Cell Physiol. 40(6): 592-603 (1999) JSPP 01999 Structural and Immunological Characterization of the pentagona L. Chloroplast

Timothy D. Sherman I, William T. pettigrew and Kevin C. Vaughn ' Department of Biological Sciences, University of South Alabama, Mobile, AL 36688, U.S.A. Crop Genetics and Production Unit, USDA-ARS, P.O. Box 345, Stoneville, MS 38776, U.S.A. ' Southern Weed Science Research Unit, USDA-ARS, P.O. Box 350, Stoneville, MS 38776, U.S.A.

Structural and immunochemical studies were used little or no need to conduct photosynthesis on their own to determine the photosynthetic potential of the dodder and, although still possessing plastids, have greatly reduced (Cuscuta pentagona) chloroplast. Ultrastructural studies or non-existent photosynthetic capacity. Only a few differ- revealed that thylakoid membranes of pre-parasitic phase ent groups have been examined extensively at the physio- Cuscuta pentagona are almost all organized into long, logical and genetic levels. Parasitic angiosperms have come overlapping grana stacks of mainly two to five thylakoids from four plant families: the Orobanchaceae (Epiphagus with little space between adjacent stacks. Immunoblots virginiana, Conopholis americana, and Orobanche spp.), reveal chloroplast proteins associated with PSI and 11, as the Scrophulariaceae (Lathraea clandestina L.) (Dela- well as cytochrome f and plastocyanin. Stromal extracts vault et al. 1996), the Raflesiaceae (Cytinus hypocistis L.) contained immmunologically-detectable RuBisCO and phos- (Thalouarn et al. 1986), and the Cuscutaceae (a few Cus- phoribulokinase. Cytochemical localizations of the oxidiz- cuta species). Of these, only the Cuscutaceae have been ing side of PSI showed product localization on the lumen shown to possess the plastidic genes necessary to conduct side of the thylakoid. Immunocytochemical localizations photosynthesis (Freyer et al. 1995, Haberhausen et al. of RuBisCO reveal exclusive labeling in the stroma, whereas 1992, Haberhausen and Zetsche 1994, Machado and antibodies to the PSII proteins, light-harvesting Chl a/b Zetsche 1990). Even within this group, it is likely that there complex and the oxygen-evolving complex of PSII, are exists a "gradient" of species with differing levels of adap- concentrated over the thylakoids. A limited capacity for tation for the parasitic lifestyle. Of the 170 species of C02 fixation was found in seedlings by monitoring Cuscuta found globally (Pazy and Plitmann 1995), only a exchange rates in the presence and absence of atrazine. handful have been examined in any detail, with most of the These data indicate that the chloroplast from this species of anatomical characterization performed more than twenty dodder contains a number of the proteins required for a years ago (reviewed by Malik (Malik and Singh 1979)). successful fixation of C02 and the proteins in the thyla- More recently, this genus has come under scrutiny as a tool koids are organized much like other higher , with the for examination of chloroplast evolutionary rates. Two exception of the large percentage of the thylakoids or- species have become the focus of this genetic characteriza- ganized into grana. tion: Cuscuta europaea (Freyer et al. 1995, Kelly 1992, Machado and Zetsche 1990, Zhelev et al. 1994) and Cus- Key words: Chloroplasts - Cuscuta pentagona - Dodder cuta reflexa (Bommer et al. 1993, Freyer et al. 1995, - Immunological characterization - Parasitic plants - Haberhausen et al. 1992, Haberhausen and Zetsche 1994, Ultrastructure. Hibberd et a]. 1998, Machado and Zetsche 1990, Sub- ramaniam and Mahadevan 1994, Subramaniam et al. 1994). From these previous works, it appears that Cuscuta europaea is a species more specialized for the parasitic The parasitic habit has evolved in a variety of vascular lifestyle with no detectable chlorophylls or capacity to in- plants. As a function of this lifestyle, these plants have corporate 14C02 (Machado and Zetsche 1990), plastids without thylakoids (Machado and Zetsche 1990), and large deletions of photosynthetically related genes (Freyer et al. Abbreviations: CER, carbon exchange rate; DAB, diamino- benzidine; DSNBT, distyryl nitro blue tetrazolium chloride; EPSP 1995). Cuscuta reflexa, on the other hand, has been shown synthase, 5-enolpyruvylshikimate 3-phosphate synthase; LHCPII, to possess a number of photosynthetically related genes light-harvesting Chl a/b complex proteins of PSII; Mr, relative with significant homology to those found in higher plants molecular mass; OEC, oxygen evolving complex of PSI; PAR, (Haberhausen et al. 1992, Haberhausen and Zetsche 1994). photosynthetically active radiation; PIPES, Piperazine-N,N-bis- The functionality of these genes has been debated, how- [2-ethanesulfonic acid]; PRK, phosphoribulokinase; RuBisCO, ever. For instance, Haberhausen et al. (1992) found that, ribulose-1,s-bisphosphate-carboxylase/oxygenase;TCNBT, thio- carbamyl nitro blue tetrazolium. although the DNA sequence for RuBisCO should code for ' To whom correspondence should be addressed. Fax 334-414- a functional gene product, they could detect only a weak 8220, Email tshermanajaguarl .usouthal.edu signal for transcription in Northern blot analysis and could Characterization of Cuscuta pentagona Chloroplast 593 not detect the enzyme's presence via immunoblots or by deionized water. The seeds were then allowed to dry briefly to light-stimulated carbon fixation. Machado and Zetsche improve separation of the seeds and germinated on a nutrient (1990), however, found light-stimulated carbon fixation in medium (Somerville and Ogren 1982) supplemented with 25 pg mll ampicillin (to suppress bacterial growth) in glass culture this same species. Recently, the inconsistent data of these dishes. Growth characteristics and protein profiles are unaffected groups has been resolved by discovery that Cuscuta reflexa, by this antibiotic treatment. Seedlings of dodder were grown for engaged actively in parasitism, contains photosynthetically up to 10 d in a non-parasitic