Parallel subfunctionalisation of PsbO protein isoforms in angiosperms revealed by phylogenetic

14 Pages·2015·1.7 MB·English

by  Miloš Duchoslav

DuchoslavandFischerBMCPlantBiology (2015) 15:133
DOI10.1186/s12870-015-0523-4
RESEARCH ARTICLE Open Access
Parallel subfunctionalisation of PsbO protein
isoforms in angiosperms revealed by
phylogenetic analysis and mapping of
sequence variability onto protein structure
Miloš Duchoslav and Lukáš Fischer*
Abstract
Background: PsbO, the manganese-stabilising protein, is anindispensable extrinsic subunit ofphotosystem II. It
plays a crucial role inthe stabilisation ofthewater-splittingMn CaO cluster, which catalyses theoxidation ofwater
4 5
to molecular oxygen by using light energy. PsbO was alsodemonstrated to have a weak GTPase activity that could
be involved in regulation of D1protein turnover. Our analysis of psbO sequences showed that many angiosperm
species express two psbO paralogs, but the pairs of isoforms in one species were not orthologous to pairs of
isoforms indistant species.
Results: Phylogenetic analysis of 91 psbO sequences from 49 land plant species revealed thatpsbO duplication
occurred many times independently, generally at therootsof modern angiosperm families. In spite of this, thelevel
ofisoformdivergence was similar in different species. Moreover, mapping of thedifferences on theprotein tertiary
structure showed thattheisoformsin individual species differ from each other on similar positions,mostly on the
luminally exposedend of theβ-barrel structure. Comparison of thesedifferenceswith the location of differences
between PsbOs from diverse angiosperm families indicated various selection pressures inPsbO evolution and
potential interaction surfaces onthe PsbO structure.
Conclusions: The analyses suggest thatsimilar subfunctionalisation ofPsbO isoformsoccurred parallelly invarious
lineages. We speculate that thepresence of two PsbO isoforms helps the plants to finely adjust thephotosynthetic
apparatus in response to variable conditions. This might be mediatedby diverse GTPase activity,sincethe isoform
differences predominate near thepredictedGTP-bindingsite.
Keywords: Gene duplication,GTPase, Homology modelling, Manganese-stabilizing protein (MSP),Oxygen evolving
complex, Parallel evolution, Protein structure, PsbO
Background plastoquinone. PsbO, also known as the manganese-
Photosynthetic conversion of light into chemical energy stabilising protein, is one of the extrinsic subunits of
in oxygenic phototrophs is accompanied with evolution photosystem II, located on the luminal side of the thyla-
ofmolecularoxygenreleasedfromwatermolecules.This koid membrane. PsbO is present in all known oxygenic
process is realized in the oxygen evolving complex of photosynthetic organisms [1]. Despite the ability of the
photosystem II present in thylakoid membranes. Photo- cyanobacterium Synechocystis sp. PCC 6803 mutant to
system II (PSII) is a multisubunit protein–cofactor com- grow photoautotrophically with deleted psbO gene [2],
plex that uses light energy to oxidize water and to reduce PsbO seems to be crucial for PSII function. Neither the
mutant of green alga Chlamydomonas reinhardtii lacking
PsbO, nor Arabidopsis thaliana (A. thaliana) with si-
lenced expression of both psbO paralogs were able to
*Correspondence:[email protected]
growphotoautotrophicallyorevenassemblePSII[3,4].
DepartmentofExperimentalPlantBiology,FacultyofScience,Charles
UniversityinPrague,Viničná5,12844Praha2,CzechRepublic
©2015DuchoslavandFischer.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
unlessotherwisestated.
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page2of14
Three-dimensionalstructureofPsbOfromcyanobacter- In A. thaliana mutants with an impaired psbO1 or
ium Thermosynechococcus was resolved as a part of PSII psbO2 gene, the compensatory upregulation of the re-
by X-ray crystallography with a resolution down to 1.9 Å maining isoform was observed. The expression level of
[5]. The crystal structure of PSII or PsbO alone from PsbO2 in psbo1 mutant was increased several times,
plants or other eukaryotes is not available. Some infor- reaching 75 % of the total amount of PsbO in wild-type.
mation about the structure of the whole PSII dimer TheexpressionlevelofPsbO1inpsbo2mutantwas125%
surrounded by antenna complexes (the PSII-LHCII of the total PsbO in wild-type. The amount of other PSII
supercomplex) from higher plants was obtained by sin- proteins was affected similarly, leading to the same stoi-
gle particle cryo-electron microscopy and cryo-electron chiometryofPsbOperPSIIasinwildtype[19].
tomography[6–8].Unfortunately,theresolutionisinsuffi- The psbo1 mutant plants have pale green leaves, re-
cient to provide any plant-specific knowledge about the duced rosette size and slower growth rate as compared
PsbO structure. Still, relatively high pairwise identity be- towild-typeplants[17,19,23].Descriptionsofthepsbo2
tween PsbO sequences of Thermosynechococcus and mutant phenotype slightly differ from each other, prob-
higher plants (around 45 %) allows construction of hom- ably because of different growth conditions and age of
ologousmodelsforplantPsbOs[9,10]. used plants [23]. Lundin et al. [19] observed growth rate
The X-ray crystallography of cyanobacterial PSII re- slower than in wild-type and the leaf weight was even
vealedthatPsbOisaβ-barrelprotein(structuralfeatures lower than that of psbo1, while Allahverdiyeva et al. [23]
of PsbOs are discussed in connection with our results reportedaphenotype verysimilartothatofwild-type.
