Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-07-10
2004-05-04
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C435S252300, C435S419000, C435S468000, C536S023600, C800S278000
Reexamination Certificate
active
06730828
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to glutathione transferase (GST) subunits, to nucleic acid sequences encoding glutathione transferase subunits, and to uses of these glutathione transferases and coding sequences, especially in the field of plant biotechnology.
BACKGROUND OF THE INVENTION
Glutathione transferases (GSTs, EC. 2.5.1.18), also referred to as glutathione S-transferases, are multifunctional enzymes capable of catalysing the conjugation of electrophilic substrates with the tripeptide glutathione (GSH, gamma-glutamylcysteinylglycine). The electrophilic substrate may be of natural or synthetic origin, examples including endogenous stress-metabolites, drugs, pesticides and pollutants. Conjugation with GSH renders the compounds non-toxic and suitable for export from the cytosol and further metabolism. In addition to their activities in GSH conjugation, GSTs may have additional activities as glutathione peroxidases, catalysing the reduction of organic hydroperoxides to the corresponding alcohol according to the reaction:
R—OOH+2GSH→R—OH+GSSG.
All known active GSTs are composed of two polypeptide subunits, with each subunit possessing a binding site for GSH and the electrophilic co-substrate. The two subunits may either be identical, giving rise to a homodimer, or dissimilar giving rise to heterodimers. GSTs may therefore be defined according to their source, or class, and their component subunits according to the nomenclature SpGST x-y, where Sp=source or class of GST; x and y describe the subunit types.
Each discrete subunit is encoded by a distinct gene, with many eukaryotes containing GST multigene families encoding multiple isoenzymes.
The plant in which GSTs have been characterised in the greatest detail is maize (
Zea mays L.
). The major maize GSTs are composed of three discrete subunits, termed I, II and III. These subunits associate together to form three isoenzymes containing the
Zea mays
GST I subunit, namely ZmGSTI-I, ZmGSTI-II and ZmGSTI-III as well as the homodimers ZmGSTII-II and ZmGSTIII-III. The nucieotide sequences of ZmGSTI, ZmGSTII and ZmGSTIII have been determined. In view of their relatedness in sequence, these maize GSTs have collectively been termed type I plant GSTs. Additional maize GSTs with activities toward herbicides have been described as ZmGSTV-V and ZmGSTV-VI. The sequence of ZmGSTV differs markedly from the other maize GSTs described to date, resembling the auxin-inducible GSTs from dicotyledenous plants which have been termed the type III GSTs.
The maize GST subunit types are associated with differing substrate specificities. The ZmGSTI subunit has broad-ranging, but low, activities toward chloro-s-triazine, chloroacetanilide and diphenyl ether herbicides. The ZmGSTII and ZmGSTIII subunits show greater specificity toward chloroacetanilides, while ZmGSTV and ZmGSTVI are highly active toward diphenyl ethers. The GST isoenzymes differ in their patterns of expression in the organs of maize. Thus, ZmGSTI-I and ZmGSTV-V are expressed in all plant parts, while ZmGSTI-II is root specific. The expression of the GST subunits is also differentially affected by herbicide safeners. These are compounds which enhance the tolerance of cereal crops to herbicides, in part, by increasing the expression of detoxifying enzymes such as GSTs. Thus, the ZmGSTII and ZmGSTV subunits accumulate in maize seedlings following treatment with the safeners dichlormid or benoxacor while the ZmGSTI and ZmGSTIII subunits are only modestly enhanced by safeners.
Far less is known regarding GSTs in plant species other than maize. GSTs with activities toward non-herbicide substrates have been identified in some plants, and mRNAs apparently encoding GSTs have been shown to be expressed in plants including carnation, tobacco and thale cress (
Arabidopsis thaliana
). However, isoenzymes with activities toward herbicides have only been definitively identified in soybean, pea and pine trees. Of these, only in soybean has the nucleotide coding sequences of the herbicide-detoxifying GST been reported.
