Predicting C4 photosynthesis evolution: modular, individually adaptive steps on a Mount Fuji fitness landscape.

An ultimate goal of evolutionary biology is the prediction and experimental verification of adaptive trajectories on macroevolutionary timescales. This aim has rarely been achieved for complex biological systems, as models usually lack clear correlates of organismal fitness. Here, we simulate the fitness landscape connecting two carbon fixation systems: C3 photosynthesis, used by most plant species, and the C4 system, which is more efficient at ambient CO2 levels and elevated temperatures and which repeatedly evolved from C3. Despite extensive sign epistasis, C4 photosynthesis is evolutionarily accessible through individually adaptive steps from any intermediate state. Simulations show that biochemical subtraits evolve in modules; the order and constitution of modules confirm and extend previous hypotheses based on species comparisons. Plant-species-designated C3-C4 intermediates lie on predicted evolutionary trajectories, indicating that they indeed represent transitory states. Contrary to expectations, we find no slowdown of adaptation and no diminishing fitness gains along evolutionary trajectories.

The C4 Ppc promoters of many C4 grass species share a common regulatory mechanism for gene expression in the mesophyll cell.

C4 photosynthetic plants have evolved from C3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C4 photosynthesis. In this study, we demonstrate that the 5'-flanking sequences of the C4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450 bp (upstream of their transcription start site) of the analyzed C4 Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C4 Ppc regulatory mechanisms of multiple closely related C4 grass species.

Reporter-based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana.

The evolution of C4 photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C4 cycle is the photosynthetic activation of the C3 bundle sheath by increasing its volume and organelle number. Therefore, to engineer C4 photosynthesis into existing C3 crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C4 -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C3  species Arabidopsis thaliana. To ensure a high-throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast-targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping-by-sequencing approach the genomic segments that contained mutated candidate genes were identified.

Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana.

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

SHORTROOT-Mediated Increase in Stomatal Density Has No Impact on Photosynthetic Efficiency.

The coordinated positioning of veins, mesophyll cells, and stomata across a leaf is crucial for efficient gas exchange and transpiration and, therefore, for overall function. In monocot leaves, stomatal cell files are positioned at the flanks of underlying longitudinal leaf veins, rather than directly above or below. This pattern suggests either that stomatal formation is inhibited in epidermal cells directly in contact with the vein or that specification is induced in cell files beyond the vein. The SHORTROOT pathway specifies distinct cell types around the vasculature in subepidermal layers of both root and shoots, with cell type identity determined by distance from the vein. To test whether the pathway has the potential to similarly pattern epidermal cell types, we expanded the expression domain of the rice (Oryza sativa ssp japonica) OsSHR2 gene, which we show is restricted to developing leaf veins, to include bundle sheath cells encircling the vein. In transgenic lines, which were generated using the orthologous ZmSHR1 gene to avoid potential silencing of OsSHR2, stomatal cell files were observed both in the normal position and in more distant positions from the vein. Contrary to theoretical predictions, and to phenotypes observed in eudicot leaves, the increase in stomatal density did not enhance photosynthetic capacity or increase mesophyll cell density. Collectively, these results suggest that the SHORTROOT pathway may coordinate the positioning of veins and stomata in monocot leaves and that distinct mechanisms may operate in monocot and eudicot leaves to coordinate stomatal patterning with the development of underlying mesophyll cells.

Efficient 2-phosphoglycolate degradation is required to maintain carbon assimilation and allocation in the C4 plant Flaveria bidentis.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C4 plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C4 plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C4 photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C3 plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants.

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 (hcf222-1) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1, the accumulation of the cytochrome b6f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis (Arabidopsis thaliana) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane.

Expression of SULTR2;2, encoding a low-affinity sulphur transporter, in the Arabidopsis bundle sheath and vein cells is mediated by a positive regulator.

The bundle sheath provides a conduit linking veins and mesophyll cells. In the C3 plant Arabidopsis thaliana, it also plays important roles in oxidative stress and sulphur metabolism. However, the mechanisms responsible for the patterns of gene expression that underpin these metabolic specializations are poorly understood. Here, we used the Arabidopsis SULTR2;2 gene as a model to better understand mechanisms that restrict expression to the bundle sheath. Deletion analysis indicated that the SULTR2;2 promoter contains a short region necessary for expression in the bundle sheath and veins. This sequence acts as a positive regulator and is tolerant to multiple consecutive deletions indicating considerable redundancy in the cis-elements involved. It is highly conserved in SULTR2;2 genes of the Brassicaceae and is functional in the distantly related C4 species Flaveria bidentis that belongs to the Asteraceae. We conclude that expression of SULTR2;2 in the bundle sheath and veins is underpinned by a highly redundant sequence that likely represents an ancient and conserved mechanism found in families as diverse as the Asteraceae and Brassicaceae.

