MFP1 Affects Relative Abundance of Chloroplast Protein Complexes
Amanda R. Havighorst1 and Annkatrin Rose2*
1South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208. 2Department of Biology, Appalachian State University, Boone, NC 28608. *Corresponding author.
Eastern Biologist, No. 6 (2018)
Abstract
Matrix Attachment Region-Binding Filament-Like Protein 1 (MFP1) is an integral thylakoid membrane protein conserved in land plant chloroplasts. Here we investigate its function in Arabidopsis thaliana on cellular, molecular and physiological levels. Immuno-detection shows its presence as a dimer and in higher molecular weight complexes of chloroplasts. Blue native polyacrylamide gel electrophoresis indicates that the relative abundance of chloroplast complexes is altered in MFP1 knock-out mutants. These results demonstrate that while MFP1 does not appear to contribute to the stacking of thylakoid membranes or affect photosynthetic efficiency under standard growth conditions, it associates with and affects higher molecular mass protein complexes in the chloroplast. This suggests a role of MFP1 in the assembly or disassembly of these complexes during thylakoid remodeling.
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2018 No. 6
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2018 EASTERN BIOLOGIST 6:1–21
MFP1 Affects Relative Abundance of
Chloroplast Protein Complexes
Amanda R. Havighorst1 and Annkatrin Rose2*
Astract – Matrix Attachment Region-Binding Filament-Like Protein 1 (MFP1) is an
integral thylakoid membrane protein conserved in land plant chloroplasts. Here we
investigate its function in Arabidopsis thaliana on cellular, molecular and physiological
levels. Immuno-detection shows its presence as a dimer and in higher molecular weight
complexes of chloroplasts. Blue native polyacrylamide gel electrophoresis indicates that
the relative abundance of chloroplast complexes is altered in MFP1 knock-out mutants.
These results demonstrate that while MFP1 does not appear to contribute to the stacking
of thylakoid membranes or affect photosynthetic efficiency under standard growth conditions,
it associates with and affects higher molecular mass protein complexes in the chloroplast.
This suggests a role of MFP1 in the assembly or disassembly of these complexes
during thylakoid remodeling.
Introduction
Matrix attachment region-binding filament-like protein 1 (MFP1) is a long
coiled-coil protein found in the thylakoid membranes and nucleoids of plant
chloroplasts (Jeong et al. 2003). It was originally isolated due to its ability to
bind DNA and later confirmed to have strong non-specific DNA binding activity
in Arabidopsis thaliana chloroplasts (Meier et al. 1996, Jeong et al. 2003).
Structurally, MFP1 resembles long coiled-coil proteins similar to intermediate
filaments, such as the nuclear lamins, and membrane-associated proteins, such
as golgins (Jeong et al. 2003). Proteins with extended coiled-coil domains are
largely absent in prokaryotes, thus making MFP1 an intriguing candidate to study
the role of a eukaryotic-type protein in an organelle derived from a prokaryotic
endosymbiont (Rose et al. 2005). However, its function has remained largely
enigmatic as despite its evolutionary conservation in plants, knock-out mutants
lacking MFP1 protein show no visible phenotype under standard growth conditions
(Jeong et al. 2003).
MFP1 belongs to the growing list of nuclear encoded proteins with dual targeting
mechanisms for the nucleus as well as plastids. It was originally predicted to be
associated with the nuclear envelope, but was later found to contain a convincing
nuclear localization signal in its C-terminus and a functional N-terminal chloroplast
targeting peptide and thylakoid localization signal (Gindullis and Meier 1999,
1South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208.
2Department of Biology, Appalachian State University, Boone, NC 28608.
*Corresponding author – rosea@appstate.edu.
Manuscript Editor: Tim Lindblom
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Jeong et al. 2003). Plastid proteomics studies have identified MFP1 in thylakoid
and nucleoid-enriched fractions (Friso et al. 2004, Majeran et al. 2012, Peltier et al.
2004), and immunolocalization experiments with antisera against MFP1 confirmed
its presence in both chloroplasts and the nuclear matrix (Samaniego et al. 2006a,
Samaniego et al. 2008). Most proteins with a dual plastid/nuclear localization are
involved in gene expression and associated with plastid DNA or RNA (Krause et
al. 2012). In support of this hypothesis, MFP1 can be detected in transcriptionally
active chromosome (TAC) fractions (Melonek et al. 2012). The association of
MFP1 with DNA appears to occur without detectable sequence specificity and can
be inhibited by phosphorylation (Jeong et al. 2004, Samaniego et al. 2006b). It has
been suggested that MFP1 may play a role in anchoring the chloroplast DNA to
the thylakoid membrane in mature chloroplasts, and the quantitative distribution of
the protein observed in proteomics studies appears consistent with such a function
(Majeran et al. 2012). However, no difference could be seen in the pattern of nucleoids
associated with thylakoids between wild-type and MFP1 knock-out mutant
plants (Jeong et al. 2003).
In addition to its DNA binding activity residing in the C-terminal quarter, the
defining feature of MFP1 is its coiled-coil domain spanning over 80% of the protein
sequence and suggesting a long rod-like domain capable of forming filaments
(Meier et al. 1996). Long coiled-coil domains play important roles in the organization
of eukaryotic cells, including formation of cytoskeletal networks, structural
maintenance of chromosomes and organization of the nuclear lamina, and organization
and fusion of membranes (Rose and Meier 2004). The coiled-coil domain
in MFP1 is preceded by a thylakoid targeting signal and transmembrane domain,
which functions to anchor the protein in the thylakoid membrane in chloroplasts
with the coiled-coil domain on the stroma side (Jeong et al. 2003). This topology of
MFP1 in the thylakoid membranes resembles that of golgins at the Golgi apparatus,
suggesting a possible role in membrane organization. The expression of MFP1
appears to be tightly correlated with the presence of thylakoid membranes. Gene
expression and proteomics studies show that MFP1 begins to accumulate during
the period of chloroplast development, while it drops off again with the transition
from chloroplasts to chromoplasts (Barsan et al. 2012, Jeong et al. 2003). Likewise,
MFP1 expression is highest when light is present and in the shoots of the plant,
where photosynthesis occurs; it is lowest in the dark and in non-photosynthetic
tissues (Jeong et al. 2003).