mode on this medium under contin- competent cells in only a thin band of cells adjacent to the uous light of lOOpmol m2s' PAR at room temperature (21- vascular tissue (Hibberd et al. 1998). In a third species, 23°C) Samples of wild dodder were collected from a local pop- ulation in Stoneville, MS, U.S.A. as green or orange colored stem Cuscuta campestris Yuncker, photosynthetic pigments and segments. These segments were either fixed directly in the field or PSII activity were examined in specimens grown under brought back to the laboratory where they were processed imme- varying environmental conditions (Dinelli et al. 1993). diately for either biochemical or structural analysis. Data from that study clearly indicates that Cuscuta PSI and PSII cytochemistry-Partial reactions of photosyn- campestris possesses the predominant pigments for pho- thetic electron transport were visualized cytochemically as previ- tosynthesis (chlorophyll a and b, as well as a and β caro- ously described (Vaughn and Outlaw 1983, Vaughn et al. 1983). tenes, and other accessory pigments) and low levels of PSII The oxidizing side of PSI was localized using diaminobenzidine (DAB) at 1 mg mll. Reaction at this concentration is specific for activity. PSI activity, preferentially staining the stroma lamellae and ends Some previously published structural studies of Cus- of grana stacks. PSII was localized using the tetrazoliums: distyryl cuta chloroplastic structure indicate that the plastids have nitro blue tetrazolium chloride (DSNBT) or thiocarbamyl nitro underdeveloped structure, consisting chiefly of lipid bodies blue tetrazolium (TCNBT) at 1 mg mll. These compounds and rudimentary thylakoids (Dodge and Lawes 1974, receive electrons from PSII. Laudi et al. 1974, Machado and Zetsche 1990). However, Immunocytochemistry-Small (-1 mm) stem pieces were fixed in 3% glutaraldehyde in 0.05 M Piperazine-N,N-bis[2- these studies were performed on plants that have parasi- ethanesulfonic acid] (PIPES) buffer (pH 7.4) for 2 h at 4OC. The tized a host and likely were from regions of tissue outside specimens were then washed in 0.05 M PIPES buffer (pH 7.4) and of the photosynthetic cells identified by Hibberd et al. dehydrated in ethanol to 70% at 4OC. Subsequent dehydration to (1998). Additionally, these studies were conducted at a 100% ethanol was carried out after transferring the specimens to developmental stage marked by reduced need for endoge- a freezer (-20°C) The samples were infiltrated with L.R. White resin (Polysciences, Warrington, PA, U.S.A., [soft formulation]) nous carbon fixation, and thus not reflective of the ultimate in 25% steps, for 2-4 h at each step and two overnight changes of photosynthetic potential of the plant. 100% resin. Polymerization was carried out in BEEM@capsules In this study, we took several approaches to investi- (Polysciences, Warrington, PA, U.S.A.) using resin to which 100 gate the photosynthetic capacity of Cuscuta pentagona, a u\ of L.R. White catalyst was added. The specimens remained at species that we have found to possess a marked green color -20° for 8-10 h to ensure complete polymerization. during certain developmental stages. First, we utilized see- Sections were cut with a Reichert Ultracut E ultramicrotome dlings grown on a nutrient medium under defined condi- to a thickness corresponding to a pale gold reflectance color and mounted on either uncoated 300 mesh gold or nickel grids. The tions as well as field grown plants in the parasitic mode grids were then processed in the steps described previously for close to the time of flowering. It is at these stages that we un-osmicated specimens (Vaughn 1989). The primary antisera observed maximal accumulation of chlorophyll (and pre- were utilized at dilutions of 1 : 40 to 1 : 160. These dilutions were sumably chloroplast development). Second, we character- determined empirically depending upon the titer of the antisera ized the presence of enzymes involved in photosynthesis preparation, the relative ability of the antisera to recognize the polypeptides on Western blots, and the lack of background la- both immunologically and cytochemically. Finally, whole beling over cell walls and intercellular spaces. These antisera were seedling measurements of carbon exchange rates were from rabbits immunized with light-harvesting Chl a/b complex conducted. This system of assays was used to examine the proteins of PSII (LHCPII), the 33 kDa protein of the oxygen- structural and biochemical characteristics of these plastids. evolving complex (OEC) of PSII (Kawata and Cheung 1990), Our data indicate that this species of Cuscuta has a com- PsaA/PsaB complex (originally described as the P7oo Chl a-bind- plete assemblage of photosynthetic pigments and protein ing protein) (Vierling and Alberte 1983), plastocyanin (Hauska et al. 1983), the chloroplastic coupling factor 1 (Lax and Vaughn complexes for both light and dark reactions, but that pre- 1986, Radunz 1980), cytochrome f (Pettigrew and Vaughn 1998), parasitic and post-parasitic tissues differ in abundance of RuBisCO (Pettigrew and Vaughn 1998). RuBisCO activase (Sal- photosynthetically-related proteins. vucci et al. 1985), or phosphoribulokinase (McKay and Gibbs 1991). These antisera were gifts from a variety of different sources. The immunolabeling experiments were repeated on different tissues (3 Materials and Methods batchedembeddings) and samples (3 samples/tissue). Morpho- metric data were collected and analyzed as described by Lax and Plant material-Seeds of Cuscuta pentagona (F&J Seeds, Vaughn (Lax and Vaughn 1991). Urbana, IL, U.S.A.) were immersed in concentrated sulfuric acid Electrophoresis and imrnunoblotting-Fresh tissue of pre- for 1 h at room temperature in a stainless steel strainer, neutral- parasitic and parasitic dodder were homogenized with a chilled ized in saturated sodium bicarbonate, and then rinsed in running mortar and pestle in 330 mM sorbitol, 10 mM HEPES (pH 7.7 at 594 Characterization of Cuscuta pentagona Chloroplast

