# US 2013/0160165 A1 · Plants Having Enhanced Yield-Related Traits and Method for Making the Same

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- Number: US 2013/0160165 A1
- Document ID: US20130160165A1
- Status: Published application
- Kind: A1
- Application number: 13/805,857
- Filed: 2011-06-21
- Published: 2013-06-20
- Earliest claimed: 2010-06-24
- Assignees: BASF Plant Science Company BmbH
- Inventors: Christophe Reuzeau; Steven Vandenabeele; Valerie Frankard
- Web page: https://patentgrep.com/patent/US20130160165A1

## CPC

- C12N 15/8262: {involving plant development}
- C07K 14/415: from plants
- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- C12N 15/8271: {for stress resistance, e.g. heavy metal resistance}
- C12N 15/8273: {for drought, cold, salt resistance}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates generally to the field of molecular biology and to the methods for enhancing various economically important yield-related traits in plants. More specifically, the present invention relates to a method for enhancing yield-related traits in plants by modulating expression of a nucleic acid encoding an OsRSZ33 RRM polypeptide or a growth-related protein (GRP) having at least 25% amino acid sequence identity to SEQ ID NO:251 or a ZPR polypeptide. The present invention also relates to plants having modulated expression of a nucleic acid encoding an OsRSZ33 RRM polypeptide or a growth-related polypeptide as defined herein or a ZPR polypeptide. Such plants have enhanced yield-related traits relative to controls. The invention also provides hitherto unknown OsRSZ33 RRM-encoding nucleic acids or GRP-encoding nucleic acids or a ZPR polypeptide, and constructs comprising the same, which are useful in performing the methods of the invention.

## Claims

### Claim at position 1

1-76. (canceled)

### Claim 77

A method for enhancing yield-related traits in a plant relative to a control plant, comprising modulating expression in a plant of a nucleic acid selected from the group consisting of:
(a) a nucleic acid encoding an OsRSZ33 RRM polypeptide, wherein said OsRSZ33 RRM polypeptide comprises from N- to C-terminus an RRM domain and two Zinc Knuckles;
(b) a nucleic acid encoding a growth-related protein (GRP) having at least 25% sequence identity to the amino acid sequence of SEQ ID NO: 251; and
(c) a nucleic acid encoding a ZPR polypeptide, wherein said ZPR polypeptide comprises a Leucine Zipper (ZIP) domain that is six heptads in length.

### Claim 78 (depends on 77)

The method of claim 77, wherein the OsRSZ33 RRM polypeptide comprises one or more of motifs 1 to 3 (SEQ ID NO: 241 to 246).

### Claim 79 (depends on 77)

The method of claim 77, wherein the growth-related protein has a motif having at least 70% sequence identity to motif 7: [NHPS][SC][CR][RK]K[NK][VST][PD][GD][TAV][ST]F[VL][SE]DL[RK]DH[IM][HD]EFI[NH]AS[ASM]DEH[MRK][TH]CF[TK][KN]T[IL][K Q][KR]MF[GD]MS[KM][TAV]V[AT] (SEQ ID NO: 742).

### Claim 80 (depends on 77)

The method of claim 77, wherein the ZPR polypeptide comprises one or more of the following motifs:
(SEQ ID NO: 929)
(i)
Motif 8:
II[EK]ENE[KR]LR[KE][KR]A[LS][LA]L[HR][QR]EN,
(SEQ ID NO: 930)
(ii)
Motif 9:
[EI]M[EK][MI]KNLKLY[EML]EN[KQ][SCI],
(SEQ ID NO: 931)
(iii)
Motif 10:
[QKL][AD]L[LF][ST][QE][LI][QI][KQ][KQ][LI]SxPx.

### Claim 81 (depends on 80)

The method of claim 80, wherein the Leucine Zipper domain is located in the central to C-terminal region of said ZPR polypeptide.

### Claim 82 (depends on 77)

The method of claim 77, wherein the ZPR polypeptide further comprises one or more of the following motifs:
(SEQ ID NO: 932)
(i)
Motif 11:
K[VL][KA]K[EI]M[EK]MKNLKLY[MEL]EN[QKR][SIC]I[IL]EENE
[KR]LR[KE][KRQ]A[LS][LA]L[HR][QR]EN[LKQ][AD]L[LF][STQ][QI][LI][QI][KQ
N][KQ][IF]S,
(SEQ ID NO: 933)
(ii)
Motif 12:
MC[HS][AG][SI]Sx[HS][LS][ES]S,
(SEQ ID NO: 934)
(iii)
Motif 13:
[VI][HS][RV]L[NK][LR]RR.

