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
to determine the photosynthetic potential of the dodder
(Cuscuta pentagona) chloroplast. Ultrastructural studies
revealed that thylakoid membranes of pre-parasitic phase
Cuscuta pentagona are almost all organized into long,
overlapping grana stacks of mainly two t o five thylakoids
with little space between adjacent stacks. Immunoblots
reveal chloroplast proteins associated with PSI and 11, as
well as cytochrome f and plastocyanin. Stromal extracts
contained immmunologically-detectable RuBisCO and phosphoribulokinase. Cytochemical localizations of the oxidizing side of PSI showed product localization on the lumen
side of the thylakoid. Immunocytochemical localizations
of RuBisCO reveal exclusive labeling in the stroma, whereas
antibodies t o the PSII proteins, light-harvesting Chl a/b
complex and the oxygen-evolving complex of PSII, are
concentrated over the thylakoids. A limited capacity for
C 0 2 fixation was found in seedlings by monitoring
exchange rates in the presence and absence of atrazine.
These data indicate that the chloroplast from this species of
dodder contains a number of the proteins required for a
successful fixation of C 0 2 and the proteins in the thylakoids are organized much like other higher plants, with the
exception of the large percentage of the thylakoids organized into grana.
Key words: Chloroplasts - Cuscuta pentagona - Dodder
- Immunological characterization - Parasitic plants -
Ultrastructure.
The parasitic habit has evolved in a variety of vascular
plants. As a function of this lifestyle, these plants have
Abbreviations: CER, carbon exchange rate; DAB, diaminobenzidine; DSNBT, distyryl nitro blue tetrazolium chloride; EPSP
synthase, 5-enolpyruvylshikimate 3-phosphate synthase; LHCPII,
light-harvesting Chl a / b complex proteins of PSII; Mr, relative
molecular mass; OEC, oxygen evolving complex of P S I ; PAR,
photosynthetically active radiation; PIPES, Piperazine-N,N-bis[2-ethanesulfonic acid]; PRK, phosphoribulokinase; RuBisCO,
ribulose-1,s-bisphosphate-carboxylase/oxygenase;
TCNBT, thiocarbamyl nitro blue tetrazolium.
To whom correspondence should be addressed. Fax 334-4148220, Email tshermanajaguarl .usouthal.edu
'
little or no need to conduct photosynthesis on their own
and, although still possessing plastids, have greatly reduced
or non-existent photosynthetic capacity. Only a few different groups have been examined extensively at the physiological and genetic levels. Parasitic angiosperms have come
from four plant families: the Orobanchaceae (Epiphagus
virginiana, Conopholis americana, and Orobanche spp.),
the Scrophulariaceae (Lathraea clandestina L.) (Delavault et al. 1996), the Raflesiaceae (Cytinus hypocistis L.)
(Thalouarn et al. 1986), and the Cuscutaceae (a few Cuscuta species). Of these, only the Cuscutaceae have been
shown to possess the plastidic genes necessary to conduct
photosynthesis (Freyer et al. 1995, Haberhausen et al.
1992, Haberhausen and Zetsche 1994, Machado and
Zetsche 1990). Even within this group, it is likely that there
exists a "gradient" of species with differing levels of adaptation for the parasitic lifestyle. Of the 170 species of
Cuscuta found globally (Pazy and Plitmann 1995), only a
handful have been examined in any detail, with most of the
anatomical characterization performed more than twenty
years ago (reviewed by Malik (Malik and Singh 1979)).
More recently, this genus has come under scrutiny as a tool
for examination of chloroplast evolutionary rates. Two
species have become the focus of this genetic characterization: Cuscuta europaea (Freyer et al. 1995, Kelly 1992,
Machado and Zetsche 1990, Zhelev et al. 1994) and Cuscuta reflexa (Bommer et al. 1993, Freyer et al. 1995,
Haberhausen et al. 1992, Haberhausen and Zetsche 1994,
Hibberd et a]. 1998, Machado and Zetsche 1990, Subramaniam and Mahadevan 1994, Subramaniam et al.
1994). From these previous works, it appears that Cuscuta
europaea is a species more specialized for the parasitic
lifestyle with no detectable chlorophylls or capacity to incorporate 14C02 (Machado and Zetsche 1990), plastids
without thylakoids (Machado and Zetsche 1990), and large
deletions of photosynthetically related genes (Freyer et al.