2OC), 1 mM EDTA, 1 mM MgC12, and 5 mM cysteine at a ratio of roplasts of the typical C3 dicot. Within the plastid, the 5 ml buffer per gram fresh weight. Homogenate was filtered arrangement of thylakoids is also unique. Towards the tip through one layer of cheesecloth and large particles removed by of the growing seedlings, virtually all of the chloroplasts centrifugation for 5 min at 500Xg and 4OC. Plastids were col- lected at 6,000xg for 15 min, and then were disrupted in the contain thylakoid doublets in which the thylakoids are ap- above buffer minus sorbitol plus proteinase inhibitors (1 mM pressed along their entire length (Fig. 1). There are essen- PMSF, lOyM pepstatin, 10,uM leupeptin, and 5 mM 1,10- tially no areas where unappressed thylakoids are noted. In phenanthroline). Thylakoids were pelleted at 13,000 x g for 15 the most elaborate thylakoid arrangement observed, a min. Stromal proteins from the supernatant were precipitated stack of five thylakoids was detected, but again without with four volumes of acetone at -20°C Precipitated proteins much in the way of unappressed lamellae. In many ways, were recovered by centrifugation at 16,000 x g for 1 min. Thyla- koids and stromal proteins were solubilized in 125 mM Tris (pH these chloroplasts are similar to what is observed in very 6.8 at 4OC) plus protease inhibitors with brief sonication. Protein immature leaves of dicots (Pettigrew and Vaughn 1998) in concentration was assayed by the method of Bradford (1976) and which the thylakoids exhibit a similar complete appression. adjusted to approximately 1 mg mlwith a concentrated buffer Evidence of plastid division in dodder (constriction of a stock to yield a final solution of 125 mM Tris (pH 6.8), 10% (v/v) relatively large plastid) is noted frequently in areas of the glycerol, 2% (w/v) lithium dodecyl sulfate, 1% (v/v) /?-mer- shoot where nuclear divisions are also noted. captoethanol, and 0.02% (w/v) bromophenol blue. Samples were heated to 37OC for 30 min and centrifuged briefly to remove in- Besides the unique arrangement of thylakoids, the soluble material. SDS-PAGE was performed according to the dodder chloroplasts often contain thylakoid-bound crys- method of Laemmli (1970) in a mini-gel format. Proteins were talline inclusions (Fig. 2). The crystal contains a regular transferred to BioTrace NT nitrocellulose (Gelman Sciences, Ann latticework of alternating electron opaque and translucent Arbor, MI, U.S.A.) in 15% ethanol, 25 mM Tris, and 192 mM material and is quite rectangular in outline. Favorable glycine for 1 h at 1 mA cm2and 1 h at 2.5 mA cm2. Unsatu- rated sites on the membranes were blocked with 0.5% (w/v) sections reveal that a single, distended thylakoid membrane Blocking Reagent (Boehringer-Mannheim, Indianapolis, IN, surrounds the inclusion. Such inclusions are typical of U.S.A.) in 10 mM Tris pH 7.5, 150 mM NaCI (TBS) for 1 h. Blots underdeveloped plastids in the epidermis, vascular tissue, were then exposed to primary antibody at a dilution of 1 : 1,000 to and roots but are relatively uncommon occurrences in 1 : 4,000 in this blocking solution for 2 h; washed in TBS con- the chloroplasts of photosynthetic tissue. An exception is taining 0.05% (v/v) Tween-20 for 3 times for 10 rnin each. This found in the chloroplasts of spinach, where a similar was followed by treatment with alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) for 1 h, washed as above, and visu- membrane bound inclusion is noted. However, in a plastid alized with 330yg ml' nitroblue tetrazolium and 165 yg ml' 5- mutant of spinach with reduced thylakoid content, the bromo-4-chloro-3-indoyl phosphate in 100 mM Tris (pH 9.5 at number of these inclusions is also increased (Vaughn et al. 24OC), 100 mM NaCI, and 5 mM MgC12. 1981). Phytoferritin aggregations are sometimes observed Net photosynthetic measurements-C02 exchange rates (CER) in the stroma, and starch grains are observed in virtually were measured using a LI-COR 6200 portable photosynthesis unit every plastid profile. Plastoglobuli are abundant and are of (LI-COR, Lincoln, NE, U.S.A.) with a 1.0 liter volume leaf chamber. Dodder seedlings used for these measurements came varying electron opacity, even within a single chloroplast. from the same culture dish. Samples of equal fresh weight (100 Ribosomes are found abundantly throughout the stroma mg) were prepared by submerging a number of plants in either a and are often associated with the tops of the small grana. herbicide solution containing 10yM atrazine or a 0.1% ethanol Clear areas of the stroma devoid of ribosomes, containing solution (control for the herbicide) for 0.5 h before the CER threadlike inclusions, probably represent areas of chlo- reading. Immediately before CER measurements, the seedlings were blotted dry. he tissue samples were complet~lyanchored in roplast DNA accumulation. the chamber during the gas exchange measurements. The experi- The chloroplast envelope is invaginated, which, in thin ments were repeated on several different groups of seedlings on sections, appears as paler, inclusions in the stroma (Fig. 3). several different sampling dates. Tubular structures about the diameter of microtubules are Spectroscopy-Shoot tips of 7-days old seedlings were im- associated with the inner envelope and extend into the mersed in 80% (v/v) acetone and placed in a sealed tube main- stroma. Frequently, these "chloroplast microtubules" oc- tained in darkness at 4OC. When the shoot tissue was devoid of pigment, the solvent was used to produce visible spectra (350-700 cur in clusters. Although such structures have been ob- nm) with a Gilford Response spectrophotometer (Oberlin, OH, served in other chloroplasts (Vaughn and Wilson 1981), U.S.A.). Leaves of Pelargonium hortorum were treated similarly their function is unknown. and used as an example of a typical higher plant pigment extract. Chloroplasts from material collected under field situ- ations (in which there was noticeable green to the tissues) Results and Discussion are structurally similar to those grown under laboratory conditions, indicating that the super-stacked thylakoid Structural studies-Chloroplasts of dodder are quite configuration is not due to growth of the plants under unlike those of typical mature C3 dicots. First, the chlo- laboratory conditions. In this greenish tissue all chlo- roplasts are much smaller, with lengths of about 2-3 pm roplasts have similar morphology. In materials collected and widths of -1 ,urn, compared to the 6-8 pm long chlo- from the field, tissues that lacked any green appearance, Characterization of Cuscuta pentagona Chloroplast 595

