Photosynthesis in the Biomass Model Species Lemna minor Displays Plant-Conserved and Species-Specific Features.

Lemnaceae are small freshwater plants with extraordinary high growth rates. We aimed to test whether this correlates with a more efficient photosynthesis, the primary energy source for growth. To this end, we compared photosynthesis properties of the duckweed Lemna minor and the terrestrial model plant Arabidopsis thaliana. Chlorophyll fluorescence analyses revealed high similarity in principle photosynthesis characteristics; however, Lemna exhibited a more effective light energy transfer into photochemistry and more stable photosynthesis parameters especially under high light intensities. Western immunoblot analyses of representative photosynthesis proteins suggested potential post-translational modifications in Lemna proteins that are possibly connected to this. Phospho-threonine phosphorylation patterns of thylakoid membrane proteins displayed a few differences between the two species. However, phosphorylation-dependent processes in Lemna such as photosystem II antenna association and the recovery from high-light-induced photoinhibition were not different from responses known from terrestrial plants. We thus hypothesize that molecular differences in Lemna photosynthesis proteins are associated with yet unidentified mechanisms that improve photosynthesis and growth efficiencies. We also developed a high-magnification video imaging approach for Lemna multiplication which is useful to assess the impact of external factors on Lemna photosynthesis and growth.

3D-printed autoclavable plant holders to facilitate large-scale protein production in plants.

The Australian tobacco plant Nicotiana benthamiana is becoming increasingly popular as a platform for protein production and metabolic engineering. In this system, gene expression is achieved transiently by infiltrating N. benthamiana plants with suspensions of Agrobacterium tumefaciens carrying vectors with the target genes. To infiltrate larger numbers of plants, vacuum infiltration is the most efficient approach known, which is already used on industrial scale. Current laboratory-scale solutions for vacuum infiltration, however, either require expensive custom-tailored equipment or produce large amounts of biologically contaminated waste. To overcome these problems and lower the burden to establish vacuum infiltration in new laboratories, we present here 3D-printed plant holders for vacuum infiltration. We demonstrate that our plant holders are simple to use and enable a throughput of around 40 plants per hour. In addition, our 3D-printed plant holders are made from autoclavable material, which tolerate at least 12 autoclave cycles, helping to limit the production of contaminated waste and thus contributing to increased sustainability in research. In conclusion, our plant holders provide a simple, robust, safe and transparent platform for laboratory-scale vacuum infiltration that can be readily adopted by new laboratories interested in protein and metabolite production in Nicotiana benthamiana. Practical application Transient expression in Nicotiana benthamiana provides a popular and rapid system for producing proteins in a plant host. To infiltrate larger numbers of plants (typically >20), vacuum infiltration is the method of choice. However, no system has been described so far which is robust to use and can be used without expensive and complex equipment. Our autoclavable 3D-printed plant holders presented here will greatly reduce the efforts required to adopt the vacuum infiltration technique in new laboratories. They are easy to use and can be autoclaved at least 12 times, which contributes to waste reduction and sustainability in research laboratories. We anticipate that the 3D printing design provided here will drastically lower the bar for new groups to employ vacuum infiltration for producing proteins and metabolites in Nicotiana benthamiana.

Effectiveness of Light-Quality and Dark-White Growth Light Shifts in Short-Term Light Acclimation of Photosynthesis in Arabidopsis.

Photosynthesis needs to run efficiently under permanently changing illumination. To achieve this, highly dynamic acclimation processes optimize photosynthetic performance under a variety of rapidly changing light conditions. Such acclimation responses are acting by a complex interplay of reversible molecular changes in the photosynthetic antenna or photosystem assemblies which dissipate excess energy and balance uneven excitation between the two photosystems. This includes a number of non-photochemical quenching processes including state transitions and photosystem II remodeling. In the laboratory such processes are typically studied by selective illumination set-ups. Two set-ups known to be effective in a highly similar manner are (i) light quality shifts (inducing a preferential excitation of one photosystem over the other) or (ii) dark-light shifts (inducing a general off-on switch of the light harvesting machinery). Both set-ups result in similar effects on the plastoquinone redox state, but their equivalence in induction of photosynthetic acclimation responses remained still open. Here, we present a comparative study in which dark-light and light-quality shifts were applied to samples of the same growth batches of plants. Both illumination set-ups caused comparable effects on the phosphorylation of LHCII complexes and, hence, on the performance of state transitions, but generated different effects on the degree of state transitions and the formation of PSII super-complexes. The two light set-ups, thus, are not fully equivalent in their physiological effectiveness potentially leading to different conclusions in mechanistic models of photosynthetic acclimation. Studies on the regulation of photosynthetic light acclimation, therefore, requires to regard the respective illumination test set-up as a critical parameter that needs to be considered in the discussion of mechanistic and regulatory aspects in this subject.

