The Plant Journal (2012) 69, 4756

doi: 10.1111/j.1365-313X.2011.04769.x

Multiple Arabidopsis genes primed for recruitment into

C4 photosynthesis
Kaisa Kajala, Naomi J. Brown, Ben P. Williams, Philippa Borrill, Lucy E. Taylor and Julian M. Hibberd*
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK
Received 4 August 2011; revised 25 August 2011; accepted 26 August 2011; published online 14 October 2011.
*
For correspondence (fax +44 (0)1223 333953; e-mail julian.hibberd@plantsci.cam.ac.uk).

SUMMARY
C4 photosynthesis occurs in the most productive crops and vegetation on the planet, and has become
widespread because it allows increased rates of photosynthesis compared with the ancestral C3 pathway.
Leaves of C4 plants typically possess complicated alterations to photosynthesis, such that its reactions are
compartmented between mesophyll and bundle sheath cells. Despite its complexity, the C4 pathway has
arisen independently in 62 separate lineages of land plants, and so represents one of the most striking
examples of convergent evolution known. We demonstrate that elements in untranslated regions (UTRs) of
multiple genes important for C4 photosynthesis contribute to the metabolic compartmentalization characteristic of a C4 leaf. Either the 5 or the 3 UTR is sufficient for cell specificity, indicating that functional
redundancy underlies this key aspect of C4 gene expression. Furthermore, we show that orthologous PPDK and
CA genes from the C3 plant Arabidopsis thaliana are primed for recruitment into the C4 pathway. Elements
sufficient for M-cell specificity in C4 leaves are also present in both the 5 and 3 UTRs of these C3 A. thaliana
genes. These data indicate functional latency within the UTRs of genes from C3 species that have been
recruited into the C4 pathway. The repeated recruitment of pre-existing cis-elements in C3 genes may have
facilitated the evolution of C4 photosynthesis. These data also highlight the importance of alterations in trans
in producing a functional C4 leaf, and so provide insight into both the evolution and molecular basis of this
important type of photosynthesis.
Keywords: C4 photosynthesis, untranslated regions, pyruvate,orthophosphate dikinase, carbonic anhydrase,
Cleome gynandra, Arabidopsis.

INTRODUCTION
Having evolved in at least 62 separate lineages of land
plants, C4 photosynthesis is considered one of the most
remarkable examples of convergent evolution (Sage et al.,
2011). Current estimates are that land plants first started to
use C4 photosynthesis around 3032 million years ago, and
that it evolved in response to high temperatures and
reductions in atmospheric CO2 content (Christin et al.,
2008; Edwards et al., 2010; Vicentini et al., 2008). The C4
pathway allows CO2 to be concentrated around the central
photosynthesis enzyme Ribulose-1,5-Bisphosphate Carboxylase Oxygenase (RuBisCO). Because RuBisCO does not
completely distinguish between CO2 and O2 (Bowes et al.,
1971), the increased supply of CO2 to RuBisCO in C4 plants
reduces the oxygenation reaction, and in turn limits the
wasteful reactions of photorespiration (Hatch, 1987). In all
cases, C4 plants use a biochemical pump to increase the
amount of CO2 in compartments within which RuBisCO is
limited. Although photosynthesis reactions are compart 2011 The Authors
The Plant Journal 2011 Blackwell Publishing Ltd

mented within a single cell in some C4 lineages (Reiskind

et al., 1989; Voznesenskaya et al., 2001), most C4 species
partition photosynthetic reactions between two distinct cell
types that are arranged in concentric circles around veins,
generating so-called Kranz anatomy (Brown et al., 2005;
Hatch, 1987; Langdale and Nelson, 1991). This separation of
metabolic reactions between bundle sheath (BS) and
mesophyll (M) cells mean that C4 acids are produced in M
cells and then diffuse to BS cells where the CalvinBenson
cycle operates (Hatch, 1987; Leegood, 2002), and is
achieved by restricting the accumulation of proteins to
either BS or M cells. For example, carbonic anhydrase (CA),
phosphoenolpyruvate carboxylase (PEPC), NADP-malate
dehydrogenase (MDH), pyruvate,orthophosphate dikinase
(PPDK) and the PPDK regulatory protein (RP) typically
accumulate in M cells in C4 plants, whereas a C4 acid
decarboxylase, phosphoribulokinase (PRK), ribose-5-phosphate isomerase (RPI) and RuBisCO are restricted to BS
47

48 Kaisa Kajala et al.

cells (Kanai and Edwards, 1999; Ku et al., 1976; Sinha and
Kellogg, 1996).
Compartmentalization of photosynthesis proteins
between M and BS cells is generated by both transcriptional
and post-transcriptional mechanisms (Hibberd and Covshoff, 2010). Mechanisms underlying the M-cell specificity of
PEPC and PPDK are probably the best studied. For example,
in maize (Zea mays), accumulation of PEPC in M cells is
related to specific changes in sequences within the
promoter, DNA methylation, and also histone modifications.
M-cell-specific accumulation of ZmPEPC transcripts in maize
is correlated with demethylation of a PvuII site 3.1 kb
upstream of the PEPC gene during greening (Langdale et al.,
1991), but this is unlikely to be the only mechanism
responsible for M-cell-specific accumulation of PEPC
because when regions of only 1.7 or 0.6 kb upstream of
ZmPEPC are fused to the uidA gene encoding b-glucoronidase, GUS staining was strong in developing and mature M
cells (Kausch et al., 2001; Taniguchi et al., 2000). Sequences
within this 0.6 kb region of ZmPEPC1 bind unidentified
proteins termed PEPIb and PEPIc (Taniguchi et al., 2000).
High trimethylation of histone H3K4 tails was detected
within ZmPEPC1 in M cells but not BS cells (Danker et al.,
2008), although the functional significance of this is not
certain. In C4 Flaveria, a cis-element that is necessary and
sufficient for M-cell-specific expression of the GUS reporter
has been identified (Akyildiz et al., 2007; Gowik et al., 2004).
This element is contained within a 41 bp region known as
mesophyll-enhancing module 1 (MEM1) from C4 species of
Flaveria. MEM1 is located in a distal region of the promoter
()1566 to )2141), and contains a CACT tetranucleotide and a
G A substitution. Placing MEM1 into the C3 Ppc promoter
from Flaveria pringlei represses accumulation of the GUS
reporter in BS cells rather than increasing it in M cells
(Akyildiz et al., 2007). Analysing the amount of Ppc transcripts in Flaveria bidentis on the basis of GUS accumulation
driven by the FbPpc promoter indicates that control is
predominantly via transcription (Stockhaus et al., 1997).
Although a mesophyll-enhancing module has been identified in the promoter to the FbCA3 gene from F. bidentis, its
function has not been confirmed (Tanz et al., 2009).
Pyruvate,orthophosphate dikinase also accumulates preferentially in M cells, and a region between )301 and )296
from the translational start site is important for this. Gelretardation assays showed that this region binds a protein
termed PPD1 (Matsuoka and Numazawa, 1991). When the
region between )370 and )76 in the maize promoter is fused
to the rice (Oryza sativa) promoter, high expression is
observed in M protoplasts (Matsuoka and Numazawa, 1991).
In stable transformants of F. bidentis, use of the region
between )1212 and +279 from the transcriptional start site
fused to the uidA gene results in high levels of GUS in M
cells, but lower amounts in BS cells (Rosche et al., 1998). In
summary, these data are consistent with transcriptional

