Evolution of C4 photosynthesis in the genus Flaveria: how many and which genes does it take to make C4?

Abstract

Selective pressure exerted by a massive decline in atmospheric CO(2) levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C(4) photosynthesis. C(4) species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C(4) trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C(3) and C(4) species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C(3) to C(3)-C(4) intermediate to C(4) species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C(4)-associated characteristics. Single gene analysis defines the C(4) cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C(4) associated pathways. Molecular evidence for a photorespiratory CO(2) pump prior to the establishment of the C(4) cycle-based CO(2) pump is provided. Cluster analysis defines the upper limit of C(4)-related gene expression changes in mature leaves of Flaveria as 3582 alterations.

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Evolution of C4Photosynthesis in the Genus Flaveria: How
Many and Which Genes Does It Take to Make C4?
W
Udo Gowik,a,1Andrea Bra ¨utigam,bKatrin L. Weber,bAndreas P.M. Weber,band Peter Westhoffa
aInstitute of Plant Molecular and Developmental Biology, Heinrich-Heine-University, 40225 Duesseldorf, Germany
bInstitute of Plant Biochemistry, Heinrich-Heine-University, 40225 Duesseldorf, Germany
Selective pressure exerted by a massive decline in atmospheric CO2levels 55 to 40 million years ago promoted the evolution
of a novel, highly efficient mode of photosynthetic carbon assimilation known as C4photosynthesis. C4species have
concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex
C4trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of
related C3and C4species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C3to C3-
C4intermediate to C4species, to quantify the differences in the transcriptomes of closely related plant species with varying
degrees of C4-associated characteristics. Single gene analysis defines the C4 cycle enzymes and transporters more
precisely and provides new candidates for yet unknown functions as well as identifies C4associated pathways. Molecular
evidence for a photorespiratory CO2pump prior to the establishment of the C4cycle-based CO2pump is provided. Cluster
analysis defines the upper limit of C4-related gene expression changes in mature leaves of Flaveria as 3582 alterations.
INTRODUCTION
C4plants are characterized by high rates of photosynthesis and
efficient use of water and nitrogen resources. High photosyn-
thetic rates are achieved by addition of a new metabolic path-
way, the C4cycle, in which the initial product of CO2fixation is a
four-carbon (C4) organic acid rather than a three-carbon (C3)
organicacid.InmostC4species,C4photosynthesisinvolvestwo
different cell types, mesophyll and bundle sheath cells. Only few
species have been described that carry out a C4cycle within a
single cell (Edwards et al., 2004). As shown in Figure 1A, in an
NADP-dependent malic enzyme type C4plant, CO2is initially
fixed in the mesophyll cells by phosphoenolpyruvate carboxyl-
ase (PEPC), which converts three-carbon phosphoenolpyruvate
(PEP) into four-carbon oxaloacetate (OAA). OAA is converted
into a transport form (malate or aspartate) by malate dehydro-
genase (MDH) or aspartate aminotransferase (Asp-AT), respec-
tively, and is then transported to the bundle sheath. Following
decarboxylation of malate by NADP-malic enzyme (NADP-ME),
the CO2is refixed by ribulose 1,5-bisphosphate carboxylase/
oxygenase (Rubisco), producing 3-phosphoglycerate that is
further converted to triose phosphate. The pyruvate produced
from malate (or its aminated form, Ala) is transferred back to the
mesophyll where PEP is regenerated by pyruvate orthophos-
phate dikinase (PPDK) (Hatch, 1987).
C4plants show drastically reduced rates of photorespiration
because CO2isconcentrated atthesiteofRubiscoandisableto
outcompete molecular oxygen, which, when used by Rubisco,
results in photorespiration. Close contact between mesophyll
andbundlesheathcellsisvitalforC4photosynthesis,andtheleaf
structure of C4plants is altered compared with most C3plants.
The bundle sheath cells are enlarged, the interveinal distance is
reduced,andtheleafthicknessislimitedtomaximizethecontact
of mesophyll and bundle sheath cells (Dengler and Nelson,
1999). This pattern is called Kranz anatomy (Haberlandt, 1904).
To guarantee the high flux of metabolites between the two cell
types,theyareconnectedvianumerousplasmodesmata(Botha,
1992). The CO2pump ensures high rates of photosynthesis even
whenCO2concentrationsarelowintheintercellularairspacesof
the leaf. Therefore, C4plants are able to limit the opening of their
stomataandminimizewaterlossduetotranspiration.AstheCO2
pump delivers saturating concentrations of CO2to the site of
Rubisco, high photosynthetic rates are maintained with less
Rubisco than is required in C3species. This is reflected in higher
nitrogen use efficiency (Long, 1999).
While the basic biochemistry of the C4cycle is well under-
stood,ourknowledgeaboutothergenesandproteinsneededfor
efficient C4photosynthesis is limited. For example, we have not
identified yet all the transporters that ensure the increased inter-
and intracellular metabolic fluxesnor the genesthat regulate and
maintain the alterations in cell and overall leaf morphology.
C4photosynthesisevolvedseveraltimesindependentlyduring
the evolution of higher plants. It originated at least 32 times in
eudicots and 16 times in monocots (Sage, 2004; Muhaidat et al.,
2007). These multiple independent origins of C4photosynthesis
suggest that the evolution of a C3into a C4species must have
beenrelativelyeasyingeneticterms(WesthoffandGowik,2010).
Recently, the C4syndrome has been investigated at the systems
level by comparing the transcriptome of a C4to a closely related
C3species(Bra ¨utigametal., 2011).Approximately600transcripts
were differentially expressed at a significant level. While many of
1Address correspondence to gowik@uni-duesseldorf.de.
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) are: Udo Gowik
(gowik@uni-duesseldorf.de) and Peter Westhoff (west@uni-duesseldorf.de).
WOnline version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.111.086264
This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
The Plant Cell Preview, www.aspb.org ã 2011 American Society of Plant Biologists. All rights reserved.1 of 19

