Planta /2001) 213: 565±574 DOI 10.1007/s004250100520

ORIGINAL ARTICLE

Luisa Moysset á Eva Ferna ndez á Nuria Cortadellas Esther Simo n Intracellular localization of phytochrome in Robinia pseudoacacia pulvini

Received: 10 June 2000 / Accepted: 12 December 2000 / Published online: 8 March 2001 Ó Springer-Verlag 2001

Abstract The intracellular localization of phytochrome chrome, red-absorbing form á Pfr: phytochrome, far-red in the pulvini of Robinia pseudoacacia L. wasanalyzed absorbing form á R: red light by immunogold electron microscopy after red /R; 15 min) and far-red /FR; 5 min) irradiation 2 h after the beginning of the photoperiod. Screening of the available Introduction antibodiesby immunoblotting demonstrated that none of the oat /Avena sativa L.) anti-phytochrome A /phy A) Robinia pseudoacacia lea¯etsshowcircadian and nycti- monoclonal antibodies) /MAbs) detected Robinia phy- nastic movements as a result of the curvature of a spe- tochrome. A putative Robinia phy A wasdetected by cialized motor organ, the pulvinus, located at the base of immunoblotting using a MAb to mustard /Sinapis alba the lea¯et blade. Lea¯etsmove from a folded position L.) phy A /CP 2/9). No cross-reactivity was observed in /closed lea¯ets) to a horizontally extended position blotsprobed with a MAb against Cucumis sativus L. phy /open lea¯ets) during dark/light cycles and the move- B /mAT1). Ultrathin sections of LR White resin-em- ments persist with a circadian rhythmicity in continuous bedded pulvini were immunolabelled with CP 2/9 MAb. darkness. As in other related legumes /Satter and Gal- The labelling wasrestricted to cortical cellsand there ston 1981), these movements are driven by turgor was no evidence of labelling either in the vascular system changesin the pulvini of the cortical motor cellswhich or in the epidermis. The pattern of labelling was the cause alterations in both the size and the shape of the same in both extensor and ¯exor cells irrespective of motor cell /Moysset and Simo n 1991). The pattern of whether phytochrome wasin the far-red-absorbing/Pfr) K+ and Cl± distribution in pulvini motor cells during state or had reverted to the red-absorbing /Pr) form. lea¯et movement suggests that the extensor and ¯exor Isolated labels and clusters of labels were randomly cell ion ¯uxes are mainly responsible for turgor changes distributed throughout the cytoplasm. Gold particles /Moysset et al. 1991). Red light /R) pulses a€ect were also found in the interior of nuclei, chloroplasts nyctinastic closure and shift the phase of rhythmic and mitochondria. movement when applied at the appropriate time. Phy- tochrome, acting throughout a low-¯uence response Keywords Leaf movement á Phytochrome /LFR), is the photoreceptor involved in these responses /immunolocalization) á Pulvinus á Robinia /pulvinus) since the e€ect of 15 min of R is reversed by 5 min of far- red light /FR; Go mez and Simo n 1995). Abbreviations FR: far-red light á MAb: monoclonal Phytochrome isa ubiquitousand well-known pho- antibody á phy: phytochrome holoprotein á Pr: phyto- toreceptor found in plants. It is a soluble chromoprotein consisting of an apoprotein covalently attached to a linear tetrapyrrole chromophore /Furuya 1993). Phyto- chrome chromoproteinsare encoded by a small L. Moysset á E. Ferna ndez á E. Simo n/&) multigene family /Mathewsand Sharrock 1997). Phy- Unit of Plant Physiology, tochrome genes/ PHY) have been detected in several Faculty of Biology, University of Barcelona, plant species. A number of remarkable di€erences in the Avda. Diagonal 645, 08028 Barcelona, Spain E-mail: [email protected] PHY family have been found within the angiosperms. Fax: +34-93-4112842 The PHY family consists of ®ve members in Arabidopsis N. Cortadellas /PHYA±PHYE; Mathewsand Sharrock 1997) and Scienti®c-Technical Services, University of Barcelona, Solanum lycopersicum /PHYA, PHYB1, PHYB2, C/. Sole i Sabaris3, 08028 Barcelona, Spain PHYE, PHYF; Pratt et al. 1997) but only three genes 566 /PHYA, PHYB, PHYB¢) have been identi®ed in Populus The aim of the present work was to study immuno- trichocarpa /Howe et al. 1998). Legumesapparently lack chemically the inter- and intracellular localization of PHYC /Mathewset al. 1995) but a putative PHYA phytochrome in Robinia pseudoacacia pulvini in order to pseudogene has been identi®ed in Pisum /Sato 1990), relate phytochrome distribution to its e€ects on lea¯et and three genes/ PHYA, PHYA¢, PHYE) have been movements. found in some members of the tribe Robinieae such as Sesbania and Hebestigma /Mathewset al. 1995). Among the various phytochromes, phy A and phy B are the best Materials and methods known. The light-labile phy A isthe mostabundant phytochrome in dark-grown plantswhile the light-stable Plant material phy B is the most abundant phytochrome species in light-grown plants. Analysis of phy A- and phy