Rod Casey

Distribution and Some Properties of Seed Globulins

This chapter is intended to highlight recent advances, generic themes and gaps in our knowledge of the seed globulins of individual groups of plants and their evolution, that emerge from the following thirteen detailed descriptions. It is not intended to be a comprehensive review of seed globulins, for which the reader is referred to Derbyshire et al. (1976), Croy and Gatehouse (1985), Higgins (1984), Casey et al. (1986), Rerie et al. (1992) and Boulter and Croy (1997).

Kinetics of thermal inactivation of pea seed lipoxygenases and the effect of additives on their thermostability

Abstract Mature pea seeds contain two major lipoxygenases (LOX) isoenzymes designated LOX-2 and LOX-3. The thermal inactivation of crude pea LOX and the recombinant LOX (rLOX) were studied. Heat-inactivation plots for crude extracts of pea LOX were linear from which thermodynamic activation parameters, ΔH # , ΔS # sand ΔG # have been estimated. The enzymatic activity was relatively stable with a respective half-life ( t 1/2 ) at 60 °C of 54.2 min for LOX from pea ( Pisum sativum L. cv. Birte) or 18.4 min for a mutant line lacking LOX-2. At 50°C the thermostability of LOX-3 present in crude extracts of the mutant strain ( t 1/2 =66.8 min) was 90% greater than purified recombinant LOX-3 (rLOX-3; t 1/2 =34.6 min). However, rLOX-3 was more heat-stable than rLOX-2. Both rLOX-3 and pea mutant line lacking LOX-2 possessed considerable thermostability at 60°C ( t 1/2 =16.5 min and 18.4 min, respectively). Even at the higher temperatures of 70°C the t 1/2 values were 84 and 51, respectively. It is suggested that LOX in crude enzyme extracts was stabilised at 50°C due to protection by other constituents, possibly including starch and proteins. Separate tests at 70°C in the presence of additives (polyols, detergents and small ions) showed that sucrose was the most effective stabiliser and increased the stability of pea LOX by 400–600%.

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers.

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Recombinant Lipoxygenases and Oxylipin Metabolism in Relation to Food Quality

Abstract Lipoxygenases catalyze the conversion of polyunsaturated fatty acids into hydroperoxides, that are in turn converted to oxylipins, which play important roles in defence reactions in plants and animals. This review describes the distribution of lipoxygenases in Nature, their diversity in terms of structure and catalytic activity, and their significance for food biotechnology. The last includes the production of flavors and aromas, the destruction of vitamins, pigments and other anti-oxidants, the improvement of dough rheology during baking, and the potential of recombinant lipoxygenases, and other enzymes of oxylipin metabolism, for food biotechnology.

Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes

BackgroundHydroperoxide lyase (HPL) is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX) to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively.ResultsTo study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two M. truncatula HPL cDNAs and the localisation of the corresponding chimeras were verified by confocal microscopy in tobacco protoplasts and leaves. Results reported here indicated a distribution of M.truncatula 9/13-HPL (HPLF) between cytosol and lipid droplets (LD) whereas, as expected, M.truncatula 13-HPL (HPLE) was targeted to plastids. Notably, such endocellular localisation has not yet been reported previously for any 9/13-HPL. To verify a possible physiological significance of such association, purified recombinant HPLF was used in activation experiments with purified seed lipid bodies. Our results showed that lipid bodies can fully activate HPLF.ConclusionWe provide evidence for the first CYP74C enzyme, to be targeted to cytosol and LD. We also showed by sedimentation and kinetic analyses that the association with LD or lipid bodies can result in the protein conformational changes required for full activation of the enzyme. This activation mechanism, which supports previous in vitro work with synthetic detergent micelle, fits well with a mechanism for regulating the rate of release of volatile aldehydes that is observed soon after wounding or tissue disruption.

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent‐free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48‐fold increase in k cat/K m (to 5.9 × 107 M−1 s−1) with concomitant changes in the spin state equilibrium of the haem‐iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9‐AOS activity and behaviour in vitro has implications for its role in planta.

Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer-micelle association

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/K(m) of up to 1.6x10(8) M(-1) x s(-1) is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

The effect of modifying carbohydrate metabolism on seed protein gene expression in peas

Summary Mutations at the r locus in peas ( Pisum sativum L.) affect starch content and composition by altering the activity of a specific isoform (SBEI) of starch-branching enzyme. There are several pleiotropic consequences of homozygosity for a mutant allele at the r locus, including seed wrinkling, and increased lipid and sugar concentrations that are thought to be, at least in part, a consequence of the re-partitioning of carbon that is incorporated into starch in the wild-type line. Mutant alleles at the r locus also have an effect on storage protein gene expression, apparently through the specific destabilization of legumin mRNAs in the high-sugar environment of the developing rr seed; the amounts of vicilin and convicilin mRNAs are unaffected. Other wrinkled-seeded mutants have been identified that perturb starch synthesis and result in elevated sugar levels in developing seeds; these include rb , a mutation in one of the structural genes for ADPglu-cose pyrophosphorylase. The recent production of a series of near-isogenic mutant and double mutant backcross Unes for r and rb , and of near-isogenic lines containing four additional mutations that influence starch synthesis ( lam , rug-3 , rug-4 and rug-5 , defective in a granule-bound starch synthase, plastidial phosphoglucomutase, sucrose synthase and a soluble starch synthase, respectively) has provided the opportunity to examine the expression of legumin and vicilin genes in a range of different near-isogenic lines with different patterns of carbohydrate metabolism. We report and discuss here analyses of legumin and vicilin deposition in pea genotypes RRRbRb (wild-type round-seeded), rrRbRb, RRrbrb, rrrbrb (all wrinkled-seeded), the homozygous wrinkled-seeded mutants rug-3 , rug-4 and rug-5 and the round-seeded low-amylose mutant lam .

Isolation of a pea (Pisum sativum) seed lipoxygenase promoter by inverse polymerase chain reaction and characterization of its expression in transgenic tobacco

Part of the 5′-flanking sequence of a pea (Pisum sativum) lipoxygenase (LOX) gene was cloned, after amplification from genomic DNA by inverse polymerase chain reaction. Translational and transcriptional fusions of 818 bp of the 5′-flanking region and its deletion derivatives (−513 and −356) were made to a β-glucuronidase (GUS)-coding sequence and introduced into tobacco. Analysis of T1 transformants showed that the 818 bp 5′-flanking sequence drove GUS expression in seeds that was temporally regulated in a fashion similar to the accumulation of LOX mRNA in developing pea seeds. Contrary to expectations, however, expression of the 818 bp promoter-GUS fusion was not seed-specific; GUS activity was highest in leaves and also present in stems and, to a lesser extent, roots. Deletion analysis identified the region between −818 and −513 as essential for high-level, temporally regulated expression in seeds and also indicated that the sequence between −513 and −356 plays a negative role in leaf/stem, but not seed, expression. Comparison of translational and transcriptional fusions indicated that the LOX initiation codon was used more efficiently than the GUS initiation codon by the tobacco leaf translational apparatus.

Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5′ end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5′-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.