GSTs in plants have also been shown to have secondary activities as glutathione peroxidases, able to reduce organic hydroperoxides, such as fatty acid hydroperoxides to the corresponding monohydroxy alcohols. GSTs with glutathione peroxidase activity have been isolated from peas, soybean,
A. thaliana
and wheat flour. Since fatty acid hydroperoxides are a cornmon result of membrane peroxidation imposed during oxidative stress, glutathione peroxidases provide an important cytoprotective function in preventing the accumulation of fatty acid hydroperoxides and their subsequent degradation to toxic aldehydes. Glutathione peroxidases may therefore have a vital function in protecting plant cells from oxidative stress. The intervention of glutathione peroxidases in lipid peroxidation has also been cited as a determinant of flour quality in wheat.
Of particular relevance to this invention is the lack of knowledge concerning the GSTs of wheat (
Triticum aestivum L
.).
Some information is available from experiments on whole plants and plant extracts. Several herbicides including examples of the chloroacetanilides, as well as dimethenamid and fenoxaprop-ethyl undergo GSH conjugation in the course of their detoxification in wheat. Also, in crude plant extracts OST activities toward chloroacetanilide herbicides, dimethenamid and fenoxaprop-ethyl have been demonstrated.
There have been very few reports of the purification of GSTs from wheat. A GST was purified from wheat flour, and described as a homodimer of 27.5 kDa polypeptides with activity toward the non-herbicide substrate 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione peroxidase activity toward fatty acid hydroperoxides. A safener-induced GST with activity toward CDNB and dimethenamid, termed GSTTaI-I, has been purified and partially sequenced from the wheat progenitor species
Triticum tauschii
, (Reichers et al, (1997), Plant Physiology, 114, pages 1461 to 1470).
Moreover, very little is known regarding GST genes in wheat. An mRNA originally described as wir5, which showed sequence similarity to the type 1 maize GSTs, was identified as accumulating in wheat leaves during the onset of acquired resistance to powdery mildew (
Erysiphe graminis
). The gene was termed gstA1 and shown to be similar in genomic organisation to maize ZmGST1. The gstA1 polypeptide was expressed in recombinant bacteria and shown to have an apparent molecular mass of 29 kDa. The respective enzyme showed GST activity towards the non-herbicide CDNB, though the activity toward other substrates and activity as a glutathione peroxidase was not reported. An antibody was raised to the recombinant GstA1 and used in Western blotting experiments to show that this GST was specifically induced in wheat leaves by pathogen attack. In contrast, a distinct class of GSTs composed of 25 kDa and 26 kDa subunits, which were recognised by an antiserum raised to undefined GSTs in maize, accumulated following exposure to cadmium and the herbicides atrazine, alachlor and paraquat. The activities of these xenobiotic-inducible GSTs in wheat and the corresponding nucleotide sequences were not reported. A cDNA correponding to am mRNA encoding a safener-inducible type III GST has been isolated from
Triticum tauschii
and had the same amino acid sequence as GSTTaI-I, (Reicher et al, (1997), Plant Physiology, 1141, page 1568).
Thus, although wheat is an important crop plant, there has been little molecular characterisation of wheat GSTs or their genes and, to date, only two purified GSTs and two GST gene sequences, gstA1 and GSTTa1 available.
Significantly, neither purified recombinant GST proteins expressed from gene gstA1 or GSTTa1 were reported to exhibit activity towards herbicides. Hence, none of the previous work on wheat GSTs actually provides any means of achieving herbicide resistance based on the function of wheat GSTs.
SUMMARY OF THE INVENTION
We have purified four GST isoenzymes with activity toward herbicides from wheat shoots treated with the h
Cole David J.
Cummins Ian
Edwards Robert
Aventis Agriculture Limited
Baker & Botts L.L.P.
Kruse David H
Nelson Amy J.
LandOfFree
Isolated nucleic acids encoding a wheat glutathione... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Isolated nucleic acids encoding a wheat glutathione..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Isolated nucleic acids encoding a wheat glutathione... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3267811