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  • Multiple Arabidopsis genes primed for recruitment intoC4 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 [email protected]).

    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 charac-

    teristic of a C4 leaf. Either the 5 or the 3 UTR is sufficient for cell specificity, indicating that functionalredundancy 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. thalianagenes. 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 C4pathway allows CO2 to be concentrated around the central

    photosynthesis enzyme Ribulose-1,5-Bisphosphate Carbo-

    xylase 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-

    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-phos-

    phate isomerase (RPI) and RuBisCO are restricted to BS

    2011 The Authors 47The Plant Journal 2011 Blackwell Publishing Ltd

    The Plant Journal (2012) 69, 4756 doi: 10.1111/j.1365-313X.2011.04769.x

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  • 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 Covs-

    hoff, 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, DNAmethylation, and also histonemodifications.

    M-cell-specific accumulation of ZmPEPC transcripts inmaize

    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-glucoron-idase, 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 aG A substitution. Placing MEM1 into the C3 Ppc promoterfrom 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 tran-

    scripts 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 identi-

    fied 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 pref-

    erentially in M cells, and a region between )301 and )296from the translational start site is important for this. Gel-

    retardation assays showed that this region binds a protein

    termed PPD1 (Matsuoka and Numazawa, 1991). When the

    region between )370 and )76 in themaize promoter is fusedto the rice (Oryza sativa) promoter, high expression is

    observed inM protoplasts (Matsuoka and Numazawa, 1991).

    In stable transformants of F. bidentis, use of the region

    between )1212 and +279 from the transcriptional start sitefused 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 impor-

    tant 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 phy-

    logenetic proximity to A. thaliana facilitates annotation of

    likely gene function, and also provides relatively simple

    resources for functional analysis of orthologous genes in aC3species. To investigate mechanisms underlying M-cell spec-

    ificity 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-CA4genes fromC3A. thaliana, wewere also ableto 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 tran-

    scriptional 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 3RACE, 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 theuidA gene encoding b-glucuronidase (GUS) and investi-gated whether it contained enhancer elements compared

    with the orthologous promoter from the closely related C3species A. thaliana (Figure S1A,B). This promoter drives

    expression of transcripts that encode the longer form of the

    48 Kaisa Kajala et al.

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

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  • 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 pro-

    moter, 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 accumu-

    lation 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 chloro-

    plasts 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. gyn-

    andra (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

    (a)

    (b) (c) (d)

    (e) (f) (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 themature protein, gray

    representing the chloroplast transit peptide, and white indicating 5 or 3 untranslated regions. The translational start and stop codons are annotated. The promoterdirecting 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 maximumMUG 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.

    C4 evolution 49

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  • 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

    CaMV 35S promoter, when the CgPPDK cDNA including 5and 3 UTRs (Figure S1D) was incorporated into this con-struct, 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 wassufficient (Figure 2f and Table S1). Compared with each UTR

    alone, the presence of both 5 and 3 UTRs slightly increasedthe proportion of M cells accumulating GUS.

    The 5 and 3 UTRs of PPDK from A. thaliana generateM-cell specificity in C. gynandra

    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 UTRsfrom A. thaliana PPDK were sufficient for preferential accu-

    mulation 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 speci-

    ficity. 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 5UTR nor the 3UTR of CgPPDK, or both together, led toa 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 aloneslightly increased the activity of GUS in leaves of A. thaliana

    compared with the CaMV 35S:uidA controls (Figure 3e).

    (a)

    (c) (e)

    (d)

    (f)

    (g)

    (b) Figure 2. PPDK accumulates in M cells of C. gynandra due to elements inboth 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 aresufficient to generate M-cell specificity in leaves of C. gynandra.

    (g) Elements within either the 5 or 3 UTR of PPDK from A. thaliana aresufficient 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.