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the transcriptional changes could be placed into a C4context, the
questionofwhich and how manyof the changes are related tothe
C4syndrome rather than to the evolutionary distance of the two
species remained open.
To get an insight how many and which genes were altered
during C4evolution, we performed a comparative transcriptome
analysis of leaves of closely related C3, C4, and C3-C4interme-
diate species of the genus Flaveria. This genus is very valuable
for investigating the evolution of the C4pathway because, in
addition to having closely related C3and C4species of the NADP-
ME type, it also contains a large range of C3-C4-intermediate
species differing in the degree of “C4ness” (Figure 1B).
Since no species of the genus Flaveria is a model organism
with a known genome sequence and consequently no micro-
arrays are available, we used massively parallel pyrosequencing
of mRNAs (RNA-Seq) to analyze the leaf transcriptomes of C3,
C4, and C3-C4intermediate Flaveria species. This digital gene
expression analysis (DGE) was based on generating random
sequence tags that were proportional to the abundance of the
corresponding transcripts in a particular sample and was shown
tobeusefulforcomparingsteadystatetranscriptlevelsinrelated
nonmodelspecies(Bra ¨utigamandGowik,2010;Bra ¨utigametal.,
2011). The leaf transcriptomes of Flaveria bidentis (C4) and
Flaveria pringlei (C3) were analyzed by the 454-FLX technology,
and thenewer454-TITANIUM technology wasusedto sequence
the leaf transcriptomes of Flaveria trinervia (C4), Flaveria robusta
(C3), and Flaveria ramosissima (C3-C4).
RESULTS
Carbon Isotope Discrimination in the Different
Flaveria Species
Plants discriminate against13CO2during CO2uptake because of
the different diffusivity of13CO2and12CO2and the preference of
Rubisco for12CO2. In C4plants, this effect is less pronounced
due to the CO2concentration mechanism. Thus, C3and C4
plants can be distinguished by the carbon isotope composition
oftheirdrymatter(O’Leary,1981).Toconfirmthephotosynthetic
types under greenhouse conditions, the carbon isotope ratios of
the five Flaveria species investigated in this study were analyzed
by determining the d13C values of dried leaf material (see Sup-
plemental Table 1 online). The d13C values of the C4species are
12 to 15‰ higher (less negative) than the d13C values of the C3
species. The d13C value of the C3-C4intermediate species F.
ramosissima is C3like (see Supplemental Table 1 online). These
results echo earlier results (Monson et al., 1986; Edwards and
Ku, 1987; Monson et al., 1988) showing that most C3-C4inter-
mediate Flaverias, including F. ramosissima, exhibit C3 like
carbon isotope ratios, although F. ramosissima fixes almost
50% of CO2via the C4pathway. Hence, the Flaverias under
investigation behave as expected under our conditions.
454 Sequencing of Flaveria Leaf Transcriptomes
Toidentifydifferencesintranscriptabundancerelatedtoaspects
of the C4syndrome, the leaf transcriptomes of F. bidentis (C4)
and F. pringlei (C3) were compared. Also, in a second exper-
iment, the leaf transcriptomes of F. trinervia (C4), F. robusta (C3),
and F. ramosissima (C3-C4) were similarly compared. The anal-
ysis of gene expression in five species rather than a species pair
reduced the probability of detecting species specific rather than
C4-specificdifferences.OnesequencingrunonaGSFLXsystem
was conducted on the cDNA libraries from both F. bidentis and
F. pringlei, leading to 135,855,412 and 114,292,070 nucleotides
of raw sequences, respectively. For F. trinervia, F. robusta, and
F. ramosissima, the more advanced 454 TITANIUM technology
wasused,leadingto285,219,596,308,800,825and333,275,756
nucleotides of raw data. After quality control and processing,
this resulted in 527,596 clean reads from F. bidentis and 448,627
clean reads from F. pringlei with a mean read length of 229
nucleotides for both species, 974,217 clean reads from
Figure 1. The Genus Flaveria as a Model Organism to Study C4Evolution.
(A) Schematicview of the NADP-ME type C4pathwayas it can be found in C4Flaveria species modified from Gowik and Westhoff (2011). See the text for
abbreviations and a detailed description of the pathway.
(B) Phylogeny of the genus Flaveria according to McKown et al. (2005).
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F. trinervia, 871,850 clean reads from F. robusta, and 1,096,348
clean reads from F. ramosissima with mean read lengths of 286,
349, and 297 bp, respectively (Table 1).
Clean reads were aligned to a minimal set of coding se-
quences of the Arabidopsis thaliana transcriptome (http://www.
Arabidopsis.org/), as described by Bra ¨utigam et al. (2011) to
minimizeerroneousreadmappingtogenesthathavearisenfrom
segmental or tandem gene duplications in the Brassicacean
lineage (Bra ¨utigam and Gowik, 2010). The alignment was per-
formed in protein space using the BLAST-like alignment tool
BLAT (Kent, 2002), and the best hit for each 454 read was
retained. Between 66.6 and 72.7% of the reads from each
Flaveria species could be mapped onto the Arabidopsis tran-
scriptome (Table 2). The quantitative data for all genes detected
can be found in Supplemental Data Set 1 online.
ESTs corresponding to 55, 55, 58, 60, and 61% of the
Arabidopsis transcripts included in the minimal coding se-
quences set were identified in the individual leaf cDNA libraries
of F. bidentis, F. pringlei, F. trinervia, F. robusta, and F. ramo-
sissima, respectively. This indicated that the leaf transcriptomes
oftheFlaveriaspeciesweresampledtoacomparableextent.We
examined the coverage of different functional gene classes to
test whether the data sets and the mappings for the different
species within each experiment were comparable. For most
functional classes transcriptsrepresenting morethan50%of the
genes were detected (Table 3) in each of the five species. The
classes putative lipid transfer protein, defense, and function
unknown were the only ones underrepresented in the F. robusta/
F. ramosissima/F. trinervia experiment as well as in the F.
pringlei/F. bidentis experiment. The coverage of the individual
functional classes was comparable for all species (Table 3).
Differential gene expression within each experiment (F. biden-
tis versus F. pringlei and F. trinervia versus F. robusta) was
determined using Poisson statistics (Audic and Claverie, 1997)
followed by a Bonferroni correction to account for multiple
parallel testing. Among the 13,574 transcripts captured in the
F. bidentis/F. pringlei experiment, the abundance of 463 tran-
scripts differed significantly (P < 0.01) between the C3and the C4
plant. Two hundred transcripts were more abundant and 263
transcripts less abundant in the C4plant F. bidentis compared
with the C3plant F. pringlei (Table 2; see Supplemental Data Set
1 online). The combined ESTs of F. trinervia (C4) and F. robusta
(C3) correspond to 14,304 transcripts. A total of 410 transcripts
were significantly (P < 0.01) more and 585 transcripts less
abundant in the C4plant.
To independently confirm the DGE results, quantitative RT-
PCR experiments were performed on three leaf RNA isolates of
F. bidentis and F. pringlei, the RNA used for 454 sequencing and
two independent isolates. The results obtained with cDNA used
for RNA-Seq as well as the mean values from three experiments
stronglycorrelatedwiththeresultsfromDGE(R2=0.95and0.86,
respectively; see Supplemental Figure 1 online), indicating the
reliability of the expression ratios estimated by RNA-Seq.
A Number of Functional Classes Differ between C4and
C3Species
Transcripts of genes known to be involved in the C4cycle, the
photosynthetic electron transport and CO2fixation, and photo-
respiration showed pronounced differences between C3and C4
(Figure 2). A high percentage of the genes contained in the
functional class of potential C4cycle genes showed strong and
significant upregulation in the C4plants in both the F. bidentis/
F. pringlei and the F. trinervia/F. robusta comparison. Other
classes with high percentages of significantly more highly ex-
pressed genes in both experiments are glycolysis and the
oxidative pentose phosphate pathway, whereas genes related
to nitrogen metabolism and the shikimate pathway were signif-
icantly downregulated in the C4species.
To complement this analysis, we searched for functional
classes showing significant differential expression between the
C3and C4species using overrepresentation analysis by the
PageMan Software (Usadel et al.,2006). Thissoftwareconsiders
the changes of all genes within a functional class. These are
compared with the changes of all genes observed within the
whole experiment to predict functional classes that exhibit
differential expression compared with all the other remaining
functional classes. In both experiments, next to the not assigned
class and classes related to photosynthesis, several classes
associated with protein metabolism, especially the ribosomal
proteins, show differential expression profiles compared with all
other functional classes (Figure 3). Since Pageman and Mapman
aredesigned forArabidopsis,aC3plant,nofunctional C4classis
annotated in these tools.
Table 1. Results of the 454 Sequencing Runs
Data F. trinervia (454 T) F. robusta (454 T) F. ramosissima (454 T) F. bidentis (454 F) F. pringlei (454 F)
Raw reads
Raw nucleotides
Raw mean length
Clean and processed reads
Clean nucleotides
Clean and processed mean length
Reads mapped on TAIR9 coding sequences
Reads mapped (%)
966,609860,8861,084,773
333,275,756
515,931444,438
285,219,596308,800,825 135,855,412114,292,070
295 358307 263257
974,217871,850 1,096,348
326,109,740
527,596448,627
278,160,925299,789,666120,768,247102,606,567
286349 297229229
648,969 634,109759,862 368,342326,108
66.672.769.369.872.7
F. trinervia, F. ramosissima, and F. robusta cDNA libraries were sequenced using 454 TITANIUM (454 T), whereas F. bidentis and F. pringlei cDNA
libraries were sequenced using 454 FLX (454 F) chemistry. The raw reads from 454 sequencing were processed (exclusion of low-quality reads,
elimination of adaptor sequences, and separation of sequence reads joined via concatemerization) to obtain clean reads.
Evolution of C4Photosynthesis3 of 19