Efficient In Vivo Screening Method for the Identification of C4 Photosynthesis Inhibitors Based on Cell Suspensions of the Single-Cell C4 Plant Bienertia sinuspersici.

The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient in vivo assay based on oxygen evolution measurements using suspensions of chlorenchyma cells isolated from the single-cell C4 plant Bienertia sinuspersici to identify and characterize inhibitors of C4 photosynthesis. This novel approach fills the gap between conventional in vitro assays for inhibitors targeting C4 key enzymes and in vivo experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C4 photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and confirm their potential to act as a new class of C4 selective inhibitors.

Agrobacterium-mediated transient transformation of Bienertia sinuspersici to assay recombinant protein distribution between dimorphic chloroplasts.

KEY MESSAGE: Bienertia sinuspersici, a species capable of performing C4 photosynthesis within individual photosynthetic cells, can be transformed with Agrobacterium allowing for the analysis of subcellular protein distribution under preservation of the unique cellular morphology.

Transit peptide elements mediate selective protein targeting to two different types of chloroplasts in the single-cell C4 species Bienertia sinuspersici.

Bienertia sinuspersici is a terrestrial plant that performs C4 photosynthesis within individual cells through operating a carbon concentrating mechanism between different subcellular domains including two types of chloroplasts. It is currently unknown how differentiation of two highly specialized chloroplasts within the same cell occurs as no similar cases have been reported. Here we show that this differentiation in photosynthetic cells of B. sinuspersici is enabled by a transit peptide (TP) mediated selective protein targeting mechanism. Mutations in the TPs cause loss of selectivity but not general loss of chloroplast import, indicating the mechanism operates by specifically blocking protein accumulation in one chloroplast type. Hybrid studies indicate that this selectivity is transferable to transit peptides of plants which perform C4 by cooperative function of chloroplasts between two photosynthetic cells. Codon swap experiments as well as introducing an artificial bait mRNA show that RNA affects are not crucial for the sorting process. In summary, our analysis shows how the mechanism of subcellular targeting to form two types of chloroplast within the same cell can be achieved. This information is not only crucial for understanding single-cell C4 photosynthesis; it provides new insights in control of subcellular protein targeting in cell biology.

Development, subcellular positioning and selective protein accumulation in the dimorphic chloroplasts of single-cell C4 species.

C4 photosynthesis is typically associated with a carbon concentrating mechanism based on close collaboration between two photosynthetic cell types (Kranz C4). Surprisingly, four species in the family Chenopodiaceae have been described, which perform all required steps for a functional and effective C4 cycle within individual photosynthetic cells. These single-cell C4 species utilize a unique subcellular compartmentation and two functionally different chloroplast types that mimic the functions of the two cell types of the Kranz C4 system. In this review, we will summarize and discuss studies on chloroplast development, positioning and selective accumulation of nuclear encoded proteins, which ultimately allow the operation of a C4 carbon concentrating mechanism within individual cells.

Developmental and Subcellular Organization of Single-Cell C4 Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

Can we learn from heterosis and epigenetics to improve photosynthesis?

Heterosis is the increase in fitness and yield of F1 hybrids derived from a cross between distantly related genotypes. The use of heterosis is one of the most successful crop breeding strategies, but the underlying molecular mechanisms are still poorly defined. There is ample evidence that heterosis is associated with increased rates of photosynthesis and recent analyses have shed light on the underlying biochemical principles. In parallel, the importance of epigenetic chromatin modifications in heterosis has now been established. The first direct links between epigenetic changes and improved photosynthesis have also been demonstrated. As epigenetic engineering is now possible, we discuss the feasibility of altering the epigenetic code to enhance photosynthesis.

One decade after the discovery of single-cell C4 species in terrestrial plants: what did we learn about the minimal requirements of C4 photosynthesis?

