Plants Having Enhanced Yield-Related Traits And A Method For Making The Same

Abstract

The present invention concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a CLC-like (Chloride Channel-like) polypeptide, an OsBURP-like (BURP-domain containing protein) polypeptide, an AP2/ERF polypeptide, or a protein fusion comprising a TPS (trehalose-6-phosphate synthase) and a TPP (trehalose-6-phosphate phosphatase) polypeptide, or modulating one or more nucleic acids encoding a TPS and a TPP enzyme, whether comprised in the same or in separate molecules. The present invention also concerns plants having modulated expression of a nucleic acid encoding a CLC-like polypeptide, an OsBURP-like polypeptide, an AP2/ERF polypeptide, or a TPP polypeptide, which plants have enhanced yield-related traits relative to control plants. Also provided are unknown TPS-encoding and TPP-encoding nucleic acids, and constructs comprising the same, and constructs a CLC-like, OsBURP-like, or AP2/ERF coding nucleic acid useful in performing the methods of the invention.

1 . 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 a CLC-like polypeptide, wherein said CLC-like polypeptide comprises a Voltage_CLC domain (Pfam entry PF00654) and C-terminally thereof a CBS domain (Pfam entry PF00571);   (b) a nucleic acid encoding an OsBURP-like polypeptide, wherein said OsBURP-like polypeptide comprises a BURP domain;   (c) a nucleic acid encoding a AP2/ERF polypeptide, wherein said AP2/ERF polypeptide comprises an AP2/ERF domain; and   (d) a nucleic acid encoding a TPS-TPP protein fusion which is a fusion between a TPS polypeptide or an active portion thereof and a TPP polypeptide or an active portion thereof, or a first nucleic acid encoding a TPS polypeptide or an active portion thereof and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules.   
     
     
         2 . The method of  claim 1 , wherein:
 (a) the CLC-like polypeptide comprises one or more of the motifs 1 to 8 (SEQ ID NO: 83 to SEQ ID NO: 90) or one or more of the motifs as represented by SEQ ID NO: 492 to SEQ ID NO: 503;   (b) the OsBURP-like polypeptide comprises one or more of the following motifs:
 (i) Motif 9 of SEQ ID NO: 140; 
 (ii) Motif 10 of SEQ ID NO: 141; 
 (iii) Motif 11 of SEQ ID NO: 142; 
   (c) the AP2/ERF polypeptide comprises one or more of the following motifs:
 (i) Motif 25 of SEQ ID NO: 265; 
 (ii) Motif 26 of SEQ ID NO: 266); 
 (iii) Motif 27 of SEQ ID NO: 267; 
 (iv) Motif 28 of SEQ ID NO: 268, 
 in particular wherein said AP2/ERF polypeptide comprises Motif 25 and at least Motif 26, or wherein said AP2/ERF polypeptide comprises Motif 25, Motif 26 and Motif 27 or Motif 28, or wherein said AP2/ERF polypeptide comprises Motif 25, Motif 26 and Motif 27, 
   or   (d) in the TPS-TPP protein fusion,
 (i) the TPS polypeptide or active portion thereof has 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%, 99% or 100% overall sequence identity to the amino acid sequence of any one of the polypeptides of Table A4 or an active portion thereof, the amino acid sequence of SEQ ID NO: 273 or an active portion thereof, or the amino acid sequence of SEQ ID NO: 275 or an active portion thereof, 
 (ii) the TPP polypeptide or an active portion thereof has 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%, 99% or 100% overall sequence identity to the amino acid sequence of any one of the polypeptides of Table A5 or an active portion thereof, the amino acid sequence of SEQ ID NO: 299 or an active portion thereof, or the amino acid sequence of SEQ ID NO: 301 or an active portion thereof. 
   
