Non-photosynthetic Tissues Research Articles

Respiration and nitrogen assimilation: targeting mitochondria-associated metabolism as a means to enhance nitrogen use efficiency

Considerable advances in our understanding of the control of mitochondrial metabolism and its interactions with nitrogen metabolism and associated carbon/nitrogen interactions have occurred in recent years, particularly highlighting important roles in cellular redox homeostasis. The tricarboxylic acid (TCA) cycle is a central metabolic hub for the interacting pathways of respiration, nitrogen assimilation, and photorespiration, with components that show considerable flexibility in relation to adaptations to the different functions of mitochondria in photosynthetic and non-photosynthetic cells. By comparison, the operation of the oxidative pentose phosphate pathway appears to represent a significant limitation to nitrogen assimilation in non-photosynthetic tissues. Valuable new insights have been gained concerning the roles of the different enzymes involved in the production of 2-oxoglutarate (2-OG) for ammonia assimilation, yielding an improved understanding of the crucial role of cellular energy balance as a broker of co-ordinate regulation. Taken together with new information on the mechanisms that co-ordinate the expression of genes involved in organellar functions, including energy metabolism, and the potential for exploiting the existing flexibility for NAD(P)H utilization in the respiratory electron transport chain to drive nitrogen assimilation, the evidence that mitochondrial metabolism and machinery are potential novel targets for the enhancement of nitrogen use efficiency (NUE) is explored.

Circadian regulation of chloroplastic f and m thioredoxins through control of the CCA1 transcription factor.

Chloroplastic thioredoxins f and m (TRX f and TRX m) mediate light regulation of carbon metabolism through the activation of Calvin cycle enzymes. The role of TRX f and m in the activation of Calvin cycle enzymes is best known among the TRX family. However, the discoveries of new potential targets extend the functions of chloroplastic TRXs to other processes in non-photosynthetic tissues. As occurs with numerous chloroplast proteins, their expression comes under light regulation. Here, the focus is on the light regulation of TRX f and TRX m in pea and Arabidopsis during the day/night cycle that is maintained during the subjective night. In pea (Pisum sativum), TRX f and TRX m1 expression is shown to be governed by a circadian oscillation exerted at both the transcriptional and protein levels. Binding shift assays indicate that this control probably involves the interaction of the CCA1 transcription factor and an evening element (EE) located in the PsTRX f and PsTRX m1 promoters. In Arabidopsis, among the multigene family of TRX f and TRX m, AtTRX f2 and AtTRX m2 mRNA showed similar circadian oscillatory regulation, suggesting that such regulation is conserved in plants. However, this oscillation was disrupted in plants overexpressing CCA1 (cca1-ox) or repressing CCA1 and LHY (cca1-lhy). The physiological role of the oscillatory regulation of chloroplastic TRX f and TRX m in plants during the day/night cycle is discussed.

Aspen shoots are carbon autonomous during bud break

Current thinking holds that carbon autonomy of branches in trees is unlikely, particularly during bud break, when the new developing shoots require significant influx of carbon resources from more distant sources. Results from recent studies indicate that the impact of bud break on overall tree reserves might be small. In two studies the independence of flushing shoots from stored carbon reserves and the photosynthesis in developing new leaves and shoots of Populus tremuloides were explored. New developing shoots quickly became a positive carbon source and only a few days into flush, the photosynthetic system of the newly developing shoots was efficient enough to achieve positive carbon gain even at low light levels. Only 14% of the stored shoot reserves, without any mobilization from more distant reserves, were used during bud break and early shoot expansion. Without any underlying stress, shoots of deciduous trees appear to be carbon autonomous during bud break when demand on stored carbon should be the highest. The development of an efficient photosynthetic system in new shoots is critical in the recovery of carbon reserves in aspen. It minimizes the cost of bud break to the overall stored carbon reserves by optimizing the assimilation of carbon in the newly developed leaves, while eliminating the cost for mobilizing carbon reserves from more distant sources. This carbon autonomy of shoots has important implications for the whole tree carbon balance particularly to the non-photosynthetic tissues which functions solely depending on carbon export from the newly developing leaves and shoots.

Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering

14C labeling experiments performed with kernel cultures showed that developing maize endosperm is more efficient than other non-photosynthetic tissues such as sunflower and maize embryos at converting maternally supplied substrates into biomass. To characterize the metabolic fluxes in endosperm, maize kernels were labeled to isotopic steady state using 13C-labeled glucose. The resultant labeling in free metabolites and biomass was analyzed by NMR and GC–MS. After taking into account the labeling of substrates supplied by the metabolically active cob, the fluxes through central metabolism were quantified by computer-aided modeling. The flux map indicates that 51–69% of the ATP produced is used for biomass synthesis and up to 47% is expended in substrate cycling. These findings point to potential engineering targets for improving yield and increasing oil contents by, respectively, reducing substrate cycling and increasing the commitment of plastidic carbon into fatty acid synthesis at the level of pyruvate kinase.

Maize Global Transcriptomics Reveals Pervasive Leaf Diurnal Rhythms but Rhythms in Developing Ears Are Largely Limited to the Core Oscillator

BackgroundPlant diurnal rhythms are vital environmental adaptations to coordinate internal physiological responses to alternating day-night cycles. A comprehensive view of diurnal biology has been lacking for maize (Zea mays), a major world crop.MethodologyA photosynthetic tissue, the leaf, and a non-photosynthetic tissue, the developing ear, were sampled under natural field conditions. Genome-wide transcript profiling was conducted on a high-density 105 K Agilent microarray to investigate diurnal rhythms.ConclusionsIn both leaves and ears, the core oscillators were intact and diurnally cycling. Maize core oscillator genes are found to be largely conserved with their Arabidopsis counterparts. Diurnal gene regulation occurs in leaves, with some 23% of expressed transcripts exhibiting a diurnal cycling pattern. These transcripts can be assigned to over 1700 gene ontology functional terms, underscoring the pervasive impact of diurnal rhythms on plant biology. Considering the peak expression time for each diurnally regulated gene, and its corresponding functional assignment, most gene functions display temporal enrichment in the day, often with distinct patterns, such as dawn or midday preferred, indicating that there is a staged procession of biological events undulating with the diurnal cycle. Notably, many gene functions display a bimodal enrichment flanking the midday photosynthetic maximum, with an initial peak in mid-morning followed by another peak during the afternoon/evening. In contrast to leaves, in developing ears as few as 47 gene transcripts are diurnally regulated, and this set of transcripts includes primarily the core oscillators. In developing ears, which are largely shielded from light, the core oscillator therefore is intact with little outward effect on transcription.

Characterization of foliar manganese (Mn) in Mn (hyper)accumulators using X‐ray absorption spectroscopy

Plant hyperaccumulation of the essential nutrient manganese (Mn) is a rare phenomenon most evident in the Western Pacific region, and differs from hyperaccumulation of other elements. Mn hyperaccumulators employ a variety of species-dependent spatial distribution patterns in sequestering excess foliar Mn, including primary sequestration in both nonphotosynthetic and photosynthetic tissues. This investigation employed synchrotron X-ray absorption spectroscopy (XAS) in a comparative study of Mn (hyper)accumulators, to elucidate in situ the chemical form(s) of foliar Mn in seven woody species from Australia, New Caledonia and Japan. Foliar Mn was found to predominate as Mn(II) in all samples, with strong evidence of the role of carboxylic acids, such as malate or citrate, as complexing ligands. Overall, the X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine-structure spectroscopy (EXAFS) data appeared weighted against previous observations that oxalate binds excess Mn in Mn-(hyper)accumulating species.

The psychedelic genes of maize redundantly promote carbohydrate export from leaves.