Fig. 1 Electron micrographs illustrating the arrangement of thylakoids in the most developed dodder chloroplasts. (A) Unlike other chloroplasts where the thylakoids are separated into regions of appressed and unappressed lamellae, the thylakoids in dodder are all present in ministacks of 2-3 thylakoids with virtually no separation between adjacent grana. S=starch. (B) Higher magnification mi- crograph showing the appressed nature of thylakoids in dodder chloroplasts. All of the thylakoids are arranged into small grana (g) of two to three thylakoids. The region between the thylakoids (marked with arrowheads) is more electron opaque than the thylakoids typical of the appressed regions. Bars=0.5 pm in A, 0.1 pm in B. however, have no chloroplasts that possess this super- the size of the plastids appear to be less than those grown stacking of thylakoids and show no immunologically de- under continuous light (Fig. 4). Prolamellar bodies, which tectable RuBisCO (data not shown). Surprisingly, when are prominent structures in etioplasts, are also found, al- dodder seedlings are germinated and grown in the dark, the beit in a much reduced state in the etioplasts of dodder. structure of the thylakoids is not too different from plants Because prolamellar bodies are the chloroplastic sites of grown in the light. The development of the thylakoids and accumulation of protochlorophyllide (and its conversion to 596 Characterization of Cuscuta pentagona Chloroplast