B-de®- Seedsof Robinia pseudoacacia L. /Parcsi Jardins,Barcelona, Spain) were sown in pots containing a mixture of soil, peat and cient mutants suggests that their modes of action are Perlite /1:1:1, by vol.) after subjecting them to a 24-h-long imbib- di€erent /Furuya and SchaÈ fer 1996). ition /boiling water waspoured over seedsand allowed to cool). Phytochrome controlsa wide range of physiological Plantswere grown in a growth chamber at 25 °C under 16-h light 8-h dark cycles. Illumination was provided by white ¯uorescent and developmental processes. Thus, phytochromes are ±2 ±1 expressed in various tissues throughout the life cycle of tubes/PhilipsF96T12/CW/VHO, 215 W) giving 100 lmol m s at the plant apex. Plantswere watered with tap water on alternate plants/Nagatani 1997). The diversity of phytochrome daysand fed from the secondweek after germination with complete action can be explained in part by the multiple molecular Hoagland nutrient solution /Hoagland and Arnon 1950) every species, which may also transduce light signals via fortnight. Four-to twelve-month-old plantswere usedfor the distinct mechanisms. Information concerning the sub- experiments. Seeds of Cucumis sativus L. /Fito Seeds, Barcelona, Spain) were imbibed for 2 h in darkness and then sown in moist cellular distribution of phytochromes in plants might vermiculite. Both germination and seedling growth took place in provide important insights into the mode of action of darkness at 25 °C. Five-day-old seedlings were submitted to a 24-h these photoreceptors. de-etiolation treatment that consisted of 15-min R pulses every 2 h Phytochrome localization in green plantsisvery of darkness. Seeds of Sinapis alba L. /ProductosPlantarum, Bar- celona, Spain) were imbibed for 24 h in darkness or in a 16-h light scarce since most work has been done in etiolated tissues. 8-h dark cycle and then sown in moist vermiculite. Subsequently, A heterogeneousdistribution of phytochrome within a seedlings were allowed to develop in the dark or in 16-h light 8-h single tissue has been frequently observed. Thus, phyto- dark cycles /light-grown seedlings) at 25 °C for 14 days. chrome isabundant in somecellsbut apparently lacking or in low abundance in adjacent cellseven if cellsare Light treatments morphologically indistinguishable /Pratt 1994). Until recently, immunochemical analysis has provided ambig- Red light wasobtained from eight nature luxe ¯uorescenttubes uousresultsconcerning the subcellular localization of /Osram 15 W/76) each wrapped in three layers of medium-red phytochromes. Thus, light /Mackenzie et al. 1975; ®lter /No. 27; Rosco Supergel, Syndenham, London, UK) to give a ¯uence rate of 17.5 lmol m±2 s±1. Far-red light wasob- McCurdy and Pratt 1986) and electron /McCurdy and tained from ®ve 150-W incandescent bulbs /Sylvania BRS) ®l- Pratt 1986; Speth et al. 1986) microscopy have shown tered through 10 cm of water, one layer of blue ®lter /Cinemoid that phytochrome isuniformly distributedthroughout No. 20; Rank Strand Electrics, London, UK) and one layer of deep-orange ®lter /Cinemoid No. 5A) to give a ¯uence rate of the cytosol in dark-grown coleoptiles. After photocon- ±2 ±1 version of the red-absorbing form /Pr) to the far-red- 26 lmol m s . The ¯uence rates were measured using a quantum radiometer photometer /188B; Li-Cor Lincoln, Neb., absorbing form /Pfr), phytochrome becomes sequestered USA), ®tted with a quantum sensor /Li-190 SB) for R and a in discrete areas. This reaction has also been found in near-infrared sensor /Li-220 SB) for FR. A dim green safelight other monocotyledonousseedlings/Epel et al. 1980) and was used in the dark [15-W daylight ¯uorescent tube /Sylvania) in pea seedlings /Saunders et al. 1983). MoÈ singer et al. covered with two layersof green ®lter /No. 90 RoscoSupergel)]. /1987) have reported data that clearly relate phy A with barley nuclei and, more recently, Sakamoto and Naga- Antibodies tani /1996) reported immunodetection of phy B in iso- lated Arabidopsis nuclei. Finally, there isevidence that 39/6 IgG polyclonal antibody raised against Avena sativa L. phy- phy A and phy B can be translocated from cytosol to tochrome A and immunoanity-puri®ed rat monoclonal antibod- ies /MAbs) raised against A. sativa L. cv. Saladin phytochrome A nuclei in an R/FR light-dependent manner /Kircher et al. /MAC50, MAC52, MAC54, MAC56), A. sativa SDS-denatured 1999; Yamaguchi et al. 1999; Hisada et al. 2000). phytochrome A /MAC197, MAC200) and S. alba phytochrome A Phytochrome and componentsof the circadian clock /CP 2/9, CP 3/289) were provided by B. Thomas/Horticulture seem to be located in the pulvini since excised pulvini Research International, Wellesbourne, UK). MAC50±56, show red-light responses and circadian rhythms /Go mez MAC197, CP 2/9 and CP 3/289 are IgG2a and MAC200 isan IgG2b. Monoclonal antibody /mAT1) raised against tobacco