ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulate PEPC1 cell-specific gene expression in maize.

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C4 photosynthesis and depends on the cell-specific accumulation of major C4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyll-specific gene expression in maize, has been shown to keep the same properties when introduced into rice (C3 plant), indicating that rice has the transcription factors (TFs) needed to confer C4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C4 regulators were co-opted from C3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo- and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.

Depletion of the "gamma-type carbonic anhydrase-like" subunits of complex I affects central mitochondrial metabolism in Arabidopsis thaliana.

"Gamma-type carbonic anhydrase-like" (CAL) proteins form part of complex I in plants. Together with "gamma carbonic anhydrase" (CA) proteins they form an extra domain which is attached to the membrane arm of complex I on its matrix exposed side. In Arabidopsis two CAL and three CA proteins are present, termed CAL1, CAL2, CA1, CA2 and CA3. It has been proposed that the carbonic anhydrase domain of complex I is involved in a process mediating efficient recycling of mitochondrial CO2 for photosynthetic carbon fixation which is especially important during growth conditions causing increased photorespiration. Depletion of CAL proteins has been shown to significantly affect plant development and photomorphogenesis. To better understand CAL function in plants we here investigated effects of CAL depletion on the mitochondrial compartment. In mutant lines and cell cultures complex I amount was reduced by 90-95% but levels of complexes III and V were unchanged. At the same time, some of the CA transcripts were less abundant. Proteome analysis of CAL depleted cells revealed significant reduction of complex I subunits as well as proteins associated with photorespiration, but increased amounts of proteins participating in amino acid catabolism and stress response reactions. Developmental delay of the mutants was slightly alleviated if plants were cultivated at high CO2. Profiling of selected metabolites revealed defined changes in intermediates of the citric acid cycle and amino acid catabolism. It is concluded that CAL proteins are essential for complex I assembly and that CAL depletion specifically affects central mitochondrial metabolism.

The 5'UTR Intron of Arabidopsis GGT1 Aminotransferase Enhances Promoter Activity by Recruiting RNA Polymerase II.

Photorespiration is essential for the detoxification of glycolate and recycling of carbon to the Calvin Benson Bassham cycle. Enzymes participating in the pathway have been identified, and investigations now focus on the regulation of photorespiration by transporters and metabolites. However, regulation of photorespiration on the gene level has not been intensively studied. Here, we show that maximum transcript abundance of Glu:glyoxylate aminotransferase 1 (GGT1) is regulated by intron-mediated enhancement (IME) of the 5' leader intron rather than by regulatory elements in the 5' upstream region. The intron is rich in CT-stretches and contains the motif TGTGATTTG that is highly similar to the IME-related motif TTNGATYTG. The GGT1 intron also confers leaf-specific expression of foreign promoters. Quantitative PCR analysis and GUS activity measurements revealed that IME of the GGT1 5'UTR intron is controlled on the transcriptional level. IME by the GGT1 5'UTR intron was at least 2-fold. Chromatin immunoprecipitation experiments showed that the abundance of RNA polymerase II binding to the intron-less construct is reduced.

A simple mechanism for the establishment of C2-specific gene expression in Brassicaceae.

The transition of C3 , via C2 towards C4 photosynthesis is an important example of stepwise evolution of a complex genetic trait. A common feature that was gradually emphasized during this trajectory is the evolution of a CO2 concentration mechanism around Rubisco. In C2 plants, this mechanism is based on tissue-specific accumulation of glycine decarboxylase (GDC) in bundle sheath (BS) cells, relative to global expression in the cells of C3 leaves. This limits photorespiratory CO2 release to BS cells. Because BS cells are surrounded by photosynthetically active mesophyll cells, this arrangement enhances the probability of re-fixation of CO2 . The restriction of GDC to BS cells was mainly achieved by confinement of its P-subunit (GLDP). Here, we provide a mechanism for the establishment of C2 -type gene expression by studying the upstream sequences of C3 Gldp genes in Arabidopsis thaliana. Deletion of 59 bp in the upstream region of AtGldp1 restricted expression of a reporter gene to BS cells and the vasculature without affecting diurnal variation. This region was named the 'M box'. Similar results were obtained for the AtGldp2 gene. Fusion of the M box to endogenous or exogenous promoters supported mesophyll expression. Nucleosome densities at the M box were low, suggesting an open chromatin structure facilitating transcription factor binding. In silico analysis defined a possible consensus for the element that was conserved across the Brassicaceae, but not in Moricandia nitens, a C2 plant. Collective results provide evidence that a simple mutation is sufficient for establishment of C2 -specific gene expression in a C3 plant.