Until about 10 years ago the general accepted textbook knowledge was that terrestrial C4 photosynthesis requires separation of photosynthetic functions into two specialized cell types, the mesophyll and bundle sheath cells forming the distinctive Kranz anatomy typical for C4 plants. This paradigm has been broken with the discovery of Suaeda aralocaspica, a chenopod from central Asia, performing C4 photosynthesis within individual chlorenchyma cells. Since then, three more single-cell C4 (SCC4) species have been discovered in the genus Bienertia. They are interesting not only because of their unusual mode of photosynthesis but also present a puzzle for cell biologists. In these species, two morphological and biochemical specialized types of chloroplasts develop within individual chlorenchyma cells, a situation that has never been observed in plants before. Here we review recent literature concerning the biochemistry, physiology, and molecular biology of SCC4 photosynthesis. Particularly, we focus on what has been learned in relation to the following questions: How does the specialized morphology required for the operation of SCC4 develop and is there a C3 intermediate type of photosynthesis during development? What is the degree of specialization between the two chloroplast types and how does this compare to the chloroplasts of Kranz C4 species? How do nucleus-encoded proteins that are targeted to chloroplasts accumulate differentially in the two chloroplast types and how efficient is the CO2 concentrating mechanism in SCC4 species compared to the Kranz C4 forms?

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.

Re-engineering of carbon fixation in plants - challenges for plant biotechnology to improve yields in a high-CO2 world.

Source and sink strength control plant carbon gain and yield. Source strength was recently engineered by modifying the large subunit of Rubisco, replacing the small subunit, and creating improved thermostable Rubisco activases. This technological breakthrough makes Rubisco engineering feasible at last. Enhancement of leaf transitory starch synthesis or induction of artificial sinks in leaves increased biomass and yield. Importantly, such approaches also had a positive feedback on source strength. In addition, novel targets for the improvement of carbon gain in crops have been identified that are especially relevant in the light of climate change.

In vitro cultures and regeneration of Bienertia sinuspersici (Chenopodiaceae) under increasing concentrations of sodium chloride and carbon dioxide.

To study the developmental transition of chloroplasts from C(3) to C(4) photosynthesis in the terrestrial single-cell C(4) species Bienertia sinuspersici, a regeneration protocol was developed. Stem explant material developed callus either with or without red nodular structures (RNS) when cultured on Murashige-Skoog (MS) salts and vitamins, supplemented with 5 mM phosphate, plus 1 mg L(-1) dichloropenoxy-acetic acid (2,4-D), and 87 mM sucrose (Stage 1 media). Only calli having RNS were able to regenerate plantlets. MS media plus phosphate was used throughout regeneration, with the Stage 2 media containing 2 mg L(-1) 6-benzylaminopurine, 43 mM sucrose and 1.5% soluble starch. Stage 3 media had no hormones or organic sources of carbon, and cultures were grown under ambient (~400 ppm) versus CO(2) enrichment (1.2% CO(2)). When calli without RNS were cultured under Stage 3 conditions with 1.2% CO(2), there was an increase in growth, protein content, and photosystem II yield, while structural and biochemical analyses indicated the cells in the calli had C(3) type photosynthesis. CO(2) enrichment during growth of RNS during Stage 3 had a large effect on regeneration success, increasing efficiency of shoot and root development, size of plantlets, leaf soluble protein, and chlorophyll concentration. Anatomical analysis of plantlets, which developed under 1.2% CO(2), showed leaves developed C(4) type chlorenchyma cells, including expression of key C(4) biochemical enzymes. Increasing salinity in the media, from 0 to 200 mM NaCl, increased tissue osmolality, average plantlet area and regeneration success, but did not affect protein or chlorophyll content.

How do single cell C4 species form dimorphic chloroplasts?

Bienertia sinuspersici is one of only three higher land plant species known to perform C 4 photosynthesis without Kranz anatomy through partitioning of photosynthetic functions between dimorphic chloroplasts in a single photosynthetic cell. We recently reported the successful separation of the two chloroplast types, and biochemical and functional analyses revealed differences in protein composition and specialization of photosynthetic functions. In Kranz type C 4 species, spatial (or cell-specific) control of transcription of nuclear genes contributes to development of dimorphic chloroplasts, but obviously this cannot be involved in formation of dimorphic chloroplasts within individual photosynthetic cells. Therefore, we address here the question of how nuclear encoded proteins could be selectively targeted to plastids within a cell to form two types of chloroplasts. We discuss current knowledge of chloroplast differentiation in single cell C 4 species and present three hypothetical mechanisms for how this could occur.

Resolving the compartmentation and function of C4 photosynthesis in the single-cell C4 species Bienertia sinuspersici.