regulation of both the PEPC and PPDK genes being important for M-cell specificity in maize and Flaveria.
It is known that genes encoding NAD-dependent malic
enzyme (NAD-ME) and NADP-dependent malic enzyme
(NADP-ME) from C3 species contain cis-elements sufficient
for BS specificity in C4 leaves, and thus cell specificity in the
C4 leaf can be mediated by changes in trans (Brown et al.,
2011). The location of cis-elements within the coding region
of these ME genes and the requirement for transcription
implies post-transcriptional regulation (Brown et al., 2011).
To determine the importance of regulation of C4 genes by
elements within their transcripts, and the extent to which
these elements are present in C3 orthologs, we used the
Cleome genus, which is closely related to Arabidopsis
thaliana and contains C3 and C4 plants (Brown et al., 2005;
Marshall et al., 2007; Voznesenskaya et al., 2007). The phylogenetic proximity to A. thaliana facilitates annotation of
likely gene function, and also provides relatively simple
resources for functional analysis of orthologous genes in a C3
species. To investigate mechanisms underlying M-cell specificity in Cleome gynandra, we isolated cDNAs and genomic
sequences encoding PPDK and CA4, and investigated
regions of each gene that were sufficient for accumulation
in M cells. By comparison with regulation of the orthologous
PPDK and b-CA4 genes from C3 A. thaliana, we were also able
to investigate the extent to which mechanisms have evolved
de novo in the C4 PPDK and CA4 genes from C. gynandra.
RESULTS
Cloning PPDK from C. gynandra
Compartmentation of photosynthesis proteins between M
and BS cells in C4 species can be generated by both transcriptional and post-transcriptional mechanisms, and in
most cases alterations in cis-elements have been linked to
this compartmentation (Akyildiz et al., 2007; Hibberd and
Covshoff, 2010; Westhoff and Gowik, 2004). To investigate
mechanisms underlying the accumulation of proteins in M
cells, we used a combination of degenerate PCR, 5 and 3
RACE, 454 sequencing and genome walking to isolate the
gene encoding PPDK from C. gynandra, the most closely
related C4 plant to the C3 model Arabidopsis thaliana (Brown
et al., 2005; Marshall et al., 2007). This allowed us to
assemble the sequence of the PPDK gene from C. gynandra
(Figure 1a). To confirm that the CgPPDK gene is highly
expressed, targeted to chloroplasts and therefore probably
important for C4 photosynthesis, we used two approaches.
First we created a fusion between the promoter sequence
(including the 5 UTR) isolated from C. gynandra and the
uidA gene encoding b-glucuronidase (GUS) and investigated whether it contained enhancer elements compared
with the orthologous promoter from the closely related C3
species A. thaliana (Figure S1A,B). This promoter drives
expression of transcripts that encode the longer form of the

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

C4 evolution 49

(a)

(b)

(c)

(e)

(f)

(d)

(g)

Figure 1. The PPDK promoter from C4 C. gynandra contains enhancer elements that are recognized in C3 A. thaliana.
(a) Structure of the C4 PPDK gene from C. gynandra. Exons are numbered and indicated by boxes, with black representing those that encode the mature protein, gray
representing the chloroplast transit peptide, and white indicating 5 or 3 untranslated regions. The translational start and stop codons are annotated. The promoter
directing expression of the chloroplastic protein is upstream of the first exon, while intron 2 contains a promoter that generates transcripts encoding the cytosolic
PPDK.
(b, c) GUS staining of stable transformants of A. thaliana containing either the endogenous A. thaliana (b) or C. gynandra (c) chloroplastic PPDK promoter fused to
the uidA gene. Scale bars are 0.5 cm.
(d) Quantitative analysis of GUS activity shows a 20-fold enhancement when the C. gynandra promoter was used. Boxes indicates upper and lower quartiles,
median values are represented by horizontal lines within the boxes, and minimum and maximum MUG activities for each construct are indicated by black dots and
whiskers.
(eg) Confocal laser scanning microscopy after microprojectile bombardment of Arabidopsis thaliana leaves shows that the PPDK::GFP fusion protein localizes to
chloroplasts. (e) Chlorophyll channel, (f) GFP channel, and (g) chlorophyll and GFP images merged. Scale bars = 10 lm.

