research paper
Research paper
Plant Signaling & Behavior 8:5, e24037; May 2013; © 2013 Landes Bioscience
Cuscuta europaea plastid apparatus in various
developmental stages
Localization of THF1 protein
Renáta Švubová,1,* Miroslav Ovečka,2,3 Andrej Pavlovič,1 Ľudmila Slováková1 and Alžbeta Blehová1
Department of Plant Physiology; Faculty of Natural Sciences; Comenius University in Bratislava; Bratislava, Slovak Republic; 2Institute of Botany; Slovak Academy of Sciences;
Bratislava, Slovak Republic; 3Centre of the Region Haná for Biotechnological and Agricultural Research; Faculty of Science; Palacký University; Olomouc, Czech Republic
Keywords: confocal microscopy, Cuscuta europaea, haustorium, plasma membrane fraction, plasmodesmata, plastid
Abbreviations: Chl, chlorophyll; FLP, flu-like protein; Fv/Fm, maximal quantum yield of PSII; GluTR, Glutamyl-tRNAreductase; LHCI, light-harvesting complex; RbcL, large rubisco subunit; THF1, thylakoid formation protein 1; ΦPSII, effective
photochemical quantum yields of PSII
It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of
chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder.
Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically
more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in
photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity
of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1)
is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling
with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial
plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane
and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte
primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein
in “get together” tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C.
europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions
of this protein in the complex relationship between C. europaea and its host.
Introduction
Parasitic plants have a specialized absorptive organ, called haustorium, which is used to penetrate through the host tissues and
connect to xylem or phloem. Cuscuta (dodder) is the only parasitic genus found in the morning-glory family Convolvulaceae.
The genus Cuscuta has been considered to be a holoparasitic
angiosperm, obtaining both water and organic nutrients from its
host plants. Some species like C. subinclusa D., C. gronovii Willd.,
C. campestris Yunck. possess thylakoids and contain both chlorophyll a and b especially in the growth tips of the seedlings.1-4
Plastids of C. grandiflora R. et al. and C. odorata H.B.K. do not
contain chlorophyll, the enzyme Rubisco and developed thylakoids.3 Using ultrastructural studies, Sherman et al.5 showed no
differences between the structure of C. pentagona thylakoids in
seedlings germinated and grown in the dark and seedlings grown
in the light. Plastids had poorly differentiated endomembrane
system with prolamellar bodies, which are prominent structures
of etioplasts. It has been shown that C. reflexa Roxb. possesses a
number of photosynthesis-related genes with significant homology to those found in higher plants.6,7 Immunoblots revealed
chloroplast proteins associated with PSI and PSII, as well as
cytochrome f and plastocyanin.5 Up to date literature provides
information that C. europaea is more specialized for the parasitic
lifestyle than other Cuscuta species, with no detectable chlorophylls, no capacity for CO2 assimilation and no thylakoids in
plastids.8 Large deletions of photosynthesis-related genes has
been documented as well.9,10
Several nucleus- and plastid-encoded Arabidopsis genes that
can ultimately affecting thylakoid formation or chloroplast development have been identified. One of many, the thf1 gene, was
found in various plant species.11-14 Based on sequence similarity
*Correspondence to: Renáta Švubová; Email: [email protected]
Submitted: 02/15/13; Accepted: 02/18/13
http://dx.doi.org/10.4161/psb.24037
Citation: Švubová R, Ovečka M, Pavlovič A, Slováková L, Blehová A. Cuscuta europaea plastid apparatus in various developmental stages: Localization of THF1
protein. Plant Signal Behav 2013; 8: e24037.
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Plant Signaling & Behavior
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1
Results
Plant material cultivation. Seeds of Cuscuta started to
germinate approximately 10 d after inoculation to the soil
around the base of 8-week-old tobacco plants, but germination was very asynchronous. After 20 d of cultivation,
dodder vines attacked their host plant. Cuscuta set up flowers approximately 90 d after the host plant-parasite connection establishment.