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C4CycleGenesAreStronglyUpregulatedintheC4Flaverias
Transcripts encoding the proteins necessary for the NADP-ME
type of C4photosynthesis were significantly upregulated in the
C4plants F. trinervia and F. bidentis compared with the C3
plants F. robusta and F. pringlei. The biggest difference, with a
180/125-fold higher transcript abundance, was PPDK followed
by the PEPC with a 134/47-fold upregulation (Figure 4A; see
Supplemental Table 2 online). Also, the abundance of tran-
scripts for NADP-ME and the chloroplastidic MDH was 6/7 and
14/23timeshigherintheC4plants.Withanabsoluteabundance
ranging from 3515/3,678 (MDH) to 34,365/15,887 (PEPC) reads
per million (rpm), all these transcripts belong to the most
abundant transcripts in F. trinervia and F. bidentis. Also, Ala
aminotransferases and one transcript encoding an ASP-AT
were upregulated in the C4plants. This confirms that amino
acids, in addition to malate, are also used as transport metab-
olites in the C4Flaverias.
The adenosine monophosphate kinase gene was found to be
strongly upregulated in both C4 species. In the F. bidentis/
F. pringlei experiment, we also identified two significantly upreg-
ulated inorganic pyrophosphatases (see Supplemental Table 2
online).
Transcripts encoding regulatory factors for C4cycle proteins,
the PEPC kinase (PEPC-K) and the PPDK regulatory protein
(PPDK-RP), were upregulated as well (4- to 46-fold for PEPC-K
and 2- to 13-fold for PPDK-RP), although their absolute abun-
dance is clearly lower than that of the C4enzymes (73 to 176 rpm
for PEPC-K and 120 to 274 rpm for PPDK-RP).
The genes encoding the enzymes necessary for the NAD-ME
or phosphoenolpyruvate carboxykinase (PEP-CK) C4subtype,
such as the mitochondrial NAD-dependent malate dehydrogen-
ase (mNAD-MDH), the mitochondrial NAD-dependent malic
enzyme (mNAD-ME), cytoplasmatic or mitochondrial ASP-ATs,
or the cytoplasmic PEP-CK, show only low or moderate expres-
sionandtheC4-toC3-associateddifferencesweresmallandnot
significant (Figure 4A; see Supplemental Table 2 online), indi-
cating that the true C4 Flaveria species exclusively use the
NADP-ME pathway as reported earlier (Drincovich et al., 1998).
This is supported by the extractable protein activities and steady
state metabolite amounts (see Supplemental Table 3 and Sup-
plemental Table 4 online). PEP-CK activity is increased in C4
albeit much less compared with the major decarboxylation
enzyme NADP-ME.
In F. ramosissima (C3-C4), Next to the NADP-ME Type C4
Cycle,TypicalNAD-METypeC4GenesAlsoAreUpregulated
In the C3-C4intermediate plant F. ramosissima, the transcripts of
the genes related to the NADP-ME type C4 photosynthesis
showed intermediate amounts compared with the C3plant F.
robustaandtheC4plantF.trinervia(Figure4A;seeSupplemental
Table 2 online). The amounts of all these transcripts were
significantly higher than in the C3plant F. robusta, implying that
in F. ramosissima, the C4cycle is working to a certain extent and
that F. ramosissima is a true intermediate based on its transcrip-
tional profile. By contrast, the transcript abundance for the Ala
aminotransferase gene in F. ramosissima was higher than in the
C4species F. trinervia.
Additionally, cytoplasmic and mitochondrial ASP-AT genes
and an mNAD-MDH were upregulated significantly in F. ra-
mosissima compared with the C3 and the C4 species. Two
mNAD-MEs were upregulated in F. ramosissima, whereas the
differences weresignificant onlyforonegene incomparison with
the C4plant F. trinervia (Figure 4A; see Supplemental Table 2
online). Accordingly, the extractable NAD-ME activity in the
Table 2. Mapping Results for the 454 Reads
Species ComparisonTranscriptomesNo. of Loci Percentage of Total Loci in TAIR9
TAIR 9 (minimalized transcriptome)
F. trinervia
F. robusta
F. ramosissima
F. trinervia + F.robusta
F. trinervia
F. robusta
F. trinervia + F. ramosissima
F. trinervia
F. ramosissima
F. ramosissima + F. robusta
F. ramosissima
F. robusta
F. bidentis
F. pringlei
F. bidentis + F. pringlei
F. bidentis
F. pringlei
21,972
12,817
13,264
13,534
14,304
410
585
14,371
344
503
14,547
385
369
12,164
12,254
13,574
200
263
58.3
60.4
61.6
65.1
1.9
2.7
65.4
1.6
2.3
66.2
1.8
1.7
55.4
55.8
61.8
0.9
1.2
Ft/Fro comparison more abundant (P < 0.01)
Ft/Fra comparison more abundant (P < 0.01)
Fra/Fro comparison more abundant (P < 0.01)
Fb/Fp comparison more abundant (P < 0.01)
Reads were mapped to a minimal version of the Arabidopsis transcriptome in the protein space using BLAT. Total numbers of transcripts detected by
at least one read and the numbers of significantly differentially abundant transcripts (P < 0.01) in all possible species-by-species comparisons are
given along with the corresponding percentage of the total loci. Ft, F. trinervia; Fra, F. ramosissima; Fro, F. robusta; Fb, F. bidentis; Fp, F. pringlei.
4 of 19The Plant Cell