One defining characteristic of the thylakoids of vascular plants is the formation
of grana stacks during chloroplast development. These have been proposed to be
an adaptation to growing on land, particularly in shaded environments and under
fluctuating light conditions, by optimizing light capture and photosynthetic capacity
(Chow et al. 2005, Mullineaux 2005). The grana stacks are highly dynamic
with stacks forming during the first few hours of the light period, but largely
disintegrating again during the dark period (Pfeiffer and Krupinska 2005). The
mechanism of the formation of grana stacks is not yet well understood and has
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been suggested to involve physicochemical forces and increased entropy in the surrounding
stroma leading to spontaneous stacking in vitro (Chow et al. 2005, Kim
et al. 2005). The stacking appears to be further controlled by electrostatic forces
and the phosphorylation of photosystem II (Fristedt et al. 2010). The stacking process
may also involve specific proteins. For example, it appears that trimeric light
harvesting complex (LHC) II is needed for grana stacks to form properly (Cui et
al. 2011). In the Golgi bodies, an organelle which represents a similarly stacked
dynamic membrane structure, the cisternae are held together by golgins, which are
long coiled-coil proteins forming proteinaceous bridges between the membranes
(Barr and Short 2003, Short et al. 2005). Most of the MFP1 produced by the cell is
located in the thylakoid membrane of the chloroplast with the coiled-coil domain on
the stroma side (Jeong et al. 2003). This topology would seem optimal for a protein
with a role in thylakoid stacking and led us to hypothesize that the protein may have
a function in membrane stacking similar to that of the golgins.
The objective of this study was to trace the evolutionary history of MFP1 and
elucidate its function in the chloroplast. To investigate whether MFP1 is involved
in thylakoid membrane stacking, A. thaliana wild-type and MFP1 knock-out
mutants were observed using transmission electron microscopy to directly view
the thylakoid membranes. To test whether MFP1 is involved in protein-protein
interactions, plants were then examined on the molecular scale using Blue-Native
Polyacrylamide Gel Electrophoresis (BN-PAGE) to detect any differences in the
presence and abundance of chloroplast protein complexes between wild-type and
mutant plants and determine whether MFP1 associates with any such complexes.
Finally, photosynthesis and chlorophyll measurements were used to test whether
lack of MFP1 affected photosynthesis rates.
Methods
Sequence analysis
Sequence similarity searches were performed using the blastp and tblastn algorithms
and databases available at NCBI (http://www.ncbi.nlm.nih.gov/). For tblastn
searches with protein sequence on nucleotide databases (WGS, whole-genome shotgun
contigs; EST, expressed sequence tags; TSA, transcriptome shotgun assembly)
to identify putative distant homologs, the scoring parameters were adjusted by
changing the matrix to BLOSUM45 and searches limited to the organisms of interest.
Pairwise alignments to verify sequence similarity were performed using the
Smith-Waterman algorithm (http://www.ebi.ac.uk/Tools/psa/emboss_water/, Smith
and Waterman 1981).
Nucleotide sequences retrieved in searches were translated into amino acid
sequences for protein domain analysis using the ExPASy Translate tool (http://
web.expasy.org/translate/). Domain and targeting predictions were performed
using ChloroP for chloroplast targeting peptides (http://www.cbs.dtu.dk/services/
ChloroP/, Emanuelsson et al. 1999), TargetP for chloroplast, mitochondrial and
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secretory pathway targeting in plants (http://www.cbs.dtu.dk/services/TargetP/,
Emanuelsson et al. 2007), PredAlgo for chloroplast and mitochondrial targeting
in algae (http://giavap-genomes.ibpc.fr/predalgo/, Tardif et al. 2012), TatP for
twin-arginine signal peptides (http://www.cbs.dtu.dk/services/TatP/, Bendtsen
et al. 2005), PSORT for general targeting predictions (http://psort.hgc.jp/, Nakai
and Horton 1999), TMHMM for transmembrane domains (http://www.cbs.dtu.
dk/services/TMHMM/, Sonnhammer et al. 1998), and MultiCoil for coiled-coil
domains (http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi, Wolf et al.
1997). Predicted targeting peptides were removed using cleavage site predictions
before proceeding with the prediction of other domains in the protein sequences.
Plants and growth conditions
Plants used in this study included Arabidopsis thaliana (L.) Heynh. (Thale
Cress) wild-type (WT) plants of Wassilewskija (Ws-2) ecotype and a previously
described MFP1 knock-out mutant (KO) in Ws-2 background, which has a T-DNA
insertion in the third intron and does not express MFP1 (Jeong et al. 2003). Seeds
were planted on peat pellets (Jiffy brand, Plantation Products, Norton, MA, USA)
or in MetroMix 360 (SunGro Horticulture, Agawam, MA, USA) and cold-stratified
at 4°C for several days. Plants were grown under long-day (16 hours light at
23°C, 8 hours dark at 21°C) or short-day (8 hours light at 23°C, 16 hours dark at
21°C) conditions with an average light intensity of 200 μmol m-2 s-1 in a Percival
Environmental Chamber E-30B (Percival Scientific Inc., Boone, IA , USA).
DNA extraction and PCR analysis of genotype
To verify genotypes, DNA was extracted from leaves and analyzed by PCR.
Tissue samples were ground using a pellet pestle in a 1.5 mL centrifuge tube
in 500 μL extraction buffer (0.2 M Tris-HCl, pH 9, 0.4 M LiCl, 25 mM EDTA,
1% SDS). After centrifuging for 5 minutes at 16,100 xg in a microcentrifuge
(Eppendorf Centrifuge 5415 D, Eppendorf International, Hamburg, Germany), the
supernatant was spun again and 400 μL of the cleared lysate was mixed with 400 μL
of isopropanol to precipitate the DNA. After 10 minutes of centrifugation, the pellet
was dried and resuspended in 100 μL of TE buffer.
PCR was performed using 2 μL of DNA extract and Taq polymerase in a standard
50 μL PCR reaction using the primers MFP1-FP (5′-GGG CTT CTG TGT TCG
ATG AAT GTC G-3′), MFP1-RP (5′-TTC TTA TGA GTT CTT CCT TCT GCT GTT
TG-3′), and JL-202 (5′-CAT TTT ATA ATA ACG CTG CGG ACA TCT AC-3′).