    50 Kaisa Kajala et al.

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

    sufficient for accumulation in M cells

    Laser capturemicroscopy 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 betweenGFP 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 con-

    sistent 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 forM accumulation (Figure 4h and Table S1). As with PPDK,

    either the 5 or the 3 UTR was sufficient for accumulation ofGUS in M cells (Figure 4i). Taken together, these data

    indicate that elements in either the 5 or the 3 UTR ofCgPPDK and CgCA4 are sufficient for preferential accumu-

    lation 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 fromA. thaliana CA4 (Figure S1Q,R) were sufficient for preferen-

    tial accumulation in theM 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. thali-

    ana 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 onNAD-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 path-

    way. As the CaMV 35S promoter drives expression in bothM

    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

    (a) (b)

    (c)

    (e)

    (d)

    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) orthe 3 UTR alone (d) in the CaMV35S::uidA fusion had little impact on thespatial accumulation of GUS in A. thaliana.

    (e) Quantitative assays show that both UTRs and the 5 UTR increaseexpression compared with the CaMV 35S control.

    Scale bars = 1 cm.

    C4 evolution 51

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  • CgBASS2

    AtBASS2

    CgCA4

    AtCA4

    ATG

    1

    1

    1

    2

    2

    2

    1 2

    3

    3

    3

    4

    4

    4

    5

    5

    5

    6

    6

    6

    7

    7

    7

    8

    8

    8

    9

    9

    9

    10

    10

    11

    11

    12

    12

    13

    13

    3 4 5 6 7 8 9

    ATG

    ATG

    ATG TAA

    TAA

    TGA

    1 kb

    TAA

    (a)

    (h)

    (i)

    (b)

    (e) (f) (g)

    (c) (d)

    Figure 4. CA accumulates in M cells of C. gyn-

    andra 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 ortho-

    logs. Exons are numbered and indicated by black

    boxes, with arrowheads representing transcrip-

    tional 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) local-

    izes 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 CA4from C. gynandra are sufficient to generate

    M-cell specificity in leaves of C. gynandra.

    (i) Elements within either the 5 or 3 UTR of CA4from C. gynandra and equivalent regions from

    A. thaliana CA4 are sufficient to generate M-cell

    specificity in leaves of C. gynandra. Cells accu-

    mulating GUS were counted. Three or more

    biological replicates were performed. Error bars

    represent standard errors. P values were deter-

    mined by a one-tailed t test.

    52 Kaisa Kajala et al.

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

  • 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 impor-

    tant 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 C4pathway 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 in-

    creased accumulation of GUS when fused to the 5 UTR ofCgPPDK alone indicates that at least part of this increased

    expression may be due to elements within the 5 UTR. Ourdata 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 chloro-

    plastic 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) andNAD-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 ofCgCA4 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 polypy-

    rimidine tract after the 5 cap (Levy et al., 1991) and thedownstream 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. biden-

    tis, the 5 and 3 UTRs of transcripts encoding the smallsubunit 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 ele-

    ments 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-cell-

    specific 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 PCRfollowed by 5 and 3 RACE using a FirstChoice RLM-RACE kit(Ambion, http://www.ambion.com/). Coding regions were ampli-fied from both cDNA and genomic DNA. Promoter regions wereisolated by genome walking using a BD GenomeWalker UniversalKit (BD Biosciences, http://www.bdbiosciences.com/). Reportergene constructs containing the gfp gene or the uidA gene weregenerated by cloning the UTRs and coding sequence (CDS) into amodified pENTR/D-TOPO vector (Invitrogen, http://www.invitro-gen.com/) containing a gfp::uidA::nosT cassette (Brown et al.,2011). The 3 UTRs were inserted between uidA and nosT. The 5UTRs were cloned together with the CgPPDK promoter or fused tothe CaMV 35S promoter by PCR and inserted in front of the cas-sette. Spliced coding sequences amplified from cDNA wereinserted between the 5 UTRs and the gfp gene. The assembledconstructs were used for microprojectile bombardment (Hibberdet al., 1998), and recombined with LR clonase (Invitrogen) into theGateway destination vector pGWB1 (Nakagawa et al., 2007),transferred into Agrobacterium tumefaciens GV3101, and theninto Arabidopsis thaliana.