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Table 3. Qualitative Patterns of Transcript Abundance in the Leaves of F.trinervia, F. ramosissima, F. robusta, F. bidentis, and F. pringlei
Functional Class Genes Detected in:
F. trinervia
58.33%
91.30%
86.96%
87.76%
37.50%
75.00%
91.67%
90.00%
94.12%
92.00%
70.23%
90.48%
93.48%
80.77%
100.00%
93.02%
91.43%
85.48%
90.32%
84.62%
100.00%
93.65%
92.23%
91.67%
89.02%
68.83%
68.33%
76.07%
100.00%
60.57%
72.00%
55.00%
82.29%
76.72%
76.71%
88.97%
73.44%
93.75%
84.84%
83.33%
60.93%
82.11%
85.77%
89.29%
60.00%
56.25%
58.75%
80.88%
81.11%
67.21%
68.24%
80.77%
50.38%
39.01%
21.11%
73.73%
42.06%
F. robusta
60.37%
91.30%
95.65%
89.80%
62.50%
85.71%
91.67%
90.00%
100.00%
92.00%
72.52%
95.24%
91.30%
84.62%
100.00%
93.02%
97.14%
86.77%
90.32%
92.31%
100.00%
93.65%
96.12%
93.75%
93.90%
74.03%
73.75%
80.48%
100.00%
63.09%
73.60%
60.00%
85.94%
80.95%
80.76%
94.12%
71.88%
93.75%
87.43%
91.67%
62.31%
82.63%
86.39%
89.88%
60.00%
62.50%
57.11%
80.88%
82.12%
68.70%
68.24%
80.77%
56.49%
45.05%
24.44%
76.27%
44.22%
F. ramosissima
61.60%
91.30%
95.65%
93.88%
50.00%
89.29%
91.67%
90.00%
94.12%
92.00%
80.92%
90.48%
93.48%
76.92%
100.00%
93.02%
85.71%
90.32%
96.77%
84.62%
100.00%
95.24%
95.15%
97.92%
93.90%
76.19%
75.83%
82.12%
100.00%
64.04%
77.07%
67.50%
86.46%
82.01%
81.67%
94.12%
70.31%
93.75%
88.72%
100.00%
63.70%
84.74%
87.64%
90.48%
66.67%
62.50%
60.79%
82.35%
84.76%
69.30%
76.35%
84.62%
56.49%
47.80%
24.44%
77.12%
44.67%
F. bidentis
55.36%
91.30%
91.30%
91.84%
75.00%
82.14%
91.67%
90.00%
94.12%
92.00%
70.99%
76.19%
93.48%
65.38%
100.00%
88.37%
82.86%
82.90%
90.32%
92.31%
100.00%
90.48%
94.17%
91.67%
90.24%
67.10%
61.67%
72.54%
100.00%
53.31%
70.40%
55.00%
81.77%
73.54%
73.91%
90.44%
68.75%
100.00%
85.58%
83.33%
58.65%
80.00%
81.02%
85.71%
56.67%
56.25%
52.12%
70.59%
76.45%
64.98%
62.16%
76.92%
54.96%
35.71%
22.22%
68.64%
39.44%
F. pringlei
55.77%
91.30%
86.96%
89.80%
50.00%
82.14%
66.67%
90.00%
94.12%
92.00%
66.41%
90.48%
93.48%
76.92%
100.00%
95.35%
88.57%
86.45%
90.32%
84.62%
100.00%
88.89%
93.20%
91.67%
90.24%
71.00%
62.50%
76.70%
100.00%
59.31%
68.80%
62.50%
82.81%
69.31%
74.90%
90.44%
62.50%
93.75%
83.92%
100.00%
59.64%
80.53%
82.02%
89.88%
56.67%
56.25%
53.20%
76.47%
77.20%
62.89%
64.19%
84.62%
48.85%
36.26%
21.11%
74.58%
39.29%
All
C4candidate
Photosynthesis, photosystem I
Photosynthesis, photosystem II
Photosynthesis, cytochrome b6/f
Photosynthesis, cef
Photosynthesis, ATPase
Photosynthesis, other
Calvin cycle
Tricarboxylic acid cycle
Mitochondrial electron transfer/ATPase
OPPP
Glycolysis
Other central carbon metabolism
Photorespiration
Starch
Suc
Lipids
Nitrogen metabolism
Shikimate pathway
One-carbon compound metabolism
Amino acid metabolism
Nucleotide metabolism
Pigment synthesis
Cofactor synthesis
Secondary metabolism
Enzyme, other
Enzyme, putative
Sulfur assimilation
Cell wall synthesis
Minor sugar metabolism
b 1.3 Glucan metabolism
Vesicle trafficking
Cytoskeleton
Other cellular processes
Chloroplast process
Mitochondrial process
Peroxisomal process
Protein synthesis
Protein modification
Protein degradation
Heat shock/protein folding
Transport
Redox regulation
Metal handling
Storage protein
Transcriptional regulation
Epigenetic regulation
Posttranslational modification (phosphate)
Other regulatory processes
Hormone metabolism
Hormone signaling
Hormone-responsive genes
Defense
Putative lipid transfer proteins
Not classified
Function unknown
Different functional classes are represented by comparable fractions of genes detected in the leaf cDNA libraries from the different species.
Evolution of C4Photosynthesis 5 of 19

Page 6

leaves of F. ramosissima was significantly higher than in the
other four Flaveria species (Figure 4B; see Supplemental Table
3 online). We further analyzed the steady state amounts of
metabolites, including those associated with the C4pathway.
In F. ramosissima, the Ala level was comparable to those found
in the two C4species; however, the Asp level exceeded those
of all other Flaverias (Figure 4C; see Supplemental Table 4
online).
C4-Related Transport
C4photosynthesis requires the transport of large amounts of
metabolites across the chloroplast envelope, and this transport
is not necessary in C3plants (Bra ¨utigam et al., 2008; Weber and
von Caemmerer, 2010; Bra ¨utigam and Weber, 2011). Our ex-
periments confirmed the importance of the plastidic phospho-
enolpyruvate phosphate translocator and the triosephosphate
Figure 2. The Quantitative Patterns of Transcript Accumulation in C3and C4Flaverias Are Distinct.
(A) Comparison of F. trinervia (Ft, C4) and F. robusta (Fro, C3).
(B) Comparison of F. bidentis (Fb, C4) and F. pringlei (Fp, C3). Shown are the percentages of genes with significantly higher abundance of transcripts in
the C4species (green bars), percentages of genes unchanged (gray bars, including genes not detected), and percentages of genes with significantly
lower abundance of transcripts in C4species (yellow bars). Percentages are based on the total number of genes in each annotation class (values in
parentheses on the y axis). TCA, tricarboxylic acid.
6 of 19The Plant Cell

Page 7

phosphate translocator for the C4 pathway since they were
upregulated in the C4Flaverias (Figure 4A; see Supplemental
Table 2 online), confirming earlier results from other C4species
(WeberandvonCaemmerer,2010;Bra ¨utigamandWeber,2011).
The Flaveria species belong to the group of pyruvate sodium
symporter C4plants (Aoki et al., 1992). A gene annotated as bile
acid sodium symporter was dramatically upregulated in the C4
compared with the C3Flaveria species. BASS 2 protein is a
pyruvate sodium symporter (T. Furumoto, T. Yamaguchi,
Y. Ohshima-Ichie, M. Nakamura, Y. Iwata, M. Shimamura,
Y. Takahashi, J. Ohnishi, S. Hata, U. Gowik, P. Westhoff,
A. Bra ¨utigam, A. Weber, and K. Izui, unpublished data). To avoid
massive sodium imbalance across the chloroplast envelope,
BASS 2 acts in concert with a sodium proton antiporter (NHD),
Figure 3. Overrepresentation Analyses of Up- and Downregulated Genes within Functional Gene Classes Defined by MapMan Bins.
Fisher’s exact test followed by the Bonferroni correction was used to identify functional categories enriched in up- or downregulated genes when
transcript abundances in F. trinervia (Ft, C4) and F. robusta (Fro, C3), F. bidentis (Fb, C4) and F. pringlei (Fp, C3), or F. ramosissima (Fra, C3-C4) and F.
robusta (Fro, C3) were compared. Blue, up- or downregulated genes are significantly overrepresented; red, up- or downregulated genes are significantly
underrepresented. aa, amino acid; LHC, light-harvesting complex; PS, photosynthesis.
Evolution of C4Photosynthesis 7 of 19

Page 8

tying pyruvate import to the proton gradient (T. Furumoto, T.
Yamaguchi, Y. Ohshima-Ichie, M. Nakamura, Y. Iwata, M.
Shimamura, Y. Takahashi, J. Ohnishi, S. Hata, U. Gowik, P.
Westhoff,A.Bra ¨utigam,A.Weber,andK.Izui,unpublisheddata).
In addition to BASS 2, a NHD was highly upregulated in both C4
species compared with the C3Flaverias. The chloroplast dicar-
boxylate transporter 1 (DiT1) catalyzes the exchange of malate
and OAA in addition to malate and 2-oxoglutarate and is ex-
pressed in the mesophyll of the NADP-ME grasses maize (Zea
mays) and sorghum (Sorghum bicolor; Kinoshita et al., 2011).
DiT1 as well as the chloroplast DiT2 were significantly upregu-
lated in the C4plants F. bidentis and F. trinervia compared with
the C3species (Figure 4A; see Supplemental Table 2 online). An
additional gene belonging to the bile acid sodium symporter
family,BASS 4wasupregulated to acomparable extentintheC4
species (see Supplemental Table 2 online).
Several other transport proteins of unknown function displayed
a C4 accumulation pattern. These candidate C4 transporters
Figure 4. Differences in C4Pathway Gene Expression for F. trinervia (C4), F. ramosissima (C3-C4), F. robusta (C3), F. bidentis (C4), and F. pringlei (C3).
(A) Schematic view of the NADP-ME type C4pathway. Relative transcript abundances are given in small inset boxes. The transcript levels for F. trinervia,
F. ramosissima, and F. robusta were normalized by setting the F. robusta transcript level to one, and the F. bidentis and F. pringlei transcript levels were
normalized by setting the F. pringlei transcript level to one for each gene.
(B) Activity of NAD-ME in the extractable enzyme fractions of leaves from all five species (+ SE; n = 3). FW, fresh weight.
(C) Ala and Asp amounts in the leaves of all five species (+ SE; n = 3).
8 of 19The Plant Cell

