Genetic mutation in Escherichia coli genome during adaptation to the murine intestine is optimized for the host diet.

Mammalian gut microbes colonize the intestinal tract of their host and adapt to establish a microbial ecosystem. The host diet changes the nutrient profile of the intestine and has a high impact on microbiota composition. Genetic mutations in Escherichia coli, a prevalent species in the human gut, allow for adaptation to the mammalian intestine, as reported in previous studies. However, the extent of colonization fitness in the intestine elevated by genetic mutation and the effects of diet change on these mutations in E. coli are still poorly known. Here, we show that notable mutations in sugar metabolism-related genes (gatC, araC, and malI) were detected in the E. coli K-12 genome just 2 weeks after colonization in the germ-free mouse intestine. In addition to elevated fitness by deletion of gatC, as previously reported, deletion of araC and malI also elevated E. coli fitness in the murine intestine in a host diet-dependent manner. In vitro cultures of medium containing nutrients abundant in the intestine (e.g., galactose, N-acetylglucosamine, and asparagine) also showed increased E. coli fitness after deletion of the genes-of-interest associated with their metabolism. Furthermore, the host diet was found to influence the developmental trajectory of gene mutations in E. coli. Taken together, we suggest that genetic mutations in E. coli are selected in response to the intestinal environment, which facilitates efficient utilization of nutrients abundant in the intestine under laboratory conditions. Our study offers some insight into the possible adaptation mechanisms of gut microbes.IMPORTANCEThe gut microbiota is closely associated with human health and is greatly impacted by the host diet. Bacteria such as Escherichia coli live in the gut all throughout the life of a human host and adapt to the intestinal environment. Adaptive mutations in E. coli are reported to enhance fitness in the mammalian intestine, but to what extent is still poorly known. It is also unknown whether the host diet affects what genes are mutated and to what extent fitness is affected. This study suggests that genetic mutations in the E. coli K-12 strain are selected in response to the intestinal environment and facilitate efficient utilization of abundant nutrients in the germ-free mouse intestine. Our study provides a better understanding of these intestinal adaptation mechanisms of gut microbes.

Metabolomics approach for the identification of bioactive compounds released from young and mature soybean upon in vitro gastrointestinal digestion and their effect on health-related bioactive properties.

The aim of the present study was to comparatively investigate the relative phytochemical profiles (phenolic content, organic and amino acids, free sugars, and other metabolites using metabolomics approach), and bioactive potentials of young (YS) and mature soybean (MS) upon in vitro simulated gastrointestinal digestion (SGID). Cumulatively, a total of 198 metabolites were identified in MS and YS, 119 metabolites in undigested YS, and a total of 136 metabolites in undigested MS, which further increased to 156 and 152 in YS and MS upon SGID, respectively. Gastric digesta of both YS and MS exhibited higher inhibitory properties towards α-amylase and DPP-IV enzymes than their intestinal digesta. Furthermore, the intestinal digesta of MS showed higher antioxidant and anti-inflammatory activities compared to the YS intestinal digesta. Overall, the results suggested that the gastrointestinal digestion of YS and MS displayed distinctive metabolic profiles together with varied bioactive potentials.

Effect of the light and dark conditions on flower opening time between cultivated rice (Oryza sativa) and a near-isogenic early-morning flowering line.

Flower opening time (FOT) is affected by genetic and environmental factors, but little is known about the effect of light and dark conditions on FOT in cereal crops. FOT of an indica rice cultivar, IR64, and its near-isogenic line carrying a QTL for an early-morning flowering trait (IR64+qEMF3) were investigated in a natural-light and temperature-controlled small greenhouse by exposing either the panicle or stem or both plant organs to different light and dark conditions. FOT did not change in either genotype when panicles were exposed to light. A large difference in FOT was found between genotypes when panicles were exposed to dark conditions; no flower opening was observed in IR64, whereas flower opening was delayed but observed in IR64+qEMF3. These results suggest that the panicle is the organ that perceives light for flower opening in both genotypes, whereas the light requirement to reach flower opening was quite different between genotypes. Flower opening of IR64 occurred concomitantly with elongation of anther filament in the light after the dark treatment stopped, whereas approximately half of flowering of IR64+qEMF3 resulted in apparent cleistogamy even during dark treatment. An extended duration of the dark treatment until 1730H (30-50 min before sunset) made FOT of IR64 spikelets on the next day shifted to a time as early as that of IR64+qEMF3, with significant advancement of FOT compared to the control IR64 spikelets. Our results indicated that different flowering responses to light and dark conditions exist between IR64 and IR64+qEMF3. These findings provide clues for understanding the unique genetic controls of flowering in an EMF line in rice. This study also showed evidence that artificial light environments can shift the FOT of IR64 to that of IR64+qEMF3.

