Non-photosynthetic Tissues Research Articles

Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC–MS/MS

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC-MS/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.

The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis

Sweet potato is an important crop because of its high yield and biomass production. We herein investigated the potential of the promoter activity of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) from sweet potato (Ipomoea batatas) in order to develop the high expression system of exogenous DNA in Arabidopsis. We isolated two different cDNAs (IbRbcS1 and IbRbcS2) encoding RbcS from sweet potato. Their predicted amino acid sequences were well conserved with the mature RbcS protein of other plants. The tissue-specific expression patterns of these two genes revealed that expression of IbRbcS1 was specific to green tissue, whereas that of IbRbcS2 was non-photosynthetic tissues such as roots and tubers. These results suggested that IbRbcS1 was predominantly expressed in the green tissue-specific of sweet potato over IbRbcS2. Therefore, the IbRbcS1 promoter was transformed into Arabidopsis along with β-glucuronidase (GUS) as a reporter gene. GUS staining and semi-quantitative RT-PCR showed that the IbRbcS1 promoter conferred the expression of the GUS reporter gene in green tissue-specific and light-inducible manners. Furthermore, qPCR showed that the expression levels of GUS reporter gene in IbRbcS1 pro:GUS were same as those in CaMV 35S pro:GUS plants. These results suggest that the IbRbcS1 promoter is a potentially strong foreign gene expression system for genetic transformation in plants.

Spectral Variability Analysis of In-Situ Hyperspectral Remote Sensing at Leaf and Branch Scales for Tree Species at Tropical Urban Forest

Spectral variability analysis has been carried out on in-situ hyperspectral remote sensing data for 20 tree species available in tropical forest in Malaysia. Five different spectral ranges have been tested to evaluate the influence of intra-species spectral variability at specific spectral range given by different spatial scales (i.e. leaf to branch scales). The degree of intra-species spectral variability was not constant among different spectral ranges where the influence of spatial scale towards intra-species spectral variability at these spectral ranges was found increasing from leaf to branch scale. The ratio of leaves to non-photosynthetic tissues has made branch scale significantly influent the intra-species spectral variability. Results have shown that a specific spectral range was species sensitive on the intra-species and inter-species spectral variability in this study. This study also suggested the use of species sensitive wavelengths extracted from specific spectral range in hyperspectral remote sensing data in order to achieve good accuracy in tree species classification.

A rapid method for landscape assessment of carbon storage and ecosystem function in moss and lichen ground layers

Mat-forming “ground layers” of mosses and lichens often have functional impacts disproportionate to their biomass, and are responsible for sequestering one-third of the world's terrestrial carbon as they regulate water tables, cool soils and inhibit microbial decomposition. Without reliable assessment tools, the potential effects of climate and land use changes on these functions remain unclear; therefore, we implemented a novel “Ground Layer Indicator” method as part of the U.S.D.A. Forest Inventory and Analysis (FIA) program. Non-destructive depth and cover measurements were used to estimate biomass, carbon and nitrogen content for nine moss and lichen functional groups among eight contrasted habitat types in Pacific Northwest and subarctic U.S.A. (N = 81 sites). Ground layer cover, volume, standing biomass, carbon content and functional group richness were greater in boreal forest and tundra habitats of Alaska compared to Oregon forest and steppe. Biomass of up to 22769 ± 2707 kg ha−1 (mean ± SE) in upland Picea mariana forests was nearly double other reports, likely because our method included viable, non-photosynthetic tissues. Functional group richness, which did not directly correspond with biomass, was greatest in lowland Picea mariana forests (7.1 ± 0.4 functional groups per site). Bootstrap resampling revealed that thirty-two microplots per site were adequate for meeting data quality objectives. Here we present a non-destructive, repeatable and efficient method (sampling time: ca. 60 min per site) for gauging ground layer functions and evaluating responses to ecosystem changes. High biomass and functional distinctiveness in Alaskan ground layers highlight the need for increased attention to currently under-sampled boreal and arctic regions, which are projected to be among the most active responders to climate change.

