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Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used in C4 Photosynthesis Is Based on an Ancient Code Found in the Ancestral C3 State

Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used... C photosynthesis has evolved repeatedly from the ancestral C state to generate a carbon concentrating mechanism that 4 3 increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C cycle is accomplished through cell-type- specific accumulation of enzymes but cis-elements and transcription factors controlling C photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcriptioninC plants evolved from ancestral components found in C species. Two basic Helix-Loop-Helix (bHLH) 4 3 transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C and C Panicoid grass species of the PACMAD clade. Our 3 4 analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C state. We conclude that C photosynthesis has co-opted an ancient C regulatory code built on G-box recognition by bHLH to 4 3 regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C photosynthesis. Key words: basic Helix-Loop-Helix, cis-element evolution, C3 and C4 photosynthesis, NADP-Malic Enzyme, PACMAD Panicoid grasses. Monson 1993). In environments such as the tropics where Introduction rates of photorespiration are high, C photosynthesis has C plants inherited a carbon fixation system developed by evolved repeatedly from the ancestral C state (Lloyd and photosynthetic bacteria, with atmospheric carbon dioxide Farquhar 1994; Osborne and Beerling 2006). Phylogenetic stud- (CO ) being incorporated into ribulose-1, 5-bisphosphate ies estimate that the first transition from C to C occurred 3 4 (RuBP) by the enzyme Ribulose Bisphosphate Carboxylase/ around 30 million years ago (MYA) (Christin et al. 2008, 2011; Oxygenase (RuBisCO) to form the three-carbon compound Vicentini et al. 2008). The ability of the C cycle to concentrate (C ) 3-phosphoglycerate (Calvin and Massini 1952). However, CO around RuBisCO limits oxygenation and so increases pho- RuBisCO can also catalyse oxygenation of RuBP, which leads tosynthetic efficiency in conditions where photorespiration is to the production of 2-phosphoglycolate, a compound that is enhanced (Hatch and Slack 1966; Maier et al. 2011; Christin toxic to the plant cell and needs to be detoxified through an and Osborne 2014; Lundgren and Christin 2017). energetically wasteful process called photorespiration (Bowes The evolution of C photosynthesis involved multiple et al. 1971; Sharkey 1988; Sage 2004). The oxygenase reaction modifications to leaf anatomy and biochemistry (Hatch of RuBisCO becomes more common as temperature 1987; Sage 2004). In most C plants, photosynthetic reactions increases and so in C plants photorespiration can reduce are partitioned between two distinct cell types known as photosynthetic output by up to 30% (Ehleringer and mesophyll (M) and bundle sheath (BS) cells (Langdale 2011). The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is Open Access properly cited. 1690 Mol. Biol. Evol. 35(7):1690–1705 doi:10.1093/molbev/msy060 Advance Access publication April 5, 2018 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE M and BS cells are arranged in concentric circles around veins cell-specific expression in the C leaf, no TFs recognizing these in the so-called Kranz anatomy (Haberlandt 1904), which cis-elements have yet been identified. enables CO pumping from M to BS where RuBisCO is spe- To address this gap in our understanding, a bottom-up cifically located. Atmospheric CO is first converted to HCO approach was initiated to identify TFs that regulate the maize 2 3 by carbonic anhydrase (CA) and then combined with phos- gene ZmC -NADP-ME (GRMZM2G085019) that encodes the phoenolpyruvate (PEP) by PEP-carboxylase (PEPC) to pro- Malic Enzyme responsible for releasing CO in the BS cells. duce oxaloacetate in the M cells. This four-carbon acid (C ) Using Yeast One-Hybrid two maize TFs belonging to the su- is subsequently converted into malate and/or aspartate that perfamily of basic Helix-Loop-Helix (bHLH), ZmbHLH128 and transport the fixed CO from M to BS cells (Kagawa and 2 ZmbHLH129, were identified and functionally characterized. Hatch 1974; Hatch 1987). Three biochemical C subtypes 4 We show that these TFs bind two cis-elements synergistically are traditionally described based on the predominant type and analysis of the NADP-ME promoters from grass species of C acid decarboxylase responsible for the CO release 4 2 from BEP and PACMAD (Panicoideae subfamily) indicated around RuBisCO in the BS: NADP-dependent Malic Enzyme that this regulation is likely derived from an ancestral G-box (NADP-ME, e.g. Zea mays), NAD-dependent Malic Enzyme that is present in C species. (NAD-ME, e.g. Gynandropsis gynandra formerly designated Results Cleome gynandra) and phosphoenolpyruvate carboxykinase (PEPCK). However, recent reports suggest that only the ZmbHLH128 and ZmbHLH129 Homeologs Bind NADP-ME and NAD-ME should be considered as distinct FAR1/FHY3 Binding Site cis-Elements in the C subtypes, which in response to environmental cues may ZmC -NADP-ME Promoter involve a supplementary PEPCK cycle (Williams et al. 2012; To identify TFs that interact with the ZmC -NADP-ME gene Wang et al. 2014; Rao and Dixon 2016). (GRMZM2G085019), we studied the promoter region com- The recruitment of multiple genes into C photosynthesis prising 1982 bp upstream of the translational start site. This involved both an increase in their transcript levels (Hibberd region was divided into six overlapping fragments ranging and Covshoff 2010) and also patterns of expression being from 235 to 482 bp in length (supplementary table S1, modified from relatively constitutive in C species (Maurino Supplementary Material online) and used in Yeast One- et al. 1997; Penfield et al. 2004; Taylor et al. 2010; Brown et al. Hybrid (Y1H). Each fragment was used to generate one yeast 2011; Maier et al. 2011) to M- or BS-specific in C plants bait strain that was then used to screen a maize cDNA ex- (Hibberd and Covshoff 2010). Therefore, considerable efforts pression library. After screening at least 1.3 million colonies for have been made to identify the transcription factors (TF) and each region of the promoter, two maize bHLH TFs known as the cis-elements they recognize that are responsible for this ZmbHLH128 and ZmbHLH129 were identified. Both of these light-dependent and cell-specific gene expression (Hibberd TFs bind the promoter between base pairs 389 and 154 in and Covshoff 2010). Various studies suggest that different relation to the predicted translational start site of ZmC - transcriptional regulatory mechanisms have been adopted NADP-ME (fig. 1A). These interactions were confirmed by during C to C evolution. One is the acquisition of novel 3 4 re-transforming yeast bait strains harbouring each of the six cis-elements in C gene promoters that can be recognized by sections of the promoter with cDNAs encoding ZmbHLH128 TFs already present in C plants (Matsuoka et al. 1994; Ku and ZmbHLH129. Consistent with the initial findings, et al. 1999; Nomura et al. 2000), and a second possibility is the ZmbHLH128 and ZmbHLH129 only activated expression of acquisition of novel or modified TFs responsible for the re- the HIS3 reporter when transformed into yeast containing cruitment of genes into the C pathway through cis-elements fragment 389 to 154 bp upstream of ZmC -NADP-ME that pre-exist in C plants (Patel et al. 2006; Brown et al. 2011; (fig. 1B, supplementary fig. S1, Supplementary Material Kajala et al. 2012). online). A small number of cis-elements found in different gene ZmbHLH128 and ZmbHLH129 possess a bHLH domain regions have been shown to be sufficient for the M- or BS- followed by a contiguous leucine zipper (ZIP) motif specific expression of C genes. For example, a 41-base pair (fig. 1C). This bHLH domain is highly conserved between (bp) Mesophyll Expression Module 1 (MEM1) cis-element both ZmbHLHs and consists of 61 amino acids that can be was identified from the PEPC promoter of C Flaveria trinervia separated into two functionally distinct regions. The first is a and shown to be necessary and sufficient for M cell-specific basic region located at the N-terminal end of the bHLH do- accumulation of PEPC transcripts in C Flaveria species main andisinvolvedinDNA binding, andthe second is a (Gowik et al. 2004). A MEM1-like cis-element has also been Helix-Loop-Helix region mediating dimerization towards the found in the C carbonic anhydrase (CA3)promoterof carboxy-terminus (fig. 1C)(Murre et al. 1989; Toledo-Ortiz Flaveria bidentis and shown to drive M cell-specific expression et al. 2003). ZmbHLH128 and ZmbHLH129 share 91% amino (Gowik et al. 2017). A second cis-element named MEM2 and acid identity (fig. 1C) and they are encoded by homeolog consisting of 9 bp from untranslated regions has also been genes located in syntenic regions of maize chromosomes 4 shown to be capable of directing M-specificity in C G. gynan- dra (Kajala et al. 2012; Williams et al. 2016). Lastly, in the case and 5 (fig. 1D, supplementary table S2, Supplementary of the NAD-ME gene from C G. gynandra aregionfromthe Material online). coding sequence generates BS-specificity (Brown et al. 2011). Although ZmbHLH128 and ZmbHLH129 both possess In contrast to these insights into cis-elements that control three amino acids involved in G-box binding (K9, E13, and 1691 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.1. ZmbHLH128 and ZmbHLH129 homeologs bind the ZmC -NADP-ME promoter. (A) Schematic representation of the ZmC -NADP-ME 4 4 promoter, divided into fragments used as baits in Y1H screenings, and the ZmbHLH TFs identified. ATG and TAG are the translational start codon and the stop codon of the ZmC -NADP-ME ORF, respectively. ZmbHLH position on the scheme indicates that they bind between the base pairs 389 and 154 in relation to the ATG. (B) Analysis of ZmbHLH-pZmC -NADP-ME binding specificity. Each of the six yeast bait strains was transformed with both ZmbHLHs (pAD-GAL4-2.1::TF vectors) and positive interactions selected on CM -HIS -LEUþ 3-AT [yeast Complete Minimal medium lacking histidine and leucine amino acids, and supplemented with 3-amino-1, 2, 4-triazole (3-AT), a competitive inhibitor of the HIS3 gene product]. (C) Schematic representation of bHLH and ZIP protein domains, and respective position in protein sequences. (D) Schematic representation of ZmbHLH128 and ZmbHLH129 (black) and four additional maize homeolog gene pairs located in syntenic regions of chromo- somes 4 and 5. Homeolog genes are indicated by colour. Arrows indicate direction of transcription of each gene. Genomic coordinates provided from the B73 RefGen_v3 assembly version. R17) (Massari and Murre 2000; Li et al. 2006), this family of TFs Fisher and Caudy 1998; Kim et al. 2016). Therefore, the ZmC - 0 0 has also been shown to bind to N-box (5 -CACGCG-3 ), NADP-ME promoter was assessed for additional cis-elements 0 0 N-box B (5 -CACNAG-3 ) and FBS (FAR1/FHY3 Binding Site, to which ZmbHLH128 and ZmbHLH129 might bind. A total 0 0 5 -CACGCGC-3 )motifs(Sasai et al. 1992; Ohsako et al. 1994; of eight such cis-elements were found, consisting of two 1692 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE FIG.2. ZmbHLH128 and ZmbHLH129 bind two FBS cis-elements present in ZmC -NADP-ME promoter. (A) Schematic representation of position and nucleotide sequence of eight cis-elements recognized by bHLH that were identified in the ZmC -NADP-ME promoter. FBS stands for FHY3/ FAR1 Binding Site and it is a N-box-containing motif. (B) EMSA probe sequences used to test in vitro binding affinity of ZmbHLH128 and ZmbHLH129 to cis-elements (highlighted in bold). Arrows indicate that the FBS cis-elements are present in opposite orientations. (C) EMSAs showing in vitro binding affinity of Trx::ZmbHLH128 (gel on the left) and Trx::ZmbHLH129 (gel on the right) to the radiolabeled probes described in (B). Arrowheads indicate uplifted ZmbHLH–DNA probe complexes. Free probe indicates unbound DNA probes. N-boxes B, two N-boxes, one G-box, two FBSs, and one E-box intensities detected when both cis-elements were combined (fig. 2A). Electrophoretic Mobility Shift Assays (EMSA) were (fig. 2C) suggests that they function synergistically. Overall, used to test whether ZmbHLH128 and ZmbHLH129 were these data indicate that ZmbHLH128 and ZmbHLH129 target able to interact with each of these cis-elements in vitro 21 bp of DNA sequence (7 bp FBS, 7 bp spacer, and 7 bp FBS). (fig. 2B and C). Consistent with the Y1H findings, EMSA showed that recombinant Trx::ZmbHLH128 and ZmbHLH128 and ZmbHLH129 Form Both Trx::ZmbHLH129 proteins caused an uplift of radiolabeled Homo- and Heterodimers and ZmbHLH129 probes containing FBS cis-elements (probes 6, 7, and 6þ 7) Impairs trans-Activation by ZmbHLH128 (fig. 2C), positioned between nucleotides 389 and 154 in Because ZmbHLH128 and ZmbHLH129 bind the FBS cis-ele- relation to the predicted translational start site (see fig. 1A). ments in close proximity but also possess domains mediating ZmbHLH128 also showed weak binding to probe 3 that con- protein dimerization, we next investigated whether these tained a N-box cis-element that was not bound by proteins form homo- and/or heterodimers. In vitro, the re- ZmbHLH128 or ZmbHLH129 in Y1H (see fig. 1B), and signal combinant Trx::ZmbHLH128 and Trx::ZmbHLH129 proteins intensity was similar to that observed from probe 7 (fig. 2C). formed homodimers (fig. 3A). To confirm this interaction We cannot exclude however that relatively weak binding to in vivo,aswellastotestfor heterodimerization, probe 7 is due to it being three nucleotides-shorter than the Bimolecular Fluorescence Complementation Assays (BiFC) other probes (fig. 2B). Trx alone and OsPIF14 (a bHLH known in maize protoplasts were performed. While negative controls to bind the N-box motif; Cordeiro et al. 2016)wereusedas produced no YFP fluorescence, ZmbHLH128 and negative controls (fig. 2C). Thetwo FBSmotifs, in probe 6þ 7, ZmbHLH129 formed both homo- and heterodimers are separated by a short 7 bp spacer sequence and are found in opposite orientations (fig. 2B). The increase in band (fig. 3B). With the exception of ZmbHLH129 homodimers 1693 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.3. ZmbHLH128 and ZmbHLH129 form both homo- and heterodimers. (A) Western blot of BN-PAGE for the recombinant proteins Trx::His::ZmbHLH128 and Trx::His::ZmbHLH129. Gel was loaded with equivalent amount of protein. Recombinant proteins were immunodetected using a-His antibody. MW indicates molecular-weight size marker. (B) Protein interactions between ZmbHLH128 and ZmbHLH129 were tested by BiFC in maize mesophyll protoplasts co-transformed with constructs expressing ZmbHLH128 and ZmbHLH129 fused to N- and C-terminal YFP N C