The PCR program consisted of an initial melt at 94°C for 5 minutes, followed by
30 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 2 minutes at 72°C, with
a final extension step at 72°C for 5 minutes (using a GeneAmp PCR system 9700
thermocycler, Applied Biosystems, Carlsbad, CA, USA). For each DNA sample,
two reactions were run, MFP1-FP + MFP-RP to amplify the wild-type allele, and
JL-202 + MFP1-RP to amplify the T-DNA insertion allele, and analyzed on 1%
agarose gels stained with ethidium bromide.
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Transmission electron microscopy
Leaf samples from four-week-old plants were fixed in a 2.5% solution of
glutaraldehyde (SPI Supplies, West Chester, PA, USA) in 1 mM sodium phosphate
buffer (pH 6.7) for two weeks to ensure proper tissue infiltration. After fixation, the
samples were washed twice for ten minutes with sodium phosphate buffer, stained
for two hours using 1% osmium tetroxide (Ted Pella, Redding, CA, USA) in sodium
phosphate buffer, washed twice for ten minutes and dehydrated in an ethanol gradient
(successively, 50% ethanol for 2 hours, followed by 70%, 85%, and 90% ethanol for
1 hour and 15 minutes each, then in 100% ethanol overnight). Samples were removed
from ethanol and soaked twice for seven minutes in propylene oxide (Electron
Microscopy Sciences, Hatfield, PA, USA), and for one hour in a 1:1 mixture of propylene
oxide and Spurr’s Low-Viscosity Resin (SPI Supplies). After allowing the
propylene oxide to evaporate overnight, the old resin was removed and new resin
added before embedding the leaf tissue in flat embedding molds (BEEM brand, Ted
Pella) at 70°C for 14–15 hours. Once hardened, the resin-embedded samples were
cut using a glass knife on a microtome (Ultracut E Ultramicrotome, Reichert-Jung,
Depew, NY, USA) into 99 nm sections and placed on 3 mm copper grids. Samples
were stained in a 1% uranyl acetate (Electron Microscopy Sciences) solution for five
minutes, rinsed 10x in distilled water, stained for five minutes using lead citrate prepared
fresh according to the protocol by Reynold (1963), and rinsed 10x in CO2-free
distilled water in a container with pellets of sodium hydroxide to prevent the lead
from precipitating. After drying the samples, they were examined under a JEM-1400
transmission electron microscope (JEOL Ltd., Peabody, MA, USA).
Chloroplast isolation and protein extraction
Chloroplast isolation was performed essentially as described previously (Kügler
et al. 1997) starting with 2.5 g leaf tissue collected from 4-week-old plants. The
chloroplasts were purified by gradient centrifugation using Percoll (Research
Organics, Cleveland, OH, USA) and processed at 4°C for protein extraction immediately
after isolation. Proteins were extracted from chloroplasts and BN-PAGE
analysis performed according to a protocol modified from Kikuchi et al. (2011).
The chloroplast pellet was resuspended in 160 μL solubilization buffer (50 mM
Bis-Tris-HCl, pH 7; 0.5 M Aminocaproid Acid; 10% w/v Glycerol; 1% w/v watersoluble
Digitonin (EMD Millipore, Darmstadt, Germany); 1% (v/v) protease
inhibitor cocktail for plant extracts (Amresco, Solon, OH, USA)) and incubated
for ten minutes. The solution was centrifuged for ten minutes at 16,100 xg and the
supernatant transferred to new tubes in 80 μL aliquots. Each aliquot received 2 μL
CBB-G solution (5% w/v Coomassie Brilliant Blue G-250, 50 mM Bis-Tris-HCl
pH 7.0, 0.5 M Aminocaproic Acid) and was stored overnight at -20°C.
Blue native polyacrylamide gel electrophoresis and immunoblotting
Proteins were analyzed on a BN-PAGE mini gel with a gradient of 4% to 14%
polyacrylamide, with a 3% stacking gel essentially as described previously (Kikuchi
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et al. 2011). The gel was poured using a gradient mixer (GM-100, C.B.S. Scientific
Inc., Del Mar, CA, USA) and a peristaltic pump (Pump 60 RPM, VWR Int., Radnor,
PA, USA) into a Bio-Rad 1.5 mm spacer gel plate for the Mini PROTEAN 3 system
(Bio-Rad Laboratories, Inc., Hercules, CA, USA), overlaid with 100% isopropanol
and allowed to polymerize before pouring the stacking gel. Gels were run at 4°C
in a Mini-PROTEAN Tetra electrophoresis cell (Bio-Rad Laboratories) at 30 V for
30 minutes, and then raised to 80 V for 6 hours.
Immediately after the run, the BN gel was photographed and lanes for 2-D
analysis were then excised and denatured before being fitted into a preparative
well in SDS-PAGE gels (1.5 mm, 12% running gel, 4% stacking gel). Any gaps
were sealed using a low-melting agarose sealing buffer (0.5% w/v agarose, 25 mM
Tris, 192 mM glycine, 0.1% w/v SDS). SDS-PAGE in Tris-glycine buffer and
immunoblotting
were performed essentially as described (Sambrook et al. 1989)
using the Mini-PROTEAN electrophoresis and Trans-Blot system. Proteins were
blotted onto PVDF membrane (Pall Life Sciences, Port Washington, NY, USA)
and detected using a 1:5,000 dilution of MFP1 antibody OSU91 (Jeong et al.
2003) and a 1:10,000 dilution of Anti-Rabbit IgG (Sigma-Aldrich Co., St. Louis,
MO, USA) followed by ECL detection on X-Ray film (Blue Ultra Autorad Film,
GeneMate, BioExpress, Kaysville, UT, USA) for 15–20 minutes. The exposed
film was developed using an X-Ray developer machine (SRX-101A, Konica
Minolta, Ramsey, NJ, USA).