Mitochondrial gamma carbonic anhydrases are required for complex I assembly and plant reproductive development.

Complex I of the mitochondrial electron transport chain (mETC) in plants contains an extra domain that is made up from proteins homologous to prokaryotic gamma-carbonic anhydrases (γCA). This domain has been suggested to participate in complex I assembly or to support transport of mitochondrial CO2 to the chloroplast. Here, we generated mutants lacking CA1 and CA2 - two out of three CA proteins in Arabidopsis thaliana. Double mutants were characterized at the developmental and physiological levels. Furthermore, the composition and activity of the mETC were determined, and mutated CA versions were used for complementation assays. Embryo development of double mutants was strongly delayed and seed development stopped before maturation. Mutant plants could only be rescued on sucrose media, showed severe stress symptoms and never produced viable seeds. By contrast, callus cultures were only slightly affected in growth. Complex I was undetectable in the double mutants, but complex II and complex IV were upregulated concomitant with increased oxygen consumption in mitochondrial respiration. Ectopic expression of inactive CA variants was sufficient to complement the mutant phenotype. Data indicate that CA proteins are structurally required for complex I assembly and that reproductive development is dependent on the presence of complex I.

Photosynthetic Genes and Genes Associated with the C4 Trait in Maize Are Characterized by a Unique Class of Highly Regulated Histone Acetylation Peaks on Upstream Promoters.

Histone modifications contribute to gene regulation in eukaryotes. We analyzed genome-wide histone H3 Lysine (Lys) 4 trimethylation and histone H3 Lys 9 acetylation (two modifications typically associated with active genes) in meristematic cells at the base and expanded cells in the blade of the maize (Zea mays) leaf. These data were compared with transcript levels of associated genes. For individual genes, regulations (fold changes) of histone modifications and transcript levels were much better correlated than absolute intensities. When focusing on regulated histone modification sites, we identified highly regulated secondary H3 Lys 9 acetylation peaks on upstream promoters (regulated secondary upstream peaks [R-SUPs]) on 10% of all genes. R-SUPs were more often found on genes that were up-regulated toward the blade than on down-regulated genes and specifically, photosynthetic genes. Among those genes, we identified six genes encoding enzymes of the C4 cycle and a significant enrichment of genes associated with the C4 trait derived from transcriptomic studies. On the DNA level, R-SUPs are frequently associated with ethylene-responsive elements. Based on these data, we suggest coevolution of epigenetic promoter elements during the establishment of C4 photosynthesis.

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex.

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'.

The carbonic anhydrase domain of plant mitochondrial complex I.

The mitochondrial NADH dehydrogenase complex (complex I) consists of several functional domains which independently arose during evolution. In higher plants, it contains an additional domain which includes proteins resembling gamma-type carbonic anhydrases. The Arabidopsis genome codes for five complex I-integrated gamma-type carbonic anhydrases (γCA1, γCA2, γCA3, γCAL1, γCAL2), but only three copies of this group of proteins form an individual extra domain. Biochemical analyses revealed that the domain is composed of one copy of either γCAL1 or γCAL2 plus two copies of the γCA1/γCA2 proteins. Thus, the carbonic anhydrase domain can have six distinct subunit configurations. Single and double mutants with respect to the γCA/γCAL proteins were employed to genetically dissect the function of the domain. New insights into complex I biology in plants will be reviewed and discussed.