domains. YFP and YFP indicate split N- and C-terminal YFP domains, respectively. whose location extended to the cytoplasm and plasma lost when this TF was co-expressed with its homeolog membrane, in each case YFP signal was specifically local- ZmbHLH129 (fig. 4D). ized to the nucleus (fig. 3B). Nuclear localization of these ZmbHLH proteins supports their roles as transcriptional regulators. The G-Box-Based cis-Element Pair Recognized by To test the capacity of ZmbHLH128 and ZmbHLH129 to ZmbHLH128 and ZmbHLH129 in NADP-ME regulate transcription, transient expression assays were per- Promoters Operates Synergistically formed in leaves of Nicotiana benthamiana.The GUS reporter To understand whether the two FBS cis-elements identified in gene driven by the fragment of pZmC -NADP-ME to which the promoter of ZmC -NADP-ME (see fig. 2) are associated ZmbHLH128 and ZmbHLH129 bind was used as reporter, with the evolution of C photosynthesis, we investigated while the full-length ZmbHLH128 and ZmbHLH129 CDS whether they are conserved in promoters of other NADP- sequences driven by the constitutive CaMV35S promoter MEs from C and C grass species. Three C species 3 4 3 were used as effectors (fig. 4A). Co-infiltration of this re- (Dichanthelium oligosanthes, Oryza sativa,and porter with the ZmbHLH128 effector resulted in an in- Brachypodium distachyon)and threeC species (Z. mays, crease in GUS activity, indicating that ZmbHLH128 can Sorghum bicolor,and Setaria italica) were assessed (fig. 5A). act as a transcriptional activator (fig. 4B). In contrast, Within the C species, Z. mays and S. bicolor possess two ZmbHLH129 showed no intrinsic trans-activation activity plastidic NADP-ME isoforms: one that is used in C photo- (fig. 4C). In order to test whether the ZmbHLH128- synthesis (C -NADP-ME, GRMZM2G085019, and ZmbHLH129 heterodimers had a different trans-activation 4 Sobic.003g036200) and a second one not involved in the C activity from ZmbHLH128 or ZmbHLH129 homodimers, 4 cycle (nonC -NADP-ME, GRMZM2G122479, and leaves were co-infiltrated with the reporter and both effec- Sobic.009g108700) (Alvarez et al. 2013; Emms et al. 2016). tors simultaneously. Interestingly, the trans-activation activity observed for the ZmbHLH128 alone (fig. 4B)was In contrast, S. italica possesses only one plastidic NADP-ME 1694 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE isoform that is used in the C cycle (C -NADP-ME, Si000645) 4 4 (Alvarez et al. 2013; Emms et al. 2016). Although in C B. distachyon no homologous cis-elements to the FBSs in the ZmC -NADP-ME promoter were detected, in O. sativa one G-box was found in the same position as FBS 1 from Z. mays. Moreover, in the other promoters, cis-elements that can bind bHLH proteins were present in pairs (fig. 5A). In both the C and C grasses these cis-element pairs flank a 3 4 spacer that is highly conserved in sequence and length (7– 9bp) (fig. 5A). The C -NADP-ME promoters from Z. mays and S. bicolor share a common mutation in the third nucleo- tide position of the alignment (A!G) (fig. 5A). Two additional mutations are specific to Z. mays (the first and last nucleotides of FBS 1 and FBS 2, respectively), while one is S. bicolor-specific (C!T at the fourth position) (fig. 5A). It is possible that muta- tions unique to Z. mays or S. bicolor are neutral and the main impact on C -NADP-ME gene expression is due to mutation in the third nucleotide in the common ancestor of Z. mays and S. bicolor. Alternatively, it is also possible that both this mutation in the last common ancestor and species-specific modifica- tions impacted on gene expression of C -NADP-ME. To test if ZmbHLH128 and ZmbHLH129 bind the cis-ele- ments identified from these additional species EMSA was per- formed on each cis-element separately as well as the cis- element pairs found in each NADP-ME promoter (fig. 5B and C, supplementary table S3, Supplementary Material online). ZmbHLH128 and ZmbHLH129 showed low binding affinity for the single G-box identified in the O. sativa promoter (probe 13) and binding affinity was not increased by mutating the G-box to a canonical N-box (probe m13) (fig. 5B and C). This low binding affinity behaviour for single G-box cis-elements was consistent for all the NADP-ME promoters containing G- boxes (probes5,7,9,and 11) (fig. 5B and C). Although both ZmbHLHs did not show binding affinity for the additional N- boxes or N-box-like alone (probes 6, 8, 10, and 12) (fig. 5B and C), when these additional motifs were acquired and formed a pair with the ancestral G-box, binding affinity was increased (probes 5þ 6, 7þ 8, 9þ 10, and 11þ 12) and led to an in- creased uplift compared with the G-boxes alone (probes 5, 7, 9, and 11) (fig. 5B and C). Given the similar length of probes 1, 2, 1þ 2, 5, 7, 9, and 11 (24–30 bp) (supplementary table S3, Supplementary Material online), it is possible that this differ- ence in migration of ZmbHLH–probe complexes results from the binding of bHLH to G-boxes in a lower oligomeric state (supplementary fig. S2, Supplementary Material online), which based on the literature must be dimers (De Masi et al. 2011). Strong binding of cis-element pairs was also observed when the ancestral G-box evolved into either FBS or FeRE1 elements FIG.4. ZmbHLH129 impairs trans-activation of the ZmC -NADP-ME promoter by ZmbHLH128. (A) Schematic representation of reporter found in C Z. mays and S. bicolor (probes 1þ 2and 3þ 4) and effector constructs used in transient expression assays in leaves of (fig. 5B and C). In the C Z. mays promoter, both ZmbHLHs N. benthamiana. Reporter construct contains GUS gene driven by the showed binding affinity for single FBS cis-elements minimal CaMV35S promoter (m35S) fused to pZmC -NADP-ME (389 to 154 bp). Effector constructs contain the ZmbHLH128 or FIG.4. Continued ZmbHLH129 CDS driven by the full CaMV35S promoter. (B–D) Box (C) ZmbHLH129, and (D) ZmbHLH128 and ZmbHLH129. Different plots (2.5–97.5 percentiles) showing GUS activity, expressed in pico- letters denote differences in experimental data that are statistically moles of the reaction product 4-methylumbelliferone (MU) gener- significant (One-way ANOVA, Tukey test, P 0.05, n¼ 10-13). EV ated per minute per microgram of protein, in leaves agro-infiltrated indicates pGWB3i empty vector (no promoter fragment cloned). with reporter and the following effector constructs: (B) ZmbHLH128, Cross inside box plots indicates mean. f.c. indicates fold-change. 1695 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.5. The G-box-based cis-element pair recognized by ZmbHLH128 and ZmbHLH129 in NADP-ME promoters operates synergistically. (A) Sequence alignment of the two FBS cis-elements present in ZmC -NADP-ME promoter against homologous cis-elements present in other promoters of genes encoding plastidic NADP-ME. C grasses: Z. mays, S. bicolor and S. italica;C grasses: D. oligosanthes, O. sativa, and B. distachyon. 4 3 Plastidic NADP-MEs are color-coded: green for C , blue for nonC and magenta for C . Cis-elements are highlighted in bold and colored according 4 4 3 to the NADP-ME they belong to. FBS stands for FHY3/FAR1 Binding Site and FeRE1 for Iron Responsive Element 1. (B) EMSA probes used to test in vitro binding affinity of ZmbHLH128 and ZmbHLH129 to each cis-element described in (A). Probe sequences are listed in supplementary table S3, Supplementary Material online. (C) EMSA assays showing in vitro binding affinity of Trx::ZmbHLH128 (gel on the left) and Trx::ZmbHLH129 (gel on the right) proteins to the probes described in (B). Arrowheads indicate uplifted ZmbHLH-DNA probe complexes. Free probe indicates unbound DNA probes. 1696 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE Acquisition of N-Box-Derived cis-Elements in (probes 1 and 2) in the highest oligomeric state (fig. 5B and C, supplementary fig. S2, Supplementary Material online). NADP-ME Promoters Facilitates ZmbHLH128 and Since ZmbHLH128 and ZmbHLH129 showed weak bind- ZmbHLH129 Binding in PACMAD Panicoid Grasses ing to single cis-elements, we tested their binding by mutating Phylogenetic analysis of the genes encoding C and C plas- 3 4 these cis-elements in probes with the pairs (supplementary tidic NADP-MEs reflects previously reported grass species fig. S3, Supplementary Material online). For each pair, three phylogeny (fig. 6A)(Grass Phylogeny Working Group II mutant probes were designed: two in which the two cis- 2012). It inferred two main clades: one formed by C BEP elements were mutated individually (keeping one cis-element species (B. distachyon and O. sativa) and a second formed wild-type) and one in which both cis-elements were mutated by C (D. oligosanthes)and C Panicoid species of the 3 4 simultaneously (supplementary table S3, Supplementary PACMAD clade (S. italica, S. bicolor,and Z. mays)(fig. 6A). Material online). Competition experiments were performed Based on the observed nucleotide modifications in cis-ele- using radiolabeled wild-type probes (with cis-element pairs) mentsrecognizedbybHLHTFs,weproposeamodel relating and 200- to 400-fold excess of unlabeled wild-type and mu- to the recruitment of NADP-ME into C photosynthesis in tant probes (supplementary fig. S3, Supplementary Material grasses (fig. 6B). This proposes that an ancestral G-box found online). Binding of both ZmbHLHs to the labeled wild-type in the NADP-ME promoter of the common ancestor of C probes could be efficiently out-competed by unlabeled wild- BEP O. sativa and C Panicoid grasses has been conserved type and mutant probes in which the following cis-elements during the evolution of C photosynthesis. However, in the were not mutated: FBS 1 (in Z. mays C -NADP-ME, probe Panicoideae subfamily of the PACMAD clade a second 1þ m2-A, supplementary fig. S3A, Supplementary Material cis-element recognized by bHLH is present such that the online); FBS 2 (in Z. mays C -NADP-ME,probe m1þ 2-B, NADP-ME gene from the C species D. oligosanthes and genes supplementary fig. S3A, Supplementary Material online); N- encoding plastidic nonC -NADP-ME from C S. bicolor and 4 4 box (in S. bicolor C -NADP-ME, probe m3þ 4-E, supplemen- Z. mays all contain a G- and N-box/N-box-like pair. In C S. tary fig. S3B, Supplementary Material online); and G-box (in S. italica this cis-code has been retained in the C -NADP-ME,but italica C -NADP-ME, probe 5þ m6-G, supplementary fig. S3C, in S. bicolor and Z. mays the original G-box has evolved to Supplementary Material online; Z. mays nonC -NADP-ME, become either a FeRE1 or a FBS element, respectively (fig. 6B). probe 7þ m8-J, supplementary fig. S3D, Supplementary No G-box motifs are, however, present in the promoter of Material online; S. bicolor nonC -NADP-ME, probe 9þ m10- genes encoding cytosolic NADP-ME from S. bicolor and Z. M, supplementary fig. S3E, Supplementary Material online; mays. Overall, these results suggest that the acquisition of and D. oligosanthes C -NADP-ME, probe 11þ m12-P, supple- N-box-derived cis-elements have facilitated ZmbHLH128 mentary fig. S3F, Supplementary Material online). These and ZmbHLH129 binding to promoters of genes encoding EMSA competition experiments thus confirmed binding of plastidic NADP-ME in the PACMAD (Panicoideae subfamily). ZmbHLH128 and ZmbHLH129 to the cis-elements described above. Taken together, the results indicate that a second cis- Discussion element recognized by bHLH TFs is acquired in the promoters ZmbHLH128 and ZmbHLH129 Homeologs Interact of genes encoding plastidic NADP-ME and that each cis- with Maize C - and nonC -NADP-ME Promoters 4 4 element pair operates synergistically to allow interaction in vitro Showing Different trans-Activation Activity in with either ZmbHLH128 or ZmbHLH129 in C and C grasses 3 4 planta (fig. 5, supplementary figs. S2 and S3, Supplementary Material In this study, we showed that ZmbHLH128 and ZmbHLH129 online). form a maize homeolog pair resulting from the recent maize Given the binding affinity in vitro of ZmbHLH128 and whole genome duplication (WGD) event that occurred 5– ZmbHLH129 to the G-box in the ZmnonC -NADP-ME pro- 12 million years ago. This WGD occurred 5–16 million years moter (probes 7 and 7þ 8, fig. 5C), we tested their binding after C photosynthesis evolved in the Andropogoneae tribe ability in planta. Transient expression assays were performed 4 of the PACMAD clade (17–21 MYA) (Christin et al. 2008, in leaves of N. benthamiana co-infiltrated with GUS reporter 2009). As the length of exons 1 and 2 and the total number gene driven by a ZmnonC -NADP-ME promoter fragment of amino acids in the mature protein of ZmbHLH128 are containing the cis-element pair G- and N-box-like (368 to more similar to sorghum ortholog SbbHLH66 (supplementary 143 bp) and the effector constructs ZmbHLH128 and fig. S5, Supplementary Material online), we propose that ZmbHLH129 (supplementary fig. S4A, Supplementary ZmbHLH129 has diverged more from the ancestral gene. Material online). Compared with the reporter alone, co- Both of these TFs bind two FBS cis-elements that are in close infiltration of ZmnonC -NADP-ME reporter and the proximity in the maize C -NADP-ME (GRMZM2G085019) ZmbHLH128 and ZmbHLH129 effectors did not impact on promoter. Although ZmbHLH128 has been predicted in silico GUS activity in tobacco system (supplementary fig. S4B–D, to regulate C photosynthesis (Wang et al. 2014), as far as we Supplementary Material online). These results suggest that although ZmbHLH128 on its own binds both the ZmC - are aware, this is the first report of its functional characteri- NADP-ME and ZmnonC -NADP-ME promoters in vitro zation. ZmbHLH128 alone activates ZmC -NADP-ME gene (probes1,2,1þ 2, 7, and 7þ 8, fig. 5B and C), this might expression, while ZmbHLH129 alone shows no trans-activa- not be the case in planta (supplementary fig. S4, tion activity on this promoter. As the duplication event that Supplementary Material online). generated ZmbHLH129 took place after the evolution of C 1697 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.6. Acquisition of N-box-derived cis-elements in NADP-ME promoters facilitates ZmbHLH128 and ZmbHLH129 binding in PACMAD Panicoid grasses. (A) Phylogenetic tree of genes encoding plastidic NADP-ME from C and C grass species. C : B. distachyon (Bd), O. sativa (Os), and D. 3 4 3 oligosanthes (Do); C : S. italica (Si), S. bicolor (Sb) and Z. mays (Zm). NADP-MEs are color-coded: magenta for C , blue for nonC and green for C . 