Figure 1. Concentration of Chl a, Chl b (A) and carotenoids (B) in C. europaea
Pigment analysis. Early developmental stages and
and N. benthamiana. (1) Cuscuta flowers, (2) 7-d-old Cuscuta seedlings, (3) 30-dflowers of C. europaea accumulated chlorophylls. Sevenold Cuscuta stems, (4) Nicotiana leaves, (5) Nicotiana stems. FM, fresh mass. Preday-old seedlings show the highest level of these photosented data are means (± standard errors) of three independent experiments.
synthetic pigments (Fig. 1A, column 2). We detected only
The small letters inside the graphs (a, b, c, d) denote significant difference at p
trace amounts of the Chls (a+b) in 30-d-old dodder stems
< 0.05 (Student’s t-test).
compared with young seedlings and flowers. The decrease
was statistically significant (Fig. 1A). Dodder flowers and
and different functions that were initially attributed to the pro- 30-d-old vines accumulate high amounts of carotenoids (Fig. 1B,
tein, various names of the same protein do exist—THF1,12,15 columns 1, 3). The lowest level of carotenoids was identified in
ToxA-binding protein16 or Psb29.17 Previously, it was suggested young dodder seedlings (Fig. 1B, column 2). The high levels of
that THF1 protein is involved in thylakoid membrane differ- Chls (a+b) and carotenoids were observed in tobacco leaves (Fig.
entiation,12 in responses to fungal and bacterial attacks18 and as 1A and B, column 4). Significantly lower content of Chls (a+b)
a G-protein (GPA1) interacting partner in sugar (D-glucose) and carotenoids were observed in tobacco stems (Fig. 1A and B,
signaling pathway.15 Huang et al.15 provided evidence that the column 5).
C-terminal 162 residues of THF1 interact with the constitutively
Chlorophyll a fluorescence. Maximum quantum yield of PSII
active form of GPA1 (GTP-bound form of GPA1). Zhang et al.14 (Fv/Fm), which is proportional to the quantum yield of O2 evoluproposed a model for G protein-mediated chloroplast development tion, was relatively high and did not differ significantly between
via FtsH proteases, which play a critical role during chloroplast Cuscuta stem and flower. Effective photochemical quantum
development in Arabidopsis.19,20 Immunofluorescence microscopy yield of PSII (ΦPSII), which measures the proportion of the light
revealed that THF1 localizes to the stroma (soluble THF1) and absorbed by chlorophylls associated with PSII, did not shown any
to the membranes of plastids in developing C. europaea haus- significant changes between Cuscuta stem and flower. However,
torium, most abundantly in the digitate cells of the endophyte the values of Fv/Fm and ΦPSII were lower in comparison to the host
primordium.21 Zhang et al.14 have postulated that the THF1- plant, as documented by the 2-D chlorophyll fluorescence imagmediated regulation of chloroplast development may be achieved ing system (Fig. 2).
via G protein-dependent and/or -independent pathways. In the
Plastid ultrastructure. Detailed ultrastructural study revealed
G protein-independent pathway, stroma- and/or thylakoid-local- differences in the plastid apparatus between young and older
ized THF1 controls chloroplast development through affect- developmental stages of the dodder stem. In growing tips of the
ing the stability of FtsH protease. In the G protein-dependent young dodder seedlings, chloroplasts with poorly differentiated
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pathway, the THF1 is at least in part, localized to the outer
membrane of plastid and to stromules,38,39 which appear
to associate with the plasma membrane. After the binding to GPA1, this activated form can transmit signals from
the plasma membrane to nuclei via second messengers and
ultimately control the expression of the FtsH genes needed
for chloroplast development.14
Our results proved that photosynthesis is not a main process in the plastids in C. europaea haustorium. Localization
of THF1 protein in plasma membrane and plasmodesmata
leading us to pronounce assumption, that this protein
has probably other functions, for example in sugar sensing within the dodder haustorial cells. Furthermore, result
from western blot showing high levels of THF1 in doder
and tobbaco tissues which are actively involved in the
development of parasitic connection. To clarify the precise
role of THF1 in host-parasitic interactions could be argued
and needs further investigations.
endomembrane system are shown (Fig. 3A). Chloro-amyloplasts
in the cortical cells of young dodder stems contained both thylakoids and large starch grains (Fig. 3B). Thirty-day-old stems
represented advanced ontogenesis stage and their cells contained
only amyloplasts with enlarged and sometimes numerous starch
grains (Fig. 3C and D).