PPDK protein targeted to chloroplasts (Parsley and Hibberd,

2006; Rosche and Westhoff, 1995; Sheen, 1991). Second we
created a translational fusion between the CgPPDK coding
sequence and GFP under the control of the CaMV 35S promoter, and used laser scanning confocal microscopy to
determine the subcellular localization of the fusion protein.
Staining stable transformants of A. thaliana indicated that
use of the A. thaliana PPDK promoter resulted in accumulation of GUS in older senescing leaves (Figure 1b) (Taylor
et al., 2010), but use of the promoter from C. gynandra
extended accumulation of GUS into mature leaves
(Figure 1c). Quantitative assays of GUS activity using
methylumbelliferyl-D-glucuronide (MUG) indicated that use
of the C. gynandra PPDK promoter increased GUS activity
by at least 20-fold in mature leaves (Figure 1d). Laser
scanning confocal microscopy showed that GFP localized to
chloroplasts after microprojectile bombardment was used

to deliver the CgPPDK::GFP fusion into leaves (Figure 1eg),

in contrast with the cytosolic localization of GFP when under
the control of the CaMV 35S promoter alone (Figure S2).
The increased accumulation of GUS driven by the PPDK
promoter from C. gynandra compared with the promoter
from A. thaliana and localization of the protein to chloroplasts are consistent with this CgPPDK gene being important
in the C4 pathway.
Untranslated regions are sufficient for accumulation of
PPDK in M cells
Hybridization in situ and quantitative RT-PCR after laser
microdissection of M and BS cells showed that CgPPDK
transcripts accumulate preferentially in M cells of C. gynandra (Figure 2a,b). To investigate the regions of the
CgPPDK gene that generate M-cell specificity, we used
microprojectile bombardment to deliver various parts of the

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

50 Kaisa Kajala et al.

(a)

(c)

(b)

Figure 2. PPDK accumulates in M cells of C. gynandra due to elements in

both the 5 and 3 UTRs.
(a) In situ hybridization shows that transcripts encoding PPDK localize to M
cells of C. gynandra leaves.
(b) Quantitative RT-PCR after extraction of RNA from either mesophyll (M) or
bundle sheath (BS) cells shows preferential accumulation of PPDK transcripts
in M cells.
(ce) The constitutive CaMV 35S promoter drives expression of the uidA
reporter in mesophyll (c) and bundle sheath (d) cells, and more BS cells than
M cells showed staining under our assay conditions (e).
(f) Elements within either the 5 or 3 UTR of PPDK from C. gynandra are
sufficient to generate M-cell specificity in leaves of C. gynandra.
(g) Elements within either the 5 or 3 UTR of PPDK from A. thaliana are
sufficient to generate M-cell specificity in leaves of C. gynandra.
Cells accumulating GUS were counted. Three or more biological replicates
were performed. Error bars represent standard errors. P values were
determined by a one-tailed t test.

(e)
CaMV 35S promoter, when the CgPPDK cDNA including 5
and 3 UTRs (Figure S1D) was incorporated into this construct, GUS accumulated preferentially in M cells (Figure 2f
and Table S1). This indicates that neither the endogenous
promoter nor the introns of CgPPDK are necessary for
preferential accumulation in M cells of C. gynandra, and that
elements contained within the transcript are sufficient for
M-cell specificity.
To investigate regions of the CgPPDK transcript that are
sufficient for preferential accumulation in M cells, we
produced a series of truncations fused to uidA
(Figure S1EG). The results showed that the CgPPDK coding
region was not necessary for preferential accumulation in M
cells, and that either the 5 UTR or 3 UTR alone was
sufficient (Figure 2f and Table S1). Compared with each UTR
alone, the presence of both 5 and 3 UTRs slightly increased
the proportion of M cells accumulating GUS.

(d)

(f)

The 5 and 3 UTRs of PPDK from A. thaliana generate

M-cell specificity in C. gynandra

(g)

CgPPDK gene fused to the uidA reporter into leaves of

C. gynandra, and counted the number of M and BS cells
accumulating GUS. When uidA was transcriptionally fused
to the constitutive CaMV 35S promoter (Figure S1C), M and
BS cells accumulated GUS (Figure 2c,d). Under our assay
conditions, this control construct labeled more BS than M
cells (Figure 2e). Despite being under the control of the

Comparison of the UTRs from the PPDK genes of A. thaliana

and C. gynandra showed regions of sequence conservation
between the two species (Figure S3). The 5 and 3 UTRs
from A. thaliana PPDK were sufficient for preferential accumulation of GUS in M cells of C. gynandra (Figure 2g and
Figure S1HJ), and, as with the orthologous regions from
C. gynandra, each UTR alone was sufficient for this specificity. These data indicate that the PPDK gene from
A. thaliana possesses cis-elements that are sufficient for
M-cell specificity when placed into a closely related C4 leaf.
We next investigated how the UTRs from CgPPDK behave in
A. thaliana. This analysis showed that, in A. thaliana, neither
the 5 UTR nor the 3 UTR of CgPPDK, or both together, led to
a large alteration in the relatively constitutive accumulation
of GUS driven by the CaMV 35S promoter (Figure 3ad).
However, quantitative assays of GUS activity using MUG
showed that both UTRs from CgPPDK and the 5 UTR alone
slightly increased the activity of GUS in leaves of A. thaliana
compared with the CaMV 35S:uidA controls (Figure 3e).

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

C4 evolution 51

(a)

(b)

(c)

(d)

(e)

Figure 3. Untranslated regions of PPDK from Cleome gynandra show

relatively constitutive accumulation of GUS in stable transformants of
A. thaliana.
(a) The CaMV 35S::uidA fusion leads to accumulation of GUS in most leaves
and cells of A. thaliana, particularly the vascular system.
(bd) Including both the 5 and 3 UTRs of CgPPDK (b), the 5 UTR alone (c) or
the 3 UTR alone (d) in the CaMV35S::uidA fusion had little impact on the
spatial accumulation of GUS in A. thaliana.
(e) Quantitative assays show that both UTRs and the 5 UTR increase
expression compared with the CaMV 35S control.
Scale bars = 1 cm.