Photosynthesis measurements and chlorophyll count
For photosynthesis measurements, two-week old plants grown in MetroMix 360
were transferred to Ray Leach Cone-tainers (model SC-7, Stuewe and Sons Inc.,
Tangent, OR, USA) and the soil surface sealed with white soft modeling clay (Sculpey
Pluffy brand, Polyform Products Company, Elk Grove Village, IL, USA). Plants
were allowed to rest for at least 12 hours before being fitted into the Whole Plant
Arabidopsis Chamber for measurements using the RGB light source and LI-6400XT
Portable Photosynthesis System (LI-COR, Lincoln, NE, USA). Conditions were
set to match those in the growth chamber ([CO2] = 400 ppm, RH = 60–70%,
temperature = 23°C), and photosynthesis-irradiance curves were recorded based on
CO2 uptake per time and leaf area as photosynthetic photon flux density (PPFD) was
decreased stepwise (1000, 750, 500, 300, 150, 100, 75, 50, 25, 0 μmol m-2 s-1).
Leaf area was determined using a CanoScan 9000F Mark II scanner (Canon,
Melville, NY, USA) and the Black Spot Leaf Area Calculator (http://www.ncbs.res.
in/blackspot.html, Varma and Osuri 2013).
To determine chlorophyll content on leaf area basis, all leaves measured from
each plant were incubated in 3 mL N,N-Dimethylformamide (DMF) for ten days
in the dark at 4°C to extract the pigments. Samples were diluted 1:10 in DMF and
absorption at 470 nm, 647 nm, and 664 nm measured on a Genesys 20 spectrophotometer
after setting the blank at 720 nm (Thermo Scientific, Waltham, MA, USA).
Total pigment amounts were calculated as described previously (Porra et al. 1989).
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Subsequent statistical analysis was done in SigmaPlot 12.5 (Systat Software,
San Jose, CA, USA) using a two-tailed T-test to calculate significance values.
Results
MFP1 is conserved and expressed in embryophytes
MFP1 has been described as conserved among higher plants (Harder et al.
2000), and homologs of A. thaliana MFP1 can be identified with high confidence
in a wide range of angiosperms through standard sequence similarity searches
(Table 1). Recent advances in genome sequencing have opened up the opportunity
to expand the search for homologs beyond the flowering plants. MFP1-like
sequences can be found in the expressed sequence tags (EST) database in eight
gymnosperm species representing all phyla (several conifers, Ginkgo biloba L.,
Cycas rumphii Miq., and Welwitschia mirabilis Hook. f.), as well as seedless
plants including ferns (Adiantum capillus-veneris L. gametophyte), the lycophyte
Selaginella moellendorffii Hieron., and the bryophyte Physcomitrella patens
(Hedwig) Bruch and Schimper. The presence of genes coding for previously
unknown MFP1 homologs in non-flowering plants was verified using a tblastn
search with the basal angiosperm Amborella trichopoda Baill. sequence on wholegenome
shotgun contigs (WGS database). This search confirmed with high confidence
DNA sequences coding for full length MFP1 homologs in several recently
sequenced conifer genomes, a putative homolog in the seedless vascular plant S.
moellendorffii, and suggested the presence of a gene coding for an MFP1-like
Table 1. Organisms with coding sequences for MFP1-like proteins identified in BLAST
sequence searches and included in the domain analysis in Figure 1.
Organism Sequences Identity Query coverage E-value Search*
Eudicots Multiple (28) 38-100% 79-100% < 10-100 A
Amborella XP_006844348.1 37% 94% 3e-106 A
Monocots Multiple (10) 33-37% 73-100% < 10-70 A
Conifers Multiple (3) 34-35% 78-94% < 10-65 B
Selaginella ADFJ01000391.1 25% 67% 2e-12 B
Physcomitrella ABEU01011440.1 25%
31%
25%
37%
6e-05
2e-14
B
C
Klebsormidium BANV01000989.1 44% 23% 0.001 D
*Searches included: (A) blastp query on nr database using NP_188221.2 (Arabidopsis);
(B) tblastn query on wgs database (excluding flowering plants) using XP_006844348.1
(Amborella); (C) tblastn query on WGS database (excluding flowering plants) using translated
peptide from a predicted full-length sequence based on ADFJ01000391.1 and GENSCAN
prediction (Selaginella, see Fig. S1; see supplemental File 1, available online at https://
eaglehill.us/ebioonline/suppl-files/ebio-006-rose-s1.pdf); (D) tblastn query on wgs database
(excluding flowering plants) using translated peptide from a predicted full-length sequence
based on ABEU01011440.1 and GENSCAN prediction (Physcomitrella, see Fig. S2).
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protein in the genome of P. patens (Table 1). All sequence similarities were supported
by reverse searches.
To confirm sequence similarities and potentially identify more distantly
related sequences, EST data and GENSCAN gene predictions were combined
to generate putative full-length MFP1 peptide sequence from S. moellendorffii
and P. patens (Figs. S1 and S2; see Supplemental File 1, available online at
https://eaglehill.us/ebioonline/suppl-files/ebio-006-rose-s1.pdf). The putative
peptide translated from the P. patens sequence was used in blastp and tblastn
searches on green algae and cyanobacterial sequences, including whole genome
Figure 1. Conservation of MFP1 domain structure in plants. Homologs of MFP1 and putative
MFP1-like proteins were identified through database searches and analyzed for predicted
targeting, transmembrane, and coiled-coil domains represented by colored boxes.
The following full length MFP1 sequences were retrieved from GenBank: Oryza sativa,
NP_001042206.1; A. thaliana, NP_188221.2; Amborella trichopoda, XP_006844348.1;
Pinus massoniana, GAQR01005672.1 (RNA, translated). The following putative full length
sequences were constructed using partial ESTs and GENSCAN predictions and are available as supplementary
material: S. moellendorffii, P. patens, and K. flaccidum (see Supplemental File 1, available
online at https://eaglehill.us/ebioonline/suppl-files/ebio-006-rose-s1.pdf). Organisms are identified
by their genus name in the figure, with the length of each protein listed in amino acids (aa). The dotted
triangle in the S. moellendorffii sequence indicates a putative exon predicted in GENSCAN
using Arabidopsis splicing models, but not with maize splicing models.
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shotgun assemblies, to query for the possible presence of MFP1-like sequences
outside the embryophytes. These searches identified a short putative MFP1-like
sequence from the terrestrial alga Klebsormidium flaccidum (Kuetz.) Silva et al.
