements The author wishes to thank Professor D. ]. CARR for critical reading of the manuscript and Ms F. SCHWEINBERGER and Miss S. C. CHEN for their competent technical assistance.

References COLBEAU, A., ]. NACHBAUR, and P. M. VIGNAIS: Biochim. Biophys. Acta, 249, 462-492 (1971). FLECK, A. and H. N. MUNRO: Biochim Biophys. Acta (Arnst.) 55, 571-583 (1962). FUAD, N.: Ph. D. thesis, Australian National University (1979). GRESSEL,]. and P. H. QUAIL: Plant Cell Physiol. 17, 771-786 (1976). PRATT, L. H.: Photochem. Photobiol. 27, 81-105 (1978). QUAIL, P. H.: Photochem. Photobiol. 22, 299-301 (1975 a). - Planta (Berl.) 123, 223-234 (1975 b). - Planta (Berl.) 123,235-246 (1975 c). QUAIL, P. H. and ]. GRESSEL: In: H. SMITH (Ed.): Light and Plant Development, pp. 111128. Butterworths, London, Boston, Sydney, Wellington, Durban, Toronto, 1975. SMITH, L.: In: D. GLICK (Ed.): Methods in biochemical analysis, Vol. 2, pp. 427-434. Interscience Publishers, New York, London, Sydney, Toronto, 1955. Yu, R.: Aust.]. Plant Physiol. 2, 273-279 (1975 a). - ]. Exptl. Bot. 26, =#= 95, 808-822 (1975 b). Yu, R. and]. CARTER: Plant Cell Physiol. 17, 1321-1328 (1976).

RICHARD Yu, Department of Developmental Biology, Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2601, Australia.

Z. P/lanzenphysiol. Ed. 99. S. 449-460. 1980.