UTRs from C. gynandra or A. thaliana CA4 genes are

sufficient for accumulation in M cells
Laser capture microscopy and quantitative RT-PCR designed
against contigs derived from 454 sequencing (Brautigam
et al., 2011) detected strong and preferential accumulation
of CgCA4 transcripts, encoding a carbonic anhydrase, and
CgBASS2 transcripts, encoding a pyruvate transporter
(Furumoto et al., 2011), in M cells (Figure S4). We used
RACE and PCR on genomic DNA to identify full-length
transcripts as well as the gene structure for each of these
genes. In both cases, coding sequences were very similar to

orthologs in A. thaliana, and the number of introns was

conserved (Figure 4a). A fusion between GFP and the coding
sequence of CgCA4 led to accumulation of GFP in close
proximity to the plasma membrane (Figure 4bd), and a
fusion between CgBASS2 and GFP labeled the outside of
chloroplasts (Figure 4eg). In addition to the high level of
their transcripts as detected by 454 sequencing (Brautigam
et al., 2011), the localization of CA4 and BASS2 to the plasma
membrane and chloroplast envelopes, respectively, is consistent with their localization in A. thaliana and their roles in
C4 photosynthesis.
Microprojectile bombardment showed that elements
present in CgCA4 transcripts were sufficient for M-cell
specificity, but elements in CgBASS2 were not (Figure 4h,
Table S1 and Figure S1KP). The coding region of CgCA4
was not necessary, but the 5 and 3 UTRs were sufficient for
M accumulation (Figure 4h and Table S1). As with PPDK,
either the 5 or the 3 UTR was sufficient for accumulation of
GUS in M cells (Figure 4i). Taken together, these data
indicate that elements in either the 5 or the 3 UTR of
CgPPDK and CgCA4 are sufficient for preferential accumulation of PPDK in M cells of C. gynandra.
Alignments of the CA4 UTR sequences from C. gynandra
and A. thaliana showed some similarities in their sequences
(Figure S5). When introduced into C. gynandra leaves by
microprojectile bombardment, the 5 and 3 UTRs from
A. thaliana CA4 (Figure S1Q,R) were sufficient for preferential accumulation in the M cells (Figure 4i and Table S1), and,
as with the orthologous regions from C. gynandra, each
UTR alone was sufficient to confer such specificity (Figure 4i
and Table S1). These data indicate that the CA4 gene from
A. thaliana possesses cis-elements that are sufficient for
M-cell specificity when placed into the C4 environment.
DISCUSSION
Functional latency within multiple C3 genes co-opted
into C4 photosynthesis
As elements in the UTRs of PPDK and CA4 from C3 A. thaliana are sufficient for preferential accumulation in M cells of
C. gynandra, alterations to cis-elements are not required to
generate M-specific function in the C4 pathway. Combined
with work on NAD-ME1 and 2 (Brown et al., 2011), these data
indicate that four genes from the last common ancestor of
A. thaliana and C. gynandra have been recruited into
cell-specific accumulation in C. gynandra without changes
to cis-elements. The fact that genes can be recruited to
M- and BS-specific function in C4 leaves without alterations
to cis-elements may have facilitated evolution of the pathway. As the CaMV 35S promoter drives expression in both M
and BS cells of C4 leaves (Bansal et al., 1992; Brown et al.,
2011; Patel et al., 2006), the reduction in the proportion of BS
cells containing GUS when PPDK or CA4 UTRs are present
implies that post-transcriptional regulation is probably

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

52 Kaisa Kajala et al.

(a)

2 3

4 5 67

8 9

CgCA4
TAA

ATG
2

5 6 7 8

AtCA4
TAA

ATG
1

3 45 6

7 8

10 11

12 13

CgBASS2
TGA

ATG
1 2 3 4 5 6

7 8 9 10 11 12

13

AtBASS2

1 kb
TAA

ATG

(b)

(c)

(d)

(e)

(f)

(g)

Figure 4. CA accumulates in M cells of C. gynandra due to elements in either the 5 or 3 UTR.

(a) Gene structures of CA4 and BASS2 genes
from C. gynandra and their A. thaliana orthologs. Exons are numbered and indicated by black
boxes, with arrowheads representing transcriptional start sites. The translational start and stop
codons are indicated.
(bg) Confocal laser scanning microscopy after
microprojectile bombardment of Arabidopsis
thaliana leaves shows that CA4::GFP (bd) localizes close to the plasma membrane and
BASS2::GFP (eg) localizes to the chloroplast
envelope. (b, e) Chlorophyll channel, (c, f) GFP
channel, and (d, g) chlorophyll and GFP images
merged. Scale bars = 10 lm.
(h) Elements within either the 5 or 3 UTR of CA4
from C. gynandra are sufficient to generate
M-cell specificity in leaves of C. gynandra.
(i) Elements within either the 5 or 3 UTR of CA4
from C. gynandra and equivalent regions from
A. thaliana CA4 are sufficient to generate M-cell
specificity in leaves of C. gynandra. Cells accumulating GUS were counted. Three or more
biological replicates were performed. Error bars
represent standard errors. P values were determined by a one-tailed t test.

(h)

(i)

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

C4 evolution 53
important. However, we cannot rule out the possibility that
transcriptional mechanisms contribute to cell specificity.
The fact that the BS specificity of NAD-ME proteins important for C4 photosynthesis in C. gynandra is mediated by
elements that are present in their coding regions, together
with the data on CgPPDK and CgCA4, suggests that
transcript sequence is important for M-cell and BS-cell
specificity in this species.
Genes recruited into M- or BS-specific function in the C4
pathway are present in C3 plants, but prior to recruitment
into C4 photosynthesis, tend to be expressed at relatively
constitutive and low levels (Aubry et al., 2011; Brown et al.,
2010; Taylor et al., 2010). When we expressed the uidA gene
under the control of the CgPPDK promoter in A. thaliana,
accumulation of GUS was increased in mature leaves
compared with use of the endogenous AtPPDK promoter.
This indicates that the C4 PPDK promoter has acquired
strong enhancer elements that extend expression from
senescent leaves in the C3 species A. thaliana (Taylor et al.,
2010) into mature photosynthetic leaves of C. gynandra.
Furthermore, our data indicate that these enhancer elements
are recognized by factors in A. thaliana, and thus trans
factors responsible for enhanced expression in the C4 plant
C. gynandra are already present in A. thaliana. The increased accumulation of GUS when fused to the 5 UTR of
CgPPDK alone indicates that at least part of this increased
expression may be due to elements within the 5 UTR. Our
data are also consistent with the PPDK gene recruited into
the C4 pathway from C. gynandra possessing the same dual
promoter system that produces either cytosolic of chloroplastic isoforms of PPDK as reported for other species
(Parsley and Hibberd, 2006; Rosche and Westhoff, 1995;
Sheen, 1991).
Mechanisms underlying cell specificity in C4 leaves
Because a large number of genes in multiple plant lineages
are co-opted into the pathway, it is not surprising that a
number of mechanisms, ranging from transcriptional
through post-transcriptional to translational, are known to
control accumulation of proteins in either M or BS cells
(Berry et al., 1987; Hibberd and Covshoff, 2010; Sheen,
1999). However, although post-transcriptional regulation
(Patel et al., 2004, 2006) and translational elongation (Berry
et al., 1986) have been implicated in the accumulation of
protein in BS cells, there have been no reports that these
mechanisms result in accumulation of proteins in M cells of
C4 leaves. The regulation of CgPPDK and CgCA4 is therefore
more similar to that of RbcS in the BS cells of F. bidentis
(Patel et al., 2004, 2006) and NAD-ME in C. gynandra (Brown
et al., 2011) than to that of PPDK or other M-cell-specific
proteins studied to date (Akyildiz et al., 2007; Gowik et al.,
2004; Kausch et al., 2001; Matsuoka and Numazawa, 1991).
The 5 or 3 UTR of CgPPDK or equivalent regions of
CgCA4 are sufficient to generate M-cell specificity in