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included a magnesium/proton exchanger, a high affinity potas-
sium transporter, and the three chloroplastic cation exchangers
CAX1, CAX3, and CAX4, which were all more highly expressed in
the C4plants. Some transporter protein genes were significantly
downregulated in the C4species: two for sugar transporters and
the two for the water channel proteins TIP2;2 and PIP2B, respec-
tively. A transcript encoding a putative voltage-dependent anion
channel 1 (VDAC1) was also less abundant in the C4plants.
PhotorespirationIsDownregulatedinC4butUpregulatedin
the C3-C4Intermediate Species
The highest percentage of genes downregulated in the C4
species in both experiments belonged to the photorespiration
class (Figure 2). Nearly all of the genes within this class were
downregulated in the C4species, and for nearly 50% of them,
the differences are statistically significant (Figure 5A). This was
also true for the genes related to the reassimilation of photo-
respiratory ammonium by the plastidic Gln synthase and the
ferredoxin-dependent Glu synthase but not for the transporters
DiT1 and DiT2, which catalyze the 2-oxoglutarate/Glu exchange
across the plastid membrane (see above). Flux through the
photorespiratory pathway is reduced in C4 plants compared
with C3plants (Leegood, 2002; Sage, 2004), and, at least for
this pathway, transcript abundance mirrors flux (Bra ¨utigam
et al., 2011; this article).
Surprisingly, the C3-C4intermediate F. ramosissima did not
show intermediate characteristics. By contrast, transcript abun-
dances for most genes related to photorespiration in the C3-C4
intermediate species F. ramosissima were higher than in the C3
species F. robusta, and for more than one half of them, this
difference was statistically significant. In addition to the tran-
script amounts, both the steady state amount of Gly as well as
the steady state amount of Ser increased, while glycolate and
glycerate amounts remained comparable to the C3 and C4
species (Figure 5B; see Supplemental Table 4 online).
Photosynthetic Electron Transport and Calvin-Benson
Cycle Were Modified during C4Evolution
Within the Calvin-Benson cycle class, most genes showed lower
transcript abundance in the C4 than in the C3 plants. The
strongest differences were found for the genes encoding the
small subunit of the Rubisco, which were downregulated 4.5-
to 12.5-fold in the C4Flaverias. In the C3-C4intermediate F.
ramosissima, the transcript abundance of most Calvin-Benson
cycle geneswasC3like withthe exception ofthesmallsubunit of
Rubisco, which was significantly downregulated, mirroring ear-
lier investigations on Rubisco protein amounts in C3-C4inter-
mediate Flaveria species (Wessinger et al., 1989).
Theclasseswithgenesinvolvedinthephotosyntheticelectron
transport showed heterogeneous characteristics (see Supple-
mental Figure 2 online). Photosystem I genes were upregulated
to a higher percentage in the C4plants, whereas more of the
photosystem II genes were downregulated in both C4species.
The class of genes related to the cyclic electron transfer was one
of the classes containing the highest fraction of significantly
upregulated genes in F. trinervia compared with F. robusta as
wellasinF.bidentiscomparedwithF.pringlei.Sincemostgenes
encoding the ATPase and the cytochrome b6f complex are
encoded on the chloroplast genome, they were not analyzed in
the experiments. In F. ramosissima, several genes related to the
cyclic electron transfer as well as of photosystem I showed
intermediate abundance compared with the C3and C4species,
and others were at the level found in the C3plant. Most tran-
scripts related to photosystem II show intermediate character-
istics as many of them are downregulated compared with the C3
plant F. robusta but not as much as much as in the C4plant F.
trinervia.
Chloroplast Biogenesis and Maintenance Is Altered in
C4Species
Several genes involved in chloroplast biogenesis and mainte-
nance were differentially expressed between the C3 and C4
Flaverias (see Supplemental Table 5 online). Among these are
genes encoding proteins of so far unknown function, which are
predicted as being localized in the plastids, making them can-
didates for further analysis.
HCF101 and HCF107 are involved in the biogenesis of pho-
tosystem I and photosystem II, respectively (Lezhneva et al.,
2004; Sane et al., 2005), and were found to be upregulated in
the C4species. Several DnaJ proteins with unknown function
behaved similarly. Plastidic DnaJ proteins are involved in the
stabilizationofthylakoidmembranecomplexeslikephotosystem
II (Chen et al., 2010). Several proteases belonging to Clp (ClpR1
and ClpP5) and FtsH (FtsH8, VAR1, and VAR2) complexes were
upregulated, too. While the Clp complex is essential for chloro-
plast biogenesis (Kim et al., 2009), the FtsH complex is mainly
involved in the maintenance of photosystem II function (Kato
et al., 2009).
The two chloroplast RNA binding proteins CSP41A and
CSP41B were downregulated in the C4species. These proteins
playaroleintheexpressionofplastidgenesandmaybeinvolved
in the biogenesis of plastidial ribosomes (Beligni and Mayfield,
2008; Bollenbach et al., 2009). Several proteins involved in
chloroplast division, namely, FtsZ1, FtsZ2, Arc5, and Cpn60B
(Gao et al., 2003), are downregulated in the C4species. Although
chloroplast division is largely completed in mature leaves, pro-
tein turnover appeared upregulated in both C4species com-
pared with the respective C3species.
The C4Syndrome Alters Nitrogen Metabolism, Amino Acid
Metabolism, and Translation
C4plants need less Rubisco in their leaves than C3species to
perform the same amount of CO2fixation leading to a better
nitrogen use efficiency by C4 compared with C3 species
(Black, 1973; Ku et al., 1979; Oaks, 1994; Brown, 1999;
Osborne and Freckleton, 2009; Ghannoum et al., 2011). Pro-
tein synthesis was altered in the C4Flaveria species, since this
MapMan bin and several of its sub-bins are enriched in
downregulated transcripts compared with all other MapMan
bins using the overrepresentation analysis of the PageMan
software (Figure 3). Downregulated transcripts representing
cytosolic ribosomes were enriched in all C4species, while
Evolution of C4Photosynthesis9 of 19

Page 10

transcripts associated with plastidic ribosomes were only
overrepresented in F. trinervia. No enrichment was detected
for downregulated components of mitochondrial ribosomes,
indicating that there was no general effect on translation but
specific for ribosomes translating photosynthetic and photo-
respiratory transcripts.
In F. ramosissima, the abundance of transcripts related to the
eukaryotic ribosomal proteins was similar to C3levels, whereas
thetranscriptsrelatedtotheplastidicribosomalproteinsshowed
amountsthatareintermediatebetweentheC3speciesF.robusta
and the C4species F. trinervia.
In accordance with these findings, elemental analysis showed
that the C4Flaverias exhibit higher carbon to nitrogen ratios (7.8
to 8.6) than the C3species (5.5 to 5.7) (see Supplemental Table 6
online). F. ramosissima had an intermediate carbon to nitrogen
ratio (6.8 to 6.9).
Figure 5. Photorespiration Is Altered between F. trinervia (C4), F. ramosissima (C3-C4), F. robusta (C3), F. bidentis (C4), and F. pringlei (C3).
(A) Schematic view of the photorespiratory pathway. Relative transcript abundances are given in small inset boxes. The transcript levels for F. trinervia,
F. ramosissima, and F. robusta were normalized by setting the F. robusta transcript level to one, and the F. bidentis and F. pringlei transcript levels were
normalized by setting the F. pringlei transcript level to one for each gene.
(B) Amounts of important photorespiratory metabolites in the leaves of all five species (6 SE; n = 3).
10 of 19 The Plant Cell

