Research ArticleResearch Article

Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes

Christian Wiludda, Stefanie Schulze, Udo Gowik, Sascha Engelmann, Maria Koczor, Monika Streubel, Hermann Bauwe, Peter Westhoff

Published January 2012. DOI: https://doi.org/10.1105/tpc.111.093872

Abstract

The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C3 plants but is restricted to bundle sheath cells in C4 species. GDC is also required for general cellular C1 metabolism. In the Asteracean C4 species Flaveria trinervia, a single functional GLDP gene, GLDPA, encodes the P-subunit of GDC, a decarboxylating Gly dehydrogenase. GLDPA promoter reporter gene fusion studies revealed that this promoter is active in bundle sheath cells and the vasculature of transgenic Flaveria bidentis (C4) and the Brassicacean C3 species Arabidopsis thaliana, suggesting the existence of an evolutionarily conserved gene regulatory system in the bundle sheath. Here, we demonstrate that GLDPA gene regulation is achieved by an intricate interplay of transcriptional and posttranscriptional mechanisms. The GLDPA promoter is composed of two tandem promoters, PR2 and PR7, that together ensure a strong bundle sheath expression. While the proximal promoter (PR7) is active in the bundle sheath and vasculature, the distal promoter (PR2) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 5′ untranslated leader of PR2-derived transcripts is inefficiently spliced and apparently suppresses the output of PR2 by eliciting RNA decay.

INTRODUCTION

C4 photosynthesis is based on the division of labor between two distinct photosynthetically active cell types: mesophyll and bundle sheath cells. After conversion to HCO3−CO2 is initially fixed in mesophyll cells by phosphoenolpyruvate carboxylase in the form of either malate or Asp and then transported into bundle sheath cells. There CO2 is released, refixed by ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco), and finally enters the Calvin-Benson cycle as it occurs in C3 plants. As a bifunctional enzyme, Rubisco is able to catalyze the carboxylation as well as the oxygenation of its substrate ribulose 1,5-bisphosphate. The fixation of O2 leads to accumulation of phosphoglycolate, which is toxic for plant cells. To regenerate phosphoglycerate from phosphoglycolate, photorespiration is essential. However, this metabolic pathway leads to the loss of previously fixed CO2 and thus decreases the efficiency of photosynthesis. The high concentration of CO2 in the bundle sheath cells, caused by the C4 cycle, suppresses the oxygenase reaction of Rubisco and, thereby, photorespiration effectively (Anderson, 1971; Ogren, 1984; Hatch, 1987; Leegood et al., 1995; Foyer et al., 2009). Nevertheless, photorespiratory processes are not completely repressed in C4 species (Yoshimura et al., 2004; Zelitch et al., 2009; Bauwe et al., 2010).

C4 plants have evolved at least 62 times independently from C3 ancestors (Sage et al., 2011), indicating that the conversion from C3 toward C4 photosynthesis did not require drastic alterations but could have been implemented rather easily in genetic terms (Sage, 2004; Brown et al., 2011; Gowik and Westhoff, 2011). It is assumed that a crucial step toward C4 photosynthesis was the establishment of a functional photorespiratory CO2 pump, which required the restriction of the Gly decarboxylase complex (GDC) to the bundle sheath cells (Bauwe and Kolukisaoglu, 2003; Sage, 2004). In C3 plants, GDC accumulates in all photosynthetically active cells. By contrast, in C4 plants, GDC occurs only in bundle sheath but not in mesophyll cells, and consequently photorespiratory activity of C4 plants is restricted to the bundle sheath. In C3-C4 intermediate species, which are considered an evolutionary link in the transition from C3 to C4 plants, GDC activity is restricted to the bundle sheath cells (Ohnishi and Kanai, 1983; Rawsthorne et al., 1988; Morgan et al., 1993; Yoshimura et al., 2004). The genus Flaveria includes C4, C3, and several C3-C4 intermediate species (Powell, 1978; McKown et al., 2005) and therefore represents a well-studied model system to study molecular mechanisms of the evolutionary transition from C3 to C4 photosynthesis (Westhoff and Gowik, 2004; Brown et al., 2005). GDC is located in the mitochondria and consists of four subunits, the L-, H-, P-, and T-proteins. Together, the four proteins cleave Gly, resulting in the release of CO2, NH3, and a tetrahydrofolate-bound C1 residue (Oliver, 1994; Douce et al., 2001). Aside from its involvement in the photorespiratory pathway, GDC also contributes to the C1 metabolism in all biosynthetic tissues that is essential for the synthesis of proteins, nucleic acids, pantothenates, and methylated molecules (Mouillon et al., 1999; Hanson and Roje, 2001).

The GLDPA gene encodes the P-subunit of GDC in the C4 plant Flaveria trinervia (Cossu and Bauwe, 1998). In situ hybridization studies showed that GLDPA transcripts accumulate only in bundle sheath cells, suggesting that GLDPA is specifically transcribed in this tissue (Engelmann et al., 2008). In agreement with this finding, the GLDPA 5′-flanking region from −1 to −1571 (with regard to the translational start site at +1; here referred to as the GLDPA promoter) directs expression of reporter genes in the bundle sheath but not in the mesophyll cells. In Flaveria bidentis (C4), the promoter is also active, although to a varying degree, in the vasculature of leaves and roots, stomata, and in the pericycle cells of roots (Figure 1). Surprisingly, the GLDPA promoter exhibited a similar activity in Arabidopsis thaliana (C3) (Engelmann et al., 2008; Figure 1), suggesting that the regulatory networks controlling bundle sheath gene expression are similar in the Brassicacean C3 species Arabidopsis and the Asteracean C4 species F. bidentis. Promoter deletion and recombination experiments identified a distal region that enhanced promoter activity and an intermediate segment that appeared to contain mesophyll-repressing sequences (Engelmann et al., 2008).

Figure 1.
Figure 1.

Fluorescence Microscopy Analysis of GLDPA Promoter Activity in Transgenic F. bidentis and Arabidopsis.

(A) Schematic presentation of the GLDPA-Ft:H2B-YFP construct that was transformed into F. bidentis or Arabidopsis to express YFP fused to histone 2B (H2B) under the control of the GLDPA promoter. The H2B-YFP fusion protein is retained in the nucleus of the expressing cell, which prevents any diffusion of the reporter protein into adjacent cells (Boisnard-Lorig et al., 2001).

(B) to (H) The localization of YFP was examined by fluorescence microscopy in longitudinal (B) and cross sections (C) of F. bidentis leaves, whole F. bidentis leaf blades in top view (D), guard cells of both the upper (E) and lower epidermis (F) of F. bidentis, and in roots of F. bidentis (G) and Arabidopsis (H). The fluorescence image is displayed underneath, and the corresponding merge of the fluorescent signal and the bright-field picture above. In the root, single endodermis (EN) and pericycle (PE) cells are indicated by an arrowhead.

In this study, we demonstrate that the functional architecture of the GLDPA promoter is much more complex than previously thought. We show that the GLDPA promoter is in fact composed of two subpromoters acting in tandem to ensure strong bundle sheath and sparse mesophyll expression of the gene. We discuss why the bundle sheath–exclusive expression of a single leaf-specific GLDP gene, as predicted and requested by the functional model of C4 photosynthesis, must allow limited expression in the mesophyll.