### Claim 83 (depends on 77)

The method of claim 77, wherein the modulated expression is effected by:
(a) introducing and expressing in a plant a nucleic acid encoding an OsRSZ33 RRM polypeptide;
(b) introducing and expressing in a plant a nucleic acid encoding a growth-related protein in a plant; or
(c) introducing and expressing in a plant a nucleic acid encoding a ZPR polypeptide.

### Claim 84 (depends on 77)

The method of claim 77, wherein the nucleic acid encoding an OsRSZ33 RRM polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid;
or wherein the nucleic acid encoding a GRP encodes any one of the polypeptides listed in Table F or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid;
or wherein the nucleic acid encoding a ZPR polypeptide encodes any one of the polypeptides listed in Table J or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.

### Claim 85 (depends on 77)

The method of claim 77, wherein the nucleic acid sequence encoding an OsRSZ33 RRM polypeptide encodes an orthologue or paralogue of any of the proteins given in Table A;
or wherein the nucleic acid encoding a GRP encodes an orthologue or paralogue of any of the polypeptides given in Table F;
or wherein the nucleic acid sequence encoding a ZPR polypeptide encodes an orthologue or paralogue of any of the polypeptides given in Table J.

### Claim 86 (depends on 77)

The method of claim 77, wherein the nucleic acid encoding a GRP encodes the polypeptide of SEQ ID NO: 251.

### Claim 87 (depends on 77)

The method of claim 77, wherein the nucleic acid encoding a ZPR polypeptide encodes the polypeptide of SEQ ID NO: 747 or a homologue thereof.

### Claim 88 (depends on 77)

The method of claim 77, wherein the enhanced yield-related traits obtained by modulating expression of a nucleic acid encoding an OsRSZ33 RRM polypeptide comprise increased yield and/or early vigour relative to a control plant;
or wherein the enhanced yield-related traits obtained by modulating expression of a nucleic acid encoding a growth-related protein (GRP) comprise increased yield relative to a control plant;
or wherein the enhanced yield-related traits obtained by modulating expression of a nucleic acid encoding a ZPR polypeptide comprise increased yield relative to a control plant, and preferably increased seed yield relative to a control plant.

### Claim 89 (depends on 88)

The method of claim 88, wherein the increased yield obtained by modulating expression of a nucleic acid encoding a growth-related protein (GRP) comprises increased seed yield relative to a control plant, and preferably said increased seed yield is selected from any one or more of:
(i) increased harvest index;
(ii) increased total seed weight;
(iii) increased number of filled seeds; and
(iv) increased thousand kernel weight.

### Claim 90 (depends on 88)

The method of claim 88, wherein the enhanced yield-related trait obtained by modulating expression of a nucleic acid encoding a growth-related protein (GRP) comprises increased biomass, and preferably increased aboveground biomass, relative to a control plant.

### Claim 91 (depends on 77)

The method of claim 77, wherein the enhanced yield-related traits are obtained under non-stress conditions.

### Claim 92 (depends on 77)

The method of claim 77, wherein the enhanced yield-related traits obtained by modulating expression in a plant of a nucleic acid encoding a growth-related protein (GRP) are obtained under conditions of drought stress, salt stress or nitrogen deficiency;
or wherein the enhanced yield-related traits obtained by modulating expression in a plant of a nucleic acid encoding a ZPR polypeptide are obtained under conditions of drought stress, salt stress or nitrogen deficiency.

### Claim 93 (depends on 83)

The method of claim 83, wherein the nucleic acid encoding an OsRSZ33 RRM polypeptide is operably linked to a constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice;
or wherein the nucleic acid encoding a growth-related protein (GRP) is operably linked to a constitutive promoter, a medium strength constitutive promoter, a plant promoter, a GOS2 promoter, or a GOS2 promoter from rice;
or wherein the nucleic acid encoding a ZPR polypeptide is operably linked to a constitutive promoter, a medium strength constitutive promoter, a plant promoter, a GOS2 promoter, or a GOS2 promoter from rice.