4 3 4 4 NADP-ME genomic sequences were aligned using MUSCLE, and the phylogenetic tree inferred by NJ method (1000 bootstrap pseudoreplicates, node numbers indicate bootstrap values). Gene encoding C plastidic NADP-ME from A. thaliana (AtC -NADP-ME) was used as outgroup. (B) 3 3 Diagram representing C to C molecular evolution of homologous bHLH binding cis-elements identified in promoters of genes encoding plastidic 3 4 NADP-ME. Dashed arrow indicates intermediate evolutionary steps from C to C . Vertical lines indicate two independent C origins of S. italica 3 4 4 and S. bicolor/Z. mays (Paniceae and Andropogoneae tribes, respectively). photosynthesis, it seems possible that this gene is not re- heterodimers (Kang et al. 2010). The latter scenario has quired for C photosynthesis. ZmbHLH128 and been reported for the maize Dof1 and Dof2 TFs. Dof1 is a ZmbHLH129 form heterodimers and despite ZmbHLH128 transcriptional activator of light-regulated genes in leaves, activating the expression of ZmC -NADP-ME its regulatory however, in stems and roots, this TF is not able to regulate activity is impaired by its homeolog ZmbHLH129. To explain those genes since the repressor Dof2 is expressed there and this impairment, we hypothesize different scenarios that may blocks Dof-specific cis-elements (Yanagisawa and Sheen occur in vivo: either ZmbHLH128 and ZmbHLH129 act as 1998). heterodimers and ZmbHLH128 loses its DNA binding activity In addition to the capacity of ZmbHLH128 and when combined with ZmbHLH129 or they act as homo- ZmbHLH129 to interact with FBSs found in the maize C - dimers and compete directly for the same FBSs, toward which NADP-ME promoter, both ZmbHLHs were shown to bind in vitrotothe promoter of maize nonC -NADP-ME ZmbHLH129 has a higher binding affinity. The former sce- nario has been described for bZIP TFs from Arabidopsis, (GRMZM2G122479) that possesses the cis-element pair G- where bZIP63 has negative effects on the formation of and N-box-like. In planta,however,ZmbHLH128 and bZIP1–DNA complexes probably due to conformational dif- ZmbHLH129 showed no trans-activation activity on this pro- ferences between bZIP1 homodimer and bZIP1-bZIP63 moter. It is well known that primary DNA sequence and its 1698 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE structural properties are determinants of DNA binding spe- encourage DNA looping. In addition to the spacer length, its cificity in vivo (Rohs et al. 2009) and so it is possible that both sequence appears highly conserved. This is consistent with ZmbHLHs display increased in vivo binding specificity for the evidence suggesting that nucleotides outside core cis-ele- FBS pair in the ZmC -NADP-ME promoter than for the G- and ments affect TF binding specificity by providing genomic con- N-box-like pair in the ZmnonC -NADP-ME promoter. text and influencing three-dimensional structure (Atchley Therefore, ZmbHLH128 seems to affect the level of expression et al. 1999; Mart ınez-Garcia et al. 2000; Grove et al. 2009; of NADP-ME as it activates the ZmC -NADP-ME promoter Gord^ an et al. 2013). For example, Cbf1 and Tye7 are yeast through the pair formed by two FBSs but the same trend bHLHs that show preference for a subset of G-boxes present was not observed for the ZmnonC -NADP-ME promoter with throughout the yeast genome (Gord^ an et al. 2013). These differences in binding preferences were observed not just the G- and N-box pair. In addition, we hypothesize that these modifications of promoter sequences may also affect light/ in vivo but also in vitro and so DNA sequences flanking circadian regulation of the ZmC -NADP-ME gene as FBS cis- core G-boxes were found to explain this differential bHLH- elements have been described in promoters of circadian- G-box binding (Gord^ an et al. 2013). clock-regulated and light-responsive genes (Lin et al. 2007, The mechanism proposed here for how bHLH TFs interact with their target cis-elements suggests that these DNA 2011; Kim et al. 2016). Themutationoftwo closeFBSsin the promoter of the circadian-clock gene EARLY sequences are not randomly arranged in gene promoters FLOWERING 4 (ELF4)provedtobesufficienttoabolishits and may affect how cis-element specificity is achieved. rhythmic expression (Li et al. 2011). More broadly, our find- Indeed, in some promoters bound by bHLH TFs two or ings also contribute to the understanding of gene regulatory more cis-elements were found to be clustered. For example, two overlapping FBSs were reported in the 400 bp upstream networks controlling C photosynthesis. of the translational start site of the gene encoding ELF4 (Li The G-Box-Based cis-Element Pair Present in et al. 2011). Also, pairs of G- and N-boxes were found to be NADP-ME Promoters Synergistically Bind Either highly enriched in promoters targeted by the bHLH PIF1 (Kim et al. 2016). It is possible that multiple cis-elements serve to ZmbHLH128 or ZmbHLH129 We identified a cis-element pair recognized by bHLH that recruit additional TFs for in vivo cooperative binding. occupy homologous positions in NADP-ME promoters from C Photosynthesis Co-Opted an Ancient C C and C grasses. These cis-elements flank a short spacer and 4 3 3 4 Cis-Regulatory Code Built on G-Box Recognition by operate synergistically to facilitate interaction with ZmbHLH128 and ZmbHLH129. We suggest a mechanism bHLH Transcription Factors by which these TFs may be recruited to the cis-elements Finally, from this study we propose a model that summarizes associated with C photosynthesis. We propose that one how molecular evolution of cis-elements recognized by bHLHs may relate to the recruitment of NADP-ME into C cis-element is sufficient to recruit a bHLH homodimer (G- box) or tetramer (N-box or FBS in promoters where the an- photosynthesis. C photosynthesis is an excellent example of cestral G-box is no longer present); however, the presence of a convergent evolution (Sage et al. 2011; Christin et al. 2013)as second cis-element in the vicinity increases bHLH binding it has evolved independently over 60 times in angiosperms affinity (supplementary fig. S2, Supplementary Material on- (Sage et al. 2011; Sage 2016) and at least 22 times in grasses (Grass Phylogeny Working Group II 2012). How this repeated line).Itispossiblethatboth cis-elements are brought together through the interaction with a bHLH tetramer formed by two evolution has come about is not fully understood. Our model dimers, which may involve DNA bending (supplementary fig. contributes to our understanding of C evolution and is based S2, Supplementary Material online). Therefore, this cis-ele- on the following findings: first, in rice, which belongs to the ment pair could operate synergistically to confer stabilization BEP clade that contains no C species, only one copy of a G- of bHLH binding. This mechanism of TF-DNA assembly has box was present in the NADP-ME promoter. In contrast, cis- previously been proposed for MADS-domain TFs that can element pairs recognized by ZmbHLH128 and ZmbHLH129 bind two nearby CArG boxes through DNA looping and for- in NADP-ME promoters seem to be common in the mation of tetrameric complexes (Theissen 2001; Theissen and Panicoideae subfamily of the PACMAD clade that contains independent C lineages. For example, in the PACMAD Saedler 2001; Melzer et al. 2009; Smaczniak et al. 2012; Smaczniak et al. 2017). In this case, and consistent with our Panicoid grasses a G- and N-box pair was identified in C D. results, MADS-domain TFs were found to bind single CArG oligosanthes (Do024386) and appears to be reasonably con- boxes either as dimers or tetramers, however, when their served in C species. However, in the case of the C -NADP- 4 4 target gene promoters contain CArG box pairs they bind as MEsfrom S. bicolor and Z. mays (Sobic.003g036200 and tetramers (Smaczniak et al. 2012). It has been proposed that GRMZM2G085019) these elements have diversified. Both of the probability of DNA loop formation increases with shorter these grass species belong to the C tribe Andropogoneae in distances between cis-elements due to the low elastic bending which the plastidic NADP-ME isoform that is used in C energy required to bring the protein dimers together photosynthesis (C -NADP-ME) evolved by duplication from (Agrawal et al. 2008). Interestingly, in all NADP-ME promoters an ancestral plastidic NADP-ME that still exists and is not assessed in this study except rice and Brachypodium the two involved in the C cycle (nonC -NADP-ME, Sobic.009g108700 4 4 cis-elements were found to be in close proximity, which may and GRMZM2G122479) (Tausta et al. 2002; Maier et al. 2011; 1699 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE Alvarez et al. 2013). In contrast, C S. italica together with C Materials and Methods 4 3 D. oligosanthes belong to the grass tribe Paniceae in which Plant Growth Conditions and Collection of Leaf only one plastidic NADP-ME isoform is known to exist Samples (Si000645 and Do024386) (Alvarez et al. 2013; Emms et al. To construct the cDNA expression library, maize plants 2016). Surprisingly, the cis-element pair identified in the C - (Z. mays L. var. B73) were grown at 16 h photoperiod with NADP-ME promoter from S. italica (G- and N-box) was found 2 1 a light intensity of 340–350 lmol m s ,atday/night tem- to be closer to those occurring in the C and nonC -NADP-ME 3 4 perature of 28/26 C, and 70% relative humidity. Two light promoters from D. oligosanthes, S. bicolor,and Z. mays (G- regimes were used: (1) nine days in 16 h photoperiod; and (2) and N-box/N-box-like) than to those occurring in the C - nine days in 16 h photoperiod followed by a 72 h dark treat- NADP-ME promoters from S. bicolor and Z. mays (FeRE1 ment. In both experiments, sample collection was performed and N-box or FBS and FBS, respectively). A similar trend under 16 h photoperiod. Third leaves grown in the former has previously been observed (Alvarez et al. 2013)and may and latter light regimes were harvested respectively at time be explained by the independent evolutionary origin of C points covering the Zeitgeber times (ZT) 0.5, 0.5, 2 h, and photosynthesis in grass tribes formed by S. italica (Paniceae) ZT 1, 2, 4, 8, 12, 15.5 h. For isolation of maize mesophyll or S. bicolor/Z. mays (Andropogoneae). protoplasts, maize plants were grown for 10 days at 25 C, Taken together, our findings suggest that an ancestral G- 2 1 16 h photoperiod (60 lmol m s ), and 70% relative box in combination with N-box-derived cis-elements form humidity. For transient expression assays in planta, the basis of the synergistic binding of either ZmbHLH128 or N. benthamiana (tobacco) plants were grown for 5 weeks ZmbHLH129 to NADP-ME promoters from PACMAD 2 1 at 22 C, 16 h photoperiod (350 lmol m s ), and 65% Panicoid grasses. Nucleotide diversity in cis-elements recog- relative humidity. After agro-infiltration of tobacco leaves, nized by bHLH TFs has been suggested as one of the mech- anisms by which these TFs are involved in complex and plants were left to grow into the same growth conditions diverse transcriptional activity (Toledo-Ortiz et al. 2003). and leaf discs (2.5 cm in diameter) collected 96 h post- We, therefore, cannot exclude the possibility that the gene infection. encoding the plastidic NADP-ME from C BEP B. distachyon (BRADI2g05620) can also be bound by ZmbHLH128 or Generation of Yeast Bait Strains ZmbHLH129 despite none of the typical cis-elements recog- Yeast bait strains were generated as previously described nized by bHLH being identified in the promoter. Given recent (Ouwerkerk and Meijer 2001; Serra et al. 2013). Yeast evidence indicating that the bHLH TF family is often recruited strain Y187 (Clontech) was used to generate six bait into C photosynthesis regulation (Huang and Brutnell 2016), strains carrying overlapping fragments of the ZmC - we suggest that the observed nucleotide modifications in the NADP-ME (GRMZM2G085019) promoter cloned into cis-element pair present in C -NADP-ME promoters from S. the yeast integrative vector pINT1-HIS3 (Ouwerkerk and bicolor and Z. mays may underlie changes in bHLH binding Meijer 2001)as NotI-SpeIor XbaI-SpeI fragments (supple- specificity in vivo and, therefore, contribute to the NADP-ME mentary table S1, Supplementary Material onlline). The recruitment into C photosynthesis in the Andropogoneae ZmC -NADP-ME promoter region was defined as the tribe from the PACMAD clade. The presence of a bHLH du- 1,982 bp upstream of the predicted translational start plicate (ZmbHLH129) that seems not to be required for C site (ATG). To assess self-activation/HIS3 leaky expression, photosynthesis and has evolved to repress the activity of its yeast bait strains were titrated in complete minimal me- homeolog (ZmbHLH128) is unique to maize as this homeolog dium (CM) lacking histidine, with increasing concentra- gene pair resulted from the maize WGD. Therefore, we hy- tions of 3-amino-1, 2, 4-triazole (3-AT, up to 75 mM). pothesize that the single orthologous bHLH in all the other Panicoid species of the PACMAD clade activates C -NADP- Construction of cDNA Expression Library ME gene expression. This agrees with the hypothesis that C Total RNA was extracted from third leaves of maize seedlings photosynthesis has on multiple occasions made use of cis- using TRIzol reagent (Invitrogen), following the manufac- regulators found in C species and, therefore, that the recruit- turer’s instructions. RNA samples from nine time points (de- ment of C genes was made through minor rewiring of pre- scribed in ‘plant growth conditions and collection of leaf existing regulatory networks (Reyna-Llorens and Hibberd samples’) were pooled in equal amounts for mRNA purifica- 2017). We conclude that regulation of C genes can be based tion using the PolyATract mRNA Isolation System IV on an ancient code founded on a G-box present in the BEP (Promega). A unidirectional cDNA expression library was pre- clade as well as the Panicoideae of the PACMAD clade. pared using the HybriZAP-2.1 XR cDNA Synthesis Kit and the Acquisition of a second cis-element recognized by bHLH in HybriZAP-2.1 XR Library Construction Kit (Stratagene), fol- Panicoid grasses appears to have facilitated synergistic binding lowing the manufacturer’s instructions. Four micrograms of by either ZmbHLH128 or ZmbHLH129. Although this G-box- mRNA were used for first strand cDNA synthesis. After in vivo based cis-code has remained similar in S. italica,ithas diverged in maize and sorghum. Thus, different C grass lineages may excision and amplification of the pAD-GAL4-2.1 phagemid vector, this maize cDNA expression library was used to trans- employ slightly different molecular circuits to regulate orthol- ogous C photosynthesis genes. form yeastbaitstrains. 