RESULTS

Analysis of 5′ Ends of Transcripts of the GLDPA Gene of F. trinervia Revealed Two Independent Transcription Start Sites

The 1571-bp GLDPA 5′-flanking region was subdivided into seven segments with region 1 being most distal and region 7 being most proximal to the translational start site (Engelmann et al., 2008; Figure 2A). Since the transcription start site (TSS) of the GLDPA gene of F. trinervia had not been determined experimentally, rapid amplification of 5′ cDNA ends (5′-RACE; Frohman et al., 1988) was used for mapping 5′ ends of GLDPA transcripts as present in total leaf extracts. The 5′-RACE analysis revealed two RNA 5′ end classes with one starting in the most proximal region 7 predominantly at nucleotide −100 upstream of the predicted translational start codon at +1 (ATG+1) and the other starting in the distal region 2 between nucleotides −1185 and −1174 (Figure 2A). Half of the analyzed transcripts starting from region 7 contained a 5′ untranslated region (5′ UTRR7) of 100 nucleotides. The remaining 5′ UTRR7s were slightly shorter with a length between 66 and 99 nucleotides. The UTRs of the two detected 5′ ends of RNAs transcribed from region 2 (5′ UTRR2s) included parts of region 2 and 3 but lacked regions 4, 5, 6, and 7 as well as the first 17 nucleotides of the predicted GLDPA open reading frame (Figure 2A). Fourteen individual and randomly selected 5′-RACE products were sequenced. Twelve of them started in region 7 and only two in region 2 (Figure 2A), indicating that the dominant TSS is that one located in region 7.

Figure 2.
Figure 2.

Analysis of Transcript 5′ Ends of the Endogenous GLDPA Gene of F. trinervia.

(A) The GLDPA promoter and its transcriptional output based on 5′-RACE. The dissection of the GLDPA promoter into seven regions has been described by Engelmann et al. (2008). The schematic structure of the 5′ UTRs of the two types of RNAs originating from region 2 (5′ UTRR2s) or region 7 (5′ UTRR7s) and their corresponding cDNA sequences are depicted below the schematic drawing of the GLDPA promoter. The TSSs within region 2 (TSSR2) and 7 (TSSR7) are indicated as well as the start codons used when transcription starts from region 2 (ATG+25) or region 7 (ATG+1) and the number (No.) of the 5′ UTRs detected for each 5′ UTR variant whose length is stated in nucleotides (nt).

(B) Diagram showing the read coverage of the GLDPA contig derived from 454 sequencing reads (Gowik et al., 2011). The coverage upstream of the translational start site (ATG+1) up to 100 nucleotides downstream was analyzed in 50-nucleotide windows. A contig corresponding to the 91-nucleotide spliced variant starting from TSSR2 that was detected by 5′-RACE (A) was represented by only two 454 reads. The TSSs (TSSR2 and TSSR7) and the start codons (ATG+1 and ATG+25) are marked by arrowheads. The different GLDPA promoter regions 2 to 7 shown as columns are allocated to their respective positions.

The comparison of the 5′ UTRR2s with the DNA sequence of the GLDPA promoter identified the signatures of a spliceosomal intron with two putative GT splice donor sites at −1103 and −1037 within region 3 and a shared AG splice acceptor site at +16 within the open reading frame. If splicing occurs, regardless of which donor site is used, the next available putative start codon at position +25 (ATG+25; Figure 2A) could be used, resulting in the shortening of the mitochondrial GLDPA presequence by eight amino acids.

The analysis of the leaf transcriptome of F. trinervia by 454 pyrosequencing confirmed the 5′-RACE data (Figure 2B; Gowik et al., 2011). The most distal reads detected for the GLDPA gene started exactly at position −1185 within region 2. Additionally, the 91-nucleotide 5′ UTRR2 splicing variant starting at −1185 (Figure 2A) was also found twice by 454 sequencing. In contrast with the low abundance of transcripts in the range from −1185 to −100, the frequency of mRNAs increased at or downstream of position −100 within region 7. This shows that region 7 is transcriptionally more active than region 2, which is consistent with the results obtained by 5′-RACE. We conclude that the GLDPA promoter contains two putative TSSs with the major and proximal TSS located in region 7 predominantly at position −100 (TSSR7) and the distal and minor TSS in region 2 around position −1185 (TSSR2).

The Proximal and Distal TSSs of the F. trinervia GLDPA Gene Are Functional in Transgenic F. bidentis and Arabidopsis

To test whether the two putative TSSs are used in a transgenic promoter-reporter gene context, 5′-RACE experiments were performed with transgenic F. bidentis and Arabidopsis both containing the GLDPA-Ft:β-glucuronidase (GUS) chimeric gene (see Supplemental Figure 1 online; Engelmann et al., 2008). In both F. bidentis and Arabidopsis, all RNA 5′ ends started between position −90 and −100 (i.e., in region 7). Transcripts that originated from region 2 at position −1185 were detected, despite their very low abundance. The 5′ UTRs of these mRNAs were not spliced. This is to be expected because the splice acceptor site that occurs in the GLDPA reading frame is not available due to the substitution of the GLDPA reading frame by the GUS sequence. Taken together, these findings demonstrate that regions 2 and 7 of the GLDPA promoter function as separate promoters in transgenic plants of both Arabidopsis and F. bidentis.

Both GLDPA Transit Peptide Variants Ensure Mitochondrial Import

The P-subunit of Gly decarboxylase is located in the mitochondria; hence, the GLDPA precursor protein should contain a mitochondrial targeting sequence (presequence) at its N terminus (Tanudji et al., 1999; Huang et al., 2009). Analysis of the GLDPA coding sequence by UniProtKB predicts a transit peptide of 63 amino acids, which is equivalent to 189 nucleotides of the nucleotide sequence (Cossu and Bauwe, 1998). The use of the distal promoter and the removal of the intron shift the putative translational start site to position +25 nucleotides. This would result in a presequence truncated by eight residues at its N terminus. Therefore, we investigated whether the full-size transit peptide of 63 amino acids can target the green fluorescent protein (GFP) to mitochondria and, if so, whether a deletion of the eight N-terminal residues would interfere with mitochondrial targeting. The two different GLDPA presequence variants were fused with the GFP reporter gene, and the various constructs were transiently expressed in leaf protoplasts of Nicotiana benthamiana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (Odell et al., 1985). The distribution of GFP throughout the cell was analyzed by confocal microscopy (Figure 3).

Figure 3.
Figure 3.

Localization Study of the Two Different Transit Peptide Variants of GLDPA.

The structures of the three constructs used for transient expression in leaves of N. benthamiana are diagrammed at the top. 35S:GLDPAmt-Ft-mgfp6 contains the full-length GLDPA sequence encoding the predicted presequence for mitochondrial targeting (GLDPAmt-Ft). In 35S:GLDPAmtΔ24-Ft-mgfp6, the transit peptide lacks the eight amino terminal residues. 35S:mgfp6 is devoid of any transit peptide sequence and served as a control. For visualizing mitochondria, MitoTracker staining was performed (+MT) or omitted as negative control (−MT). Three different channels were used to separate the fluorescence signals of MitoTracker-labeled mitochondria (magenta color), GFP (green color), and chlorophyll of chloroplasts (blue color) from each other. When merging MitoTracker and GFP fluorescence (M + GFP), white color indicates overlapping of both signals. All three fluorescence signals are merged in the last column (M + GFP + CP). C, chloroplasts; M, mitochondria; MT, MitoTracker; WT, wild type.

