US2016068854A1PendingUtilityA1

Increasing plant growth by modulating omega-amidase expression in plants

47
Assignee: LOS ALAMOS NAT LAB LC IP A 187Priority: Feb 28, 2010Filed: Jun 19, 2015Published: Mar 10, 2016
Est. expiryFeb 28, 2030(~3.6 yrs left)· nominal 20-yr term from priority
Inventors:Pat J. UnkeferPenelope S. AndersonThomas J. Knight
C12N 15/8261C12N 15/8227C12N 9/80Y02A40/146C12N 15/8262
47
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Claims

Abstract

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.

Claims

exact text as granted — not AI-modified
1 - 41 . (canceled) 
     
     
         42 . A method for generating transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production to thereby improve growth characteristics of the transgenic plants, comprising:
 (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein the Ω-amidase transgene is operably linked to a heterologous promoter;   (b) generating a transgenic plant from the plurality of plant cells; and   (c) expressing the Ω-amidase transgene in root tissue of the transgenic plant or the progeny of the transgenic plant,   wherein said transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramate production relative to an analogous wild type or untransformed plant.   
     
     
         43 . The method of  claim 42 , wherein the Ω-amidase transgene codes for a polypeptide having Ω-amidase catalytic activity and an amino acid sequence selected from the group consisting of
 (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and 
 (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. 
 
     
     
         44 . The method according to  claim 42 , wherein the Ω-amidase transgene is incorporated into the genome of the plant. 
     
     
         45 . The method according to  claim 42 , 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, 35S 2 promoter, GI4 promoter, GI5 promoter, and GRP promoter. 
     
     
         46 . The method according to  claim 42 , wherein endogenous Ω-amidase expression in leaf tissue is inhibited. 
     
     
         47 . The method according to  claim 46 , wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor. 
     
     
         48 . The method according to  claim 46 , 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. 
     
     
         49 . The method according to  claim 46 , 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. 
     
     
         50 . The method according to  claim 46 , wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from the group consisting of 6-diazo-5-oxonor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate. 
     
     
         51 . The method according to  claim 42 , wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type plant or untransformed plant. 
     
     
         52 . The method according to  claim 42 , wherein the transgenic plant further comprises a GPT transgene. 
     
     
         53 . The method according to  claim 52 , wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1. 
     
     
         54 . The method according to  claim 42 , wherein the transgenic plant further comprises a GPT transgene and a GS transgene. 
     
     
         55 . The method according to  claim 54 , wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter. 
     
     
         56 . The method according to  claim 42 , wherein endogenous GPT expression in the transgenic plant is increased by gene activation. 
     
     
         57 . The method according to  claim 42 , wherein endogenous GS expression in the transgenic plant is increased by gene activation. 
     
     
         58 . The method according to  claim 42 , wherein the Ω-amidase transgene is codon optimized for expression in the plant. 
     
     
         59 - 72 . (canceled) 
     
     
         73 . The method according to  claim 42 , wherein said transgenic plant or said progeny of said transgenic plant has at least one increased growth characteristic selected from the group consisting of increased biomass yield, increased NO 3  uptake, increased chlorophyll per unit weight, increased CO 2  fixation, and increased seed yield. 
     
     
         74 . The method according to  claim 42 , further comprising selecting a progeny said transgenic plant having increased growth characteristic relative to an analogous wild type or untransformed plant, wherein the growth characteristic is selected from the group consisting of increased biomass yield, increased NO 3  uptake, increased chlorophyll per unit weight, increased CO 2  fixation, and increased seed yield. 
     
     
         75 . The method according to  claim 42 , wherein the plant is a monocotyledonous plant. 
     
     
         76 . The method according to  claim 42 , wherein the plant is a dicotyledonous plant. 
     
     
         77 . The method according to  claim 42 , wherein the plurality of plant cells are taken from a plant selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa,  Arabidopsis , grasses, fruits, vegetables, flowering plants, and trees. 
     
     
         78 . The method according to  claim 42 , wherein the promoter is a root-preferred promoter. 
     
     
         79 . The method according to  claim 42 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 3. 
     
     
         80 . The method according to  claim 42 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 3. 
     
     
         81 . The method according to  claim 42 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 3. 
     
     
         82 . A progeny of any generation of the transgenic plant generated according to  claim 42 , wherein the progeny comprises the Ω-amidase transgene. 
     
     
         83 . A seed of any generation of the transgenic plant generated according to  claim 42 , wherein the progeny comprises the Ω-amidase transgene. 
     
     
         84 . A method for generating and selecting transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production and improved growth characteristics of the transgenic plants, comprising:
 (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein said Ω-amidase transgene includes a heterologous plant promoter;   (b) generating a plurality of transgenic plants from the plant cells;   (c) expressing the Ω-amidase transgene in root tissue of the plant or the progeny of the plant; and   (d) selecting a transgenic plant having an increased leaf-to-root ratio of 2-oxo-glutaramate production relative to an analogous wild type or untransformed plant.   
     