Fig. 2 Types of inclusions in dodder chloroplasts. Dodder chloroplasts contain inclusions found in other plastid types (A & B) as well as those rather unique to dodder. (A) Plastoglobuli (p) are of varying electron opacity and are present throughout the chloroplast, although most often associated with the thylakoids. (B) Phytoferritin crystals, which may represent a storage form of iron in these plastids, occurs in clusters in the stroma and is not membrane bound like the other inclusion type (C & D). (C) Membrane-bound crystalline inclusions are about 0.5 pm in length and contain a regular, repeating structure of electron opaque and translucent areas. (D) Higher magnification electron micrograph of the crystalline inclusion showing that these bodies are membrane-bound (arrowheads note the distended thylakoid that surrounds the crystal). Bars=0.5pm in A-C, 0.1 pm in D.

Chl in light), it would appear that dodder chloroplasts electron transport system close to that of plastocyanin contain similar mechanisms in dealing with the etioplast/ (Vaughn and Duke 1981, Vaughn et al. 1983). When dod- chloroplast conversion to that found in other higher der chloroplasts are exposed to DAB, a strong reaction is plants. noted in the thylakoids with more reaction towards the Cytochemistry-Partial reactions of photosynthetic edge of the thylakoid stacks than in the middle (Fig. SA, electron transport may be visualized cytochemically, which B). These are consistent with the results of previous studies allows for determination of activity as well as presence of that show a majority of PSI activity is present in unstacked a particular thylakoid component (Vaughn and Outlaw areas of lamellae (Vaughn et al. 1983). In contrast, incu- 1983, Vaughn et al. 1983). PSI partial reactions involve bation of shoot segments in the tetrazoliums, DSNBT and the oxidation of DAB via donation of electrons into the TCNBT, (which receive electrons from PSII) results in a Characterization of Cuscuta pentagona Chloroplast 597

Fig. 3 Imaginations from inner envelope (e) in dodder chloroplast are not unlike microtubules both in longitudinal-section (A-arrows) and cross-section (B-asterisk). These structures may be present to position the thylakoids away from the plastid envelope. Bars= 0.1 urn. reaction that is primarily present in the stacked areas of PSII staining in dodder. thylakoid membranes (not shown). The latter reaction is Immunocytochemistry-The light harvesting complex weak relative to PSII cytochemical localizations in other of PSII and RuBisCO are the major proteins of the higher plants (e.g., Vaughn et al. 1983), but this may be thylakoids and stroma, respectively. Immunocytochemical due to the poor penetration of the cytochemical reagents or localizations with antibodies to these proteins reveal spe- the greater sensitivity of the PSII reactions to aldehyde cific labeling of the thylakoids and stroma. Labeling of fixation. Only cells that are cut open react with the reagent, RuBisCO is noted throughout the stroma (Fig. 6). A rela- indicating that poor penetration is the major obstacle in tively strong localization is also noted for the OEC of PSII

Fig. 4 Ultrastructure of plastids from dark-grown dodder seedlings. (A) Dodder etioplasts are relatively simple organelles with the thylakoids less extensive and present more as single membranes than in the light-grown material. (B) In addition, the etioplasts contain very small prolamellar bodies, indicative of an accumulation of protochlorophyllide. Bar=0.5 pm in A, 0.1 pm in B. Characterization of Cuscuta pentagona Chloroplast

Fig. 5 Cytochemical detection of PSI (DAB oxidation by PSI) partial reactions in dodder chloroplasts. (A) Strong PSI reaction fills the lumen of the thylakoids when DAB is utilized as an electron donor to PSI. (B) Higher magnification micrograph allowing comparison between the positive reaction on the thylakoids (t) and the absence of reaction on the envelope membrane. S=starch, pg=plastoglobuli; Bars =1.0 pm in A; 0.5 pm in B.