When the 35S:GLDPAmt-Ft-mgfp6 construct containing the full-length GLDPA presequence (GLDPAmt-Ft) fused to GFP was analyzed, GFP fluorescence was exclusively detected in the mitochondrial network. As expected, in the absence of any mitochondrial targeting peptide (construct 35S:mgfp6), GFP was evenly distributed throughout the cytoplasm with no visible association to any cellular organelle. When the truncated transit peptide was investigated (35S:GLDPAmtΔ24-Ft-mgfp6), the cellular pattern of GFP fluorescence was indistinguishable from that obtained with the 35S:GLDPAmt-Ft-mgfp6 construct. Therefore, the absence of the first eight amino acids from the GLDPA presequence did not affect the mitochondrial targeting of the passenger protein. We conclude that both transit peptide variants are capable of targeting the GLDPA protein into mitochondria.

Region 7 of the GLDPA Promoter Directs Bundle Sheath– and Vasculature-Specific Gene Expression in Both F. bidentis and Arabidopsis

The presence of the putative TSS identified in region 7 predominantly at position −100 (TSSR7) and in region 2 at position −1185 (TSSR2), respectively, raises the question whether regions 2 and 7 function as promoters that initiate transcription at these positions. To test the promoter function of region 7, the corresponding segment was fused to GUS (construct GLDPA-Ft-7; Figure 4A), and the expression pattern of the chimeric gene was analyzed in leaves of both transgenic F. bidentis and Arabidopsis (Figure 4).

Figure 4.
Figure 4.

Functional Analysis of Region 7 of the GLDPA Promoter in Leaves of Transgenic F. bidentis and Arabidopsis.

(A) Schematic presentation of the GLDPA-Ft-7 construct. 5′ UTRR7100, 100-bp 5′ untranslated region of GLDPA region 7.

(B) to (G) Histochemical localization of GUS activity in cross sections ([B], [C], [E], and [F]) and leaf blades in top view ([D] and [G]) of leaves of transgenic F. bidentis ([B] to [D]) and Arabidopsis ([E] to [G]). Single bundle sheath cells are indicated by arrowheads. Incubation times for the GUS staining were 17 h ([E] and [F]), 29 h (G), 43 h (B), 66 h (C), and 70.5 h (D).

(H) Fluorometrical quantification of GUS activities of transgenic F. bidentis and Arabidopsis plants transformed with the GLDPA-Ft-7 construct. Each single dot represents one independent transgenic line. The number of lines examined (n) is indicated above as well as the median of all values (x̃), also displayed as a black line in the diagram. MU, 4-methylumbelliferone.

Transgenic GLDPA-Ft-7 plants of F. bidentis exhibited GUS activity only in bundle sheath cells and vascular bundles (Figures 4B to 4D). This expression pattern was indistinguishable from that of the full-length GLDPA promoter (Figure 1; Engelmann et al., 2008). However, the promoter activity of GLDPA-Ft-7 was much lower than that of the full-length promoter (Figure 4H; Engelmann et al., 2008), indicating that other regions of the GLDPA promoter enhance the activity of region 7.

An almost identical expression pattern was observed in leaves of transgenic GLDPA-Ft-7 plants of Arabidopsis (Figures 4E to 4G). The two species differed only in the extent of GUS staining within the vasculature, which was less in the C4 plant compared with the C3 plant. As in F. bidentis, the GLDPA-Ft-7 promoter activity in Arabidopsis was very low (Figure 4H).

These findings demonstrate that region 7 is a functional promoter (PR7) that can initiate transcription on its own. Furthermore, PR7 directs gene expression specifically in bundle sheath cells and the vascular bundles like the full-length GLDPA promoter.

The 5′ UTR in PR7-Derived Transcripts Does Not Contribute to Gene Expression Specificity

RNAs transcribed from PR7 at TSSR7 contain a 100-nucleotide-long 5′ UTR (5′ UTRR7100) that is part of the subpromoter PR7 as defined above. It has been reported that the 5′ UTRs of transcripts from C4 genes can be responsible for the bundle sheath–specific accumulation of the corresponding RNAs (Patel et al., 2006). To analyze whether the 5′ UTRR7100 of the GLDPA gene contributes to or may be even responsible for the observed bundle sheath specificity of GLDPA expression, the 5′ UTRR7100 was fused to the GUS coding sequence, and the transgene was stably expressed in Arabidopsis driven by the CaMV 35S promoter (35S:GLDPA-Ft-5′UTRR7100-GUS). Transgenic Arabidopsis plants containing a 35S:GUS gene served as controls (Figure 5A).

Figure 5.
Figure 5.

Analysis of the Gene Regulatory Properties of the 100-bp 5′ Untranslated Region of GLDPA Region 7 (5′ UTRR7100) in Transgenic Arabidopsis.

(A) Schematic structure of the two constructs used for transformation. 35S:GUS consists of the CaMV 35S promoter and the GUS reporter gene, while 35S:GLDPA-Ft-5′UTRR7100-GUS additionally contains the 100-bp long 5′ UTRR7.

(B) to (I) Histochemical GUS staining of Arabidopsis transformed with 35S:GUS or 35S:GLDPA-Ft-5′UTRR7100-GUS in seedlings ([B] and [F]), young leaf blades ([C] and [G]), and cross sections of mature rosette leaves ([D], [E], [H], and [I]). Staining was for 1 h ([H] and [I]), 3 h ([C] and [G]), 4 h ([B] and [F]), or 16 h ([D] and [E]).

(J) Quantitative measurements of expression strength by analyzing GUS activities in leaf extracts of transgenic Arabidopsis plants transformed with the 35S:GUS or 35S:GLDPA-Ft-5′UTRR7100-GUS construct. Each single dot represents one independent transgenic Arabidopsis line. The number of lines examined (n) is indicated above as well as the median of all values (x̃), which is additionally charted as a black line in the diagram. The Mann-Whitney U test was used to test for significantly different GUS activities (***P < 0.001). MU, 4-methylumbelliferone.

Independently of whether the 5′ UTRR7100 was inserted between the 35S promoter and the GUS gene or not, the GUS gene was expressed in all inner tissues of mature rosette leaves as well as in cotyledons, roots, and partially in hypocotyls of young seedlings (Figures 5B to 5I). Thus, the 5′ UTRR7100 did not alter the expression pattern of the reporter gene. Transgenic F. bidentis and Arabidopsis plants harboring the construct GLDPA-Ft-7 expressed the GUS reporter gene specifically in the bundle sheath and the vasculature (Figure 4). Since the construct GLDPA-Ft-7 contains the proximal promoter and the 5′ UTRR7100, we conclude that the 5′ UTRR7100 most likely does not contribute to bundle sheath–specific expression in either Arabidopsis or F. bidentis. Transgenic Arabidopsis plants showed a sevenfold higher GUS activity in total leaf extracts when the 5′ UTRR7100 was present compared with those plants expressing the 5′ UTRR7100-less GUS variant (Figure 5J). In addition, young seedlings harboring 35S:GLDPA-Ft-5′UTRR7100-GUS exhibited much stronger GUS staining within the whole primary root than did 35S:GUS seedlings (Figures 5B and 5F).

These findings indicate that the 5′ UTRR7100 is not involved in the bundle sheath specificity of transcript accumulation. It neither destabilizes transcript accumulation in the mesophyll cells nor enhances transcript accumulation in the bundle sheath cells and the vasculature. We conclude that the bundle sheath– and vasculature-specific expression of genes driven by PR7 is regulated transcriptionally.