1700 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE 0 0 Yeast One-Hybrid (Y1H) Screening and Validation GCGCTGGA-3 /5 -GGGGACCACTTTGTACAAGAAAGCT Yeast bait strains were transformed with 1 lgofmaize cDNA GGGTNCTAAGCATTTGGGGGCCAG-3 (underlined expression library according to Ouwerkerk and Meijer (2001) sequences indicate attB Gateway adaptors). ZmbHLH128 and Serra et al. (2013). At least, 1.3 million yeast colonies of and ZmbHLH129 CDS were recombined into pDONR221 each yeast bait strain transformed with the maize cDNA ex- (Invitrogen) to obtain Entry clones through BP-Gateway re- pression library were screened in CM -HIS -LEU supple- action (Invitrogen), following the manufacturer’s instructions. N C mented with 3-AT: 5 mM (1982 to 1524 bp), 20 mM CDSwerethenrecombinedintovectors YFP 43 and YFP 43 (389 to 154 bp, 776 to 334 bp) or 75 mM (973 to through LR-Gateway reaction (Invitrogen) to raise a transla- 702 bp, 1225 to 891 bp, 1617 to 1135 bp). Plasmids tional fusion with N- and C-terminal domains of yellow fluo- from yeast clones that actively grew on selective medium rescent protein (YFP), respectively. Final BiFC constructs were N N were extracted. To know whether the isolated clones denominated as YFP ::ZmbHLH128, YFP ::ZmbHLH129, C C encoded transcription factors (TFs), the cDNA insert was se- YFP ::ZmbHLH128, and YFP ::ZmbHLH129. Maize mesophyll quenced and the results analyzed using BLAST programes. To protoplasts were transformed with 6 lg of each of the BiFC validate DNA-TF interactions in yeast, isolated plasmids constructs. Protoplasts transformed with YFP ::Akin10 encoding TFs were re-transformed into the yeast bait strain (Arabidopsis SNF1 Kinase Homolog 10), YFP ::Akin3 in which they were found to bind. To assess TF binding (Arabidopsis SNF1 Kinase Homolog 3) and YFP 43 and specificity, plasmids encoding TFs were also transformed YFP 43 empty vectors were used as negative controls. into the yeast bait strains to which they do not bind. Transformations were performed in triplicate. YFP fluo- rescence and chlorophyll autofluorescence signals were Yeast Cell Spotting observed under a confocal microscope (Leica SP5). Yeast bait strains transformed with plasmids encoding TFs were grown overnight until log or mid-log phase at 30 Cin Transient Expression Assays in planta liquid yeast CM medium supplemented with Histidine For the transient expression assays in tobacco leaves, reporter (CMþHIS -LEU). Cultures were normalized to an OD of and effector constructs were generated in the Gateway binary 0.4, spotted onto solid medium CMþHIS -LEU or CM -HIS - vectors pGWB3i [pGWB3 containing an intron-tagged b-glu- LEUþ 3-AT, and grown for 3 days at 30 C. curonidase (GUS) open reading frame (Berger et al. 2007)] and pGWB2 (Tanaka et al. 2012), respectively. Isolation and Transformation of Maize Mesophyll To construct the reporter plasmids, promoter fragments of Protoplasts ZmC -NADP-ME (GRMZM2G085019, from 389 to Maize mesophyll protoplasts were isolated from 10-day-old 154 bp) and ZmnonC -NADP-ME (GRMZM2G122479, maize greening plants and transformed according to from 368 to 143 bp) were fused to a 136 bp minimal Lourenc ¸o et al. (2013) with minor modifications. Mid- CaMV35S promoter (m35S) in a 3-step PCR reaction: (1) section of newly matured second leaves was digested in a promoter sequences were amplified with long chimeric pri- cell wall digestive medium containing 1.5% (w/v) cellulase mers to introduce overlapping ends (reverse primer of R-10 (Duchefa), 0.3% (w/v) macerozyme R-10 (Duchefa), pZmC -NADP-ME/pZmnonC -NADP-ME was designed to be 4 4 10 mM MES (pH 5.7), 0.4 M mannitol, 1 mM CaCl ,0.1% complementary to the forward primer of the m35S)(supple- (w/v) BSA and 5 mM b-mercaptoethanol. Several leaf blades mentary table S4, Supplementary Material online); (2) pro- were stacked and cut perpendicularly to the long axis into moter sequences amplified by PCR in (1) were mixed 0.5–1 mm slices and quickly transferred to digestive medium according to the fusion products of interest in a ratio of 1:1 (25 ml digestive medium for each set of 10 leaf blades). Purity [ZmC -NADP-ME (389 to 154 bp)::m35S and ZmnonC - 4 4 and integrity of isolated protoplasts were examined under NADP-ME (368 to 143 bp)::m35S]and 10 PCRcycles light microscopy. Mesophyll protoplasts were quantified 6 1 were run without primers (denaturation at 98 Cfor 10s, and its abundance adjusted to 2 10 protoplasts ml . 55 Cfor 30 s, and72 C for 1 min); and (3) fusion products Transformed protoplasts were resuspended in 1.25 ml of in- of interest were amplified with attB-containing primers (sup- cubation solution [0.6 M mannitol, 4 mM MES (pH 5.7) and plementary table S4, Supplementary Material online). To ob- 4 mM KCl] and incubated in 24-well plates for 18 h at room tain Entry clones, promoter fragments fused to m35S were temperature under dark. cloned into pDONR221 (Invitrogen) through BP-Gateway Bimolecular Fluorescence Complementation (BiFC) reaction (Invitrogen), following the manufacturer’s instructions. Promoter sequences were then recombined Assay into the binary vector pGWB3i through LR-Gateway reac- To generate BiFC constructs, full-length coding sequences tion (Invitrogen) to obtain the final reporter constructs for (CDS) of ZmbHLH128 (GRMZM2G314882) and promoter::GUS analysis (pZmC -NADP-ME and ZmbHLH129 (GRMZM5G856837) were PCR-amplified using 4 pZmnonC -NADP-ME). For the effector constructs (TF respectively the following pairs of attB-containing primers: 5 - driven by the CaMV35S promoter), ZmbHLH128 and GGGGACAAGTTTGTACAAAAAAGCAGGCTNNATGATG 0 0 ZmbHLH129 Entry clones previously generated (see BiFC AACTGCGCCGGA-3 /5 -GGGGACCACTTTGTACAAGAAA 0 0 GCTGGGTNCTAAGCATTAGGCGGCCAG-3 ,and 5 -GGGG assay) were directly recombined into the binary vector ACAAGTTTGTACAAAAAAGCAGGCTNNATGATGGACT pGWB2 through LR-Gateway reaction (Invitrogen). 1701 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE Blue Native-Polyacrylamide Gel Electrophoresis Reporter and effector constructs together with a construct harboring the silencing suppressor P1b (Valli et al. 2006)were (BN-PAGE) and Western Blotting transformed into the Agrobacterium tumefaciens strain Molecular mass of oligomers co-existing in purified GV301. Overnight cultures of Agrobacterium harboring re- ZmbHLH128 and ZmbHLH129 recombinant proteins was determined by blue native polyacrylamide gel electrophoresis porter, effector and P1b constructs were sedimented (5000 (BN-PAGE). Two micrograms of the recombinant proteins gfor 15 min, at 4 C) and resuspended in infiltration medium (Trx::His::ZmbHLH128 or Trx::His::ZmbHLH129) were re- (10 mM MgCl , 10 mM MES (pH 5.6), 200 lMacetosyrin- solved on a 3–12% Novex Bis–Tris NativePAGE mini gel gone) to an OD of 0.3, 1, and 0.5, respectively, and mixed (Life Technologies), following the manufacturer’s instructions. in a ratio of 1:1:1. Mixed Agrobacterium cultures were incu- HMW Native Marker Kit (66–669 kDa, GE Healthcare) was bated for 2 h at 28 C and used to spot-infiltrate the abaxial used to estimate molecular mass. Resolved proteins were side of 5-week-old tobacco leaves. As controls, tobacco leaves transferred to a polyvinylidene difluoride membrane (GE were agro-infiltrated with mixed cultures carrying the re- Healthcare). The membrane was destained with a 50% porter construct alone or the empty vector pGWB3i and (v/v) methanol and 10% (v/v) acid acetic solution followed effector constructs. Infected leaves were analyzed at 96 h by pure methanol. For immunodetection of post-infiltration. Leaf discs of 2.5 cm in diameter were col- Trx::His::ZmbHLH128 and Trx::His::ZmbHLH129, the mem- lected from the infiltrated spots and used for the quantifica- brane was incubated with a-His antibody (GE Healthcare) tion of GUS activity. GUS activity was quantified by followed by a-mouse horseradish peroxidase-conjugated an- measuring the rate of 4-methylumbelliferyl-b-D-glucuronide tibody (abcam) for 1 h each at room temperature. (MUG) conversion to 4-methylumbelliferone (MU) as de- scribed in Jefferson et al. (1987) and Williams et al. (2016). Electrophoretic Mobility Shift Assay (EMSA) In brief, soluble protein was extracted from agro-infiltrated DNA probes were generated by annealing oligonucleotide tobacco leaf discs by freezing in liquid nitrogen and macera- pairs in a thermocycler followed by radiolabeling as described tion, followed by addition of protein extraction buffer. Diluted in Serra et al. (2013). DNA probe sequences and respective protein extracts (1:2) were incubated with 1 mM MUG for 30, annealing temperatures are listed in supplementary table S3, 60, 90, and 120 min at 37 C in a 96-well plate. GUS activity Supplementary Material online. EMSAs were performed using was terminated at the end of each time point by the addition 400 ng of the recombinant proteins Trx::ZmbHLH128 or of 200 mM Na CO and MU fluorescence measured by ex- Trx::ZmbHLH129, and 50 fmol of radiolabeled probes. 2 3 citing at 365 nm and measuring emission at 455 nm. The Competition assays were performed adding 200- to 400- fold molar excess of the unlabeled probe. Trx::OsPIF14 concentration of MU/unit fluorescence in each sample was (LOC_Os07g05010) and Trx protein, both purified by interpolated using a concentration gradient of MU from 1.5 Cordeiro et al. (2016), were used as negative controls. Each to 800 lMMU. protein was mixed with probes in a 10 ll reaction containing 10 mM HEPES (pH 7.9), 40 mM KCl, 1 mM EDTA (pH 8), Production of Recombinant ZmbHLH128 and 1mM DTT, 50ng herring sperm DNA, 15 lg BSA and 10% (v/v) glycerol. Binding reactions were incubated for 1 h on ice ZmbHLH129 and the bound complexes resolved on a native 5% polyacryl- ZmbHLH128 and ZmbHLH129 full-length CDSwerePCR- amide gel (37.5:1). Gel electrophoresis and detection of radio- amplified using, respectively, the following pairs of gene 0 0 active signal were performed as described in Serra et al. specific primers 5 -GAATTCATGATGAACTGCGCCGGA-3 / 0 0 0 (2013). 5 -CTCGAGCTAAGCATTAGGCGGCCAG-3 and 5 -GAA 0 0 TTCATGATGGACTGCGCTGGA-3 /5 -CTCGAGCTAAGCA 0 Synteny Analysis TTTGGGGGCCAG-3 (underlined sequences indicate adap- SynFind (Tang et al. 2015) was used to identify maize syntenic tors with restriction enzyme sites). ZmbHLH128 and chromosomal regions for ZmbHLH128 (GRMZM2G314882) ZmbHLH129 were cloned as EcoRI-XhoI fragments into and ZmbHLH129 (GRMZM5G856837) genes against Z. mays the expression vector pET32a (Novagen), generating B73 RefGen_v3 genome. A table containing maize syntelog N-terminal Trx-tagged fusions. pET32a-Trx::ZmbHLH128 gene pairs was retrieved using SynFind tool (supplementary and pET32a-Trx::ZmbHLH129 constructs were confirmed table S2, Supplementary Material online). by sequencing and transformed into Rosetta (DE3)pLysS competent cells (Invitrogen) for protein expression. Cells Phylogenetic Analyses transformed with pET32a-Trx::ZmbHLH128 and pET32a- ZmbHLH128 and ZmbHLH129 were used as references to Trx::ZmbHLH129 constructs were, respectively, grown in identify closely related bHLH genes of Z. mays, S. bicolor, Terrific Broth (TB) and Luria-Bertani (LB) medium to an Setaria viridis, S. italica, O. sativa,and B. distachyon,through OD of 0.5. Protein expression was induced with 4 mM 600 Phytozome database (Goodstein et al. 2012). Predicted CDS isopropyl-D-1-thiogalactopyranoside (IPTG) and allowed to were aligned using MUSCLE. The resulting alignment was occur for 3 h (ZmbHLH128) or 5 h (ZmbHLH129) at 30 C. used to infer a maximum likelihood phylogenetic tree, using Protein purification was performed as described in Cordeiro GTRþ GþI nucleotide substitution model (1,000 bootstrap et al. (2016). pseudoreplicates) in MEGA 7 software (Kumar et al. 2016). 1702 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE Christin P-A, Boxall SF, Gregory R, Edwards EJ, Hartwell J, Osborne CP. Phylogenetic analysis of genes encoding C and C plastidic 3 4 2013. Parallel recruitment of multiple genes into C photosynthesis. NADP-ME isoforms from B. distachyon (BRADI2g05620), O. Genome Biol Evol. 5(11):2174–2187. sativa (LOC_Os01g09320), D. oligosanthes (Do024386), S. ital- Christin P-A, Osborne CP. 2014. The evolutionary ecology of C plants. ica (Si000645), S. bicolor (Sobic.003g036200, New Phytol. 204(4):765–781. Sobic.009G108700), and Z. mays (GRMZM2G085019, Christin P-A, Osborne CP, Sage RF, Arakaki M, Edwards EJ. 2011. C eudicots are not younger than C monocots. JExp Bot. GRMZM2G122479) was performed using Geneious Pro 5.3.6 4 62(9):3171–3181. software (Kearse et al. 2012). Full-length genomic sequences Christin P-A, Salamin N, Kellogg EA, Vicentini A, Besnard G. 2009. were aligned using MUSCLE. Phylogenetic tree was inferred Integrating phylogeny into studies of C variation in the grasses. using the Neighbor Joining (1,000 bootstrap pseudoreplicates) New Phytol. 149(1):82–87. and rooted using the gene encoding C plastidic NADP-ME Cordeiro AM, Figueiredo DD, Tepperman J, Borba AR, Lourenc ¸o T, Abreu IA, Ouwerkerk PBF, Quail PH, Margarida Oliveira M, Saibo (At1g79750) from Arabidopsis thaliana,adicotangiosperm. NJM. 2016. Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box Supplementary Material and interacts preferentially with the active form of phytochrome B. Supplementary data areavailableat Molecular Biology and Biochim Biophys Acta 1859(2):393–404. Evolution online. De Masi F, Grove CA, Vedenko A, Alib es A, Gisselbrecht SS, Serrano L, Bulyk ML, Walhout AJM. 2011. Using a structural and logics systems Acknowledgments approach to infer bHLH-DNA binding specificity determinants. Nucleic Acids Res. 39(11):4553–4563. We thank Lisete Fernandes (Escola Superior de Tecnologia da Ehleringer JR, Monson RK. 1993. Evolutionary and ecological aspects of Sau de de Lisboa, Portugal) for discussions and advice photosynthetic pathway variation. Annu RevEcolEvolSyst. 24(1):411–439. concerning EMSA experiments, Cec ılia Arraiano Lab (ITQB- Emms DM, Covshoff S, Hibberd JM, Kelly S. 2016. Independent and NOVA, Oeiras, Portugal) for material used in EMSA parallel evolution of new genes by gene duplication in two origins experiments, Myriam Goudet and Samuel Brockington of C photosynthesis provides new insight into the mechanism of (Department of Plant Sciences, University of Cambridge, phloem loading in C species. MolBiolEvol. 33(7):1796–1806. UK) for assistance with phylogenetic analyses. This study Fisher A, Caudy M. 1998. The function of hairy-related bHLH repressor was supported by European Union project 3to4 (Grant agree- proteins in cell fate decisions. BioEssays 20(4):298–306. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, ment no: 289582) and Fundac ¸~ ao paraaCi^ enciaeTecnologia DirksW,HellstenU,PutnamN,Rokhsar DS.2012. Phytozome: a (FCT) through research unit GREEN-it ‘Bioresources for comparative platform for green plant genomics. Nucleic Acids Res. Sustainability’ (UID/Multi/04551/2013). ARB (SFRH/BD/ 40(D1):D1178–D1186. 105739/2014), AG (SFRH/BD/89743/2012), AMC (SFRH/BD/ Gord^ an R, Shen N, Dror I, Zhou T, Horton J, Rohs R, Bulyk ML. 2013. Genomic regions flanking E-box binding sites influence DNA binding 74946/2010), PMB (SFRH/BPD/86742/2012), IAA (IF/00960/ specificity of bHLH transcription factors through DNA shape. Cell 2013—POPH-QREN), and NJMS (IF/01126/2012—POPH- Rep. 3(4):1093–1104. QREN) were funded by FCT, TSS and PG by European GowikU,BurscheidtJ,AkyildizM,SchlueU,KoczorM,StreubelM, Union project 3to4 (Grant agreement no: 289582), and IR-L Westhoff P. 2004. Cis-regulatory elements for mesophyll-specific by BBSRC grant (BB/L014130). gene expression in the C plant Flaveria trinervia, the promoter of the C phosphoenolpyruvate carboxylase gene. Plant Cell 16(5):1077–1090. References Gowik U, Schulze S, Saladi e M, Rolland V, Tanz SK, Westhoff P, Ludwig Agrawal NJ, Radhakrishnan R, Purohit PK. 2008. Geometry of mediating M. 2017. 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Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used in C4 Photosynthesis Is Based on an Ancient Code Found in the Ancestral C3 State