Whole-plant carbohydrate partitioning involves the assimilation of carbon in leaves and its translocation to nonphotosynthetic tissues. This process is fundamental to plant growth and development, but its regulation is poorly understood. To identify genes controlling carbohydrate partitioning, we isolated mutants that are defective in exporting fixed carbon from leaves. Here we describe psychedelic (psc), a new mutant of maize (Zea mays) that is perturbed in carbohydrate partitioning. psc mutants exhibit stable, discrete chlorotic and green regions within their leaves. psc chlorotic tissues hyperaccumulate starch and soluble sugars, while psc green tissues appear comparable to wild-type leaves. The psc chlorotic and green tissue boundaries are usually delineated by larger veins, suggesting that translocation of a mobile compound through the veins may influence the tissue phenotype. psc mutants display altered biomass partitioning, which is consistent with reduced carbohydrate export from leaves to developing tissues. We determined that the psc mutation is unlinked to previously characterized maize leaf carbohydrate hyperaccumulation mutants. Additionally, we found that the psc mutant phenotype is inherited as a recessive, duplicate-factor trait in some inbred lines. Genetic analyses with other maize mutants with variegated leaves and impaired carbohydrate partitioning suggest that Psc defines an independent pathway. Therefore, investigations into the psc mutation have uncovered two previously unknown genes that redundantly function to regulate carbohydrate partitioning in maize.

The gene encoding the catalytically inactive β-amylase BAM4 involved in starch breakdown in Arabidopsis leaves is expressed preferentially in vascular tissues in source and sink organs

Genetic studies in Arabidopsis thaliana have shown that two members of the beta-amylase (BAM) family BAM3 and BAM4 are required for leaf starch breakdown at night. Both are plastid proteins and while BAM3 encodes an active BAM, BAM4 is not an active alpha-1,4-glucan hydrolase. To gain further insight into the possible function of BAM4 we constructed reporter genes using promoters for both BAM3 and BAM4 genes, driving beta-glucuronidase (GUS) and luciferase (LUC) expression in transgenic Arabidopsis plants. Both promoters directed expression in vascular tissue throughout the plant including cotyledons, leaves, petioles, stems, petals, siliques and roots. Tissue sections showed expression to be focused in phloem cells in stem and petiole. The BAM3 promoter was also expressed strongly throughout the photosynthetic tissues of leaves, sepals and siliques, whereas the BAM4 promoter was not. Conversely, the BAM4 promoter was active in root tip but the BAM3 promoter was not. To confirm these expression patterns and to compare with expression of other starch genes we carried-out RT-PCR analysis on RNA from vascular (replum) and non-vascular (valve) tissues of siliques. This confirmed that BAM4 expression together with RAM1 (BAM5) and GWD2 genes is stronger in the replum than the valve, whereas BAM3 is strong in both tissues. These results show that even though BAM3 and BAM4 genes apparently interact genetically in leaf starch metabolism, BAM4 is preferentially expressed in non-photosynthetic vascular tissue, so revealing a potentially greater level of complexity in the control of starch breakdown than had previously been recognised.

Starch Synthesis in Arabidopsis Is Achieved by Spatial Cotranscription of Core Starch Metabolism Genes

Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-beta-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls.

NTRC links built-in thioredoxin to light and sucrose in regulating starch synthesis in chloroplasts and amyloplasts