C. gynandra. The most parsimonious explanation is that

all four UTRs share a characteristic that is recognized by a
common trans factor to generate M-cell specificity. In
eukaryotes, high AU content of UTRs is important to induce
instability and degradation of mRNAs (Chen and Shyu,
1995). However, we detected no clear difference in the AU
content of the CgCA4 and CgPPDK UTRs compared with
those from CgBASS2, which were not sufficient for M-cell
specificity. This implies that AU content per se is not
sufficient for M-cell specificity in C. gynandra. A polypyrimidine tract after the 5 cap (Levy et al., 1991) and the
downstream element (DST) motif (Newman et al., 1993) are
also known to regulate transcript stability; however, neither
of these elements were present in the UTRs of the PPDK
and CA4 genes that we studied. In the C4 species F. bidentis, the 5 and 3 UTRs of transcripts encoding the small
subunit of RuBisCO can specify accumulation of the GFP
reporter in BS cells, and it was proposed that RNA-binding
proteins mediate turnover of transcripts via AU-rich elements identified in these UTRs (Patel et al., 2006). This is
also a possible mechanism in accumulation of CgPPDK and
CgCA4 transcripts in M cells of C. gynandra, although a
clear UUAUU motif (Patel et al., 2006) is not present.
Irrespective of the exact mechanism, our data show that
M-cell specificity in C4 leaves is under the control of
elements in UTRs, and importantly that this has occurred
for multiple genes. Our data also show that elements in the
UTRs of PPDK and CA4 from C3 A. thaliana are sufficient for
accumulation in M cells of C. gynandra, and demonstrate
that alterations to cis-elements are not required for M-cellspecific function in the C4 pathway. The results also suggest
that post-transcriptional regulation is particularly important
in maintaining the C4 pathway in leaves of this species.
EXPERIMENTAL PROCEDURES
Generation of constructs
cDNA sequences were obtained after performing degenerate PCR
followed by 5 and 3 RACE using a FirstChoice RLM-RACE kit
(Ambion, http://www.ambion.com/). Coding regions were amplified from both cDNA and genomic DNA. Promoter regions were
isolated by genome walking using a BD GenomeWalker Universal
Kit (BD Biosciences, http://www.bdbiosciences.com/). Reporter
gene constructs containing the gfp gene or the uidA gene were
generated by cloning the UTRs and coding sequence (CDS) into a
modified pENTR/D-TOPO vector (Invitrogen, http://www.invitrogen.com/) containing a gfp::uidA::nosT cassette (Brown et al.,
2011). The 3 UTRs were inserted between uidA and nosT. The 5
UTRs were cloned together with the CgPPDK promoter or fused to
the CaMV 35S promoter by PCR and inserted in front of the cassette. Spliced coding sequences amplified from cDNA were
inserted between the 5 UTRs and the gfp gene. The assembled
constructs were used for microprojectile bombardment (Hibberd
et al., 1998), and recombined with LR clonase (Invitrogen) into the
Gateway destination vector pGWB1 (Nakagawa et al., 2007),
transferred into Agrobacterium tumefaciens GV3101, and then
into Arabidopsis thaliana.

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

54 Kaisa Kajala et al.

Plant transformation
Transient expression of the constructs in C. gynandra was achieved
by microprojectile bombardment as described by Brown et al.
(2011). C. gynandra seeds from intact pods were germinated on
moist filter paper in darkness at 30C for 24 h, transferred onto MS
medium with 1% w/v sucrose and 0.8% w/v agar (pH 5.8), and
grown for 13 days at 22C, 200 lmol m)2 sec)1 photon flux density
with a 16 h photoperiod. Gold particles (1.0 lm diameter; Bio-Rad,
http://www.bio-rad.com/) were coated with 0.5 pmol plasmid DNA
of pENTRY/D-TOPO-based constructs. Two-week-old seedlings
were positioned abaxial side up on filter paper moistened with halfstrength MS medium, 6 cm below the stopping screen (Bio-Rad),
and bombarded three times (at 1100 psi) using a Bio-Rad PDS-1000/
He particle delivery system. After bombardment, seedlings were
held vertically with the base of their stems in half-strength MS
medium, and incubated for 48 h at 22C, 200 lmol m)2 sec)1 photon flux density with a 16 h photoperiod, prior to GUS staining or
laser scanning confocal microscopy.
Transgenic A. thaliana Col-0 plants were generated by floral
dipping (Clough and Bent, 1998). Primary transformants were
identified by selection on 50 lg ml)1 kanamycin for 7 days (Harrison et al., 2006), and grown for a further 3 weeks on a 3:1 mixture of
compost and vermiculite at 20C, 200 lmol m)2 sec)1 photon flux
density with a 16 h photoperiod, prior to GUS staining.