C3 cotyledons are followed by C4 leaves: intra-individual transcriptome analysis of Salsola soda (Chenopodiaceae).

Some species of Salsoleae (Chenopodiaceae) convert from C3 photosynthesis during the seedling stage to the C4 pathway in adult leaves. This unique developmental transition of photosynthetic pathways offers the exceptional opportunity to follow the development of the derived C4 syndrome from the C3 condition within individual plants, avoiding phylogenetic noise. Here we investigate Salsola soda, a little-studied species from tribe Salsoleae, using an ontogenetic approach. Anatomical sections, carbon isotope (δ13C) values, transcriptome analysis by means of mRNA sequencing, and protein levels of the key C4 enzyme phosphoenolpyruvate carboxylase (PEPC) were examined from seed to adult plant stages. Despite a previous report, our results based on δ13C values, anatomy and transcriptomics clearly indicate a C3 phase during the cotyledon stage. During this stage, the entire transcriptional repertoire of the C4 NADP-malic enzyme type is detected at low levels compared to a significant increase in true leaves. In contrast, abundance of transcripts encoding most of the major photorespiratory enzymes is not significantly decreased in leaves compared to cotyledons. PEPC polypeptide was detected only in leaves, correlating with increased PEPC transcript abundance from the cotyledon to leaf stage.

A MEM1-like motif directs mesophyll cell-specific expression of the gene encoding the C4 carbonic anhydrase in Flaveria.

The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter-reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.

A plastidial sodium-dependent pyruvate transporter.

Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids, terpenoids and branched-chain amino acids. In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers, which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C(3) and C(4) plants of the genera Flaveria and Cleome, here we have identified a novel gene that is abundant in C(4) species, named BASS2 (BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C(4) plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.

Evolution of C4 photosynthesis in the genus flaveria: establishment of a photorespiratory CO2 pump.

C4 photosynthesis is nature's most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO(2) by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO(2) release in plants and is active in all photosynthetic tissues of C(3) plants, but only in the bundle sheath cells of C(4) plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C(4) photosynthesis, leading to a photorespiratory CO(2) concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C(3) to C(4) photosynthesis in the genus Flaveria. We found that C(3) Flaveria species already contain a bundle sheath-expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C(4) Flaveria species. Analyses of C(3)-C(4) intermediate Flaveria species revealed that the photorespiratory CO(2) pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C(4) evolution.

Expression of a nuclear-encoded psbH gene complements the plastidic RNA processing defect in the PSII mutant hcf107 in Arabidopsis thaliana.

The helical-repeat RNA-binding protein HCF107 is required for processing, stabilization and translation of plastid-encoded psbH mRNA. The psbH gene encodes a small, hydrophilic subunit of the PSII complex and is part of the plastidic psbB-psbT-psbH-petB-petD transcription unit. In Arabidopsis hcf107 mutants, only trace amounts of PSII proteins can be detected. Beside drastically reduced synthesis of PsbH, the synthesis of CP47 was also reduced in these mutants, although the corresponding psbB transcripts accumulate to wild type levels. This situation raises the question, whether the reduction of CP47 is a direct consequence of the mutation, based on targeting of HCF107 to the psbB mRNA, or a secondary affect due to the absent PsbH. To clarify this issue we introduced a chimeric psbH construct comprising a sequence encoding a chloroplast transit peptide into the hcf107-2 background. We found that the nucleus-localized psbH was able to complement the mutant defect resulting in photoautotrophic plants. The PSII proteins CP47 and D1 accumulated to barely half of the wild type level. Further experiments showed that cytosolically synthesized PsbH was imported into chloroplasts and assembled into PSII complexes. Using this approach, we showed that the tetratricopeptide repeat protein HCF107 of Arabidopsis is only responsible for expression of PsbH and not for synthesis of CP47. In addition the data suggest the necessity of the small, one-helix membrane spanning protein PsbH for the accumulation of CP47 in higher plants.

RNA-Seq based phylogeny recapitulates previous phylogeny of the genus Flaveria (Asteraceae) with some modifications.

BACKGROUND: The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the genus Flaveria contains 21 of the 23 known Flaveria species and has been previously constructed using a combination of morphological data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnL-F). RESULTS: Here we developed a new strategy to update the phylogenetic tree of 16 Flaveria species based on RNA-Seq data. The updated phylogeny is largely congruent with the previously published tree but with some modifications. We propose that the data collection method provided in this study can be used as a generic method for phylogenetic tree reconstruction if the target species has no genomic information. We also showed that a "F. pringlei" genotype recently used in a number of labs may be a hybrid between F. pringlei (C3) and F. angustifolia (C3-C4). CONCLUSIONS: We propose that the new strategy of obtaining phylogenetic sequences outlined in this study can be used to construct robust trees in a larger number of taxa. The updated Flaveria phylogenetic tree also supports a hypothesis of stepwise and parallel evolution of C4 photosynthesis in the Flavaria clade.