Effects of feed crops and boiling on chicken egg yolk and white determined by a metabolome analysis.

Compositional analyses of eggs have primarily focused on nutritional components, including large molecules, such as proteins. However, few reports have investigated the effects of heating and hen feed crops on taste components, such as free amino acids and sugars. Herein, water-soluble metabolites in raw and boiled eggs produced from chickens raised with corn- or rice-fed were analyzed. Each egg was separated into yolk and white, and freeze-dried samples were analyzed by CE-MS and LC-MS. Abundant metabolites included amino acids in yolks and sugars in whites. Compared to corn-fed, rice-fed resulted in three times higher betaine and uridine monophosphate concentrations in yolks and whites, respectively. Boiled whites contained more than four times higher concentrations of amino acids and fructose than raw whites. Metabolites in yolks exhibited minimal changes after boiling. Our findings support the use of water-soluble metabolomics to evaluate the effects of heating and feed crops on taste components.

Metabolome profiling of various seaweed species discriminates between brown, red, and green algae.

MAIN CONCLUSION: Among seaweed groups, brown algae had characteristically high concentrations of mannitol, and green algae were characterised by fructose. In red algae, metabolite profiles of individual species should be evaluated. Seaweeds are metabolically different from terrestrial plants. However, general metabolite profiles of the three major seaweed groups, the brown, red, and green algae, and the effect of various extraction methods on metabolite profiling results have not been comprehensively explored. In this study, we evaluated the water-soluble metabolites in four brown, five red, and two green algae species collected from two sites in northern Japan, located in the Sea of Japan and the Pacific Ocean. Freeze-dried seaweed samples were processed by methanol-water extraction with or without chloroform and analysed by capillary electrophoresis- and liquid chromatography-mass spectrometry for metabolite characterisation. The metabolite concentration profiles showed distinctive characteristic depends on species and taxonomic groups, whereas the extraction methods did not have a significant effect. Taxonomic differences between the various seaweed metabolite profiles were well defined using only sugar metabolites but no other major compound types. Mannitol was the main sugar metabolites in brown algae, whereas fructose, sucrose, and glucose were found at high concentrations in green algae. In red algae, individual species had some characteristic metabolites, such as sorbitol in Pyropia pseudolinearis and panose in Dasya sessilis. The metabolite profiles generated in this study will be a resource and provide guidance for nutraceutical research studies because the information about metabolites in seaweeds is still very limited compared to that of terrestrial plants.

Genomic comparison between Staphylococcus aureus GN strains clinically isolated from a familial infection case: IS1272 transposition through a novel inverted repeat-replacing mechanism.