     
         85 . The method according to  claim 84 , wherein said transgenic plant or said progeny of the transgenic plant has at least one increased growth characteristic selected from the group consisting of increased biomass yield, increased NO 3  uptake, increased chlorophyll per unit weight, increased CO 2  fixation, and increased seed yield. 
     
     
         86 . The method according to  claim 84 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 3. 
     
     
         87 . The method according to  claim 84 , wherein the heterologous plant promoter is a root-preferred promoter. 
     
     
         88 . The method according to  claim 87 , 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, 35S 2 promoter, GI4 promoter, GI5 promoter, and GRP promoter. 
     
     
         89 . The method according to  claim 84 , wherein the Ω-amidase transgene encodes a polypeptide having Ω-amidase catalytic activity and an amino acid sequence selected from the group consisting of
 (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and 
 (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. 
 
     
     
         90 . The method according to  claim 84 , wherein the Ω-amidase transgene is incorporated into the genome of the plant. 
     
     
         91 . The method according to  claim 84 , wherein the plant is a monocotyledonous plant. 
     
     
         92 . The method according to  claim 84 , wherein the plant is a dicotyledonous plant. 
     
     
         93 . The method according to  claim 84 , wherein the plurality of plant cells are taken from a plant selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa,  Arabidopsis , grasses, fruits, vegetables, flowering plants, and trees. 
     
     
         94 . The method according to  claim 84 , wherein said transgenic plant produces more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant. 
     
     
         95 . The method according to  claim 84 , wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant. 
     
     
         96 . The method according to  claim 84 , wherein the transgenic plant has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant. 
     
     
         97 . The method according to  claim 84 , wherein the plant further comprises a GPT transgene. 
     
     
         98 . The method according to  claim 97 , wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1. 
     
     
         99 . The method according to  claim 84 , wherein the plant further comprises a GPT transgene and a GS transgene. 
     
     
         100 . The method according to  claim 99 , wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter. 
     
     
         101 . The method according to  claim 84 , wherein endogenous GPT expression in the plant is increased by gene activation. 
     
     
         102 . The method according to  claim 84 , wherein endogenous GS expression in the plant is increased by gene activation. 
     
     
         103 . The method according to  claim 84 , wherein the transgene is codon optimized for expression in the plant. 
     
     
         104 . The method according to  claim 84 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 3. 
     
     
         105 . The method according to  claim 84 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 3. 
     
     
         106 . A method for generating and selecting transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production and increased growth characteristics of the transgenic plants, comprising:
 (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein said Ω-amidase transgene includes a heterologous plant promoter;   (b) generating a plurality of transgenic plants from the plant cells; and   (c) selecting from said plurality of transgenic plants a transgenic plant having an increased biomass yield relative to an analogous wild type or untransformed plant, wherein a polypeptide encoded by said Ω-amidase transgene catalyzes the synthesis of 2-oxo-glutaramate.   
     
     
         107 . The method according to  claim 106 , wherein said transgenic plant or said progeny of the transgenic plant has at least one additional increased growth characteristic selected from the group consisting of increased NO 3  uptake, increased chlorophyll per unit weight, increased CO 2  fixation, and increased seed yield. 
     
     
         108 . The method according to  claim 106 , wherein said polypeptide has an amino acid sequence according to SEQ ID NO: 3. 
     
     
         109 . The method according to  claim 106 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 3. 
     
     
         110 . The method according to  claim 106 , wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 3. 
     
     
         111 . The method according to  claim 106 , wherein the heterologous plant promoter is a root-preferred promoter. 
     
     
         112 . The method according to  claim 111 , 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, 35S 2 promoter, GI4 promoter, GI5 promoter, and GRP promoter. 
     
     
         113 . The method according to  claim 106 , wherein said polypeptide has an amino acid sequence selected from the group consisting of
 (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and   (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38.   
     
     
         114 . The method according to  claim 106 , wherein the Ω-amidase transgene is incorporated into the genome of the transgenic plant. 
     
     
         115 . The method according to  claim 106 , wherein the transgenic plant is a monocotyledonous plant. 
     
     
         116 . The method according to  claim 106 , wherein the transgenic plant is a dicotyledonous plant. 
     
     
         117 . The method according to  claim 106 , 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. 
     
     
         118 . The method according to  claim 106 , wherein said transgenic plant produces more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant. 
     
     
         119 . The method according to  claim 106 , wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant. 
     
     
         120 . The method according to  claim 106 , wherein the transgenic plant has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant. 
     
     
         121 . The method according to  claim 106 , wherein the transgenic plant further comprises a GPT transgene. 
     
     
         122 . The method according to  claim 121 , wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1. 
     
     
         123 . The method according to  claim 106 , wherein the transgenic plant further comprises a GPT transgene and a GS transgene. 
     
     
         124 . The method according to  claim 123 , wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter. 
     
     
         125 . The method according to  claim 106 , wherein endogenous GPT expression in the transgenic plant is increased by gene activation. 
     
     
         126 . The method according to  claim 106 , wherein endogenous GS expression in the transgenic plant is increased by gene activation. 
     
     
         127 . The method according to  claim 106 , wherein the Ω-amidase transgene is codon optimized for expression in the plant.

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