and PsbO functions in chapter Discussion). It is located Under growth light (120 μmol photons m−2 s−1), the
in the vicinity of the water splitting Mn CaO cluster, psbo2 mutant had characteristics of electron transport
4 5
but it is not directly involved in binding of the cluster chain very similar to wild-type, whereas investigation of
[9]. The main function of the PsbO is to stabilise the the psbo1 mutant showed malfunction of both the donor
Mn CaO cluster, in particular to modulate the calcium and acceptor sides of PSII and high sensitivity of PSII
4 5
and chloride requirements for efficient water splitting centres to photodamage [23]. Bricker and Frankel [24]
(for review see [11]). Besides this “basic” function, reported that many of the defects of psbo1 photosystems
PsbO seems to be involved also in other processes (for are reverted by higher concentration of CaCl , but
2
review see [12, 13]). Spinach PsbO was shown to be Allahverdiyeva et al. [23] did not observe similar effect.
able to bind GTP [14] and also to hydrolyse it, al- Nevertheless, the importance of the PsbO2 seems to be
though very slowly[10].ItwasproposedthattheGTPase exhibited under high light conditions. For example, the
activity of PsbO in plants might be involved in D1 repair maximum quantum efficiency (F /F ) values of wild-
V M
cycle[10]. type and mutant plants became similar after 3 weeks of
In plants and algae, the PsbO protein is encoded by a moderate light (500 μmol photons m−2 s−1) [23]. Lundin
nuclear psbO gene [1]. Transport to chloroplasts and et al. [19] reported that after 15 days of high light
thylakoids is ensured by two consecutive N-terminal (1000 μmol photons m−2 s−1), the psbo1 mutant did not
transit peptides, that are cleaved to produce the mature have significantly reduced leaf weight, whereas the leaf
PsbO [15]. A. thaliana expresses two psbO genes, psbO1 weight ofpsbo2mutantwasreduced drastically.
[TAIR:At5g66570] and psbO2 [TAIR:At3g50820], encod- Lundin et al. [19] also showed that psbo2 mutant has
ing for PsbO1 and PsbO2 proteins [16, 17]. The two iso- lower level of phosphorylation of D1 and D2 subunits
forms differ in only 11 amino acids [18]; nevertheless, and that the degradation of photo-damaged D1 protein
their function seems to be slightly different. Murakami is impaired in this mutant. This, together with a finding
et al. [18] reported that A. thaliana PsbO2 recovered that PSII membranes with PsbO2 have higher GTPase
oxygen evolution of PsbO-depleted spinach PSII parti- activity than PSII membranes with PsbO1 [21], led to a
cles less efficiently than PsbO1. The activity with PsbO2 conclusion,thatPsbO1hasamainfunctioninthestabil-
reached only 80 % of that with PsbO1, while the binding isation of Mn CaO cluster and the facilitation of the
4 5
efficiency of the isoforms was very similar. In contrast, water oxidation reaction, whereas PsbO2 regulates the
the oxygen evolution of PSII membranes isolated from turnoverofD1subunit[19,21,23].
A. thaliana mutants lacking PsbO1 or PsbO2 was simi- The presence of two PsbO isoforms is not unique for
larwhencorrected forthe amount ofPSII[19]. A. thaliana. Our previous study focused on the analysis
The amount of PsbO1 in wild-type A. thaliana plants of a spontaneously tuberising potato mutant revealed
is higher than that of PsbO2 [18–20]. The expression of that potato plants also express two PsbO isoforms, one
the isoforms stays similar during plant development and of which is missing in the mutant [25]. A comparison of
duringvariousshorttimestresses[21].Onlyafter40days the two characterised A. thaliana and two potato PsbO
ofcold stress,noticeable change inrelativeabundance of isoforms showed that sequences of the two paralogs in
isoformswasobservedinfavourofPsbO2 [22]. each species are more related than isoforms coming
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page3of14
from different species. It indicated independent duplica- Brassicaceae family and Oryza sativa, Physcomitrella
tion of psbO gene in these two species. To understand patens, Populus trichocarpa, Solanum lycopersicum, and
this unexpected phylogeny and evolution of PsbO iso- Vitisviniferafrom otherfamilies.
forms,wedidadetailedanalysisofpsbOsequencesfrom
a number of land plant species. Mapping the sequence EvaluationofPsbOsequencevariability
differences between PsbO proteins from various species The frequency of differences between isoforms, between
and families and between PsbO isoforms in individual species and between families were calculated for each
species on their tertiary structure, we found that the position in the alignment independently using scripts
evolution of the two isoforms was parallel in numerous written in R language [35] and partially using SeqinR
angiosperm lineages. Based on the location of isoform- package [36]. Plant families represented with just a sin-
specific differences and literature data about A. thaliana gle PsbO sequence were not included in the calculation.
and spinach PsbOs, we hypothesise that the pairs of iso- Only two most divergent isoforms were considered in
forms present in many species differ in GTPase activity case of species expressing more than two isoforms. All
and that the presence of proteins diversified in this way sequences excluded from calculation are marked with an
helps to improve photosynthetic performance under asterisk in Additional file 2. To estimate the between-
varyingconditions. isoform and between-species variability across all angio-
sperms, both types of differences were first calculated
Materials and methods for every family independently and afterwards the values
RetrievalandanalysisofpsbOsequences were averaged, in order to avoid bias caused by different
Sequences of expressed psbO genes were retrieved as numbers ofanalysed species within eachfamily.