Region 2 Activates Gene Expression in the Leaf Chlorenchyma and Vascular Tissues in Both F. bidentis and Arabidopsis

To investigate the promoter activity contained in region 2 of the GLDPA promoter, the sequence of region 2 was fused to the GUS reporter gene and the expression pattern and strength of the resulting GLDPA-Ft-2:GUS gene (construct GLDPA-Ft-2; Figure 6A) was analyzed in transgenic F. bidentis and Arabidopsis (Figure 6). Transgenic GLDPA-Ft-2 plants of both F. bidentis and Arabidopsis showed an indistinguishable GUS expression pattern. Uniform GUS staining was detectable in all inner leaf tissues, namely, the chlorenchyma (mesophyll and bundle sheath cells) and the vascular bundles. No tissue was stained preferentially (Figures 6B to 6E). Therefore, region 2 of the GLDPA promoter is essentially a general leaf promoter (PR2) that functions in both F. bidentis and Arabidopsis with no obvious cell or tissue preference. The promoter activity of region 2 was much stronger than that of region 7 alone in both F. bidentis (~400-fold) and Arabidopsis (~1000-fold) (Figures 4H and 6F), reaching almost the promoter activity of the full-length GLDPA promoter at least in Arabidopsis (Engelmann et al., 2008).

Figure 6.
Figure 6.

Functional Analysis of Region 2 of the GLDPA Promoter in Leaves of Transgenic F. bidentis and Arabidopsis.

(A) Schematic structure of the GLDPA-Ft-2 construct. 5′ UTRR2, 5′ untranslated region of GLDPA region 2.

(B) to (E) Histochemical GUS staining in cross sections of leaves of transgenic F. bidentis or Arabidopsis harboring the GLDPA-Ft-2 construct. Incubation times for the GUS staining procedure were 1.5 h (E) and 2 h ([B] to [D]).

(F) Fluorometrical quantification of GUS activities of transgenic F. bidentis and Arabidopsis plants transformed with the GLDPA-Ft-2 construct. Each single dot represents one independent transgenic line. The number of lines examined (n) is indicated above as well as the median of all values (x̃), which is also displayed as a black line in the diagram. MU, 4-methylumbelliferone.

Region 1 Enhances the Promoter Activities of Regions 2 and 7 of the GLDPA Promoter

Regions 1 and 2 together were previously suggested to act as a general transcriptional enhancing module of the GLDPA promoter (Engelmann et al., 2008). However, our findings revealed that region 2 alone is a strong autonomous promoter. To investigate whether region 1 alone enhances transcriptional activity, it was combined with either the proximal promoter, region 7 (PR7), or the distal promoter, region 2 (PR2), fused to GUS, and analyzed in transgenic F. bidentis and Arabidopsis plants. The activity of PR7 or PR2 in the presence of region 1 (constructs GLDPA-Ft-1-7 and GLDPA-Ft-1-2) was then compared with that of the constructs GLDPA-Ft-7 and GLDPA-Ft-2, which lack region 1 (see Supplemental Figure 2 online).

In both F. bidentis and Arabidopsis, the addition of region 1 to the PR2 and PR7 promoter segments caused similar effects. In combination with PR7, region 1 enhanced promoter activity 15- to 18-fold, while the enhancing effect of region 1 on PR2 was small to moderate (approximately twofold). In both cases, the addition of region 1 did not alter the spatial expression patterns of the attached promoters. Therefore, region 1 exhibits only a quantitative enhancing effect but contains no cell or tissue specificity component.

Region 7 Represses the Promoter Activity of Region 2 of the GLDPA Promoter Stably in F. bidentis but Only Partially in Arabidopsis

Our data showed that the full-length GLDPA promoter functions essentially as a bundle sheath–and vasculature-specific promoter with minute amounts of transcripts derived from the nonspecific distal subpromoter PR2 (region 2). The question arose as to how this expression pattern could be achieved in view of the fact that the nonspecific subpromoter PR2 alone is about two to three magnitudes stronger than the specific proximal subpromoter PR7 (region 7) alone. Previous experiments had shown that a recombined promoter consisting of regions 1, 2, 3, and 7 in the order given (GLDPA-Ft-1-2-3-7) directed an expression pattern that was indistinguishable from that of the full-length promoter (Engelmann et al., 2008). A plausible hypothesis is that the activity of PR2 within the GLDPA promoter is repressed by the proximal promoter PR7 and/or region 3. To identify the component in the GLDPA promoter that suppresses its activity in the mesophyll tissue, various combinations of regions 1, 2, 3, and 7 were analyzed with regard to their expression specificities in both F. bidentis and Arabidopsis.

Transgenic F. bidentis plants expressing GLDPA-Ft-1-2-7:GUS (GLDPA-Ft-1-2-7; Figure 7A) retained the expression specificity in bundle sheath cells (Figure 7B), suggesting that, in F. bidentis, region 3 is not required for promoter specificity. As expected, omission of region 1 (GLDPA-Ft-2-7; Figure 7A) did not affect the spatial GUS staining pattern (Figure 7C). Therefore, in F. bidentis, the presence of PR7 alone suffices to repress the activity of PR2.

Figure 7.
Figure 7.

Functional Analysis of the Interactions of Regions 2 and 7 of the GLDPA Promoter in Transgenic F. bidentis.

(A) Schematic structure of the constructs GLDPA-Ft-1-2-7 and GLDPA-Ft-2-7.

(B) and (C) Histochemical GUS staining in leaf cross sections of transgenic F. bidentis transformed with either GLDPA-Ft-1-2-7 or GLDPA-Ft-2-7. Incubation times for the GUS staining procedure were 2 h (B) and 6 h (C).

By contrast, in Arabidopsis, the absence of region 3 in the promoter constructs caused a loss of bundle sheath and vasculature specificity. All transgenic Arabidopsis plants harboring GLDPA-Ft-1-2-7 showed GUS expression in all inner leaf tissues (Engelmann et al., 2008). Thus, the expression pattern resembles that of PR2 alone (Figure 6). In contrast with the stable GUS expression pattern of transgenic GLDPA-Ft-1-2-7 plants, Arabidopsis lines containing the GLDPA-Ft-2-7 construct varied in their GUS expression patterns between two extremes: bundle sheath/vasculature-specific GUS staining to an expression in all inner leaf tissues (Figure 8). In the presence of region 3 (GLDPA-Ft-2-3-7; Figure 9A), all transgenic Arabidopsis plants exhibited GUS expression in the bundle sheath and the vasculature (Figures 9C and 9G). In Arabidopsis, therefore, PR7 is not sufficient to suppress PR2 but needs region 3 for stable repression. With regard to GLDPA-Ft-1-2-7 Arabidopsis plants, the presence of region 1 enhances the activity of PR2, and the partially repressive function of PR7 is overcome.

Figure 8.
Figure 8.

Functional Analysis of the Interactions of Regions 2 and 7 of the GLDPA Promoter in Transgenic Arabidopsis.

(A) Schematic structure of the promoter-reporter gene construct GLDPA-Ft-2-7.

(B) to (K) Analysis of GUS staining patterns in leaf cross sections of five independent transgenic Arabidopsis lines ([B]/[G], [C]/[H], [D]/[I], [E]/[J], and [F]/[K]) carrying the GLDPA-Ft-2-7 construct. Incubation times for the GUS staining procedure were 3.5 h ([C], [E], [H], and [J]), 4 h ([B], [D], [F], [I], and [K]), and 5 h (G).