1995). Cuscuta reflexa, on the other hand, has been shown
to possess a number of photosynthetically related genes
with significant homology to those found in higher plants
(Haberhausen et al. 1992, Haberhausen and Zetsche 1994).
The functionality of these genes has been debated, however. For instance, Haberhausen et al. (1992) found that,
although the DNA sequence for RuBisCO should code for
a functional gene product, they could detect only a weak
signal for transcription in Northern blot analysis and could
Characterization of Cuscuta pentagona Chloroplast
not detect the enzyme's presence via immunoblots or by
light-stimulated carbon fixation. Machado and Zetsche
(1990), however, found light-stimulated carbon fixation in
this same species. Recently, the inconsistent data of these
groups has been resolved by discovery that Cuscuta reflexa,
engaged actively in parasitism, contains photosynthetically
competent cells in only a thin band of cells adjacent to the
vascular tissue (Hibberd et al. 1998). In a third species,
Cuscuta campestris Yuncker, photosynthetic pigments and
PSII activity were examined in specimens grown under
varying environmental conditions (Dinelli et al. 1993).
Data from that study clearly indicates that Cuscuta
campestris possesses the predominant pigments for photosynthesis (chlorophyll a and b, as well as a and β carotenes, and other accessory pigments) and low levels of PSII
activity.
Some previously published structural studies of Cuscuta chloroplastic structure indicate that the plastids have
underdeveloped structure, consisting chiefly of lipid bodies
and rudimentary thylakoids (Dodge and Lawes 1974,
Laudi et al. 1974, Machado and Zetsche 1990). However,
these studies were performed on plants that have parasitized a host and likely were from regions of tissue outside
of the photosynthetic cells identified by Hibberd et al.
(1998). Additionally, these studies were conducted at a
developmental stage marked by reduced need for endogenous carbon fixation, and thus not reflective of the ultimate
photosynthetic potential of the plant.
In this study, we took several approaches to investigate the photosynthetic capacity of Cuscuta pentagona, a
species that we have found to possess a marked green color
during certain developmental stages. First, we utilized seedlings grown on a nutrient medium under defined conditions as well as field grown plants in the parasitic mode
close to the time of flowering. It is at these stages that we
observed maximal accumulation of chlorophyll (and presumably chloroplast development). Second, we characterized the presence of enzymes involved in photosynthesis
both immunologically and cytochemically. Finally, whole
seedling measurements of carbon exchange rates were
conducted. This system of assays was used to examine the
structural and biochemical characteristics of these plastids.
Our data indicate that this species of Cuscuta has a complete assemblage of photosynthetic pigments and protein
complexes for both light and dark reactions, but that preparasitic and post-parasitic tissues differ in abundance of
photosynthetically-related proteins.
Materials and Methods
Plant material-Seeds of Cuscuta pentagona (F&J Seeds,
Urbana, IL, U.S.A.) were immersed in concentrated sulfuric acid
for 1 h at room temperature in a stainless steel strainer, neutralized in saturated sodium bicarbonate, and then rinsed in running
593
deionized water. The seeds were then allowed to dry briefly to
improve separation of the seeds and germinated on a nutrient
medium (Somerville and Ogren 1982) supplemented with 25 pg
m l l ampicillin (to suppress bacterial growth) in glass culture
dishes. Growth characteristics and protein profiles are unaffected
by this antibiotic treatment. Seedlings of dodder were grown for
up to 10 d in a non-parasitic mode on this medium under continuous light of lOOpmol m 2 s ' PAR at room temperature (2123°C) Samples of wild dodder were collected from a local population in Stoneville, MS, U.S.A. as green or orange colored stem
segments. These segments were either fixed directly in the field or
brought back to the laboratory where they were processed immediately for either biochemical or structural analysis.
P S I and PSII cytochemistry-Partial reactions of photosynthetic electron transport were visualized cytochemically as previously described (Vaughn and Outlaw 1983, Vaughn et al. 1983).
