Maize Bundle Sheath Research Articles

Epi-Brassinolide Regulates ZmC4 NADP-ME Expression through the Transcription Factors ZmbHLH157 and ZmNF-YC2

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO2 from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the -1616 bp and -1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Light-Dependent Reactions of Photosynthesis in Mesophyll and Bundle Sheath Chloroplasts of C4 Plant Maize. How Our Views Have Changed in Recent Years

Abstract Plants experience a range of light intensities and qualities in their environment. Leaves are subjected to spatial and temporal gradients in incident light, which has major consequences in the photosynthetic carbon assimilation. Plants acclimate to light by developing a range of mechanisms, from adjustments in leaf morphology to changes in the photosynthetic apparatus stoichiometry. In C4 plants, light intensity is a major limiting factor for photosynthesis at optimum temperatures. Under limiting light, it is not clear if all of factors (e.g., temperature, mineral nutrition, water supply) are co-limiting or if there is one primary limitation. Differences in light quality and intensity have a profound impact on C4 photosynthesis, where pathways require metabolic coordination of the mesophyll and bundle sheath cycles. Changes in the linear versus cyclic electron flux in maize (NADP-malic enzyme C4 subtype) in the mesophyll and bundle sheath chloroplasts in response to light may lead to an imbalance in the coordination of the C3 and C4 pathways. Additionally, the rearrangement of the thylakoid complexes of both types of chloroplasts in maize optimizes the light energy distribution between the mesophyll and bundle sheath cells and may also participate in energy dissipation. This review aims to highlight the changes in the understanding of the functions of photosystem II in maize bundle sheath chloroplasts and the role of super and megacomplexes in the thylakoids.

Qualitative and quantitative evaluation of thylakoid complexes separated by Blue Native PAGE

BackgroundBlue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging.ResultsUsing a detergent mixture of 1% (w/V) n-dodecyl-β-d-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3–8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed.ConclusionsHere we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes.

Network and epigenetic characterization of subsets of genes specifically expressed in maize bundle sheath cells

Bundle sheath (BS) cells exhibit dramatically structural differences and functional variations at physiological, biochemical and epigenetic levels as compared to mesophyll (M) cells in maize. The regulatory mechanisms controlling functional divergences between M and BS have been extensively investigated. However, BS cell-related regulatory networks are still not completely characterized. To address this, we herein conducted WGCNA-related co-expression assays using bulk M and BS cell RNA-seq data sets and identified a module containing 384 genes highly expressed in BS cells (including 20 hub TFs) instead of M cells. According to the hub TF centered regulatory network, we found that Dof22 and Dof30 might act as key regulators in the regulation of expression of BS-specific genes, and several MYB TFs exhibited a high collaboration with Dof TFs. By comparing the enrichment levels of histone modifications, we found that genes in the aforementioned module were more enriched with histone acetylation as compared to other BS-enriched DEGs with similar expression levels. Moreover, we found that a subset of genes functioning in photosynthesis, protein auto processing and enzymatic activities were significantly enriched with broad H3K4me3. Thus, our study provides evidence showing that regulatory network and histone modifications may play vital roles in the regulation of a subset of genes with important functions in BS cells.

Biochemical properties and ultrastructure of mesophyll and bundle sheath thylakoids from maize (Zea mays) chloroplasts.