and Simo n 1995). However, nothing isknown about /Nicotiana tabacum L.) phytochrome B wasprovided by E. Lo pez- phytochrome distribution in the pulvinus. Since lea¯et Juez and A. Nagatani /Riken Institute, Wako-shi, Japan). Pro- movementsdepend on oppositefunctional changesin duction of rabbit anti-phytochrome IgG and MAbsMAC50 to extensor and ¯exor cells it is reasonable to suppose that MAC56 wasasdescribedby Thomaset al. /1984a, b). Production and characterization of monoclonal anti-tobacco phytochrome B phytochrome might be di€erentially expressed in the two antibody wasdescribedin Lo  pez-Juez et al. /1992). For immu- partsof the pulvinus. noblotting, anti-phytochrome A MAbswere usedat concentrations 567 of 0.5±50 lgml±1. For immunolocalization, antibody CP 2/9 was stained for alkaline phosphatase with BCIP/NTB premixed solu- used at concentrations of 15 and 30 lgml±1. Lyophilized hybrid- tion /0.48 mM nitro blue tetrazolium, 0.56 mM 5-bromo-4-chloro- oma culture supernatant of mAT1 was diluted 1:1 in TBS /Tris- 3-indolylphosphate, 10 mM Tris-HCl and 59.3 mM MgCl2, pH 9.2; bu€ered saline solution) containing 1% /w/v) fat-free milk powder B-6404, Sigma) according to the manufacturer'sinstructionsfor /Lo pez-Juez et al. 1992). 20±30 min. Reactions were stopped with distilled water and membraneswere air-dried at room temperature. Each immunoblot wasprobed with only one antibody. Experimentswere repeated at Phytochrome extraction least three times independently with fresh extractions. The speci- ®city of labelling wastested:/i) by omitting the primary antibody, Lea¯ets/0.5±1 g FW) and secondarypulvini /0.2±0.5 g FW) of /ii) by using non-immune rat IgG /I-4131, Sigma) and non-immune R. pseudoacacia were harvested at di€erent times of the light/dark mouse IgG /I-5381, Sigma) at concentrations identical to those of cycle. All samples were collected from the third to the ®fth fully experimental antibodies, and /iii) by using phytochrome extracts developed leaf /counting from the apex). Apical fragments/0.5±1 g from Cucumis for mAT1 antibody and Sinapis for CP 2/9 antibody. FW), including cotyledonsand one-third of the hypocotyl, of etiolated and light-grown 14-day-old S. alba seedlings and de- etiolated C. sativus seedlings were harvested. Samples were irradi- Immunocytochemistry and electron microscopy ated on ice with 5-min R exposure and immediately frozen in liquid nitrogen. Thisirradiation convertsabout 87% of total phyto- Lateral secondary pulvini from the third to the ®fth fully developed chrome to Pfr, which is more resistant to proteolysis /Vierstra and leaf /counting from the apex) of R. pseudoacacia were used for Quail 1983). Frozen tissues were powdered at liquid-nitrogen immunolocalization experiments. Pulvini were cut from lea¯et and temperature by meansof a mortar and pestleand homogenized at a rachis. A small section of the lea¯et was left attached to distinguish ±1 ratio of 1 ml /g FW) in an extraction bu€er consisting of 100 mM between the proximal and distal zones. For immunolocalization Tris-HCl /pH 8.3), 50% ethylene glycol, 140 mM ammonium sul- during nyctinastic closure, pulvini were cut 2 h after the beginning of phate, 10 mM Na4EDTA. Just prior to use, protease inhibitors the photoperiod, ¯oated on 5 ml distilled water in Petri dishes and were added to a ®nal concentration of 20 mM sodium bisulphite, transferred to darkness after R /15 min) or 15 min R plus 5 min FR. 4 mM phenylmethylsulphonyl ¯uoride /PMSF), 52 mM 2-mer- Irradiation with FR wasapplied immediately after R or 10 min later. ±1 ±1 captoethanol, 10 mM iodoacetamide, 2 lgml leupeptin, 2 lgml Pulvini were placed in ®xative solution immediately after R or FR ±1 aprotinin and 1 lgml pepstatin A. Before homogenization, in- irradiationsor 10±30 min later. Until the ®xation wascomplete, all ±1 soluble polyvinylpyrrolidone at a ratio of 0.1 g /g FW) wasadded manipulations were done under dim green safelight not absorbed by to the extraction bu€er. The crude homogenateswere centrifuged phytochrome /Pratt 1984). Two sets of pulvini were collected from at 20,000 g for 20 min at 4 °C and the supernatants were boiled 8±10 plantsand were randomly distributedfor each light treatment. /5 min) after mixing with an equal volume of boiling, 2´ sample For immunoelectron microscopy, pulvini were vacuum-in®l- bu€er /Laemmli 1970). The mixtureswere spunfor 1 min at room trated with 0.5% /v/v) glutaraldehyde-4% /w/v) formaldehyde in temperature and the supernatants were used for electrophoresis and 0.1 M sodium phosphate /pH 7.4) at 4 °C and ®xed for 20±24 h in immunoblotting. All manipulationswere performed under green the same solution. After dehydration through a cold graded-etha- light with samples kept on ice unless otherwise speci®ed. nol series /30±100%) the samples were embedded in Lowicryl K4M or LR White resin /London Resin