Sucrose synthase activity and carbohydrates content in relation to phosphorylation status of Vicia faba root meristems during reactivation from sugar depletion.

Carbohydrate starvation of Vicia faba root meristems leads to readjustment of carbohydrate metabolism and blocks the cell cycle in two principal control points (PCP1/2). The cell cycle reactivation is possible after sucrose provision, although with a delay of about 12h. During this period, the cells are sensitive to 6-dimethylaminopurine (6-DMAP) and okadaic acid (OA), inhibitors of protein kinases and phosphatases, respectively. The aim of the present study was to investigate whether those inhibitors are involved in inhibition of cell cycle revival through interference with the activities of two sucrose-cleaving enzymes: sucrose synthase (SuSy; EC 2.4.1.13) and invertase (INV; EC 3.2.1.26). In sugar-starved cells, the in situ activity of both enzymes decreased significantly. Following supplementation of root meristems with sugar, INV remained inactive, but SuSy activity increased. Despite the lack of INV activity, glucose was present in meristem cells, but its content was low in cells treated with OA. In the latter case, the size of plastids was reduced, they had less starch, and Golgi structures were affected. In sugar-starved cells, SuSy activity was induced more by exogenous sucrose than by glucose. The sucrose-induced activity was strongly inhibited by OA (less by 6-DMAP) at early stages of regeneration, but not at the stages preceding DNA replication or mitotic activities. The results indicate that prolongation of regeneration and a marked decrease in the number of cells resuming proliferation (observed in previous studies) and resulting from the action of inhibitors, are correlated with the process of SuSy activation at the beginning of regeneration from sugar starvation.

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹4C]glucose 1-phosphate, [U-¹4C]sucrose, [U-¹4C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹4C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹4-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹4C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹4C]glucose 1-phosphate or adenosine-[U-¹4C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹4C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

A dual-targeted purple acid phosphatase in Arabidopsis thaliana moderates carbon metabolism and its overexpression leads to faster plant growth and higher seed yield.

• Overexpression of AtPAP2, a purple acid phosphatase (PAP) with a unique C-terminal hydrophobic motif in Arabidopsis, resulted in earlier bolting and a higher seed yield. Metabolite analysis showed that the shoots of AtPAP2 overexpression lines contained higher levels of sugars and tricarboxylic acid (TCA) metabolites. Enzyme assays showed that sucrose phosphate synthase (SPS) activity was significantly upregulated in the overexpression lines. The higher SPS activity arose from a higher level of SPS protein, and was independent of SnRK1. • AtPAP2 was found to be targeted to both plastids and mitochondria via its C-terminal hydrophobic motif. Ectopic expression of a truncated AtPAP2 without this C-terminal motif in Arabidopsis indicated that the subcellular localization of AtPAP2 is essential for its biological actions. • Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution. AtPAP2 is the first PAP shown to modulate carbon metabolism and the first shown to be dual-targeted to both plastids and mitochondria by a C-terminal targeting signal. • One PAP-like sequence carrying a hydrophobic C-terminal motif could be identified in the genome of the smallest free-living photosynthetic eukaryote, Ostreococcus tauri. This might reflect a common ancestral function of AtPAP2-like sequences in the regulation of carbon metabolism.

Differential responses of the antioxidant defence system and ultrastructure in a salt-adapted potato cell line.

Changes in lipid peroxidation and ion content and the possible involvement of the antioxidant system in salt tolerance at the cellular level was studied in a potato (Solanum tuberosum L.) callus line grown on 150 mM NaCl (salt-adapted) and in a non-adapted line exposed to 150 mM NaCl (salt-stressed). Salinity reduced the growth rate and increased lipid peroxidation in salt-stressed line, which remained unaltered in the adapted line. Na⁺ and Cl⁻ content increased due to salinity in both lines, but the adapted line displayed greater K⁺/Na⁺ ratio than the stressed one. Total superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2) activities decreased in both salt-exposed lines; catalase (CAT, EC 1.11.1.6) activity did not change in the adapted line, but decreased in the stressed cell line. Salinity caused the suppression of one GR isoform, while the isozyme patterns of SOD, APX, and CAT were not affected. Ascorbate and reduced glutathione increased in both salt-exposed calli lines. α-Tocopherol increased as a result of salt exposure, with higher levels found in adapted calli. Electron microscopy showed that neither the structural integrity of the cells nor membrane structure were affected by salinity, but plastids from adapted cells had higher starch content. The results suggest that the enzymic and non-enzymic components of the antioxidant system are differentially modulated by salt. Different concentrations of antioxidant metabolites are more relevant to the adaptive response to salinity in potato calli than the differences in activity of the antioxidant enzymes.

Plastidial glyceraldehyde-3-phosphate dehydrogenase deficiency leads to altered root development and affects the sugar and amino acid balance in Arabidopsis.

Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD(+) to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

[Ultramorphometric study of the plastids in apical tuber cells of original and transgenic potato plants treated with ambiol].

Ultramorphometric characteristics of plastids in cells of apical tuber meristems of original and defensin gene-transfected potato (Solanum tuberosum L.) plants, either maintained under normal conditions or subjected to treatment with the antioxidant ambiol, were compared. Under normal conditions, the tuber cells of the original and transgenic potato plants differed in neither the number nor size of the plastids. Only certain quantitative distinctions in the development of individual ultrastructural characteristics of plastids were detected. Treatment with ambiol enhanced the differentiation of the internal membrane system of plastids in the cells of original and transgenic plants, especially the tubular membrane systems. Certain differences in the responses to ambiol of cell plastids of original and transgenic plants were related to plastid sizes and development of individual intraplastid structures. The results comply with earlier data on varying responses of mitochondria of original and transgenic plants to ambiol treatment.

Mitochondrial and plastid functions as antimalarial drug targets.

Malaria parasites possess three genomes: the nuclear chromosomes, the mitochondrial genome, and the plastid genome. Realization that the parasites contain a plastid remnant with its own genome has created much excitement not only from a basic biological point of view but also from the prospects for developing new antimalarial drugs. Both the mitochondrial and the plastid genomes are the smallest examples of their kind known to date. The plastid appears to be derived from an ancestral secondary endosymbiotic event. Interestingly, the main functions usually associated with a mitochondrion and a plastid, i.e. oxidative phosphorylation and photosynthesis, do not appear to be conserved in malaria parasites. Completion of the parasite genome sequence has provided the opportunity to assess functions assigned to these highly derivatized organelles. It is clear that these organelles serve vital functions since interference with their activity is incompatible with parasite growth. A number of antimalarial compounds target functions of either the mitochondrion or the plastid. This review will survey our current understanding of mitochondrial and plastid functions with a view to identify processes that are or have a potential to be targets for antimalarial drugs.

Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D.

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

[Geoperception in the lentil root cap]. / La perception geotropique dans la coiffe des racines de Lentille.

Previous analysis showed that, in its initial phase, the geotropic response of Lens culinaris L. roots cannot be explained by a simple action by sliding, pressure or contact of amyloplasts on a sensitive surface located along the longitudinal wall. In this study another mode of action is tested by considering the following parameters as functions of the roots inclination: (1) the distance (d) which the amyloplasts move; (2) their number of contacts (mean c) with parietal cytoplasm; (3) the variable (sin alpha) of the transversal component of the statolith weight (mean M x g sin alpha). It is shown that the initial rate of curvature (mean V), at the various angles, is related to the sedimentation of the amyloplasts by the equation mean V = a log b mean d mean c sin alpha (where a and b are constants). The results obtained prove that the geotropic stimulation is dependent upon the sine of the angle (alpha) of the root inclination and explain the sine rule deviation. The role of statoliths is discussed in the light of recent literature on growth inhibitors which are involved in the geotropic reaction.