A transcription factor module mediating C2photosynthesis

A transcription factor module mediating C2 photosynthesis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A transcription factor module mediating C 2 photosynthesis View ORCID Profile Patrick J. Dickinson , View ORCID Profile Sebastian Triesch , View ORCID Profile Urte Schlüter , View ORCID Profile Andreas P.M. Weber , View ORCID Profile Julian M. Hibberd doi: https://doi.org/10.1101/2023.09.05.556297 Patrick J. Dickinson 1 Department of Plant Sciences, University of Cambridge , Downing Street, Cambridge CB2 3EA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick J. Dickinson Sebastian Triesch 2 Institute of Biochemistry, Heinrich-Heine University , 40225 Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sebastian Triesch Urte Schlüter 2 Institute of Biochemistry, Heinrich-Heine University , 40225 Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Urte Schlüter Andreas P.M. Weber 2 Institute of Biochemistry, Heinrich-Heine University , 40225 Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andreas P.M. Weber Julian M. Hibberd 1 Department of Plant Sciences, University of Cambridge , Downing Street, Cambridge CB2 3EA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Julian M. Hibberd For correspondence: jmh65{at}cam.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT C 4 photosynthesis has arisen from the ancestral C 3 state in over sixty lineages of angio-sperms. It is widely accepted that an early step in C 4 evolution is restriction of glycine decarboxylase activity to bundle sheath cells to generate the so-called C 2 pathway. In C 2 Moricandia species, changes to the cis -regulatory region controlling expression of the P-subunit of GLYCINE DECARBOXYLASE ( GLDP ) in mesophyll cells enables this trait, but the mechanism underpinning GLDP expression in the bundle sheath is not known. We identify a MYC-MYB transcription factor module previously associated with the control of glucosinolate bio-synthesis as the basis of GLDP expression in bundle sheath cells. In C 3 Arabidopsis thaliana this module drives GLDP expression in bundle sheath cells along with as yet unidentified factors driving expression in mesophyll cells. In the C 2 species Moricandia arvensis, GLDP expression is lost from mesophyll cells and the MYC-MYB dependent expression in the bundle sheath is revealed. Evolution of C 2 photosynthesis is thus associated with a MYC-MYB based transcriptional network already present in the C 3 state. This work identifies a molecular genetic mechanism underlying the bundle sheath accumulation of glycine decarboxylase required for C 2 photosynthesis and thus a foundational step in the evolution of C 4 photosynthesis. Introduction Fixation of CO 2 during photosynthesis is central to life. In plants this is dependent on Ribulose 1,5-Bisphosphate Carboxylase Oxygenase (RuBisCO) operating as part of the Calvin-Benson-Bassham cycle. However, in addition to reacting with CO 2 RuBisCO catalyses a side-reaction with O 2 to produce the toxic metabolite phosphoglycolate ( Bowes et al., 1971 ). The photorespiratory pathway metabolises phosphoglycolate, but CO 2 is lost and ATP, NADPH and amino acids are required ( Tolbert, 1971 ). As temperatures increase the ratio of oxygenation to carboxylation reactions at the RuBisCO active site increases and so losses from photorespiration become more significant ( Jordan and Ogren, 1984 ). It is widely thought that carbon concentrating mechanisms such as C 4 photosynthesis evolved to reduce the metabolic costs of photorespiration. In the case of the C 4 pathway this involves modifications to leaf anatomy, cell biology and biochemistry ( Hatch, 1987 ). Typically, C 4 biochemistry enables initial fixation of bicarbonate by the enzyme phospho enol pyruvate carboxylase in mesophyll cells. Subsequent decarboxylation of C 4 acids releases high concentrations of CO 2 in a compartment such as the bundle sheath ( Sage, 2001 ; Christin et al., 2013 ) and so the oxygenase activity of RuBisCO is reduced ( Leegood, 2002 ; Carmo-Silva