Co.,, Hampshire, UK). In®l- tration with K4M wasconducted at ±35 °C and polymerization was Electrophoresis, electroblotting and immunostaining carried out by heat /at 60 °C) or by UV light /360 nm) at ±35 °C. In®ltration with LR White resin was conducted at ±20 °Corat4°C. After separation by discontinuous SDS-PAGE in 7.5% acrylamide In the ®rst case, the resin was polymerized for 24 h in gelatin gelsaccording to Laemmli /1970), proteinswere electroblotted to capsules at 60 °C in the absence of O2 or at ±20 °C by UV light. In nitrocellulose membranes /Bio-Rad) at 40 V for 3 h in transfer the second case, LR White resin was polymerized only by heat. To bu€er [25 mM Tris-HCl /pH 8.3), 192 mM glycine and 20% avoid variation due to tissue processing, 8±10 pulvini were treated methanol]. Molecular-weight standards were obtained from simultaneously by the same embedding procedure. For conven- Bio-Rad /161-0303 SDS-PAGE standards, high range). After tional electron microscopy, pulvini were ®xed in 2.5% /v/v) electroblotting, membranes were stained /15 s) by immersion in glutaraldehyde-2% /w/v) formaldehyde in 0.1 M sodium phosphate Ponceau working solution [0.2% /w/v) Ponceau in 3% trichloro- /pH 7.4), post-®xed in 2% OsO4 and embedded in Spurr'sresinas acetic acid and 3% /w/v) sulfosalicylic acid], rinsed with water, cut described by Moysset and Simo n /1991). Cross-sections of pulvini into strips and immunostained. Following Pratt /1984), membranes were obtained on an Ultracut ultramicrotome /Reichert, Vienna, were blocked for 1 h at room temperature in phosphate-bu€ered Austria). Thick sections were stained with 0.5% methylene blue. saline /PBS) solution [10 mM sodium phosphate /pH 7.4), 0.9% Ultrathin sections were collected on Formvar-coated copper or NaCl] containing 1% bovine serum albumin /BSA), 0.02% sodium gold grids. Sections on gold grids were processed for immunogold azide and 1% /w/v) fat-free milk powder. Incubation wasdone with labelling. All sections were double-stained with uranyl acetate and the primary antibody diluted in PBS containing 0.05% Tween 20, lead citrate and viewed with a PhilipsEM 301 microscopeat 80 kV. 1% BSA, 0.02% sodium azide and 1% /w/v) fat-free milk powder For immunolabelling with CP 2/9 MAb, sections were pro- for 1 h at room temperature. After washing /3´10 min) with the cessed as described by McCurdy and Pratt /1986). Grids were ®rst wash solution [10 mM Tris-HCl /pH 8, 25 °C), 0.05% Tween 20, incubated for 30 min in TBS-BSA [10 mM Tris-HCl /pH 7.4), 1% BSA, 0.02% sodium azide and 1% /w/v) fat-free milk powder] 500 mM NaCl, 0.3% /v/v) Tween 20, 1% /w/v) BSA] and 5% /v/v) membraneswere incubated in a secondaryantibody [1:500 dilution lamb serum at room temperature and then incubated for 1 h in the in PBS containing 0.05% Tween 20, 1% BSA, 0.02% sodium azide rat MAb diluted in TBS-BSA and 1% /v/v) lamb serum. After and 1% /w/v) fat-free milk powder] for 1 h at room temperature. three 10-min washes with TBS-BSA, grids were incubated in goat The following secondary antibodies were used: anti-rabbit IgG antibodiesto rat IgG [R-5130; Sigma; 5 lgml±1 in TBS-BSA plus alkaline phosphatase conjugate /A-3687, Sigma) for anti-phyto- 1% /v/v) lamb serum] for 1 h at room temperature. Following three chrome A polyclonal antibody, anti-rat IgG /R-5130, Sigma) for all 10-min washes in TBS-BSA, grids were incubated for 1 h in anti- anti-phytochrome A MAbsand anti-mouseIgG /M-8642, Sigma) goat IgG coupled with 10-nm colloidal gold /G-5402; Sigma) for anti-phytochrome B MAb. After washing /3´10 min), mem- diluted 1:50 in TBS-BSA plus 1% lamb serum and then rinsed branespreviouslylabelled with MAbswere incubated with the consecutively in TBS /10 min) and deionized and distilled water third antibody [1:1000 dilution in 0.05 M Tris-HCl /pH 8, 25 °C), /3´10 min). Non-immune rat IgG /I-4131; Sigma), which was 1% BSA, 1 mM MgCl2 and 0.1% sodium azide] conjugated to diluted to the same concentration as MAbs in TBS-BSA plus 1% alkaline phosphatase /A-4187, Sigma) for 1 h at room temperature. lamb serum, and TBS-BSA, plus 1% lamb serum were used in place After washing /3´10 min) in wash solution, membranes were of the MAbs for control sections. Two blocks for each set of 568 samples were sectioned, and three to ®ve grids, corresponding to gold localization of phytochrome in sections of Robinia extensor and ¯exor zones, from each block were immunostained pulvini. independently. The density of labelling over selected areas was determined by None of the antibodiesagainst Avena sativa phy A manually counting the gold particleson electron micrographsat recognized Robinia phytochrome and no cross-reactivity 16,000´ magni®cation and calculating the number per unit area was observed in blots probed with MAb against /lm2). We made between 5 and 15 individual measurements from Cucumis phy B /data not shown). several sections of at least 3 pulvini from 3 plants for each light treatment. Preservation of the structure and ultrastructure Results of motor cells

