Heterotrophic Tissues Research Articles

Morphology, epidermal features and δ13C signature of Lopingian (late Permian) conifers

Conifers, the most successful group of Permian gymnosperms, dominate the famous Bletterbach (Dolomites, NE-Italy) plant fossil assemblage, a highly diverse and well-documented late Permian (Lopingian) flora. An integrated analysis of morphology, cuticles and isotope geochemistry was carried out on approximately 50 conifer shoots across five genera (Ortiseia, Majonica, Dolomitia, Pseudovoltzia and Quadrocladus) and eight species, including three (Pseudovoltzia sjerpii, Quadrocladus solmsii, Quadrocladus cf. orobiformis) described for the first time from Bletterbach. Taxon-specific carbon isotope analyses reveal intra-specific and/or intra-generic variability, identifying a unique geochemical composition for Majonica alpina, which may reflect a possible species-specific geochemical signature or adaptation to particular environmental conditions. The isotopic differences observed between leaves and axes indicate the preservation of distinct isotopic ratios in photosynthetic versus heterotrophic tissues, underscoring the importance of sampling multiple plant parts to accurately capture individual and taxonomic isotopic variability. The study of stable isotopes of organic carbon on well-preserved plant remains is enhanced the paleoenvironmental reconstruction of the Bletterbach flora.

Evidence for dual targeting control of Arabidopsis 6-phosphogluconate dehydrogenase isoforms by N-terminal phosphorylation.

The oxidative pentose-phosphate pathway (OPPP) retrieves NADPH from glucose-6-phosphate, which is important in chloroplasts at night and in plastids of heterotrophic tissues. We previously studied how OPPP enzymes may transiently locate to peroxisomes, but how this is achieved for the third enzyme remained unclear. By extending our genetic approach, we demonstrated that Arabidopsis isoform 6-phosphogluconate dehydrogenase 2 (PGD2) is indispensable in peroxisomes during fertilization, and investigated why all PGD-reporter fusions show a mostly cytosolic pattern. A previously published interaction of a plant PGD with thioredoxin m was confirmed using Trxm2 for yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays, and medial reporter fusions (with both ends accessible) proved to be beneficial for studying peroxisomal targeting of PGD2. Of special importance were phosphomimetic changes at Thr6, resulting in a clear targeting switch to peroxisomes, while a similar change at position Ser7 in PGD1 conferred plastid import. Apparently, efficient subcellular localization can be achieved by activating an unknown kinase, either early after or during translation. N-terminal phosphorylation of PGD2 interfered with dimerization in the cytosol, thus allowing accessibility of the C-terminal peroxisomal targeting signal (PTS1). Notably, we identified amino acid positions that are conserved among plant PGD homologues, with PTS1 motifs first appearing in ferns, suggesting a functional link to fertilization during the evolution of seed plants.

Source‐sink patterns on coffee trees related to annual climate variability: An approach through stable isotopes analysis

AbstractStable isotopic determination constitutes a useful tool to identify the processes that control the dynamics of the carbon and nitrogen flow in plants, unravelling the mechanisms of their differential investment under different environments. This work aimed to evaluate the spatiotemporal variation of source‐sink patterns of coffee trees under field conditions in response to climatic conditions through the assessment of stable isotopes. For this purpose, stems, leaves, and fruit samples from coffee trees were collected following a temporal pattern based on the region's climatic characteristics and the plant's phenology and a spatial pattern considering different parts of the canopy. The carbon and nitrogen percentage content, the C/N ratio, and the carbon and nitrogen isotopic compositions (δ13C and δ15N) were determined for all samples. The basal portion of the orthotropic branch was also considered for the isotopic analysis of the tree's growth rings. The results obtained were correlated with the climatic variables of the region through a Pearson correlation analysis (p < .05). Coffee plants showed traditional δ13C values of C3 plants. Temporal δ13C variation was associated with the different growth rates between phenological stages and the use of substrates produced at different times under different environmental conditions leading to differences in photosynthetic discrimination. Spatial δ13C variation was observed with heterotrophic tissues isotopically heavier than leaves, with a significant decrease trend in δ13C values from the top (upper third) to the bottom (lower third), associated with ecophysiological differences between the canopy, isotopic fractionation processes downstream of photosynthetic carbon discrimination, and the fixation of C from other pools. Temporal δ15N variation was associated with the precipitation rates in the region and the fertilization distribution across the tree, while the spatial variation was with the plant's nitrogen assimilation and translocation patterns. The tree growth rings isotopic analyses showed isotopic differences between growth rings of the same plant addressed by the climatic conditions, with precipitation being the primary climatic determinant influencing the fixation and discrimination against 13C. Our results highlight the importance of using stable isotope analysis as a reference point for coffee ecophysiological studies to characterize how the temporal and spatial patterns of δ13C and δ15N emerge and signal the influence of climate on the source‐sink relationship of coffee trees under field conditions.

