Ian Cummins

Glutathione-mediated detoxification systems in plants

Recent work has highlighted the presence of diverse glutathione-dependent enzymes in plants with potential roles in the detoxification of both xenobiotic and endogenous compounds. In particular, studies on glutathione transferases are further characterising their role in xenobiotic metabolism, and also raising intriguing possible roles in endogenous metabolism. The solution of their three-dimensional structures together with studies on their molecular diversity and substrate specificity is providing new insights into the function and classification of these enigmatic enzymes.

Multiple roles for plant glutathione transferases in xenobiotic detoxification

Discovered 40 years ago, plant glutathione transferases (GSTs) now have a well-established role in determining herbicide metabolism and selectivity in crops and weeds. Within the GST superfamily, the numerous and plant-specific phi (F) and tau (U) classes are largely responsible for catalyzing glutathione-dependent reactions with xenobiotics, notably conjugation leading to detoxification and, more rarely, bioactivating isomerizations. In total, the crystal structures of 10 plant GSTs have been solved and a highly conserved N-terminal glutathione binding domain and structurally diverse C-terminal hydrophobic domain identified, along with key coordinating residues. Unlike drug-detoxifying mammalian GSTs, plant enzymes utlilize a catalytic serine in place of a tyrosine residue. Both GSTFs and GSTUs undergo changes in structure during catalysis indicative of an induced fit mechanism on substrate binding, with an understanding of plant GST structure/function allowing these proteins to be engineered for novel functions in detoxification and ligand recognition. Several major crops produce alternative thiols, with GSTUs shown to use homoglutathione in preference to glutathione, in herbicide detoxification reactions in soybeans. Similarly, hydroxymethylglutathione is used, in addition to glutathione in detoxifying the herbicide fenoxaprop in wheat. Following GST action, plants are able to rapidly process glutathione conjugates by at least two distinct pathways, with the available evidence suggesting these function in an organ- and species-specific manner. Roles for GSTs in endogenous metabolism are less well defined, with the enzymes linked to a diverse range of functions, including signaling, counteracting oxidative stress, and detoxifying and transporting secondary metabolites.

Key role for a glutathione transferase in multiple-herbicide resistance in grass weeds

Multiple-herbicide resistance (MHR) in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides (Am) AmGSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, AmGSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward AmGSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.

Biosynthesis of seed storage products during embryogenesis in rapeseed, Brassica napus

Summary The timing of the synthesis of storage oils and storage proteins during embryogenesis in field-grown rapeseed, Brassica napus L. has been examined. For the first two weeks after anthesis the embryos grew by cell division until they were about 1 mm in length. Storage oil accumulation was apparent even in very young embryos but only became significant when cell division ceased and cell expansion commenced at 2-3 weeks after anthesis. The most rapid phase of oil deposition was between 4 and 6 weeks after anthesis. The bulk of the storage oil, which accumulated initially as large oil-bodies of 2-4pm diameter, was deposited before the onset of storage protein synthesis. The major seed storage proteins, cruciferin and napin, were synthesised from week 5 and the maximal rate of synthesis was between weeks 5 and 7 after anthesis. The hydrophobic protein, oleosin, which accounted for 20% of total protein in mature seeds, was synthesised between 7 and 10 weeks after anthesis. It has recently been shown that this protein is the major component of the osmiophilic membrane which develops around the storage oil-bodies at this stage of embryogenesis [Murphy et al. (1989) Biochem. J. 258,285-293]. The acquisition of a proteinaceous membrane by the oil-bodies coincided with a reduction in their average diameter from 2-4Im to 0.3 -1 p,m. It is proposed that there are three major phases in storage product formation in rapeseed embryos, (i) oil deposition into large storage bodies, (ii) polar protein deposition into vacuoles, (iii) oleosin synthesis and assembly onto oil-bodies causing their reduction in size. The significance of these results for the regulation of storage product synthesis in oilseeds is discussed.

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.