Immunoblotting analysis Before phytochrome immunolocalization, some embed- ding media and protocolswere testedto evaluate the The cross-reactivity between phytochromes from Robinia structural and ultrastructural preservation of pulvinar green pulvini and antibodiesagainstphy A and phy B motor cells and the possible adverse e€ects on antigen from other species was tested by western blotting. Fig- reactivity of resin polymerization by heat and UV. The ure 1a shows that CP 2/9, a Sinapis alba anti-phy A results /Figs. 2, 3) were compared with previously MAb, recognized phytochrome isolated from green described structural and ultrastructural features of pulvini by generating a 115-kDa band on the blots. The R. pseudoacacia secondary pulvini /Moysset and Simo n size of this band was the same regardless of whether 1991). phytochrome waspuri®ed after 8 h of darkness/Fig.1a, The Lowicryl K4M-embedded pulvini were dicult lane 1), or after 2±14 h of light /Fig. 1a, lanes2 and 3). No to cut and sections were folded and unstable in the cross-reactivity was observed in controls in which CP 2/9 electron beam. The integrity of cellsand organelleswas wasomitted or wasreplaced with non-immune IgG disrupted, irrespective of whether polymerization was by /Fig. 1a, lane 4). To test the speci®city of this antibody, heat or by UV light. The cytoplasm was separated from control experiments/Fig. 1b) were carried out using the wall in many regionsand it appeared very or mod- phytochrome isolated from etiolated and green S. alba erately electron-dense. Plasmalemma and tonoplast were seedlings. CP 2/9 cross-reacted with phytochrome destroyed. Cellular organelles were identi®ed by shape isolated from etiolated seedlings, giving a band /Fig. 1b, and electron-density although there was a loss of mem- lane 1) of 121 kDa. Thisantibody failed completely to brane structure. Tannin vacuoles appeared as granular detect phytochrome from green mustard seedlings or amorphous material. The chloroplasts were distended /Fig. 1b, lane 2). No cross-reactivity was observed in and the thylakoid system was poorly preserved al- blotsprobed with non-immune IgG /Fig. 1b, lane 3). though, occasionally, one or more grana-like structures These results thus provide a reliable basis for immuno- were identi®able. The mitochondria were swollen and the mitochondrial matrix had a relatively high degree of electron density, but cristae were not clearly seen.

Fig. 1 Immunoblot analysis of phytochrome isolated from Robinia pseudoacacia pulvini /a) and from etiolated and light-grown Sinapis alba seedlings /b). Phytochrome was subjected to electrophoresis on 7.5% SDS polyacrylamide gels, electrotransferred to nitrocellulose and immunostained with 1.28 lgml±1 CP 2/9. a Robinia phytochrome was isolated after 8 h of darkness /lane 1) and after 2h/lane 2) and 14 h /lane 3) of light. An extract at 8 h of darkness Fig. 2 Cross-section of a Robinia secondary pulvinus. The motor wasstainedwith non-immune IgG / lane 4). b Sinapis phytochrome organ was®xed in 2.5% glutaraldehyde-2%formaldehyde, post- wasisolatedfrom etiolated / lane 1) and light-grown /lane 2) ®xed in osmium and embedded in Spurr resin. The pulvinus has a seedlings. An extract from etiolated seedlings was stained with non- central vascular bundle /CC) bordered by thick-walled collenchyma immune IgG /lane 3). Only the region around the phytochrome cells, which in turn are surrounded by several layers of cortical band is shown. The position of a polypeptide of 116 kDa is shown motor cells/ CM) and an epidermis/ E). Motor cellschange in size on the left and shape during leaf movement. Bar = 0.15 mm 569 localization studies. In comparison with the previous protocol, the cytoplasmic matrix had a uniform, gran- ular, highly electron-dense appearance and there was little or no apparent swelling of chloroplasts and mito- chondria. The chloroplasts were elongated and had an electron-dense stroma and variable degrees of swelling of the thylakoids, which were partially visible, although chloroplast envelopes were lost. Mitochondria had an electron-dense matrix and cristae were identi®able, as scattered electron-translucent areas. Tannin vacuoles contained a compact and electron-dense material but, occasionally, they appeared granular. Nuclei were also present; they had a ®ne ®brogranular appearance and were identi®able in the cytoplasmic matrix as more electron-dense areas. Similar structural and ultrastruc- tural featureswere observed in pulvinar motor cells embedded in UV-polymerized LR White resin /data not shown). However, sections were less electron-beam stable, many cells were disrupted by cutting, and tannin vacuoles were separated from the cytoplasm by an electron-translucent layer.