The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield.

We have increased the productivity and yield of potato (Solanum tuberosum) by developing a novel method to enhance photosynthetic carbon fixation based on expression of a polyprotein (DEFp) comprising all three subunits (D, E and F) of Escherichia coli glycolate dehydrogenase (GlcDH). The engineered polyprotein retained the functionality of the native GlcDH complex when expressed in E. coli and was able to complement mutants deficient for the D, E and F subunits. Transgenic plants accumulated DEFp in the plastids, and the recombinant protein was active in planta, reducing photorespiration and improving CO2 uptake with a significant impact on carbon metabolism. Transgenic lines with the highest DEFp levels and GlcDH activity produced significantly higher levels of glucose (5.8-fold), fructose (3.8-fold), sucrose (1.6-fold) and transitory starch (threefold), resulting in a substantial increase in shoot and leaf biomass. The higher carbohydrate levels produced in potato leaves were utilized by the sink capacity of the tubers, increasing the tuber yield by 2.3-fold. This novel approach therefore has the potential to increase the biomass and yield of diverse crops.

A Common histone modification code on C4 genes in maize and its conservation in Sorghum and Setaria italica.

C4 photosynthesis evolved more than 60 times independently in different plant lineages. Each time, multiple genes were recruited into C4 metabolism. The corresponding promoters acquired new regulatory features such as high expression, light induction, or cell type-specific expression in mesophyll or bundle sheath cells. We have previously shown that histone modifications contribute to the regulation of the model C4 phosphoenolpyruvate carboxylase (C4-Pepc) promoter in maize (Zea mays). We here tested the light- and cell type-specific responses of three selected histone acetylations and two histone methylations on five additional C4 genes (C4-Ca, C4-Ppdk, C4-Me, C4-Pepck, and C4-RbcS2) in maize. Histone acetylation and nucleosome occupancy assays indicated extended promoter regions with regulatory upstream regions more than 1,000 bp from the transcription initiation site for most of these genes. Despite any detectable homology of the promoters on the primary sequence level, histone modification patterns were highly coregulated. Specifically, H3K9ac was regulated by illumination, whereas H3K4me3 was regulated in a cell type-specific manner. We further compared histone modifications on the C4-Pepc and C4-Me genes from maize and the homologous genes from sorghum (Sorghum bicolor) and Setaria italica. Whereas sorghum and maize share a common C4 origin, C4 metabolism evolved independently in S. italica. The distribution of histone modifications over the promoters differed between the species, but differential regulation of light-induced histone acetylation and cell type-specific histone methylation were evident in all three species. We propose that a preexisting histone code was recruited into C4 promoter control during the evolution of C4 metabolism.

Photorespiratory bypasses: how can they work?

Photorespiration has been suggested as a target for increasing photosynthesis for decades. Within the last few years, three bypass pathways or reactions have been designed and tested in plants. The three reactions bypass photorespiration either in the chloroplast or in the peroxisome, or oxidize glycolate completely to CO(2) in the chloroplast. The reactions differ in their demand for energy and reducing power as well as in the catabolic fate of glycolate. The design, energy balance, and reported benefits of the three bypasses are compared here, and an outlook on further optimization is given.

Chromatin modification acts as a memory for systemic acquired resistance in the plant stress response.

Priming of defence genes for amplified response to secondary stress can be induced by application of the plant hormone salicylic acid or its synthetic analogue acibenzolar S-methyl. In this study, we show that treatment with acibenzolar S-methyl or pathogen infection of distal leaves induce chromatin modifications on defence gene promoters that are normally found on active genes, although the genes remain inactive. This is associated with an amplified gene response on challenge exposure to stress. Mutant analyses reveal a tight correlation between histone modification patterns and gene priming. The data suggest a histone memory for information storage in the plant stress response.