A characteristic feature of C4 plants is the differentiation of the photosynthetic leaf tissues into two distinct cell types: mesophyll (M) and bundle sheath (BS) cells. We have investigated several biochemical parameters, including pigment composition, polypeptide patterns, fluorescence at 77K, the activity of photosystems and ultrastructure of mesophyll and bundle sheath chloroplasts of maize (Zea mays L.) plants. It is shown that the BS chloroplasts have ~2-fold higher chlorophyll a/b ratio than M chloroplasts, 6.15 and 3.12 respectively. The PSI apoprotein (68 kDa) was more abundant in BS than in M thylakoids. Polypeptides belonging to PSII core antenna, are in similar amounts in both types of membranes, but the 45kDa band is more intensive in M thylakoids. Polypeptides in the region of 28-24 kDa of the light-harvesting complex of PSII (LHCII) are also present in both types of chloroplasts, though their amounts are reduced in BS thylakoids. The chlorophyll fluorescence emission spectra in M cells showed the presence of three bands at 686, 695 and 735 nm characteristics of LHCII, PSII core and PSI complexes, respectively. However, in the fluorescence spectrum of agranal plastids, there are almost traces of the band at 695 nm, which belongs to the PSII core complex. The research results revealed that the photochemical activity of PSII in BS chloroplasts is ~5 times less than in the chloroplasts of M cells. The highest PSI activity was found in maize BS chloroplasts.

Tissue-specific Hi-C analyses of rice, foxtail millet and maize suggest non-canonical function of plant chromatin domains.

Chromatins are not randomly packaged in the nucleus and their organization plays important roles in transcription regulation, which is best studied in the mammalian models. Using in situ Hi-C, we have compared the 3D chromatin architectures of rice mesophyll and endosperm, foxtail millet bundle sheath and mesophyll, and maize bundle sheath, mesophyll and endosperm tissues. We found that their global A/B compartment partitions are stable across tissues, while local A/B compartment has tissue-specific dynamic associated with differential gene expression. Plant domains are largely stable across tissues, while new domain border formations are often associated with transcriptional activation in the region. Genes inside plant domains are not conserved across species, and lack significant co-expression behavior unlike those in mammalian TADs. Although we only observed chromatin loops between gene islands in the large genomes, the maize loop gene pairs' syntenic orthologs have shorter physical distances in small genome monocots, suggesting that loops instead of domains might have conserved biological function. Our study showed that plants' chromatin features might not have conserved biological functions as the mammalian ones.

Maize bundle sheath chloroplasts - a unique model of permanent State 2

The effect of high intensity of far red (FR) light on the energy distribution between PSII and PSI, measured as a change in the fluorescence emission in mesophyll (M) and bundle sheath (BS) chloroplasts of Zea mays was investigated in this paper. In bundle sheath thylakoids, FR light (730önm) significantly stimulates 77öK fluorescence of PSII but does not change the PSI fluorescence. In mesophyll thylakoids, the FR light used after the white light increased and decreased the PSII and PSI fluorescence, respectively. This indicates that in M chloroplasts light quality controls photosynthetic state transitions. The blue native (BN) PAGE, followed by SDS PAGE demonstrated that LHCII proteins associated with PSI were phosphorylated in the BS thylakoids in FR light. In M chloroplasts FR-light induced dephosphorylation of LHCII and the detachment of antenna from PSI occurred. In the BS thylakoids, the FR light initiated dephosphorylation of free and aggregated LHCII antenna, the dephosphorylated antenna can bound to PSII, thereby strongly increase its fluorescence, while part of LHCII pool stays associated with PSI. Thus aggregates of LHCII tended to be resolubilized in FR light and this allowed LHCII to connect to PSII, without changes in the pool of PSI-LHCI-LHCII. Our data indicate that PSI is locked in State 2 in agranal bundle sheath chloroplasts of maize. These results allow us to better understand the complexity of regulatory mechanisms of state transitions activated in response to light changes in M and BS chloroplasts of maize.

Corrigendum: Freeze-quenched maize mesophyll and bundle sheath separation uncovers bias in previous tissue-specific RNA-Seq data.

