Increasing plant growth by modulating omega-amidase expression in plants
View Patent ↗The present disclosure relates to compositions and methods for increasing the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules in plants by modulating levels of ω-amidase to increase nitrogen use efficiency, resulting in enhanced growth, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.
1. A transgenic plant comprising an ω-amidase transgene, wherein the ω-amidase transgene is operably linked to a root-preferred promoter and wherein the ω-amidase transgene encodes an ω-amidase protein having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, wherein the transgenic plant has increased biomass relative to a plant of the same species that does not comprise the ω-amidase transgene.
2. The transgenic plant according to claim 1 , wherein the ω-amidase transgene is incorporated into the genome of the plant.
3. The transgenic plant according to claim 1 , wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2 promoter, GI4 promoter, GI5 promoter, and GRP promoter.
4. The transgenic plant according to claim 1 , wherein endogenous ω-amidase expression in leaf tissue is inhibited.
5. The transgenic plant according to claim 4 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor.
6. The transgenic plant according to claim 5 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected from the group consisting of a mutant ω-amidase gene that eliminates endogenous ω-amidase expression, an endogenous ω-amidase knockout mutant, and an endogenous ω-amidase knockdown mutant.
7. The transgenic plant according to claim 4 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous ω-amidase gene.
8. The transgenic plant according to claim 4 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from the group consisting of 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
9. The transgenic plant according to claim 1 , wherein root-preferred expression of the ω-amidase transgene results in an increased leaf-to-root ratio of 2-oxoglutaramate relative to a plant of the same species that does not comprise the ω-amidase transgene.
10. The transgenic plant according to claim 9 , wherein the leaf-to-root ratio of 2-oxoglutaramate is at least two times higher than that of a plant of the same species that does not comprise an ω-amidase transgene.
11. The transgenic plant according to claim 1 , further comprising a GPT transgene.
12. The transgenic plant according to claim 11 , wherein the GPT transgene is a GPT/F:V mutant given by SEQ ID NO:1.
13. The transgenic plant according to claim 1 , further comprising a GPT transgene and a GS transgene.
14. The transgenic plant according to claim 13 , wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
15. The transgenic plant according to claim 1 , wherein the transgene is codon optimized for expression in the plant.
16. The transgenic plant according to claim 1 , wherein the transgenic plant has increased nitrogen use efficiency.
17. The transgenic plant according to claim 1 , wherein the transgenic plant is selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis , grasses, fruits, vegetables, flowering plants, and trees.
18. A progeny of any generation of the transgenic plant according to claim 1 , wherein the progeny comprises an ω-amidase transgene operably linked to a root-preferred promoter and wherein the ω-amidase transgene encodes an ω-amidase protein having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, wherein the transgenic plant has increased biomass relative to a plant of the same species that does not comprise the ω-amidase transgene.
19. A seed of any generation of the transgenic plant according to claim 1 , wherein the seed comprises an ω-amidase transgene operably linked to a root-preferred promoter and wherein the ω-amidase transgene encodes an ω-amidase protein having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, wherein the seed germinates to produce a transgenic plant that has increased biomass relative to a plant of the same species that does not comprise the ω-amidase transgene.
20. A method for increasing biomass of a plant relative to a wild type or untransformed plant of the same species, comprising:
(a) introducing an ω-amidase transgene into the plant, wherein the ω-amidase transgene is operably linked to a root-preferred promoter and wherein the ω-amidase transgene encodes an ω-amidase protein having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3;
(b) expressing the ω-amidase transgene in root tissue of the plant or the progeny of the plant; and
(c) selecting a plant having an increased biomass relative to a plant of the same species that does not comprise the ω-amidase transgene.
21. The method according to claim 20 , wherein the ω-amidase transgene is incorporated into the genome of the plant.
22. The method according to claim 20 , wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2 promoter, GI4 promoter, GI5 promoter, and GRP promoter.
23. The method according to claim 20 , wherein endogenous ω-amidase expression in leaf tissue is inhibited.
24. The method according to claim 23 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor.
25. The method according to claim 23 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected from the group consisting of a mutant ω-amidase gene that eliminates endogenous ω-amidase expression, an endogenous ω-amidase knockout mutant, and an endogenous ω-amidase knockdown mutant.
26. The method according to claim 23 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous ω-amidase gene.
27. The method according to claim 23 , wherein the endogenous ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from the group consisting of 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
28. The method according to claim 20 , wherein the leaf-to-root ratio of 2-oxoglutaramate is at least two times higher than that of a progenitor or wild type plant of the same species.
29. The method according to claim 20 , wherein the plant further comprises a GPT transgene.
30. The method according to claim 29 , wherein the GPT transgene is a GPT/F:V mutant given by SEQ ID NO:1.
31. The method according to claim 20 , wherein the plant further comprises a GPT transgene and a GS transgene.
32. The method according to claim 31 , wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
33. The method according to claim 20 , wherein the transgene is codon optimized for expression in the plant.