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Abstract

C photosynthesis has evolved repeatedly from the ancestral C state to generate a carbon concentrating mechanism that 4 3 increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C cycle is accomplished through cell-type- specific accumulation of enzymes but cis-elements and transcription factors controlling C photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcriptioninC plants evolved from ancestral components found in C species. Two basic Helix-Loop-Helix (bHLH) 4 3 transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C and C Panicoid grass species of the PACMAD clade. Our 3 4 analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C state. We conclude that C photosynthesis has co-opted an ancient C regulatory code built on G-box recognition by bHLH to 4 3 regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C photosynthesis. Key words: basic Helix-Loop-Helix, cis-element evolution, C3 and C4 photosynthesis, NADP-Malic Enzyme, PACMAD Panicoid grasses. Monson 1993). In environments such as the tropics where Introduction rates of photorespiration are high, C photosynthesis has C plants inherited a carbon fixation system developed by evolved repeatedly from the ancestral C state (Lloyd and photosynthetic bacteria, with atmospheric carbon dioxide Farquhar 1994; Osborne and Beerling 2006). Phylogenetic stud- (CO ) being incorporated into ribulose-1, 5-bisphosphate ies estimate that the first transition from C to C occurred 3 4 (RuBP) by the enzyme Ribulose Bisphosphate Carboxylase/ around 30 million years ago (MYA) (Christin et al. 2008, 2011; Oxygenase (RuBisCO) to form the three-carbon compound Vicentini et al. 2008). The ability of the C cycle to concentrate (C ) 3-phosphoglycerate (Calvin and Massini 1952). However, CO around RuBisCO limits oxygenation and so increases pho- RuBisCO can also catalyse oxygenation of RuBP, which leads tosynthetic efficiency in conditions where photorespiration is to the production of 2-phosphoglycolate, a compound that is enhanced (Hatch and Slack 1966; Maier et al. 2011; Christin toxic to the plant cell and needs to be detoxified through an and Osborne 2014; Lundgren and Christin 2017). energetically wasteful process called photorespiration (Bowes The evolution of C photosynthesis involved multiple et al. 1971; Sharkey 1988; Sage 2004). The oxygenase reaction modifications to leaf anatomy and biochemistry (Hatch of RuBisCO becomes more common as temperature 1987; Sage 2004). In most C plants, photosynthetic reactions increases and so in C plants photorespiration can reduce are partitioned between two distinct cell types known as photosynthetic output by up to 30% (Ehleringer and mesophyll (M) and bundle sheath (BS) cells (Langdale 2011). The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is Open Access properly cited. 1690 Mol. Biol. Evol. 35(7):1690–1705 doi:10.1093/molbev/msy060 Advance Access publication April 5, 2018 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE M and BS cells are arranged in concentric circles around veins cell-specific expression in the C leaf, no TFs recognizing these in the so-called Kranz anatomy (Haberlandt 1904), which cis-elements have yet been identified. enables CO pumping from M to BS where RuBisCO is spe- To address this gap in our understanding, a bottom-up cifically located. Atmospheric CO is first converted to HCO approach was initiated to identify TFs that regulate the maize 2 3 by carbonic anhydrase (CA) and then combined with phos- gene ZmC -NADP-ME (GRMZM2G085019) that encodes the phoenolpyruvate (PEP) by PEP-carboxylase (PEPC) to pro- Malic Enzyme responsible for releasing CO in the BS cells. duce oxaloacetate in the M cells. This four-carbon acid (C ) Using Yeast One-Hybrid two maize TFs belonging to the su- is subsequently converted into malate and/or aspartate that perfamily of basic Helix-Loop-Helix (bHLH), ZmbHLH128 and transport the fixed CO from M to BS cells (Kagawa and 2 ZmbHLH129, were identified and functionally characterized. Hatch 1974; Hatch 1987). Three biochemical C subtypes 4 We show that these TFs bind two cis-elements synergistically are traditionally described based on the predominant type and analysis of the NADP-ME promoters from grass species of C acid decarboxylase responsible for the CO release 4 2 from BEP and PACMAD (Panicoideae subfamily) indicated around RuBisCO in the BS: NADP-dependent Malic Enzyme that this regulation is likely derived from an ancestral G-box (NADP-ME, e.g. Zea mays), NAD-dependent Malic Enzyme that is present in C species. (NAD-ME, e.g. Gynandropsis gynandra formerly designated Results Cleome gynandra) and phosphoenolpyruvate carboxykinase (PEPCK). However, recent reports suggest that only the ZmbHLH128 and ZmbHLH129 Homeologs Bind NADP-ME and NAD-ME should be considered as distinct FAR1/FHY3 Binding Site cis-Elements in the C subtypes, which in response to environmental cues may ZmC -NADP-ME Promoter involve a supplementary PEPCK cycle (Williams et al. 2012; To identify TFs that interact with the ZmC -NADP-ME gene Wang et al. 2014; Rao and Dixon 2016). (GRMZM2G085019), we studied the promoter region com- The recruitment of multiple genes into C photosynthesis prising 1982 bp upstream of the translational start site. This involved both an increase in their transcript levels (Hibberd region was divided into six overlapping fragments ranging and Covshoff 2010) and also patterns of expression being from 235 to 482 bp in length (supplementary table S1, modified from relatively constitutive in C species (Maurino Supplementary Material online) and used in Yeast One- et al. 1997; Penfield et al. 2004; Taylor et al. 2010; Brown et al. Hybrid (Y1H). Each fragment was used to generate one yeast 2011; Maier et al. 2011) to M- or BS-specific in C plants bait strain that was then used to screen a maize cDNA ex- (Hibberd and Covshoff 2010). Therefore, considerable efforts pression library. After screening at least 1.3 million colonies for have been made to identify the transcription factors (TF) and each region of the promoter, two maize bHLH TFs known as the cis-elements they recognize that are responsible for this ZmbHLH128 and ZmbHLH129 were identified. Both of these light-dependent and cell-specific gene expression (Hibberd TFs bind the promoter between base pairs 389 and 154 in and Covshoff 2010). Various studies suggest that different relation to the predicted translational start site of ZmC - transcriptional regulatory mechanisms have been adopted NADP-ME (fig. 1A). These interactions were confirmed by during C to C evolution. One is the acquisition of novel 3 4 re-transforming yeast bait strains harbouring each of the six cis-elements in C gene promoters that can be recognized by sections of the promoter with cDNAs encoding ZmbHLH128 TFs already present in C plants (Matsuoka et al. 1994; Ku and ZmbHLH129. Consistent with the initial findings, et al. 1999; Nomura et al. 2000), and a second possibility is the ZmbHLH128 and ZmbHLH129 only activated expression of acquisition of novel or modified TFs responsible for the re- the HIS3 reporter when transformed into yeast containing cruitment of genes into the C pathway through cis-elements fragment 389 to 154 bp upstream of ZmC -NADP-ME that pre-exist in C plants (Patel et al. 2006; Brown et al. 2011; (fig. 1B, supplementary fig. S1, Supplementary Material Kajala et al. 2012). online). A small number of cis-elements found in different gene ZmbHLH128 and ZmbHLH129 possess a bHLH domain regions have been shown to be sufficient for the M- or BS- followed by a contiguous leucine zipper (ZIP) motif specific expression of C genes. For example, a 41-base pair (fig. 1C). This bHLH domain is highly conserved between (bp) Mesophyll Expression Module 1 (MEM1) cis-element both ZmbHLHs and consists of 61 amino acids that can be was identified from the PEPC promoter of C Flaveria trinervia separated into two functionally distinct regions. The first is a and shown to be necessary and sufficient for M cell-specific basic region located at the N-terminal end of the bHLH do- accumulation of PEPC transcripts in C Flaveria species main andisinvolvedinDNA binding, andthe second is a (Gowik et al. 2004). A MEM1-like cis-element has also been Helix-Loop-Helix region mediating dimerization towards the found in the C carbonic anhydrase (CA3)promoterof carboxy-terminus (fig. 1C)(Murre et al. 1989; Toledo-Ortiz Flaveria bidentis and shown to drive M cell-specific expression et al. 2003). ZmbHLH128 and ZmbHLH129 share 91% amino (Gowik et al. 2017). A second cis-element named MEM2 and acid identity (fig. 1C) and they are encoded by homeolog consisting of 9 bp from untranslated regions has also been genes located in syntenic regions of maize chromosomes 4 shown to be capable of directing M-specificity in C G. gynan- dra (Kajala et al. 2012; Williams et al. 2016). Lastly, in the case and 5 (fig. 1D, supplementary table S2, Supplementary of the NAD-ME gene from C G. gynandra aregionfromthe Material online). coding sequence generates BS-specificity (Brown et al. 2011). Although ZmbHLH128 and ZmbHLH129 both possess In contrast to these insights into cis-elements that control three amino acids involved in G-box binding (K9, E13, and 1691 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.1. ZmbHLH128 and ZmbHLH129 homeologs bind the ZmC -NADP-ME promoter. (A) Schematic representation of the ZmC -NADP-ME 4 4 promoter, divided into fragments used as baits in Y1H screenings, and the ZmbHLH TFs identified. ATG and TAG are the translational start codon and the stop codon of the ZmC -NADP-ME ORF, respectively. ZmbHLH position on the scheme indicates that they bind between the base pairs 389 and 154 in relation to the ATG. (B) Analysis of ZmbHLH-pZmC -NADP-ME binding specificity. Each of the six yeast bait strains was transformed with both ZmbHLHs (pAD-GAL4-2.1::TF vectors) and positive interactions selected on CM -HIS -LEUþ 3-AT [yeast Complete Minimal medium lacking histidine and leucine amino acids, and supplemented with 3-amino-1, 2, 4-triazole (3-AT), a competitive inhibitor of the HIS3 gene product]. (C) Schematic representation of bHLH and ZIP protein domains, and respective position in protein sequences. (D) Schematic representation of ZmbHLH128 and ZmbHLH129 (black) and four additional maize homeolog gene pairs located in syntenic regions of chromo- somes 4 and 5. Homeolog genes are indicated by colour. Arrows indicate direction of transcription of each gene. Genomic coordinates provided from the B73 RefGen_v3 assembly version. R17) (Massari and Murre 2000; Li et al. 2006), this family of TFs Fisher and Caudy 1998; Kim et al. 2016). Therefore, the ZmC - 0 0 has also been shown to bind to N-box (5 -CACGCG-3 ), NADP-ME promoter was assessed for additional cis-elements 0 0 N-box B (5 -CACNAG-3 ) and FBS (FAR1/FHY3 Binding Site, to which ZmbHLH128 and ZmbHLH129 might bind. A total 0 0 5 -CACGCGC-3 )motifs(Sasai et al. 1992; Ohsako et al. 1994; of eight such cis-elements were found, consisting of two 1692 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE FIG.2. ZmbHLH128 and ZmbHLH129 bind two FBS cis-elements present in ZmC -NADP-ME promoter. (A) Schematic representation of position and nucleotide sequence of eight cis-elements recognized by bHLH that were identified in the ZmC -NADP-ME promoter. FBS stands for FHY3/ FAR1 Binding Site and it is a N-box-containing motif. (B) EMSA probe sequences used to test in vitro binding affinity of ZmbHLH128 and ZmbHLH129 to cis-elements (highlighted in bold). Arrows indicate that the FBS cis-elements are present in opposite orientations. (C) EMSAs showing in vitro binding affinity of Trx::ZmbHLH128 (gel on the left) and Trx::ZmbHLH129 (gel on the right) to the radiolabeled probes described in (B). Arrowheads indicate uplifted ZmbHLH–DNA probe complexes. Free probe indicates unbound DNA probes. N-boxes B, two N-boxes, one G-box, two FBSs, and one E-box intensities detected when both cis-elements were combined (fig. 2A). Electrophoretic Mobility Shift Assays (EMSA) were (fig. 2C) suggests that they function synergistically. Overall, used to test whether ZmbHLH128 and ZmbHLH129 were these data indicate that ZmbHLH128 and ZmbHLH129 target able to interact with each of these cis-elements in vitro 21 bp of DNA sequence (7 bp FBS, 7 bp spacer, and 7 bp FBS). (fig. 2B and C). Consistent with the Y1H findings, EMSA showed that recombinant Trx::ZmbHLH128 and ZmbHLH128 and ZmbHLH129 Form Both Trx::ZmbHLH129 proteins caused an uplift of radiolabeled Homo- and Heterodimers and ZmbHLH129 probes containing FBS cis-elements (probes 6, 7, and 6þ 7) Impairs trans-Activation by ZmbHLH128 (fig. 2C), positioned between nucleotides 389 and 154 in Because ZmbHLH128 and ZmbHLH129 bind the FBS cis-ele- relation to the predicted translational start site (see fig. 1A). ments in close proximity but also possess domains mediating ZmbHLH128 also showed weak binding to probe 3 that con- protein dimerization, we next investigated whether these tained a N-box cis-element that was not bound by proteins form homo- and/or heterodimers. In vitro, the re- ZmbHLH128 or ZmbHLH129 in Y1H (see fig. 1B), and signal combinant Trx::ZmbHLH128 and Trx::ZmbHLH129 proteins intensity was similar to that observed from probe 7 (fig. 2C). formed homodimers (fig. 3A). To confirm this interaction We cannot exclude however that relatively weak binding to in vivo,aswellastotestfor heterodimerization, probe 7 is due to it being three nucleotides-shorter than the Bimolecular Fluorescence Complementation Assays (BiFC) other probes (fig. 2B). Trx alone and OsPIF14 (a bHLH known in maize protoplasts were performed. While negative controls to bind the N-box motif; Cordeiro et al. 2016)wereusedas produced no YFP fluorescence, ZmbHLH128 and negative controls (fig. 2C). Thetwo FBSmotifs, in probe 6þ 7, ZmbHLH129 formed both homo- and heterodimers are separated by a short 7 bp spacer sequence and are found in opposite orientations (fig. 2B). The increase in band (fig. 3B). With the exception of ZmbHLH129 homodimers 1693 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.3. ZmbHLH128 and ZmbHLH129 form both homo- and heterodimers. (A) Western blot of BN-PAGE for the recombinant proteins Trx::His::ZmbHLH128 and Trx::His::ZmbHLH129. Gel