The oxidizing side of PSI was localized using diaminobenzidine
(DAB) at 1 mg m l l . Reaction at this concentration is specific for
PSI activity, preferentially staining the stroma lamellae and ends
of grana stacks. PSII was localized using the tetrazoliums: distyryl
nitro blue tetrazolium chloride (DSNBT) or thiocarbamyl nitro
blue tetrazolium (TCNBT) at 1 mg m l l . These compounds
receive electrons from PSII.
Immunocytochemistry-Small (-1 mm) stem pieces were
fixed in 3% glutaraldehyde in 0.05 M Piperazine-N,N-bis[2ethanesulfonic acid] (PIPES) buffer (pH 7.4) for 2 h at 4OC. The
specimens were then washed in 0.05 M PIPES buffer (pH 7.4) and
dehydrated in ethanol to 70% at 4OC. Subsequent dehydration to
100% ethanol was carried out after transferring the specimens to
a freezer (-20°C) The samples were infiltrated with L.R. White
resin (Polysciences, Warrington, PA, U.S.A., [soft formulation])
in 25% steps, for 2-4 h at each step and two overnight changes of
100% resin. Polymerization was carried out in BEEM@capsules
(Polysciences, Warrington, PA, U.S.A.) using resin to which 100
u\ of L.R. White catalyst was added. The specimens remained at
-20° for 8-10 h to ensure complete polymerization.
Sections were cut with a Reichert Ultracut E ultramicrotome
to a thickness corresponding to a pale gold reflectance color and
mounted on either uncoated 300 mesh gold or nickel grids. The
grids were then processed in the steps described previously for
un-osmicated specimens (Vaughn 1989). The primary antisera
were utilized at dilutions of 1 : 40 to 1 : 160. These dilutions were
determined empirically depending upon the titer of the antisera
preparation, the relative ability of the antisera to recognize the
polypeptides on Western blots, and the lack of background labeling over cell walls and intercellular spaces. These antisera were
from rabbits immunized with light-harvesting Chl a/b complex
proteins of PSII (LHCPII), the 33 kDa protein of the oxygenevolving complex (OEC) of PSII (Kawata and Cheung 1990),
PsaA/PsaB complex (originally described as the P7oo Chl a-binding protein) (Vierling and Alberte 1983), plastocyanin (Hauska et
al. 1983), the chloroplastic coupling factor 1 (Lax and Vaughn
1986, Radunz 1980), cytochrome f (Pettigrew and Vaughn 1998),
RuBisCO (Pettigrew and Vaughn 1998). RuBisCO activase (Salvucci 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
batchedembeddings) and samples (3 samples/tissue). Morphometric data were collected and analyzed as described by Lax and
Vaughn (Lax and Vaughn 1991).
Electrophoresis and imrnunoblotting-Fresh
tissue of preparasitic and parasitic dodder were homogenized with a chilled
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
5 ml buffer per gram fresh weight. Homogenate was filtered
through one layer of cheesecloth and large particles removed by
centrifugation for 5 min at 500Xg and 4OC. Plastids were collected at 6,000xg for 15 min, and then were disrupted in the
above buffer minus sorbitol plus proteinase inhibitors (1 mM
PMSF, lOyM pepstatin, 10,uM leupeptin, and 5 mM 1,10phenanthroline). Thylakoids were pelleted at 13,000 x g for 15
min. Stromal proteins from the supernatant were precipitated
with four volumes of acetone at -20°C Precipitated proteins
were recovered by centrifugation at 16,000 x g for 1 min. Thylakoids and stromal proteins were solubilized in 125 mM Tris (pH
6.8 at 4OC) plus protease inhibitors with brief sonication. Protein
concentration was assayed by the method of Bradford (1976) and
adjusted to approximately 1 mg m l with a concentrated buffer
stock to yield a final solution of 125 mM Tris (pH 6.8), 10% (v/v)
glycerol, 2% (w/v) lithium dodecyl sulfate, 1% (v/v) /?-mercaptoethanol, and 0.02% (w/v) bromophenol blue. Samples were
heated to 37OC for 30 min and centrifuged briefly to remove insoluble material. SDS-PAGE was performed according to the
method of Laemmli (1970) in a mini-gel format. Proteins were
transferred to BioTrace NT nitrocellulose (Gelman Sciences, Ann
Arbor, MI, U.S.A.) in 15% ethanol, 25 mM Tris, and 192 mM
glycine for 1 h at 1 mA c m 2 and 1 h at 2.5 mA c m 2 . Unsaturated sites on the membranes were blocked with 0.5% (w/v)
Blocking Reagent (Boehringer-Mannheim, Indianapolis, IN,
U.S.A.) in 10 mM Tris pH 7.5, 150 mM NaCI (TBS) for 1 h. Blots
were then exposed to primary antibody at a dilution of 1 : 1,000 to
1 : 4,000 in this blocking solution for 2 h; washed in TBS containing 0.05% (v/v) Tween-20 for 3 times for 10 rnin each. This
was followed by treatment with alkaline phosphatase-conjugated
goat anti-rabbit IgG (H+L) for 1 h, washed as above, and visualized with 330yg m l ' nitroblue tetrazolium and 165 yg m l ' 5bromo-4-chloro-3-indoyl phosphate in 100 mM Tris (pH 9.5 at
24OC), 100 mM NaCI, and 5 mM MgC12.
