Pierre Gadal

Evidence for the existence of several enzyme-specific thioredoxins in plants

Thioredoxin, a small molecular weight protein, functioning as hydrogen carrier in DNA synthesis has recently been identified to be an indispensable factor in the light activation of certain regulatory enzymes of plant chloroplasts [l] . In the presence of thioredoxin and ferredoxin-thioredoxin reductase, photochemically-reduced ferredoxin is able to activate enzymeglike NADP malate dehydrogenase [2] and fructose 1,6-bisphosphatase (FBPase) [3] , both enzymes restricted to the chloroplasts [3,4]. In vitro reduced ferredoxin and ferredoxin-thioredoxin reductase can be replaced by the non-physiological sulfhydryl reagent dithiothreitol (DTT), thus eliminating the need for light, but not the need for thioredoxin [3,5,6] . During our efforts to isolate and purify thioredoxin from different plant species (spinach, sorghum and French beans) and plant materials (leaves and roots) we have realized that there are several forms of thioredoxin which are specific for the activation of either NADP malate dehydrogenase or FBPase. Similar findings have recently been reported by [7] . We describe here a simple method for the separation of different forms of thioredoxin which is applicable to various plant materials. In addition our results indicate that the different forms of thioredoxin are enzyme specific. However a given form of thioredoxin isolated from one plant species can activate the respective enzyme isolated from another plant species and vice versa.

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis.

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.

Recovery of active, highly purified phosphoenolpyruvate carboxylase from specific immunoadsorbent column

Since its discovery in 1953 [ 11, PEP carboxylase (orthophosphate:oxaloacetate carboxylase, EC 4.1 -1.3 1) has been the subject of numerous studies. Indeed, the carboxylase, which is ubiquitous among bacteria and plants, represents a means of incorporating inorganic carbon in a non-photosynthetic way. It has been implicated in numerous physiological roles such as, anaplerotic pathways [2], Cq photosynthesis and crassulacean acid metabolism [3,4], stomata1 aperture [5], ionic equilibrium in root cells [6], pH stat [7] and biological rhythms [8]. A number of isoenzymes of PEP carboxylase have been reported [9-l 31. Many experiments have been and are being performed on the functional and regulatory properties of the enzyme and on its localization in cells and tissues [ 141. We have been concerned with the purification of PEP carboxylase isoforms in bean and sorghum leaves and roots [ 15 ,161. Immunochemical techniques provide an efficient method for enzyme purification and localization; unfortunately such techniques usually result in the complete loss of biological activity. Here we describe a new and simple method based on the elution of an active, highly purified enzyme from an immunoadsorbent column.

Occurrence and influence of light on the relative proportions of two glutamine sythetases in rice leaves

Two glutamine synthetases (GS1 and GS2) were identified in green rice leaves by chromatography on a DEAE-cellulose column. The contribution of GS2 to the total activity was high compared to GS1. When an extract from dark-grown leaves was chromatographed, two peaks of enzymatic activity were also detected but in this case GS1 was predominant. During the greening of etiolated leaves GS1 decreased while GS2 increased, and after a light exposure of 48 h the typical pattern of green leaves was obtained. When GS1 and GS2 were rechromatographed no interconversion occurred. The Km values for glutamate and the heat stabilities of the two enzymes were shown to be different.

Intracellular distribution of enzymes associated with nitrogen assimilation in roots

The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.

Relation between protoplast division, cell-cycle stage and nuclear chromatin structure

SummaryUsing different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.

A CDC2 GENE OF PETUNIA-HYBRIDA IS DIFFERENTIALLY EXPRESSED IN LEAVES, PROTOPLASTS AND DURING VARIOUS CELL-CYCLE PHASES

Analysis of p34cdc2 kinase in higher eukaryotes has demonstrated that p34cdc2 function is conserved in all eukaryotic cells. The p34cdc2 kinase (the product of the cdc2 gene) is required during the G1 cell cycle phase at the initiation of DNA replication and also in G2-M phases for entry into mitosis. In this paper we report the isolation and characterization of a cdc2 Petunia hybrida PCR fragment (cdc2Pet). Using a DNA probe based on this fragment and a p34cdc2-specific antibody, cdc2Pet transcript and p34 protein levels were found to be constant both in 2C nuclei of highly proliferating mesophyll 2C cells derived from protoplasts and in 2C nuclei isolated directly from expanded petunia leaves.Both the cdc2Pet transcript and p34cdc2 protein levels were found to be higher in nuclei at 4C than in those at 2C, even when these 4C nuclei were from non-proliferating tissue. Thus cdc2Pet mRNA and protein levels measured in different tissues should not be interpreted to reflect exclusively the proliferative state of the tissue but also the frequency of G2 cells including those in the differentiated state.

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.

Immunocytochemical study of phosphoenolpyruvate carboxylase in nodulated Alnus glutinosa

The activities of phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.3.1) have been investigated in various organs of young nodulated Alnus glutinosa. The root nodules exhibited the highest specific enzyme activity when compared with the one in roots and leaves. Furthermore, in the root nodules the PEP carboxylase was predominantly localized in the cytosol of the large cortical cells containing the endophyte vesicles.

Fatty acid composition of roots and root nodules of Alnus species

Abstract Fatty acid composition of roots and root nodules from Alnus species is reported. Differences in total lipid content and in fatty acid composition w