RNA hybridization and GUS staining

RNA in situ hybridization was performed using digoxigenin-labeled
RNA probes on 7 lm transverse sections of C. gynandra leaf primordia as previously described (Brown et al., 2011). Riboprobe
template DNA of the PPDK coding sequence was amplified by PCR
using primers with T3 and T7 polymerase binding sites, allowing
generation of both antisense and sense probes.
For analysis of GUS accumulation in transgenic plants, tissue was
placed in 0.1 M Na2HPO4 (Fisher Scientific, http://www.fishersci.
com/), 0.5 mM K ferricyanide (Fisher Scientific), 0.5 mM K ferrocyanide (Sigma-Aldrich, http://www.sigmaaldrich.com/), 10 mM
EDTA, 0.06% v/v Triton X-100 (Sigma-Aldrich) and 2 mM X-GlcA
(5-bromo-4-chloro-3-indolyl beta-D-glucuronide) (Sigma-Aldrich),
vacuum infiltrated (2 30 sec for A. thaliana, 3 3 min for C. gynandra), and incubated at 37C for 20 h. Tissue was then fixed in 3:1
ethanol/acetic acid for 30 min at room temperature, and chlorophyll
was cleared using 70% v/v ethanol at 37C for 24 h and then 5% w/v
sodium hydroxide at 37C for 2 h. M and BS cells containing GUS
were identified and counted using phase-contrast microscopy. In
each experiment, at least 50 cells were counted, and at least three
independent repetitions of the bombardment were performed for
each construct. MUG activity assays were performed on 3-week-old
seedlings. Samples were frozen in liquid nitrogen and then ground
in 200 ll 50 mM NaH2PO4, 0.07% b-mercaptoethanol, 0.1% Triton X-100 prior to centrifugation at 13 000 g for 5 min. A dilution
series was run for each sample to ensure linearity of the assay. The
assay was stopped using 200 mM Na2CO3 after various time
intervals in order to ensure linearity.

sion objective. Images were collected using separate channels for

GFP (500545 nm), chlorophyll fluorescence (630720 nm) and
transmission microscopy, and these channels were overlaid using
LAS AF software (Leica).

Quantitative PCR and laser capture microscopy

For quantitative RT-PCR, C. gynandra was grown a 3:1 compost/
vermiculite mixture, at 22C, 350 lmol m)2 sec)1 photon flux density, 65% relative humidity and a 16 h photoperiod. Mature leaf
samples were collected from 4-week-old plants, cut into 2 mm
strips, fixed in 3:1 ethanol/acetic acid for 16 h, and treated for 1 h
each in 75, 85, 100, 100 and 100% v/v ethanol, followed by 1 h each
in 75:25, 50:50, 25:75, 0:100, 0:100 and 0:100% v/v ethanol/Histoclear
(Thermo Scientific, http://www.thermoscientific.com), all at 4C
(Kerk et al., 2003). Paraplast X-tra (Sigma-Aldrich) was added to the
final Histoclear wash, and the samples were incubated at 58C for
5 days, washing the sample with molten Paraplast X-tra every 12 h.
The sections were positioned upright in Paraplast X-tra in Peel-AWay S22 moulds (Agar Scientific, http://www.agarscientific.com/),
allowed to cool and stored at 4C. The sample blocks were sectioned
using a rotary microtome (Jung) and Shandon MX35+ Premier
microtome blades (Thermo Scientific) to 7 lm thickness. The sections were floated in sterile water on Probe-On Plus microscope
slides (Fisher Scientific) to straighten them, the water was drained
off, and the slides were dried at 42C for 16 h. The slides were stored
at 4C and used within 4 weeks after sectioning. Prior to laser capture microdissection, slides were washed in Histoclear for 1 h and
then air-dried for 15 min.
Laser capture microdissection was performed using a Veritas
microdissection instrument model 704 and CapSure HS LCM caps
(both MDS Analytical Technologies, http://www.moleculardevices.
com/) according to the manufacturers instructions. Approximately 500 cells were captured per cap. Immediately after LCM,
RNA was extracted directly from the caps using a PicoPure RNA
extraction kit (MDS Analytical Technologies), and synthesized into
cDNA using SuperScript II and random primers (Promega, http://
www.promega.com/). Quantitative RT-PCR was performed exactly
as described by Brautigam et al. (2011) using a DNA Engine
thermal cycler, a Chromo4 real-time detector (Bio-Rad, http://
www.bio-rad.com) and SYBR Green JumpStart Taq Ready Mix
(Sigma-Aldrich). The PCR program comprised initial denaturation at 94C for 2 min, followed by 40 cycles of 94C for 20 sec,
60C for 30 sec, 72C for 30 sec and 75C for 5 sec. CT values
were generated for three technical replicates for three independent biological replicates per cell type. Standard errors were
calculated from 2DDC T values of each combination of biological
replicates.

ACKNOWLEDGEMENTS
We thank the Frank Smart fund for a studentship to K.K., the Biotechnology and Biology Sciences Research Council for funding, and
Samart Wanchana (C4 Rice Centre, International Rice Research
Institute) for advice.

SUPPORTING INFORMATION
Confocal laser scanning microscopy
The subcellular localization of translational fusions of coding
sequences of PPDK and CA4 to GFP was visualized by laser scanning confocal microscopy. Sections of transformed leaves were
placed on microscope slides adaxial side up, a drop of water was
added, and a cover slip was placed on top. The cells were viewed
using a Leica TCS SP5 confocal microscope (http://www.leica.com/)
with an excitation wavelength of 488 nm and a 63 water-immer-

Additional Supporting Information may be found in the online

version of this article:
Figure S1. Schematics of each construct used for regulation of gene
expression in C. gynandra and A. thaliana.
Figure S2. Confocal laser scanning microscopy shows that GFP
expressed under the control of the CaMV 35S promoter localizes to
the cytosol after microprojectile bombardment of A. thaliana
leaves.

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

C4 evolution 55
Figure S3. Alignment of the 5 UTRs and 3 UTRs of C. gynandra and
A. thaliana PPDK genes.
Figure S4. Quantitative assessment of CA4 and BASS2 transcripts
from mesophyll and bundle sheath cells of C. gynandra.
Figure S5. Alignment of the 5 UTRs and 3 UTRs of C. gynandra and
A. thaliana CA4 genes.
Table S1. Number of cells showing GUS staining after microprojectile bombardment.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.