Profiling of spatial metabolite distributions in wheat leaves under normal and nitrate limiting conditions

The control and interaction between nitrogen and carbon assimilatory pathways is essential in both photosynthetic and non-photosynthetic tissue in order to support metabolic processes without compromising growth. Physiological differences between the basal and mature region of wheat (Triticum aestivum) primary leaves confirmed that there was a change from heterotrophic to autotrophic metabolism. Fourier Transform Infrared (FT-IR) spectroscopy confirmed the suitability and phenotypic reproducibility of the leaf growth conditions. Principal Component–Discriminant Function Analysis (PC–DFA) revealed distinct clustering between base, and tip sections of the developing wheat leaf, and from plants grown in the presence or absence of nitrate. Gas Chromatography-Time of Flight/Mass Spectrometry (GC-TOF/MS) combined with multivariate and univariate analyses, and Bayesian network (BN) analysis, distinguished different tissues and confirmed the physiological switch from high rates of respiration to photosynthesis along the leaf. The operation of nitrogen metabolism impacted on the levels and distribution of amino acids, organic acids and carbohydrates within the wheat leaf. In plants grown in the presence of nitrate there was reduced levels of a number of sugar metabolites in the leaf base and an increase in maltose levels, possibly reflecting an increase in starch turnover. The value of using this combined metabolomics analysis for further functional investigations in the future are discussed.

Impact of phenanthrene exposure on activities of nitrate reductase, phosphoenolpyruvate carboxylase, vacuolar H+-pyrophosphatase and plasma membrane H+-ATPase in roots of soybean, wheat and carrot

Crops differ in uptake of polycyclic aromatic hydrocarbons (PAHs). However, the physiological mechanism underlying the differences in PAH uptake among crop species is unclear. As PAH transport by H+-coupled symporters may affect cytosolic pH, and intracellular pH is precisely regulated for metabolism, four enzymes linked with cytosolic pH regulation in root cells were determined at different concentrations of phenanthrene (PHE) and pHs. Activity of nitrate reductase (NRase) is stimulated by PHE and follows the order: wheat>carrot>soybean. The optimum pH of NRase is 7.5 in wheat and carrot, while 6.5 in soybean. The activity of phosphoenolpyruvate carboxylase (PEPCase) is generally decreased by PHE and always lower in soybean than in wheat and carrot. PEPCase peaks its maximum activity at pH 7.0 and wheat shows the highest value with high PHE (1.2mgL−1) at pHs of 7.0 and 8.0 in comparison with carrot and soybean. Vacuolar H+-pyrophosphatase (V-H+-PPase) is promoted by PHE in wheat and carrot, while it does not change significantly in soybean (P>0.05). V-H+-PPase reaches its maximum value at pH 7.5 and follows the order of wheat>carrot>soybean at alkaline medium with high PHE. Plasma membrane (PM) H+-ATPase is enhanced by K+ and follows the order: wheat>soybean>carrot. The orders of activity of these enzymes are not consistent with that of PHE uptake, indicating their various functions. However, in soybean, PM H+-ATPase activity under high PHE and the uptake of PHE are always higher than those in wheat and carrot, suggesting that PM H+-ATPase plays an essential role in PHE uptake. Therefore, the changes of PM H+-ATPase activity seem to be a direct consequence responding to PHE stress and those of NRase, PEPCase and V-H+-PPase might be the downstream accidents regulated by cytosoilc pH. Our study provides insight into the roles of these enzymes in non-photosynthetic tissues and supports their importance in the physiological responses to PHE stress among different crops.

Tocochromanols in wood: a potential new tool for dendrometabolomics.

Tocochromanols are the most abundant lipid-soluble antioxidants in plants. Among them, α-tocopherol (α-Toc) shows a particularly high sensitivity to environmental stressors and its content is used as a stress biomarker even in non-photosynthetic tissues. Nevertheless, the presence of tocochromanols has not been described yet in the xylem of woody plants, even when their functions regarding cell membrane protection and the transport of photoassimilates may be crucial in this tissue and despite its potential utility in dendrometabolomics. Considering all these, we aimed to determine the presence and distribution of tocochromanols in the xylem of woody plants, to examine their responsiveness to high temperature and to evaluate their potential as environmental bioindicators. The analysis of 29 phyllogenetically diverse species showed that α-Toc is the most abundant and frequent tocochromanol in the xylem and is ubiquitously present in all the studied species, with a concentration ranging from 0.5 to 39.3 μg g(-1) of dry weight. α-Tocopherol appeared to be mainly located in the parenchyma rays and was found in both the sapwood and the heartwood, suggesting that it is present even in dead parenchyma cells. The levels of α-Toc in the xylem did not change in response to locally induced xylem heating, but responded positively to the 3-year moving average of annual precipitation. The present findings suggest that α-Toc may be linked to changes in climatic stress. This should enhance further research on the environmental controls of α-Toc variation in the xylem as a first step towards a deeper understanding of dendrometabolomics.

