Plant Cell Physiol. 40(6): 592-603 (1999) JSPP 01999 Structural and Immunological Characterization of the Cuscuta 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 plants, 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