δ13C in above-ground and below-ground organs of Spinulum annotinum (Lycopodiaceae)

• 12 C and 13 C isotope abundance in S. annotinum has been investigated for the first time. • δ 13 C value in S. annotinum is lower than that reported in other C 3 plants. • Different above- and below-ground S. annotinum organs have distinct δ 13 C values. • δ 13 C values of S. annotinum are connected with local plant diversity. Physiological processes in plants and plant–environment interactions can be analysed from the composition of stable carbon isotopes ( 13 C/ 12 C). This ratio can be indicated using the δ (delta) notation. Substantial evidence has been published in recent years demonstrating that post-photosynthetic fractionation occurs in plants and leads to differences in 13 C-enrichment in heterotrophic (as compared with autotrophic) organs. Yet our understanding of processes leading to 13 C divergence between leaves and heterotrophic tissues remains low. Large part of the isotope analysis is concerned with angiosperms, but not spore-bearing tracheophytes. In this study, we explored the 12 C and 13 C isotope distribution in above-ground and below-ground organs of an archaic lycophyte Spinulum annotinum (L.) A.Haines (interrupted clubmoss). The main objectives of this study were to determine the natural distribution of stable carbon isotopes in sporophytes of S. annotinum and to discuss possible causes for differences in δ 13 C. The study revealed that there are significant differences between δ 13 C in different S. annotinum organs. The δ 13 C values varied by about 2.4‰. The highest δ 13 C values were −30.0‰ in roots and the lowest were −32.4‰ in plagiotropic shoot leaves. The distance to a body of water had no significant effect on δ 13 C values in S. annotinum . Outgrowth size did not have a clear relationship with δ 13 C in S. annotinum . The Shannon Diversity Index was a significant predictor for δ 13 C values in S. annotinum . The uncovered δ 13 C and the relationship with abiotic habitat conditions and neighbouring plant species in the community provide a base for future lycophyte ecophysiology research.

Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues.

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.

Perforated Meckel’s Diverticulum with Pancreatic and Gastric Heterotropia and Acute Peritonitis - A Case Report

Meckel’s diverticulum, the most common congenital anomaly of small bowel although usually silent, can cause complications like intestinal obstruction, bleeding, diverticulitis, perforation etc. We report a case, which presented with acute onset of severe pain in right iliac fossa which was clinically and sonographically diagnosed as acute appendicitis. Histopathological report of appendix was non specific findings. Four days after appendectomy patient again came with features of acute abdomen. X-ray abdomen showed free gas under diaphragm. Abdomen was explored with a midline incision, a perforated Meckel’sdiverticulum was found which was managed by wedge resection and repair of the ileum. Histopathological examination of specimen revealed diverticular wall with normal appearing intestinal mucosa and muscle coat which showed two heterotrophic tissues (pancreatic and gastric ) in the wall.These also showed features of perforation and acute peritonitis. This is probably the first case of Meckel’s diverticulitis with heterotropic pancreatic and gastric tissue in Bangladesh. J Shaheed Suhrawardy Med Coll 2020; 12(2): 115-118

Identification of new proteins in mature sieve elements.

The phloem enables vascular plants to transport photoassimilates from source tissues to heterotrophic sink tissues. In the phloem, unbroken strings of enucleated sieve elements, which lose the majority of their cellular contents upon maturation, provide a low resistance path for mass flow. The protein machinery in mature sieve elements performs vital functions to maintain the flow, transmit systemic signals and defend the sugar stream against pests. However, our knowledge of this particular protein population is very limited since mature sieve elements are difficult to isolate and not amenable to transcriptomic analysis due to their enucleate nature. Here, we used co-expression analysis and published gene clusters from transcriptomic studies to generate a list of sieve element proteins that potentially survive the enucleation process to reside in mature sieve elements. We selected seven candidates and show that they all localize in sieve elements in Arabidopsis roots and six of them in bolting stems. Our results support the idea that nascent sieve elements prior to enucleation translate part of the protein machinery found in mature sieve elements. Our co-expression list and the publicly available gene clusters expressed in late proto- and meta-phloem sieve elements are valuable resources for uncharacterized genes that may function in mature sieve elements.

