Non-photosynthetic Cells Research Articles

Developmental and photosynthetic regulation of δ-endotoxin reveals that engineered sugarcane conferring resistance to ‘dead heart’ contains no toxins in cane juice

The phosphoenolpyruvate (PEP) carboxylase is regulated at the levels of transcription and post-translation in C4 plants in light and abundantly accumulates in leaf mesophyll cells. We report here developmental and photosynthetic regulation of stably accumulated Bacillus thuringiensis δ-endotoxin under the control of PEP-C promoter in transgenic sugarcane. In young leaves of plants, the transprotein is accumulated to 39% of the levels in mature leaves (135 ng mg(-1)), and is induced with the cell development, from base to tip. Nevertheless, these levels are decreased up to 99.98% in non-photosynthetic cells as cane matures, from top to bottom, suggesting the photosynthesis regulation of δ-endotoxin in cane cells. Further, transgenic plants are highly resistant to 'dead heart'. In these studies, Scirpophaga nivela larvae causing 'dead heart' were killed within one hour of release to the transgenic plants. Therefore, this report may be regarded as the first report that provides a better strategy for developing transgenic sugarcane lines with absolute protection against invading larvae and no toxin residues in cane juice.

Bacterial responses to photo-oxidative stress

Singlet oxygen is one of several reactive oxygen species that can destroy biomolecules, microorganisms and other cells. Traditionally, the response to singlet oxygen has been termed photo-oxidative stress, as light-dependent processes in photosynthetic cells are major biological sources of singlet oxygen. Recent work identifying a core set of singlet oxygen stress response genes across various bacterial species highlights the importance of this response for survival by both photosynthetic and non-photosynthetic cells. Here, we review how bacterial cells mount a transcriptional response to photo-oxidative stress in the context of what is known about bacterial stress responses to other reactive oxygen species.

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells. These complex organelles also play essential roles in apoptotic cell death, cell survival, mammalian development, neuronal development and function, intracellular signalling, and longevity regulation. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

Effects of chloroplast dysfunction in a subpopulation of leaf mesophyll cells on photosynthetic and respiratory activities of a whole leaf: A study using variegated leaves of Hedera helix L.

A variegated leaf of Hedera helix is composed of green and white mesophyll cells with and without photosynthetic capacity, respectively. We measured the photosynthesis and dark respiration rates, as well as CO2 compensation points, of H. helix leaves with various extents of variegation. The photosynthetic rate (on an area basis) of the variegated leaves increased almost linearly according to the increase in the proportion of green area to total leaf area. In contrast, dark respiration rate was nearly constant irrespective of the extent of leaf variegation. These results suggest that chloroplast dysfunction in white mesophyll cells did not drastically affect photosynthetic activity of green mesophyll cells, or respiratory rates of both green and white mesophyll cells. CO2 compensation point was elevated when the proportion of green area became extremely low, indicating that the proportion of non-photosynthetic cells within a photosynthetic organ could affect its CO2 compensation point.

Low-Intensity Light Therapy: Exploring the Role of Redox Mechanisms

Low-intensity light therapy (LILT) appears to be working through newly recognized photoacceptor systems. The mitochondrial electron transport chain has been shown to be photosensitive to red and near-infrared (NIR) light. Although the underlying mechanisms have not yet been clearly elucidated, mitochondrial photostimulation has been shown to increase ATP production and cause transient increases in reactive oxygen species (ROS). In some cells, this process appears to participate in reduction/oxidation (redox) signaling. Redox mechanisms are known to be involved in cellular homeostasis and proliferative control. In plants, photostimulation of the analogous photosynthetic electron transport chain leads to redox signaling known to be integral to cellular function. In gene therapy research, ultraviolet lasers are being used to photostimulate cells through a process that also appears to involve redox signaling. It seems that visible and near visible low-intensity light can be used to modulate cellular physiology in some nonphotosynthetic cells, acting through existing redox mechanisms of cellular physiology. In this manner, LILT may act to promote proliferation and/or cellular homeostasis. Understanding the role of redox state and signaling in LILT may be useful in guiding future therapies, particularly in conditions associated with pro-oxidant conditions.

