F. Ambard-Bretteville

Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate–regulated by interaction of PII with the biotin carboxyl carrier subunit

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the Vmax of the ACCase reaction without altering the Km for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.

Study of the supramolecular organization of light-harvesting chlorophyll protein (LHCP): Conversion of the oligomeric form into the monomeric one by phospholipase A2 and reconstitution with liposomes

The photosynthetic membranes are composed mainly of lipids and proteins arranged in lipo-protein complexes. Among these complexes, the light-harvesting chlorophyll protein (LHCP) represents 40-50% of the total chlorophyll found in the membrane. Consequently, it is supposed to play a fundamental role in the structure of thylakoids, e.g., in grana stacking (reviews [l-4]). Due to its heterogeneous composition including pigments, lipids and polypeptides, it cannot be established with certainty which of its constituents plays the key role in the stacking process. sidered as a passive support. In [7] a more functional role of lipids was proposed and it was suggested that specific arrangements of components necessary for the functioning of the light reactions of photosynthesis can be regulated to a large extent by the lipid content of the membrane [7].

Lipid composition of chlorophyl-protein complexes: Specific enrichment in trans-hexadecenoic acid of an oligomeric form of light-harvesting chlorophyll aa/b protein

Thylakoid membranes of higher plants and green algae can be resolved by SDS-polyacrylamide gel electrophoresis into at least 2 different chlorophyll-protein complexes and -30 polypeptides. The 2 chlorophy~-protein complexes correspond to the P700 chI a--protein complex (CPI) assumed to originate from the reaction centre of photosystem I and to the lightharvesting chl a&protein complex named LHCP or CP II [ 1 ] which is considered to represent the antenna chlorophyll of photosystem II. It is now well known that using milder conditions of solubilization, additional &l-proteins can be obtained. They correspond to a new chla-protein complex generally termed CPa, which may represent the photosystem II reaction centre complex 12-41, and to several oligomeric forms of CP 1 and LHCP f3-131. Although these different &l-protein complexes are characterized by some of their physiological, biochemical and physical properties (reviews [ 1,14--16]), little is known about their lipid composition other than pigments. The &-protein complexes were firstly considered to contain only traces of lipids [ 171. Two recent analyse? -’ -d the lipid composition of sub-

Cross-phosphorylation between Arabidopsis thaliana Sucrose Nonfermenting 1-related Protein Kinase 1 (AtSnRK1) and Its Activating Kinase (AtSnAK) Determines Their Catalytic Activities

Arabidopsis thaliana sucrose nonfermenting 1-related protein kinase 1 complexes belong to the SNF1/AMPK/SnRK1 protein kinase family that shares an ancestral function as central regulators of metabolism. In A. thaliana, the products of AtSnAK1 and AtSnAK2, orthologous to yeast genes, have been shown to autophosphorylate and to phosphorylate/activate the AtSnRK1.1 catalytic subunit on Thr175. The phosphorylation of these kinases has been investigated by site-directed mutagenesis and tandem mass spectrometry. The autophosphorylation site of AtSnAK2 was identified as Thr154, and it was shown to be required for AtSnAK catalytic activity. Interestingly, activated AtSnRK1 exerted a negative feedback phosphorylation on AtSnAK2 at Ser261 (Ser260 of AtSnAK1) that was dependent on AtSnAK autophosphorylation. The dynamics of these reciprocal phosphorylation events on the different kinases was established, and structural modeling allowed clarification of the topography of the AtSnAK phosphorylation sites. A mechanism is proposed to explain the observed changes in the enzymatic properties of each kinase triggered by these phosphorylation events.

Evidence for a chlorophyll a/b—protein complex associated with Photosystem II

A new chlorophyll a/b—protein complex designated as LHCPx was separated by SDS—PAGE. This complex differs from the light‐harvesting chl a/b—protein of PS II, by its chl a/b ratio, its fluorescence properties and its apoprotein composition. Evidence is provided that this complex corresponds to a part of PS I antenna.