Fig. 3 Motor cellsof a Robinia secondary pulvinus embedded in heat-polymerized LR White resin. In®ltration was carried out at Phytochrome distribution in green pulvini ±20 °C. Pulvinuswas®xed in 0.5% glutaraldehyde-4% formalde- hyde in 0.1 M sodium phosphate /pH 7.4). Note vacuoles /V), Sinapis alba anti-phyA /CP 2/9) MAb reacted positively tannin vacuoles/ TV), nuclei /N) and chloroplasts /arrows). Bar = 4 lm when it was assayed against Robinia phytochrome by immunoblotting. Thus, we used this antibody at concentrationsof 15±30 lgml±1 for immunocytochem- The LR White resin-embedded pulvini were easier to ical applications in transverse sections of pulvini. cut and sections were relatively electron-beam stable. Remarkable labelling wasobtained both in heat- and Appreciable di€erences in structural and ultrastructural UV-cured LR White resin-embedded pulvini but only preservation were noted depending on the in®ltration the former results are presented here. Table 1 shows the temperature and polymerization. When in®ltration was labelling density of gold particles observed in selected conducted at 4 °C, the continuity of the plasmalemma areasof Robinia pulvini. and the tonoplast was frequently disrupted. In com- In pulvini ®xed immediately after a 15-min R pulse, parison with K4M-embedded pulvini the cytoplasmic CP 2/9 clearly labelled Robinia cortical motor cellsbut matrix was dispersed and, in some cells, lacunate areas there wasno evidence of labelling either in vascular were commonly observed. The structural integrity of tissue or in the epidermis. The labelling was present in mitochondria and chloroplast envelopes was not main- the cytosol, chloroplasts, mitochondria and nuclei in all tained and both organellesshowed variable degreesof examined sections but the level of labelling was low swelling. The thylakoid system retained a loose ap- both in extensor and ¯exor motor cells. Substantial pearance and grana were not evident. When in®ltration accumulation of labelling wasfound in the cytosol. wascarried out at ±20 °C and the resin was polymerized Single gold labelsand clustersoflabelswere randomly by heat /Fig. 3), ultrastructural preservation improved, distributed throughout the cytoplasm /Fig. 4a) and and thisprotocol wastherefore selectedfor immuno- considerable variation of labelling density was found in

Table 1 Immunogold labelling of phytochrome in Robinia show the number of areas over which the values were obtained. pseudoacacia pulvinar motor cellsafter R /15 min) and R-FR /15- Control sections with the primary antibody omitted or incubated min R plus5-min FR) irradiations.Numbersof gold particlesper with non-immune IgG alwaysgave lessthan0.5 gold particles lm±2 unit area /lm2) are given asmeans‹ SE. Numbersin parentheses Cell type Light treatmentsNumber of gold particles lm±2

Cytosol Chloroplast Mitochondria Nucleus

Extensor R 7.72‹0.85 /11) 4.71‹0.99 /7) 2.00‹0.94 /5) 4.41‹0.86 /6) Flexor R 8.40‹1.90 /9) 4.25‹0.64 /8) 1.31‹0.83 /6) 3.87‹1.10 /8) Extensor R-FR 8.11‹1.35 /10) 5.75‹1.23 /10) 2.42‹1.36 /5) 4.22‹0.86 /9) Flexor R-FR 7.25‹1.07 /12) 5.62‹0.96 /8) 2.44‹0.72 /7) 6.53‹1.56 /8) 570

Fig. 4a±c Immunogold localization of phytochrome in cross- sections of Robinia cortical extensor motor cells from pulvini ®xed Discussion after R /15 min). Phytochrome wasdetected by using S. alba anti- ±1 phyA MAb, CP 2/9 /15 lgml ). Arrowheads indicate the 10-nm Immunoblot analysis indicated that all monocot MAbs gold particles. Immunolabelling was found in the cytoplasm, near the cell wall /a) or associated with some granular material /b). against phytochrome tested fail to cross-react with Ro- Control sections were incubated with non-immune rat IgG /c). binia phytochrome /data not shown). This is consistent Note the absence of labelling in cell walls /a, c) and tannin vacuole with earlier observations that MAbs directed to phyto- /b). TV Tannin vacuole, W cell wall. Bar = 0.2 lm chrome from etiolated oatsdid not stainphytochrome from dicots/Cordonnier et al. 1985). By using CP 2/9 the molecular mass of the polypeptide detected from the same cell. The gold particles, in some cells, were Robinia /about 115 kDa) waslower than that of the 121- associated with a granular material /Fig. 4b). Similar kDa phytochrome from etiolated Sinapis alba.This granular structures were found both in R- and FR- di€erence in mass could arise from a proteolytic cleav- irradiated pulvini. Some gold particleswere alsoob- age of a small fragment /about 6 kDa) rather than an served in chloroplasts /Fig. 5a, b, d) and, occasionally, inherent di€erence between Robinia phytochrome and in the inter-cristae space of mitochondria /Fig. 5c). We Sinapis phy A. Although Robinia phytochrome wasex- observed a greater degree of labelling in the plastids tracted under conditionsto inhibit proteolysiswe cannot than in the mitochondria /Table 1). Appreciable la- exclude the possibility of proteolytic cleavage of a small belling wasfound in nuclei /Figs.5d, 6b), although not fragment /between 4 and 9 kDa), ashasbeen found in all the nuclei in a given section were labeled. No labels other species /Vierstra et al. 1984). As results from were found in cell walls, vacuoles, or starch grains. The comparative immunostaining of both etiolated and size and distribution pattern of labelling was similar in green-mustard