et al., 2015 ). Some genera contain species that possess biochemical and anatomical characteristics associated with both C 3 and C 4 photosynthesis. Such plants have become known as C 3 -C 4 intermediates or more recently C 2 species ( Sage et al., 2012 ; Lundgren, 2020 ), and although statistical modelling predicted that the order of C 4 trait acquisition is flexible ( Williams et al., 2013 ), a consistent and early event is considered a shift of glycine decarboxylase from mesophyll cells such that its activity is restricted to the bundle sheath ( Rawsthorne et al., 1988 ; Morgan et al., 1993 ). Repositioning of glycine decarboxylase to the bundle sheath is conjectured to initiate greater rates of CO 2 release and thus increased photosynthetic activation of this tissue. The two-carbon glycine molecule thus provides CO 2 for photosynthesis and led to the term C 2 photosynthesis. Glycine decarboxylase is made up of four subunits and loss of expression of the P-subunit ( GLDP ) from the mesophyll has repeatedly driven the appearance of C 2 photosynthesis ( Rawsthorne et al., 1988 ; Morgan et al., 1993 ; Schulze et al., 2016 ). One example of this is found in the Brassicaceae family where the Moricandia genus contains both C 3 and C 2 species ( Schlüter et al., 2017 ; Schlüter et al., 2023 ). In Brassicaceae species a DNA region referred to as the mesophyll (M) box is highly conserved in promoters of the GLDP1 gene from C 3 and C 2 species ( Adwy et al., 2015 ). Promoter deletion analysis showed that this region is involved in driving expression in mesophyll cells in A. thaliana and C 3 M. moricandioides ( Adwy et al., 2015 , Adwy et al., 2019 ). Insertion of transposable elements between the M box and the core promoter is thought to abolish mesophyll expression of GLDP1 in C 2 species leading to bundle sheath preferential expression ( Triesch et al., 2022 ). In contrast to our understanding of how loss of mesophyll expression of GLDP1 is brought about, the molecular architecture enabling the emergence of bundle sheath GLDP1 expression in the Brassicaceae has not yet been defined. Using C 3 Arabidopsis thaliana we first show that a bipartite MYC and MYB transcription factor module responsible for directing the transcription factor MYB76 and thus glucosinolate biosynthesis genes to the bundle sheath is also able to pattern GLDP1 to this tissue. In the C 3 state this MYC-MYB module operates in parallel with the M box ( Adwy et al 2015 ) to ensure expression in both mesophyll and bundle sheath cells. The MYC-MYB binding sites are conserved in C 2 M. arvensis whereas the insertion of transposable elements has shifted the M box so that it’s function is disrupted ( Triesch et al., 2022 ). Therefore, this MYC-MYB module allows expression of GLDP1 and assembly of the glycine decarboxylase holoprotein specifically in bundle sheath cells. We thus identify a molecular architecture in the C 3 state operating in both cis and trans that underpins a foundational trait associated with the evolution of C 2 and C 4 photosynthesis. Results and Discussion In C 3 A. thaliana MYC and MYB transcription factors drive expression in the bundle sheath which combined with a mesophyll module generates broad expression across the leaf A. thaliana contains two copies of GLDP , both of which are expressed in leaves (Supplemental Fig. 1A) ( Aubry et al., 2013 ). All Brassicaceae lineages containing C 2 species are members of the monophyletic Brassiceae tribe. In the Brassiceae, GLDP2 is lost leaving GLDP1 as the only GLDP copy, preconditioning the evolution of C 2 photosynthesis ( Schlüter et al., 2017 ). As GLDP1 is the copy of GLDP involved in C 2 photosynthesis we focussed on understanding the expression of GLDP1 . As expected, the GLDP1 promoter from C 3 A. thaliana drove constitutive expression in leaves ( Fig. 1A , Supplemental Fig. 2). Consistent with previous analysis ( Adwy et al., 2015 ), a 5’ deletion removing the M box revealed that the proximal promoter is sufficient to generate expression in bundle sheath strands ( Fig. 1B , Supplemental Fig. 3). Download figure Open in new tab Figure 1. MYC and MYB TF binding motifs control bundle sheath strand expression in Arabidopsis thaliana . 