Western blot analysis. The highest levels of THF1 protein
were detected in Cuscuta stems attached to the host, Cuscuta
haustorium and tobacco stems attacked by the parasite (Fig. 4A,
columns 2, 4 and 5). In dodder seedlings and tobacco stem unattacked by Cuscuta we observed notable decrease in THF1 protein accumulation (Fig. 4A, columns 1 and 3). Presence of RbcL
and D1 proteins were proved in dodder seedlings, tobacco leaves,
tobacco stems and stems attacked by dodder, although the levels
of these proteins were lower in dodder seedlings (Fig. 4A, columns 1, 3, 5 and 6). LHC complex polypeptides were observed in
all examined samples, the highest accumulation was in tobacco
tissues. FLP protein was detected only in tobacco. We found the
accumulation of the GluTR in dodder young seedlings and stems
attached to the host (Fig. 4A, columns 1 and 2), but it was not
detected in haustorium (Fig. 4A, column 4).
Using western blot analysis we detected the THF1 protein
accumulation in plastid membrane and stromal fractions isolated
Discussion
The current literature provides some information about in vitro
and in vivo cultivation of parasitic plants.4,22-26 It is generally
known, that before inoculation of dodder seeds on the cultivation
medium or in the soil, it is necessary to treat dodder seeds with
concentrated H2SO4. After this step, the germination is running
Figure 3. Ultrastructure of plastids in dodder stems. (A) Chloroplasts in growth tips, (B) chloro-amyloplasts in cortex of 7-d-old seedlings (C and D) amyloplasts in cortical cells of 30-d-old stem. Arrows denote thylakoids, stars denote starch grains. Electron microscopy. Bars: 200 nm, (A); 500 nm, (B–D).
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Figure 2. Chlorophyll a fluorescence in dodder (C. europaea, f-flowers,
c-stems) attached to the host (N. benthamiana, n-stems). Fv/Fm, maximum quantum yield of PSII; ΦPSII, effective photochemical quantum
yield of PSII.
from chloroplasts of 7-d-old seedlings (Fig. 4B, columns 2 and
3). In plasma membrane fractions originated from dodder haustorium, we confirmed lower levels of THF1 protein (Fig. 4B,
column 1).
THF1 protein in situ immulocalization. Microscopical
analysis and THF1 protein immulocalization were performed on
C. europaea stems attached to the host plant (N. benthamiana)
at the stage of dodder haustorium penetration into the phloem
area of the host plant (Fig. 5). Based on immunofluorescence
microscopy results we found high accumulation of THF1 protein
in endophytic tissue of the haustorium and in cortical cells of
the parasite. Tissues of the host plant accumulated low levels of
THF1 protein (Fig. 5B and C).
Subcellular localization by confocal microscopy revealed
plastid-related but also plastid-unrelated (extra-plastidial) distribution of the THF1 protein. In host tobacco plants the protein
was found mainly in the stroma of plastids in cells of the stem,
however in parasite cells, in addition of plastid-localized protein,
the plasma membrane expressed strong labeling (Fig. 6). In general, a higher accumulation of this protein was detected in dodder tissues. Chloro-amyloplasts with one starch grain in cortical
cells of the host stem, as well as amyloplasts with 2 to 3 small
starch grains in parasite cortex and haustorial cells were evenly
distributed at the cell periphery (Fig. 6). Amyloplasts in digitate haustorial cells comprised 4 to 5 large starch grains which
depressed THF1-positive stroma to margins and to the center of
plastids (Fig. 6G–I). In cortex and haustorial cells of the C. europaea stems the THF1 protein was distributed in stroma and outer
membrane of plastids and in the plasma membrane (Fig. 6G–I).
Especially in digitate haustorial cells of the dodder, the protein
was detected also in plasmodesmata (Fig. 6J-L).
faster and more effectively. Lee26 scarified seeds of C. japonica
Choisy with concentrated H2SO4 for 45 min and after washing
with distilled water, seeds were placed on moist paper. Seeds of
C. europaea were not able to germinate after this procedure and
thus, we shortened the time period for H2SO4 treatment to 15
min, which yielded effective germination.