Page 11

Consequently, the genes involved in amino acid synthesis were
downregulated in the C4Flaveria species, since downregulated
transcripts were overrepresented within the bins “amino acid
metabolism” and “amino acid metabolism synthesis.” In the F.
bidentis/F. pringlei experiment, the bins “nitrogen metabolism”
and “ammonia metabolism” were enriched in downregulated
genes (Figure 3).
Expression Changes Related to C4
To discover additional genes that might be associated with the
C4trait,allFlaveriatranscriptomedatawereclustered andtested
for C4-related patterns. Hierarchical clustering showed that the
two C4species are more similar to each other than to the other
three analyzed Flaveria species with respect to their overall leaf
transcript profile (Figure 6A). K-means clustering identified 20
clusters with species-related gene expression changes, which
are unrelated to a C4pattern (see Supplemental Figure 3 online).
Six clusters show patterns related to C4photosynthesis, either
highinC4versuslowinC3(threeclusters)orhighinC3versuslow
in C4(three clusters) (Figure 6B). The clusters vary in regard to F.
ramosissima expression as exemplified for C4transcripts (inter-
mediate) or photorespiratory transcripts (higher in F. ramosis-
sima) above. Taken together, the C4clusters contained 3582
transcripts (Figure 6B; see Supplemental Data Set 2 online). A
total of 1418 of these genes were in clusters with C4upregulated
genes, whereas 2164 genes were downregulated during C4
evolution.
Early Evolutionary Changes
Clusters one and two contained 1213 genes, which were
upregulated in the two C4species and C4-like or intermediate
in the C3-C4intermediate species F. ramosissima. The genes
encoding the core C4 enzymes and known or putative C4
transporters were all part of cluster one (see Supplemental
Data Set 2 online). Additional functional classes that were
enriched within cluster one and two were minor carbohydrate
metabolism, glycolysis, the tricarboxylic acid cycle, abscisic
acid metabolism, posttranslational modification of proteins, and
phosphoinositol and light signaling (Figure 6c; see Supplemental
Figure 4 online). No cluster was formed that contains transcripts
downregulated both in the C4species and in the intermediate.
The number of thesetranscripts was thussmall. Wesuggest that
the changes in the C4species and the intermediate were C4
changes in the narrow sense.
Late Evolutionary Changes
Cluster three contains transcripts that were more highly ex-
pressed in the C4species compared with the C3species but not
in the C3-C4intermediate species F. ramosissima. In this cluster,
photosynthesis and light reaction transcripts as well as tran-
scripts related to abscisic acid, auxin, and ethylene metabolism,
several families of transcription factors and phosphorelay sig-
nalingwereenriched.Clustersfour,five,andsixcontainedgenes
that were downregulated in the C4compared with the C3spe-
cies. In the C3-C4intermediate species F. ramosissima, genes
fromthethreeclustersweremainlyexpressedonC3level(Figure
6B). Within these clusters, genes related to major carbohydrate
metabolism (including the Calvin Benson cycle and photorespi-
ration) and minor carbohydrate metabolism, tricarboxylic acid
cycle, C1 metabolism, and tetrapyrrole synthesis were enriched
(Figure 6C). The cluster analysis confirmed the overrepresenta-
tion analysis based on the species by species comparisons with
respect to the protein synthesis and nitrogen metabolism and
indicated these changes are late changes. A total of 2369
changes were late and we suggested that these changes were
C4changes in the wider sense.
Regulatory Genes
In clusters one and two, the C4clusters in the narrow sense, we
found 151 genes encoding transcriptional regulators and 35
genes related to signaling pathways (see Supplemental Data Set
3 online). Among the transcriptional regulators, we identified two
plastidal Sigma70-like factors, SIG1 and SIG5, which were
furthermoresignificantlyupregulatedintheF.trinervia/F.robusta
experiment. Plastidial sigma factors are encoded in the nuclear
genome and control plastid gene expression by guiding RNA
polymerase to the promoter (Lerbs-Mache, 2011). In F. ramo-
sissima, SIG5 showed an abundance that was intermediate
compared with the F. robusta and F. trinervia, whereas the
transcript abundance of SIG1 was comparable to the C4plant
F. trinervia in the C3-C4intermediate. In the C4plant Cleome
gynandra,adifferentSigma70-likefactor,SIG6,wasupregulated
significantly compared with the C3 plant Cleome spinosa
(Bra ¨utigam et al., 2011). Thus, it might be possible that also the
different abundance of plastidic sigma factors in C3 and C4
species differentially regulate chloroplast gene expression and
thus alter the abundance of the complexes of the photosynthetic
electron transfer chain observed in these species. Another tran-
scription factor that was expressed significantly differential in the
F. trinervia and F. robusta was the auxin response factor ARF2.
With 185 to 516 rpm, the ARF2 gene was highly expressed for a
regulatoryfactor inthe leavesofallfive Flaveriaspecies.Inthe C3-
C4intermediate F. ramosissima, the abundance of ARF2 tran-
scriptswasintermediatecomparedwithF.trinerviaandF.robusta.
Homozygous Arabidopsis ARF2 mutants show a pleiotropic
phenotype. Among others, the leaf size is enlarged caused by
an increase of both cell division and cell expansion (Okushima
et al., 2005; Gonzalez et al., 2010). Thus, one can assume that
ARF2 is involved in the establishment and maintenance of the
typical C4 leaf anatomy. GOLDEN2 LIKE (GLK) transcription
factors are known to be involved in the chloroplast dimorphism
inmesophyllandbundlesheathcellsofmaize(Watersetal.,2009).
GLK2 was a member of cluster two, indicating that changes to
theGLKproteinsplayedalsoanimportantroleinthedevelopment
of the C4pathway in Flaveria. Interestingly, in Cleome, the GLK2
counterpart GLK1 was upregulated in the C4species (Bra ¨utigam
et al., 2011).
A total of 183 transcription factors and 91 genes related to
signaling were foundin clusters threeto six,the C4clustersin the
wider sense (see Supplemental Data Set 3 online). Most strik-
ingly, one can find 73 signaling receptor kinase genes, including
CLAVATA1 and ERECTA in clusters four, five, and six, meaning
Evolution of C4Photosynthesis 11 of 19

Page 12

Figure 6. Cluster Analysis of Transcript Abundances in F. bidentis (C4), F. trinervia (C4), F. ramosissima (C3-C4), F. robusta (C3), and F. pringlei (C3).
(A) Hierarchical sample clustering of all expressed transcripts. The tree was calculated with the MEV program using the HCL module with the Euclidean
distance criterion and the average linkage method. According to their transcript profiles, the two C4species are more closely related to each other than
to the other three Flaveria species.
(B) C4-related clusters. K-means analysis was used to define 26 clusters identifying different expression profiles. The six clusters with a C4-related
pattern are shown. All 26 clusters can be found in Supplemental Figure 2 online.
(C) Functional category (MapMan bins) enrichment among the six C4-related clusters. Enrichment of genes belonging to distinct functional categories
was analyzed with the Wilcoxon statistic followed by the Benjamini-Hochberg correction. Blue, significantly overrepresented; red, significantly
underrepresented. The complete enrichment analysis for all 26 clusters is shown in Supplemental Figure 4 online. aa, amino acid; CHO, carbohydrate;
PS, photosynthesis; TCA, tricarboxylic acid cycle.
12 of 19The Plant Cell