1A) Schematic and representative GUS staining image of the full length -1458 bp A. thaliana GLDP1 promoter upstream of the translational start site (ATG) from 19 independent T1 lines. B) Schematic and representative GUS staining image of the -561 bp A. thaliana GLDP1 promoter upstream of the ATG from 13 independent T1 lines C) Predicted TF binding motifs in the M-box and from -561 bp upstream to the ATG of the A. thaliana GLDP1 promoter. The position in the promoter (bp) is on the x axis, and the predicted binding affinity (p-values calculated from the log-likelihood score by the FIMO tool is on the y axis). The motifs are coloured by the motif clusters shown underneath the plots (Supplemental Table 1). D to F) Transcription factor binding motifs and representative GUS staining images of nucleotides - 561 to -295 bp upstream of the ATG fused to CaMV35sMin (D), -347 bp upstream to the ATG (E) and -100 bp sequence from -347 to -247 bp upstream of the translational start site fused to CaMV35sMin (F) from 17, 7 and 14 independent T1 lines respectively. Distance from the ATG (bp) is on the x-axis, and the predicted binding affinity (p-values calculated from the log-likelihood score by the FIMO tool ( Grant et al., 2011 )) is on the y axis. On GUS images, leaves were stained for 24 (A, B, E, and F) or 48 (D) hrs, scale bars are 200 μm and a zoom in of a region of the image is marked by a dashed white box. Bundle sheath cells marked with a dashed white box on the zoomed in inlay. To better understand mechanisms controlling the expression of GLDP1 in bundle sheath strands of A. thaliana we first used publicly available data to identify potential transcription factor binding sites in the promoter ( Fig. 1C ). Because not all transcription factor binding sites have been defined and some transcription factors are predicted to bind to the same or very similar sequences, we clustered motifs from the JASPAR database (Fornes, 2020) by similarity (Supplemental table 1). This provided an indication of transcription factor families likely able to bind the GLDP1 promoter. In the 59 base pair M box, binding sites for C2H2, MADS, bZIP and BPC transcription factor families were present ( Fig. 1C ) suggesting that members of these families could be responsible for generating expression in the mesophyll. Although previous work had shown that nucleotides -561 to -295 upstream of the translational start site of GLDP1 are necessary for expression in bundle sheath strands ( Adwy et al., 2015 ) it is not known if they are sufficient for this patterning. We therefore searched for transcription factor binding sites in this region but also in the sequence up to the translational start site ( Fig. 1C ). Motifs associated with sixteen families of transcription factor families were identified, and this included closely spaced MYELOCYTOMATOSIS (MYC - belonging to the bHLH family) and MYOBLASTOMA (MYB) binding sites ( Fig. 1C ). A bipartite module involving MYC2,3&4 and MYB28&29 directs expression of the MYB76 transcription factor and glucosinolate biosynthesis genes to the bundle sheath of A. thaliana ( Dickinson and Knerova et al., 2020 ). Re-analysis of publicly available data showed that GLDP1 ex pression was not reduced in leaves of the triple my c2/3/4 mutant ( Major et al., 2017 ). However, in the double myb28/29 mutant ( Burow et al., 2015 ) a small reduction in GLDP1 transcript abundance was apparent (Supplemental Fig. 1B). As the bundle sheath of A. thaliana comprises only ∼15% of all cells in the leaf ( Kinsman and Pyke, 1998 ), expression of GLDP1 in the mesophyll will dominate signal from whole leaves. We thus considered this small change in GLDP1 expression in the myb28/29 mutant allele as consistent with MYB transcription factors controlling GLDP1 expression in the bundle sheath. We hypothesised that the closely spaced MYC and MYB motifs drive expression of AtGLDP1 in bundle sheath strands that becomes