The genus Cuscuta is considered to be a holoparasitic angiosperm, obtaining both water and organic nutrients from its host
plants. Previously, it has been shown that pre-parasitic seedlings
of C. campestris, C. subinclusa and C. gronovii contain chlorophylls, enzyme Rubisco and thylakoids, especially in their
growth tips.1,2 According to Haberhausen and Zetsche,6 C.
reflexa possesses a number of photosynthesis-related genes with
significant homology to those found in higher plants. Machado
and Zetsche8 reported a very low chlorophyll content for C.
reflexa {112 ± 24 [μg (g FW) -1]}. C. odorata and C. grandiflora
had an amoeboid-like shape of proplastid and did not contain
visible thylakoid structures. Western blotting revealed that LSU,
the product of rbcL gene, was not detected in protein extracts of
C. odorata and C. grandiflora.3 It has been stated that C. europaea appears to be the best adapted to a parasitic lifestyle with
a number of deletions in photosynthesis-related genes, without
development of thylakoid membranes and with no detectable
levels of chlorophylls.8-10,40 However, we found out that young
developmental stages of C. europaea accumulate relatively high
levels of chlorophyll a and b. Significantly lower content of chlorophylls and increase in carotenoids concentration were observed
only in 30-d-old dodder stems. These facts are supported by electron microscopy observations. Ultrastructural analysis has shown
presence of chloroplasts in growth tips and chloro-amyloplasts in
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Figure 4. (A) Western blot analysis of the THF1, GluTR, FLP, D1, LHCI
complex and RbcL in C. europaea: (1) 7-d-old seedlings, (2) 30-d-old Cuscuta stems attached to the host, (4) haustorium and in N. benthamiana,
(3) stems, (5) stems attacked by Cuscuta, and (6) leaves. Twenty-five μg
proteins have been loaded. (B) Western blot analysis of the THF1 protein
in C. europaea, (1) plasma membrane fraction isolated from dodder haustoria. Purity of fraction was tested using anti-LHCI antibody, (2) plastid
membrane fraction isolated from chloroplasts of 7-d-old dodder seedlings, (3) plastid soluble (stromal) fraction isolated from chloroplasts of
7-d-old dodder seedlings. Purity of fractions were tested using anti-D1
and anti-RbcL antibodies. Fifteen microgram proteins have been loaded.
cortical cells of young dodder stems, while no chloroplasts were
present in cells of older dodder stems. At points of contact with
the host, the coiled dodder stems produces haustoria that penetrate host tissues and form vascular connections.27 Haustorial
cells contained only amyloplasts with 4 to 5 starch grains. Our
results shown low photochemical activity of PSII (Fig. 2) as well
as low content of examined photosynthesis-related proteins (Fig.
4A, column 4). GluTR, the key regulatory enzyme in biosynthesis of 5-aminolevulinic acid, universal precursor of tetrapyrroles,
is tightly controlled at transcriptional and posttranslational levels.28 In young dodder seedlings (7-d-old) we observed relatively
high GluTR accumulation. These results are in correlation with
measuring of Chls (a+b) concentrations. FLP protein, which is
thought to bind to GluTR and represses its activity to prevent
overproduction of Pchlide,29 was not detected in dodder seedlings,
stems, haustoria. Photosystem I (PSI) is a membrane-bound multisubunit protein complex located in the chloroplast thylakoids.
It utilizes light energy to oxidize plastocyanin or cytochrome c
and to reduce ferredoxin or flavodoxin. In higher plants, green
and red algae, the outer light-harvesting system associated with
PSI is made up of LHCa proteins that are encoded by the cab
genes and known collectively as LHCI (light-harvesting complex I). Some studies have explored the structural basis by which
LHCI act as the outer light-harvesting system of PSI.30 LHCI
polypeptide complex was observed in all examined Cuscuta and
tobbaco samples, but the highest accumulation was detected in
tobacco. Accumulation of D1 protein, which represents a reaction center protein of photosystem II (PSII), was observed in 7-dold dodder seedlings and in tobacco (Fig. 4A) and never in older
dodder stems and in haustoria. Results of Zhang et al.14 strongly
suggest that THF1 and G proteins are the new regulators for
FtsH protease and THF1 probably functions as a regulator for
FtsH expression. The FtsH, a well-characterized family of membrane bound proteases, are required for damaged D1 protein
degradation, which is a core protein of the PSII reaction center.
