Anthony R. Ashton

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis.

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO4) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds(tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3,tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutantstds1, tds2, tds3, andtds5 (which do not produce PA) and tds6(6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 andtt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.

3 - Enzymes of C4 Photosynthesis

[Extract] The history of the resolution of C4 photosynthesis follows a pattern of demonstrating the operation of unique photosynthetic biochemistry by various means and then identifying the enzymes necessary to support that biochemistry. Critical to the developing understanding of this process was the recognition of two types of photosynthetic cells in C4 plants (mesophyll and bundle sheath) with quite different enzyme complements and distinct biochemical roles (see Fig. 3.1). As currently interpreted (see Edwards and Walker, 1983; Hatch, 1987) the reactions unique to the C4 pathway serve, in association with some remarkable modifications of leaf anatomy and ultrastructure, to concentrate CO2 in bundle sheath cells for utilisatiqn by the photosynthetic carbon reduction cycle carboxylase, ribulose L5-bisphosphate carboxylase-oxygenase (Rubisco). The Rubisco-mediated oxygenase reaction and associated photorespiration are thereby eliminated.

Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction.

BACKGROUND NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. RESULTS The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 A resolution. The core structure is homologous to AND-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP+, hydrogen bonding to the catalytic His225 as well as obstructing access of the C4 acid substrate. Two loops flanking the active site, termed the Arg2 and Trp loops, that contain the C4 acid substrate binding residues are prevented from closing by the C-terminal extension. CONCLUSIONS The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP+ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP+ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP+ but not NADPH.

Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat.

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH4)2SO4, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; Km values at pH 8.5 are oxaloacetate, 18 microM; malate, 24 mM; NADPH, 50 microM; and NADP, 45 microM. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, Ki = 50 microM). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of Mr 38,000. Active enzyme is much more sensitive (greater than 50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10(2) from the binding affinities for active NADP-malate dehydrogenase and 10(4) for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.

Molecular cloning of two different cDNAs for maize acetyl CoA carboxylase.

Acetyl CoA carboxylase (EC 6.4.1.2) in plants is a chloroplast-localized, biotin-containing enzyme that catalyses the carboxylation of acetyl CoA to malonyl CoA, the first committed step of the fatty acid biosynthesis pathway. Acetyl CoA carboxylase is the target site for the monocotyledon-specific aryloxy-phenoxypropionate and cyclohexanedione groups of herbicides. We have purifed a herbicide-sensitive acetyl CoA carboxylase from maize leaves to homogeneity (specific activity 7 μmol min-1 mg-1), separating it during the purification from a minor herbicide-resistant acetyl CoA carboxylase. The purified enzyme is a dimer of 230 kDa subunits. Antibodies raised to the purified acetyl CoA carboxylase detected three cross-reacting clones in a maize leaf cDNA expression library, each having an insert of 4–4.5 kb. Restriction analysis and sequencing showed that the cDNAs were derived from two different transcripts. Comparison of the deduced amino acid sequences with those of chicken and yeast acetyl CoA carboxylases confirmed that both types encoded acetyl CoA carboxylase, corresponding to the C-terminal half of the enzyme. The overall identity of the maize and chicken sequences was 37% (58% similarity) but for some shorter regions was much higher. Analysis of six other acetyl CoA carboxylase clones recovered from the maize cDNA library showed four belonged to one type and two to the other. The nucleotide sequence similarity between the two types of cDNA was approximately 95% in the coding region but considerably less in the 3′-untranslated region. Northern blot analysis of maize RNA showed a single band of 8.2–8.5 kb for acetyl CoA carboxylase mRNA. Southern blot hybridisations indicated that there are probably no more than two genes in maize for acetyl CoA carboxylase. The possible significance of two different cDNAs for acetyl CoA carboxylase is discussed.

Regulation of C4 photosynthesis: Regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.

A role for ribulose-1,5-bisphosphate carboxylase as a metabolite buffer

Ribulose1 ,

Isolation and characterization of four genes encoding pyruvate, phosphate dikinase in the oomycete plant pathogen Phytophthora cinnamomi.

bisphosphate carboxylase, the most abundant protein known, is localized within the stromal phase of the chloroplast. The concentration of Ru-1,5-P2 carboxylase in this compartment has been estimated to be 3-4 mM (active site) or 250-300 mg/ml [1,2]. In fact the concentration of Ru-1,5-P2 carboxylase in the stroma is similar to the concentration of the enzyme in some crystals of the purified protein (type I crystals contain 266 mg protein/ml [3]). The extraordinary abundance of this protein is largely due to the requirement for high rates of photosynthetic carbon assimilation and the low catalytic turnover rate of the enzyme (2.8 pmol . min-l . mg -I or 3 s-l, [4]). Some enzymes are present in tissues at greater concentrations than their substrates [5,6]. This is also the case for Ru-1,5-P2 carboxylase where [CO21 is 11 PM while RU-1,5-P2 levels normally range from 0.2 4 mM depending upon experimental conditions [7]. Conditions such as these do not fulfill conditions normally assumed for enzymic catalysis where the substrate concentration is far in excess of the enzyme concentration. The consequences of these conditions upon the kinetics of Ru-1,5-P2 carboxylase have been studied in detail [8]. Thus, although the K,,, of Ru-1,5-P2 carboxylase for Ru-1,5-P2 is 20-30 yM the half-maximal rate of carboxylation can only be achieved when there is 3 1 Ru-1,5-P2/2 Abbreviations: RU-1,5-P2, ribulose-1,5-bisphosphate; Fru-1,6-PI, fructose-l,&bisphosphate; Sed-1,7-P2, sedoheptulose-1,7-bisphosphate active sites, i.e., 2 mM (or lOO-times the K,,, for Ru-1,5-P2). The sheer abundance of Ru-1,5-P2 carboxylase can also shed light on potential allosteric regulation of the enzyme. Some metabolites including Sed-1,7-P2 and NADPH may regulate Ru-1,5-P2 carboxylase activity in vivo [9]. However, the concentration of enzyme is significantly greater than the concentration of most of the proposed effecters [ 1,101. Thus, if all the Fru1,6-q of the chloroplast ( 0.4 mM) was bound to Ru-1,5-q carboxylase most of the Ru-1,5-P2 carboxylase molecules would still not be affected. What was not been explicitly discussed is the corollaly to these observations, i.e., what effect will the presence of mM Ru-1,5-P2 carboxylase have upon the metabolism of these effecters? Ru-1,5-P2 carboxylase could potentially bind a large fraction of the total chloroplast Fru-1,6-4, Sed-1,7-P2 or NADPH which would effectively reduce the free concentration of these metabolites by 2 IO-fold. This metabolite buffering would have profound effects upon photosynthetic carbon metabolism and our interpretation of it since the redox potential of the NADPH/NADP+ couple can influence numerous reactions directly and indirectly while the 2 bisphosphatase reactions are important sites for regulation of the photosynthetic carbon reduction cycle.