Stable carbon isotope discrimination is under genetic control in the C4 species maize with several genomic regions influencing trait expression.

In plants with C4 photosynthesis, physiological mechanisms underlying variation in stable carbon isotope discrimination (Δ(13)C) are largely unknown, and genetic components influencing Δ(13)C have not been described. We analyzed a maize (Zea mays) introgression library derived from two elite parents to investigate whether Δ(13)C is under genetic control in this C4 species. High-density genotyping with the Illumina MaizeSNP50 Bead Chip was used for a detailed structural characterization of 89 introgression lines. Phenotypic analyses were conducted in the field and in the greenhouse for kernel Δ(13)C as well as plant developmental and photosynthesis-related traits. Highly heritable significant genetic variation for Δ(13)C was detected under field and greenhouse conditions. For several introgression library lines, Δ(13)C values consistently differed from the recurrent parent within and across the two phenotyping platforms. Δ(13)C was significantly associated with 22 out of 164 analyzed genomic regions, indicating a complex genetic architecture of Δ(13)C. The five genomic regions with the largest effects were located on chromosomes 1, 2, 6, 7, and 9 and explained 55% of the phenotypic variation for Δ(13)C. Plant development stage had no effect on Δ(13)C expression, as phenotypic as well as genotypic correlations between Δ(13)C, flowering time, and plant height were not significant. To our knowledge, this is the first study demonstrating Δ(13)C to be under polygenic control in the C4 species maize.

Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.

The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4 Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4 Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4 Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode.

Regulation of the photorespiratory GLDPA gene in C(4) flaveria: an intricate interplay of transcriptional and posttranscriptional processes.

The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C(3) plants but is restricted to bundle sheath cells in C(4) species. GDC is also required for general cellular C(1) metabolism. In the Asteracean C(4) 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 (C(4)) and the Brassicacean C(3) 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, P(R2) and P(R7), that together ensure a strong bundle sheath expression. While the proximal promoter (P(R7)) is active in the bundle sheath and vasculature, the distal promoter (P(R2)) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 5' untranslated leader of P(R2)-derived transcripts is inefficiently spliced and apparently suppresses the output of P(R2) by eliciting RNA decay.

The Sorghum bicolor genome and the diversification of grasses.

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the approximately 730-megabase Sorghum bicolor (L.) Moench genome, placing approximately 98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the approximately 75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization approximately 70 million years ago, most duplicated gene sets lost one member before the sorghum-rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum's drought tolerance.

Evolution of C4 photosynthesis in the genus Flaveria: how many and which genes does it take to make C4?

Selective pressure exerted by a massive decline in atmospheric CO(2) levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C(4) photosynthesis. C(4) species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C(4) trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C(3) and C(4) species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C(3) to C(3)-C(4) intermediate to C(4) species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C(4)-associated characteristics. Single gene analysis defines the C(4) cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C(4) associated pathways. Molecular evidence for a photorespiratory CO(2) pump prior to the establishment of the C(4) cycle-based CO(2) pump is provided. Cluster analysis defines the upper limit of C(4)-related gene expression changes in mature leaves of Flaveria as 3582 alterations.

Recruitment of a ribosomal release factor for light- and stress-dependent regulation of petB transcript stability in Arabidopsis chloroplasts.

Land plant genomes encode four functional ribosomal peptide chain release factors (Prf) of eubacterial origin, two (PrfA and PrfB homologs) for each endosymbiotic organelle. Formerly, we have shown that the Arabidopsis thaliana chloroplast-localized PrfB homolog, PrfB1, is required not only for termination of translation but also for stabilization of UGA stop codon-containing chloroplast transcripts. A previously undiscovered PrfB-like protein, PrfB3, is localized to the chloroplast stroma in a petB RNA-containing complex and found only in vascular plants. Highly conserved positions of introns unequivocally indicate that PrfB3 arose from a duplication of PrfB1. Notably, PrfB3 is lacking the two most important tripeptide motifs characteristic for all eubacterial and organellar PrfB homologs described so far: the stop codon recognition motif SPF and the catalytic center GGQ for peptidyl-tRNA hydrolysis. Complementation studies, as well as functional and molecular analyses of two allelic mutations in Arabidopsis, both of which lead to a specific deficiency of the cytochrome b6f complex, revealed that PrfB3 is essentially required for photoautotrophic growth. Plastid transcript, polysome, and translation analyses indicate that PrfB3 has been recruited in vascular plants for light- and stress-dependent regulation of stability of 3' processed petB transcripts to adjust cytochrome b6 levels.