A bacterial insertion sequence (IS) is a mobile DNA sequence carrying only the transposase gene (tnp) that acts as a mutator to disrupt genes, alter gene expressions, and cause genomic rearrangements. "Canonical" ISs have historically been characterized by their terminal inverted repeats (IRs), which may form a stem-loop structure, and duplications of a short (non-IR) target sequence at both ends, called target site duplications (TSDs). The IS distributions and virulence potentials of Staphylococcus aureus genomes in familial infection cases are unclear. Here, we determined the complete circular genome sequences of familial strains from a Panton-Valentine leukocidin (PVL)-positive ST50/agr4 S. aureus (GN) infection of a 4-year old boy with skin abscesses. The genomes of the patient strain (GN1) and parent strain (GN3) were rich for "canonical" IS1272 with terminal IRs, both having 13 commonly-existing copies (ce-IS1272). Moreover, GN1 had a newly-inserted IS1272 (ni-IS1272) on the PVL-converting prophage, while GN3 had two copies of ni-IS1272 within the DNA helicase gene and near rot. The GN3 genome also had a small deletion. The targets of ni-IS1272 transposition were IR structures, in contrast with previous "canonical" ISs. There were no TSDs. Based on a database search, the targets for ce-IS1272 were IRs or "non-IRs". IS1272 included a larger structure with tandem duplications of the left (IRL) side sequence; tnp included minor cases of a long fusion form and truncated form. One ce-IS1272 was associated with the segments responsible for immune evasion and drug resistance. Regarding virulence, GN1 expressed cytolytic peptides (phenol-soluble modulin α and δ-hemolysin) and PVL more strongly than some other familial strains. These results suggest that IS1272 transposes through an IR-replacing mechanism, with an irreversible process unlike that of "canonical" transpositions, resulting in genomic variations, and that, among the familial strains, the patient strain has strong virulence potential based on community-associated virulence factors.

Metabolomic Profiling as a Possible Reverse Engineering Tool for Estimating Processing Conditions of Dry-Cured Hams.

Dry-cured hams are popular among consumers. To increase the attractiveness of the product, objective analytical methods and algorithms to evaluate the relationship between observable properties and consumer acceptability are required. In this study, metabolomics, which is used for quantitative profiling of hundreds of small molecules, was applied to 12 kinds of dry-cured hams from Japan and Europe. In total, 203 charged metabolites, including amino acids, organic acids, nucleotides, and peptides, were successfully identified and quantified. Metabolite profiles were compared for the samples with different countries of origin and processing methods (e.g., smoking or use of a starter culture). Principal component analysis of the metabolite profiles with sensory properties revealed significant correlations for redness, homogeneity, and fat whiteness. This approach could be used to design new ham products by objective evaluation of various features.

Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and (13)C metabolic flux analysis.

BACKGROUND: Glycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. However, the main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ΔptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. The mutant can recover normal growth by co-utilization of glycerol and glucose after loss of glucose PTS transporter. To reveal the metabolic potential of the ΔptsGglpK* mutant, this study examined the flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant. RESULTS: By labeling experiments using [1,3-(13)C]glycerol and [1-(13)C]glucose, (13)C metabolic flux analysis was employed to decipher the metabolisms of both the wild-type strain and the ΔptsGglpK* mutant in chemostat cultures. When cells were maintained at a low dilution rate (0.1 h(-1)), the two strains showed similar fluxome profiles. When the dilution rate was increased, both strains upgraded their pentose phosphate pathway, glycolysis and anaplerotic reactions, while the ΔptsGglpK* mutant was able to catabolize much more glycerol than glucose (more than tenfold higher). Compared with the wild-type strain, the mutant repressed its flux through the TCA cycle, resulting in higher acetate overflow. The regulation of fluxomes was consistent with transcriptional profiling of several key genes relevant to the TCA cycle and transhydrogenase, namely gltA, icdA, sdhA and pntA. In addition, cofactor fluxes and their pool sizes were determined. The ΔptsGglpK* mutant affected the redox NADPH/NADH state and reduced the ATP level. Redox signaling activated the ArcA regulatory system, which was responsible for TCA cycle repression. CONCLUSIONS: This work employs both (13)C-MFA and transcription/metabolite analysis for quantitative investigation of the co-metabolism of glycerol and glucose in the ΔptsGglpK* mutant. The ArcA regulatory system dominates the control of flux redistribution. The ΔptsGglpK* mutant can be used as a platform for microbial cell factories for the production of biofuels and biochemicals, since most of fuel molecule (e.g., alcohols) synthesis requires excess reducing equivalents.

Simultaneous analysis of consumer variables, acceptability and sensory characteristics of dry-cured ham.