The high efficiency of C4 photosynthesis relies on spatial division of labor, classically with initial carbon fixation in the mesophyll and carbon reduction in the bundle sheath. By employing grinding and serial filtration over liquid nitrogen, we enriched C4 tissues along a developing leaf gradient. This method treats both C4 tissues in an integrity-preserving and consistent manner, while allowing complementary measurements of metabolite abundance and enzyme activity, thus providing a comprehensive data set. Meta-analysis of this and the previous studies highlights the strengths and weaknesses of different C4 tissue separation techniques. While the method reported here achieves the least enrichment, it is the only one that shows neither strong 3' (degradation) bias, nor different severity of 3' bias between samples. The meta-analysis highlighted previously unappreciated observations, such as an accumulation of evidence that aspartate aminotransferase is more mesophyll specific than expected from the current NADP-ME C4 cycle model, and a shift in enrichment of protein synthesis genes from bundle sheath to mesophyll during development. The full comparative dataset is available for download, and a web visualization tool (available at http://www.plant-biochemistry.hhu.de/resources.html) facilitates comparison of the the Z. mays bundle sheath and mesophyll studies, their consistencies and their conflicts.

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells.

In maize (Zea mays), Bundle Sheath Defective2 (BSD2) plays an essential role in Rubisco biogenesis and is required for correct bundle sheath (BS) cell differentiation. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. This raises the possibility of additional BSD2 roles in cell development. To test this hypothesis, transgenic lines were created that overexpress and underexpress BSD2 in both BS and M cells, driven by the cell type-specific Rubisco Small Subunit (RBCS) or Phosphoenolpyruvate Carboxylase (PEPC) promoters or the ubiquitin promoter. Genetic crosses showed that each of the transgenes could complement Rubisco deficiency and seedling lethality conferred by the bsd2 mutation. This was unexpected, as RBCS-BSD2 lines lacked BSD2 in M chloroplasts and PEPC-BSD2 lines expressed half the wild-type BSD2 level in BS chloroplasts. We conclude that BSD2 does not play a vital role in M cells and that BS BSD2 is in excess of requirements for Rubisco accumulation. BSD2 levels did affect chloroplast coverage in BS cells. In PEPC-BSD2 lines, chloroplast coverage decreased 30% to 50%, whereas lines with increased BSD2 content exhibited a 25% increase. This suggests that BSD2 has an ancillary role in BS cells related to chloroplast size. Gas exchange showed decreased photosynthetic rates in PEPC-BSD2 lines despite restored Rubisco function, correlating with reduced chloroplast coverage and pointing to CO2 diffusion changes. Conversely, increased chloroplast coverage did not result in increased Rubisco abundance or photosynthetic rates. This suggests another limitation beyond chloroplast volume, most likely Rubisco biogenesis and/or turnover rates.

A maize bundle sheath defective mutation mapped on chromosome 1 between SSR markers umc1395 and umc1603

The bsd-pg (bundle sheath defective pale green) mutant is a novel maize mutation, controlled by a single recessive gene, which was isolated from offspring of maize plantlets regenerated from tissue callus of the maize inbred line 501. The characterization was that the biogenesis and development of the chloroplasts was mainly interfered in bundle sheath cells rather than in mesophyll cells. For mapping the bsd-pg, an F2 population was derived from a cross between the mutant bsd-pg and an inbred line Xianzao 17. Using specific locus amplified fragment sequencing (SLAF-Seq) technology, a total of 5 783 polymorphic SLAFs were analysed with 1 771 homozygous alleles between maternal and paternal parents. There were 49 SLAFs, which had a ratio of paternal to maternal alleles of 2:1 in bulked normal lines, and three trait-related candidate regions were obtained on chromosome 1 with a size of 3.945 Mb. For the fine mapping, new simple sequence repeats (SSRs) markers were designed by utilizing information of the B73 genome and the candidate regions were localized a size of 850 934 bp on chromosome 1 between umc1603 and umc1395, including 35 candidate genes. These results provide a foundation for the cloning of bsd-pg by map-based strategy, which is essential for revealing the functional differentiation and coordination of the two cell types, and helps to elucidate a comprehensive understanding of the C4 photosynthesis pathway and related processes in maize leaves.

The short-term response of Arabidopsis thaliana (C3) and Zea mays (C4) chloroplasts to red and far red light.