Heterogeneity of the Mitochondrial Proteome for Photosynthetic and Non-photosynthetic Arabidopsis Metabolism

Heterogeneity of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles in different tissues. We quantitatively compared the mitochondrial proteome isolated from a non-photosynthetic cell culture model with more specialized mitochondria isolated from photosynthetic shoots. Differences in intact mitochondrial respiratory rates with various substrates and activities of specific enzymes provided a backdrop of the functional variation between these mitochondrial populations. Proteomics comparisons provided a deep insight into the different steady-state abundances of specific mitochondrial proteins. Combined these data showed the elevated level of the photorespiratory apparatus and its complex interplay with glycolate, cysteine, formate, and one-carbon metabolism as well as the decrease of selected parts of the tricarboxylic acid cycle, alterations in amino acid metabolism focused on 2-oxoglutarate generation, and degradation of branched chain amino acids. Comparisons with microarray analysis of these tissue types showed a positive, mild correlation between mRNA and mitochondrial protein abundance, a tighter correlation for specific biochemical pathways, but over 78% concordance in direction between changes in protein and transcript abundance in the two tissues. Overall these results indicated that the majority of the variation in the plant mitochondrial proteome occurred in the matrix, highlighted the constitutive nature of the respiratory apparatus, and showed the differences in substrate choice and/or availability during photosynthetic and non-photosynthetic metabolism.

Structure and Function of Plant-Type Ferredoxins

The plant-type ferredoxins (Fds) are the [2Fe-2S] proteins that function primarily in photosynthesis; they transfer electrons from photoreduced Photosystem I to ferredoxin NADP(+) reductase in which NADPH is produced for CO(2) assimilation. In addition, Fds partition electrons to various ferredoxin-dependent enzymes not only for assimilations of inorganic nitrogen and sulfur and N(2) fixation but also for regulation of CO(2) assimilation cycle. Although Fds are small iron-sulfur proteins with molecular weight of 11 KDa, they are expected to interact with surprisingly many enzymes. Several Fd isoforms were found in non-photosynthetic cells as well as Fds in photosynthetic cells, leading to the recognition that they have differentiated physiological roles. In a quarter of century, X-ray crystallography and NMR spectroscopy provided wealth of structural data, which shed light on the structure-function relationship of the plant-type Fds and gave structural basis for the biochemical and spectroscopic properties so far accumulated. Thus the structural studies of Fds have come to a new era in which different roles of Fds and interactions with various enzymes are clarified on the basis of the tertiary and quaternary structures, although they are premature at present. This article reviews briefly the structures of the plant-type Fds together with their functions, properties, and interactions with Fd related enzymes. Lastly the folding motif of Fd, that has grown to be a large family by including many functionally unrelated proteins, is noted.

Light and Nitrate Utilization by Soybean Callus Cells

We studied the role of light during exogenous assimilation of nitrate (as the only source of nitrogen) by the callus cells of soybean (Glycine max). The nitrate absorbed and assimilated by the photosynthetic (mixotrophic) and non-photosynthetic cells (heterotrophic and chlorophyll-containing cells cultivated in the light in the same medium with diuron) was quantified. The assimilated nitrate was quantified at the final stage of the growth cycle as the difference between the amount of nitrogen absorbed from the medium and the amount of endogenous nitrate in the cells. Comparison of the assimilated nitrate quantities per accumulated dry biomass between the photosynthetic and non-photosynthetic cells demonstrated that nearly 30% of nitrate is assimilated with the help of photosynthesis in a mixotrophic culture when nitrate is the only source of nitrogen.

Cell-specific measurement of cytosolic glutathione in poplar leaves.

The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves or roots; however, there is little information on the effect that such asymmetries have on the actual GSH concentration in each compartment or cell type. In the present study in situ labelling with monochlorobimane (MCB) in combination with confocal laser scanning microscopy was used to quantify GSH in each of the main cell types of poplar leaves from fluorescence of the GSB conjugate formed. Comparison of results from the in situ approach with total GSH levels measured in vitro by high-performance liquid chromatography suggested that only the cytosolic GSH pool was labelled using this approach. This suggests that an appropriate GST was not present within the chloroplasts to catalyse the conjugation reaction and that chloroplastic GSH does not rapidly exchange with the cytoplasmic pool under the conditions of the assay. Cytosolic GSH levels were between 0.2 and 0.3 mm for both photosynthetic and non-photosynthetic (epidermal) cell types in wild-type poplar leaves. Cytosolic levels increased by around two-fold in transgenic poplars over-expressing bacterial gamma-glutamylsynthetase (gamma-ECS) in the cytosol of all cell types, but there was no concomitant increase in the chloroplastic GSH pool.

