Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Acclimation of photosynthesis to increasing atmospheric CO2: The gas exchange perspective

The nature of photosynthetic acclimation to elevated CO₂ is evaluated from the results of over 40 studies focusing on the effect of long-term CO₂ enrichment on the short-term response of photosynthesis to intercellular CO₂ (the A/C_i response). The effect of CO₂ enrichment on the A/C_i response was dependent on growth conditions, with plants grown in small pots (< 5 L) or low nutrients usually exhibiting a reduction of A at a given C_i, while plants grown without nutrient deficiency in large pots or in the field tended to exhibit either little reduction or an enhancement of A at a given C_i following a doubling or tripling of atmospheric CO₂ during growth. Using theoretical interpretations of A/C_i curves to assess acclimation, it was found that when pot size or nutrient deficiency was not a factor, changes in the shape of A/C_i curves which are indicative of a reallocation of resources within the photosynthetic apparatus typically were not observed. Long-term CO₂ enrichment usually had little effect or increased the value of A at all C_i. However, a minority of species grown at elevated CO₂ exhibited gas exchange responses indicative of a reduced amount of Rubisco and an enhanced capacity to metabolize photosynthetic products. This type of response was considered beneficial because it enhanced both photosynthetic capacity at high CO₂ and reduced resource investment in excessive Rubisco capacity. The ratio of intercellular to ambient CO₂ (the C_i/C_a ratio) was used to evaluate stomatal acclimation. Except under water and humidity stress, C_i/C_a exhibited no consistent change in a variety of C₃ species, indicating no stomatal acclimation. Under drought or humidity stress, C_i/C_a declined in high-CO₂ grown plants, indicating stomata will become more conservative during stress episodes in future high CO₂ environments.Abbreviations A net CO₂ assimilation rate - C_i (C_a) intercellular (ambient) partial pressure of CO₂ - operational C_i intercellular partial pressure of CO₂ at a given ambient partial pressure of CO₂ - g_s stomatal conductance - normal CO₂ current atmospheric mole fraction of CO₂ (330 to 355 mol mol^–1) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO₂ assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO₂ fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO₂ fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO₂ fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the malate valve prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The malate valve is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the malate valve functions at various redox states of the acceptor side of Photosystem I.Abbreviations ARC anabolic reduction charge (NADPH/(NADP+NADPH)) - Chl chlorophyll - DTT dithiothreitol; Fd-ferredoxin - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetate - PS photosystem - qN non-photochemical quenching - qP photochemical quenching - _E quantum efficiency of PS II Dedicated to Prof. Dr. Hans Walter Heldt on the occasion of his 60th birthday.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization,purification and kinetic properties

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, -kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where -kg is the first substrate to bind followed by the binding of O₂ and desacetoxyvindoline. Their K _m values for -kg, O₂ and desacetoxyvindoline are 45 M, 45 M and 0.03 M, respectively. The first product to be released was deacetylvindoline followed by CO₂ and succinate, respectively.Abbreviations -kg -ketoglutarate or 2-oxoglutarate - NMT N-methyltransferase - SAM S-adenosyl-l-methionine - TLC thin layer chromatography - VBL vinblastine - VCR vincristine     

Expression of a single gene encoding microbody NAD-malate dehydrogenase during glyoxysome and peroxisome development in cucumber

A full-length cDNA clone encoding microbody NAD⁺-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD⁺-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.