Light quality has various effects on photochemistry and protein phosphorylation in Zea mays and Arabidopsis thaliana thylakoids due to different degrees of light penetration across leaves and redox status in chloroplasts. The effect of the spectral quality of light (red, R and far red, FR) on the function of thylakoid proteins in Zea mays and Arabidopsis thaliana was investigated. It was concluded that red light stimulates PSII activity in A. thaliana thylakoids and in maize bundle sheath (BS) thylakoids, but not in mesophyll (M) thylakoids. The light quality did not change PSI activity in M thylakoids of maize. FR used after a white light period increased PSI activity significantly in maize BS and only slightly in A. thaliana thylakoids. As shown by blue native (BN)-PAGE followed by SDS-PAGE, proteins were differently phosphorylated in the thylakoids, indicating their different functions. FR light increased dephosphorylation of LHCII proteins in A. thaliana thylakoids, whereas in maize, dephosphorylation did not occur at all. The rate of phosphorylation was higher in maize BS than in M thylakoids. D1 protein phosphorylation increased in maize and decreased in A. thaliana upon irradiation with both R and growth light (white light, W). Light variations did not change the level of proteins in thylakoids. Our data strongly suggest that response to light quality is a species-dependent phenomenon. We concluded that the maize chloroplasts were differently stimulated, probably due to different degrees of light penetration across the leaf and thereby the redox status in the chloroplasts. These acclimation changes induced by light quality are important in the regulation of chloroplast membrane flexibility and thus its function.

The end of the line: can ferredoxin and ferredoxin NADP(H) oxidoreductase determine the fate of photosynthetic electrons?

At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems.

Scarecrow Plays a Role in Establishing Kranz Anatomy in Maize Leaves

More than a quarter of the primary productivity on land, and a large fraction of the food that humans consume, is contributed by plants that fix atmospheric CO(2) by C(4) photosynthesis. It has been estimated that transferring the C(4) pathway to C(3) crops could boost yield by 50% and also increase water use efficiency and reduce the need for fertilizer, particularly in dry, hot environments. The high productivity of maize (Zea mays), sugarcane (Saccharum spp.) and several emerging bioenergy grasses is due largely to C(4) photosynthesis, which is enabled by the orderly arrangement, in concentric rings, of specialized bundle sheath and mesophyll cells in leaves in a pattern known as Kranz anatomy. Here we show that PIN, the auxin efflux protein, is present in the end walls of maize bundle sheath cells, as it is in the endodermis of the root. Since this marker suggests the expression of endodermal genetic programs in bundle sheath cells, we determined whether the transcription factor SCARECROW, which regulates structural differentiation of the root endodermis, also plays a role in the development of Kranz anatomy in maize. Mutations in the Scarecrow gene result in proliferation of bundle sheath cells, abnormal differentiation of bundle sheath chloroplasts, vein disorientation, loss of minor veins and reduction of vein density. Further characterization of this signal transduction pathway should facilitate the transfer of the C(4) trait into C(3) crop species, including rice.

Response of the malate dehydrogenase system of maize mesophyll and bundle sheath to salt stress

In maize (Zea mays L., cv. Voronezhskaya-76) seedlings subjected to salinity, the values of indicators of stress response development (contents of proline and lactate, activity of peroxidase) were higher in the cells of mesophyll than in the bundle sheath. At short-term NaCl (150 mM) action, the main reactions of the total adaptation syndrome were located in the cell of mesophyll. At salinity, substantial rearrangements of the isoenzyme composition of the malate dehydrogenase (MDH) system main enzymes occurred, which determined cell energization, the synthesis of reducing equivalents, maintenance of the osmotic balance, and functioning of the Hatch-Slake cycle. The changes in some intermediate concentrations and MDH-system enzyme functioning occurring under stress conditions permit a suggestion that, in maize tissues subjected to salt stress, an additional metabolic pathway related to aspartate synthesis and transport is induced.