ESTs (expressed sequence tags) and assembled ESTs The frequency of between-isoform differences within a
(PUTs, PlantGDB-assembled unique transcripts) inpublic family was calculated as follows; first, each position in
sequence databases NCBI GenBank [26] and PlantGDB the alignment was assigned 0 or 1 (for the same or dif-
(Plant Genome Database) [27], respectively. The database ferent amino acids in the two compared isoforms, re-
searches were performed using tBLASTn [28, 29] with spectively) for each species and then the values were
potatoPsbOproteinsequence(sequence“Solanumtuber- averaged within a family. To get the frequency of
osum 2”, translation of [PlantGDB:PUT-157a-Solanum_ between-species differences, all species within a family
tuberosum-55973153]) as a query. ESTs werealigned into were compared pair wise with each other, giving the
contigsforeachspeciesusing“DeNovoAssemble”toolof values 0,0.5or 1(for aminoacidsinboth isoformsiden-
Geneious R6 [30]. Formation of consensus sequences tical, amino acid in one isoform identical or no identical
from multiple overlapping ESTs strongly increased reli- amino acid) for each position and each comparison.
ability of analysed sequences compared to individually Values for each position were averaged within a family.
submitted annotated cDNAs, some of which contain evi- As the dependency of this average variability value on
dent errors. All retrieved sequences were aligned using the proportion of species that have certain amino acid
MAFFT v7.017 [31] and incomplete and unreliable se- different from the consensus is not linear, it was line-
quenceswereexcludedfromfurtheranalyses(seeanalysed arised usingtheequation
sequences in Additional file 1). Spinach psbO sequence
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
(cid:3) (cid:4)
was retrieved as cDNA [GenBank:X05548.1] because of
ð2n−1Þ− 4 1−Δ nðn−1Þþ1
the lack of ESTs and included in alignment for compari- Δ ¼ species
son(Additionalfile2).Indexingofisoformsineachfamily specieslinear 2ðn−1Þ
was random and does not reflect relation to A. thaliana
isoforms. where n is the numberofcomparedspecies, Δ isthe
species
Phylogenetic trees were built from psbO coding se- non-linear average value of between-species variability
quences by maximum likelihood (ML) method using (themeanfrompairwisecomparisons)andΔ is
specieslinear
CIPRES Science Gateway [32]. ML analysis was imple- thelinearisedvalueofthebetween-speciesvariability.
mented in tool RAxML v7.6.6 [33] using GTRGAMMA To estimate the between-family variability, the above
approximationwith 1000bootstrapreplicates. mentioned method for the calculation of the between-
The presence and position of introns was analysed by species differences was applied on sets containing se-
comparing psbO cDNAs (Additional file 1) and corre- quences from just one species from each family. A mean
sponding genomic sequences, obtained using BLASTn values obtained from all such combinations of species
[28, 29] searches in Phytozome database [34] for the (53,760 in total) included both between-species and
followingrepresentativespecieswitheasilyavailablegen- between-family differences, so the values of between-
omic sequence: Arabidopsis lyrata, Arabidopsis thali- species differences were subtracted from it, giving the
ana, Brassica rapa, and Thellungiella halophila from netbetween-family differences.
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page4of14
Homologymodellingandmappingofvariabilityonthe found also in the genomic sequence of Oryza sativa.
proteinstructure Zea mays, a recent tetraploid, expresses two isoforms
Homology model of potato PsbO (sequence “Solanum with littledivergence(Additional file3).
tuberosum2”)wasbuiltusingSwiss-Modelserver[37,38] Among dicots, Malvaceae, Myrtaceae, Phrymaceae and
based on PsbO from cyanobacterium Thermosynechococ- RutaceaeseemtoexpressonlyonepsbOgene.Asteraceae,
cus vulcanus [PDB:3ARC] (chain O) [5]. Extra 13 amino Euphorbiaceae,Fabaceae,Salicaceae,SolanaceaeandVita-
acids present on the N-terminus of potato PsbO were ceae seem to express two psbO genes (or four in the case
pasted to the model manually using Swiss-PdbViewer of recent tetraploids such as Glycine max or Nicotiana
v4.1.0[39]withoutattempttoshowanyfolding. tabacum).BrassicaceaehavevariousnumbersofpsbOiso-
Homology model of potato PsbO was coloured ac- forms; however, most of them can be sorted into two
cording to the frequency of the respective type of vari- groups. While Arabidospsis thaliana expresses just two
ability using Swiss-PdbViewer v4.1.0 [39] and scripts isoforms (psbO1, psbO2), each from one group, genus
written in R language [35]. The images were rendered Brassica expresses three to five genes - one gene corre-
usingPOV-Rayv3.6[40]. sponds to psbO2 of A. thaliana, while the gene ortholo-
gous to psbO1 of A. thaliana is present in several very
Determinationofspatialcentresofdifferences similar sub-isoforms (4in B. napus, 3 in B. rapa and 2 in
Spatial centres of the differences were calculated using B. oleracea; Additional files 3 and 4). Thellungiella halo-
coordinates of α-carbon atoms of amino acids in the phila expresses three psbO genes, two of which corres-
PsbO homology model using scripts written in R lan- pondtopsbO1 and psbO2 of A. thaliana, the third one is
guage [35]. The arithmetic mean of the coordinates was most similar to pseudogenes that can be found in gen-
weighed by frequency of the respective difference on omic sequences of A. thaliana [TAIR:At4g37230], Arabi-
each position. The 13 N-terminal amino acids with dopsis lyrata [GenBank:XM_002866937] and Brassica
unknown folding were excluded from the calculation. rapa [Phytozome:Bra017790] (data not shown).