Plants have an unusual plastid-localized NADP-thioredoxin reductase C (NTRC) containing both an NADP-thioredoxin reductase (NTR) and a thioredoxin (Trx) domain in a single polypeptide. Although NTRC is known to supply reductant for detoxifying hydrogen peroxide in the dark, its other functions are unknown. We now report that NTRC plays a previously unrecognized role in the redox regulation of ADP-glucose pyrophosphorylase (AGPase), a central enzyme of starch synthesis. When supplied NADPH, NTRC activated AGPase in vitro in a redox reaction that required the active site cysteines of both domains of the enzyme. In leaves, AGPase was activated in planta either by light or external feeding of sucrose in the dark. Leaves of an Arabidopsis NTRC KO mutant showed a decrease both in the extent of redox activation of AGPase and in the enhancement of starch synthesis either in the light (by 40-60%) or in the dark after treatment with external sucrose (by almost 100%). The light-dependent activation of AGPase in isolated chloroplasts, by contrast, was unaffected. In nonphotosynthetic tissue (roots), KO of NTRC decreased redox activation of AGPase and starch synthesis in response to light or external sucrose by almost 90%. The results provide biochemical and genetic evidence for a role of NTRC in regulating starch synthesis in response to either light or sucrose. The data also suggest that the Trx domain of NTRC and, to a lesser extent, free Trxs linked to ferredoxin enable amyloplasts of distant sink tissues to sense light used in photosynthesis by leaf chloroplasts and adjust heterotrophic starch synthesis accordingly.

Shoot-Specific Down-Regulation of Protein Farnesyltransferase (α-Subunit) for Yield Protection against Drought in Canola

Canola (Brassica napus L.) is one of the most important oilseed crops in the world and its seed yield and quality are significantly affected by drought stress. As an innate and adaptive response to water deficit, land plants avoid potential damage by rapid biosynthesis of the phytohormone abscisic acid (ABA), which triggers stomatal closure to reduce transpirational water loss. The ABA-mediated stomatal response is a dosage-dependent process; thus, one genetic engineering approach for achieving drought avoidance could be to sensitize the guard cell's responsiveness to this hormone. Recent genetic studies have pinpointed protein farnesyltransferase as a key negative regulator controlling ABA sensitivity in the guard cells. We have previously shown that down-regulation of the gene encoding Arabidopsis beta-subunit of farnesyltransferase (ERA1) enhances the plant's sensitivity to ABA and drought tolerance. Although the alpha-subunit of farnesyltransferase (AtFTA) is also implicated in ABA sensing, the effectiveness of using such a gene target for improving drought tolerance in a crop plant has not been validated. Here, we report the identification and characterization of the promoter of Arabidopsis hydroxypyruvate reductase (AtHPR1), which expresses specifically in the shoot and not in non-photosynthetic tissues such as root. The promoter region of AtHPR1 contains the core motif of the well characterized dehydration-responsive cis-acting element and we have confirmed that AtHPR1 expression is inducible by drought stress. Conditional and specific down-regulation of FTA in canola using the AtHPR1 promoter driving an RNAi construct resulted in yield protection against drought stress in the field. Using this molecular strategy, we have made significant progress in engineering drought tolerance in this important crop species.

Detecting seasonal change of broad-leaved woody canopy leaf area density profile using 3D portable LIDAR imaging.

Seasonal change of vertical leaf area density (LAD) profiles of woody canopy broad-leaved trees (Zelkova serrata [Thunberg] Makino) was estimated using 3D portable scanning light detection and ranging (LIDAR) imaging. First, 3D point cloud data for the canopy were collected using a portable LIDAR in spring, summer, autumn and winter. For data collection, the canopy was evenly scanned by the LIDAR from three positions 10 m above the ground. Next, the vertical LAD profile in each season was computed from the LIDAR data using the voxel-based canopy profiling (VCP) method. For the computation, non-photosynthetic tissues were eliminated using the LIDAR data obtained during winter. Influence of leaf inclination angle (LIA) on LAD estimation was corrected by LIA data measured by a high-resolution portable scanning LIDAR. The resultant profiles showed that LAD values tended to increase at the upper canopy from spring to summer and decrease at the middle and lower canopy from summer to autumn. Moreover, LIDAR-derived LIA distributions were compared among different seasons. LIA showed an even distribution in spring but changed to a planophile distribution in summer. In autumn, the angles in the <30° class decreased and those between the 30 and 40°classes increased.