REFERENCES
Akyildiz, M., Gowik, U., Engelmann, S., Koczor, M., Streubel, M. and Westhoff, P. (2007) Evolution and function of a cis-regulatory module for
mesophyll-specific gene expression in the C4 dicot Flaveria trinervia. Plant
Cell, 19, 33913402.
Aubry, S., Brown, N.J. and Hibberd, J.M. (2011) The role of proteins in C3
plants prior to their recruitment into the C4 pathway. J. Exp. Bot. 62, 3049
3059.
Bansal, K.C., Viret, J.F., Haley, J., Khan, B.M., Schantz, R. and Bogorad, L.
(1992) Transient expression from cab-m1 and rbcS-m3 promoter sequences is different in mesophyll and bundle sheath cells in maize leaves.
Proc. Natl Acad. Sci. USA, 89, 36543658.
Berry, J.O., Nikolau, B.J., Carr, J.P. and Klessig, D.F. (1986) Translational
regulation of light-induced ribulose-1,5-bisphosphate carboxylase gene
expression in amaranth. Mol. Cell. Biol. 6, 23472353.
Berry, J.O., Carr, J.P. and Klessig, D.F. (1987) Translational control of ribulose1,5-bisphosphate carboxylase synthesis in amaranth. J. Cell. Biochem. 33,
4856.
Bowes, G., Ogren, W.L. and Hageman, R.H. (1971) Phosphoglycolate production catalyzed by ribulose diphosphate carboxylase. Biochem. Biophys.
Res. Commun. 45, 716722.
Brautigam, A., Kajala, K., Wullenweber, J. et al. (2011) An mRNA blueprint for
C4 photosynthesis derived from comparative transcriptomics of closely
related C3 and C4 species. Plant Physiol. 155, 142156.
Brown, N.J., Parsley, K. and Hibberd, J.M. (2005) The future of C4 research
maize, Flaveria or Cleome? Trends Plant Sci. 10, 215221.
Brown, N.J., Palmer, B.G., Stanley, S. et al. (2010) C4 acid decarboxylases
required for C4 photosynthesis are active in the mid-vein of the C3 species
Arabidopsis thaliana, and are important in sugar and amino acid metabolism. Plant J. 61, 122133.
Brown, N.J., Newell, C.A., Stanley, S., Chen, J.E., Perrin A., J., Kajala, K. and
Hibberd, J.M. (2011) Independent and parallel recruitment of pre-existing
mechanisms underlying C4 photosynthesis. Science, 331, 14361439.
Chen, C.-Y.A. and Shyu, A.-B. (1995) AU-rich elements: characterization and
importance in mRNA degradation. Trends Biochem. Sci. 20, 465470.
Christin, P.A., Besnard, G., Samaritan, E., Duvall, M.R., Hodkinson, T.R.,
Savolainen, V. and Salamin, N. (2008) Oligocene CO2 decline promoted C4
photosynthesis in grasses. Curr. Biol. 18, 3743.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,
735743.
Danker, T., Dreesen, B., Offermann, S., Horst, I. and Peterhansel, C. (2008)
Developmental information but not promoter activity controls the methylation state of histone H3 lysine 4 on two photosynthetic genes in maize.
Plant J. 53, 465474.
Edwards, E.J., Osborne, C.P., Stromberg, C.A.E., Smith, S.A. and Consortium,
C.G. (2010) The origins of C4 grasslands: integrating evolutionary and
ecosystem science. Science, 328, 587591.
Furumoto, T., Yamaguchi, T., Ohshima-Ichie, Y. et al. (2011) A plastidial sodium-dependent pyruvate transporter. Nature, 476, 472475.
Gowik, U., Burscheidt, J., Akyildiz, M., Schlue, U., Koczor, M., Streubel, M.
and Westhoff, P. (2004) cis-Regulatory elements for mesophyll-specific
gene expression in the C4 plant Flaveria trinervia, the promoter of the C4
phosphoenolpyruvate carboxylase gene. Plant Cell, 16, 10771090.