### Claim 94 (depends on 77)

The method of claim 77, wherein the nucleic acid encoding an OsRSZ33 RRM polypeptide is of plant origin, from a monocotyledonous plant, from the family Poaceae, from the genus Oryza, or from Oryza sativa;
or wherein the nucleic acid encoding a GRP is of plant origin, from a monocotyledonous plant, from the family Poaceae, from the genus Oryza, or from Oryza sativa;
or wherein the nucleic acid encoding a ZPR polypeptide is of plant origin, from a dicotyledonous plant, from the family Solanaceae, from the genus Solanum, or from Solanum lycopersicum.

### Claim 95 (depends on 77)

A plant, plant cell or plant part, including seeds, obtained by the method of claim 77, wherein said plant, plant cell or plant part comprises:
(i) a recombinant nucleic acid encoding the OsRSZ33 RRM polypeptide;
(ii) a recombinant nucleic acid encoding the GRP; or
(iii) a recombinant nucleic acid encoding the ZPR polypeptide.

### Claim 96 (depends on 77)

A construct comprising:
(i) the nucleic acid encoding an OsRSZ33 RRM polypeptide, a GRP, or a ZPR polypeptide as defined in claim 77;
(ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
(iii) a transcription termination sequence.

### Claim 97 (depends on 96)

The construct of claim 96, wherein one of the control sequences capable of driving expression of the nucleic acid encoding an OsRSZ33 RRM polypeptide of (i) is a constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice;
or wherein one of the control sequences capable of driving expression of the nucleic acid encoding a GRP of (i) is a constitutive promoter, a medium strength constitutive promoter, a plant promoter, a GOS2 promoter, or a GOS2 promoter from rice;
or wherein one of the control sequences capable of driving expression of the nucleic acid encoding a ZPR polypeptide of (i) is a constitutive promoter, a medium strength constitutive promoter, a plant promoter, a GOS2 promoter, or a GOS2 promoter from rice.

### Claim 98 (depends on 96)

A method for making a plant having enhanced yield-related traits relative to a control plant, comprising utilizing the construct of claim 96,
wherein the nucleic acid encodes an OsRSZ33 RRM polypeptide and the plant has increased yield, particularly increased biomass and/or increased seed yield, relative to a control plant;
or wherein the nucleic acid encodes a GRP and the plant has enhanced yield-related traits relative to a control plant;
or wherein the nucleic acid encodes a ZPR polypeptide and the plant has enhanced yield-related traits, preferably increased yield or increased seed yield, relative to a control plant.

### Claim 99 (depends on 96)

A plant, plant cell or plant part transformed with the construct of claim 96.

### Claim 100 (depends on 77)

A method for the production of a transgenic plant having enhanced yield-related traits or increased yield, particularly increased biomass and/or increased seed yield, relative to a control plant, comprising:
(i) introducing and expressing in a plant a nucleic acid encoding an OsRSZ33 RRM polypeptide, a GRP, or a ZPR polypeptide as defined in claim 77, or a genetic construct comprising said nucleic acid; and
(ii) cultivating the plant under conditions promoting plant growth and development.

### Claim 101 (depends on 100)

The method of claim 100, wherein the enhanced yield-related traits obtained upon modulating expression in a plant of a nucleic acid encoding a GRP comprise increased yield relative to a control plant.

### Claim 102 (depends on 77)

A transgenic plant or a transgenic plant cell derived from said transgenic plant, wherein:
said transgenic plant has increased yield, particularly increased biomass and/or increased seed yield, relative to a control plant resulting from modulated expression of a nucleic acid encoding an OsRSZ33 RRM polypeptide as defined in claim 77;
(ii) said transgenic plant has enhanced yield-related traits or increased yield relative to a control plant resulting from modulated expression of a nucleic acid encoding a GRP as defined in claim 77; or
(iii) said transgenic plant has enhanced yield-related traits, preferably increased yield or increased seed yield, relative to a control plant resulting from modulated expression of a nucleic acid encoding a ZPR polypeptide as defined in claim 77.

### Claim 103 (depends on 102)

The transgenic plant of claim 102, or a transgenic plant cell derived thereof, wherein said plant is a crop plant, such as beet or sugarbeet or alfalfa, or a monocot such as sugarcane, or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum emmer, spelt, secale, einkorn, teff, milo and oats

### Claim 104 (depends on 102)

Harvestable parts of the transgenic plant of claim 102, wherein said harvestable parts are preferably shoot biomass and/or seeds.