Overall spatial centres of the differences between iso-
forms and the differences between species in angio- PairsofPsbOisoformsevolvedineveryangiosperm
sperms were calculated as an arithmetic mean of spatial familyindependently
centres calculated for all families. The statistical signifi- The majority of analysed angiosperm species have just
cance of the divergence in the location of the spatial two PsbO isoforms (Additional file 3). Such situation
centres of the between-isoform and the between-species could likely results from a gene duplication event in a
differences was assessed using a randomisation test. common ancestor followed by functional divergence of
Variable positions in the alignment were randomly shuf- the paralogs. The paralogous genes encoding the func-
fled and the spatial centres for the between-isoform and tionally divergent isoforms can be inherited by descen-
the between-species variability were calculated. Differ- dants or potentially lost. However, the phylogenetic tree
ence between means of the two types of spatial centres derived from coding sequences of psbOs indicates a dif-
projected ontheaxis of highestvariability wascompared ferent evolutionaryscenario (Fig.1).
with the value obtained for real alignment. The p-value The basic topology of the phylogenetic tree does not
wascalculatedfrom 50,000 randomisations. contain dichotomous branching to two groups of func-
tionallydivergedorthologsatthetreebase,butitreflects
Results basic phylogeny of land plant families. The branching to
ThemajorityofangiospermspeciesexpresstwopsbO two isoforms is also absent at the base of angiosperms.
genes Instead, the branching events are clearly present at the
Searching public databases for expressed sequences of bases of several families (for example Solanaceae, Faba-
psbOgenes from land plants (Embryophyta) we obtained ceae,Brassicaceae,Zingiberaceae;Fig.1).Thisunexpected
91 sequences from 49 species and 36 genera. Analysis of topology indicates that duplications of psbO gene oc-
these sequences showed that the majority of the ana- curred independently in each plant family that contains
lysed angiosperm species express more than one, in species with multiple PsbO isoforms. Moreover, these
mostcasestwopsbOisoforms(Additionalfile3).Incon- families do not form any cluster in the phylogenetic tree
trast, all analysed representatives of gymnosperms (from ofpsbOorintheconsensualphylogenyofangiosperms.
both Cycadophyta and Coniferophyta groups) seem to To further confirm the independent duplication of
expressonlyone psbOisoform. psbO genes in ancestor of each angiosperm family, the
In monocots, psbOsequences were available from only presence and position of introns was analysed in avail-
two families: Zingiberaceae species have two psbO iso- ablegenomicsequencesofpsbOgenes.Accordingtothis
forms,whereasmostPoaceaespecies withavailable ESTs analysis,alllandplantshaveanintronataconservedsite,
express only one psbO gene. A single psbO gene was 12nucleotidesupstreamtheboundarybetweensequences
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page5of14
79 Capsicum annuum 1
100
89 Solanum tuberosum 1
Nicotiana tabacum 1
98 Nicotiana tabacum 4
100 Capsicum annuum 2
74 92100 NSicooltaiannuam t atubbaecruomsu 2m 2
38 Nicotiana tabacum 3
100 Artemisia annua 2
Artemisia annua 1
Mimulus guttatus 1
99 Theobroma cacao 1
17 Gossypium raimondii 1
100 Populus trichocarpa 2
26 4148 Populus trichocarpa 1
100 Manihot esculenta 1
23 Manihot esculenta 2
Citrus sinensis 1
71 Vitis vinifera 2
Vitis vinifera 1
21
46 Lotus japonicus 1
88 Glycine max 2
31 100 Glycine max 1
100 Phaseolus vulgaris 1
76 Medicago truncatula 2
51
Medicago truncatula 1
30 Lotus japonicus 2
18 10079 Glycine max 4
Glycine max 3
Phaseolus vulgaris 2
88 Fragaria vesca 1
100 Malus domestica 1
94 Malus domestica 2
100 Linum usitatissimum 1
Linum usitatissimum 2
Thellungiella halophila 3
60 Brassica rapa 2
52 100
Arabidopsis thaliana 2
99
Thellungiella halophila 2
100 53 Brassica rapa 1
89
39 Brassica rapa 3
78 Brassica rapa 4
Thellungiella halophila 1
61 Arabidopsis thaliana 1
Eucalyptus grandis 1
100 Triticum aestivum 1
41 Hordeum vulgare 1
98 100 Zea mays 1
Zea mays 2
37 44 Oryza sativa 1
100 Curcuma longa 2
96 Zingiber officinale 2
97 Zingiber officinale 1
Curcuma longa 1
100 100 Sequoia sempervirens 1
100
96 Cryptomeria japonica 1
100 Taxus baccata 1
Cycas rumphii 1
98 Pinus sylvestris 1
Picea abies 1
Selaginella moellendorffii 1
100 Physcomitrella patens 2
92 Physcomitrella patens 3
94 Physcomitrella patens 1
Physcomitrella patens 4
0.2
Fig.1AphylogenetictreefromcodingsequencesofpsbOgenesfrom36generaoflandplants.Eachgenusisrepresentedbysequencesfrom
onlyonespeciesforthesakeofsimplicity.Sequencesfromdifferentspeciesbelongingtothesamegenusareverysimilarandtheirinclusion
doesnotchangethephylogenetictreetopology(seethefullphylogenetictreeinAdditionalfile4).Thetreewasconstructedbythemaximum
likelihoodmethod,numbersatbranchesdenotebootstrappercentages
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page6of14
encoding the transit peptide and the mature protein. In theisoformsaresimilarinmultiplefamiliesdespitethein-