Developmental and cell type characterization of bundle sheath and mesophyll chloroplast transcript abundance in maize

The C4 grass Zea mays separates light and light-independent photosynthetic processes into two leaf cell types: bundle sheath (BS) and mesophyll (M). When mature, BS and M cells have anatomically and biochemically distinct chloroplasts that must cooperate to complete the process of photosynthesis. This report compares changes in transcript abundance between young and mature maize BS and M chloroplasts from specific segments of the leaf developmental gradient. Representative transcripts encoding components of Photosystem I, Photosystem II, Cytochrome b (6) f, thylakoidal NADH dehydrogenase; and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase as well as nine nuclear-coded transcripts encoding chloroplast proteins were measured using quantitative RT-PCR. In addition, 887 nuclear genes encoding plastid-localized proteins, as well as 64 chloroplast and 34 mitochondrial genes were assayed utilizing a cDNA microarray. In 9 out of the 18 chloroplast-encoded genes and 84 genes from the 985 element microarray revealed greater than twofold transcript abundance differences between developmental stages and/or cell types. Patterns for transcripts associated with operons and gene clusters suggest differing regulatory mechanisms for particular polycistronic stretches. In summary, this report provides evidence that cell type-specific transcript abundance varies more in the young developing chloroplast, and differences plateau or subside as chloroplasts mature.

Cytokinin-binding protein (70 kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and its putative function

Cytokinins regulate chloroplast differentiation and functioning, but their targets in plastids are not known. In this connection, the plastid localization of the 70 kDa cytokinin-binding protein (CBP70) was studied immunocytochemically in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus) using monoclonal antibodies (mAbs) against CBP70 recognizing this protein not only in nuclei and cytoplasm, but also in plastids. CBP70 was detected in the amyloplasts of the root cap and etioplasts of the mesocotyl, stem apex, and leaves encircling the stem axis in the node. Immunogold electron microscopy demonstrated CBP70 localization in amyloplasts outside starch grains and revealed a dependence of CBP70 content in etioplasts on the degree of their inner membrane differentiation: the low CBP70 amount in etioplasts at the early stages of membrane development, the high content in etioplasts with actively developing membranes, and a considerable decrease in plastids with the formed prolamellar body. This suggests that CBP70 is involved in etioplast structure development. CBP70 was also observed in chloroplasts of the bundle sheath of green maize leaves. CBP70 purified from etioplasts mediated trans-zeatin-dependent activation of transcription elongation in vitro in the transcription systems of maize etioplasts and barley chloroplasts, suggesting that CBP70 is a plastid transcription elongation factor or a modulator of plastid elongation factor activity. CBP70 involvement in the cytokinin-dependent regulation of plastid transcription elongation could be essential for the cytokinin control of the biogenesis of this organelle.

Reconstruction of Metabolic Pathways, Protein Expression, and Homeostasis Machineries across Maize Bundle Sheath and Mesophyll Chloroplasts: Large-Scale Quantitative Proteomics Using the First Maize Genome Assembly

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C(4) photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.

Physical, chemical, and regulatory properties of glycolate oxidase in C3 and C4 plants

Physical, chemical, and regulatory properties of glycolate oxidase (GO) isolated from the leaves of C4 and C3 plants (Zea mays L., cv. Voronezhskaya 76 and Glycine max (L.) Merr., cv. Pripyat’, respectively) were studied. The homogenous preparations were obtained by multistage enzyme purification from soybean leaves and maize mesophyll and bundle sheath. The glycolate oxidase (GO) preparations obtained consisted of two types of subunits, 37 and 44 kD. The GO isolated from C3 plant leaves had many in common with that extracted from C4 plant bundle sheath as regards physical, chemical, and catalytic properties. The primary function of GO in both plant types is metabolism of glycolate, which is a product of ribulosebisphosphate oxalacetic acid oxidation and is used by plants for biosynthesis of hydrocarbons and amino acids.

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

Implication of the glutamine synthetase/glutamate synthase pathway in conditioning the amino acid metabolism in bundle sheath and mesophyll cells of maize leaves

We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADH-GOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L. The plants responded to a light/dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ. The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase. We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP(+) oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor. The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf. The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates. Taken together, we conclude that the GS1/ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization.