Georgios Aivalakis

Spatial and Temporal Organization of Sucrose Metabolism in Lotus japonicus Nitrogen-Fixing Nodules Suggests a Role for the Elusive Alkaline/Neutral Invertase

Symbiotic nitrogen fixation (SNF) in legume nodules is a highly energy demanding process, fuelled by plant-supplied carbohydrates mainly in the form of sucrose. In this study, we have combined molecular and biochemical approaches in order to study the spatial and temporal organisation of sucrose metabolism in nitrogen-fixing nodules of the model legume Lotus japonicus, with an emphasis on the neglected role of alkaline/neutral invertase. For this purpose, a full-length cDNA clone coding for an alkaline/neutral invertase isoform, termed LjInv1, was identified in a L. japonicus mature nodule cDNA libraries. Alkaline/neutral invertase activity was also found to be the predominant invertase activity in mature nodules. Real-time reverse-transcription polymerase chain reaction analysis was used in order to study the temporal expression patterns of LjInv1 in parallel with genes encoding acid invertase and sucrose synthase (SuSy) isoforms, and enzymes involved in the subsequent hexose partitioning including hexokinase, phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI). The spatial organisation of sucrose metabolism was studied by in situ localisation of LjInv1 transcripts and alkaline/neutral invertase activity, and SuSy protein during nodule development. Furthermore, the spatial organisation of hexose metabolism was investigated by histochemical localisation of hexokinase, PGM and PGI activities in mature nodules. The results considered together indicate that alkaline/neutral invertase could contribute to both the Glc-1-P and Glc-6-P pools in nodules, fuelling both biosynthetic processes and SNF. Furthermore, transcript profiling analysis revealed that genes coding for hexokinase and putative plastidic PGM and PGI isoforms are upregulated during the early stages of nodule development, while the levels of transcripts corresponding to cytosolic PGM and PGI isoforms remained similar to uninfected roots, indicating a possible role of LjInv1 in producing hexoses for starch production and other biosynthetic processes in developing nodules.

Carbon Metabolism in Developing Soybean Root Nodules: The Role of Carbonic Anhydrase

A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development.

Induction and spatial organization of polyamine biosynthesis during nodule development in Lotus japonicus

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an alpha-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

A Lotus japonicus β-type carbonic anhydrase gene expression pattern suggests distinct physiological roles during nodule development☆

A full-length cDNA clone, designated Ljca1, coding for a beta-type carbonic anhydrase (CA; EC: 4.2.1.1) was isolated from a Lotus japonicus nodule cDNA library. Semi-quantitative RT-PCR analysis revealed that Ljca1 codes for a nodule-specific CA, transcripts of which accumulate at maximum levels in young nodules at 14 days post-infection (d.p.i.). In situ hybridization and immunolocalization revealed that Ljca1 transcripts and LjCA1 polypeptides were present at high levels in all cell types of young nodules. In contrast, in mature nodules both transcripts and polypeptides were confined in a few cell layers of the nodules inner cortex. However, the central infected tissue of both young and mature nodules exhibited high CA activity, indicating the presence of additional CA isoforms of plant and/or microbial origin. This was supported by the finding that a putative Mesorhizobium loti CA gene was transiently expressed during nodule development. In addition, the temporal and spatial accumulation of phosphoenolpyruvate carboxylase (PEPC; EC: 4.1.1.31) was determined by semi-quantitative RT-PCR and immunolocalization. The results suggest that LjCA1 might fulfill different physiological needs during L. japonicus nodule development.

Nodulation enhances dark CO2 fixation and recycling in the model legume Lotus japonicus

During symbiotic nitrogen fixation (SNF), the nodule becomes a strong sink for photosynthetic carbon. Here, it was studied whether nodule dark CO(2) fixation could participate in a mechanism for CO(2) recycling through C(4)-type photosynthesis. Differences in the natural δ(13)C abundance between Lotus japonicus inoculated or not with the N-fixing Mesorhizobium loti were assessed. (13)C labelling and gene expression of key enzymes of CO(2) metabolism were applied in plants inoculated with wild-type or mutant fix(-) (deficient in N fixation) strains of M. loti, and in non-inoculated plants. Compared with non-inoculated legumes, inoculated legumes had higher natural δ(13)C abundance and total C in their hypergeous organs and nodules. In stems, (13)C accumulation and expression of genes coding for enzymes of malate metabolism were greater in inoculated compared with non-inoculated plants. Malate-oxidizing activity was localized in stem xylem parenchyma, sieve tubes, and photosynthetic outer cortex parenchyma of inoculated plants. In stems of plants inoculated with fix(-) M. loti strains, (13)C accumulation remained high, while accumulation of transcripts coding for malic enzyme isoforms increased. A potential mechanism is proposed for reducing carbon losses during SNF by the direct reincorporation of CO(2) respired by nodules and the transport and metabolism of C-containing metabolites in hypergeous organs.

l -Ascorbic acid metabolism in parthenocarpic and seeded cherry tomatoes

The auxin treatment in tomato plants during anthesis has been extensively used for setting fruits in adverse climatic conditions (e.g., low temperatures and inadequate light), which is well known that reduces pollen availability and fertility. Since auxin application may affect fruit composition and quality, we examined l-ascorbic acid metabolism in seeded fruit (set by natural pollination) and parthenocarpic fruit (set by auxin) in cherry tomato cv. Conchita. Specifically, we studied the oxidized and total ascorbic acid contents, the expression of all characterized genes of l-ascorbic acid metabolism, the activity of ascorbate peroxidase and dehydroascorbate reductase and the immunolocalization of ascorbate peroxidase. Differences were detected between seeded and parthenocarpic fruits, in the expression of some of the genes of ascorbic acid metabolism. However, strong presence of l-ascorbic acid peroxidase protein was detected on the developing seeds. Our data indicate that induced parthenocarpy in auxin treated plants has a significant influence in ascorbic acid metabolism comparing to seeded tomato fruits.

Effect of nitrogen fertilization on distribution profiles of selected macronutrients in oriental field‐grown tobacco plants

Abstract As nitrogen (N) fertilization is considered incompatible with oriental tobacco agricultural practice, we studied the influence of N fertilization [no fertilization or ammonium nitrate (NH4NO3) fertilizer applied at either 50 or 100 kg N ha‐1], the growth stage (plant age) and the stalk position (basal, middle, and upper) on the macronutrient phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) concentration distribution in leaves of the oriental tobacco cv. Myrodata Agriniou. The distribution profiles of leaf P showed an increasing trend from the lower to the upper nodes in all treatments during the vegetative stage up to the fruit set while almost uniform profiles were detected after that. Leaf K, Ca, and Mg accumulation profiles showed decreasing trends from the base to the top over the season. All of the examined macronutrients were accumulated in the lower leaves of the higher fertilized plants late in the season. Leaf dry matter accumulation increased by N fertilization and plant ...

Bromoethane-induced changes in respiration rate, ethylene synthesis, and enzyme activities in potato tubers in relation to dormancy breakage

Summary This paper describes the effect of bromoethane, which promotes the breakage of dormancy of potato tuber buds, on the metabolic activity of tubers, up to the stage of visible sprouting. The respiratory activity of treated tubers increased to a maximum 2 d after treatment, which, despite a subsequent decline, remained at a higher level than that of untreated controls for a further 8 d. Similarly, bromoethane induced a higher level of ethylene release from tubers over 10 d from its application compared to the controls. With the exception of invertases (acid and alkaline), all the other enzymes studied (i.e., hexokinase, fructokinase, glucose-6-phosphate dehydrogenase, uridine-5-diphosphoglucose pyrophosphorylase, adenosine-5-diphosphoglucose pyrophosphorylase and -glucosidase) exhibited higher rates of activity in bromoethane-treated tubers than in the controls. From the results of this study, it appears that the application of bromoethane increased the metabolic activity of potato tubers prior to the first visible sign of bud sprouting, which occurred approx. 10 d after treatment.

Induced parthenocarpic cherry tomato fruits did not shown significant differences in l -ascorbate content but showed different pattern in GalLDH and GME expression

Parthenocarpy in tomato is often induced by auxins to overcome fertilization problems due to low temperatures. To estimate the effect of this agronomical practice on the physiology and dietary value of cherry tomato fruits we determined l-ascorbic acid, the expression and immunolocalization of galactono 1,4 lactone dehydrogenase and the expression of GDP-mannose 3′,5′-epimerase, key genes in l-ascorbic acid biosynthesis. The levels of l-ascorbic acid did not differ between seeded and parthenocarpic fruits while the relative expression of galactono 1,4 lactone dehydrogenase and GDP-mannose 3′,5′-epimerase gene transcripts showed some significant differences between seeded and parthenocarpic fruits. The galactono 1,4 lactone dehydrogenase immunohistolocalization signal was stronger in the ovules and mature embryos of seed-containing fruits. Our data suggest that although there were differences in the expression of the studied genes and in enzyme localization, these did not cause differences in the l-ascorbic acid content of parthenocarpic fruits produced by auxin application.

Co-localization of Carbonic Anhydrase and Phosphoenol-pyruvate Carboxylase and Localization of Pyruvate Kinase in Roots and Hypocotyls of Etiolated Glycine max Seedlings

We investigated the presence of carbonic anhydrase in root and hypocotyl of etiolated soybean using enzymatic, histochemical, immunohistochemical and in situ hybridization approaches. In parallel, we used in situ hybridization and immunolocalization to determine the expression pattern and localization of phosphoenolpyruvate carboxylase. Their co-localization in the root tip as well as in the central cylinder, suggests that a large fraction of the CO2 may be re-introduced into C4 compounds. GmPK3 expression, coding for a cytoplasmic isoform of pyruvate kinase, was detected in all different root cell types, suggesting that both phosphoenolpyruvate-utilizing enzymes are involved in phosphoenolpyruvate metabolism in etiolated soybean roots; a case indicative of the necessary flexibility plant metabolism has to adopt in order to compensate various physiological conditions.