This sequence failed to pick up any plant MFP1 sequences outside of the mosses
when used in a reverse search on whole genome shotgun sequences, but a more
detailed analysis of its protein domains showed structural similarity to MFP1
(Figs. 1 and S3).
Analysis of predicted targeting and structural domains on the identified or
predicted full-length MFP1 and MFP1-like sequences demonstrates that the
MFP1 domain structure is well conserved throughout the plant kingdom (Fig.
1). Similarities between the sequences selected for Figure 1 are summarized in
Table 2. All angiosperm and gymnosperm sequences analyzed showed the pattern
of a chloroplast targeting peptide followed by a twin-arginine (TAT pathway)
thylakoid targeting peptide, a transmembrane domain, and a long coiled-coil
domain. Seedless plants and bryophytes shared the coiled-coil domain with significant
similarity scores to seed plants, but were more variable in their N-terminal
domains. Specifically, while most of the non-seed plant sequences had a predicted
chloroplast targeting peptide and a hydrophobic domain that could act as
Table 2. Sequence similarities between full-length MFP1 and MFP1-like proteins. Pairwise
sequence alignments were performed using the Smith-Waterman algorithm. Percent identity
is given (with percent similarity in parenthesis) above the diagonal, and the length of the top
scoring local alignment in amino acids (aa) below the diagonal.
Rice
A. thaliana
A. trichopoda
Pine
S. moellendorffii
P. patens
K. flaccidum
Rice 100%
766 aa
35.1%
(71.5%)
35.5%
(72.7%)
31.6%
(68.8%)
22.8%
(60.0%)
24.1%
(66.2%)
22.4%
(58.3%)
A. thaliana 706 aa 100%
726 aa
37.2%
(71.0%)
33.0%
(66.8%)
24.0%
(64.2%)
25.0%
(59.8%)
21.6%
(53.0%)
A. trichopoda 670 aa 710 aa 100%
728 aa
34.4%
(70.0%)
23.8%
(64.8%)
21.6%
(62.1%)
22.2%
(57.2%)
Pine 693 aa 702 aa 727 aa 100%
806 aa
26.6%
(67.0%)
25.9%
(61.6%)
22.4%
(56.8%)
S. moellendorffii 715 aa 682 aa 651 aa 730 aa 100%
805 aa
26.6%
(64.0%)
24.1%
(59.7%)
P. patens 515 aa 564 aa 657 aa 560 aa 583 aa 100%
583 aa
29.8%
(60.4%)
K. flaccidum 633 aa 670 aa 540 aa 553 aa 444 aa 439 aa 100%
663
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a transmembrane anchor, they failed to score positive for twin-arginine thylakoid
targeting signals. The K. flaccidum sequence was predicted to contain a putative
chloroplast targeting signal by ChloroP with a threshold score of 0.5, but failed to
be predicted as a chloroplast protein in PredAlgo, a program trained to detect algae
targeting signals.
MFP1 knock-out mutants show a delay in bolting under short days
To investigate the possible function of MFP1 using reverse genetics, wild-type
A. thaliana plants and a knock-out mutant lacking MFP1 protein were examined
for phenotypes. Under standard long-day conditions, plants grew at the same rate
and bolted at the same time as wild-type, essentially showing no mutant phenotype
as previously reported (Jeong et al. 2003). However, when grown under short-day
photoperiod, plants repeatedly showed a slight difference in bolting time compared
to wild-type (Fig. 2). Plants were analyzed by PCR to verify their genotypes.
Grana stacks appear normal in the absence of MFP1
Based on the similarity of MFP1 structure and topology with golgins, the
hypothesis was tested that MFP1 might be involved in membrane stacking events
in the chloroplast similarly to golgins facilitating the stacking of the Golgi cisternae
(Fig. 3A). Transmission electron microscopy was used to compare the thylakoid
membranes in wild-type and mutant plants grown under short-day conditions for
four weeks. However, electron micrographs of mutant chloroplasts showed no
significant difference in the grana stacking compared to the wild-type; both had
healthy, abundant grana stacks with no apparent abnormalities (Fig. 3B–C). The
number of grana stacks and the number of layers present per grana stack were
counted in a limited number of chloroplast sections. A “grana stack” was considered
a section of thylakoid membrane containing three or more layers; sections
with two or fewer layers were not counted. The knock-out mutant was found to
contain a slightly greater number of grana stacks (23.5 stacks/chloroplast, n = 2)
than the wild-type (20 stacks/chloroplast, n = 3), but the grana stacks in the mutant
had fewer layers (3.73 layers/granum, n = 30) than did the stacks in the wild-type
(4.41 layers/granum, n = 49).
Lack of MFP1 is associated with changes in relative abundance of chloroplast
protein complexes
After rejecting the thylakoid stacking hypothesis, we investigated the possibility
of MFP1 being involved in protein complex formation based on the fact that coiledcoil
domains often serve as protein-protein interaction domains. Chloroplasts were
isolated from wild-type and knock-out mutant plants grown under short-day conditions,
solubilized with 1% digitonin, and analyzed using blue native polyacrylamide
gel electrophoresis (BN-PAGE). A 1D BN-PAGE gel was used to compare
the protein complexes present in the chloroplasts of both the wild-type and the
mutant (Fig. 4A). Bands that appeared different in intensity between wild-type and
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A.R. Havighorst and A. Rose
2018 No. 6
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Figure 3. MFP1 does not appear
to be involved in thylakoid stacking.
(A) Model of the stacking
hypothesis of MFP1 function.
The diagram on the left shows
the topology of MFP1 as an
integral thylakoid membrane
protein with the transmembrane
domain in yellow and coiled-coil
domain in blue. The C-terminal
domain of MFP1 binds to chloroplast
DNA. The diagram on
the right illustrates a possible
mode of action for MFP1 being
involved in thylakoid stacking
via dimerization of its coiled-coil
domains. Wild-type and mutant
plants lacking MFP1 were subjected
to transmission electron
microscopy to test the stacking
hypothesis. (B) TEM micrograph
of thylakoid membranes from a
wild-type plant. (C) TEM micrograph of thylakoid membranes from a knock-out mutant
plant. Bars = 500 nm. No differences in thylakoid stacking were observed between wildtype
and mutant. Plant genotypes were confirmed through PCR.