(L) GUS staining in leaf blades of four different GLDPA-Ft-2-7 Arabidopsis lines representative of the smooth transition of the various expression patterns detected. This transition is schematically depicted as blue bars representing the varying intensity of GUS staining of the mesophyll (MC) and the bundle sheath cells, including the vascular bundles (BS + VB). Incubation times for the GUS staining procedure were 6, 3, 3.5, and 6 h (from left to right).

Figure 9.
Figure 9.

Functional Analysis of Region 3 of the GLDPA Promoter in Transgenic Arabidopsis.

(A) Schematic structure of the constructs GLDPA-Ft-3-7, GLDPA-Ft-2-3-7, GLDPA-Ft-2-3, and GLDPA-Ft-3-2.

(B) to (I) Histochemical GUS staining in cross sections of leaves of transgenic Arabidopsis transformed with GLDPA-Ft-3-7, GLDPA-Ft-2-3-7, GLDPA-Ft-2-3, or GLDPA-Ft-3-2. Incubation times for GUS staining were 0.5 h ([D] and [H]), 1 h (E), 2.5 h (I), 3.5 h ([C] and [G]), 5 h (B), or 6 h (F).

(J) Fluorometrical measurement of GUS activities in transgenic Arabidopsis transformed with GLDPA-Ft-3-7, GLDPA-Ft-2-3-7, GLDPA-Ft-2-3, or GLDPA-Ft-3-2. Each single dot represents one independent transgenic line. The number of plants analyzed (n) is indicated at the top of each diagram as well as the median values (x̃), which are also added as black lines in the diagrams. The Mann-Whitney U test was used to test for significantly different GUS activities (***P < 0.001; **P < 0.01). MU, 4-methylumbelliferone.

In Arabidopsis, Region 3 Cannot Maintain Bundle Sheath Specificity on Its Own and Needs the Presence of PR7

Since region 3 of the GLDPA promoter is absolutely required for the suppression of PR2 activity in Arabidopsis, the question arose whether in Arabidopsis PR7 also is required for region 3 to be functional. In the natural context of the GLDPA promoter, region 3 is located 3′ to the subpromoter PR2 followed further downstream by PR7. Therefore, we wanted to know whether the position of region 3 with respect to PR2 is important for its suppressing activity and whether region 3 can also influence the activity of PR7.

To investigate whether region 3 alone could repress PR2 activity, it was fused downstream (GLDPA-Ft-2-3) as well as upstream (GLDPA-Ft-3-2) of PR2 (Figure 9A). In both cases, all transgenic Arabidopsis plants exhibited the same uniform GUS expression pattern in the chlorenchyma and vasculature as detected for PR2 alone (Figures 9D, 9E, 9H, and 9I). By contrast, the combination of PR2, region 3, and PR7 (GLDPA-Ft-2-3-7; Figure 9A) caused specific GUS expression in the bundle sheath and the vasculature (Figures 9C and 9G). Therefore, region 3 alone is not sufficient to suppress PR2, independent of its location down- or upstream of PR2, but the presence of PR7, in addition, is necessary.

To examine whether region 3 could affect also PR7 activity, the corresponding transgene (GLDPA-Ft-3-7; Figure 9A) was constructed and analyzed in transgenic Arabidopsis. As expected, the combination of region 3 and PR7 led to the same bundle sheath– and vasculature-specific expression pattern as PR7 alone (Figures 9B and 9F), indicating that region 3 has no influence on the spatial activity of PR7.

However, the GLDPA-Ft-3-7 construct was 20-fold more active than GLDPA-Ft-7 (Figures 4H and 9J). Interestingly, region 3 was also able to enhance PR2 activity (cf. constructs GLDPA-Ft-3-2 [Figure 9J] and GLDPA-Ft-2 [Figure 6F]), although to a much less degree than observed for PR7 (twofold versus 20-fold). Thus, region 3 can enhance transcription of PR7 and PR2 with a comparable strength as detected for region 1.

DISCUSSION

The commonly believed evolutionary scenario of C4 photosynthesis predicts that relatively early along the path toward C4 photosynthesis, a photorespiratory CO2 pump was established by compartmentalization of Gly decarboxylase activity in the bundle sheath (Sage, 2004; Bauwe, 2011). All available experimental data confirm the final outcome of this evolutionary process, namely, that in present C4 species, Gly decarboxylase accumulates predominantly in the bundle sheath (Li et al., 2010; Majeran et al., 2010). Along these lines, the activity of the GLDPA promoter of the Asteracean C4 species F. trinervia was found to be restricted to the bundle sheath cells and vasculature in leaves of both transgenic F. bidentis (C4) and Arabidopsis (C3) (Engelmann et al., 2008). This suggested that the bundle sheath–specific accumulation of GLDPA mRNAs should be essentially regulated by transcription (Engelmann et al., 2008). Data presented in this article show that GLDPA gene regulation is much more complex than previously thought. We discuss and provide evidence that the mRNA output of this gene is determined by a combination of transcriptional and posttranscriptional means.

The GLDPA Promoter Consists of Two Promoters in Tandem

The GLDPA promoter is composed of tandem promoters, PR2 and PR7, that together ensure a strong bundle sheath expression. The two subpromoters are not easily recognized by inspection of their corresponding nucleotide sequences. No reliable candidates for TATA boxes can be detected in the predicted distance of 25 to 40 bp upstream of the two transcriptional initiation sites (Joshi, 1987; Bernard et al., 2010; Zuo and Li, 2011). This is not surprising since, for instance in Arabidopsis, only 20 to 30% of all promoters contain a TATA box/variant (Molina and Grotewold, 2005; Yamamoto et al., 2009; Bernard et al., 2010). Recently, TC elements have been proposed as a novel class of regulatory elements that control transcription in plants (Bernard et al., 2010). Indeed, several TC elements are located around TSSR2 but predominantly around TSSR7 within the predicted range of 50 bp up- and/or downstream of the corresponding TSS (see Supplemental Figure 3 online; Zuo and Li, 2011). The possible importance of TC elements for transcriptional regulation of PR7 is supported by the fact that the motifs CCCTTT, CCTTCT, and TCTTCT are unique to region 7 within the GLDPA promoter and that TCTTCT is one of the three TC element types most frequently observed (Bernard et al., 2010). TSSR2 is flanked by a sequence repeat that is very similar to the predicted Initiator (Inr) motif shown to be essential for the light-dependent activity of the psaDb promoter from Nicotiana sylvestris (Nakamura et al., 2002; see Supplemental Figure 3 online). According to the YR rule (YR, Y = C or T, R = A or G, TSS underlined), most of the Arabidopsis promoters contain the CA dimer sequence around their TSSs (Yamamoto et al., 2007), which is also true for TSSR2. Yamamoto et al. (2007) consider this YR rule to represent a less stringent form of Inr. This indicates that Inr elements might be crucial for transcriptional activity of PR2.

The GLDPA Subpromoters Diverge in Their Specificities

When analyzed separately, the two subpromoters diverge in their spatial expression profiles. The proximal promoter (PR7) alone, defined by region 7, is relatively weak, and, as observed for the full-length GLDPA promoter, specifically active in the bundle sheath and the vasculature (Figure 4). The distal promoter (PR2) alone, defined by region 2, is strong and drives expression in all inner leaf cells, including the mesophyll (Figure 6). By contrast, in the context of the full-length GLDPA promoter, which exhibits strong specific activity, the final RNA output from both promoters, as measured by 5′-RACE and RNA sequencing experiments, is just the reverse. RNAs transcribed from the proximal promoter dominate the GLDPA transcript population, and RNAs derived from PR2 are in the minority (Figure 2). We could show that the activity of PR7 is enhanced in the presence of regions 1 and 3 of the GLDPA promoter, which might explain the high accumulation of PR7-derived transcripts. When the two subpromoters (i.e., regions 2 and 7) are combined and fused to the GUS reporter gene, the readout of this chimeric gene in F. bidentis is similar to that of the complete GLDPA promoter (cf. Figures 1 and 7). This finding suggests that the proximal bundle sheath–specific promoter PR7 turns off the activity of the unspecific distal promoter PR2. How could a downstream promoter interfere with the activity of an upstream promoter and even disable it?