     
     
         3 . The method of  claim 1 , wherein
 (a) the nucleic acid encodes a CLC-like polypeptide, and wherein said modulated expression is effected by introducing and expressing in a plant said nucleic acid,   (b) the nucleic acid encodes an OsBURP-like polypeptide, and wherein said modulated expression is effected by introducing and expressing in a plant said nucleic acid, or   (c) the nucleic acid encodes an AP2/ERF-like polypeptide, and wherein said modulated expression is effected by introducing and expressing in a plant said nucleic acid,   or wherein said modulated expression is effected by introducing and expressing in a plant:   (i) a nucleic acid encoding a TPS-TPP protein fusion 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%, 99% or 100% overall sequence identity to the amino acid sequence of any one of the polypeptides of Table A6 or an active portion thereof, the amino acid sequence of SEQ ID NO: 440 or an active portion thereof, the amino acid sequence of SEQ ID NO: 442 or an active portion thereof, or the amino acid sequence of SEQ ID NO: 444 or an active portion thereof; or   (ii) a first nucleic acid encoding a TPS polypeptide or an active portion thereof and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules,   in particular, wherein said one or more nucleic acids as set forth in (i) and (ii) are expressed in the chloroplast of a plant cell, plant or plant part thereof and preferably comprise a sequence encoding a chloroplast targeting signal, further preferably said sequence is located at the 5′ terminus of the coding region, even more preferably encodes any one of the polypeptide chloroplast targeting signals of Table 3, most preferably encodes SEQ ID NO: 456 or a portion thereof.   
     
     
         4 . The method of  claim 1 , wherein
 (a) the nucleic acid encoding a CLC-like polypeptide encodes any one of the proteins listed in Table A1 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridizing with such a nucleic acid,   (b) the nucleic acid encoding an OsBURP-like polypeptide encodes any one of the proteins listed in Table A2 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridizing with such a nucleic acid, or   (c) the nucleic acid encoding an AP2/ERF polypeptide encodes any one of the proteins listed in Table A3 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridizing with such a nucleic acid.   
     
     
         5 . The method of  claim 1 , wherein the nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A1, A2 or A3. 
     
     
         6 . The method of  claim 1 , wherein
 (a) the expression of the nucleic acid encoding a CLC-like polypeptide is modulated, and wherein said enhanced yield-related traits comprise increased yield, preferably increased biomass and/or increased seed yield, relative to a control plant,   (b) the expression of the nucleic acid encoding an OsBURP-like polypeptide is modulated, and wherein said enhanced yield-related traits comprise increased yield and/or early vigour, preferably increased biomass and/or increased seed yield, relative to a control plant,   (c) the expression of the nucleic acid encoding an AP2/ERF polypeptide is modulated, and wherein said enhanced yield-related traits comprise increased yield, preferably increased biomass and/or increased seed yield, relative to a control plant, in particular wherein said increased biomass relative to a control plant comprises early vigour, maximum height, total root biomass and harvest index, and/or wherein said increased seed yield relative to a control plant comprises total seed weight, number of filled seeds and fill rate, or   (d) the expression of the nucleic acid encoding a TPS-TPP protein fusion is modulated, or wherein the expression of a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof is modulated, and wherein said enhanced yield-related traits comprise any one or more of increased size or number of floral meristems, increased number of flowers, increased number of seeds, increased yield, preferably increased biomass and/or increased seed weight, relative to a control plant.   
     
     
         7 . The method of  claim 1 , wherein:
 (a) the expression of the nucleic acid encoding a CLC-like polypeptide is modulated, and wherein said enhanced yield-related traits are obtained under conditions of drought stress or salt stress,   (b) the expression of the nucleic acid encoding an OsBURP-like polypeptide is modulated, and wherein said enhanced yield-related traits are obtained under drought stress conditions,   (c) the expression of the nucleic acid encoding an AP2/ERF polypeptide is modulated, and wherein said enhanced yield-related traits are obtained under conditions of drought stress, salt stress or nitrogen deficiency, in particular wherein said enhanced yield-related traits are obtained under drought-stress conditions, or   (d) the expression of the nucleic acid encoding a TPS-TPP protein fusion is modulated, or wherein the expression of a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof is modulated, and wherein said enhanced yield-related traits are obtained under plant cell, plant culturing conditions where nitrogen would be limiting for growth for the control plant cell, plant or a part thereof, and/or wherein said enhanced yield-related traits are obtained under non-stress conditions or under stress conditions selected from any one or more of drought, salt and cold stress.   
     
     
         8 . The method of  claim 1 , wherein said nucleic acid or said one or more nucleic acids is/are operably linked to a constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice. 
     