Why are non-photosynthetic tissues generally 13C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses.

Non-photosynthetic, or heterotrophic, tissues in C3 plants tend to be enriched in 13C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO2 fixation. Unlike in C3 plants, roots of herbaceous C4 plants are generally not 13C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C3 plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against 13C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively 13C-depleted and night sucrose 13C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against 13C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.

Silicon amelioration of manganese toxicity in Mn-sensitive and Mn-tolerant maize varieties

Differences in tolerance to Mn excess and amelioration by Si were evaluated in two maize varieties. Dry weight, callose accumulation, chloroplast ultrastructure, and photosynthesis parameters were used as stress indicators. Variety Kneja 605 was much more Mn-sensitive than variety Kneja 434. In Kneja 605 excess Mn caused severe chloroplast damage and enhanced carotenoid production, symptoms similar to those triggered by photoinhibiton. In Mn-tolerant Kneja 434, in contrast, a Mn-induced decrease of the carotenoid concentrations, and only slight alterations in the chloroplasts were observed. These effects were similar to light Fe-deficiency symptoms. The threshold tissue concentration for Mn-induced callose accumulation was much lower in Kneja 605 than in Kneja 434. Therefore tolerance to excess Mn in Kneja 434 was not due to more efficient exclusion but to more efficient detoxification and compartmentation of Mn. The constitutively thicker epidermal layers in Kneja 434 and the observation that Si-induced amelioration of Mn toxicity in Kneja 605 substantially increased the thickness of the epidermal layers suggest that Mn storage in non-photosynthetic tissue could be a Mn tolerance mechanism in maize.

Differential regulation of two glutamine synthetase genes by a single Dof transcription factor

The PpDof5 transcription factor from maritime pine (Pinus pinaster) is a regulator of the expression of glutamine synthetase (GS) genes in photosynthetic and non-photosynthetic tissues. PpDof5 mRNA is detected almost ubiquitously during pine development with low levels of gene expression in green tissues and much higher levels in roots and lignified shoots. The PpDof5 protein expressed in bacteria binds to oligonucleotide probes containing the AAAG core sequence derived from the promoters of GS1a and GS1b genes. Transient expression experiments in agroinfiltrated tobacco leaves and in pine protoplasts demonstrated that PpDof5 is able to trans-regulate differentially the transcription of both GS1a and GS1b. PpDof5 activated transcription of the GS1b promoter and, in contrast, behaved as a transcriptional repressor of the GS1a promoter. These results support a regulatory mechanism for the transcriptional control of the spatial distribution of cytosolic GS isoforms in pine. Considering the precise expression patterns of GS1 genes required to fulfil the ammonium assimilation requirements during tree development, we hypothesize that PpDof5 could have a key role in the control of ammonium assimilation for glutamine biosynthesis in conifers. A regulatory model of GS1 gene expression in pine is proposed.

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot ( Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis (Southern and Western) of callus tissue (non-photosynthetic tissue) to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.

Molecular characterization of Cassava (Manihot esculenta Crantz) with yellow-orange roots for beta-carotene improvement