We conducted a consumer acceptability analysis of dry-cured ham based on sensory evaluation. Consumer acceptability data are rendered heterogeneous by the diverse backgrounds and assessment abilities of the participants, requiring versatile analytical methods for their interpretation. Totally, 9 sensory attributes of 12 kinds of dry-cured ham samples collected from Japan (n=9), Italy (n=1), Spain (n=1), and Germany (n=1) were tasted by 117 Japanese consumers who showed acceptable evaluation abilities during blind sampling. Common techniques, such as hierarchical clustering, principal component analysis, and external preference mapping, were simultaneously utilized to analyze each characteristics scored in modified hedonic scale. These analyses revealed the relationships between the features and preferences of the assessors. For example, consumers aged 20-30 with smoking and drinking habits preferred sweetness and saltiness, and gave high ratings to Spanish Jómon serrano and Italian prosciutto. Our approach could assist ham marketers to identify potential purchasers and the preferred characteristics of their products.

Capillary electrophoresis-mass spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) has proven to be useful for metabolomics studies. Charged metabolites are first separated by CE based on charge and size and are subsequently selectively detected using MS. The major advantages of CE-MS are its high resolution and the fact that almost any charged species can be analyzed by two methods, both cationic and anionic. This technique can readily be applied to various types of biological samples originating from bacteria, plants, mammals, and body fluids. This chapter highlights detailed practical procedures for using this technology.

Structure and immunocytochemical localization of photosynthetic enzymes in the lamina joint and sheath pulvinus of the C4 grass Arundinella hirta.

The C(4) grass Arundinella hirta exhibits a unique C(4) anatomy, with isolated Kranz cells (distinctive cells) and C(4)-type expression of photosynthetic enzymes in the leaf sheath and stem as well as in the leaf blade. The border zones between these organs are pale green. Those between the leaf blade and sheath and between the sheath and stem are called the lamina joint and sheath pulvinus, respectively, and are involved in gravity sensing. We investigated the structure and localization of C(3) and C(4) photosynthetic enzymes in these tissues. In both zones the epidermis lacked stomata. The inner tissue was composed of parenchyma cells and vascular bundles. The parenchyma cells were densely packed with small intercellular spaces and contained granal chloroplasts with large starch grains. No C(4)-type cellular differentiation was recognized. Western blot analysis showed that the lamina joint and pulvinus accumulated substantial amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate,Pi dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Immunogold electron microscopy revealed PEPC in the cytosol and both PPDK and rubisco in the chloroplasts of parenchyma cells, suggesting the occurrence of C(3) and C(4) enzymes within a single type of chlorenchyma cell. These data indicate that the lamina joint and pulvinus have unique expression patterns of C(3) and C(4) enzymes, unlike those in C(4)-type anatomy.

Introduction of the ZmDof1 gene into rice enhances carbon and nitrogen assimilation under low-nitrogen conditions.

The excessive application of nitrogen fertilizer to maximize crop yields causes negative environmental effects such as pollution and ecological imbalance. To overcome this problem, researchers have attempted to improve the nitrogen assimilation capacity of crops. Maize Dof1 (ZmDof1) is a plant-specific transcription factor shown to promote nitrogen assimilation in Arabidopsis thaliana (Arabidopsis) even under nitrogen-deficient conditions. The present study examines the effect of the introduction of the ZmDof1 gene on carbon and nitrogen assimilation in rice. ZmDof1 induced the expression of phosphoenolpyruvate carboxylase (PEPC) genes in transgenic rice plants and transactivated the PEPC promoters in protoplast transient assays, showing similar effects in rice as in Arabidopsis. Transgenic rice expressing ZmDof1 and grown in the presence of 360 µm (nitrogen-sufficient) or 90 µm (nitrogen-deficient) of nitrogen concentrations showed modulation of metabolite content and gene expression associated with the anaplerotic pathway for the TCA cycle, suggesting an increased carbon flow towards nitrogen assimilation. Furthermore, increases in carbon and nitrogen amounts per seedling were found in Dof1 rice grown under nitrogen-deficient conditions. Nitrogen deficiency also resulted in the predominant distribution of nitrogen to roots, accompanied by significant increases in root biomass and modification of the shoot-to-root ratio. Measurement of the CO2 gas exchange rate showed a significant increase in the net photosynthesis rate in Dof1 rice under nitrogen-deficient conditions. Taken these together, the present study displayed that ZmDof1 expression in rice could induce gene expressions such as PEPC genes, modulate carbon and nitrogen metabolites, increase nitrogen assimilation and enhance growth under low-nitrogen conditions.