was loaded with equivalent amount of protein. Recombinant proteins were immunodetected using a-His antibody. MW indicates molecular-weight size marker. (B) Protein interactions between ZmbHLH128 and ZmbHLH129 were tested by BiFC in maize mesophyll protoplasts co-transformed with constructs expressing ZmbHLH128 and ZmbHLH129 fused to N- and C-terminal YFP N C domains. YFP and YFP indicate split N- and C-terminal YFP domains, respectively. whose location extended to the cytoplasm and plasma lost when this TF was co-expressed with its homeolog membrane, in each case YFP signal was specifically local- ZmbHLH129 (fig. 4D). ized to the nucleus (fig. 3B). Nuclear localization of these ZmbHLH proteins supports their roles as transcriptional regulators. The G-Box-Based cis-Element Pair Recognized by To test the capacity of ZmbHLH128 and ZmbHLH129 to ZmbHLH128 and ZmbHLH129 in NADP-ME regulate transcription, transient expression assays were per- Promoters Operates Synergistically formed in leaves of Nicotiana benthamiana.The GUS reporter To understand whether the two FBS cis-elements identified in gene driven by the fragment of pZmC -NADP-ME to which the promoter of ZmC -NADP-ME (see fig. 2) are associated ZmbHLH128 and ZmbHLH129 bind was used as reporter, with the evolution of C photosynthesis, we investigated while the full-length ZmbHLH128 and ZmbHLH129 CDS whether they are conserved in promoters of other NADP- sequences driven by the constitutive CaMV35S promoter MEs from C and C grass species. Three C species 3 4 3 were used as effectors (fig. 4A). Co-infiltration of this re- (Dichanthelium oligosanthes, Oryza sativa,and porter with the ZmbHLH128 effector resulted in an in- Brachypodium distachyon)and threeC species (Z. mays, crease in GUS activity, indicating that ZmbHLH128 can Sorghum bicolor,and Setaria italica) were assessed (fig. 5A). act as a transcriptional activator (fig. 4B). In contrast, Within the C species, Z. mays and S. bicolor possess two ZmbHLH129 showed no intrinsic trans-activation activity plastidic NADP-ME isoforms: one that is used in C photo- (fig. 4C). In order to test whether the ZmbHLH128- synthesis (C -NADP-ME, GRMZM2G085019, and ZmbHLH129 heterodimers had a different trans-activation 4 Sobic.003g036200) and a second one not involved in the C activity from ZmbHLH128 or ZmbHLH129 homodimers, 4 cycle (nonC -NADP-ME, GRMZM2G122479, and leaves were co-infiltrated with the reporter and both effec- Sobic.009g108700) (Alvarez et al. 2013; Emms et al. 2016). tors simultaneously. Interestingly, the trans-activation activity observed for the ZmbHLH128 alone (fig. 4B)was In contrast, S. italica possesses only one plastidic NADP-ME 1694 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE isoform that is used in the C cycle (C -NADP-ME, Si000645) 4 4 (Alvarez et al. 2013; Emms et al. 2016). Although in C B. distachyon no homologous cis-elements to the FBSs in the ZmC -NADP-ME promoter were detected, in O. sativa one G-box was found in the same position as FBS 1 from Z. mays. Moreover, in the other promoters, cis-elements that can bind bHLH proteins were present in pairs (fig. 5A). In both the C and C grasses these cis-element pairs flank a 3 4 spacer that is highly conserved in sequence and length (7– 9bp) (fig. 5A). The C -NADP-ME promoters from Z. mays and S. bicolor share a common mutation in the third nucleo- tide position of the alignment (A!G) (fig. 5A). Two additional mutations are specific to Z. mays (the first and last nucleotides of FBS 1 and FBS 2, respectively), while one is S. bicolor-specific (C!T at the fourth position) (fig. 5A). It is possible that muta- tions unique to Z. mays or S. bicolor are neutral and the main impact on C -NADP-ME gene expression is due to mutation in the third nucleotide in the common ancestor of Z. mays and S. bicolor. Alternatively, it is also possible that both this mutation in the last common ancestor and species-specific modifica- tions impacted on gene expression of C -NADP-ME. To test if ZmbHLH128 and ZmbHLH129 bind the cis-ele- ments identified from these additional species EMSA was per- formed on each cis-element separately as well as the cis- element pairs found in each NADP-ME promoter (fig. 5B and C, supplementary table S3, Supplementary Material online). ZmbHLH128 and ZmbHLH129 showed low binding affinity for the single G-box identified in the O. sativa promoter (probe 13) and binding affinity was not increased by mutating the G-box to a canonical N-box (probe m13) (fig. 5B and C). This low binding affinity behaviour for single G-box cis-elements was consistent for all the NADP-ME promoters containing G- boxes (probes5,7,9,and 11) (fig. 5B and C). Although both ZmbHLHs did not show binding affinity for the additional N- boxes or N-box-like alone (probes 6, 8, 10, and 12) (fig. 5B and C), when these additional motifs were acquired and formed a pair with the ancestral G-box, binding affinity was increased (probes 5þ 6, 7þ 8, 9þ 10, and 11þ 12) and led to an in- creased uplift compared with the G-boxes alone (probes 5, 7, 9, and 11) (fig. 5B and C). Given the similar length of probes 1, 2, 1þ 2, 5, 7, 9, and 11 (24–30 bp) (supplementary table S3, Supplementary Material online), it is possible that this differ- ence in migration of ZmbHLH–probe complexes results from the binding of bHLH to G-boxes in a lower oligomeric state (supplementary fig. S2, Supplementary Material online), which based on the literature must be dimers (De Masi et al. 2011). Strong binding of cis-element pairs was also observed when the ancestral G-box evolved into either FBS or FeRE1 elements FIG.4. ZmbHLH129 impairs trans-activation of the ZmC -NADP-ME promoter by ZmbHLH128. (A) Schematic representation of reporter found in C Z. mays and S. bicolor (probes 1þ 2and 3þ 4) and effector constructs used in transient expression assays in leaves of (fig. 5B and C). In the C Z. mays promoter, both ZmbHLHs N. benthamiana. Reporter construct contains GUS gene driven by the showed binding affinity for single FBS cis-elements minimal CaMV35S promoter (m35S) fused to pZmC -NADP-ME (389 to 154 bp). Effector constructs contain the ZmbHLH128 or FIG.4. Continued ZmbHLH129 CDS driven by the full CaMV35S promoter. (B–D) Box (C) ZmbHLH129, and (D) ZmbHLH128 and ZmbHLH129. Different plots (2.5–97.5 percentiles) showing GUS activity, expressed in pico- letters denote differences in experimental data that are statistically moles of the reaction product 4-methylumbelliferone (MU) gener- significant (One-way ANOVA, Tukey test, P 0.05, n¼ 10-13). EV ated per minute per microgram of protein, in leaves agro-infiltrated indicates pGWB3i empty vector (no promoter fragment cloned). with reporter and the following effector constructs: (B) ZmbHLH128, Cross inside box plots indicates mean. f.c. indicates fold-change. 1695 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.5. The G-box-based cis-element pair recognized by ZmbHLH128 and ZmbHLH129 in NADP-ME promoters operates synergistically. (A) Sequence alignment of the two FBS cis-elements present in ZmC -NADP-ME promoter against homologous cis-elements present in other promoters of genes encoding plastidic NADP-ME. C grasses: Z. mays, S. bicolor and S. italica;C grasses: D. oligosanthes, O. sativa, and B. distachyon. 4 3 Plastidic NADP-MEs are color-coded: green for C , blue for nonC and magenta for C . Cis-elements are highlighted in bold and colored according 4 4 3 to the NADP-ME they belong to. FBS stands for FHY3/FAR1 Binding Site and FeRE1 for Iron Responsive Element 1. (B) EMSA probes used to test in vitro binding affinity of ZmbHLH128 and ZmbHLH129 to each cis-element described in (A). Probe sequences are listed in supplementary table S3, Supplementary Material online. (C) EMSA assays showing in vitro binding affinity of Trx::ZmbHLH128 (gel on the left) and Trx::ZmbHLH129 (gel on the right) proteins to the probes described in (B). Arrowheads indicate uplifted ZmbHLH-DNA probe complexes. Free probe indicates unbound DNA probes. 1696 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE Acquisition of N-Box-Derived cis-Elements in (probes 1 and 2) in the highest oligomeric state (fig. 5B and C, supplementary fig. S2, Supplementary Material online). NADP-ME Promoters Facilitates ZmbHLH128 and Since ZmbHLH128 and ZmbHLH129 showed weak bind- ZmbHLH129 Binding in PACMAD Panicoid Grasses ing to single cis-elements, we tested their binding by mutating Phylogenetic analysis of the genes encoding C and C plas- 3 4 these cis-elements in probes with the pairs (supplementary tidic NADP-MEs reflects previously reported grass species fig. S3, Supplementary Material online). For each pair, three phylogeny (fig. 6A)(Grass Phylogeny Working Group II mutant probes were designed: two in which the two cis- 2012). It inferred two main clades: one formed by C BEP elements were mutated individually (keeping one cis-element species (B. distachyon and O. sativa) and a second formed wild-type) and one in which both cis-elements were mutated by C (D. oligosanthes)and C Panicoid species of the 3 4 simultaneously (supplementary table S3, Supplementary PACMAD clade (S. italica, S. bicolor,and Z. mays)(fig. 6A). Material online). Competition experiments were performed Based on the observed nucleotide modifications in cis-ele- using radiolabeled wild-type probes (with cis-element pairs) mentsrecognizedbybHLHTFs,weproposeamodel relating and 200- to 400-fold excess of unlabeled wild-type and mu- to the recruitment of NADP-ME into C photosynthesis in tant probes (supplementary fig. S3, Supplementary Material grasses (fig. 6B). This proposes that an ancestral G-box found online). Binding of both ZmbHLHs to the labeled wild-type in the NADP-ME promoter of the common ancestor of C probes could be efficiently out-competed by unlabeled wild- BEP O. sativa and C Panicoid grasses has been conserved type and mutant probes in which the following cis-elements during the evolution of C photosynthesis. However, in the were not mutated: FBS 1 (in Z. mays C -NADP-ME, probe Panicoideae subfamily of the PACMAD clade a second 1þ m2-A, supplementary fig. S3A, Supplementary Material cis-element recognized by bHLH is present such that the online); FBS 2 (in Z. mays C -NADP-ME,probe m1þ 2-B, NADP-ME gene from the C species D. oligosanthes and genes supplementary fig. S3A, Supplementary Material online); N- encoding plastidic nonC -NADP-ME from C S. bicolor and 4 4 box (in S. bicolor C -NADP-ME, probe m3þ 4-E, supplemen- Z. mays all contain a G- and N-box/N-box-like pair. In C S. tary fig. S3B, Supplementary Material online); and G-box (in S. italica this cis-code has been retained in the C -NADP-ME,but italica C -NADP-ME, probe 5þ m6-G, supplementary fig. S3C, in S. bicolor and Z. mays the original G-box has evolved to Supplementary Material online; Z. mays nonC -NADP-ME, become either a FeRE1 or a FBS element, respectively (fig. 6B). probe 7þ m8-J, supplementary fig. S3D, Supplementary No G-box motifs are, however, present in the promoter of Material online; S. bicolor nonC -NADP-ME, probe 9þ m10- genes encoding cytosolic NADP-ME from S. bicolor and Z. M, supplementary fig. S3E, Supplementary Material online; mays. Overall, these results suggest that the acquisition of and D. oligosanthes C -NADP-ME, probe 11þ m12-P, supple- N-box-derived cis-elements have facilitated ZmbHLH128 mentary fig. S3F, Supplementary Material online). These and ZmbHLH129 binding to promoters of genes encoding EMSA competition experiments thus confirmed binding of plastidic NADP-ME in the PACMAD (Panicoideae subfamily). ZmbHLH128 and ZmbHLH129 to the cis-elements described above. Taken together, the results indicate that a second cis- Discussion element recognized by bHLH TFs is acquired in the promoters ZmbHLH128 and ZmbHLH129 Homeologs Interact of genes encoding plastidic NADP-ME and that each cis- with Maize C - and nonC -NADP-ME Promoters 4 4 element pair operates synergistically to allow interaction in vitro Showing Different trans-Activation Activity in with either ZmbHLH128 or ZmbHLH129 in C and C grasses 3 4 planta (fig. 5, supplementary figs. S2 and S3, Supplementary Material In this study, we showed that ZmbHLH128 and ZmbHLH129 online). form a maize homeolog pair resulting from the recent maize Given the binding affinity in vitro of ZmbHLH128 and whole genome duplication (WGD) event that occurred 5– ZmbHLH129 to the G-box in the ZmnonC -NADP-ME pro- 12 million years ago. This WGD occurred 5–16 million years moter (probes 7 and 7þ 8, fig. 5C), we tested their binding after C photosynthesis evolved in the Andropogoneae tribe ability in planta. Transient expression assays were performed 4 of the PACMAD clade (17–21 MYA) (Christin et al. 2008, in leaves of N. benthamiana co-infiltrated with GUS reporter 2009). As the length of exons 1 and 2 and the total number gene driven by a ZmnonC -NADP-ME promoter fragment of amino acids in the mature protein of ZmbHLH128 are containing the cis-element pair G- and N-box-like (368 to more similar to sorghum ortholog SbbHLH66 (supplementary 143 bp) and the effector constructs ZmbHLH128 and fig. S5, Supplementary Material online), we propose that ZmbHLH129 (supplementary fig. S4A, Supplementary ZmbHLH129 has diverged more from the ancestral gene. Material online). Compared with the reporter alone, co- Both of these TFs bind two FBS cis-elements that are in close infiltration of ZmnonC -NADP-ME reporter and the proximity in the maize C -NADP-ME (GRMZM2G085019) ZmbHLH128 and ZmbHLH129 effectors did not impact on promoter. Although ZmbHLH128 has been predicted in silico GUS activity in tobacco system (supplementary fig. S4B–D, to regulate C photosynthesis (Wang et al. 2014), as far as we Supplementary Material online). These results suggest that although ZmbHLH128 on its own binds both the ZmC - are aware, this is the first report of its functional characteri- NADP-ME and ZmnonC -NADP-ME promoters in vitro zation. ZmbHLH128 alone activates ZmC -NADP-ME gene (probes1,2,1þ 2, 7, and 7þ 8, fig. 5B and C), this might expression, while ZmbHLH129 alone shows no trans-activa- not be the case in planta (supplementary fig. S4, tion activity on this promoter. As the duplication event that Supplementary Material online). generated ZmbHLH129 took place after the evolution of C 1697 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE FIG.6. Acquisition of N-box-derived cis-elements in NADP-ME promoters facilitates ZmbHLH128 and ZmbHLH129 binding in PACMAD Panicoid grasses. (A) Phylogenetic tree of genes encoding plastidic NADP-ME from C and C grass species. C : B. distachyon (Bd), O. sativa (Os), and D. 3 4 3 oligosanthes (Do); C : S. italica (Si), S. bicolor (Sb) and Z. mays (Zm). NADP-MEs are color-coded: magenta for C , blue for nonC and green for C . 