Casssava (Manihot esculenta Crantz) is one of the main food and income sources of about 500 million people in the tropics. The crop is mainly cultivated by small farmers in tropical Africa, Asia and Latin America. Embrapa Mandioca e Fruticultura Tropical, based in Cruz das Almas, Bahia, maintains one of the largest cassava genebanks of Latin America. Among the accessions it contains, those with yellow-orange root color are particularly interesting. The objective of this study was to characterize 30 cassava accessions with yellow-orange root color by RAPD markers. The genetic distances of the 47 analyzed primers varied from 9.0 to 31.7 %, demonstrating the existing genetic variability to be exploited for the development of cassava varieties with higher beta-carotene contents. Molecular characterization of Cassava (Manihot esculenta Crantz) with yellow-orange roots for beta-carotene improvement Claudia Fortes Ferreira1*, Elaine Alves2, Katia Nogueira Pestana3, Davi Theodoro Junghans1, Adilson Kenji Kobayashi4, Vania de Jesus Santos3, Raimundo Pereira Silva1, Paulo Henrique Silva3, Ediclan Soares3, and Wania Fukuda1 1 Embrapa Mandioca e Fruticultura Tropical, s/n, C.P. 007, 44.380-000, Cruz das Almas, BA, Brasil, *E-mail: claudiaf@cnpmf.embrapa.br 2 Universidade Federal da Bahia (UFB), Rua Basilio da Gama, s/n, Canela, 40.110-907, Salvador, BA, Brasil 3 Universidade Federal do Reconcavo Baiano (UFRB), Campus Universitario da UFRB, 44.380-000, Cruz das Almas, BA, Brasil 4 Embrapa Meio-Norte, Av. Duque de Caxias, 5650, Buenos Aires, 64.006-220, Teresina, PI, Brasil INTRODUCTION Manihot esculenta Crantz is a species native to tropical America (Olsen and Schaal 2001), initially cultivated by native Latin Americans and later introduced into the African and Asian continents. The worldwide cassava production, on an area of 17.870.626 hectares is approximately 195.574.112 tons (FAO 2004). It is considered one of the most important sources of calories and is an inexpensive staple food in Latin America (Montero 2003), mainly in the northeastern region of Brazil (Mendes et al. 2006). The genetic diversity of this species is wide (Nassar 2006), concentrated mainly in Latin America and the Caribbean. Approximately 8500 cassava accessions are maintained worldwide in different collections, of which 7500 in South America (Costa and Morales 1994). Received 24 January 2007 Accepted 07 November 2007 In Brazil, 4132 accessions have been collected and are maintained in genebanks across the country (Fukuda 2000). Carotenes (-carotene, b-carotene, lycopene) represent the most multifaceted group of pigments in nature, with colors varying from yellow to red, found in photosynthetic and non-photosynthetic tissues, such as roots, seeds and fruits. Once ingested, b-carotene is transformed, in the liver, into Vitamin A. Vitamin A is a micro-nutrient with functions related to vision, cell differentiation, growth development, reproduction and the immune system. Vitamin A deficiency (VAD) can cause severe diseases, e.g., ocular syndrome, xerophthalmia, and advance to irreversible blindness (Underwood et al. 1999). Although the lack of vitamin A can be prevented, xerophthalmia is still a public health problem in many developing countries (Welch and Graham 2002).

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues.

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.

Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.

Novel pattern of foliar metal distribution in a manganese hyperaccumulator.

The primary sequestration of foliar manganese (Mn) in Mn-hyperaccumulating plants can occur in either their photosynthetic or non-photosynthetic tissues, depending on the species. To date, only non-photosynthetic tissues have been found to be the major sinks in other hyperaccumulators. Here, electron (SEM) and proton (PIXE) microprobes were used to generate qualitative energy dispersive (EDS) X-ray maps of leaf cross sections. Two Mn hyperaccumulators, Garcinia amplexicaulis Vieill. (Clusiaceae) and Maytenus fournieri (Panch. and Sebert) Loesn. (Celastraceae), and the Mn accumulator Grevillea exul Lindley (Proteaceae) were studied. PIXE/EDS data obtained here for M. fournieri were in agreement with existing SEM/EDS data showing that the highest localised foliar Mn concentrations were in the epidermal tissues. However, this is the first in situ microprobe investigation of G. amplexicaulis and G. exul. The Mn X-ray maps of G. amplexicaulis revealed a previously undescribed third spatial distribution pattern among Mn-hyperaccumulating species. Manganese was relatively evenly distributed throughout the leaf photosynthetic and non-photosynthetic tissues, while in G. exul it was most highly concentrated in the epidermal cells.