An Updated Review on the Modulation of Carbon Partitioning and Allocation in Arbuscular Mycorrhizal Plants.

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that supply mineral nutrients to the host plant in exchange for carbon derived from photosynthesis. Sucrose is the end-product of photosynthesis and the main compound used by plants to translocate photosynthates to non-photosynthetic tissues. AMF alter carbon distribution in plants by modifying the expression and activity of key enzymes of sucrose biosynthesis, transport, and/or catabolism. Since sucrose is essential for the maintenance of all metabolic and physiological processes, the modifications addressed by AMF can significantly affect plant development and stress responses. AMF also modulate plant lipid biosynthesis to acquire storage reserves, generate biomass, and fulfill its life cycle. In this review we address the most relevant aspects of the influence of AMF on sucrose and lipid metabolism in plants, including its effects on sucrose biosynthesis both in photosynthetic and heterotrophic tissues, and the influence of sucrose on lipid biosynthesis in the context of the symbiosis. We present a hypothetical model of carbon partitioning between plants and AMF in which the coordinated action of sucrose biosynthesis, transport, and catabolism plays a role in the generation of hexose gradients to supply carbon to AMF, and to control the amount of carbon assigned to the fungus.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Inactivation of mitochondrial complex I stimulates chloroplast ATPase inPhyscomitrium patens

Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

Rice Glucose 6-Phosphate/Phosphate Translocator 1 is required for tapetum function and pollen development

In plants, non-green plastids in heterotrophic tissues are sites for starch and fatty acids biosynthesis, which are essential for plant development and reproduction. Distinct from chloroplasts, the metabolites for these processes in non-green plastids have to be imported through specific transporters. Glucose 6-Phosphate/Phosphate Translocator 1 is required for the uptake of cytosolic Glucose 6-Phosphate into non-green plastids. In Arabidopsis, GPT1 has been demonstrated to play essential roles in male, female gametophyte and embryo development. However, the roles of GPTs in other species are yet largely unknown. Here, we reported that rice OsGPT1 is indispensable for normal tapetal degeneration and pollen exine formation during anther and pollen development. OsGPT1 is localized in the plastid and distributed in the anther wall layers and late-stage pollen grains. Different from the gametic defects caused by mutation in AtGPT1, disruption of OsGPT1 does not affect male and female gamete transmission as well as embryo development. On the contrary, osgpt1 mutant exhibits delayed tapetum degeneration, decreased Ubisch bodies formation and thinner pollen exine, leading to pollen abortion at the mature stage. Furthermore, the expression of several genes involved in tapetal programmed cell death (PCD) and sporopollenin formation is decreased in osgpt1. Our study suggests that OsGPT1 coordinates the development of anther sporophytic tissues and the male gametophyte by integrating carbohydrate and fatty acid metabolism in the plastid.

Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis.

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.

Plasma membrane phylloquinone biosynthesis in nonphotosynthetic parasitic plants.

Nonphotosynthetic holoparasites exploit flexible targeting of phylloquinone biosynthesis to facilitate plasma membrane redox signaling. Phylloquinone is a lipophilic naphthoquinone found predominantly in chloroplasts and best known for its function in photosystem I electron transport and disulfide bridge formation of photosystem II subunits. Phylloquinone has also been detected in plasma membrane (PM) preparations of heterotrophic tissues with potential transmembrane redox function, but the molecular basis for this noncanonical pathway is unknown. Here, we provide evidence of PM phylloquinone biosynthesis in a nonphotosynthetic holoparasite Phelipanche aegyptiaca. A nonphotosynthetic and nonplastidial role for phylloquinone is supported by transcription of phylloquinone biosynthetic genes during seed germination and haustorium development, by PM-localization of alternative terminal enzymes, and by detection of phylloquinone in germinated seeds. Comparative gene network analysis with photosynthetically competent parasites revealed a bias of P. aegyptiaca phylloquinone genes toward coexpression with oxidoreductases involved in PM electron transport. Genes encoding the PM phylloquinone pathway are also present in several photoautotrophic taxa of Asterids, suggesting an ancient origin of multifunctionality. Our findings suggest that nonphotosynthetic holoparasites exploit alternative targeting of phylloquinone for transmembrane redox signaling associated with parasitism.