Net photosynthetic measurements-C02 exchange rates (CER)
were measured using a LI-COR 6200 portable photosynthesis unit
(LI-COR, Lincoln, NE, U.S.A.) with a 1.0 liter volume leaf
chamber. Dodder seedlings used for these measurements came
from the same culture dish. Samples of equal fresh weight (100
mg) were prepared by submerging a number of plants in either a
herbicide solution containing 10yM atrazine or a 0.1% ethanol
solution (control for the herbicide) for 0.5 h before the CER
reading. Immediately before CER measurements, the seedlings
were blotted dry. he tissue samples were complet~ly
anchored in
the chamber during the gas exchange measurements. The experiments were repeated on several different groups of seedlings on
several different sampling dates.
Spectroscopy-Shoot tips of 7-days old seedlings were immersed in 80% (v/v) acetone and placed in a sealed tube maintained in darkness at 4OC. When the shoot tissue was devoid of
pigment, the solvent was used to produce visible spectra (350-700
nm) with a Gilford Response spectrophotometer (Oberlin, OH,
U.S.A.). Leaves of Pelargonium hortorum were treated similarly
and used as an example of a typical higher plant pigment extract.
Results and Discussion
Structural studies-Chloroplasts of dodder are quite
unlike those of typical mature C3 dicots. First, the chloroplasts are much smaller, with lengths of about 2-3 p m
and widths of -1 ,urn, compared t o the 6-8 p m long chlo-
roplasts of the typical C3 dicot. Within the plastid, the
arrangement of thylakoids is also unique. Towards the tip
of the growing seedlings, virtually all of the chloroplasts
contain thylakoid doublets in which the thylakoids are appressed along their entire length (Fig. 1). There are essentially no areas where unappressed thylakoids are noted. In
the most elaborate thylakoid arrangement observed, a
stack of five thylakoids was detected, but again without
much in the way of unappressed lamellae. In many ways,
these chloroplasts are similar to what is observed in very
immature leaves of dicots (Pettigrew and Vaughn 1998) in
which the thylakoids exhibit a similar complete appression.
Evidence of plastid division in dodder (constriction of a
relatively large plastid) is noted frequently in areas of the
shoot where nuclear divisions are also noted.
Besides the unique arrangement of thylakoids, the
dodder chloroplasts often contain thylakoid-bound crystalline inclusions (Fig. 2). The crystal contains a regular
latticework of alternating electron opaque and translucent
material and is quite rectangular in outline. Favorable
sections reveal that a single, distended thylakoid membrane
surrounds the inclusion. Such inclusions are typical of
underdeveloped plastids in the epidermis, vascular tissue,
and roots but are relatively uncommon occurrences in
the chloroplasts of photosynthetic tissue. A n exception is
found in the chloroplasts of spinach, where a similar
membrane bound inclusion is noted. However, in a plastid
mutant of spinach with reduced thylakoid content, the
number of these inclusions is also increased (Vaughn et al.
1981). Phytoferritin aggregations are sometimes observed
in the stroma, and starch grains are observed in virtually
every plastid profile. Plastoglobuli are abundant and are of
varying electron opacity, even within a single chloroplast.
Ribosomes are found abundantly throughout the stroma
and are often associated with the tops of the small grana.