The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

Differential, temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation

The temporal and spatial expression of oleosin and Δ9-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height.In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins. Oil-body fractions prepared from young (2.5 mg) embryos contained very little oleosin protein, as examined by SDS-PAGE and western blotting, whereas identically prepared fractions from dry seeds contained over 10% (w/w) oleosin. Dehydration of oil bodies from young embryos resulted in their breakdown and coalescence into large clumps of oil which could not be re-emulsified, even after rehydration. In contrast, the oleosin-rich oil bodies from mature embryos were stable to dehydration and subsequent rehydration. It is suggested that, in developing rapeseed embryos, the accumulation of storage oil and oleosins is not concomitant but that the eventual deposition of oleosins onto the surfaces of storage oil bodies is essential for their stability during seed desiccation.

Nucleotide sequence and temporal regulation of a seed-specific Brassica napus cDNA encoding a stearoyl-acyl carrier protein (ACP) desaturase.

The nucleotide sequence is reported for a cDNA containing the entire coding region of a stearoyl-ACP desaturase (EC 1.14.99.6) fromBrassica napus L. cv. Jet neuf. The cDNA was obtained from a library constructed from poly(A)+ RNA purified from embryo tissue. The derived amino acid sequence demonstrates substantial similarity with those from other plant Δ9-desaturases. Comparative RNA-dot blot analyses using the Δ9-desaturase cDNA and a rapessed oleosin cDNA as probes showed that although both these transcripts were seed-specific, they exhibited distinct patterns of temporal regulation. The desaturase message was induced by 25 days after anthesis (DAA), peaking at 45 DAA but decreasing considerably thereafter. In contrast, the oleosin transcript did not increase until 45–50 DAA, reaching a peak much later at about 70 DAA.

Safener responsiveness and multiple herbicide resistance in the weed black‐grass (Alopecurus myosuroides)

Safeners enhance the selectivity of graminicidal herbicides such as fenoxaprop ethyl in cereals, by increasing their rates of detoxification in the crop. While studying the selectivity of fenoxaprop ethyl in wheat, we determined that the safeners mefenpyr diethyl and fenchlorazole ethyl also enhanced herbicide tolerance in the competing weed black-grass (Alopecurus myosuroides). Fenoxaprop ethyl was detoxified by conjugation with glutathione in both wheat and black-grass, with the resulting metabolites processed to the respective cysteine derivatives, which were then N-glycosylated. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabolism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydroxymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respectively. These safener-induced changes closely mirrored those determined in two independent black-grass populations that had acquired multiple herbicide resistance (MHR) in the field. In addition to enhanced glutathione metabolism, both safener treatment and MHR resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone-C-glycosides and anthocyanins. Our results demonstrate that safening in a grass weed is associated with an inducible activation in antioxidant and secondary metabolism which mirrors the biochemical phenotype exhibited in plants that are resistant to multiple classes of herbicides.

Glutathione transferases in herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides)†

Glutathione transferase (GST) activities toward the selective herbicide fenoxaprop-ethyl, together with thiol contents, have been compared in seedlings of wheat (Triticum aestivum) and two populations of black-grass (Alopecurus myosuroides) which are resistant to a range of herbicides (Peldon and Lincs E1), and a black-grass population which is susceptible to herbicides (Rothamsted). GST activities toward the non-cereal herbicides metolachlor and fluorodifen were also determined. On the basis of enzyme specific activity, GST activities toward fenoxaprop-ethyl in the leaves were in the order wheat > Peldon = Lincs E1 > Rothamsted, while with fluorodifen and metolachlor the order was Peldon = Lincs E1 > Rothamsted > wheat. Using an antibody raised to the major GST from wheat, which is composed of 25-kDa subunits, it was shown that the enhanced GST activities in both Peldon and Lincs E1 correlated with an increased expression of a 25-kDa polypeptide and the appearance of novel 27-kDa and 28-kDa polypeptides. Leaves of both wheat and black-grass contained glutathione and hydroxymethylglutathione, with the concentrations of glutathione being in the order Peldon > Lincs E1 = Rothamsted = wheat. However, in glasshouse dose-response assays, the Lincs E1 population showed much greater resistance to fenoxaprop-ethyl than Peldon. We conclude that high GST activities and the availability of glutathione may contribute partially to the relative tolerance of black-grass to herbicides detoxified by glutathione conjugation. Although herbicide-resistant populations show enhanced GST expression, in the case of fenoxaprop-ethyl the associated increased detoxifying activities alone cannot explain the differences between populations in the degree of resistance seen at the whole plant level.