That a strong upstream promoter can shut off the activity of a downstream promoter is well documented (Mazo et al., 2007). This phenomenon is called transcriptional interference and may be defined as “the in cis suppression of one transcriptional process by another” (Palmer et al., 2011). Transcriptional interference has been documented as a general regulatory process affecting the transcription from adjacent convergent or tandem promoters (Palmer et al., 2011). Promoters can impede or even block one another by different ways. One possibility is the transcription from a strong regulatory promoter that might impair the recruitment of the transcription initiation complex or the transcriptional elongation of a neighboring target gene (Mazo et al., 2007; Palmer et al., 2011). Transcriptional interference has been reported for plants. The strong 35S promoter of a T-DNA inserted upstream of the Arabidopsis RibA1 gene results in large transcripts that run over the RibA1 promoter and thereby inhibit RibA1 transcription (Hedtke and Grimm, 2009).

In all known cases of transcriptional interference occurring with tandem promoters, the upstream promoter blocks the activity of the downstream promoter. However, with respect to the GLDPA promoter, just the opposite is true: The downstream promoter PR7 inhibits the output from the upstream promoter PR2. Could a roadblock mechanism explain the transcriptional interference between the GLDPA subpromoters (i.e., a pausing RNA polymerase II) (Levine, 2011) or a scaffold of general transcription factors (Yudkovsky et al., 2000) residing at the downstream promoter inhibit the progress of RNA polymerase II from the upstream promoter? There is increasing evidence that RNA polymerase II pauses quite often after having initiated transcription and having produced a nascent transcript of ~30 to 50 nucleotides (Levine, 2011). Therefore, it is conceivable that an RNA polymerase II pausing at PR7 might represent a roadblock that impedes the elongation of transcripts originating from PR2 and is involved in the suppression of the PR2 output.

The GLDPA 5′ Flanking Region: A Player in Posttranscriptional Control

One hallmark of the GLDPA gene of F. trinervia is an intron located within the 5′ UTR of PR2-derived transcripts. Depending on the splice donor site used, the intron commences 84 or 139 nucleotides behind the respective TSS of the distal promoter PR2 within region 3 and ends 17 nucleotides behind the first nucleotide of the GLDPA reading frame (Figure 2). RNA sequencing experiments using the 454 technology (Gowik et al., 2011) revealed that this 5′ intron is present in GLDPA transcripts, and the sequence reads cover the intron region uniformly (see Supplemental Figure 4 online). By contrast, sequence reads from the gene-internal introns are not detectable. The accumulation of unspliced PR2-derived transcripts with respect to their 5′ UTR demonstrates that the splicing efficiency of the 5′ intron is drastically lower than that of the gene-internal ones.

To prevent the accumulation of aberrant mRNAs (i.e., mRNAs that are erroneously or not completely spliced), eukaryotes have developed various quality control systems (Egecioglu and Chanfreau, 2011). Spliceosomal DExD/H box ATPases provide the first layer of defense. They act as kinetic proofreading systems and limit the escape of unspliced or erroneously spliced RNAs from the spliceosome (Egecioglu and Chanfreau, 2011). Despite the accuracy of these proofreading activities, unspliced mRNAs may escape detection; therefore, external quality control systems have been built up. The nuclear exosome takes part in the degradation of unspliced RNAs (Houseley et al., 2006; Fasken and Corbett, 2009), although the molecular mechanisms by which unspliced RNAs are recognized are not yet clear. Exon-junction complexes that are deposited on spliced RNAs might be involved in the recognition mechanism (Egecioglu and Chanfreau, 2011). These protein complexes also play a prominent role in nonsense-mediated mRNA decay. Nonsense-mediated mRNA decay is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs containing premature termination codons (Chang et al., 2007; Brogna and Wen, 2009). In plants, long 3′ UTRs, introns that are located in 3′ UTRs, and upstream open reading frames (uORFs) within the 5′ UTR can trigger nonsense-mediated mRNA decay (Kertész et al., 2006; Hori and Watanabe, 2007; Nyikó et al., 2009).

The 5′ intron sequence that is present in unspliced transcripts derived from PR2 contains several uORFs. The one that starts directly upstream of TSSR7 encodes more than 35 amino acids (see Supplemental Figure 5 online). uORFs are considered to have the potential to elicit the nonsense-mediated mRNA decay response when they give rise to proteins that are larger than the critical threshold of 35 amino acids (Nyikó et al., 2009). When PR2 is combined with PR7 (GLDPA-Ft-2-7), a 135-nucleotide uORF commencing directly upstream of TSSR7 might encode a protein of 45 amino acids that should promote nonsense-mediated mRNA decay (see Supplemental Figure 6 online). This 135-nucleotide uORF is present only in transcripts originating from PR2. The 5′-RACE analyses of transgenic GLDPA-Ft-2-7 plants of Arabidopsis and F. bidentis showed that transcription started from either TSSR2 (PR2) or TSSR7 (PR7). While PR7-derived RNAs exhibited more or less the same length, PR2-derived transcripts appeared to be destabilized because many RNAs started randomly between TSSR2 and TSSR7, indicating RNA degradation (see Supplemental Figure 6 online). Taken together, the presented data demonstrate that the mRNA output of the GLDPA gene of F. trinervia is regulated by an intricate interplay of transcriptional and posttranscriptional mechanisms.

Evolution of the GLDPA Promoter: The Necessity of Being Bundle Sheath Specific, but Not Completely

The presence of the 5′ intron in PR2-derived transcripts and of an alternative ATG codon 25 nucleotides behind the major translational start site results in a GLDPA protein variant whose mitochondrial targeting peptide is truncated by eight amino acids. Our import experiments showed that, nevertheless, the truncated transit peptide is capable of directing an attached passenger protein to the mitochondria (Figure 3). This shows that PR2- and PR7-derived mRNAs yield a GLDPA protein that accumulates in the mitochondria. One wonders why the GLDPA promoter contains one subpromoter with the desired specificity in the bundle sheath and a second subpromoter that is active in all internal leaf cells, including the mesophyll. Moreover, the second, nonspecific promoter is not allowed to express its full potential but is almost, even though not completely, switched off by a combination of transcriptional and posttranscriptional means.