addition, all psbO genes from Brassicaceae family contain dependentduplicationsofpsbOgenes.Detailedanalysisof
an additional intron, 282 nucleotides downstream the the sequence alignment failed to identify any compact re-
boundarybetweenthetransitpeptideandthematurepro- gion in the primary sequence that would be specific for
tein. The intron is present at a conserved site in all psbO one or the other isoform across the analysed plant fam-
genes in thisfamily, includingthe most divergent isoform ilies. Also, single positions with similar differences be-
of Thellungiella halophila. This indicates that all these tween isoforms in the majority of species were rare (see
psbO genes evolved from one common Brassicaceae- thealignmentinAdditionalfile2).
specific ancestor gene containing the additional intron, To analyse the character of the differences in PsbO se-
absentinpsbOsinotherfamilies. quences in detail, we assorted the variability into three
classes: i) variability between isoforms (within a species),
ExtentofdivergenceofPsbOisoformsissimilarinall ii) variability between species (within a family) and iii)
species variability between families (Fig. 2). Frequencies of these
The extent of differences between protein sequences of three classes of variability were calculated for each pos-
PsbO isoformsin everyspecies isin the same range, even ition of the primary sequence (Additional file 2; see
though the duplication seems to have occurred in each Materials and methods section for details). In the align-
family independently. The numbers of different amino ment of mature PsbO sequences from angiosperms
acid residues range from six in a recent tetraploid Zea (Additional file 2), 59 % of positions are fully identical,
maysto23inPopulusdeltoidesandPopulusxcanadensis 77 % of positions can be described as conserved (with
(2–9 % of total residues; Additional file 3). Interestingly, lowlevelof variabilitybelow 10 %). The variability inthe
similar divergence between isoforms can be found also in remaining23%ofpositionscouldstemfromeitherselec-
the moss Physcomitrella patens (24 different amino acid tionpressurefavouringaspecificsubstitution(positivese-
residuesbetweentwomostdivergentisoforms). lection), or, on the contrary, from the lack of strong
The level of differences between PsbO isoforms is kept selectionpressuretokeepthepositioninvariable(negative
withinthisrangeeveniftheduplicationeventsofpsbOoc- selection). The lack of selection pressure should result in
curred at different times in evolutionary history. For in- frequent random changes and a high level of variability
stance, pairwise identity of nucleotide sequences encoding in all three classes. When analysing the PsbO sequences,
maturePsbOsofV.vinifera(80%)ismuchlowerthanthat it was obvious that a certain class of variability predomi-
of Populus trichocarpa (92 %). This indicates that the du- nated at many positions and that the overlap between
plication of the Vitis psbO gene probably occurred earlier theclassesatagivenpositionwasonlypartial(Additional
compared to that of the Populus gene. However, pairwise file5).
identity of the protein sequences of PsbO isoforms of V.
vinifera(93%)issimilartothatofP.trichocarpa(92%). Aminoacidresiduesvaryingbetweenisoformsdiffer
predominantlyinthelengthofsidechains
ThreeclassesofPsbOsequencevariability Analyses of substitutions at positions variable between
Consideringthatmanyangiospermspeciesexpresstwoiso- isoforms showed that some substitutions were more fre-
forms of psbO, we asked whether the differences between quent thanothers.Themostfrequentdifferencesbetween
family α family β
differences
between
families
isoform 1 isoform 2 isoform 1 isoform 2
differences
duplication duplication
between
isoforms
differences
between
species
species A B A B C D C D
Fig.2AschemeofthepsbOphyllogenyshowingthreeclassesofPsbOsequencevariability.Differencesbetweenfamiliesareinblue,differences
betweenisoformsingreenanddifferencesbetweenspeciesinred
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page7of14
isoforms resided in mutualexchanges of glutamic (E) and between residues, which differed only in the length of the
aspartic(D)acidresidues(morethan20%ofallsubstitu- side chain and had similar physicochemical properties,
tions;Fig.3).Distributionofthesetworesidueswithinthe predominated over substitutions between residues with
isoformpairswasusuallyunequal.InPsbOpairincertain more variable character. The three most frequent amino
species,glutamicacidoftenpredominatedinmostofvari- acid substitutions (D-E, I-V and S-T; Fig. 3) match these
ablepositionsinoneisoform,whereasasparticacidinthe criteria and comprise together almost 50 % of all ex-
other (Additional file 6). The total number of these two changes. Though seemingly synonymous, these substitu-
residueswasmoreorlessconstant. Accordingtothisdis- tions are strongly conserved in orthologous isoforms
tributionandtheresiduepresentonposition140(E139in withinfamiliesandinsomecasesevensharedacrossmore
spinach), almost each pair of isoforms could be divided families(seethealignmentinAdditionalfile2).