Harrison, S.J., Mott, E.K., Parsley, K., Aspinall, S., Gray, J.C. and Cottage, A.
(2006) A rapid and robust method of identifying transformed Arabidopsis
thaliana seedlings following floral dip transformation. Plant Methods, 2,
19.
Hatch, M.D. (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim. Biophys. Acta, 895, 81106.
Hibberd, J.M. and Covshoff, S. (2010) The regulation of gene expression
required for C4 photosynthesis. Annu. Rev. Plant Biol. 61, 181207.
Hibberd, J.M., Linley, P.J., Khan, M.S. and Gray, J.C. (1998) Transient
expression of green fluorescent protein in various plastid types following
microprojectile bombardment. Plant J. 16, 627632.
Kanai, R. and Edwards, G.E. (1999) The biochemistry of C4 photosynthesis. In
C4 Plant Biology (Sage, R.F. and Monson, R.K., eds). San Diego: Academic
Press, pp. 4987.
Kausch, A.P., Owen, T.P., Zachwieja, S.J., Flynn, A.R. and Sheen, J. (2001)
Mesophyll-specific, light and metabolic regulation of the C4 PPCZm1 promoter in transgenic maize. Plant Mol. Biol. 45, 115.
Kerk, N.M., Ceserani, T., Tausta, S.L., Sussex, I.M. and Nelson, T.M. (2003)
Laser capture microdissection of cells from plant tissues. Plant Physiol.
132, 2735.
Ku, S.B., Edwards, G.E. and Kanai, R. (1976) Distribution of enzymes related to
C3 and C4 pathway of photosynthesis between mesophyll and bundle
sheath cells of Panicum hians and Panicum miloides. Plant Cell Physiol. 17,
615620.
Langdale, J.A. and Nelson, T. (1991) Spatial regulation of photosynthetic
development in C4 plants. Trends Genet. 7, 191196.
Langdale, J.A., Taylor, W.C. and Nelson, T. (1991) Cell-specific accumulation
of maize phosphoenolpyruvate carboxylase is correlated with demethylation at a specific site greater than 3 kb upstream of the gene. Mol. Gen.
Genet. 225, 4955.
Leegood, R.C. (2002) C4 photosynthesis: principles of CO2 concentration and
prospects for its introduction into C3 plants. J. Exp. Bot. 53, 581590.
Levy, S., Avni, D., Hariharan, N., Perry, R.P. and Meyuhas, O. (1991) Oligopyrimidine tract at the 5 end of mammalian ribosomal protein mRNAs is
required for their translational control. Proc. Natl Acad. Sci. USA, 88, 3319
3323.
Marshall, D.M., Muhaidat, R., Brown, N.J., Liu, Z., Stanley, S., Griffiths, H.G.,
Sage, R.F. and Hibberd, J.M. (2007) Cleome, a genus closely related to
Arabidopsis, contains species spanning a developmental progression from
C3 to C4 photosynthesis. Plant J. 51, 886896.
Matsuoka, M. and Numazawa, T. (1991) Cis-acting elements in the pyruvate,orthophosphate dikinase gene from maize. Mol. Gen. Genet. 228, 143
152.
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y.,
Toyooka, K., Matsuoka, K., Jinbo, T. and Kimura, T. (2007) Development of
series of Gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104, 3441.
Newman, T.C., Ohme-Takagi, M., Taylor, C.B. and Green, P.J. (1993) DST
sequences, highly conserved among plant SAUR genes, target reporter
transcripts for rapid decay in tobacco. Plant Cell, 5, 701714.
Parsley, K. and Hibberd, J.M. (2006) The Arabidopsis PPDK gene is transcribed
from two promoters to produce differentially expressed transcripts
responsible for cytosolic and plastidic proteins. Plant Mol. Biol. 62, 339349.
Patel, M., Corey, A.C., Yin, L.P., Ali, S.J., Taylor, W.C. and Berry, J.O. (2004)
Untranslated regions from C4 amaranth AhRbcS1 mRNAs confer translational enhancement and preferential bundle sheath cell expression in
transgenic C4 Flaveria bidentis. Plant Physiol. 136, 35503561.
Patel, M., Siegel, A.J. and Berry, J.O. (2006) Untranslated regions of FbRbcS1
mRNA mediate bundle sheath cell-specific gene expression in leaves of a
C4 plant. J. Biol. Chem. 281, 2548525491.
Reiskind, J.B., Berg, R.H., Salvucci, M.E. and Bowes, G. (1989) Immunogold
localization of primary carboxylases in leaves of aquatic and a C3C4
intermediate species. Plant Sci. Lett. 61, 4352.
Rosche, E. and Westhoff, P. (1995) Genomic structure and expression of the
pyruvate,orthophosphate dikinase gene of the dicotyledonous C4 plant
Flaveria trinervia (Asteraceae). Plant Mol. Biol. 29, 663678.
Rosche, E., Chitty, J., Westhoff, P. and Taylor, W.C. (1998) Analysis of promoter activity for the gene encoding pyruvate orthophosphate dikinase in
stably transformed C4 Flaveria species. Plant Physiol. 117, 821829.
Sage, R.F., Christin, P.A. and Edwards, E.J. (2011) The C4 lineages of planet
Earth. J. Exp. Bot. 62, 31553169.

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

56 Kaisa Kajala et al.

Sheen, J.-Y. (1991) Molecular mechanisms underlying the differential
expression of maize PPDK genes. Plant Cell, 3, 225245.
Sheen, J. (1999) C4 gene expression. Annu. Rev. Plant Physiol. Plant Mol. Biol.
50, 187217.
Sinha, N.R. and Kellogg, E.A. (1996) Parallelism and diversity in multiple
origins of C4 photosynthesis in grasses. Am. J. Bot. 83, 14581470.
Stockhaus, J., Schlue, U., Koczor, M., Chitty, J.A., Taylor, W.C. and Westhoff,
P. (1997) The promoter of the gene encoding the C4 form of
phosphoenolpyruvate carboxylase directs mesophyll-specific expression
in transgenic C4 Flaveria spp. Plant Cell, 9, 479489.
Taniguchi, M., Izawa, K., Ku, M.S.B., Lin, J.H., Saito, H., Ishida, Y., Ohta, S.,
Komari, T., Matsuoka, M. and Sugiyama, T. (2000) Binding of cell typespecific nuclear proteins to the 5-flanking region of maize C4
phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells. Plant Mol. Biol. 44, 543557.
Tanz, S.K., Tetu, S.G., Vella, N.G.F. and Ludwig, M. (2009) Loss of the transit
peptide and an increase in gene expression of an ancestral chloroplastic
carbonic anhydrase were instrumental in the evolution of the cytosolic C4
carbonic anhydrase in Flaveria. Plant Physiol. 150, 15151529.

Taylor, L., Nunes-Nesi, A., Parsley, K., Leiss, A., Leach, G., Coates, C., Wingler,
A., Fernie, A.R. and Hibberd, J.M. (2010) Cytosolic pyruvate,orthophosphate
dikinase functions in nitrogen remobilization during leaf senescence and
limits individual seed growth and nitrogen content. Plant J. 62, 641652.
Vicentini, A., Barber, J.C., Aliscioni, S.S., Giussani, L.M. and Kellogg, E.A.
(2008) The age of the grasses and clusters of origins of C4 photosynthesis.
Global Change Biol. 14, 29632977.
Voznesenskaya, E.V., Franceschi, V.R., Kiirats, O., Freitag, H. and Edwards,
G.E. (2001) Kranz anatomy is not essential for terrestrial C4 plant photosynthesis. Nature, 414, 543546.
Voznesenskaya, E.V., Koteyeva, N.K., Chuong, S.D.X., Ivanova, A.N., Barroca,
J., Craven, L.A. and Edwards, G.E. (2007) Physiological, anatomical and
biochemical characterisation of photosynthetic types in genus Cleome
(Cleomaceae). Funct. Plant Biol. 34, 247267.
Westhoff, P. and Gowik, U. (2004) Evolution of C4 phosphoenolpyruvate
carboxylase. Genes and proteins: a case study with the genus Flaveria.
Ann. Bot. 93, 1323.

2011 The Authors

The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 4756

Copyright of Plant Journal is the property of Wiley-Blackwell and its content may not be copied or emailed to
multiple sites or posted to a listserv without the copyright holder's express written permission. However, users
may print, download, or email articles for individual use.