### Claim 105 (depends on 102)

Products derived from the transgenic plant of claim 102 and/or from harvestable parts of said transgenic plant.

### Claim 106

An isolated nucleic acid selected from the group consisting of:
(i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 266, 286, 316, 374, 376, 380, 424, 456, 458, 460, 462, 648, 650, 670 or 736;
(ii) the complement of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 266, 286, 316, 374, 376, 380, 424, 456, 458, 460, 462, 648, 650, 670 or 736;
(iii) a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 267, 287, 317, 375, 377, 381, 425, 457, 459, 461, 463, 649, 651, 671 or 737, preferably as a result of the degeneracy of the genetic code, said nucleic acid can be derived from the amino acid sequence of SEQ ID NO: 267, 287, 317, 375, 377, 381, 425, 457, 459, 461, 463, 649, 651, 671 or 737, and further preferably confers enhanced yield-related traits relative to control plants;
(iv) a nucleic acid having at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the nucleotide sequences of SEQ ID NO: 266, 286, 316, 374, 376, 380, 424, 456, 458, 460, 462, 648, 650, 670 and 736, or any of the other nucleic acid sequences in Table F, and further preferably conferring enhanced yield-related traits relative to control plants;
(v) a nucleic acid which hybridizes with any of the nucleic acids of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants; and
(vi) a nucleic acid encoding a polypeptide having at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the amino acid sequences of SEQ ID NO: 267, 287, 317, 375, 377, 381, 425, 457, 549, 461, 463, 649, 651, 671 and 737, and any of the other amino acid sequences in Table F, and preferably conferring enhanced yield-related traits relative to control plants.

### Claim 107

An isolated polypeptide comprising:
(i) the amino acid sequence of SEQ ID NO: 267, 287, 317, 375, 377, 381, 425, 457, 459, 461, 463, 649, 651, 671 or 737;
(ii) an amino acid sequence having at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the amino acid sequences of SEQ ID NO: 267, 287, 317, 375, 377, 381, 425, 457, 459, 461, 463, 649, 651, 671 and 737, and any of the other amino acid sequences in Table F, and preferably conferring enhanced yield-related traits relative to control plants; or
(iii) derivatives of any of the amino acid sequences of (i) or (ii) above.

### Claim 108

An isolated nucleic acid selected from the group consisting of:
(i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 748, 790, 792, 862, 864, 870, 878, 902, 912 or 918;
(ii) the complement of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 748, 790, 792, 862, 864, 870, 878, 902, 912 or 918;
(iii) a nucleic acid encoding a ZPR polypeptide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 749, 791, 793, 863, 865 or 871; and additionally or alternatively comprising one or more motifs having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one or more of motifs 1 to 6 as given in SEQ ID NO: 929 to SEQ ID NO: 934, and further preferably conferring enhanced yield-related traits relative to control plants;
(iv) a nucleic acid encoding a ZPR polypeptide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 878, 902, 912 or 918; and additionally or alternatively comprising one or more motifs having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one or more of motifs 8 to 10 as given in SEQ ID NO: 929 to SEQ ID NO: 931, and further preferably conferring enhanced yield-related traits relative to control plants; and
(v) a nucleic acid which hybridizes with any of the nucleic acid molecules of (i) to (iv) under high stringency hybridization conditions and preferably confers enhanced yield-related traits relative to control plants.

### Claim 109

An isolated polypeptide comprising:
(i) the amino acid sequence of SEQ ID NO: 749, 791, 793, 863, 865, 871, 879, 903, 913 or 919;
(ii) an amino acid sequence having at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 749, 791, 793, 863, 865 or 871; and additionally or alternatively comprising one or more motifs having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one or more of motifs 8 to 13 given in SEQ ID NO: 929 to SEQ ID NO: 934, and further preferably conferring enhanced yield-related traits relative to control plants;
(iii) an amino acid sequence having at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 879, 903, 913 or 919; and additionally or alternatively comprising one or more motifs having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one or more of motifs 8 to 10 given in SEQ ID NO: 929 to SEQ ID NO: 931, and further preferably conferring enhanced yield-related traits relative to control plants;
(iv) derivatives of any of the amino acid sequences of (i) to (iii) above.