into the E-type isoform (with predominating longer glu-
tamate) and the D-type isoform (with prevailing shorter Residuesvaryingbetweenisoformsclustertogetheron
aspartate).Accordingtothis, A.thalianaPsbO1clustered thetertiarystructureofPsbO
into D-type isoforms, whereas PsbO2 into E-type, though The positions with amino acids varying predominantly
the divergence in D/E ratio between isoforms was not as between isoforms did not cluster together in the primary
strong as in many other species. PsbOs in the ana- sequence. As protein function is tightly connected with
lysed species with single isoform were either closer to tertiary structure, we decided to analyse spatial location
the E-type or to the D-type isoforms or were in the of amino acid substitutions between PsbO isoforms on
mid-way, e.g. PsbOs from Poaceae speciesorLinumusi- the protein structure. Because no crystal structure of
tatissimum clustered with E-type isoforms, whereas eukaryote PsbO is available, we constructed homologous
PsbOsfromnon-herbaceousRutaceaeorMyrthaceaespe- model of PsbO2 from Solanum tuberosum using PsbO
cieswereclosetoD-typeisoforms(Additionalfile6).The structure from Thermosynechococcus vulcanus [5] as a
D-type isoforms were also often prolonged at C-terminus template (identity of the protein sequences is 47 %). All
withanadditionalaminoacidresidue. PsbO sequences of angiosperms are well comparable on
Exchanges in other amino acid residues were less con- a single model of structure thanks to avery high conser-
servedamongvariousfamilies.Butgenerally,substitutions vation of both the amino acid sequence and the length
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F
0.00
DEIVSTASGSKNNSNTAGAPPSAVIL−Q−SATFIFLKQLMLVTV−TAEAIALDGEIEQFSFVFYGRHQIMIYKSKTLTNQSV
Fig.3Frequencyofaminoacidsubstitutionsbetweenisoforms.Theaminoacidresiduesdifferingoncertainpositioninisoformpairsofanalysed
speciesaregivenbelowthebars,thehyphen(−)representsagap.Fortheanalysis,onerepresentativespecieswithtwoisoformswaschosen
fromeachfamilyinordertoavoidthebiascausedbyvariousnumbersofspecieswithavailabledataineachfamily(analysedspecies:Arabidopsis
thaliana,Artemisiaannua,Lotusjaponicus,Malusdomestica,Manihotesculenta,Populustrichocarpa,Solanumtuberosum,Vitisvinifera,Zeamays,
Zingiberofficinale)
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page8of14
ofthe chain.In the alignmentof 78proteinsequences of the moss Physcomitrella patens with four PsbO isoforms
PsbOs from angiosperms, 59 % of positions are fully (Additional file7).
identical and the length of the chain of mature proteins Before drawing any conclusions, we had to prove that
varies mostly between 247 and 248 amino acid residues this spatial location is specific for differences between
(Additional file2). isoformsanddoesnotreflectahighlevelofgeneralvari-
The isoforms diverged independently in every family, ability in these regions. We compared the position of
so we first mapped the isoform differences on the model isoform differences with between-species differences in
in each family separately. Fig. 4a shows the model of all families (Fig. 4c). We found that differences between
PsbO coloured according to the frequency of differences species (red-coloured in the figure) are more dispersed
between isoforms in species of the Solanaceae family. over the PsbO structure. To allow statistical analysis, we
The differences are situated mostly on the luminal end calculated spatial centres of between-isoform differences
of the β-barrel structure and some differences can be and between-species differences (green and red spheres
found also on the β1-β2 loop. Comparing this location in Fig. 4c, respectively) for each family. The spatial cen-
with positions of differences between isoforms averaged tres of isoform differences are shifted towards the lu-
across all angiosperm families, we can see that the gen- minal end of the β-barrel (with one exception, the
eral pattern is shared (Fig. 4b). Interestingly, the same Salicaceae family, which has the centre of differences be-
pattern is exhibited also in the recently diverged iso- tweenisoformsshiftedtowardstheβ1-β2loopduetohigh
forms of maize with only 6 different amino acids and in frequencyofdifferences in thispartofthestructure). The
a N-terminus 1- 2 loop b
(unknown folding)
5- 6 loop
PSII
luminally exposed
lumen end of -barrel 0.0 0.4 0.8
c d
8
0.
4
0.
0
0.