Sequences upstream of the YRTA core region are essential for transcription of the tobacco atpB NEP promoter in chloroplasts in vivo

Transcription of the plastid atpB gene is accomplished by plastid-encoded (PEP) and nuclear-encoded (NEP) RNA polymerases. In contrast to NEP promoters of many other plastid genes, the tobacco atpB NEP promoter exhibits robust activity in chloroplasts in vivo. Previously, in vitro transcription assays using extracts from non-photosynthetic cells identified two elements required for full atpB NEP promoter activity, a core sequence and an upstream GAA box. Of these, only the core sequence containing the motif YRTA is conserved in the majority of NEP promoters. We used plastid transformation to examine the requirements for atpB NEP promoter activity in chloroplasts. Our results demonstrate that sequences upstream of the core element are essential for promoter activity in vivo, and that transcription of the NEP promoter in chloroplasts is not dependent on activity from an overlapping PEP promoter.

Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.

From chloroplast to the nucleus and back again

Recent data suggest that alterations of biosynthetic pathways could guide plants to produce more of a desired dietary component. In this case, tobacco produced ten times its usual amount of tryptophan, an amino acid often in short supply in the human diet. Scientists inserted a gene encoding the enzyme that controls tryptophan production into the chloroplast genome. The approach is essentially a reversal of evolution. ‘The biosynthesis of tryptophan and other essential amino acids occurs in these plastids,’ said author Archie R. Portis, Jr, a University of Illinois crop scientist. ‘However, the genes encoding these enzymes are located in the nucleus and the proteins are imported into the plastids.’ Plastids in today's plants are believed to have evolved some 2 billion years ago from a unicellular, photosynthetic cyanobacteria that was engulfed by non-photosynthetic cells. ‘Most of the genes originally located in these early plastids moved to the nucleus,’ Portis said. ‘It is likely that those required for tryptophan biosynthesis were among these.’ The gene the researchers inserted was isolated previously from a tobacco suspension culture that had been selected for resistance to an inhibitor of tryptophan biosynthesis. (Zhang, X.H. et al. [2001] Plant Physiology 127, 131–141) PL

Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy.

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues

sketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to between phosphorylated 3-, 4-, 5-, 6-, and 7-carbon sugars catalysed by the enzymes ribose 5-phosphate isomerase the plastid. In tobacco root and leaf, however, the non-oxidative enzymes were found in the cytosolic as (Rib5P isomerase), ribulose 5-phosphate 3-epimerase (Rul5P 3-epimerase), transaldolase ( TA), and transketolwell as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose ase ( TK ). With the exception of the transaldolase, these reversible enzymes are amphibolic, and also play a part 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose in the Calvin cycle. Their function as part of the pentose phosphate pathway is to provide carbon skeletons for the 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was sup- shikimate pathway via erythrose 4-phosphate, and nucleotide synthesis utilizing ribose 5-phosphate, as well as plied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. recycling of sugar phosphate intermediates for use in the glycolytic pathway (ap Rees, 1985). The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that The enzymes of the oxidative portion of the pathway are commonly found in both the cytosolic and plastidic the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, fractions of photosynthetic (Herbert et al., 1979) and non-photosynthetic cells (Ashihara and Komamine, 1976; indicating the presence of a translocator capable of transporting pentose phosphates.

Immunogold Localization and Quantitative Distribution of Two Proteins of the Glycine Decarboxylase Complex in Developing Leaflets of Pea (Pisum sativum L.)

We investigated the distribution of the P- and H-proteins of the glycine decarboxylase complex (EC 2.1.2.10) in developing pea (Pisum sativum L.) leaflets using immunogold techniques. Antibodies to these two proteins did not bind to young, nonphotosynthetic cells. Once the leaflet turned green, the labeling density in the mitochondria of the mesophyll cells increased substantially. Few particles were found in the mitochondria of the paraveinal mesophyll and guard cells. None were found either in the epidermis or in the parenchyma of the vascular tissue. Thus leaflets not yet exposed to light, and nonphotosynthetic cells, do not express these two proteins needed for photorespiration. The process of photorespiration seems to be correlated with the photosynthetic state of the cell and of the organ. It was also found to be positively linked with the integrity of the chloroplast since the highest label density obtained in the mitochondria was found just before the onset of ultrastructural senescence in the chl...