The low activity of FtsH proteases leads to the accumulation of
damaged D1 protein, and to the inhibition of photosynthesis.
We can conclude that 7-d-old seedlings of C. europaea have some
characteristics of photosynthetic plants, while older plants have
lost this ability and are not photosynthetic.
Development of thylakoid membranes depends upon the
transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the
plastid.31 Several nuclear- and plastid-encoded Arabidopsis genes
have been identified as they can ultimately affect thylakoid formation or chloroplast development. One of them, the thf1 gene,
has been previously identified in various plant species. The
Arabidopsis thf1 gene product controls specifically an important
step required for leaf development, the normal organization of
vesicles into mature thylakoid stacks. Disruption of the thf1 gene
via T-DNA insertion results in variegated leaf patterns. Nongreen sectors of variegated leaves lacking thf1 expression contain plastids that accumulate membrane vesicles on the interior,
but lack organized thylakoid structures.12 It has been reported,
that THF1 protein localizes to the outer plastid membrane,
stroma, thylakoids and stromules.11-15,38,39 Our results have shown
presence of THF1 protein in both, parasite and host plant tissues
(Fig. 4A). Confocal microscopy revealed localization of THF1
protein to outer plastid membrane and stroma of haustorial cells
(Fig. 6G–L), what agree with previous observations of other
authors,12,15 but in addition to this, we observed localization of
this protein also in plasma membrane and plasmodesmata (Fig.
6J–L). The western blot of haustorial plasma membrane fractions
verified the presence of THF1 protein in the plasma membrane
(Fig. 4B, column 1). Huang et al.15 predicted that THF1 protein
plays a role as a G-protein (GPA1) interacting partner in sugar
(D-glucose) signaling pathway. This interaction requires proximity of the plastid or its stromule with the plasma membrane.
Stromules may also play a role in stress response.39 Wangdi et al.18
considered that THF1 have a role in COR signaling pathway in
bacterial speck disease development.
In conclusion, localization of THF1 protein in plasma membrane and plasmodesmata leading us to pronounce assumption,
that this protein has probably other functions, for example in
sugar sensing within the dodder haustorial cells. Furthermore,
result from western blot showing high levels of THF1 in doder
and tobacco tissues, which are actively involved in the development of parasitic connection. To clarify the precise role of THF1
in host-parasitic interactions could be argued and needs further
investigations.
Materials and Methods
Seeds sterilization and growth conditions. In vivo cultivated stems of parasitic dodder (Cuscuta europaea) and tobacco
(Nicotiana benthamiana) as a host plant were used in our study.
Seeds of C. europaea originate from the locality Ivanka pri Dunaji
(2009, Slovak Republic), the N. benthamiana seeds were obtained
from Gene Bank in Gatersleben, Germany. Dodder seeds were
scarified by soaking in concentrated H2SO4 for 15 min, washed
with distilled water and planted in soil around the base of
8-week-old tobacco plants. The plant material in soil (tobacco
plants with dodder) was cultivated in the growth chamber (16/8
h photoperiod) at 23 ± 2°C, 40 μmolm-2s-1 PAR.
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Protein isolation and western blot analysis. Samples (100
mg) were ground in liquid nitrogen and suspended in protein
extraction buffer [28 mM dithiotreitol, 175 mM sucrose, 28 mM
Na 2CO3, 10 mM EDTA, 5% (w/v) SDS, chemicals from SigmaAldrich] with antiprotease pill (Roche). After 30 min incubation
at 70°C and 15 min centrifugation (12,100 × g), supernatant was
used for determination of protein concentration using Bichinonic
Acid Kit for Protein Determination (Sigma-Aldrich). Protein
samples (25 μg) were separated on a 12% SDS-polyacrylamide
gel and transferred to nitrocellulose membrane (Millipore) using
Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (BioRad). For protein immunodetection, specific primary antibodies
were used. Antibodies against THF1, GluTR, FLP, D1, LHCI
and RbcL were purchased from Agrisera. Secondary antibody
Goat Anti-Rabbit IgG (H+L)-HRP Conjugate (Bio-Rad) was
used. Signal was revealed using chemiluminiscent kit Immobilon
Western (Millipore).