(Fig. 7B) and this thylakoid distribution is identical to that for these polypeptides [RuBisCO, 52 kDa; OEC, 16 to 32 obtained for the LHCPII. However, the level of the label- kDa; LHCPII, 26 kDa; and cytochrome f, 32 to 34 kDa ing with all of these sera is much less than is observed in (Chia et al. 1986); glutamine synthetase, 46 to 50 kDa developed chloroplasts or even rather underdeveloped ones (Hopfner et al. 1988); EPSP synthase, 48 kDa (Kishore of other higher plants (e.g., Pettigrew and Vaughn 1998). et al. 1988); phosphoribulokinase, 39.2 kDa (McKay and None the less, the localization of these proteins in the Gibbs 1991); plastocyanin, 10.4 kDa (Ramshaw et al. 1974); chloroplasts demonstrates that: (1) the uptake mechanisms GroEL, 60 kDa (Vierling 1991)]. Protein extracts prepared for chloroplast proteins encoded by nuclear DNA is oper- in a like manner from spinach were electrophoresed along ative, (2) signaling mechanisms that send each component with those from dodder to allow for direct comparisons of to the proper final site must be functioning, and (3) the chloroplast protein sizes (Fig. 8). Mass values for proteins chloroplast ribosomes observed are making a functional from spinach extracts closely parallel those from the dod- product (i.e. the large subunit of RuBisCO). der extracts (Fig. 9, 10). It is not possible to make state- Immunochemical studies-Chloroplast-enriched frac- ments concerning relative abundance of these proteins tions of pre-parasitic and parasitic dodder were analyzed based on the intensity of immunoreactions, however, be- by a number of single and gradient gel formats with their cause the antibodies utilized were not prepared to dodder proteins subsequently blotted to a number of membrane antigens. The immunoblots are consistent with immu- types. Proteins of LHCPII and enzymes of the carbon nogold labeling experiments in revealing that photosyn- fixation pathway are present in extracts of dodder and have thetically-related proteins are present but not to the extent relative molecular masses consistent with literature values found in a dicot such as spinach. Although comparisons of Characterization of Cuscuta pentagona Chloroplast 599

Fig. 6 Immunogold localization of RuBisCO in un-osmicated sections of 5-day old green stem material embedded in L.R. White resin. Gold particles are found throughout the stroma. S=starch; v=vacuole. Bar=0.5 pm. protein abundance between dodder and spinach extracts used in this work (which was raised to monocot PsaA/ may be problematic due to species differences in the anti- PsaB complex) or that the antiserum had lost activity over genic similarity of the polypeptides, changes in protein time with storage. Alternatively, the limited number of PSI abundance between pre- and post-parasitic dodder are evi- complexes found in dodder are much more active in these dent from the blots that were loaded with equal amounts chloroplasts that have such small regions of unstacked of total protein. There appear to be developmental changes lamellae (where PSI activity is normally found (Vaughn et in abundance of photosynthetically related proteins be- al. 1983)). There are some notable proteins that were de- tween pre- and post parasitic dodder. There is little effect tected on immunoblots of spinach chloroplast membrane of parasitism on abundance of cytochrome f, LHCPII, and soluble proteins but not on those of dodder. These and OEC, but the carbon-fixing enzymes, RuBisCO and include coupling factor I, RuBisCO activase, and PsaA/ phosphoribulokinase, are greatly reduced, as is the lumen- PsaB complex. Antisera to these proteins produced weak localized plastocyanin (Fig. 9). This differential presence signals on spinach immunoblots and so it is possible that extends to other chloroplast-localized, but non-photosyn- these proteins are present in Cuscuta pentagona, but in thetic proteins, with EPSP synthase and the chaperonin amounts that are below the detection limit with the GroEL showing reduced abundance in post-parasitic tis- methodology utilized here. sues (Fig. 10). Strangely, glutamine synthetase seems to be Spectroscopy-Acetone extracts of dodder grown un- present in greater abundance after parasitism and may der laboratory conditions produced absorption spectra reflect changes in nitrogen metabolism associated with qualitatively similar to those of geranium (Pelargonium the changed lifestyle. Immunostaining of the anti-PsaA/ hortorum). Characteristic peaks for carotenoids (400 nm- PsaB complex is consistently weak, whereas the staining 470 nm) and Chls (600 nm-665 nm) are seen in both ex- for the two PSII related proteins are quite strong. This is in tracts, although at lower abundance in the dodder tissue. contrast to the cytochemical studies that indicated little This is in agreement with a more in-depth pigment analysis PSII activity relative to that of PSI. These results are con- of these pigments in the related species, Cuscuta campestris fusing. The discrepancy may result from poor recognition (Dinelli et al. 1993). Their study showed that field-grown of the PsaA/PsaB complex of dodder by the antiserum dodder contains a full complement of photosynthetic pig- 600 Characterization of Cuscuta pentagonu Chloroplast