Page 13

that they were downregulated in the C4species. CLAVATA1 and
ERECTA are known to be involved in cell and also organ differen-
tiation by mediating cell–cell communication (Shiu and Bleecker,
2001;vanZantenetal.,2009).Althoughthefunctionofthemajority
of the other proteins is unknown, their cumulative appearance
suggests a relationship to the different types of photosynthesis
or leaf architecture (see Supplemental Data Set 3 online).
DISCUSSION
Comparison of Flaveria Leaf Transcriptomes by
Next-Generation Sequencing
We used 454 sequencing to analyze the leaf transcriptomes of
five Flaveria species exhibiting different modes of photosynthe-
sisandidentified ESTscorresponding tobetween55and61%of
the Arabidopsis transcripts included in the minimal coding se-
quences set we used for mapping in the individual leaf cDNA
libraries of the five species. Approximately 60% of the known
33,282 Arabidopsis genes show a detectable expression in the
aboveground part of Arabidopsis seedlings (Weber et al., 2007).
Assuming that comparable fractions of genes were expressed in
the leavesof theinvestigated Flaveria species, alargeproportion
of the leaf transcriptomes of all five species was captured. This
assumption was supported by the fact that the number of
captured transcripts only slightly increased in the F. trinervia/
F. ramosissima/F. robusta experiment compared with the F.
bidentis/F. pringlei experiment, although nearly twice as many
reads were available for the former.
The coverage of the individual functional gene classes was
>50% for most classes and comparable for all species, indicat-
ing that the leaf transcriptomes of the Flaveria species were
sampled to a comparable extent. Two complementary analyses
were conducted using these data: (1) a gene-by-gene compar-
ison using statistical tests based on the two experiments and (2)
a global analysis using clustering tools.
The gene-by-gene comparison resulted in 463 differentially
expressed genes in the F. bidentis/F. pringlei experiment (cor-
responding to 3.4% of the transcripts detected within these two
species) and 995 genes in the F. trinervia/F. robusta experiment
(corresponding to 6.9% of the genes detected with this exper-
iment). Since the more advanced GS TITANIUM sequencing
technology, which was used for the F. trinervia/F. robusta ex-
periment, created more reads and, thus, more statistical power
for the Audic and Claverie algorithm, more differences were
identified in this second experiment compared with the GS FLX
experiment conducted on F. bidentis and F. pringlei. The tran-
script abundance of 213 genes was significantly different in both
C3to C4comparisons, and many genes changed in the same
direction without reaching a significant level. Only 31 genes
exhibited opposing significant differential transcript abundances
in both experiments. This was equivalent to 0.21% of the
transcriptsdetectedwithintheF.trinervia/F.robustaexperiment,
indicating that the vast majority of differences in transcript
abundances found in this study is related to the different modes
of photosynthesis rather than to the phylogenetic distance of the
analyzed Flaveria species.
Leaf Transcriptomes Changed during C4Evolution
The cluster analysis resulted in six clusters with a C4-related
pattern. Taken together, these C4clusters contained 3582 tran-
scripts. A total of 1418 of these genes were in clusters with C4
upregulated genes, whereas 2164 genes are downregulated
duringC4evolution.Thesenumbersarethecurrentbestestimate
fortranscriptabundancechangesrelatedtoC4.Untilafunctional
C4cycle is introduced into a C3plant, it will remain unknown how
many of these transcript changes are necessary and sufficient to
establish a C4cycle. Based on the multiple concurrent and
parallel successful evolution of the C4trait in manyplant families,
it is likely that many of the changes will either be controlled by
common gene regulatory networks (Westhoff and Gowik, 2010)
or may have evolved after successful establishment of the C4
cycle. In this experiment, the evolutionary progression can be
established by comparing the intermediate species with the C4
and the C3species based on PageMan analysis. While all known
core C4genes were changed early during C4evolution, other
majorchangeshappenedaftertheestablishmentoftheC4cycle.
In case of the nitrogen metabolism, amino acid synthesis and
transcriptionalmachinery,whichwerereducedintheC4species,
this is logically consistent, since first the highly abundant tran-
scripts of the functional classes “Calvin-Benson cycle” or “pho-
torespiration” had to be reduced. These reductions on the other
hand require the existence of a fully functional C4cycle.
The majority of C4-related genes are regulated at least in part
at the level of transcript abundance (see above; Bra ¨utigam et al.,
2011). While the simple overrepresentation analysis based on
the species by species comparisons suggests that changes
within in the regulatory genes are statistically underrepresented
the cluster analysis discovers a multitude of regulatory genes
with C4-related transcript patterns. They may be involved in the
development and maintenance of C4leaves and are prime can-
didates for further analysis.
The Transcription of C4Cycle Genes Was Altered during C4
Evolution in Flaveria
The C4 Flaveria species are assigned to the NADP-ME C4
photosynthesis type (Ku et al., 1991). This is reflected by our
study. Transcript data, extractable enzyme activities, and the
metabolite levels confirmed that the two C4Flaveria species
F. trinervia and F. bidentis exclusively use the NADP-ME C4
pathway. In addition to the classical NADP-ME genes, we found
astrongupregulationofanAlaandanAsp-AT,indicatingthatthe
C4Flaverias also use amino acids as transport metabolites. The
protein encoded by the contig of the upregulated ASP-AT, as
well as its Arabidopsis counterpart (AT4G31990), is predicted to
be localized to the chloroplast (ChloroP,AtASP5, 0.547;FtASP5,
0.539). This confirmed earlier results showing that C4species
F.bidentisandF.trinerviauseAsptoavariableextentastransport
metabolite (Moore et al., 1986; Meister et al., 1996), whereas the
majority of ASP-AT activity is localized to the chloroplasts in
mesophyll as well as in bundle sheath cells (Moore et al., 1984;
Meister et al., 1996).
Two additional enzymes are key in C4photosynthesis. We
detected the strong and significant upregulation of an adenosine
Evolution of C4Photosynthesis 13 of 19