easily detectable once function of the mesophyll box is lost. Loss of the MYC binding site between nucleotides -305 and -299 upstream of the ATG abolished expression in bundle sheath strands ( Adwy et al., 2015 ). However, the importance of of the MYB site between nucleotides -284 and -277 has not been investigated ( Fig. 1C ). We therefore conjectured that the region containing only the MYC binding site would not be sufficient for expression in bundle sheath strands. Consistent with this, when nucleotides -561 to -295 were fused to the minimal CaMV35S promoter, GUS activity was not detected ( Fig. 1D , Supplemental Fig. 4). In contrast, when the MYC and MYB binding sites were both present, bundle sheath expression was restored ( Fig. 1E , Supplemental Fig. 5). This indicates that sequence upstream of the MYC binding site is not necessary for expression in bundle sheath strands. When only the MYC and MYB binding sites were fused to the minimal CaMV35S promoter, GUS activity was detected in bundle sheath strands ( Fig. 1F , Supplemental Fig. 6). We conclude that closely spaced MYC and MYB binding sites, from nucleotide -305 to -277, in the C 3 A. thaliana GLDP1 promoter are necessary and sufficient for bundle sheath strand expression. Thus, this MYC-MYB module can act alone to generate expression of genes such as MYB76 in bundle sheath strands ( Dickinson and Knerova et al., 2020 ) but as seen for AtGLDP1 , it can also act in concert with other elements such as the mesophyll box to ensure expression in both bundle sheath and mesophyll cells. We next sought to test whether the MYC-MYB module is conserved in GLDP1 genes from C 2 species. C 2 Moricandia species contain conserved and functional MYC and MYB binding sites in the GLDP1 promoter The Brassicaceae contains at least five independent origins of C 2 photosynthesis ( Schlüter et al., 2023 ). We hypothesized that conservation of MYC and MYB binding sites driving bundle sheath expression of GLDP1 across the Brassicaceae underpins the repeated evolution of this trait. To test this, we aligned GLDP1 promoter sequences from 17 species across the Brassicaceae including nine C 3 and eight C 2 species representing the five independent origins of C 2 photosynthesis ( Guerreiro et al., 2023 ). The MYC binding site ( CACGTG ) is perfectly conserved in all 17 species analysed and the MYB binding site ( CACCAAC ) is perfectly conserved in all species except B. gravinae and D. tenuifolia where a single substitution at position five of the motif replaced thymine with adenine ( Fig. 2A ). This suggests that the MYC and MYB binding sites responsible for driving bundle sheath strand expression of GLDP1 in A. thaliana may be functional across these C 3 and C 2 Brassicaceae species. These data indicate that cis -elements allowing expression in bundle sheath strands have remained stable for at least 20.8 Ma since the divergence of Arabidopsis and Moricandia ( Schlüter et al., 2017 ). Download figure Open in new tab Figure 2. MYC and MYB binding sites are conserved in the Brassicaceae and drive vein and bundle sheath preferential expression of Moricandia GLDP1 genes. A) Sequence alignments of the region of Brassicaceae GLDP1 promoters containing MYC and MYB TF binding sites. MYC and MYB TF biding sites are coloured in gold and blue and marked above the alignment. B) Position of MYC and MYB binding sites in the M. moricandioides GLDP1 promoter. C) Position of MYC and MYB binding sites and representative GUS staining images from 18 and 11 independent T1 lines respectively for M. moricandioides -293 bp and (D) -220 bp promoters. E) Position of MYC and MYB binding sites in the M. arvensis GLDP1 promoter. F) Position of MYC and MYB binding sites and representative GUS staining images from 19 and 9 independent T1 lines respectively for M. arvensis -318 bp and (G) -245 bp promoters. Distance from the ATG (bp) is on the x axis, and the predicted binding affinity (P values calculated from the log-likelihood score by the FIMO tool ( Grant et al., 2011 ) is on the y axis. On GUS images, leaves were