The Plastid-encoded ccsA Gene Is Required for Heme Attachment to Chloroplast c-type Cytochromes

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Effect of Paraquat on Dark-GrownPhaseolus vulgarisCells

Paraquat is known to affect all green plants and other eukaryotic organisms including mammalian cells. The aim of this study was to improve the understanding of paraquat toxicity in nonphotosynthetic plant cells using dark-grown kidney bean cells in tissue culture. It is shown that uptake of paraquat is an active process, and that paraquat inhibits cell growth, reduces DNA synthesis, and inhibits the activity of hydroxypyruvate reductase while enhancing the activity of glutathione reductase which is involved in cellular defense against oxidant stress. Additionally, it is demonstrated that iron ions are involved in paraquat toxicity. We conclude that uptake of paraquat into cells is via polyamine channels and that the deleterious effects of paraquat on these nonphotosynthetic cells are mediated by iron.

Promoter and leader regions involved in the expression of the Arabidopsis ferredoxin A gene.

Ferredoxin is a nuclear-encoded protein that is involved in a variety of electron transfer reactions in both photosynthetic and non-photosynthetic plastids. We show here that the expression of the ferredoxin A gene (FedA) in Arabidopsis thaliana is light-regulated, with its mRNA level increased 4.5-fold by transfer of dark-grown seedlings to white light for 3 h. A portion of this light regulation is mediated by phytochrome through a very low fluence type of response. In addition, it is likely that another photoreceptor(s) is also involved. The FedA promoter confers a light- and tissue-regulated expression pattern when fused to the beta-glucuronidase (GUS) and luciferase reporter genes, indicating that the gene is transcriptionally regulated. No evidence of cis-acting light-regulatory elements within the 5' untranslated leader region of the gene was detected. Nevertheless, elements within this leader are required for full activity since its deletion reduces expression both in the light and dark by 25-fold. This region includes a sequence, ACAAAA, which is also present in the 5' untranslated leader of the other three ferredoxin leaders that have been sequenced and in the leaders of 31 other plant genes or about 8% of all plant genes in the GenBank database. In addition to mediating light regulation, the FedA promoter also directs GUS expression in aerial tissues at 70-fold higher levels than in roots. GUS activity staining in aerial tissues is observed in both photosynthetic and non-photosynthetic cells. These data indicate that the FedA promoter also carries the information for expression of this gene in a highly tissue- and cell-specific manner.

Conservation and Structural Divergence of Organellar DNA and Gene Expression in Non‐Photosynthetic Plastids During Ontogenetic Differentiation and Phylogenetic Adaptation

Plastids of non-photosynthetic cells or tissues, such as chromoplasts or leukoplasts, which develop during the course of ontogenetic differentiation contain DNA which is identical to chloroplast DNA with respect to size, organization and gene content. Also in ribosome-deficient bleached plastids, produced in leaves by experimental treatments or mutation, chloroplast DNA remains unaltered. The chloroplast DNA of various bleached mutant strains of Euglena has suffered major deletions or rearrangements, but is, however, never totally lost. Also leukoplasts of parasitic higher plants contain DNA. In the organellar DNA of several parasitic plants photosynthetic genes are conserved. In the heterotrophic flagellate Astasia and in the holoparasite Epifagus virginiana (Orobanchaceae) the size of the plastid DNA is greatly reduced by major deletions and most or all photosynthetic genes or genes related to the chloroplastic respiratory chain are lost. The residual plastid genomes have, however, retained genes for RNAs, tRNAs and ribosomal polypeptides and these are transcribed, although plastidic RNA-polymerase genes are lost in Epifagus. These findings demand the existence of a nuclear-encoded RNA-polymerase. The relevance of the conservation of plastid DNA and of plastidic gene expression in non-photosynthetic cells is discussed, remains, however, at present elusive. Open reading frames of unknown function might be of particular significance for non-photosynthetic plastids.