    C4 evolution 53

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

    RosalbaResaltado

    RosalbaResaltado

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  • Plant transformation

    Transient expression of the constructs in C. gynandrawas achievedby microprojectile bombardment as described by Brown et al.(2011). C. gynandra seeds from intact pods were germinated onmoist filter paper in darkness at 30C for 24 h, transferred onto MSmedium with 1% w/v sucrose and 0.8% w/v agar (pH 5.8), andgrown for 13 days at 22C, 200 lmol m)2 sec)1 photon flux densitywith a 16 h photoperiod. Gold particles (1.0 lm diameter; Bio-Rad,http://www.bio-rad.com/) were coated with 0.5 pmol plasmid DNAof pENTRY/D-TOPO-based constructs. Two-week-old seedlingswere positioned abaxial side up on filter paper moistened with half-strength 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 wereheld vertically with the base of their stems in half-strength MSmedium, and incubated for 48 h at 22C, 200 lmol m)2 sec)1 pho-ton flux density with a 16 h photoperiod, prior to GUS staining orlaser scanning confocal microscopy.Transgenic A. thaliana Col-0 plants were generated by floral

    dipping (Clough and Bent, 1998). Primary transformants wereidentified by selection on 50 lg ml)1 kanamycin for 7 days (Harri-son et al., 2006), and grown for a further 3 weeks on a 3:1 mixture ofcompost and vermiculite at 20C, 200 lmol m)2 sec)1 photon fluxdensity with a 16 h photoperiod, prior to GUS staining.

    RNA hybridization and GUS staining

    RNA in situ hybridization was performed using digoxigenin-labeledRNA probes on 7 lm transverse sections of C. gynandra leaf pri-mordia as previously described (Brown et al., 2011). Riboprobetemplate DNA of the PPDK coding sequence was amplified by PCRusing primers with T3 and T7 polymerase binding sites, allowinggeneration 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 ferro-cyanide (Sigma-Aldrich, http://www.sigmaaldrich.com/), 10 mMEDTA, 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. gyn-andra), and incubated at 37C for 20 h. Tissue was then fixed in 3:1ethanol/acetic acid for 30 min at room temperature, and chlorophyllwas cleared using 70% v/v ethanol at 37C for 24 h and then 5% w/vsodium hydroxide at 37C for 2 h. M and BS cells containing GUSwere identified and counted using phase-contrast microscopy. Ineach experiment, at least 50 cells were counted, and at least threeindependent repetitions of the bombardment were performed foreach construct. MUG activity assays were performed on 3-week-oldseedlings. Samples were frozen in liquid nitrogen and then groundin 200 ll 50 mM NaH2PO4, 0.07% b-mercaptoethanol, 0.1% Tri-ton X-100 prior to centrifugation at 13 000 g for 5 min. A dilutionseries was run for each sample to ensure linearity of the assay. Theassay was stopped using 200 mM Na2CO3 after various timeintervals in order to ensure linearity.

    Confocal laser scanning microscopy

    The subcellular localization of translational fusions of codingsequences of PPDK and CA4 to GFP was visualized by laser scan-ning confocal microscopy. Sections of transformed leaves wereplaced on microscope slides adaxial side up, a drop of water wasadded, and a cover slip was placed on top. The cells were viewedusing a Leica TCS SP5 confocal microscope (http://www.leica.com/)with an excitation wavelength of 488 nm and a 63 water-immer-