Photosynthetic ecophysiology of evergreen leaves in the woody angiosperms – a review

Evergreen plants are an important component of many ecosystems of the world and occur in nu- merous evolutionary lineages. In this article we review phenotypic traits of evergreen woody angiosperms occurring in habitats that regularly experience frost. Leaf anatomical traits such as sclerenchymatic tissues or prominent cuticles ensure mechanical strength while often enhancing tolerance of water deficit. The low ratio of photosynthetic to nonphotosynthetic tissues as well as modified cell wall structure and nitrogen allocation patterns in evergreen leaves result in lower mass-based photosynthetic rate and photosynthetic nitrogen use efficiency in comparison with deciduous leaves. Their photosynthetic apparatus is adapted for the survival of frost in a down-regulated state with potential for photosynthetic activity in winter during periods of permissive temperatures. Leaf structure interacts with the mechanisms of frost survival. Stem xylem in evergreen plants tends to contain smaller diameter conduits incurring greater resistance to freeze/ thaw induced cavitation than in deciduous plants, although at the cost of reduced hydraulic efficiency. In contrast, no such differences in hydraulic conductivity have been documented at the leaf level. There is ev- idence for reduced structural plasticity of evergreen leaves in response to variability in irradiance, however photosynthetic downregulation occurs in mature leaves in response to self shading. Some evergreen species exhibit slow leaf development and delayed greening, while in many species aging is also a very protracted process. Finally, evergreen leaves may participate in carbohydrate and, less obviously, in nitrogen storage for the support of spring shoot and foliage growth, although the importance of this function is under debate. In conclusion, the evergreen leaf habit is correlated with numerous structural and functional traits at the leaf and also at the stem level. These correlations may generate trade-offs that shape the ecological strategies of evergreen plants.

Effective leaf area index retrieving from terrestrial point cloud data: coupling computational geometry application and Gaussian mixture model clustering

Abstract. Leaf area index (LAI) is one of the most important structural parameters of forestry studies which manifests the ability of the green vegetation interacted with the solar illumination. Classic understanding about LAI is to consider the green canopy as integration of horizontal leaf layers. Since multi-angle remote sensing technique developed, LAI obliged to be deliberated according to the observation geometry. Effective LAI could formulate the leaf-light interaction virtually and precisely. To retrieve the LAI/effective LAI from remotely sensed data therefore becomes a challenge during the past decades. Laser scanning technique can provide accurate surface echoed coordinates with densely scanned intervals. To utilize the density based statistical algorithm for analyzing the voluminous amount of the 3-D points data is one of the subjects of the laser scanning applications. Computational geometry also provides some mature applications for point cloud data (PCD) processing and analysing. In this paper, authors investigated the feasibility of a new application for retrieving the effective LAI of an isolated broad leaf tree. Simplified curvature was calculated for each point in order to remove those non-photosynthetic tissues. Then PCD were discretized into voxel, and clustered by using Gaussian mixture model. Subsequently the area of each cluster was calculated by employing the computational geometry applications. In order to validate our application, we chose an indoor plant to estimate the leaf area, the correlation coefficient between calculation and measurement was 98.28 %. We finally calculated the effective LAI of the tree with 6 × 6 assumed observation directions.

Molecular cloning, sequence analyses, and expression studies of sucrose-phosphate synthase in the potato (Solanum tuberosum L.) cultivars

Sucrose-phosphate synthase (SPS, EC 2.4.1.14) refers to a key enzyme in sucrose biosynthesis in both photosynthetic and nonphotosynthetic tissues of plants. It is encoded by different gene families. SPS exists in multiple forms which show differential distributions and functional specializations in the plant tissues. SPS activity is highly regulated by hierarchy of mechanisms including transcriptional control. Here, we report an isolation of a cDNA clone (3,591 bp) encoding full-length SPS A form consisting of 1,054 amino acids (designated KC-SPS1) from a commercially important Indian potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 by RT-PCR approach from tuber total RNA. KC-SPS1 shared 99 % sequence identity with 1,053-amino acid SPS from potato cv. Desiree. Apart from some prominent amino acid substitutions, one extra Met residue at position 235 made KC-SPS1 a distinct member of SPS A family in potato. Full-length SPS sequences from taxonomically different plant species were used in making a phylogenetic tree which showed both evolutionary relatedness, and also their grouping into different SPS families. Hydropathy characters and secondary structures were predicted in various SPS forms. Sequence analyses and comparison of the SPS sequences from the Solanaceae family members revealed many distinct features within and between the SPS gene families which were not documented earlier. SPS A form-specific expression patterns were studied in the leaves and tubers of different potato cultivars based on semi-quantitative RT-PCR and protein blot analyses. SPS activities particularly in the cold-stored tubers were probably due to altered kinetic properties. This report would be useful for in-depth studies on various SPS isoforms in potato and other Solanaceae family members.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Molecular characterization and expression analysis of chloroplast protein import components in tomato (Solanum lycopersicum).

The translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4) and two Toc34 homologues (slToc34-1 and -2) in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues.

Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses

The production of singlet oxygen is typically associated with inefficient dissipation of photosynthetic energy or can arise from light reactions as a result of accumulation of chlorophyll precursors as observed in fluorescent (flu)-like mutants. Such photodynamic production of singlet oxygen is thought to be involved in stress signaling and programmed cell death. Here we show that transcriptomes of multiple stresses, whether from light or dark treatments, were correlated with the transcriptome of the flu mutant. A core gene set of 118 genes, common to singlet oxygen, biotic and abiotic stresses was defined and confirmed to be activated photodynamically by the photosensitizer Rose Bengal. In addition, induction of the core gene set by abiotic and biotic selected stresses was shown to occur in the dark and in nonphotosynthetic tissue. Furthermore, when subjected to various biotic and abiotic stresses in the dark, the singlet oxygen-specific probe Singlet Oxygen Sensor Green detected rapid production of singlet oxygen in the Arabidopsis (Arabidopsis thaliana) root. Subcellular localization of Singlet Oxygen Sensor Green fluorescence showed its accumulation in mitochondria, peroxisomes, and the nucleus, suggesting several compartments as the possible origins or targets for singlet oxygen. Collectively, the results show that singlet oxygen can be produced by multiple stress pathways and can emanate from compartments other than the chloroplast in a light-independent manner. The results imply that the role of singlet oxygen in plant stress regulation and response is more ubiquitous than previously thought.

Plastidial NAD-Dependent Malate Dehydrogenase Is Critical for Embryo Development and Heterotrophic Metabolism in Arabidopsis

In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis.

Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signalling to enhance crop yield and food security

Sucrose is produced in, and translocated from, photosynthetically active leaves (sources) to support non-photosynthetic tissues (sinks), such as developing seeds, fruits, and tubers. Different plants can utilize distinct mechanisms to transport sucrose into the phloem sieve tubes in source leaves. While phloem loading mechanisms have been extensively studied in dicot plants, there is less information about phloem loading in monocots. Maize and rice are major dietary staples, which have previously been proposed to use different cellular routes to transport sucrose from photosynthetic cells into the translocation stream. The anatomical, physiological, and genetic evidence supporting these conflicting hypotheses is examined. Upon entering sink cells, sucrose often is degraded into hexoses for a wide range of metabolic and storage processes, including biosynthesis of starch, protein, and cellulose, which are all major constituents for food, fibre, and fuel. Sucrose, glucose, fructose, and their derivate, trehalose-6-phosphate, also serve as signalling molecules to regulate gene expression either directly or through cross-talk with other signalling pathways. As such, sugar transport and metabolism play pivotal roles in plant development and realization of crop yield that needs to be increased substantially to meet the projected population demand in the foreseeable future. This review will discuss the current understanding of the control of carbon partitioning from the cellular to whole-plant levels, focusing on (i) the pathways employed for phloem loading in source leaves, particularly in grasses, and the routes used in sink organs for phloem unloading; (ii) the transporter proteins responsible for sugar efflux and influx across plasma membranes; and (iii) the key enzymes regulating sucrose metabolism, signalling, and utilization. Examples of how sugar transport and metabolism can be manipulated to improve crop productivity and stress tolerance are discussed.

Contribution of stem CO<sub>2</sub> fixation to whole-plant carbon balance in nonsucculent species

In many plant species that remain leafless part of the year, CO2 fixation occurring in green stems represents an important carbon gain. Traditionally, a distinction has been made between stem photosynthesis and corticular photosynthesis. All stem photosynthesis is, sensu stricto, cortical, since it is carried out largely by the stem cortex. We proposed the following nomenclature: stem net photosynthesis (SNP), which includes net CO2 fixation by stems with stomata in the epidermis and net corticular CO2 fixation in suberized stems, and stem recycling photosynthesis (SRP), which defines CO2 ling in suberized stems. The proposed terms should reflect differences in anatomical and physiological traits. SNP takes place in the chlorenchyma below the epidermis with stomata, where the net CO2 uptake occurs, and it resembles leaf photosynthesis in many characteristics. SRP is found in species where the chlorenchyma is beneath a well-developed stomata-free periderm and where reassimilation of internally respired CO2 occurs. SNP is common in plants from desert ecosystems, rates reaching up to 60% of the leaf photosynthetic rate. SRP has been demonstrated in trees from temperate forests and it offsets partially a carbon loss by respiration of stem nonphotosynthetic tissues. Reassimilation can vary between 7 and 123% of respired CO2, the latter figure implying net CO2 uptake from the atmosphere. Both types of stem photosynthesis contribute positively to the carbon economy of the species, in which they occur; they are advantageous to the plant because they allow the maintenance of physiological activity during stress, an increase of integrated water use efficiency, and they provide the carbon source used in the production of new organs.