Preparation of plasma membrane, plastid membrane and
stromal fractions. The plasma membrane fraction from 6 g of
dodder haustorium (4 mo-long picking) was enriched by partitioning microsomes in an aqueous dextran/polyethylene glycol two-phase system. The purified plasma membrane fraction
was treated with 40 mM CHAPS {3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate} for 1 h at 4°C and centrifuged at 50.000 g for 30 min at 4°C.32 Purity of plasma membrane fraction was tested using anti-LHC antibody (Agrisera).
Plastids were isolated and fractionated as described previously
by Inaba et al.33 Essentially, chloroplasts were isolated from 10 g
of 7-d-old seedlings (2 mo-long picking). Isolated intact chloroplasts were separated into membrane and soluble fractions by lysis
in 0.1 M Na 2CO3, pH 11.5, followed by centrifugation at 200.000
g for 20 min. The pellets (membrane fraction) were directly dissolved in SDS-PAGE sample buffer. The soluble proteins were
recovered by precipitation with trichloroacetic acid and dissolved
into SDS-PAGE sample buffer. Purity of membrane fraction was
tested using anti-RbcL antibody and purity of soluble (stroma)
fraction was tested using anti-D1 antibody (Agrisera). Samples
(15 μg) were separated on a 12% SDS-polyacrylamide gel and
Plant Signaling & Behavior
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Figure 5. Light microscopy and Immunolocalization of THF1 protein in C. europaea and N. benthamiana. Stem segments of C. europaea and their host
were fixed and embedded in Steedman’s wax, 10 μm-thick cross sections were used for anti-THF1:IgG-FITC immunostaining. (A) C. europaea (c) and
their host N. benthamiana (n) interconnected by dodder haustorium (h). (B) Overall immunofluorescence localization of the THF1 protein in stems of
dodder and tobacco closely associated together. (C) Detailed view to vascular connection of the host and the parasite. Arrows denote host plant tissue, stars denote parasite cortical cells. Bars: 200 μm, (A and B); 100 μm, (C).
©2013 Landes Bioscience. Do not distribute.
Figure 6. For figure legend, see opposite page.
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transferred to nitrocellulose membrane using Trans-Blot® SD
Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). THF1 protein was detected by western blot analysis using rabbit polyclonal
anti-THF1 antibody (Agrisera). Secondary antibody Goat AntiRabbit IgG (H+L)-HRP Conjugate (Bio-Rad) was used. Signal
was revealed using chemiluminiscent kit Immobilon Western
(Millipore). Protein concentrations were determined according to
Bichinonic Acid Kit for Protein Determination (Sigma-Aldrich).
Pigment analysis. Chlorophyll (a+b) and carotenoids were
extracted with 80% (v/v) chilled acetone and spectrophotometrically (Jenway 6400) quantified: Chl a at 663.2 nm, Chl b at
646.8 nm, carotenoids at 470 nm. Concentration was calculated
according to Lichtenthaler.34
Measurements of chlorophyll a fluorescence. Prior to measurements, the parasitic plants with their hosts were dark-adapted
for 30 min. Chlorophyll a fluorescence was measured by fluorcam
FC1000-LC (Photon Systems Instruments). Minimal fluorescence (F0, < 0.1 μmolm-2s-1 PAR) and maximal fluorescence (Fm)
were measured using a saturation pulse (4.000 μmolm-2s-1 PAR,
800-ms duration, λ = 620 nm) and maximal quantum yield of
PSII (Fv/Fm) was calculated as (Fm - F0)/Fm.35,36 Then an actinic
light of 100 μmolm-2s-1 PAR (λ = 620 nm) was switched on for
induction of photosynthesis and steady-state fluorescence was
measured (Ft). Saturation pulses were applied in 60 sec intervals,
for estimation of maximum chlorophyll fluorescence in the lightadapted state (F’m). Effective photochemical quantum yields of
photosystem II (ΦPSII) were calculated according to Rohácek 35
and to Maxwell and Johnson36 as (F’m - Ft)/F’m.