Mutational analysis with Arabidopsis revealed that a GLDP double mutant in which both of the two GLDP genes were knocked out is lethal, even under nonphotorespiratory conditions (Engel et al., 2007). This indicates that the activity of GDC is indispensable and that all biosynthetically active cells need Gly decarboxylase activity for one-carbon metabolism (Hanson and Roje, 2001). Although GLDPA transcripts could not be observed in mesophyll cells by in situ hybridization (Engelmann et al., 2008), more sensitive methods like laser microdissection followed by RNA sequencing or immunogold labeling allow the detection of traces of GLDP transcripts and GLDP protein in mesophyll cells of C4 and C3-C4 intermediate plants (Rawsthorne et al., 1988; Yoshimura et al., 2004; Li et al., 2010). According to 454 pyrosequencing data, in C4 Flaveria species, the GLDPA gene is the only active leaf GLDP gene (Gowik et al., 2011). Therefore, we hypothesize that the GLDPA gene of F. trinervia (C4) must fulfill two purposes: First, it has to serve the requirements of the photorespiratory pathway, and its activity should therefore be restricted to the bundle sheath cells; second, small amounts of GDC activity are needed in all biosynthetically active cells, and in the mesophyll cells; therefore, the regulatory system of the GLDPA gene has to be somewhat leaky. This is achieved by a complex interplay of transcriptional and posttranscriptional mechanisms (Figure 10). The GLDPA promoter is in fact composed of two subpromoters acting in tandem. The proximal promoter PR7 is weak but suffices for bundle sheath– and vasculature-specific gene expression. To ensure strong transcriptional activity of PR7, it is enhanced by regions 1 and 3. The strong distal promoter PR2 is active in the vasculature and all chlorenchyma tissues of the leaf, including the mesophyll. Its activity is also raised in the presence of regions 1 and 3. However, PR7 suppresses the activity of PR2 apparently posttranscriptionally by destabilizing PR2-derived transcripts when they contain an intron, including the sequence of region 7, within their 5′ UTRs. Thus, the removal of this intron is essential for generating GLDPA protein. Inefficient splicing in the 5′ UTR drastically reduces the amounts of GLDPA transcripts starting from PR2.

Figure 10.
Figure 10.

The Expression of the GLDPA Gene of F. trinervia Is Regulated by an Intricate Interplay of Transcriptional and Posttranscriptional Mechanisms.

The proximal promoter PR7 is sufficient to confer expression specifically in bundle sheath cells and the vascular bundles of leaves but can be effectively enhanced by regions 1 and 3. Transcripts generated at TSSR7 are presumably stabilized by their 5′ UTR (5′ UTRR7), finally resulting in the accumulation of GLDPA protein in the distinct cell types to contribute to photorespiration. The activity of the distal promoter PR2 in all inner leaf tissues is also enhanced by regions 1 and 3. Transcripts from TSSR2 are supposed to be destabilized when they contain the sequence of region 7, which impedes RNA accumulation. The problem of RNA instability can be overcome by splicing out impairing elements, assuring at least small amounts of stable GLDPA transcript, and, thus, GLDPA protein, additionally in the mesophyll cells to serve the C1 metabolic pathway.

We provided conclusive evidence that an intricate combination of transcriptional and posttranscriptional regulation ensures small amounts of GLDPA mRNAs accumulate in the mesophyll cells of C4 Flaveria species. It remains to be investigated how this pattern of gene expression regulation evolved in the genus Flaveria. These studies are underway and should elucidate the adaptive changes in gene expression that are a central component of C4 evolution.

METHODS

Generation of Chimeric Promoters and Cloning of Promoter-Reporter Gene Constructs

The DNA amplification and cloning procedures were accomplished according to Sambrook and Russell (2001). The dissection of the GLDPA promoter from Flaveria trinervia into seven regions and the cloning of the constructs GLDPA-Ft-7 (previously referred to as GLDPA-Ft-Δ6), GLDPA-Ft-1-2-7, and GLDPA-Ft:H2B-yellow fluorescent protein (YFP) (previously referred to as GLDPA-Ft::H2B:YFP) have been described by Engelmann et al. (2008). All GLDPA promoter regions were amplified by PCR by means of the Phusion High-Fidelity DNA Polymerase (New England Biolabs) or the Pfu DNA Polymerase (Stratagene) using the GLDPA-Ft construct (Cossu, 1997; Engelmann et al., 2008) as template and the corresponding oligonucleotides containing the respective restriction sites (see Supplemental Tables 1 and 2 online). For GLDPA-Ft-2-3-7, region 2-3 as XbaI-BcuI and 7 as a BcuI-XmaI fragment were cloned together into the XbaI-XmaI–digested binary plant transformation vector pBI121 (Clontech Laboratories; Jefferson et al., 1987; Chen et al., 2003) lacking the CaMV 35S promoter. GLDPA-Ft-2-7 and GLDPA-Ft-3-7 were constructed by exchanging region 2-3 of GLDPA-Ft-2-3-7 with regions 2 and 3 as XbaI-BcuI fragments, respectively. For cloning of GLDPA-Ft-2 and GLDPA-Ft-2-3, regions 2 and 2-3 were ligated as XbaI-XmaI fragments into the XbaI-XmaI–digested pBI121 vector lacking the 35S promoter. Region 3 was cloned as an XbaI-XbaI fragment into GLDPA-Ft-2 previously cut with XbaI to generate GLDPA-Ft-3-2. The correct orientation of region 3 was confirmed by DNA sequencing. 35S:GUS was constructed by inserting the 35S promoter amplified from pBI121-35S:H2B:YFP (Boisnard-Lorig et al., 2001) as a HindIII-XbaI-XmaI fragment into the HindIII-XmaI–digested pBI121 vector, whereas for 35S:GLDPA-Ft-5′UTRR7100-GUS, the 35S promoter was directly excised from pBI121-35S:H2B:YFP as a HindIII-XbaI fragment to ligate it together with the XbaI-XmaI–digested 100-bp 5′ UTR of region 7 (5′ UTRR7100) into the HindIII-XmaI–restricted pBI121 vector. For 35S:mgfp6, 35S:GLDPAmt-Ft-mgfp6, and 35S:GLDPAmtΔ24-Ft-mgfp6, Gateway Technology (Invitrogen) was applied starting with the generation of Gateway-compatible recombination fragments by PCR. Regarding 35S:mgfp6, the primers ATG-5′-attB1 and ATG-3′-attB2 (see Supplemental Table 1 online) were used for simple primer dimer formation, elongation, and amplification generating a start codon (ATG) with flanking attB sites, whereas for 35S:GLDPAmt-Ft-mgfp6 and 35S:GLDPAmtΔ24-Ft-mgfp6, F. trinervia cDNA was used for the amplification of the full-length N-terminal presequence of the GLDPA gene (GLDPAmt, 189 bp; primers: GLDPAmt-5′-attB1/GLDPAmt-3′-attB2 and attB1/attB2 adapter) as annotated by GenBank (accession number Z99767) or a shorter version lacking the first 24 bp (GLDPAmtΔ24, 165 bp; primers: GLDPAmtΔ24-5′-attB1/GLDPAmt-3′-attB2 and attB1/attB2 adapter). The attB-flanked PCR products were recombined into pDONRTM221 (Invitrogen) and afterwards into pMDC83 (Curtis and Grossniklaus, 2003). All generated constructs were verified by DNA sequencing.

Transformation of Arabidopsis thaliana and Flaveria bidentis

All chimeric promoter-reporter gene constructs were transformed into either the Agrobacterium tumefaciens strain AGL1 (Lazo et al., 1991) or GV3101 (pMP90) (Koncz and Schell, 1986) via electroporation. Transgenic F. bidentis was generated according to Chitty et al. (1994) by means of Agrobacterium AGL1 containing the respective construct. Arabidopsis (ecotype Columbia) was transformed by the floral dip method (Clough and Bent, 1998) modified according to Logemann et al. (2006) using the Agrobacterium strain GV3101 harboring the appropriate construct. The presence of the respective transgene within the genome of each single independent F. bidentis T0 and Arabidopsis T1 line was verified by PCR after DNA isolation as described by Edwards et al. (1991).

