Compositions and methods for improved plant feedstock

• US 8,901,371 B2
• Filed: 05/02/2012
• Issued: 12/02/2014
• Est. Priority Date: 05/06/2011
• Status: Active Grant

• Current Assignee: Noble Research Institute, LLC
• Original Assignee: The Samuel Roberts Noble Foundation, Inc.
• Inventors: Shen, Hui, Chen, Fang, Dixon, Richard A.
• Primary Examiner(s): Ibrahim, Medina A
• Assistant Examiner(s): DEVEAU ROSEN, JASON
• Application Number:
  US13/462,583
• Publication Number:
  US 20120322122A1
• Time in Patent Office: 944 Days
• Field of Search: None
• US Class Current: 800/278
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8255 involving lignin biosynthesis
• CPC Class Codes: C12N 15/8261 with agronomic (input) traits, e.g. crop yield
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

The invention provides methods for modifying lignin content and composition in plants and achieving associated benefits therefrom involving altered expression of newly discovered MYB4 transcription factors. Nucleic acid constructs for modifying MYB4 transcription factor expression are described. By over-expressing the identified MYB4 transcription factors, for example, an accompanying decrease in lignin content may be achieved. Plants are provided by the invention comprising such modifications, as are methods for their preparation and use.

Claims

1. A DNA construct comprising a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid comprising the sequence of SEQ ID NO;

1;

SEQ ID NO;

7;

SEQ ID NO;

9;

SEQ ID NO;

11;

or SEQ ID NO;

13;

(b) a nucleic acid sequence exhibiting at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

1;

SEQ ID NO;

7;

SEQ ID NO;

9;

SEQ ID NO;

11;

or SEQ ID NO;

13;

(c) a nucleic acid sequence that encodes a polypeptide at least 90% identical to the polypeptide sequence of SEQ ID NO;

2;

SEQ ID NO;

8;

SEQ ID NO;

10;

SEQ ID NO;

12;

or SEQ ID NO;

14; and

(d) a nucleic acid sequence comprising the full complement of (a)-(c), wherein the nucleic acid sequence is operably linked to a heterologous promoter sequence, wherein the nucleic acid sequence encodes a protein comprising a R2-R3 domain, and wherein introduction of the nucleic acid sequence in a transgenic plant reduces the lignin content of the plant.

2. The DNA construct of claim 1, wherein the heterologous promoter sequence is a developmentally-regulated, organelle-specific, inducible, tissue-specific, constitutive, cell-specific, seed specific, or germination-specific promoter.

3. A transgenic plant or plant part thereof comprising the DNA construct of claim 1.

4. A transgenic plant cell comprising the DNA construct of claim 1.

5. The transgenic plant of claim 3, wherein the plant is forage plant, a biofuel crop, or a cereal crop.

6. The transgenic plant of claim 5, wherein the biofuel crop is switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus×

giganteus, Miscanthus sp., sericea lespedeza (Lespedeza cuneata), corn, sugarcane, sorghum, millet, ryegrass, timothy, Kochia (Kochia scoparia), soybean, alfalfa, clover, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.), Eremochloa ophiuroides (centipede grass), Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, Kentucky bluegrass, poplar, rice, cotton, Salvia miltiorrhiza (red sage), apple, Vitis vinifera (common grape), Ricinus communis (castor oil plant), Humulus lupulus (hops), Dahlia, Dendrobium (orchid), Brassica rapa (mustard), kudzu (Pueraria lobata) or wheat.

7. The transgenic plant of claim 6, wherein the biofuel crop is switchgrass (Panicum virgatum).

8. The transgenic plant of claim 3, further defined as an R0 transgenic plant.

9. The transgenic plant of claim 3, further defined as a progeny plant of any generation of an R0 transgenic plant, wherein the transgenic plant has inherited the DNA construct.

10. The transgenic plant of claim 3, further comprising at least a second nucleic acid sequence that down-regulates lignin biosynthesis.

11. The transgenic plant of claim 10, wherein the second nucleic acid DNA sequence down-regulates coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl coenzyme A:

shikimate hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA 3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), or 4-coumarate-CoA ligase (4CL).

12. The transgenic plant of claim 10, wherein the second nucleic acid sequence comprises an antisense or RNAi construct.

15. A method of decreasing a lignin content of a plant, comprising expressing in the plant the DNA construct of claim 1, wherein the lignin content is decreased in the plant.

17. A method for producing biomass or feedstock comprising obtaining the transgenic plant or part thereof according to claim 3 and harvesting feedstock or biomass therefrom.

18. The method of claim 17, further comprising producing a biofuel from said biomass or feedstock.

13. A method of modifying the lignin content of a plant comprising introducing in the plant a MYB4 transcription factor that functions to suppress lignin biosynthesis, wherein the MYB4 transcription factor is selected from the group consisting of:

(a) a nucleic acid comprising the sequence of SEQ ID NO;

1;

SEQ ID NO;

7;

SEQ ID NO;

9;

SEQ ID NO;

11;

or SEQ ID NO;

13;

(b) a nucleic acid sequence exhibiting at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

1;

SEQ ID NO;

7;

SEQ ID NO;

9;

SEQ ID NO;

11;

or SEQ ID NO;

13;

(c) a nucleic acid sequence that encodes a polypeptide at least 90% identical to the polypeptide sequence of SEQ ID NO;

2;

SEQ ID NO;

8;

SEQ ID NO;

10;

SEQ ID NO;

12;

or SEQ ID NO;

14; and

(d) a nucleic acid sequence comprising the full complement of (a)-(c), wherein the nucleic acid sequence is operably linked to a heterologous promoter sequence, wherein the nucleic acid sequence encodes a protein comprising a R2-R3 domain, and wherein introduction of the nucleic acid sequence in a transgenic plant reduces the lignin content of the plant,

wherein the plant is switchgrass (Panicum virgatum).

14. The method of claim 13, comprising over-expressing in the plant the MYB4 transcription factor, wherein lignin content is decreased in the plant.

16. The method of claim 14, wherein the content of fermentable carbohydrates is increased in the plant.

19. A method of modifying the lignin content of a plant comprising down-regulating in the plant a MYB4 transcription factor that functions to increase lignin biosynthesis, wherein the plant is switchgrass (Panicum virgatum).

20. The method of claim 19, wherein down-regulating MYB4 transcription factor comprises introducing in the plant a RNAi or antisense construct comprising all or a part of the nucleic acid sequence of SEQ ID NO:

1;

SEQ ID NO;

7;

SEQ ID NO;

9;

SEQ ID NO;

11;

SEQ ID NO;

13;

wherein the expression of the RNAi or antisense construct down-regulates said MYB4 transcription factor.