Michael Hodges

Respiration and nitrogen assimilation: targeting mitochondria-associated metabolism as a means to enhance nitrogen use efficiency

Considerable advances in our understanding of the control of mitochondrial metabolism and its interactions with nitrogen metabolism and associated carbon/nitrogen interactions have occurred in recent years, particularly highlighting important roles in cellular redox homeostasis. The tricarboxylic acid (TCA) cycle is a central metabolic hub for the interacting pathways of respiration, nitrogen assimilation, and photorespiration, with components that show considerable flexibility in relation to adaptations to the different functions of mitochondria in photosynthetic and non-photosynthetic cells. By comparison, the operation of the oxidative pentose phosphate pathway appears to represent a significant limitation to nitrogen assimilation in non-photosynthetic tissues. Valuable new insights have been gained concerning the roles of the different enzymes involved in the production of 2-oxoglutarate (2-OG) for ammonia assimilation, yielding an improved understanding of the crucial role of cellular energy balance as a broker of co-ordinate regulation. Taken together with new information on the mechanisms that co-ordinate the expression of genes involved in organellar functions, including energy metabolism, and the potential for exploiting the existing flexibility for NAD(P)H utilization in the respiratory electron transport chain to drive nitrogen assimilation, the evidence that mitochondrial metabolism and machinery are potential novel targets for the enhancement of nitrogen use efficiency (NUE) is explored.

Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves

Cytosolic NADP-dependent isocitrate dehydrogenase (cICDH) produces 2-oxoglutarate (2-OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. Three allelic lines carrying T-DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild-type Col0. Metabolite profiling by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) and high-performance liquid chromatography (HPLC) revealed that loss of cICDH function produced only small effects on leaf compounds involved in carbon and nitrogen assimilation. To analyse whether cICDH contributes to NADPH production under conditions of oxidative stress, the icdh mutation was introduced into the cat2 background, in which increased availability of H(2)O(2) causes perturbed redox homeostasis and induction of stress-related genes. Accumulation of oxidized glutathione and pathogen-related responses were enhanced in double cat2 icdh mutants compared to cat2. Single icdh mutants presented constitutive induction of PR genes, and enhanced resistance to bacteria in icdh, cat2 and cat2 icdh was quantitatively correlated with PR gene expression. However, the effect of icdh in both Col0 and cat2 backgrounds was not associated with enhanced accumulation of salicylic acid (SA). The results suggest that cICDH, previously considered mainly as an enzyme involved in amino acid synthesis, plays a role in redox signalling linked to pathogen responses.

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15 101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.

IDENTIFICATION OF A TOBACCO CDNA ENCODING A CYTOSOLIC NADP-ISOCITRATE DEHYDROGENASE

A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues. To assign this cDNA to a specific ICDH isoenzyme the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced. The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1. The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves. Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern. This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3′ non-coding region of the ICDH1 cDNA. A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (>60%) but a poor homology is observed when compared to Escherichia coli ICDH (<20%). However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E. coli NADP-ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now.

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N‐ and C‐termini of the proteins amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr‐7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.

The Regulation of Plant Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

Phosphoenolpyruvate carboxylase (PEPc) is a multifaceted enzyme that serves different physiological functions in plants. In C3 plants, an important role is in the anaplerotic supply of carbon skeletons for biosynthetic functions such as amino acid synthesis, whereas C4 and crassulacean acid metabolism (CAM) species also have a specific, highly active isoform that catalyses primary CO2 fixation in the photosynthesis pathway. More effort has been concentrated to date on the regulation of the latter, photosynthetic form of PEPc. It has long been known that this form of the enzyme is subject to allosteric control by opposing photosynthesis-related metabolites in the cytosol of the mesophyll cells. The discovery of a phosphorylation process acting on photosynthetic PEPc revitalized interest in this enzyme and the ensuing wealth of data has highlighted signaling mechanisms acting in the regulation of plant metabolism. In C4 plants, the cascade depends upon a cross-talk between the two neighboring photosynthetic cell types, involves classical second messengers like pH, phosphoinositide-specific phospholipase C, inositol-1,4,5-trisphosphate and calcium, leading to up-regulation of the activity of a Ca2+-independent, C4 PEPc-specific protein-serine/threonine kinase (PEPcK), which finally phosphorylates PEPc. The final activity of C4 PEPc and the resulting carbon flux to bundle sheath cells are dependent on the mutual interaction between metabolite and covalent control mechanisms acting on this enzyme. Recent results have suggested that a similar regulatory circuit is operative at night in mesophyll cells of CAM leaves. It has become clear that the anaplerotic PEPc which is found in all plant types, is also regulated by a PEPcK and that phosphorylation of PEPc in C3 plant leaves functions in the coordination of carbon and nitrogen assimilation. We discuss the extent to which parallels can be drawn between the regulation of the different isoforms of PEPc.

The in vitro effects of ATP and protein phosphorylation on the activity of ferredoxin: NADP+ oxidoreductase from spinach chloroplasts

Abstract When ferredoxin:NADP+ oxidoreductase (FNR) was preincubated with [γ-32P]ATP-Mg and separated by native PAGE it was found to be radioactive. This was not seen on SDS-PAGE, unless FNR was preincubated with a crude protein kinase extract with FNR kinase activity under phosphorylating conditions. These observations suggest that FNR contains an ATP-binding domain. It was found that ATP caused an inhibition of FNR diaphorase activity, which when analysed by Lineweaver-Burk plots indicated that ATP was a non-competitive inhibitor with respect to NADPH. This shows that the ATP site is distinct from the NADP(H) active site. The in vitro phosphorylation of FNR on a serine residue(s) by the crude FNR kinase extract led to a modification of ferredoxin (Fd)-dependent FNR activity. An analysis of the data showed that after phosphorylation the apparent Km for Fd and Vmax both increased. This observation suggests that the Fd-FNR interaction is modified after FNR phosphorylation in vitro. Limited proteolysis of phosphorylated FNR followed by SDS-PAGE infers that the phosphorylated amino acid(s) is located near the N-terminus.