In Situ Analysis of GUS and Detection of Its Activity

The fifth leaf from the top of 40- to 50-cm-tall transgenic F. bidentis T0 plants or three mature rosette leaves of 3- to 4-week-old transgenic Arabidopsis T1 plants prior to flowering were used for the fluorometrical quantification of GUS activity according to Jefferson et al. (1987) and Kosugi et al. (1990). The statistical significance of the difference between two data sets was analyzed by means of the Mann-Whitney U test (http://elegans.som.vcu.edu/~leon/stats/utest.html). Histochemical GUS analyses were performed as described by Engelmann et al. (2008). The fifth leaf from the top of transgenic F. bidentis T0 plants (40 to 50 cm) and single rosette leaves as whole blades or manually cut cross sections as well as young seedlings of transgenic T1 Arabidopsis plants were used for the histochemical GUS analysis in situ, respectively.

Transient Gene Expression in Leaves of Nicotiana benthamiana and Isolation of Protoplasts

The Agrobacterium-mediated infiltration of leaves of N. benthamiana was performed according to Waadt and Kudla (2008) by means of Agrobacterium GV3101 (pMP90) containing the respective construct and the Agrobacterium strain p19 for suppression of gene silencing (Voinnet et al., 2003). After 4 d, two infiltrated leaves per plant were harvested for protoplast isolation. Four leaf pieces (~0.7 × 0.7 cm each) per blade were cut out with a razor blade, transferred into 5 mL of enzyme solution (Yoo et al., 2007), vacuum-infiltrated three times for 30 s, and then incubated at room temperature for 2 h. After light shaking to release protoplasts, remaining leaf pieces were removed, and MitoTracker Orange CMTMRos (Invitrogen) was added to a final concentration of 150 nM to the suspension of protoplasts for labeling mitochondria. After incubation for 15 min at 37°C, protoplasts were centrifuged at 500g for 1 min, the supernatant was removed, and the sedimented leaf cells were resuspended in 100 μL W5 solution (Yoo et al., 2007) for analysis by confocal microscopy.

Confocal Laser Scanning Microscopy and Fluorescence Microscopy

The analysis of protoplasts by confocal laser scanning microscopy was performed with the LSM 510 (Carl Zeiss). Protoplasts were excited at 488 nm (for detection of GFP and chlorophyll fluorescence) and at 561 nm (for detection of MitoTracker fluorescence), respectively. To visualize specifically GFP fluorescence, a 505- to 550-nm band-pass emission filter was used. The fluorescence of MitoTracker Orange CMTMRos-labeled mitochondria was observed using a 575- to 615-nm band-pass filter, and the autofluorescence of chlorophyll was recorded by means of a 650-nm long-pass filter.

Leaf cross sections, complete leaf blades, and roots of transgenic F. bidentis as well as roots of transgenic Arabidopsis carrying the GLDPA-Ft:H2B-YFP chimeric gene were analyzed with the aid of an Axiophot fluorescence microscope (Carl Zeiss) that was equipped with an integrated HBO-UV lamp (Carl Zeiss) and a DP50-CU camera (Olympus Optical) using the filter set F41-028 (excitation, HQ 500/20; beam splitter, Q 515 LP; emission, HQ 535/30; AHF Analysentechnik). Bright-field and fluorescence pictures were merged with Adobe Photoshop 7.0 (Adobe Systems). Prior to fluorescence microscopy, leaf blades were extracted with 95% ethanol according to Zhou et al. (2005). Leaf blades were harvested from 30-cm-tall F. bidentis grown in the greenhouse. Roots of young seedlings of Arabidopsis and F. bidentis were taken from plants cultivated on agar medium in a climate chamber.

Analysis of mRNA 5′ Ends by 5′-RACE and 454 Sequencing

Total RNA from leaves of F. trinervia was isolated according to Westhoff et al. (1991). After enrichment by the Oligotex mRNA Midi Kit (Qiagen) 0.5 μg of poly(A+) mRNA was used for cDNA first-strand synthesis performed with the SMART RACE cDNA amplification kit (Clontech Laboratories) and PowerScript reverse transcriptase (Clontech Laboratories) according to the manufacturers’ protocols. For PCR amplification of 5′ UTRs with the Phusion High-Fidelity DNA polymerase (New England Biolabs), the gene-specific 3′ oligonucleotide GLDPA-RACE4 (5′-GAGATCTTGGACTTGTACTGTC-3′) and the SMART-II-A-Primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) were used. The PCR fragment was cloned subsequently using the CloneJET PCR cloning kit (Fermentas). Sixty independent clones were analyzed by colony PCR using the SMART-II-A-Primer and the gene-specific GLDPA-RACE6 oligonucleotide (5′-ACACCGTACATAGCAGCCATG-3′). These PCR products were verified by restriction endonuclease analyses, leading to the identification of 51 potentially correct clones. Plasmid DNA was isolated from 14 of them for DNA sequencing.

The generation of 454 reads was described by Gowik et al. (2011). The F. trinervia reads were mapped on the GLDPA gene sequence with the CLC Genomic Workbench (version 4.8). CAP3 (Huang and Madan, 1999) was used for the de novo assembly of the F. trinervia 454 reads. The GLDPA contig was identified with BLAST (Altschul et al., 1997), and the read coverage was determined in 50-nucleotide windows.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number Z99767 (F. trinervia GLDPA).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Analysis of mRNA 5′ Ends in Leaves of Transgenic Arabidopsis and F. bidentis Harboring the GLDPA-Ft:GUS Transgene.

  • Supplemental Figure 2. Functional Analysis of Region 1 of the GLDPA Promoter in Transgenic Arabidopsis and F. bidentis.

  • Supplemental Figure 3. Distribution of TC-Rich Elements and Initiator-Like Motifs within Region 2 and Region 7 of the GLDPA Promoter.

  • Supplemental Figure 4. Splicing Pattern of the GLDPA Transcript Analyzed by 454 Sequencing.

  • Supplemental Figure 5. Distribution of Upstream Open Reading Frames in PR2-Derived Transcripts.

  • Supplemental Figure 6. Analysis of mRNA 5′ Ends in Leaves of Transgenic Arabidopsis and F. bidentis Harboring GLDPA-Ft-2-7.

  • Supplemental Table 1. Oligonucleotides Used for the Amplification of the Different GLDPA-Ft Promoter Regions or the GLDPA-Ft Presequence.

  • Supplemental Table 2. Oligonucleotide Combinations for the Amplification of the Appropriate Promoter Parts or Gateway-Compatible Fragments.

Acknowledgments

This work was supported by the “Sonderforschungsbereich 590–Inhärente und adaptive Differenzierungsprozesse” and the “Forschergruppe 1186” funded by the Deutsche Forschungsgemeinshaft.

AUTHOR CONTRIBUTIONS

C.W., S.S., U.G., H.B., and P.W. designed the research. C.W., S.S., U.G., S.E., M.K., and M.S. performed the research. C.W., S.S., U.G., and P.W. analyzed data. C.W., U.G., and P.W. wrote the article.

Footnotes

  • Received November 16, 2011.
  • Revised December 23, 2011.
  • Accepted January 12, 2012.
  • Published January 31, 2012.

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Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes
Christian Wiludda, Stefanie Schulze, Udo Gowik, Sascha Engelmann, Maria Koczor, Monika Streubel, Hermann Bauwe, Peter Westhoff
The Plant Cell Jan 2012, 24 (1) 137-151; DOI: 10.1105/tpc.111.093872
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