4 3 4 4 NADP-ME genomic sequences were aligned using MUSCLE, and the phylogenetic tree inferred by NJ method (1000 bootstrap pseudoreplicates, node numbers indicate bootstrap values). Gene encoding C plastidic NADP-ME from A. thaliana (AtC -NADP-ME) was used as outgroup. (B) 3 3 Diagram representing C to C molecular evolution of homologous bHLH binding cis-elements identified in promoters of genes encoding plastidic 3 4 NADP-ME. Dashed arrow indicates intermediate evolutionary steps from C to C . Vertical lines indicate two independent C origins of S. italica 3 4 4 and S. bicolor/Z. mays (Paniceae and Andropogoneae tribes, respectively). photosynthesis, it seems possible that this gene is not re- heterodimers (Kang et al. 2010). The latter scenario has quired for C photosynthesis. ZmbHLH128 and been reported for the maize Dof1 and Dof2 TFs. Dof1 is a ZmbHLH129 form heterodimers and despite ZmbHLH128 transcriptional activator of light-regulated genes in leaves, activating the expression of ZmC -NADP-ME its regulatory however, in stems and roots, this TF is not able to regulate activity is impaired by its homeolog ZmbHLH129. To explain those genes since the repressor Dof2 is expressed there and this impairment, we hypothesize different scenarios that may blocks Dof-specific cis-elements (Yanagisawa and Sheen occur in vivo: either ZmbHLH128 and ZmbHLH129 act as 1998). heterodimers and ZmbHLH128 loses its DNA binding activity In addition to the capacity of ZmbHLH128 and when combined with ZmbHLH129 or they act as homo- ZmbHLH129 to interact with FBSs found in the maize C - dimers and compete directly for the same FBSs, toward which NADP-ME promoter, both ZmbHLHs were shown to bind in vitrotothe promoter of maize nonC -NADP-ME ZmbHLH129 has a higher binding affinity. The former sce- nario has been described for bZIP TFs from Arabidopsis, (GRMZM2G122479) that possesses the cis-element pair G- where bZIP63 has negative effects on the formation of and N-box-like. In planta,however,ZmbHLH128 and bZIP1–DNA complexes probably due to conformational dif- ZmbHLH129 showed no trans-activation activity on this pro- ferences between bZIP1 homodimer and bZIP1-bZIP63 moter. It is well known that primary DNA sequence and its 1698 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE structural properties are determinants of DNA binding spe- encourage DNA looping. In addition to the spacer length, its cificity in vivo (Rohs et al. 2009) and so it is possible that both sequence appears highly conserved. This is consistent with ZmbHLHs display increased in vivo binding specificity for the evidence suggesting that nucleotides outside core cis-ele- FBS pair in the ZmC -NADP-ME promoter than for the G- and ments affect TF binding specificity by providing genomic con- N-box-like pair in the ZmnonC -NADP-ME promoter. text and influencing three-dimensional structure (Atchley Therefore, ZmbHLH128 seems to affect the level of expression et al. 1999; Mart ınez-Garcia et al. 2000; Grove et al. 2009; of NADP-ME as it activates the ZmC -NADP-ME promoter Gord^ an et al. 2013). For example, Cbf1 and Tye7 are yeast through the pair formed by two FBSs but the same trend bHLHs that show preference for a subset of G-boxes present was not observed for the ZmnonC -NADP-ME promoter with throughout the yeast genome (Gord^ an et al. 2013). These differences in binding preferences were observed not just the G- and N-box pair. In addition, we hypothesize that these modifications of promoter sequences may also affect light/ in vivo but also in vitro and so DNA sequences flanking circadian regulation of the ZmC -NADP-ME gene as FBS cis- core G-boxes were found to explain this differential bHLH- elements have been described in promoters of circadian- G-box binding (Gord^ an et al. 2013). clock-regulated and light-responsive genes (Lin et al. 2007, The mechanism proposed here for how bHLH TFs interact with their target cis-elements suggests that these DNA 2011; Kim et al. 2016). Themutationoftwo closeFBSsin the promoter of the circadian-clock gene EARLY sequences are not randomly arranged in gene promoters FLOWERING 4 (ELF4)provedtobesufficienttoabolishits and may affect how cis-element specificity is achieved. rhythmic expression (Li et al. 2011). More broadly, our find- Indeed, in some promoters bound by bHLH TFs two or ings also contribute to the understanding of gene regulatory more cis-elements were found to be clustered. For example, two overlapping FBSs were reported in the 400 bp upstream networks controlling C photosynthesis. of the translational start site of the gene encoding ELF4 (Li The G-Box-Based cis-Element Pair Present in et al. 2011). Also, pairs of G- and N-boxes were found to be NADP-ME Promoters Synergistically Bind Either highly enriched in promoters targeted by the bHLH PIF1 (Kim et al. 2016). It is possible that multiple cis-elements serve to ZmbHLH128 or ZmbHLH129 We identified a cis-element pair recognized by bHLH that recruit additional TFs for in vivo cooperative binding. occupy homologous positions in NADP-ME promoters from C Photosynthesis Co-Opted an Ancient C C and C grasses. These cis-elements flank a short spacer and 4 3 3 4 Cis-Regulatory Code Built on G-Box Recognition by operate synergistically to facilitate interaction with ZmbHLH128 and ZmbHLH129. We suggest a mechanism bHLH Transcription Factors by which these TFs may be recruited to the cis-elements Finally, from this study we propose a model that summarizes associated with C photosynthesis. We propose that one how molecular evolution of cis-elements recognized by bHLHs may relate to the recruitment of NADP-ME into C cis-element is sufficient to recruit a bHLH homodimer (G- box) or tetramer (N-box or FBS in promoters where the an- photosynthesis. C photosynthesis is an excellent example of cestral G-box is no longer present); however, the presence of a convergent evolution (Sage et al. 2011; Christin et al. 2013)as second cis-element in the vicinity increases bHLH binding it has evolved independently over 60 times in angiosperms affinity (supplementary fig. S2, Supplementary Material on- (Sage et al. 2011; Sage 2016) and at least 22 times in grasses (Grass Phylogeny Working Group II 2012). How this repeated line).Itispossiblethatboth cis-elements are brought together through the interaction with a bHLH tetramer formed by two evolution has come about is not fully understood. Our model dimers, which may involve DNA bending (supplementary fig. contributes to our understanding of C evolution and is based S2, Supplementary Material online). Therefore, this cis-ele- on the following findings: first, in rice, which belongs to the ment pair could operate synergistically to confer stabilization BEP clade that contains no C species, only one copy of a G- of bHLH binding. This mechanism of TF-DNA assembly has box was present in the NADP-ME promoter. In contrast, cis- previously been proposed for MADS-domain TFs that can element pairs recognized by ZmbHLH128 and ZmbHLH129 bind two nearby CArG boxes through DNA looping and for- in NADP-ME promoters seem to be common in the mation of tetrameric complexes (Theissen 2001; Theissen and Panicoideae subfamily of the PACMAD clade that contains independent C lineages. For example, in the PACMAD Saedler 2001; Melzer et al. 2009; Smaczniak et al. 2012; Smaczniak et al. 2017). In this case, and consistent with our Panicoid grasses a G- and N-box pair was identified in C D. results, MADS-domain TFs were found to bind single CArG oligosanthes (Do024386) and appears to be reasonably con- boxes either as dimers or tetramers, however, when their served in C species. However, in the case of the C -NADP- 4 4 target gene promoters contain CArG box pairs they bind as MEsfrom S. bicolor and Z. mays (Sobic.003g036200 and tetramers (Smaczniak et al. 2012). It has been proposed that GRMZM2G085019) these elements have diversified. Both of the probability of DNA loop formation increases with shorter these grass species belong to the C tribe Andropogoneae in distances between cis-elements due to the low elastic bending which the plastidic NADP-ME isoform that is used in C energy required to bring the protein dimers together photosynthesis (C -NADP-ME) evolved by duplication from (Agrawal et al. 2008). Interestingly, in all NADP-ME promoters an ancestral plastidic NADP-ME that still exists and is not assessed in this study except rice and Brachypodium the two involved in the C cycle (nonC -NADP-ME, Sobic.009g108700 4 4 cis-elements were found to be in close proximity, which may and GRMZM2G122479) (Tausta et al. 2002; Maier et al. 2011; 1699 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE Alvarez et al. 2013). In contrast, C S. italica together with C Materials and Methods 4 3 D. oligosanthes belong to the grass tribe Paniceae in which Plant Growth Conditions and Collection of Leaf only one plastidic NADP-ME isoform is known to exist Samples (Si000645 and Do024386) (Alvarez et al. 2013; Emms et al. To construct the cDNA expression library, maize plants 2016). Surprisingly, the cis-element pair identified in the C - (Z. mays L. var. B73) were grown at 16 h photoperiod with NADP-ME promoter from S. italica (G- and N-box) was found 2 1 a light intensity of 340–350 lmol m s ,atday/night tem- to be closer to those occurring in the C and nonC -NADP-ME 3 4 perature of 28/26 C, and 70% relative humidity. Two light promoters from D. oligosanthes, S. bicolor,and Z. mays (G- regimes were used: (1) nine days in 16 h photoperiod; and (2) and N-box/N-box-like) than to those occurring in the C - nine days in 16 h photoperiod followed by a 72 h dark treat- NADP-ME promoters from S. bicolor and Z. mays (FeRE1 ment. In both experiments, sample collection was performed and N-box or FBS and FBS, respectively). A similar trend under 16 h photoperiod. Third leaves grown in the former has previously been observed (Alvarez et al. 2013)and may and latter light regimes were harvested respectively at time be explained by the independent evolutionary origin of C points covering the Zeitgeber times (ZT) 0.5, 0.5, 2 h, and photosynthesis in grass tribes formed by S. italica (Paniceae) ZT 1, 2, 4, 8, 12, 15.5 h. For isolation of maize mesophyll or S. bicolor/Z. mays (Andropogoneae). protoplasts, maize plants were grown for 10 days at 25 C, Taken together, our findings suggest that an ancestral G- 2 1 16 h photoperiod (60 lmol m s ), and 70% relative box in combination with N-box-derived cis-elements form humidity. For transient expression assays in planta, the basis of the synergistic binding of either ZmbHLH128 or N. benthamiana (tobacco) plants were grown for 5 weeks ZmbHLH129 to NADP-ME promoters from PACMAD 2 1 at 22 C, 16 h photoperiod (350 lmol m s ), and 65% Panicoid grasses. Nucleotide diversity in cis-elements recog- relative humidity. After agro-infiltration of tobacco leaves, nized by bHLH TFs has been suggested as one of the mech- anisms by which these TFs are involved in complex and plants were left to grow into the same growth conditions diverse transcriptional activity (Toledo-Ortiz et al. 2003). and leaf discs (2.5 cm in diameter) collected 96 h post- We, therefore, cannot exclude the possibility that the gene infection. encoding the plastidic NADP-ME from C BEP B. distachyon (BRADI2g05620) can also be bound by ZmbHLH128 or Generation of Yeast Bait Strains ZmbHLH129 despite none of the typical cis-elements recog- Yeast bait strains were generated as previously described nized by bHLH being identified in the promoter. Given recent (Ouwerkerk and Meijer 2001; Serra et al. 2013). Yeast evidence indicating that the bHLH TF family is often recruited strain Y187 (Clontech) was used to generate six bait into C photosynthesis regulation (Huang and Brutnell 2016), strains carrying overlapping fragments of the ZmC - we suggest that the observed nucleotide modifications in the NADP-ME (GRMZM2G085019) promoter cloned into cis-element pair present in C -NADP-ME promoters from S. the yeast integrative vector pINT1-HIS3 (Ouwerkerk and bicolor and Z. mays may underlie changes in bHLH binding Meijer 2001)as NotI-SpeIor XbaI-SpeI fragments (supple- specificity in vivo and, therefore, contribute to the NADP-ME mentary table S1, Supplementary Material onlline). The recruitment into C photosynthesis in the Andropogoneae ZmC -NADP-ME promoter region was defined as the tribe from the PACMAD clade. The presence of a bHLH du- 1,982 bp upstream of the predicted translational start plicate (ZmbHLH129) that seems not to be required for C site (ATG). To assess self-activation/HIS3 leaky expression, photosynthesis and has evolved to repress the activity of its yeast bait strains were titrated in complete minimal me- homeolog (ZmbHLH128) is unique to maize as this homeolog dium (CM) lacking histidine, with increasing concentra- gene pair resulted from the maize WGD. Therefore, we hy- tions of 3-amino-1, 2, 4-triazole (3-AT, up to 75 mM). pothesize that the single orthologous bHLH in all the other Panicoid species of the PACMAD clade activates C -NADP- Construction of cDNA Expression Library ME gene expression. This agrees with the hypothesis that C Total RNA was extracted from third leaves of maize seedlings photosynthesis has on multiple occasions made use of cis- using TRIzol reagent (Invitrogen), following the manufac- regulators found in C species and, therefore, that the recruit- turer’s instructions. RNA samples from nine time points (de- ment of C genes was made through minor rewiring of pre- scribed in ‘plant growth conditions and collection of leaf existing regulatory networks (Reyna-Llorens and Hibberd samples’) were pooled in equal amounts for mRNA purifica- 2017). We conclude that regulation of C genes can be based tion using the PolyATract mRNA Isolation System IV on an ancient code founded on a G-box present in the BEP (Promega). A unidirectional cDNA expression library was pre- clade as well as the Panicoideae of the PACMAD clade. pared using the HybriZAP-2.1 XR cDNA Synthesis Kit and the Acquisition of a second cis-element recognized by bHLH in HybriZAP-2.1 XR Library Construction Kit (Stratagene), fol- Panicoid grasses appears to have facilitated synergistic binding lowing the manufacturer’s instructions. Four micrograms of by either ZmbHLH128 or ZmbHLH129. Although this G-box- mRNA were used for first strand cDNA synthesis. After in vivo based cis-code has remained similar in S. italica,ithas diverged in maize and sorghum. Thus, different C grass lineages may excision and amplification of the pAD-GAL4-2.1 phagemid vector, this maize cDNA expression library was used to trans- employ slightly different molecular circuits to regulate orthol- ogous C photosynthesis genes. form yeastbaitstrains. 