Clear areas of the stroma devoid of ribosomes, containing
threadlike inclusions, probably represent areas of chloroplast DNA accumulation.
The chloroplast envelope is invaginated, which, in thin
sections, appears as paler, inclusions in the stroma (Fig. 3).
Tubular structures about the diameter of microtubules are
associated with the inner envelope and extend into the
stroma. Frequently, these "chloroplast microtubules" occur in clusters. Although such structures have been observed in other chloroplasts (Vaughn and Wilson 1981),
their function is unknown.
Chloroplasts from material collected under field situations (in which there was noticeable green t o the tissues)
are structurally similar to those grown under laboratory
conditions, indicating that the super-stacked thylakoid
configuration is not due to growth of the plants under
laboratory conditions. In this greenish tissue all chloroplasts have similar morphology. In materials collected
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 micrograph 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 superstacking of thylakoids and show no immunologically detectable RuBisCO (data not shown). Surprisingly, when
dodder seedlings are germinated and grown in the dark, the
structure of the thylakoids is not too different from plants
grown in the light. The development of the thylakoids and
the size of the plastids appear to be less than those grown
under continuous light (Fig. 4). Prolamellar bodies, which
are prominent structures in etioplasts, are also found, albeit in a much reduced state in the etioplasts of dodder.
Because prolamellar bodies are the chloroplastic sites of
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
contain similar mechanisms in dealing with the etioplast/
chloroplast conversion to that found in other higher
plants.
Cytochemistry-Partial reactions of photosynthetic
electron transport may be visualized cytochemically, which
allows for determination of activity as well as presence of
a particular thylakoid component (Vaughn and Outlaw
1983, Vaughn et al. 1983). PSI partial reactions involve
the oxidation of DAB via donation of electrons into the
electron transport system close to that of plastocyanin
(Vaughn and Duke 1981, Vaughn et al. 1983). When dodder chloroplasts are exposed to DAB, a strong reaction is
noted in the thylakoids with more reaction towards the
edge of the thylakoid stacks than in the middle (Fig. SA,
B). These are consistent with the results of previous studies
that show a majority of PSI activity is present in unstacked
areas of lamellae (Vaughn et al. 1983). In contrast, incubation of shoot segments in the tetrazoliums, DSNBT and
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
thylakoid membranes (not shown). The latter reaction is
weak relative to PSII cytochemical localizations in other
higher plants (e.g., Vaughn et al. 1983), but this may be
due to the poor penetration of the cytochemical reagents or
the greater sensitivity of the PSII reactions to aldehyde
fixation. Only cells that are cut open react with the reagent,
indicating that poor penetration is the major obstacle in
PSII staining in dodder.
Immunocytochemistry-The light harvesting complex
of PSII and RuBisCO are the major proteins of the
thylakoids and stroma, respectively. Immunocytochemical
localizations with antibodies to these proteins reveal specific labeling of the thylakoids and stroma. Labeling of
RuBisCO is noted throughout the stroma (Fig. 6 ) . A relatively 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
obtained for the LHCPII. However, the level of the labeling with all of these sera is much less than is observed in
developed chloroplasts or even rather underdeveloped ones
of other higher plants (e.g., Pettigrew and Vaughn 1998).
None the less, the localization of these proteins in the
chloroplasts demonstrates that: (1) the uptake mechanisms
for chloroplast proteins encoded by nuclear DNA is operative, (2) signaling mechanisms that send each component
to the proper final site must be functioning, and (3) the
chloroplast ribosomes observed are making a functional
product (i.e. the large subunit of RuBisCO).