Complex I-complex II ratio strongly differs in various organs of Arabidopsis thaliana.

In most studies, amounts of protein complexes of the oxidative phosphorylation (OXPHOS) system in different organs or tissues are quantified on the basis of isolated mitochondrial fractions. However, yield of mitochondrial isolations might differ with respect to tissue type due to varying efficiencies of cell disruption during organelle isolation procedures or due to tissue-specific properties of organelles. Here we report an immunological investigation on the ratio of the OXPHOS complexes in different tissues of Arabidopsis thaliana which is based on total protein fractions isolated from five Arabidopsis organs (leaves, stems, flowers, roots and seeds) and from callus. Antibodies were generated against one surface exposed subunit of each of the five OXPHOS complexes and used for systematic immunoblotting experiments. Amounts of all complexes are highest in flowers (likewise with respect to organ fresh weight or total protein content of the flower fraction). Relative amounts of protein complexes in all other fractions were determined with respect to their amounts in flowers. Our investigation reveals high relative amounts of complex I in green organs (leaves and stems) but much lower amounts in non-green organs (roots, callus tissue). In contrast, complex II only is represented by low relative amounts in green organs but by significantly higher amounts in non-green organs, especially in seeds. In fact, the complex I-complex II ratio differs by factor 37 between callus and leaf, indicating drastic differences in electron entry into the respiratory chain in these two fractions. Variation in amounts concerning complexes III, IV and V was less pronounced in different Arabidopsis tissues (quantification of complex V in leaves was not meaningful due to a cross-reaction of the antibody with the chloroplast form of this enzyme). Analyses were complemented by in gel activity measurements for the protein complexes of the OXPHOS system and comparative 2D blue native/SDS PAGE analyses using isolated mitochondria. We suggest that complex I has an especially important role in the context of photosynthesis which might be due to its indirect involvement in photorespiration and its numerous enzymatic side activities in plants.

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice.

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.

Best practice procedures for the establishment of a C(4) cycle in transgenic C(3) plants.

C(4) plants established a mechanism for the concentration of CO(2) in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase in order to saturate the enzyme with substrate and substantially to reduce the alternative fixation of O(2) that results in energy losses. Transfer of the C(4) mechanism to C(3) plants has been repeatedly tested, but none of the approaches so far resulted in transgenic plants with enhanced photosynthesis or growth. Instead, often deleterious effects were observed. A true C(4) cycle requires the co-ordinated activity of multiple enzymes in different cell types and in response to diverse environmental and metabolic stimuli. This review summarizes our current knowledge about the most appropriate regulatory elements and coding sequences for the establishment of C(4) protein activities in C(3) plants. In addition, technological breakthroughs for the efficient transfer of the numerous genes probably required to transform a C(3) plant into a C(4) plant will be discussed.

ZmOrphan94 Transcription Factor Downregulates ZmPEPC1 Gene Expression in Maize Bundle Sheath Cells.