Figure 2. MFP1 knock-out mutants bolt later than wild-type under certain conditions. (A)
Wild-type Arabidopsis and a T-DNA insertion line lacking MFP1 were grown and observed
for phenotypes under 200 μmol m-2 s-1 light intensity in a growth chamber. MFP1 mutants
(left) show delayed bolting compared to wild-type (right) under long-day conditions in
four-week old plants grown from seeds after six years of storage. This phenotype was only
observed with old seeds, but partially reproducible under short-day conditions. (B) Fourweek
old wild-type seedlings grown from recently harvested seeds under short-day conditions.
(C) Four-week old MFP1 knock-out mutant plants grown under identical conditions
alongside the wild-type plants in (B).
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mutant are marked with an asterisk. The putative identity of the observed bands was
determined by comparing the banding pattern and apparent sizes of protein complexes
with previously published BN-PAGE data of digitonin-solubilized A. thaliana
chloroplast or thylakoid proteins (Heinemeyer et al. 2004, Järvi et al. 2011).
Bands in the size range of 300–370 kDa corresponding to photosystem II monomers
(PSII-M), cytochrome b6f, and possibly F1-ATP synthase and light-harvesting complex
II (LHCII) on comparable gels, were just barely visible in the mutant when
compared to the same bands in the wild-type. On the other hand, a band around
850 kDa, corresponding to the apparent molecular weight of the state-transitionspecific
PSI-LHCII complex or a photosystem II supercomplex, appeared more
intense in the mutant than in the wild-type. The apparent molecular mass of the
LHCII trimer (LHCII-T) and the shape of the corresponding band differed between
Figure 4. MFP1 is associated with protein complexes and affects the relative abundance of
complexes in chloroplasts. Chloroplast protein extracts were solubilized with 1% digitonin
and separated by BN-PAGE. (A) Coomassie staining of chloroplast proteins observed in
gradient gel (BN-PAGE, left) and detection of MFP1 using immunoblotting (Western,
right). The blue color of the BN-PAGE gel has been digitally removed using a blue filter
for better contrast and visibility of bands. The gel picture and antibody signal have been
lined up using the positions of the 440 and 880 kDa marker bands (Ferritin, 80 μg). Putative
protein complexes corresponding to the visible bands were deduced from comparison of
banding patterns with published data (Heinemeyer et al. 2004, Järvi et al. 2011). Cyt. b6f,
cytochrome b6f; LHCI, light-harvesting complex I; LHCII, light-harvesting complex II;
LHCII-T, light-harvesting complex II trimer; PSI, photosystem I; PSII-M, photosystem
II monomer. Bands that show differential intensity in wild-type and mutant samples are
highlighted by asterisks. WT, wild-type protein extract (20 μL); KO, knock-out mutant
protein extract (20 μL). (B) Comparison of MFP1 detected by immunoblotting in wild-type
samples separated in 1D BN-PAGE and the corresponding 2D SDS-PAGE. Marker sizes for
the SDS-PAGE are indicated on the left. MFP1 signal is detected at the size of the mature
protein (~75 kDa) and several smaller fragments.
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the wild-type and mutant, showing an “inverse smile” effect and lower molecular
mass in the mutant sample (Fig. 4A). However, this effect did not occur if lower
amounts of protein were loaded.
To determine whether MFP1 might directly associate with chloroplast protein
complexes, the protein was detected using immunoblotting with an antibody
raised against the MFP1 coiled-coil domain (Fig. 4A, Western). The size of A.
thaliana MFP1 protein in SDS-PAGE gels is around 75–80 kDa (Jeong et al. 2003,
Samaniego et al. 2006a), however none of the protein was observed at that apparent
size during BN-PAGE. On the 1D BN gel (Fig. 4A, WT Western), a strong MFP1
signal is detected in wild-type extracts between the bands corresponding to the
apparent molecular size of LHCII trimer (~110 kDa) and cytochrome b6f/PSII-M
(~300 kDa), with signal trailing to around 500 kDa. MFP1 signal also appeared
above the 880 kDa marker position with a pronounced band in the range reported
for PSI multimers and PSII-LHCII supercomplexes (Heinemeyer et al. 2004, Järvi
et al. 2011). No signal was detected in mutant protein extracts, confirming the lack
of MFP1 in the knock-out plants (Fig. 4A, KO Western).
Strips of 1D BN gels containing wild-type protein were further separated by SDSPAGE
and the resulting 2D gel subjected to an immunoblot with MFP1 antibody (Fig.
4B). The strongest signals were located around 75 kDa, which is the approximate
size of the mature MFP1 protein after cleavage of the transit peptide, with several
distinct smaller sizes around 50–55 kDa and approximately 27 and 10 kDa for the
lowest mass complex. MFP1 signal in the higher molecular weight complexes mostly
represented mature MFP1 with a secondary band around 50 kDa (Fig. 4B).
Photosynthesis rates and chlorophyll content are not affected in MFP1 mutants
To test whether MFP1 affects photosynthesis or pigment composition, we
performed photosynthesis rate measurements on two-week old plants, as well as
determined chlorophyll a, b, and carotenoid content of the leaves. However, no difference
between wild-type and mutant plants could be found for either CO2 uptake
(Fig. 5A) or chlorophyll content (Fig. 5B). When comparing photosynthesis rates
of six biological replicates for each genotype at light intensities of 100, 150, and
300 μmol m-2 s-1, no statistically significant difference could be observed (P values
ranging from 0.6 to 0.9). Similarly, no difference was seen for the same plants in
either pigment content (total chlorophyll for WT = 23.18 μg cm-2 = 258.28 μmol
m-2, KO = 23.04 μg cm-2 = 256.68 μmol m-2; P = 0.83) or chlorophyll a to b ratio
(WT = 2.276, KO = 2.255; P = 0.78).