Immunochemical studies-Chloroplast-enriched fractions of pre-parasitic and parasitic dodder were analyzed
by a number of single and gradient gel formats with their
proteins subsequently blotted to a number of membrane
types. Proteins of LHCPII and enzymes of the carbon
fixation pathway are present in extracts of dodder and have
relative molecular masses consistent with literature values
for these polypeptides [RuBisCO, 52 kDa; OEC, 16 to 32
kDa; LHCPII, 26 kDa; and cytochrome f, 32 to 34 kDa
(Chia et al. 1986); glutamine synthetase, 46 to 50 kDa
(Hopfner et al. 1988); EPSP synthase, 48 kDa (Kishore
et al. 1988); phosphoribulokinase, 39.2 kDa (McKay and
Gibbs 1991); plastocyanin, 10.4 kDa (Ramshaw et al. 1974);
GroEL, 60 kDa (Vierling 1991)]. Protein extracts prepared
in a like manner from spinach were electrophoresed along
with those from dodder to allow for direct comparisons of
chloroplast protein sizes (Fig. 8). Mass values for proteins
from spinach extracts closely parallel those from the dodder extracts (Fig. 9, 10). It is not possible to make statements concerning relative abundance of these proteins
based on the intensity of immunoreactions, however, because the antibodies utilized were not prepared to dodder
antigens. The immunoblots are consistent with immunogold labeling experiments in revealing that photosynthetically-related proteins are present but not to the extent
found in a dicot such as spinach. Although comparisons of
Characterization of Cuscuta pentagona Chloroplast
599
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.
Fig. 6
protein abundance between dodder and spinach extracts
may be problematic due to species differences in the antigenic similarity of the polypeptides, changes in protein
abundance between pre- and post-parasitic dodder are evident from the blots that were loaded with equal amounts
of total protein. There appear to be developmental changes
in abundance of photosynthetically related proteins between pre- and post parasitic dodder. There is little effect
of parasitism on abundance of cytochrome f, LHCPII,
and OEC, but the carbon-fixing enzymes, RuBisCO and
phosphoribulokinase, are greatly reduced, as is the lumenlocalized plastocyanin (Fig. 9). This differential presence
extends to other chloroplast-localized, but non-photosynthetic proteins, with EPSP synthase and the chaperonin
GroEL showing reduced abundance in post-parasitic tissues (Fig. 10). Strangely, glutamine synthetase seems to be
present in greater abundance after parasitism and may
reflect changes in nitrogen metabolism associated with
the changed lifestyle. Immunostaining of the anti-PsaA/
PsaB complex is consistently weak, whereas the staining
for the two PSII related proteins are quite strong. This is in
contrast to the cytochemical studies that indicated little
PSII activity relative to that of PSI. These results are confusing. The discrepancy may result from poor recognition
of the PsaA/PsaB complex of dodder by the antiserum
used in this work (which was raised to monocot PsaA/
PsaB complex) or that the antiserum had lost activity over
time with storage. Alternatively, the limited number of PSI
complexes found in dodder are much more active in these
chloroplasts that have such small regions of unstacked
lamellae (where PSI activity is normally found (Vaughn et
al. 1983)). There are some notable proteins that were detected on immunoblots of spinach chloroplast membrane
and soluble proteins but not on those of dodder. These
include coupling factor I, RuBisCO activase, and PsaA/
PsaB complex. Antisera to these proteins produced weak
signals on spinach immunoblots and so it is possible that
these proteins are present in Cuscuta pentagona, but in
amounts that are below the detection limit with the
methodology utilized here.
Spectroscopy-Acetone extracts of dodder grown under laboratory conditions produced absorption spectra
qualitatively similar to those of geranium (Pelargonium
hortorum). Characteristic peaks for carotenoids (400 nm470 nm) and Chls (600 nm-665 nm) are seen in both extracts, although at lower abundance in the dodder tissue.
This is in agreement with a more in-depth pigment analysis
of these pigments in the related species, Cuscuta campestris
(Dinelli et al. 1993). Their study showed that field-grown
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,
antheroxanthin, and violaxanthin.
Net photosynthetic measurements-Although other
evidence presented here indicates that Cuscuta pentagona
seedlings contain the necessary components for a functional photosynthetic system, the gas exchange of these
plants is dominated by respiration. A net evolution of
C 0 2 from dodder under light conditions rather than a net
uptake of C 0 2 evidence this. The functionality of the
Cuscutapentagona carbon-fixing system was demonstrated
by comparing gas exchange measurements from atrazinetreated and untreated control dodder samples. The atrazine-treated dodder evolved C 0 2at approximately a 2-fold
greater rate than the untreated control (0.017 pmol C 0 2
sK1g FWK1and 0.009,umol C 0 2 sK1g FWK1,respectively.