    sion objective. Images were collected using separate channels forGFP (500545 nm), chlorophyll fluorescence (630720 nm) andtransmission microscopy, and these channels were overlaid usingLAS 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 den-sity, 65% relative humidity and a 16 h photoperiod. Mature leafsamples were collected from 4-week-old plants, cut into 2 mmstrips, fixed in 3:1 ethanol/acetic acid for 16 h, and treated for 1 heach in 75, 85, 100, 100 and 100% v/v ethanol, followed by 1 h eachin 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 thefinal Histoclear wash, and the samples were incubated at 58C for5 days, washing the sample with molten Paraplast X-tra every 12 h.The sections were positioned upright in Paraplast X-tra in Peel-A-Way S22 moulds (Agar Scientific, http://www.agarscientific.com/),allowed to cool and stored at 4C. The sample blocks were sectionedusing a rotary microtome (Jung) and Shandon MX35+ Premiermicrotome blades (Thermo Scientific) to 7 lm thickness. The sec-tions were floated in sterile water on Probe-On Plus microscopeslides (Fisher Scientific) to straighten them, the water was drainedoff, and the slides were dried at 42C for 16 h. The slides were storedat 4C and used within 4 weeks after sectioning. Prior to laser cap-ture microdissection, slides were washed in Histoclear for 1 h andthen 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. Approxi-mately 500 cells were captured per cap. Immediately after LCM,RNA was extracted directly from the caps using a PicoPure RNAextraction kit (MDS Analytical Technologies), and synthesized intocDNA using SuperScript II and random primers (Promega, http://www.promega.com/). Quantitative RT-PCR was performed exactlyas described by Brautigam et al. (2011) using a DNA Enginethermal 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 denatur-ation 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 valueswere generated for three technical replicates for three indepen-dent biological replicates per cell type. Standard errors werecalculated from 2DDCT values of each combination of biologicalreplicates.

    ACKNOWLEDGEMENTS

    We thank the Frank Smart fund for a studentship to K.K., the Bio-technology and Biology Sciences Research Council for funding, andSamart Wanchana (C4 Rice Centre, International Rice ResearchInstitute) for advice.

    SUPPORTING INFORMATION

    Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Schematics of each construct used for regulation of geneexpression in C. gynandra and A. thaliana.Figure S2. Confocal laser scanning microscopy shows that GFPexpressed under the control of the CaMV 35S promoter localizes tothe cytosol after microprojectile bombardment of A. thalianaleaves.

    54 Kaisa Kajala et al.

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

  • Figure S3. Alignment of the 5UTRs and 3UTRs of C. gynandra andA. thaliana PPDK genes.Figure S4. Quantitative assessment of CA4 and BASS2 transcriptsfrom mesophyll and bundle sheath cells of C. gynandra.Figure S5. Alignment of the 5UTRs and 3UTRs of C. gynandra andA. thaliana CA4 genes.Table S1. Number of cells showing GUS staining after micropro-jectile bombardment.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

    REFERENCES

    Akyildiz, M., Gowik, U., Engelmann, S., Koczor, M., Streubel, M. and West-

    hoff, 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 C3plants 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 se-

    quences 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 ribulose-

    1,5-bisphosphate carboxylase synthesis in amaranth. J. Cell. Biochem. 33,

    4856.

    Bowes, G., Ogren, W.L. and Hageman, R.H. (1971) Phosphoglycolate pro-

    duction catalyzed by ribulose diphosphate carboxylase. Biochem. Biophys.

    Res. Commun. 45, 716722.

    Brautigam, A., Kajala, K., Wullenweber, J. et al. (2011) AnmRNA 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 metabo-

    lism. 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 C4photosynthesis in grasses. Curr. Biol. 18, 3743.

    Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agro-

    bacterium-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 meth-

    ylation 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 so-

    dium-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 C4phosphoenolpyruvate 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 biochem-

    istry, 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 pro-

    moter 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 Panicummiloides. 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 demethyla-

    tion 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) Oligo-

    pyrimidine tract at the 5 end of mammalian ribosomal protein mRNAs isrequired 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 pyru-

    vate,orthophosphate dikinase gene frommaize.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 construc-

    tion 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. PlantMol. 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 transla-

    tional 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 C3C4intermediate 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 pro-

    moter 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.

    C4 evolution 55

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

  • 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 type-

    specific nuclear proteins to the 5-flanking region of maize C4phosphoenolpyruvate carboxylase gene confers its differential transcrip-

    tion 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 C4carbonic 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. andHibberd, 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 photo-

    synthesis. 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.

    56 Kaisa Kajala et al.

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

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