Fig. 7 Immunogold localization of the light-harvesting Chl a/b complex of PSII (A) and the 33 kDa protein of the oxygen-evolving complex of PSII (B) in un-osmicated sections of 5-day old green stem material embedded in L.R. White resin. Unlike the stromal reaction found for RuBisCO (Fig. 6), all of the immunogold reaction is found on the thylakoids (t). Clear spaces in the stroma between the rows of thylakoid are probably areas containing plastid DNA. Bars=0.5 Dm. ments including α and carotenes, Chls a and b, luthein, reflexa; C02 evolution decreased with increasing light in- antheroxanthin, and violaxanthin. tensity but never rose above the C02compensation point Net photosynthetic measurements-Although other for any light intensity tested (Hibberd et al. 1998). evidence presented here indicates that Cuscuta pentagona The data we present here are consistent with the seedlings contain the necessary components for a func- growth behavior of dodder seedlings, which grow vigor- tional photosynthetic system, the gas exchange of these ously for a week to ten days in the absence of a host, and plants is dominated by respiration. A net evolution of then senesce quickly thereafter. It is possible that the low C02from dodder under light conditions rather than a net level of photosynthetic carbon fixation that was measured uptake of C02 evidence this. The functionality of the here is simply a supplement to stored reserves that increase Cuscutapentagona carbon-fixing system was demonstrated the time in which the young seedling can locate and by comparing gas exchange measurements from atrazine- parasitize a suitable host. treated and untreated control dodder samples. The atra- Collectively, along with other published reports, these zine-treated dodder evolved C02at approximately a 2-fold data indicate that there is likely to be a spectrum of the greater rate than the untreated control (0.017 pmol C02 chloroplast development among dodder species. Clearly, sK1g FWK1and 0.009,umol C02sK1 g FWK1,respectively. of the dodder that have been investigated, Cuscuta Treatments are significantly different at the 0.01 probabili- europaea appears to be the most adapted to a parasitic ty level). Atrazine inhibited what little photosynthesis was lifestyle with a number of deletions of photosynthetically- occurring in the dodder and thereby resulted in a net in- related genes and essentially no development of thylakoid crease in C02evolution from the treated sample. This is in membranes (Freyer et al. 1995, Machado and Zetsche agreement with results found for mature tissues of Cuscuta 1990). Developmentally, Cuscuta reflaa may be next in Characterization of Cuscuta pentagona Chloroplast

A B C Std.

- .. -.?a-e b

. .

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Fig. 10 Immunoblots of various non-photosynthetic proteins from chloroplast extracts from young dodder seedlings, parasitic field-grown dodder, and spinach. Extracts prepared as described in Figure 9. Stromal extracts were used for all blots. Fig. 8 Representative protein profiles and a typical immunoblot of chloroplast extracts from young dodder seedlings (A), parasitic field-grown dodder (B), and spinach (C). Plastids were isolated this species, photosynthesis is localized to a ring of cells from tissues by differential centrifugation. These were lysed and separated into thylakoid and stromal fractions by further centrif- adjacent to the vascular tissue, between the pith and the ugation. Thylakoid fractions were used for these gels. Lanes were cortex. No ultrastructure of this tissue was presented in normalized for equal total protein load. Samples were subjected that work, and so it is not possible to determine what to SDS-PAGE (12.5% gel) and either stained for total protein similarities these photosynthetic cells hold to those of with Coomassie blue R-250 (right panel) or transferred to nitro- Cuscutapentagona tissues that we have characterized here. cellulose and probed with antibodies to light-harvesting Chl a/b complex of PSI1 (left panel). We have found that material collected from the field have differing chloroplasts consistent with the tissue in which they are present. Those in greenish tissue (i.e., those asso- line. A number of photosynthetically-related genes have ciated with floral development and those near haustorial been identified in the genome of this species and light- junctions) have chloroplasts with stacking of thylakoid stimulated carbon fixation was shown to be present, but membranes, as well as cytochemical and immunological only weak transcription of mRNA for RuBisCO and no evidence for active photosynthesis. Tissues that are orange immunologically detectable protein for this enzyme could in color have limited thylakoid stacking and no detectable be demonstrated (Haberhausen et al. 1992, Machado and photosynthetic ability. In Cuscutapentagona, we have not Zetsche 1990). Recently, however, Hibberd et al. (1998) identified the ring of photosynthetic cells found in by have shown that in internodal areas of mature tissue from Hibberd et al. (1998) in Cuscuta reflexa. It is also of interest