Discussion
The evolutionary origin of MFP1 coincides with a terrestrial life style
Chloroplast proteins fall into two categories based on their evolutionary origin:
one represented by proteins of prokaryotic origin that were present in the ancestor
of modern chloroplasts, and the other group consisting of proteins of eukaryotic
origin that were introduced from the host cell during chloroplast evolution. The
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long coiled-coil domain and results from database searches suggest that MFP1 falls
into the latter category and is a protein of eukaryotic origin. Prediction of chloroplast
and thylakoid targeting signals as well as transmembrane and coiled-coil
domains revealed that the domain structure of MFP1 is well conserved throughout
the seed plants (Fig. 1). The N-terminus in seedless plants is less well conserved,
but usually contains a putative chloroplast targeting signal and a hydrophobic
domain. All MFP1-like proteins share the long coiled-coil domain, with more distantly
related sequences showing significant local similarity in the C-terminal half
of the coiled-coil domain implicated in DNA binding.
Identifying more distantly related sequences to MFP1 presented a challenge
due to the long coiled-coil domain of the protein. The repetitive nature of coiledcoil
sequences often results in unrelated sequence similarities due to structural
constraints that do not indicate homology (Rose et al. 2004). Therefore, consecutive
and reverse searches were performed to confirm MFP1-like sequences and
distinguish them from random matches to unrelated long coiled-coil sequences.
Gene predictions were used to predict full-length mRNA and peptide sequences
where such information was not yet available in the database. It is likely that these
predictions will have to be adjusted as more expressed sequence information
becomes available. For example, two different versions of the S. moellendorffii
MFP1-like sequence were predicted depending on the splice site model used, and
combined 5′ and 3′ ESTs in this case covered only the coiled-coil domain. This
Figure 5. Photosynthesis rates and chlorophyll content are unaffected by lack of MFP1. (A)
Light response curves for photosynthesis rates were measured as net CO2 uptake dependent
on photosynthetic photon flux density (PPFD) in two-week old pla nts. WT, wild-type indicated
in blue with open circles; KO, knock-out mutant measurements in green with cross
marks. Each curve represents the average of three biological replicates. (B) Chlorophyll a
and b content was measured using DMF extraction and spectrophotometry. WT, wild-type
in yellow; KO, knock-out mutant in green. Each bar represents the average and standard
deviation for six biological replicates. Statistical analysis (T-test) indicated no difference
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suggests that alternative splicing might occur, with the coiled-coil domain being
more commonly expressed, or that the gene prediction is not entirely accurate.
For the P. patens sequence, gene prediction failed to identify exons corresponding
to the N-terminus of the protein in other plants and only the coiled-coil domain
could be predicted.
A search with the moss (P. patens) sequence with stringent E-value cutoffs
succeeded in identifying MFP1 from higher plants, indicating that MFP1 is present
in all phyla of embryophytes including bryophytes and may represent a novel
protein sequence adaptive to plants moving onto land around 475 million years
ago. A tblastn search with the moss sequence on whole genome shotgun contigs
identified a short peptide match translated from genomic DNA of the charophyte
alga K. flaccidum with an E-value of 0.001 close to the E-values found for clearly
unrelated sequences. Additional database searches and pairwise sequence alignments
provided evidence that this sequence represents an MFP1-like algal protein
and not a random match, supported by almost 30% identity to the moss sequence
in pairwise alignments (Table 2). Furthermore, gene prediction on the K. flaccidum
genomic sequence surrounding the match revealed a putative gene coding for a protein
strongly resembling the domain structure of MFP1 (Fig. 1), again supporting
the notion that this sequence may indeed represent a distant MFP1-like homolog.
The identification of a putative MFP1-like precursor in the alga K. flaccidum has
intriguing implications for the evolutionary origin of MFP1 and may provide a clue
to its function. K. flaccidum belongs to the charophytes, the group of green algae
most closely related to plants, and has a terrestrial growth habit. Recent analysis of
its genome revealed that it acquired many genes specific to land plants, including
ABA signaling to deal with drought stress and a primitive system for cyclic electron
flow to protect against high light intensity (Hori et al. 2014). No sequences with
significant similarities to this putative algal MFP1-like protein could be identified
from other algae, such as Chlamydomonas, or cyanobacteria, therefore it appears
to represent a novel protein in the streptophyte lineage. The presence of a putative
MFP1-ancestor sequence in K. flaccidum, but not other fully sequenced algae
genomes, proposes that the acquisition of this early form of MFP1 coincided with
the transition from an aquatic to a terrestrial life style and suggests an adaptive
function for the presence of a membrane-bound coiled-coil protein in chloroplasts
for growth on land. Subsequently, this early MFP1-like protein appears to have
undergone stabilizing selection for a TAT pathway thylakoid targeting signal during
the transition from seedless vascular plants to seed plants.
MFP1 does not play a role in thylakoid stacking or photosynthesis
One characteristic of the thylakoids of charophyte algae and land plants is the
formation of tightly appressed grana stacks. Our initial hypothesis for the function
of MFP1 was that it might be involved either in forming or maintaining the structure
of the grana stacks, based on the protein’s structural similarity to that of the
golgins, which maintain the stacks in the Golgi apparatus (Rose et al. 2004). The
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results obtained here, however, do not support this hypothesis. TEM micrographs
of the thylakoid membranes in MFP1 knock-out plants show no deficiency in
thylakoid stacking in comparison with wild-type plants grown under the same conditions
(Fig. 3). Slight differences in the number of grana stacks and grana layers
can probably be attributed simply to the natural dynamics of the membrane system
within the chloroplast. It is possible that MFP1 acts only under specific conditions,
such as in remodeling of the thylakoids during state transitions, which may involve
membrane fission and fusion events (Chuartzman et al. 2008). Interestingly, maize
MFP1 was found to be two-fold enriched in bundle sheath cells over mesophyll
cells (Majeran et al. 2008, Friso et al. 2010), suggesting it may play a role in or be
affected by thylakoid changes associated with C4 photosynthesis.
Knock-out mutants lacking MFP1 protein appear indistinguishable from
wild-type under standard growth conditions, but we found delayed bolting under
short-day photoperiod (Fig. 2). A. thaliana MFP1 appears to be a target for the transcription
factor PIL5 that mediates gene regulation through phytochrome during
seed germination and regulates genes involved in hormone signaling (Oh et al.
2009). In addition to a possible link with phytochrome response and photoperiod,
recent evidence suggests that stress can affect MFP1 expression. In cotton, MFP1
mRNA expression was found to be up-regulated in plants grown under various
stress conditions including ABA, cold, drought, high salinity and pH (Zhu et al.