The impact of PEPC phosphorylation on growth and development of Arabidopsis thaliana: Molecular and physiological characterization of PEPC kinase mutants

Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T‐DNA‐insertional mutants showed very little (dln1), or no (csi8) light‐induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass‐spectrometry‐based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism.

Separation of phosphorylated from non-phosphorylated LHCP polypeptides by two-dimensional electrophoresis

‘In vitro’ phosphorylated thylakoid polypeptides were studied by means of different electrophoretic techniques. A highly resolving two‐dimensional electrophoresis method, recently developed in the laboratory using CHAPS and SDS as detergent for electrofocusing, allows the separation of each of the LHCP apoproteins into several molecular species. Those having more acidic isoelectric points correspond to the phosphorylated forms.

Lipid Induced Transformation of the Monomeric to Oligomeric Form of the LHCP

It is now clearly established that all the chlorophyll and carotenoids in the thylakoids of higher plants are non-covalently bound to specific proteins associated in supramolecular complexes. The improvment of solubilization and analysis technics of thylakoid membranes allowed to extract these supramolecular complexes closer to the “in vivo” state. Oligomeric forms of P 700-chl a protein and light harvesting chlorophyll a/b protein were isolated (Remy et al., 1977, 1978). Recently, the presence of lipids in these chlorophyll-proteins was reported by different groups (Selstam, 1980; Tremolieres et al., 1981; Ryrie et al., 1980). Rawyler et al., 1980, Tremolieres et al., 1981 have shown that LHCP was rich in phospha-tidylcerol (PG) containing a high amount of 3-trans-hexadecenoic acid, an acyl lipid which has been implicated as the LHCP in the formation of the grana stacks (Dubacq, Tremolieres, 1983). Moreover, we reported recently that this PG enrichment was more significant in an oligomeric form of LHCP and that it was possible to convert partially monomeric LHCP into oligomeric LHCP using PG liposomes (Remy et al., 1982).

Normal and Cytoplasmic Male Sterile (Raphanus Sativus Cytoplasm) Lines of Brassica Napus Present Different Chloroplast DNA Restriction Maps and Different CF1-ß Subunits

Cytoplasmic male sterility of the rapeseed B. napus arose from intergeneric crosses involving a cytoplasmic male sterile (cms) radish (Vedel et al., 1982). The cms lines of B. napus present two maternally inherited traits, cytoplasmic male sterility and chlorophyll deficiency both resulting from interactions between the nuclear genetic system of B. napus and organellar genetic ones of radish. Preliminary data on restriction endonuclease analysis of cp DNA and 2-D electrophoresis of chloroplast polypeptides have revealed marked differences between normal (N) and cms lines (Vedel and Mathieu, 1983; Remy and Ambard-Bretteville, 1983). In this paper, we present (1) physical maps for each type of B. napus cp DNA using five restriction enzymes and gene mapping of rRNA and of five protein genes; (2) bidimensional electrophoresis of thylakoid polypeptides with an attempt to explain different IEF mobilities between N and cms s subunits of the CF1.

EcoRI analysis of chloroplastic DNAs and polypeptidic composition of thylakoids from wheat and related species

Abstract Electrophoregrams of thylakoid polypeptides isolated from diploid, tetraploid and hexaploid wheat species are compared. All hexaploids and tetraploids so far analysed except in Aegilops crassa are characterized by similar polypeptidic electrophoregrams. Among diploids, Ae. speltoides presents a polypeptidic pattern similar to that observed with tetra- and hexaploids but Ae. squarrosa and Triticum monococcum differ solely by one polypeptide called x . This polypeptide of unknown function possesses a stable apparent mol. wt. of 17 kdaltons in Ae. speltoides and all hexaploids and tetraploids with the exception found in Ae. crassa (4 x and 6 x ) where this polypeptide presents a molecular weight of 16 kdaltons similar to that of T. monococcum . In Ae. squarrosa polypeptide x has a mol. wt. of 16.5 kdaltons. For each wheat species a great analogy is found between the molecular weight variations of polypeptide x and the differences observed on electrophoregrams of DNA fragments obtained after the EcoRI specific cleavage of chloroplast DNA.