1700 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE 0 0 Yeast One-Hybrid (Y1H) Screening and Validation GCGCTGGA-3 /5 -GGGGACCACTTTGTACAAGAAAGCT Yeast bait strains were transformed with 1 lgofmaize cDNA GGGTNCTAAGCATTTGGGGGCCAG-3 (underlined expression library according to Ouwerkerk and Meijer (2001) sequences indicate attB Gateway adaptors). ZmbHLH128 and Serra et al. (2013). At least, 1.3 million yeast colonies of and ZmbHLH129 CDS were recombined into pDONR221 each yeast bait strain transformed with the maize cDNA ex- (Invitrogen) to obtain Entry clones through BP-Gateway re- pression library were screened in CM -HIS -LEU supple- action (Invitrogen), following the manufacturer’s instructions. N C mented with 3-AT: 5 mM (1982 to 1524 bp), 20 mM CDSwerethenrecombinedintovectors YFP 43 and YFP 43 (389 to 154 bp, 776 to 334 bp) or 75 mM (973 to through LR-Gateway reaction (Invitrogen) to raise a transla- 702 bp, 1225 to 891 bp, 1617 to 1135 bp). Plasmids tional fusion with N- and C-terminal domains of yellow fluo- from yeast clones that actively grew on selective medium rescent protein (YFP), respectively. Final BiFC constructs were N N were extracted. To know whether the isolated clones denominated as YFP ::ZmbHLH128, YFP ::ZmbHLH129, C C encoded transcription factors (TFs), the cDNA insert was se- YFP ::ZmbHLH128, and YFP ::ZmbHLH129. Maize mesophyll quenced and the results analyzed using BLAST programes. To protoplasts were transformed with 6 lg of each of the BiFC validate DNA-TF interactions in yeast, isolated plasmids constructs. Protoplasts transformed with YFP ::Akin10 encoding TFs were re-transformed into the yeast bait strain (Arabidopsis SNF1 Kinase Homolog 10), YFP ::Akin3 in which they were found to bind. To assess TF binding (Arabidopsis SNF1 Kinase Homolog 3) and YFP 43 and specificity, plasmids encoding TFs were also transformed YFP 43 empty vectors were used as negative controls. into the yeast bait strains to which they do not bind. Transformations were performed in triplicate. YFP fluo- rescence and chlorophyll autofluorescence signals were Yeast Cell Spotting observed under a confocal microscope (Leica SP5). Yeast bait strains transformed with plasmids encoding TFs were grown overnight until log or mid-log phase at 30 Cin Transient Expression Assays in planta liquid yeast CM medium supplemented with Histidine For the transient expression assays in tobacco leaves, reporter (CMþHIS -LEU). Cultures were normalized to an OD of and effector constructs were generated in the Gateway binary 0.4, spotted onto solid medium CMþHIS -LEU or CM -HIS - vectors pGWB3i [pGWB3 containing an intron-tagged b-glu- LEUþ 3-AT, and grown for 3 days at 30 C. curonidase (GUS) open reading frame (Berger et al. 2007)] and pGWB2 (Tanaka et al. 2012), respectively. Isolation and Transformation of Maize Mesophyll To construct the reporter plasmids, promoter fragments of Protoplasts ZmC -NADP-ME (GRMZM2G085019, from 389 to Maize mesophyll protoplasts were isolated from 10-day-old 154 bp) and ZmnonC -NADP-ME (GRMZM2G122479, maize greening plants and transformed according to from 368 to 143 bp) were fused to a 136 bp minimal Lourenc ¸o et al. (2013) with minor modifications. Mid- CaMV35S promoter (m35S) in a 3-step PCR reaction: (1) section of newly matured second leaves was digested in a promoter sequences were amplified with long chimeric pri- cell wall digestive medium containing 1.5% (w/v) cellulase mers to introduce overlapping ends (reverse primer of R-10 (Duchefa), 0.3% (w/v) macerozyme R-10 (Duchefa), pZmC -NADP-ME/pZmnonC -NADP-ME was designed to be 4 4 10 mM MES (pH 5.7), 0.4 M mannitol, 1 mM CaCl ,0.1% complementary to the forward primer of the m35S)(supple- (w/v) BSA and 5 mM b-mercaptoethanol. Several leaf blades mentary table S4, Supplementary Material online); (2) pro- were stacked and cut perpendicularly to the long axis into moter sequences amplified by PCR in (1) were mixed 0.5–1 mm slices and quickly transferred to digestive medium according to the fusion products of interest in a ratio of 1:1 (25 ml digestive medium for each set of 10 leaf blades). Purity [ZmC -NADP-ME (389 to 154 bp)::m35S and ZmnonC - 4 4 and integrity of isolated protoplasts were examined under NADP-ME (368 to 143 bp)::m35S]and 10 PCRcycles light microscopy. Mesophyll protoplasts were quantified 6 1 were run without primers (denaturation at 98 Cfor 10s, and its abundance adjusted to 2 10 protoplasts ml . 55 Cfor 30 s, and72 C for 1 min); and (3) fusion products Transformed protoplasts were resuspended in 1.25 ml of in- of interest were amplified with attB-containing primers (sup- cubation solution [0.6 M mannitol, 4 mM MES (pH 5.7) and plementary table S4, Supplementary Material online). To ob- 4 mM KCl] and incubated in 24-well plates for 18 h at room tain Entry clones, promoter fragments fused to m35S were temperature under dark. cloned into pDONR221 (Invitrogen) through BP-Gateway Bimolecular Fluorescence Complementation (BiFC) reaction (Invitrogen), following the manufacturer’s instructions. Promoter sequences were then recombined Assay into the binary vector pGWB3i through LR-Gateway reac- To generate BiFC constructs, full-length coding sequences tion (Invitrogen) to obtain the final reporter constructs for (CDS) of ZmbHLH128 (GRMZM2G314882) and promoter::GUS analysis (pZmC -NADP-ME and ZmbHLH129 (GRMZM5G856837) were PCR-amplified using 4 pZmnonC -NADP-ME). For the effector constructs (TF respectively the following pairs of attB-containing primers: 5 - driven by the CaMV35S promoter), ZmbHLH128 and GGGGACAAGTTTGTACAAAAAAGCAGGCTNNATGATG 0 0 ZmbHLH129 Entry clones previously generated (see BiFC AACTGCGCCGGA-3 /5 -GGGGACCACTTTGTACAAGAAA 0 0 GCTGGGTNCTAAGCATTAGGCGGCCAG-3 ,and 5 -GGGG assay) were directly recombined into the binary vector ACAAGTTTGTACAAAAAAGCAGGCTNNATGATGGACT pGWB2 through LR-Gateway reaction (Invitrogen). 1701 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Borba et al. doi:10.1093/molbev/msy060 MBE Blue Native-Polyacrylamide Gel Electrophoresis Reporter and effector constructs together with a construct harboring the silencing suppressor P1b (Valli et al. 2006)were (BN-PAGE) and Western Blotting transformed into the Agrobacterium tumefaciens strain Molecular mass of oligomers co-existing in purified GV301. Overnight cultures of Agrobacterium harboring re- ZmbHLH128 and ZmbHLH129 recombinant proteins was determined by blue native polyacrylamide gel electrophoresis porter, effector and P1b constructs were sedimented (5000 (BN-PAGE). Two micrograms of the recombinant proteins gfor 15 min, at 4 C) and resuspended in infiltration medium (Trx::His::ZmbHLH128 or Trx::His::ZmbHLH129) were re- (10 mM MgCl , 10 mM MES (pH 5.6), 200 lMacetosyrin- solved on a 3–12% Novex Bis–Tris NativePAGE mini gel gone) to an OD of 0.3, 1, and 0.5, respectively, and mixed (Life Technologies), following the manufacturer’s instructions. in a ratio of 1:1:1. Mixed Agrobacterium cultures were incu- HMW Native Marker Kit (66–669 kDa, GE Healthcare) was bated for 2 h at 28 C and used to spot-infiltrate the abaxial used to estimate molecular mass. Resolved proteins were side of 5-week-old tobacco leaves. As controls, tobacco leaves transferred to a polyvinylidene difluoride membrane (GE were agro-infiltrated with mixed cultures carrying the re- Healthcare). The membrane was destained with a 50% porter construct alone or the empty vector pGWB3i and (v/v) methanol and 10% (v/v) acid acetic solution followed effector constructs. Infected leaves were analyzed at 96 h by pure methanol. For immunodetection of post-infiltration. Leaf discs of 2.5 cm in diameter were col- Trx::His::ZmbHLH128 and Trx::His::ZmbHLH129, the mem- lected from the infiltrated spots and used for the quantifica- brane was incubated with a-His antibody (GE Healthcare) tion of GUS activity. GUS activity was quantified by followed by a-mouse horseradish peroxidase-conjugated an- measuring the rate of 4-methylumbelliferyl-b-D-glucuronide tibody (abcam) for 1 h each at room temperature. (MUG) conversion to 4-methylumbelliferone (MU) as de- scribed in Jefferson et al. (1987) and Williams et al. (2016). Electrophoretic Mobility Shift Assay (EMSA) In brief, soluble protein was extracted from agro-infiltrated DNA probes were generated by annealing oligonucleotide tobacco leaf discs by freezing in liquid nitrogen and macera- pairs in a thermocycler followed by radiolabeling as described tion, followed by addition of protein extraction buffer. Diluted in Serra et al. (2013). DNA probe sequences and respective protein extracts (1:2) were incubated with 1 mM MUG for 30, annealing temperatures are listed in supplementary table S3, 60, 90, and 120 min at 37 C in a 96-well plate. GUS activity Supplementary Material online. EMSAs were performed using was terminated at the end of each time point by the addition 400 ng of the recombinant proteins Trx::ZmbHLH128 or of 200 mM Na CO and MU fluorescence measured by ex- Trx::ZmbHLH129, and 50 fmol of radiolabeled probes. 2 3 citing at 365 nm and measuring emission at 455 nm. The Competition assays were performed adding 200- to 400- fold molar excess of the unlabeled probe. Trx::OsPIF14 concentration of MU/unit fluorescence in each sample was (LOC_Os07g05010) and Trx protein, both purified by interpolated using a concentration gradient of MU from 1.5 Cordeiro et al. (2016), were used as negative controls. Each to 800 lMMU. protein was mixed with probes in a 10 ll reaction containing 10 mM HEPES (pH 7.9), 40 mM KCl, 1 mM EDTA (pH 8), Production of Recombinant ZmbHLH128 and 1mM DTT, 50ng herring sperm DNA, 15 lg BSA and 10% (v/v) glycerol. Binding reactions were incubated for 1 h on ice ZmbHLH129 and the bound complexes resolved on a native 5% polyacryl- ZmbHLH128 and ZmbHLH129 full-length CDSwerePCR- amide gel (37.5:1). Gel electrophoresis and detection of radio- amplified using, respectively, the following pairs of gene 0 0 active signal were performed as described in Serra et al. specific primers 5 -GAATTCATGATGAACTGCGCCGGA-3 / 0 0 0 (2013). 5 -CTCGAGCTAAGCATTAGGCGGCCAG-3 and 5 -GAA 0 0 TTCATGATGGACTGCGCTGGA-3 /5 -CTCGAGCTAAGCA 0 Synteny Analysis TTTGGGGGCCAG-3 (underlined sequences indicate adap- SynFind (Tang et al. 2015) was used to identify maize syntenic tors with restriction enzyme sites). ZmbHLH128 and chromosomal regions for ZmbHLH128 (GRMZM2G314882) ZmbHLH129 were cloned as EcoRI-XhoI fragments into and ZmbHLH129 (GRMZM5G856837) genes against Z. mays the expression vector pET32a (Novagen), generating B73 RefGen_v3 genome. A table containing maize syntelog N-terminal Trx-tagged fusions. pET32a-Trx::ZmbHLH128 gene pairs was retrieved using SynFind tool (supplementary and pET32a-Trx::ZmbHLH129 constructs were confirmed table S2, Supplementary Material online). by sequencing and transformed into Rosetta (DE3)pLysS competent cells (Invitrogen) for protein expression. Cells Phylogenetic Analyses transformed with pET32a-Trx::ZmbHLH128 and pET32a- ZmbHLH128 and ZmbHLH129 were used as references to Trx::ZmbHLH129 constructs were, respectively, grown in identify closely related bHLH genes of Z. mays, S. bicolor, Terrific Broth (TB) and Luria-Bertani (LB) medium to an Setaria viridis, S. italica, O. sativa,and B. distachyon,through OD of 0.5. Protein expression was induced with 4 mM 600 Phytozome database (Goodstein et al. 2012). Predicted CDS isopropyl-D-1-thiogalactopyranoside (IPTG) and allowed to were aligned using MUSCLE. The resulting alignment was occur for 3 h (ZmbHLH128) or 5 h (ZmbHLH129) at 30 C. used to infer a maximum likelihood phylogenetic tree, using Protein purification was performed as described in Cordeiro GTRþ GþI nucleotide substitution model (1,000 bootstrap et al. (2016). pseudoreplicates) in MEGA 7 software (Kumar et al. 2016). 1702 Downloaded from https://academic.oup.com/mbe/article/35/7/1690/4962173 by DeepDyve user on 19 July 2022 Synergistic Binding of bHLH Transcription Factors doi:10.1093/molbev/msy060 MBE Christin P-A, Boxall SF, Gregory R, Edwards EJ, Hartwell J, Osborne CP. Phylogenetic analysis of genes encoding C and C plastidic 3 4 2013. Parallel recruitment of multiple genes into C photosynthesis. NADP-ME isoforms from B. distachyon (BRADI2g05620), O. Genome Biol Evol. 5(11):2174–2187. sativa (LOC_Os01g09320), D. oligosanthes (Do024386), S. ital- Christin P-A, Osborne CP. 2014. The evolutionary ecology of C plants. ica (Si000645), S. bicolor (Sobic.003g036200, New Phytol. 204(4):765–781. Sobic.009G108700), and Z. mays (GRMZM2G085019, Christin P-A, Osborne CP, Sage RF, Arakaki M, Edwards EJ. 2011. C eudicots are not younger than C monocots. JExp Bot. GRMZM2G122479) was performed using Geneious Pro 5.3.6 4 62(9):3171–3181. software (Kearse et al. 2012). Full-length genomic sequences Christin P-A, Salamin N, Kellogg EA, Vicentini A, Besnard G. 2009. were aligned using MUSCLE. Phylogenetic tree was inferred Integrating phylogeny into studies of C variation in the grasses. using the Neighbor Joining (1,000 bootstrap pseudoreplicates) New Phytol. 149(1):82–87. and rooted using the gene encoding C plastidic NADP-ME Cordeiro AM, Figueiredo DD, Tepperman J, Borba AR, Lourenc ¸o T, Abreu IA, Ouwerkerk PBF, Quail PH, Margarida Oliveira M, Saibo (At1g79750) from Arabidopsis thaliana,adicotangiosperm. NJM. 2016. Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box Supplementary Material and interacts preferentially with the active form of phytochrome B. Supplementary data areavailableat Molecular Biology and Biochim Biophys Acta 1859(2):393–404. Evolution online. De Masi F, Grove CA, Vedenko A, Alib es A, Gisselbrecht SS, Serrano L, Bulyk ML, Walhout AJM. 2011. Using a structural and logics systems Acknowledgments approach to infer bHLH-DNA binding specificity determinants. Nucleic Acids Res. 39(11):4553–4563. We thank Lisete Fernandes (Escola Superior de Tecnologia da Ehleringer JR, Monson RK. 1993. Evolutionary and ecological aspects of Sau de de Lisboa, Portugal) for discussions and advice photosynthetic pathway variation. Annu RevEcolEvolSyst. 24(1):411–439. concerning EMSA experiments, Cec ılia Arraiano Lab (ITQB- Emms DM, Covshoff S, Hibberd JM, Kelly S. 2016. Independent and NOVA, Oeiras, Portugal) for material used in EMSA parallel evolution of new genes by gene duplication in two origins experiments, Myriam Goudet and Samuel Brockington of C photosynthesis provides new insight into the mechanism of (Department of Plant Sciences, University of Cambridge, phloem loading in C species. MolBiolEvol. 33(7):1796–1806. UK) for assistance with phylogenetic analyses. This study Fisher A, Caudy M. 1998. The function of hairy-related bHLH repressor was supported by European Union project 3to4 (Grant agree- proteins in cell fate decisions. BioEssays 20(4):298–306. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, ment no: 289582) and Fundac ¸~ ao paraaCi^ enciaeTecnologia DirksW,HellstenU,PutnamN,Rokhsar DS.2012. Phytozome: a (FCT) through research unit GREEN-it ‘Bioresources for comparative platform for green plant genomics. Nucleic Acids Res. Sustainability’ (UID/Multi/04551/2013). 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