stained for 24 (C and F) or 48 (D and G) hrs scale bars are 200 μm and a zoom in of a region of the image is marked by a dashed white box. Bundle sheath cells marked with a dashed white box on the zoomed in inlay. H) Schematic showing model for the control of GLDP1 expression in C 3 A. thaliana and M. moricandioides (top) and C 2 M. arvensis (bottom). In C 3 species constitutive expression is driven by unknown transcription factor(s) activating mesophyll expression from the M box ( Adwy et al., 2015 ), potentially through binding to motifs from C2H2, MADS, bZIP and/or BPC families ( Fig. 1C ), and MYC and MYB TFs binding to closely spaced TF binding motifs to activate expression in the vein and bundle sheath. In C 2 species the M-box is unable to activate expression in the mesophyll however MYC and MYB binding sites are conserved leading to bundle sheath strand specific expression of GLDP1 . To test whether these motifs are functional in Brassicaceae species in addition to A. thaliana we used the Moricandia genus for further investigation. Moricandia contains C 3 and C 2 species and previous work has shown that a promoter region, containing the predicted MYC and MYB binding sites is necessary for expression in the bundle sheath strand ( Adwy et al., 2015 ; Adwy et al., 2019 ). To test whether MYC and MYB binding sites were able to drive expression in bundle sheath strands we cloned fragments from GLDP1 promoters of C 3 M. moricandioides and C 2 M. arvensis . In C 3 M. moricandioides closely spaced MYC and MYB sites are found between nucleotides -293 and -220 upstream of the predicted translational start site ( Fig. 2B ). Promoter deletions that removed all upstream sequence but retained the MYC and MYB sites, or also removed the MYC and MYB sites themselves were generated. When these motifs were present GUS activity was detected in bundle sheath strands ( Fig. 2C , Supplemental Fig.7) but when they were absent this was not the case ( Fig. 2D , Supplemental Fig. 8). Therefore, this C 3 member of Moricandia contains sequence in the GLDP1 promoter that is recognised by the MYC-MYB module of A. thaliana and it is able to pattern gene expression to bundle sheath strands. The GLDP1 promoter from C 2 M. arvensis also has closely spaced MYC and MYB motifs ( Fig. 2E ). When they were present, GUS activity was detected in A. thaliana bundle sheath stands ( Fig. 2F , Supplemental Fig. 9) but when they are absent it was not ( Fig. 2G , Supplemental Fig. 10). Taken together, these data show that the bipartite MYC and MYB transcription factor module responsible for directing MYB76 and glucosinolate biosynthesis genes to bundle sheath strands of A. thaliana is also used to pattern expression of GLDP1 to this tissue. Moreover, the cis -code that is necessary and sufficient for bundle sheath strand expression is found in GLDP1 genes from C 3 and C 2 species of Moricandia . The evolution of C 2 photosynthesis in the Brassicaceae is thus associated with a shift of the M-box which disrupts its function ( Triesch et al., 2022 ) and retention of closely spaced MYC and MYB binding sites such that GLDP1 is expressed specifically in bundle sheath strands ( Fig. 2H ). Overall, this reveals a molecular genetic mechanism underpinning the bundle sheath accumulation of glycine decarboxylase required for C 2 photosynthesis, and thus for a foundational step in the evolution of the C 4 photosynthetic pathway. Further analysis will be required to establish whether other C 2 and C 4 lineages have made use of this MYC-MYB transcription factor module or whether evolution has convergently recruited other transcription factors to pattern genes to the bundle sheath. Materials and methods Plant materials and growth conditions A. thaliana was grown on Levington F2 soil in growth chambers set at 20 °C, with a 16-hour-photoperiod with a light intensity of 150 μmol m −2 s −1 photon flux density and 60% relative humidity. Transcription factor binding site prediction, sequence alignments, cloning and GUS assays Motif clustering was performed on plant transcription