Spatial separation of the photosynthetic reactions is a key feature of C4 metabolism. In most C4 plants, this separation requires compartmentation of photosynthetic enzymes between mesophyll (M) and bundle sheath (BS) cells. The upstream region of the gene encoding the maize PHOSPHOENOLPYRUVATE CARBOXYLASE 1 (ZmPEPC1) has been shown sufficient to drive M-specific ZmPEPC1 gene expression. Although this region has been well characterized, to date, only few trans-factors involved in the ZmPEPC1 gene regulation were identified. Here, using a yeast one-hybrid approach, we have identified three novel maize transcription factors ZmHB87, ZmCPP8, and ZmOrphan94 as binding to the ZmPEPC1 upstream region. Bimolecular fluorescence complementation assays in maize M protoplasts unveiled that ZmOrphan94 forms homodimers and interacts with ZmCPP8 and with two other ZmPEPC1 regulators previously reported, ZmbHLH80 and ZmbHLH90. Trans-activation assays in maize M protoplasts unveiled that ZmHB87 does not have a clear transcriptional activity, whereas ZmCPP8 and ZmOrphan94 act as activator and repressor, respectively. Moreover, we observed that ZmOrphan94 reduces the trans-activation activity of both activators ZmCPP8 and ZmbHLH90. Using the electromobility shift assay, we showed that ZmOrphan94 binds to several cis-elements present in the ZmPEPC1 upstream region and one of these cis-elements overlaps with the ZmbHLH90 binding site. Gene expression analysis revealed that ZmOrphan94 is preferentially expressed in the BS cells, suggesting that ZmOrphan94 is part of a transcriptional regulatory network downregulating ZmPEPC1 transcript level in the BS cells. Based on both this and our previous work, we propose a model underpinning the importance of a regulatory mechanism within BS cells that contributes to the M-specific ZmPEPC1 gene expression.

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana.

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.

Developmental and environmental signals induce distinct histone acetylation profiles on distal and proximal promoter elements of the C4-Pepc gene in maize.

The maize C(4)-Pepc gene is expressed in an organ- and cell-type-specific manner, inducible by light and modulated by nutrient availability and the metabolic state of the cell. We studied the contribution of histone acetylation at five lysine residues to the integration of these signals into a graduated promoter response. In roots and coleoptiles, where the gene is constitutively inactive, three of the five lysines were acetylated and the modifications showed unique patterns with respect to their distribution on the gene. A similar pattern was observed in etiolated leaves, where the gene is poised for activation by light. Here, illumination selectively induced the acetylation of histone H4 lysine 5 and histone H3 lysine 9 in both the promoter and the transcribed region, again with unique distribution patterns. Induction was independent of transcription and fully reversible in the dark. Nitrate and hexose availability modulated acetylation of all five lysines restricted to a distal promoter region, whereas proximal promoter acetylation was highly resistant to these stimuli. Our data suggest that light induction of acetylation is controlled by regulating HDAC activity, whereas metabolic signals regulate HAT activity. Acetylation turnover rates were high in the distal promoter and the transcribed regions, but low on the proximal promoter. On the basis of these results, we propose a model with three levels of stimulus-induced histone modifications that collectively adjust promoter activity. The results support a charge neutralization model for the distal promoter and a stimulus-mediated, but transcription-independent, histone acetylation pattern on the core promoter, which might be part of a more complex histone code.

Low CO2 induces urea cycle intermediate accumulation in Arabidopsis thaliana.

The non-proteinogenic amino acid ornithine links several stress response pathways. From a previous study we know that ornithine accumulates in response to low CO2. To investigate ornithine accumulation in plants, we shifted plants to either low CO2 or low light. Both conditions increased carbon limitation, but only low CO2 also increased the rate of photorespiration. Changes in metabolite profiles of light- and CO2-limited plants were quite similar. Several amino acids that are known markers of senescence accumulated strongly under both conditions. However, urea cycle intermediates respond differently between the two treatments. While the levels of both ornithine and citrulline were much higher in plants shifted to 100 ppm CO2 compared to those kept in 400 ppm CO2, their metabolite abundance did not significantly change in response to a light limitation. Furthermore, both ornithine and citrulline accumulation is independent from sugar starvation. Exogenous supplied sugar did not significantly change the accumulation of the two metabolites in low CO2-stressed plants, while the accumulation of other amino acids was reduced by about 50%. Gene expression measurements showed a reduction of the entire arginine biosynthetic pathway in response to low CO2. Genes in both proline biosynthesis and degradation were induced. Hence, proline did not accumulate in response to low CO2 like observed for many other stresses. We propose that excess of nitrogen re-fixed during photorespiration can be alternatively stored in ornithine and citrulline under low CO2 conditions. Furthermore, ornithine is converted to pyrroline-5-carboxylate by the action of δOAT.