Unexpectedly low δ 13C in leaves, branches, stems and roots of three acacia species growing in hyper-arid environments

AbstractAimsIn plant eco-physiology, less negative (enriched) carbon 13 (13C) in the leaves indicates conditions of reducing leaf gas exchange through stomata, e.g. under drought. In addition, 13C is expected to be less negative in non-photosynthetic tissues as compared with leaves. However, these relationships in δ 13C from leaves (photosynthetic organs) to branches, stems and roots (non-photosynthetic organs) are rarely tested across multiple closely related tree species, multiple compartments, or in trees growing under extreme heat and drought.MethodsWe measured leaf-to-root 13C in three closely related desert acacia species (Acacia tortilis, A. raddiana and A. pachyceras). We measured δ 13C in leaf tissues from mature trees in southern Israel. In parallel, a 7-year irrigation experiment with 0.5, 1.0 or 4.0 L day−1 was conducted in an experimental orchard. At the end of the experiment, growth parameters and δ 13C were measured in leaves, branches, stems and roots.Important FindingsThe δ 13C in leaf tissues sampled from mature trees was ca. −27‰, far more depleted than expected from a desert tree growing in one of the Earth’s driest and hottest environments. Across acacia species and compartments, δ 13C was not enriched at all irrigation levels (−28‰ to ca. −27‰), confirming our measurements in the mature trees. Among compartments, leaf δ 13C was unexpectedly similar to branch and root δ 13C, and surprisingly, even less negative than stem δ 13C. The highly depleted leaf δ 13C suggests that these trees have high stomatal gas exchange, despite growing in extremely dry habitats. The lack of δ 13C enrichment in non-photosynthetic tissues might be related to the seasonal coupling of growth of leaves and heterotrophic tissues.

Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana.

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5'-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

Downregulation of a Mitochondrial NAD+ Transporter (NDT2) Alters Seed Production and Germination in Arabidopsis.

Despite the fundamental importance of nicotinamide adenine dinucleotide (NAD+) for metabolism, the physiological roles of NAD+ carriers in plants remain unclear. We previously characterized the Arabidopsis thaliana gene (At1g25380), named AtNDT2, encoding a protein located in the mitochondrial inner membrane, which imports NAD+ from the cytosol using ADP and AMP as counter-exchange substrates for NAD+. Here, we further investigated the physiological roles of NDT2, by isolating a T-DNA insertion line, generating an antisense line and characterizing these genotypes in detail. Reduced NDT2 expression affected reproductive phase by reducing total seed yield. In addition, reduced seed germination and retardation in seedling establishment were observed in the mutant lines. Moreover, remarkable changes in primary metabolism were observed in dry and germinated seeds and an increase in fatty acid levels was verified during seedling establishment. Furthermore, flowers and seedlings of NDT2 mutants displayed upregulation of de novo and salvage pathway genes encoding NAD+ biosynthesis enzymes, demonstrating the transcriptional control mediated by NDT2 activity over these genes. Taken together, our results suggest that NDT2 expression is fundamental for maintaining NAD+ balance amongst organelles that modulate metabolism, physiology and developmental processes of heterotrophic tissues.

Structure and photosynthetic metabolism in green prop roots of C4 sorghum

ABSTRACTPlants contain chloroplasts in various organs exposed to sunlight. The C4 crop, sorghum (Sorghum bicolor), develops prop roots on stems above the soil level with the progression of growth. These roots penetrate in the soil and form green above-ground (AG) and non-green below-ground (BG) portions. We investigated the structure and photosynthetic metabolism in the AG portion of prop roots in comparison with leaf blades and the BG portion. The AG portion lacked stomata on the epidermis and showed typical root structure as in the BG portion. However, they contained granal chloroplasts in the cortex and stele parenchyma. The chlorophyll content was much lower in the AG portion than in leaf blades. Western blot analysis showed that the AG portion accumulates ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) but lacked substantially phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase. An immunolocalization study confirmed that Rubisco is accumulated in the chloroplasts of AG portion. The AG portion accumulated only small amount of malate without diurnal change as in other organs, indicating that crassulacean acid metabolism (CAM) is inactive. The δ13C values of all organs including the AG portion were within the C4 range, suggesting that their tissue carbon is derived from C4 photosynthesis of leaves. These data suggest that the AG portion of prop roots could re-fix internally respired CO2 via C3 cycle, whereas this photosynthetic function may provide O2 to heterotrophic tissues of prop roots. This study also demonstrates that different photosynthetic types can function in different organs of a single plant.

Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures.

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1-2H]glucose or D2O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes.

NTRC Plays a Crucial Role in Starch Metabolism, Redox Balance, and Tomato Fruit Growth.

NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.

The Arabidopsis E1 subunit of the 2-oxoglutarate dehydrogenase complex modulates plant growth and seed production

Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDHenzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.