factor motifs downloaded from JASPAR using the RSAT tool ( Castro-Mondragon et al., 2017 ) as reported previously ( Dickinson and Knerova et al., 2020 ). The FIMO tool ( Grant et al., 2011 ) was used to scan DNA sequences for matches to A. thaliana transcription factor binding motifs found in the JASPAR motif database (Fornes, 2020). To account for input sequence composition, a background model was generated using the fasta-get-markov tool from the MEME suite ( Bailey et al., 2009 ). FIMO was then run with the default parameters and a P value cut-off of 1 × 10 −4 . Brassicaceae GLDP1 promoter sequences were retrieved from phytozome ( Goodstein et al., 2012 ) and promoters of Moricandia species were taken from Adwy et al., (2019) . Sequences were aligned using MUSCLE ( Edgar, 2004 ) with default settings and alignments visualised with the UGENE tool ( Okonechnikov et al., 2012 ). Promoter GUS constructs were assembled using the Golden Gate system ( Weber et al., 2011 ). Arabidopsis promoter fragments were isolated from genomic DNA by PCR (primers in supplemental table 1) and cloned into level 0 modules (module information in supplementary table 2). Moricandia promoter fragments were initially amplified from genomic DNA using primers, adding a 5’ ClaI and a 3’ XbaI overhang. The amplified promoter sequences were subcloned into the pJET1.2 cloning vector using the Thermo Scientific CloneJET PCR Cloning Kit following the manufacturer’s instructions. Moricandia promoter fragments for Golden Gate cloning were then amplified from these pJET vectors and cloning into level 0 modules. Level 1 constructs were then assembled to fuse the promoter fragments with the CaMV 35sMinimal promoter were required, the GUS reporter and Nos terminator. Level 2 constructs were then assembled to add the FastR selectable marker ( Shimada et al., 2010 ) to allow selection of positive transformants. Level 2 constructs were then placed into Agrobacterium tumefaciens strain GV3101 and introduced into A. thaliana Col-0 by floral dipping ( Clough and Bent, 1998 ). To take into account position effects associated with the transgene insertion site, GUS staining was undertaken on at least six randomly selected T1 plants for each uidA fusion. The staining solution contained 0.1 M Na 2 HPO 4 (pH 7.0), 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 10 mM EDTA (pH 8.0), 0.06% (v/v) Triton X-100 and 0.5 mg ml −1 X-gluc. Leaves from three-week-old plants were vacuum-infiltrated three times in GUS solution for one minute and then incubated at 37 °C for 24 h. Next, stained samples were fixed in 3:1 (v/v) ethanol:acetic acid for 30 minutes at room temperature, cleared in 70% (v/v) ethanol at 37 °C and then placed in 5 M NaOH for 2 h. The samples were stored in 70% (v/v) ethanol at 4 °C. The samples were imaged with an Olympus BX41 light microscope with Q Capture Pro 7 software and a QImaging MicroPublisher 3.3 RTV camera. Acknowledgements The work was funded by the Advanced European Research Council Grant 694733 REVOLUTION, BBSRC grant BBW00013X1 to JMH and European Union Program (project GAIND4CROPS GA number 862087) to JMH and APMW. Work in the group of APMW was funded by ERA-CAPS project “C4BREED” under Project ID WE 2231/20–1, the Cluster of Excellence for Plant Sciences (CEPLAS) under Germany’s Excellence Strategy EXC-2048/1 under project ID 390686111, and the CRC TRR 341 “Plant Ecological Genetics” grant by the German Research Foundation (DFG). For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising from this submission. Footnotes Email addresses PD: pd373{at}cam.ac.uk , JMH: jmh65{at}cam.ac.uk References ↵ Adwy , W. , Laxa , M. , and Peterhansel , C. ( 2015 ). A simple mechanism for the establishment of C2-specific gene expression in Brassicaceae . The Plant Journal . 84 : 1231 – 1238 . OpenUrl CrossRef PubMed ↵ Adwy , W. , Schlüter , U. , Papenbrock , J. , Peterhansel , C. , and Offermann , S. ( 2019 ). Loss of the M-box from the glycine decarboxylase P-subunit promoter in C2 Moricandia species . 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