Treatments are significantly different at the 0.01 probability level). Atrazine inhibited what little photosynthesis was
occurring in the dodder and thereby resulted in a net increase in C 0 2evolution from the treated sample. This is in
agreement with results found for mature tissues of Cuscuta
reflexa; C 0 2 evolution decreased with increasing light intensity but never rose above the C 0 2 compensation point
for any light intensity tested (Hibberd et al. 1998).
The data we present here are consistent with the
growth behavior of dodder seedlings, which grow vigorously for a week to ten days in the absence of a host, and
then senesce quickly thereafter. It is possible that the low
level of photosynthetic carbon fixation that was measured
here is simply a supplement to stored reserves that increase
the time in which the young seedling can locate and
parasitize a suitable host.
Collectively, along with other published reports, these
data indicate that there is likely to be a spectrum of the
chloroplast development among dodder species. Clearly,
of the dodder that have been investigated, Cuscuta
europaea appears to be the most adapted to a parasitic
lifestyle with a number of deletions of photosyntheticallyrelated genes and essentially no development of thylakoid
membranes (Freyer et al. 1995, Machado and Zetsche
1990). Developmentally, Cuscuta reflaa may be next in
Characterization of Cuscuta pentagona Chloroplast
A
B
C
Std.
-
-.?ae
.
b
..
EPSP
..
aro EL
Fig. 10 Immunoblots of various non-photosynthetic proteins
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
from tissues by differential centrifugation. These were lysed and
separated into thylakoid and stromal fractions by further centrifugation. Thylakoid fractions were used for these gels. Lanes were
normalized for equal total protein load. Samples were subjected
to SDS-PAGE (12.5% gel) and either stained for total protein
with Coomassie blue R-250 (right panel) or transferred to nitrocellulose and probed with antibodies to light-harvesting Chl a / b
complex of PSI1 (left panel).
line. A number of photosynthetically-related genes have
been identified in the genome of this species and lightstimulated carbon fixation was shown t o be present, but
only weak transcription of mRNA for RuBisCO and no
immunologically detectable protein for this enzyme could
be demonstrated (Haberhausen et al. 1992, Machado and
Zetsche 1990). Recently, however, Hibberd et al. (1998)
have shown that in internodal areas of mature tissue from
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.
this species, photosynthesis is localized t o a ring of cells
adjacent to the vascular tissue, between the pith and the
cortex. No ultrastructure of this tissue was presented in
that work, and so it is not possible to determine what
similarities these photosynthetic cells hold t o those of
Cuscutapentagona tissues that we have characterized here.
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 associated with floral development and those near haustorial
junctions) have chloroplasts with stacking of thylakoid
membranes, as well as cytochemical and immunological
evidence for active photosynthesis. Tissues that are orange
in color have limited thylakoid stacking and n o detectable
photosynthetic ability. In Cuscutapentagona, we have not
identified the ring of photosynthetic cells found in by
Hibberd et al. (1998) in Cuscuta reflexa. It is also of interest
A
B
C
A
B
C
-
PRK
-
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.
Characterization of Cuscuta pentagona Chloroplast
602
that the dodder pictured in the work of Hibberd et al.
(1998) is markedly greener than any of the Cuscuta pentagona tissue that we have found. It is quite likely that Cuscuta pentagona (a new world species) is physiologically
distinct from Cuscuta reflexa (an old world species) and
may have adapted differently to the parasitic lifestyle. Alternatively, the specialized anatomy found in Cuscuta
reflexa may be indicative of a greater degree of adaptation
to the parasitic habit than that of Cuscuta pentagona. It is
likely that further investigation into other members of the
genus Cuscuta may produce a spectrum of individuals with
varying degrees of adaptation to the parasitic lifestyle that
are intermediate to C. europaea and these other two species
(or perhaps even more extremely or less adapted than these
characterized species). As such, these intermediate species
would serve as useful tools for the study of chloroplast
function and chloroplast genome evolution.
W e would like t o extend thanks t o L y n n Libous-Bailey f o r
excellent technical assistance. Mention of a trademark, propriet a r y product, o r vendor does n o t constitute a guarantee or warranty o f t h e product by t h e United States Department of
Agriculture a n d does n o t imply its approval t o t h e exclusion o f
other products or vendors t h a t m a y also b e suitable.
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(Received August 10, 1998; Accepted April 3, 1999)