Bienertia sinuspersici is a land plant known to perform C(4) photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C(4) cycle. Western-blot analyses indicated that the CC has Rubisco from the C(3) cycle, the C(4) decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photorespiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C(3) cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C(4) and C(3) cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C(4) species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C(4) species.

The effects of salinity on photosynthesis and growth of the single-cell C4 species Bienertia sinuspersici (Chenopodiaceae).

Recent research on the photosynthetic mechanisms of plant species in the Chenopodiaceae family revealed that three species, including Bienertia sinuspersici, can carry out C(4) photosynthesis within individual photosynthetic cells, through the development of two cytoplasmic domains having dimorphic chloroplasts. These unusual single-cell C(4) species grow in semi-arid saline conditions and have semi-terete succulent leaves. The effects of salinity on growth and photosynthesis of B. sinuspersici were studied. The results show that NaCl is not required for development of the single-cell C(4) system. There is a large enhancement of growth in culture with 50-200 mM NaCl, while there is severe inhibition at 400 mM NaCl. With increasing salinity, the carbon isotope values (δ(13)C) of leaves increased from -17.3(o)/(oo) (C(4)-like) without NaCl to -14.6(o)/(oo) (C(4)) with 200 mM NaCl, possibly due to increased capture of CO(2) from the C(4) cycle by Rubisco and reduced leakiness. Compared to growth without NaCl, leaves of plants grown under saline conditions were much larger (~2 fold) and more succulent, and the leaf solute levels increased up to ~2000 mmol kg solvent(-1). Photosynthesis on an incident leaf area basis (CO(2) saturated rates, and carboxylation efficiency under limiting CO(2)) and stomatal conductance declined with increasing salinity. On a leaf area basis, there was some decline in Rubisco content with increasing salinity up to 200 mM NaCl, but there was a marked increase in the levels of pyruvate, Pi dikinase, and phosphoenolpyruvate carboxylase (possibly in response to sensitivity of these enzymes and C(4) cycle function to increasing salinity). The decline in photosynthesis on a leaf area basis was compensated for on a per leaf basis, up to 200 mM NaCl, by the increase in leaf size. The influence of salinity on plant development and the C(4) system in Bienertia is discussed.

Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize.

BACKGROUND: Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc) promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant. RESULTS: We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm. CONCLUSION: Our results suggest a central role of upstream promoter acetylation in the quantitative regulation of gene expression in this model gene. Induced core promoter acetylation is dispensable for the highest gene expression in the diurnal and circadian rhythm.

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.

Leaf development in the single-cell C4 system in Bienertia sinuspersici: expression of genes and peptide levels for C4 metabolism in relation to chlorenchyma structure under different light conditions.

Bienertia sinuspersici performs C(4) photosynthesis in individual chlorenchyma cells by the development of two cytoplasmic domains (peripheral and central) with dimorphic chloroplasts, an arrangement that spatially separates the fixation of atmospheric CO(2) into C(4) acids and the donation of CO(2) from C(4) acids to Rubisco in the C(3) cycle. In association with the formation of these cytoplasmic domains during leaf maturation, developmental stages were analyzed for the expression of a number of photosynthetic genes, including Rubisco small and large subunits and key enzymes of the C(4) cycle. Early in development, Rubisco subunits and Gly decarboxylase and Ser hydroxymethyltransferase of the glycolate pathway accumulated more rapidly than enzymes associated with the C(4) cycle. The levels of pyruvate,Pi dikinase and phosphoenolpyruvate carboxylase were especially low until spatial cytoplasmic domains developed and leaves reached maturity, indicating a developmental transition toward C(4) photosynthesis. In most cases, there was a correlation between the accumulation of mRNA transcripts and the respective peptides, indicating at least partial control of the development of photosynthesis at the transcriptional level. During growth under moderate light, when branches containing mature leaves were enclosed in darkness for 1 month, spatial domains were maintained and there was high retention of a number of photosynthetic peptides, including Rubisco subunits and pyruvate,Pi dikinase, despite a reduction in transcript levels. When plants were transferred from moderate to low light conditions for 1 month, there was a striking shift of the central cytoplasmic compartment toward the periphery of chlorenchyma cells; the mature leaves showed strong acclimation with a shade-type photosynthetic response to light while retaining C(4) features indicative of low photorespiration. These results indicate a progressive development of C(4) photosynthesis with differences in the control mechanisms for the expression of photosynthetic genes and peptide synthesis during leaf maturation and in response to light conditions.