0.0 0.4 0.8
Fig.4MappingvariableaminoacidresiduesonthePsbOstructure.aDifferencesbetweenisoformsinSolanaceaespeciesand(b)differences
betweenisoformsaveragedacrossallangiospermfamilies.Thevaryingpositionsaregreen-coloureddependingonfrequencyofdifferences
amongtheanalysedpairsofisoforms.cMergeddifferencesbetweenisoforms(ingreen)andbetweenspecies(inred)withequallycoloured
spheresindicatingspatialcentresofthesedifferencescalculatedseparatelyforeachangiospermfamily,thefrequencyofparticulardifferenceson
eachpositionisindicatedbycolourgradient.dMergedaverageddifferencesbetweenisoforms(ingreen),betweenplantfamilies(inblue)or
bothtypes(incyan);onlypositionswithavalueofvariabilityaboveagiventhreshold(0.24)areshowntogetherwithoverallspatialcentresof
differencesbetweenisoforms,species(withinfamilies)andfamilies(green,redandbluespheres,respectively).Thehomologymodelofthe
SolanumtuberosumPsbO2basedontheX-raystructureofcyanobacterialPsbO[PDB:3ARC][5]wasconstructedusingSwiss-Modelprogram[38];
thefirst13N-terminalaminoacidswerenotpresentinthetemplatestructure,sotheywerepastedinthemodelwithoutattemptstoshowany
foldingandtheywerenotincludedincalculationofthespatialcentres
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page9of14
shift of spatial centres of isoform differences compared merged withthe differences between isoforms and overall
with the centres of between-species differences is signifi- spatial centres of the three classes of differences (repre-
cantaccordingtoarandomizationtest(p=0.002). sented with green, red and blue spheres). The differences
betweenfamiliesaremorespreadoverthePsbOstructure
PSII-exposedsurfaceisconserved,whiledifferences compared to the differences between isoforms, similarly
betweenfamiliesaremainlyontheluminalsideofthe to the differences between species within families. The
β5-β6loop highest frequency ofdifferences between familiesis inthe
Mapping of all variable positions on the model of PsbO part of β5-β6 loop that is not interacting with PSII core
structure also showed that the PsbO surface interacting proteins (the amino acid side chains are pointing towards
with PSIIcoreproteins isfully conservedinangiosperms thylakoidlumen)andtheadjoiningpartoftheβ6strand.
with the exception of the β1-β2 loop (see Fig. 5). β1-β2
loop interacts with CP47 protein from the other mono- Discussion
merofPSII[5,9]. MechanismofduplicationandsubfunctionalisationofpsbO
Differences between families are the most frequent Several studies demonstrated that A. thaliana expresses
class of differences among PsbO sequences (Additional two psbO paralogs [17–19, 21]. Here we show that A.
files 2 and 5). Fig. 4d depicts differences between families thaliana is not an exception and that species from 9 out
of 15 investigated angiosperm families also express two
distinct psbOgenes (Additionalfile3). Unexpectedly,the
a phylogenetic analysis revealed that the psbO gene was
1- 2 loop not duplicated in the common ancestor of angiosperms,
but the duplication occurred many times independently
inindividual families(Fig. 1).
There are various mechanisms by which gene duplica-
tion can occur. In terms of its extent, duplication can in-
volve single genes, larger segments, chromosomes or
entire genomes [41]. In A. thaliana and Populus tricho-
carpa we found that chromosomal segments containing
psbOparalogsarecollinear(i.e.containhomologousgenes
in a similar order; Duchoslav,Vosolsobě, and Fischer, un-
published results), which suggests that psbO was dupli-
N-terminus (unknown folding)
catedwithinthecontextofalarger-scaleduplication.
The phylogenetic tree topology indicates that the du-
b
plication event occurred in ancestors of numerous fam-
ilies prior to extensive species radiation. The radiation
that involved many extant plant lineages in Paleogene,
was likely facilitated by the whole genome duplications
(WGD) dated to the last global extinction period at the
Cretaceous–Paleogene boundary about 66 million years
ago [42, 43]. Based on this indirect evidence, we suggest
that the psbO duplication was not gene specific, but ra-
8 ther that the paralogs were in many cases retained after
0.
WGD events that occurred independently in ancestors
4
0. ofmanysuccessfulangiosperm families.