2013). However, no significant difference in expression could be observed in A.
thaliana within 24 hours of such stress treatments (Killian et al. 2007), suggesting
that MFP1 expression changes as part of a slow, long-term adaptive response. These
pathways are of particular importance to land plants, matching well with a possible
adaptive role for MFP1 during the transition from water to land. If MFP1 was
involved in adaptation to stress or different light conditions, its lack could be causing
subtle changes and a delay in development that are not detectable under normal
growth conditions. Thus, further insight might be gained by observing chloroplast
and thylakoid ultrastructure as well as photosynthetic performance in plants grown
under different light and stress conditions.
MFP1 is a member of multiple chloroplast protein complexes
The results of the BN-PAGE experiments support the idea that MFP1 is involved
in protein complex formation and homeostasis in chloroplasts. Two strong signals
stand out when detecting MFP1 in immunoblots of 1D BN gels: A wide band in the
range of about 150–500 kDa, and a well-defined band in the megadalton range surrounded
by more diffuse signal spread nearly to the start of the running gel. No signal
was observed between the lower molecular weight complexes (~150–500 kDa) and
these higher molecular weight complexes (>880 kDa), suggesting that MFP1 is part
of multiple distinct protein complexes in the chloroplast (Fig. 4A, Western). The
presence of MFP1 in several high molecular mass complexes is also corroborated
by chloroplast membrane proteomics studies in maize, which identified MFP1
homologs in complexes of ~250, 500, and 700 kDa (Majeran et al. 2008). These
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MFP1-containing complexes likely represent thylakoid-associated complexes,
based on the known integral thylakoid membrane localization of MFP1 and a clear
lack of MFP1 observed in stroma protein complexes (Friso et al. 2004, Jeong et al.
2003, Olinares et al. 2010).
Full-length A. thaliana MFP1 has a predicted size of 82 kDa, and processed,
mature plastidic MFP1 with the targeting peptides cleaved off is predicted to be
72 kDa in size. However, no signal was detected below 150–160 kDa, suggesting
that MFP1 does not exist as a monomer in the chloroplast, but is always found
within a complex or as a homodimer (Fig. 4A). Dimerization of MFP1 has also
been previously reported from SDS-PAGE results, which indicated the presence of
MFP1 homodimer in onion nuclear matrix fractions (Samaniego et al. 2008). The
2D BN/SDS-PAGE showed a variety of partial MFP1 sizes around 50–55 kDa and
smaller in addition to the mature protein, which may indicate alternative splice
forms, modification, or cleavage of MFP1 within the complex it is forming. The
MFP1 signal detected in higher molecular mass complexes above the 880 kDa
marker size is markedly concentrated in a strong band in the megadalton range.
This signal may represent the A. thaliana transcription complex (1500–1700 kDa,
Behrens et al. 2013), based on MFP1’s known association with the nucleoid.
Lack of MFP1 affects relative abundance of chloroplast protein complexes
Due to MFP1’s location in the thylakoid membrane, we tested whether the
protein may be involved in organizing photosynthetic complexes of the thylakoid
membrane. No difference could be detected between mutant and wild-type plants
grown under standard laboratory conditions in either photosynthesis rates or chlorophyll
content (Fig. 5). However, changes in the relative abundance of several
complexes in the MFP1 knock-out mutant compared to wild-type suggest that
MFP1 may be indirectly involved in the formation or maintenance of chloroplast
protein complexes (Fig. 4A). To identify the nature of these protein complexes, the
apparent size and relative positions of the bands were compared with previously
published BN-PAGE data obtained after solubilization of chloroplasts or thylakoids
with digitonin (Heinemeyer et al. 2004, Järvi et al. 2011). The results suggest that
lack of MFP1 reduces the relative abundance of complexes in the size range for
PSII monomers, LHCII assembly, cytochrome b6f and F1 ATP synthase. On the
other hand, a band in the range of PSI-LHCII complexes or PSII supercomplexes
appears stronger in the mutant. PSI-LHCII complexes are only observed after
digitonin-solubilization and not with other detergents (Järvi et al. 2011). Since the
abundance of these complexes appears to change between wild-type and mutant,
MFP1 might facilitate some aspect of the dynamics of the system by affecting protein
complex formation or stability.
Conclusions
MFP1 is an enigmatic integral thylakoid and nucleoid protein found in all fully
sequenced plant genomes analyzed to-date. It is especially well conserved in seed
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A.R. Havighorst and A. Rose
2018 No. 6
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plants, and MFP1-like sequences can also be detected in seedless plants. Sequence
similarities and domain structures suggest that MFP1 is likely a membrane-bound
long coiled-coil protein of eukaryotic origin that was sequestered into plastids
using a chloroplast targeting signal early in the evolution of land plants. Its structure
and localization appear unique; no other protein encoded by the A. thaliana
genome is similar in sequence or even shares the same domain structure with MFP1
(Jeong et al. 2003). However, knock-out mutants lacking MFP1 are phenotypically
indistinguishable from wild-type under standard growth conditions, which is
surprising as one would expect to find a well conserved function for such a unique
and evolutionarily ancient protein. It has previously been shown that despite its
association with the chloroplast genome, MFP1 does not appear to be essential in
anchoring the nucleoid to the thylakoid membrane (Jeong et al. 2003). This study
has demonstrated that MFP1 also does not appear to be involved in thylakoid membrane
stacking or photosynthesis, but instead associates with multiple chloroplast
protein complexes. Furthermore, lack of MPF1 affects the relative abundance of
complexes in the size range of prominent thylakoid complexes. Identification of
the protein interaction partners of MFP1 in the chloroplast should provide further
insight into its function.
Acknowledgements
We are very grateful to Dr. Iris Meier (The Ohio State University) for generously
sharing seeds and antibodies used in this study, and would like to thank Dr. Guichuan
Hou (CAS Microscopy Center, Appalachian State University) for instruction and user
time on the transmission electron microscope, Dr. Howard Neufeld (Appalachian
State University) for sharing the LI-COR photosynthesis system, and Alyssa Teat for
valuable help in conducting the measurements and analyzing the data. This study was
supported with funds from the Department of Biology, College of Arts and Sciences,
and the Office of Student Research at Appalachian State University.
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