phytochrome by CP 2/9 indicate that this both extensor and ¯exor motor cells /Table 1). Control MAb isspeci®cfor Sinapis phy A, and control experi- sections with the primary antibody omitted /Fig. 5e) or mentswith non-immune IgG or by omitting CP 2/9 incubated with non-immune IgG /Figs. 4c, 5f) always exhibit no bands, it seems likely that this 115-kDa gave less than 0.5 gold particles lm±2. The speci®city of Robinia polypeptide is a putative phy A. Nevertheless, immunolabelling was also shown by its absence in thisremainsto be con®rmed. Given that the CP 2/9 calcium oxalate crystals /Fig. 6a, b). MAb wasdirected towardsa singleepitope of S. alba To test whether the localization of phytochrome is phy A it cannot be ruled out that thisantibody can dependent on the state of the photoreceptor, nyctinastic closure was induced 2 h after onset of the photoperiod c and pulvini were irradiated with R followed by FR and Fig. 5a±f Immunogold localization of phytochrome with anti- then transferred to darkness. Both in extensor and ¯exor phyA MAb of S. alba in cross-sections of Robinia cortical extensor motor cellsfrom pulvini ®xed after R /15 min). a±d Immunolabel- motor cells, the size and pattern of labelling was similar ling /arrowheads) wasfound in the chloroplast/ a, b, d), irrespective of whether phytochrome was in the Pfr state mitochondria /c) and nuclei /d). b A higher magni®cation of a or had reverted to Pr /Table 1). Thus, the labelling was part of the chloroplast viewed in a. Cell wallswere not visible after the end of R irradiation and it was not immunolabelled. e, f Control sections with the primary antibody omitted /e) or incubated with non-immune rat IgG /f). Note the reversible by FR light. Furthermore, insertion of a dark absence of labelling in mitochondria /e) and part of a chloroplast period /10±30 min) after the R pulse did not change the /f). Chl Chloroplast, M mitochondrion, N nucleus, S starch, labelling size or pattern. t thylakoid, W cell wall. Bar = 0.3 lm 571 572

Fig. 6a, b Immunogold localization of phytochrome with anti- /McCurdy and Pratt 1986; Speth et al. 1986, 1987) it phyA MAb of S. alba in an inner cortical ¯exor motor cell of modi®ed the ultrastructural features of pulvinar cortical Robinia containing a calcium oxalate crystal /Cr). Pulvini were ®xed 10 min after an R pulse. b isan higher magni®cation of a cells. In comparison, ultrastructural preservation of LR portion /i) of the nucleus/ N) viewed in a. Note the absence of White resin-embedded pulvini was good enough to allow labelling over the calcium oxalate crystal /a). Some label wasin the exact localization, mainly when in®ltration wascarried nucleus. Note /b) that labelling /arrowheads) ismore electron dense out at ±20 °C. In addition, low-temperature dehydration than the surrounding granular material. V Vacuole, W cell wall. and embedding are known to reduce antigen denatur- Bars= 1 lm/a), 0.5 lm/b) ation and enhance antigenic preservation /Vandenbosch 1991). Assectionsof pulvini embedded in UV-cured LR detect a di€erent phy in Robinia, either in addition to or White resin were more electron-beam unstable than in place of phy A, if they share the same epitope. In those of pulvini embedded in heat-polymerized LR addition, it must be kept in mind that, although mon- White resin and the antibody response was similar in oclonal antibodies are highly speci®c, they may also bind both cases, it was considered desirable to use the former to polypeptidesother than phytochrome /Cordonnier et for immunolocalization of phytochrome. al. 1983). A detailed characterization of the PHY family The reliability of immunolocalization results depends in Robinia hasnot been carried out to date. However in on the following factors: /i) speci®city of antibody, other membersof the Robinieae, PHYA, PHYA¢ and /ii) absence of artifacts caused by antigen mobility dur- PHYE have been found /Mathewset al. 1995). ing sample preparation and labelling, and /iii) absence of Most work on phytochrome immunolocalization has non-speci®c antibody binding. The speci®city of the been done with etiolated coleoptiles®xed in 0.5% glu- CP 2/9 antibody is high, as suggested by western-blot taraldehyde-4% paraformaldehyde and embedded in analysis /Fig. 1). To minimize artifacts caused by antigen LR White resin /McCurdy and Pratt 1986; Speth et al. movement, pulvini were ®xed, dehydrated and embed- 1986, 1987). We followed the same ®xation method and ded at low temperature. Also, all the results reported tested di€erent resins and embedding protocols in order here were repeated in separate experiments. Although to improve ultrastructural preservation and to maintain the number of gold particleswaslow, in all sections phytochrome antigenicity. The ultrathin sections of observed they showed a similar distribution. Further- Lowicryl K4M-embedded pulvini were electron-beam more, no labelling wasobserved in the central vacuole or unstable and showed a poor ultrastructural preservation inside the starch grains and calcium oxalate crystals. that did not allow exact localization. Similar results had Thisprovidesevidence that labelling isspeci®cand that been found in LR White-embedded pulvini when in®l- it isnot likely to be an artifact of samplepreparation. tration wasconducted at 4 °C. Although thisprotocol Our results indicate that a