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary.

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA).

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4-10.2 times higher V(max) value and 15.9-43.1 times higher K(m) value for NH(4)(+), compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with (15)NH(4)(+) showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH(4)(+) absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta.

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades--namely, the leaf sheath, stem, scale leaf, and constituents of the spike--also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

Leaf vascular systems in C(3) and C(4) grasses: a two-dimensional analysis.

BACKGROUND AND AIMS: It is well documented that C(4) grasses have a shorter distance between longitudinal veins in the leaves than C(3) grasses. In grass leaves, however, veins with different structures and functions are differentiated: large longitudinal veins, small longitudinal veins and transverse veins. Thus, the densities of the three types of vein in leaves of C(3) and C(4) grasses were investigated from a two-dimensional perspective. METHODS: Vein densities in cleared leaves of 15 C(3) and 26 C(4) grasses representing different taxonomic groups and photosynthetic subtypes were analysed. KEY RESULTS: The C(4) grasses had denser transverse veins and denser small longitudinal veins than the C(3) grasses (1.9 and 2.1 times in interveinal distance), but there was no significant difference in large longitudinal veins. The total length of the three vein types per unit area in the C(4) grasses was 2.1 times that in the C(3) grasses. The ratio of transverse vein length to total vein length was 14.3 % in C(3) grasses and 9.9 % in C(4) grasses. The C(3) grasses generally had greater species variation in the vascular distances than the C(4) grasses. The bambusoid and panicoid C(3) grasses tended to have a denser vascular system than the festucoid C(3) grasses. There were no significant differences in the interveinal distances of the three vein types between C(4) subtypes, although the NADP-malic enzyme grasses tended to have a shorter distance between small longitudinal veins than the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses. CONCLUSIONS: It seems that C(4) grasses have structurally a superior photosynthate translocation and water distribution system by developing denser networks of small longitudinal and transverse veins, while keeping a constant density of large longitudinal veins. The bambusoid and panicoid C(3) grasses have a vascular system that is more similar to that in C(4) grasses than to that in the festucoid C(3) grasses.

Cellular expression of C3 and C4 photosynthetic enzymes in the amphibious sedge Eleocharis retroflexa ssp. chaetaria.

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C(4)-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C(3) and C(4) enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C(4) pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C(4) pattern expression in this plant.

Photosynthetic enzyme accumulation during leaf development of Arundinella hirta, a C4 grass having Kranz cells not associated with veins.

The leaf of the NADP-malic enzyme type C(4) grass, Arundinella hirta, has not only mesophyll cells (MCs) and bundle sheath cells (BSCs, usual Kranz cells) but also another type of Kranz cells (distinctive cells; DCs) that are not associated with vascular bundles. We investigated photosynthetic enzyme accumulation along the base-to-tip maturation gradient of developing leaves by immunogold electron microscopy. In mature leaves, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were detected in the MC cytosol and in the BSC and DC chloroplasts, respectively. Pyruvate, P(i) dikinase (PPDK) was present in the chloroplasts of all photosynthetic cells but with higher levels in the MCs. Rubisco was first detected in the basal region of emerging leaf blades where the BSCs and DCs became discernable. Subsequently, the accumulation of PEPC and PPDK was initiated in the region where the granal proliferation in the chloroplasts was conspicuous; and, suberized lamellae were formed in the cell walls of the Kranz cells. There was no difference in the patterns of cellular development and enzyme accumulation between the BSCs and DCs or between the MCs adjacent to each type of Kranz cells. These results demonstrate that, although the DCs are not associated with veins, they behaved like BSCs with respect to enzyme induction and cellular differentiation.