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Fig. 9 Immunoblots of various photosynthetic proteins from chloroplast extracts of young dodder seedlings, parasitic field-grown dodder, and spinach. Plastids were isolated from tissues by differential centrifugation. These were lysed and separated into thylakoid and stromal fractions by further centrifugation. Stromal extracts were used to produce blots shown in the panel on the right. Thylakoid extracts were used for blots shown in the panel on the left. Each blot contained extracts from non-parasitic dodder (A), parasitic dodder (B), and spinach (C). Lanes were normalized for equal total protein load. Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to the indicated protein. 602 Characterization of Cuscuta pentagona Chloroplast that the dodder pictured in the work of Hibberd et al. 194-198. (1998) is markedly greener than any of the Cuscuta penta- Kawata, E.E. and Cheung, A.Y. (1990) Molecular analysis of an aurea photosynthetic mutant (Su/Su) in tobacco: LHCP depletion leads to gona tissue that we have found. It is quite likely that Cus- pleiotropic mutant phenotypes. EMBO J. 9: 4197-4203. cuta pentagona (a new world species) is physiologically Kelly, C.K. (1992) Resource choice in Cuscuta europaea. Proc. Natl. Acad. distinct from Cuscuta reflexa (an old world species) and Sci. USA 89: 12194-12197. Kishore, G., Shah, D., Padgette, S., della-Cioppa, G., Gasser, C., Re, D., may have adapted differently to the parasitic lifestyle. Al- Hironaka, C., Taylor, M., Wibbenmeyer, J., Eichholtz, D., Hayford, ternatively, the specialized anatomy found in Cuscuta M., Hoffman, N., Delannay, X., Horsch, R., Klee, H., et al. (1988) 5- reflexa may be indicative of a greater degree of adaptation enolpyruvylshikimate 3-phosphate synthase: from biochemistry to ge- netic engineering of glyphosate tolerance. In Biotechnology for Crop to the parasitic habit than that of Cuscuta pentagona. It is Protection. Edited by Hedrin, P.A., Menn, J.J. and Hollingworth, likely that further investigation into other members of the R.M. Vol. 379. American Chemical Society. genus Cuscuta may produce a spectrum of individuals with Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly varying degrees of adaptation to the parasitic lifestyle that of the head of bacteriophage T4. Nature 227: 680-685. Laudi, G., Medeghini Bonatti, P. and Fricano, G. (1974) Ultrastructure of are intermediate to C. europaea and these other two species plastids of parasitic higher plants. V. Influence of light on Cuscuta (or perhaps even more extremely or less adapted than these plastids. Isr. J. Bot. 23: 145-150. characterized species). As such, these intermediate species Lax, A.R. and Vaughn, K.C. (1986) Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure. would serve as useful tools for the study of chloroplast Physiol. Plant. 66: 384-391. function and chloroplast genome evolution. Lax, A.R. and Vaughn, K.C. (1991) Co-localization of polyphenol oxidase and photosystem II proteins. Plant Physiol. 96: 26-31. We would like to extend thanks to Lynn Libous-Bailey for Machado, M.A. and Zetsche, K. (1990) A structural, functional and mo- excellent technical assistance. Mention of a trademark, proprie- lecular analysis of plastids of the holoparasites Cuscuta reflexa and Cuscuta europaea. Planta 18 1: 91- 96. tary product, or vendor does not constitute a guarantee or war- Malik, C.P. and Singh, M.B. (1979) Physiological and biochemical aspects ranty of the product by the United States Department of of parasitism in Cuscuta-a review. In Annual Review of Plant Sciences. Agriculture and does not imply its approval to the exclusion of Edited by Malik, C.P. pp. 67-113. Vol. 1979. Kalyani Publishers, New other products or vendors that may also be suitable. Delhi, India. McKay, R.M.L. and Gibbs, S.P. (1991) Immunocytochemical localization of phosphoribulokinase in microalgae. Bot. Acta 104: 367-373. References Pazy, B. and Plitmann, U. (1995) Chromosome divergence in the genus Cuscuta and its systematic implications. Caryologia 48: 173-180. Bommer, D., Haberhausen, G. and Zetsche, K. (1993) A large deletion in Pettigrew, W.T. and Vaughn, K.C. (1998) Physiological, structural, and the plastid DNA of the holoparasitic Cuscuta reflexa immunological characterization of leaf and chloroplast development in concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trn1) cotton. Protoplasma 202: 23-37. and an ORF 2280 homologue. Curr. Genet. 24: 171-176. Radunz, A. (1980) Binding of antibodies onto the thylakoid membrane. Bradford, M.M. 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424. Vierling, E. and Alberte, R.S. (1983) The P700 chlorophyll a-protein: pu- Vaughn, K.C., Vierling, E., Duke, S.O. and Alberte, R.S. (1983) Im- rification, characterization and antibody preparation. Plant Physiol. 72: munocytochemical and cytochemical localization of Photosystem I and 625-633. II Plant Physiol. 73: 203-207. Zhelev, Z., Stanilova, S. and Carpenter, B. (1994) Isolation, partial char- Vaughn, K.C. and Wilson, K.G. (1981) Improved visualization of plastid acterization and complement inhibiting activity of a new glycoprotein fine structure: plastid microtubules. Protoplasma 108: 21-27. from Cuscuta europea. Biochem. Biophys. Res. Commun. 202: 186- Vierling, E. (1991) The roles of heat shock proteins in plants. Annu. Rev. 194. Plant Physiol. Mol. Biol. 42: 570-620. (Received August 10, 1998; Accepted April 3, 1999)