putative phy A is located gave good ultrastructural preservation in Avena sativa in the cytoplasm, nuclei, chloroplasts and mitochondria 573 of pulvinar motor cells. This cytoplasmic phytochrome thank the Scienti®c-Technical Servicesof the Universityof Barce- localization agreeswith the general acceptance that phy lona and especially Almudena Garcõ a for her valuable technical assistance and Robin Rycroft /Servei Assessorament LinguÈ õ stic- A is uniformly distributed in the cytosol in dark-grown Escola d'Idiomes Moderns, University of Barcelona, Barcelona, seedlings /McCurdy and Pratt 1986; Hisada et al. 2000). Spain) for correcting the English text. This work was supported However, in contrast to observations made with etio- by grant PB97±0928 from the Ministry of Education and Science, lated Glycine max seedlings /Cope and Pratt 1992), DGICYT, Spain. Pisum sativum epicotyls/Saunderset al. 1983) and A. sativa coleoptiles/McCurdy and Pratt 1986; Speth et al. 1986), R light did not induce a sequestered References distribution of phytochrome in green pulvini. The nuclear localization of a putative phy A in cor- Cope M, Pratt LE /1992) Intracellular redistribution of phyto- tical cellsof Robinia pulvini con®rmsthe recent report of chrome in etiolated soybean /Glycine max L.) seedlings. Planta Kircher et al. /1999) that phy A islocalized in the nuclei 188:115±122 Cordonnier M-M, Smith C, Greppin H, Pratt LH /1983) Produc- and cytosol of tobacco plants grown under light and tion and puri®cation of monoclonal antibodiesto Pisum and dark cycles. The same authors state that in dark-adapted Avena phytochrome. Planta 158:369±376 plants, R and FR light induced nuclear import of phy A Cordonnier M-M, Greppin H, Pratt LH /1985) Monoclonal through a very-low-¯uence response. Thus, our results antibodieswith di€ering anitiesto the red-absorbingand showing that R and FR do not appear to disturb the far-red-absorbing forms of phytochrome. Biochemistry 24:3246±3253 phytochrome distribution pattern in Robinia green Epel BL, Butler WL, Pratt LH, Tokuyasu KT /1980) Immuno¯u- pulvini are consistent with these observations in tobac- orescence localization studies of the Pr and Pfr forms of phy- co. Thisnuclear localization of phytochrome isalso tochrome in the coleoptile tipsof oats,corn and wheat. In: De consistent with the phytochrome-regulated protein Greef J /ed) Photoreceptorsand plant development. Antwerp University Press, Antwerp, pp 121±133 phosphorylation reported in isolated nuclei /Romero Furuya M /1993) Phytochromes: their molecular species, gene et al. 1991) and with the nuclear localization activity of families and functions. Annu Rev Plant Physiol Plant Mol Biol phytochrome-interacting factor 3 /PIF3; Ni et al. 1998). 44:617±645 Immunological data indicated no di€erencesbetween Furuya M, SchaÈ fer E /1996) Photoperception and signalling of induction reactionsby di€erent phytochromes.TrendsPlant Sci the distribution pattern of phytochrome after R and FR 1:301±307 irradiationsin Robinia motor cells. However, the possi- Go mez LA, Simo n E /1995) Circadian rhythm of Robinia pseudo- bility of phytochrome distribution changes comparable acacia lea¯et movements: role of calcium and phytochrome. to those observed in other material /Cope and Pratt Photochem Photobiol 61:210±215 1992) cannot be ruled out since there was no informa- Hisada A, Hanzawa H, Weller JL, Nagatani A, Reid JB, Furuya M /2000) Light-induced nuclear translocation of endogenous pea tion indicating that CP 2/9 discriminates between Pr and phytochrome A visualized by immunocytochemical procedures. Pfr. Alternatively, phytochrome redistribution in Plant Cell 12:1063±1078 Robinia pulvini might require a dark period of more Hoagland DR, Arnon DI /1950) The water culture method for than 30 min to become detectable. It isknown that the growing plantswithout soil.Calif Agric Exp St Circ No 347 Howe GT, Bucciaglia PA, Hackett WP, Furnier GR, Cordonnier- kineticsof the intracellular redistribution of phyto- Pratt MM, Gardner G /1998) Evidence that the phytochrome chrome di€ersbetween plant speciesand experimental gene family in black cottonwood hasone PHYA locusand two conditions/Speth et al. 1987; Cope and Pratt 1992; PHYB loci but lacksmembersof the PHYC/F and PHYE Hisada et al. 2000). subfamilies. Mol Biol Evol 15:160±175 Kircher S, Kozma-Bognar L, Kim L, Adam E, Harter K, SchaÈ fer The present study, while not conclusive, indicates the E, Nagy F /1999) Light quality-dependent nuclear import of the possible presence of phy A in green Robinia pulvini and plant photoreceptorsphytochrome A and B. Plant Cell the results are consistent with the hypothesis that phy A 11:1445±1456 interactswith the circadian oscillatorin the control of Laemmli UK /1970) Cleavage of structural proteins during the lea¯et circadian rhythmic movement in Robinia. In ad- assembly of the head of bacteriophage T4. Nature 227:680±685 Lo pez-Juez E, Nagatani A, Tomizawa K-I, Deak M, Kern R, dition it hasalsobeen reported that phy A isimplicated Kendrick R, Furuya M /1992) The cucumber long hypocotyl in the detection of daylength /Whitelam et al. 1998). mutant lacksa light-stablePHYB-like phytochrome. Plant Cell However, our results do not allow us to exclude the 4:241±251 presence of other phytochromes. 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