Page 14

monophosphate kinase gene in both C4species. This gene was
also found to be upregulated in the C4 plant C. gynandra
(Bra ¨utigam et al., 2011) and is thought to be involved in the
processing of the adenosine monophosphate produced by the
PPDK (Hatch and Slack, 1968). In the F. bidentis/F. pringlei
experiment, we also identified two significantly upregulated
inorganic pyrophosphatases (see Supplemental Table 2 online),
which were also upregulated in C. gynandra (Bra ¨utigam et al.,
2011). The upregulation at the transcript level is consistently
detected in different species and different genera, reinforcing
physiological analysis in that the processing of the AMP and
pyrophosphate generated by PPDK is an integral part of the C4
cycle (Slack et al., 1969).
Thenext-generationsequencinganalysisprovidedamodelfor
the transport processes at the mesophyll chloroplast envelope.
In addition to translocators for PEP and inorganic phosphate,
triose phosphates, 3-phosphoglycerate, inorganic phosphate,
pyruvate, sodium ions, and protons (BASS 2/NHD), we found a
strong upregulation of the chloroplast DiT1 and DiT2. Thus, the
pattern of DiT1 expression in the different Flaveria species was
similar to the pattern of other genes directly involved in the C4
cycle, supporting the assumption that DiT1 is indeed involved in
the C4photosynthesis as the OAA/malate shuttle of mesophyll
chloroplasts. The upregulation of plastidic Asp-AT in the C4
Flaveriaspointedtoaroleforaseconddicarboxylatetransporter,
DiT2. DiT2 has a broader substrate spectrum than DiT1 and
prefers Asp (Renne ´ et al., 2003). Upregulation of DiT2 in the C4
Flaverias prompted the hypothesis that DiT2 was involved in the
exchange of Asp across mesophyll and bundle sheath chloro-
plast envelopes as part of the C4cycle. The DiT genes were not
upregulated in the C4species C. gynandra. This coincides with
the proposed function for the DiTs, since C. gynandra is a NAD-
ME type plant and does not have to shuttle OAA, malate, or Asp
across its chloroplast envelope.
Bundle sheath chloroplasts play a key role in NADP-ME C4
photosynthesis. Next to the inorganic phosphate, which must
exhibit high activities also in the bundle sheath chloroplasts, no
further C4-related bundle sheath chloroplast transporter is
known to date. No candidates exist for the malate importer or
the pyruvate exporter. Flaveria contains an additional gene
belonging to the bile acid sodium symporter family, BASS 4,
which was upregulated to an extent comparable with other C4
genes. In contrast with the chloroplastic pyruvate transporter
BASS 2, this gene was not upregulated in the C4species C.
gynandra when compared with the C3species C. spinosa. Since
neither pyruvate export nor malate import at NAD-ME bundle
sheath chloroplasts is required, it is tempting to hypothesize that
this transporter might be involved in either pyruvate export from
or malate import into the bundle sheath chloroplast.
The comparison also revealed a number of transport proteins
with unknown or predicted functions only. These may play
accessory roles in transport by creating or dissipating gradients
needed for or caused by C4related transport, much as adeno-
sine monophosphate kinase and pyrophosphatase are needed
to balance metabolism. Two of these transport proteins, VDAC
and AVP1, were also significantly altered in the C4 plant
C. gynandra when compared with the C3plant C. spinosa
(Bra ¨utigam et al., 2011), indicating a potential relevance of these
genes for C4photosynthesis. The AVP1 transcripts are signifi-
cantly more abundant in F. trinervia and C. gynandra than in
the respective C3 species (Bra ¨utigam et al., 2011). Also, in
F. bidentis/F. pringlei, AVP1 was upregulated, although the dif-
ference was not significant. In the C3-C4 intermediate plant
F. ramosissima, AVP1 abundance was intermediate compared
with F. trinervia and F. robusta. Arabidopsis AVP1 mutants show
defects in leaf and root development since AVP1 affects polar
auxin transport (Li et al., 2005).
Up to Three Distinct CO2Concentration Mechanisms
Operate in the C3-C4Intermediate F. ramosissima
In F. ramosissima, the transcripts of the genes related to the
NADP-ME type C4photosynthesis showed intermediate levels
compared with the C3 plant F. robusta and the C4 plant
F. trinervia. This implies that in F. ramosissima, the C4cycle is
working to a certain extent and that F. ramosissima is a true
intermediate based on its transcriptional profile. This is in agree-
ment with earlier results showing that, in F. ramosissima, more
than 40% of the CO2is directly fixed into the C4acids malate
and Asp (Ku et al., 1991). Based on the transcriptional profile,
F.ramosissimaisintermediatewithregardtoaNADP-MEtypeC4
cycle. This is also reflected in the changes of photosynthetic
electron transport chain gene expression.
F. ramosissima is also intermediate with regard to protein
synthesis. The only downregulated transcripts related to the
Calvin-Benson cycle were those of Rubisco, while in C4species,
the majority of Calvin-Benson cycle transcripts were down-
regulated. Unlike C4 species, which had a downregulated
photorespiratory cycle, F. ramosissima accumulated more pho-
torespiratory transcripts. In consequence, only plastidic but not
cytosolic elements of the protein biosynthesis machinery were
downregulated, and no changes in amino acid metabolism were
detected.Hence,F.ramosissimawasnotcapableoffullyreaping
the nitrogen benefits of C4photosynthesis, as indicated by its
intermediate C/N ratio.
Wefoundsignificantupregulation ofgenesrelatedtotheNAD-
ME type C4pathway like cytoplasmic and mitochondrial ASP-AT
genes, an mNAD-MDH, or two mNAD-ME. This transcript profile
provokes the hypothesis that a (partial?) NAD-ME type C4cycle
is active in addition to the NADP-ME type C4pathway in the C3-
C4intermediate. This was confirmed by the extractable NAD-ME
activity that was significantly higher in the leaves of F. ramosis-
sima than in the other four Flaveria species. Also, an analysis of
the steady state metabolite levels suggested a similar conclu-
sion. While the Ala level in F. ramosissima was comparable to
those found in the two C4species, the Asp level exceeded those
of all other Flaverias. Since Ala and Asp are the predominant
transport metabolites in NAD-ME plants (Hatch, 1987), these
findings supported the hypothesis of a NAD-ME type C4cycle in
F. ramosissima. This finding was surprising since all true C4
Flaveria species belong exclusively to the NADP-ME C4plants
(Drincovich et al., 1998; this article). It is not clear if this reflects
plasticityinthephotosynthetic metabolismof FlaveriasduringC4
evolution that was lost after a fully developed NADP-ME cycle
was established or if F. ramosissima developed the NAD-ME
cycle after splitting from the Flaveria lineage leading to true C4
14 of 19 The Plant Cell

Page 15

species. Thiswillbeclarifiedin thefuturebyanalyzingfurtherC3-
C4intermediate Flaveria species.
Thisstudyalsoprovided comprehensive molecularevidencefor
a photorespiratory CO2concentration mechanism in the C3-C4
intermediate species, which was previously hypothesized to rep-
resent abiochemical CO2pump (Rawsthorne etal., 1988a,1988b)
and might have been an intermediate step toward the evolution of
the C4pathway (Bauwe et al., 1987; Sage, 2004; Bauwe, 2011).
Photorespiratory genes were expressed at a higher level than
even in the C3species and, importantly, also the steady state
levels of Gly and Ser, the transport metabolites of the photo-
respiratory CO2pump, were higher in F. ramosissima compared
with the C3and C4Flaveria species. Hence, a photorespiratory
CO2pump may still operate in F. ramosissima. Based on the
available data, three distinct CO2concentrating mechanisms,
the NADP-ME-, the NAD-ME-type, and the photorespiratory Gly
shuttle, operate in parallel in F. ramosissima (Figure 7).
To produce a consistent model (Figure 7), it is critical to
consider the ammonia balance between the cell types. The
photorespiratory CO2pump moves two molecules of Gly to the
bundle sheath cells where they are decarboxylated, leading to
one molecule each of Ser, CO2, and ammonium. Without com-
pensation, this would lead to a massive accumulation of am-
monia in the bundle sheath cells, even if the resulting Ser is
transported back to the mesophyll cells for phosphoglycerate
regeneration as proposed in Figure 7 and as supported by the
high steady state Ser levels in F. ramosissima leaves. In case of
F. ramosissima with a working C4 cycle, ammonia can be
balanced by adjusting the ratios of the transport metabolites
Ala/pyruvate and Asp/malate. For less advanced C3-C4inter-
mediates, which solely rely on the photorespiratory CO2con-
centration mechanism, the imbalance also needs to be solved.
Means to transport ammonia from bundle sheath to mesophyll
cells, like a Glu-oxoglutarate shuttle, an Ala-pyruvate shuttle, or
an aspartate-malate shuttle, would be required in these less
advanced intermediates. One of the latter two might have been
a starting point for the evolution of a metabolite transport
frameworkneededfortheC4cycle.IfanAla-pyruvateshuttleand
an Asp-malate shuttle would exist in parallel in a single species,
only minor alterations to these pathways, in a way that malate
andAsparetransportedfromthemesophylltothebundlesheath
cells and Ala and pyruvate are transferred back would be
necessary to establish a C4-like CO2transport pathway that
could replace the photorespiratory Gly/Ser pump.
Comparison to C4Photosynthesis in the Genus Cleome:
Common Themes of C4Evolution
Two comparative transcriptome studies on closely related C3
and C4 species from the dicot genera Flaveria and Cleome
Figure 7. Schematic of the CO2Concentrating and Photorespiratory Pathways in the C3-C4Intermediate Species F. ramosissima.
Three distinct CO2concentrating mechanisms, the NADP-ME type (green), the NAD-ME type (blue) C4pathway, and the photorespiratory Gly shuttle
(orange), operate in parallel in this C3-C4intermediate. F. ramosissima, with a working C4cycle, can compensate for the massive ammonia imbalance
introduced by the photorespiratory CO2pump, by adjusting the ratios of the transport metabolites Ala/pyruvate and Asp/malate.
Evolution of C4Photosynthesis 15 of 19