0
0. After WGD, most duplicated genes gradually accumu-
0.0 0.4 0.8
late deleterious mutations and vanish from the genome
Fig.5MappingvariableaminoacidresiduesonthePsbOstructure. (within millions of years). More rarely the duplication
aViewfromthylakoidlumen,(b)viewfromPSII.Differencesbetween leads to neo- or subfunctionalisation of the paralogs if
isoforms(ingreen)aremergedwithdifferencesbetweenspecies(in
these changes improve fitness [41]. Currently, one of the
red);thefrequencyofparticulardifferencesoneachpositionis
best models explaining stabilisation of duplicated genes
indicatedbycolourgradient.ThehomologymodeloftheSolanum
tuberosumPsbO2basedontheX-raystructureofcyanobacterialPsbO is the EAC model of subfunctionalisation (escape from
[PDB:3ARC][5]wasconstructedusingSwiss-Modelprogram[38];the adaptive conflict) [44] based on the fact that a single
first13N-terminalaminoacidswerenotpresentinthetemplate protein can perform multiple catalytic or structural
structure,sotheywerepastedinthemodelwithoutattemptstoshow
functions. In such case, the selective optimization of one
anyfolding
function may lead to a decline in another function,
DuchoslavandFischerBMCPlantBiology (2015) 15:133 Page10of14
creating an adaptive conflict that preserves the single and between-species) is in the part of β5-β6 loop that is
copy gene/protein in an intermediate state. Casual gene not interacting with PSII core proteins (the amino acid
duplication can provide a solution – escape from the side chainsarepointingtowardslumen)andtheadjoining
adaptive conflict via functional specialisation of the part of the β6 strand. In this part of PsbO, there is a very
resultingparalogs[41]. high frequency of between-family differences and a high
Multiple angiosperm species contain just two psbO frequencyofbetween-speciesdifferences,whereasbetween-
paralogs with similar extent of diversification, so we as- isoform differences are nearly absent. This suggests that
sume that the presence of two different PsbO proteins thispartofthePsbOsurfacemightbeinvolvedinbinding
givesanadvantagetothesespecies.Althoughmanyplants ofsomeotherprotein,whoseinteractionsurfacecandiffer
prosper with a single PsbO gene, in species with two iso- inindividualspeciesorfamilies. As isoformsdonot differ
forms, the loss of one isoform negatively affects growth in this region, it seems that both isoforms need to retain
and photosynthesis, e.g. in A. thaliana [18, 19, 23, 45] or this interaction identical. The presence of a hypothetical
potato [25]. It indicates that functions of current diversi- interactor is further supported by the fact that an un-
fied PsbO isoforms are no more equivalent due to sub- assigned density was detected in this part of PSII super-
functionalisationaftertheduplication. complexstructurebycryo-electrontomography[8].
The between-isoform differences were located mostly
StructuralaspectsinPsbOdiversification at the end of the β-barrel protruding into the lumen and
Protein functions are connected with protein structure. on the β1-β2 loop. This pattern was similar in all ana-
Therefore, identification of common structural differ- lysed families and even in the moss Physcomitrella and
ences between isoforms in multiple species can indicate the relatively recently duplicated psbO in maize (Fig. 4,
common functional adaptation. If various plants used Additional file 7). This indicates that the differences be-
duplicated psbOs to solve the same adaptive conflict, the tween isoforms probably enabled the same or similar
structural and functional differentiation of PsbO iso- functional adaptation of PsbOs in all analysed families.
forms would be similar or identical irrespective of inde- Since the psbO duplications were independent, the func-
pendentduplicationinindividualfamilies. tional divergence of PsbO isoforms likely represents a
To evaluate the between-isoform differences, we first parallel evolution, further supporting the impact of ob-
divided the overall variability of PsbOsequences on each serveddiversification ofPsbOisoforms.
position of the primary structure into three classes. The
variability in current PsbO sequences reflects both dif- FunctionaldifferencesbetweenPsbOisoforms
ferences present already in the ancestor species before We found that the location of the differences between
psbO duplication (between-families variability) and dif- PsbO isoforms of A. thaliana fits the pattern found in
ferencesobtainedaftertheduplication,includingspecific other angiosperms. Nine out of 11 different amino acids
diversification of isoforms (between-isoforms variability) are located at the luminal base of β-barrel and one is lo-
and species-specific changes (between-species variability; cated on the β1-β2 loop (Additional file 7). Both PsbO
Fig.2;seequantificationbellowthealignmentinAdditional isoformsofA.thalianaareabletostabilisethemanganese-
file 2). The frequency of each variability class on specific calciumclusterandenablewatersplitting[18,19].PsbO1
positions was mapped on the homology model of PsbO was demonstrated to provide more efficient water split-
(Fig.4).Themodelcorrespondedtootherpublishedhom- ting [18], whereas PsbO2 was reported to have higher
ologymodelsof higherplants’PsbO[9,10].Themapping GTPase activity and was proposed to participate in D1
showed that occupancy of the differences on the PsbO repaircycle[19,21,23].
surfacewas unequal and the locations ofthe three classes Thehighestfrequencyof between-isoformdifferencesis
ofvariabilitysignificantlydiffer. located just around the hypothetic GTP-binding site pre-
There are practically no differences between isoforms dicted by Lundin et al. [10], which is situated inside the
on the PSII-binding surface of PsbO (Fig. 5). However, luminal end of the β-barrel. Lundin et al. found hypo-
Murakami et al. [18] reported that PsbO2 of A. thaliana thetic non-canonical GTP-binding domains in spinach
is less efficient in reconstitution of oxygen evolution [10] and A. thaliana PsbO sequence [21]. G1 domain,
in vitro compared to PsbO1. Our analysis showed that binding α-phosphate, was predicted in β1 sheet, G2-G3
the PSII-binding surface is highly conserved in all angio- domain,bindingγ-phosphate,inβ2sheetandG4domain,
sperms. It indicates that the differences in water oxida- bindingguanine ring, in β4-β5 loop (marked in the align-
tion observed by Murakami et al. [18] were not caused ment in Additional file 2). Regions surrounding the G2-
by direct modulation of water oxidation on Mn CaO G3 domain, i.e. β1-β2 and β2-β3 loops, were predicted to
4 5
cluster,butratherbysomeindirect effect. be Switches I and II, respectively. These switches could
The biggest contrast in localisation of between-isoform have different conformations in GDP- and GTP-bound
differences and other types of differences (between-family state.