Anthocyanin-dependent anoxygenic photosynthesis in coloured flower petals?

Chlorophylless flower petals are known to be composed of non-photosynthetic tissues. Here, we show that the light energy storage that can be photoacoustically measured in flower petals of Petunia hybrida is approximately 10-12%. We found that the supposed chlorophylless photosynthesis is an anoxygenic, anthocyanin-dependent process occurring in blue flower petals (ADAPFP), accompanied by non-respiratory light-dependent oxygen uptake and a 1.5-fold photoinduced increase in ATP levels. Using a simple, adhesive tape stripping technique, we have obtained a backside image of an intact flower petal epidermis, revealing sword-shaped ingrowths connecting the cell wall and vacuole, which is of interest for the further study of possible vacuole-related photosynthesis. Approaches to the interpretations of ADAPFP are discussed, and we conclude that these results are not impossible in terms of the known photochemistry of anthocyanins.

Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues

RNA editing is one of the post-transcriptional processes that commonly occur in plant plastids and mitochondria. In Arabidopsis, 34 C-to-U RNA editing events, affecting transcripts of 18 plastid genes, have been identified. Here, we examined the editing and expression of these transcripts in different organs, and in green and non-green seedlings (etiolated, cia5-2, ispF and ispG albino mutants, lincomycin-, and norflurazon-treated). The editing efficiency of Arabidopsis plastid transcripts varies from site to site, and may be specifically regulated in different tissues. Steady state levels of plastid transcripts are low or undetectable in etiolated seedlings, but most editing sites are edited with efficiencies similar to those observed in green seedlings. By contrast, the editing of some sites is completely lost or significantly reduced in other non-green tissues; for instance, the editing of ndhB-149, ndhB-1255, and ndhD-2 is completely lost in roots and in lincomycin-treated seedlings. The editing of ndhD-2 is also completely lost in albino mutants and norflurazon-treated seedlings. However, matK-640 is completely edited, and accD-794, atpF-92, psbE-214, psbF-77, psbZ-50, and rps14-50 are completely or highly edited in both green and non-green tissues. In addition, the expression of nucleus-encoded RNA polymerase dependent transcripts is specifically induced by lincomycin, and the splicing of ndhB transcripts is significantly reduced in the albino mutants and inhibitor-treated seedlings. Our results indicate that plastid gene expression, and the splicing and editing of plastid transcripts are specifically and differentially regulated in various types of non-green tissues.

Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid

Ferredoxin:NADP+ oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP+), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates’ radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.

Plastid Structure, Diversification and Interconversions II. Land Plants

In the course of endosymbiogenesis, the photosynthetic prokaryotes engulfed and retained by different heterotrophic organisms have partially lost their independence during evolution and became semi-autonomous organelles, the chloroplasts. The chloroplast represents the most ancestral form of plastids that has parallelly evolved in several algal groups (reviewed in [1]) and in land plants. After briefly discussing plastid morphology, we review the most important ultrastructural features of the plastids of land plants. Then we discuss how plastids got gradually specialized in parallel with the increasing developmental and/or organizational complexity of the plant body. The plastids of non-photosynthetic tissues and cells do not need to produce and maintain a photosynthetic apparatus, but have adjusted their metabolism to the major function of the host cell (and tissue). This way, different plastid forms specialized for other functions such as storage (e.g. starch storing leucoplasts called amyloplast) or carotenoid synthesis (chromoplasts) have developed. However, the classical ultrastructural characterization and classification of plastids is often problematic. First of all, the term plastid refers to the extremely high plasticity of this organelle, and its capacity to be readily transformed from one type into another one upon different environmental or developmental stimuli. Therefore, transitional (or if persistent, intermediate) plastids with morphological features characteristic for two different plastid types can be often observed. Sometimes plastids with similar ultrastructure can have different specific functions and basically different metabolism, and should be therefore treated separately. After having recalled the different plastid types of land plants we present a dynamic model about their interconversions.