THF1 protein in situ immunolocalization. Steedman’s wax
embedding for THF1 visualization in dodder plants attached to
host was performed according to Vitha et al.37 with some modifications: sections (10 μm-thick) were incubated for 1 h at room
temperature in the primary antibody (1:200 dilution in PBS buffer: 0.14 M NaCl, 2.7 mM KCl, 6.5 mM Na 2HPO4, 1.5 mM
KH2PO4, pH 7.3, chemicals from Sigma-Aldrich), washed 2 ×
10 min in stabilizing buffer (SB buffer: 50 mM PIPES, 5 mM
MgSO4, 5 mM EGTA, pH 6.9, chemicals from Sigma-Aldrich)
References
1. Panda MM, Choudhury NK. Effect of irradiance
and nutrients on chlorophyll and carotenoid content and Hill reaction activity in Cuscuta reflexa.
Photosynthetica 1992; 26:585-92.
2. Dawson JH, Musselman LJ, Wolswinkel P, Dörr I.
Biology and control of Cuscuta. Rev Weed Sci 1994;
6:265-317.
3. van der Kooij TAW, Krause K, Dörr I, Krupinska
K. Molecular, functional and ultrastructural characterisation of plastids from six species of the parasitic
flowering plant genus Cuscuta. Planta 2000; 210:7017; PMID:10805440; http://dx.doi.org/10.1007/
s004250050670.
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and incubated overnight at RT, in the dark in secondary antibody
(1:100 dilution in PBS). Antibody against THF1 protein was
purchased from Agrisera. Secondary antibody Goat Anti-Rabbit
IgG (whole molecule) F(ab’)fragment-FITC was purchased from
Sigma-Aldrich. Sections were examined under the fluorescence
microscope (Axioskop 2 plus, Zeiss), equipped with 485/20 nm
exciter filter, 510 nm beamsplitter and 515 nm LP barrier filter
(filter Zeiss set 16, 25) and using confocal microscope (CLSM
Fluoview FV1000, Olympus) with the excitation laser line 488
nm and BA505-550 barrier emission filter.
Electron microscopy. Samples were fixed in 5% (v/v) glutaraldehyde in 0.06% (w/v) cacodylate buffer (pH 6.8) for 4 h,
washed with 0.06% (w/v) cacodylate buffer (pH 6.8) 6 × 10 min
and postfixed in 1% (v/v) osmium tetroxide in the same buffer
overnight. Fixed specimens were dehydrated in ethanol and propyleneoxide series and embedded in Spurr medium (BIOTECH).
Ultrathin sections were cut on ultramicrotome Reichert-Jung
Ultracut E and observed with JEOL 2000 FX electron microscope (Jeol Ltd.).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We would like to extend thanks to MSc. Katarína Breznenová
from the Institute of Experimental Botany AS CR, Dr. Boris
Breznen for critical reading of this manuscript, MSc Ivan
Kulich from the Department of Experimental Biology, Charles
University in Prague, CR, MSc Roman Pleskot from Institute
of Experimental Botany AS CR and Jarmila Šramková for excellent technical assistance with electron microscopy. This study
was supported by the Slovak Grant Agency APVV-0140-10 and
SPP Grant Foundation 2011 and by structural research grants
from EU and the Czech Republic to the Centre of the Region
Haná for Biotechnological and Agricultural Research, Faculty
of Science, Palacký University, Olomouc, Czech Republic (grant
no. ED0007/01/01).
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e24037-7
©2013 Landes Bioscience. Do not distribute.
Figure 6. (See previous page.) THF1 protein immunofluorescence localization by confocal microscopy in N. benthamiana (A–C) and C. europaea
(D–L) cells. (A–C) Cortical cells of N. benthamiana stem. (D–F) Cortical cells of dodder at the zone of haustorium penetration into the host plant. (G–L)
Digitate cells of the endophyte primordium. Arrows denote chloro-amyloplasts in cortex cells of N. benthamiana (A–C) and plasmodesmata in digitate
haustorial cells of the endophyte primordium (J–L). Stars denote starch grains in amyloplasts in digitate haustorial cells of the endophyte primordium
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