     
         9 . The method of  claim 1 , wherein
 (a) the nucleic acid encoding a CLC-like polypeptide is of plant origin, from a dicotyledonous plant, from the family Poaceae, from the genus  Oryza , or from  Oryza sativa,      (b) the nucleic acid encoding an OsBURP-like polypeptide is of plant origin, from a monocotyledonous plant, from the family Poaceae, from the genus  Oryza , or from  Oryza sativa,      (c) the nucleic acid encoding an AP2/ERF polypeptide is of plant origin, from a dicotyledonous plant, from the family Fabaceae, from the genus  Medicago , or from  M. truncatula , or   (d) the one or more said nucleic acids encoding the TPS or TPP protein is of plant origin, from a dicotyledonous plant, from the family Brassicaceae, from the genus  Arabidopsis , from  Arabidopsis thaliana , of bacterial origin, from  Saccharomyces , or from  Saccharomyces cerevisiae.      
     
     
         10 . A plant obtained by the method of  claim 1 , or a plant cell, plant part, seed, or progeny of said plant, wherein said plant, or said plant cell, plant part, seed, or progeny, comprises:
 (a) a recombinant nucleic acid encoding a CLC-like polypeptide,   (b) a recombinant nucleic acid encoding an OsBURP-like polypeptide,   (c) a recombinant nucleic acid encoding an AP2/ERF polypeptide,   (d) (i) a nucleic acid encoding a TPS-TPP protein fusion;    (ii) a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules; or    (iii) said one or more nucleic acids of (i) and (ii) further comprising a sequence encoding a chloroplast targeting signal.   
     
     
         11 . A construct comprising:
 (a) a nucleic acid encoding:
 (i) the CLC-like polypeptide as defined in  claim 1 , 
 (ii) the OsBURP-like polypeptide as defined in  claim 1 , 
 (iii) the AP2/ERF polypeptide as defined in  claim 1 ; or 
 (iv) a1) a nucleic acid encoding a TPS-TPP protein fusion, 
  b1) a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules, or 
  c1) said one or more nucleic acids of a1) and b1) further comprising a sequence encoding a chloroplast targeting signal, 
   (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a), and optionally   (c) a transcription termination sequence.   
     
     
         12 . The construct of  claim 11 , wherein one of said control sequences is a constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice. 
     
     
         13 . A method for the production of a transgenic plant, comprising transforming the construct of  claim 11  into a plant, wherein
 (a) the nucleic acid encodes the CLC-like polypeptide, the OsBURP-like polypeptide, or the AP2/ERF polypeptide, and wherein the transgenic plant has increased yield, particularly increased biomass and/or increased seed yield, relative to a control plant, or 
 (b) the nucleic acid encodes the TPS-TPP protein fusion, or wherein the first nucleic acid encodes the TPS polypeptide or an active portion thereof, and the second nucleic acid encodes the TPP polypeptide or an active portion thereof, and wherein the transgenic plant has increased size or number of floral meristems, increased number of flowers, increased number of seeds, increased yield, preferably increased biomass and/or increased seed weight, relative to a control plant. 
 
     
     
         14 . A plant, plant part or plant cell transformed with the construct of  claim 11 . 
     
     
         15 . A method for the production of a transgenic plant, comprising:
 (a) introducing and expressing in a plant
 (i) the nucleic acid encoding a CLC-like polypeptide as defined in  claim 1 ; 
 (ii) the nucleic acid encoding an OsBURP-like polypeptide as defined in  claim 1 ; 
 (iii) the nucleic acid encoding an AP2/ERP-like polypeptide as defined in  claim 1 , 
 (iv) a nucleic acid encoding a TPS-TPP protein fusion, 
 (v) a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules, 
 (vi) a nucleic acid encoding a TPS-TPP protein fusion further comprising a sequence encoding a chloroplast targeting signal, or 
 (vii) a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules, said one or more nucleic acids further comprising a sequence encoding a chloroplast targeting signal; 
   and   (b) cultivating the plant under conditions promoting plant growth and development,   wherein the transgenic plant has increased yield, particularly increased biomass and/or increased seed yield, relative to a control plant if the nucleic acid is a nucleic acid as defined in any one of (i) to (iii), or   wherein the transgenic plant has increased size or number of floral meristems, increased number of flowers, increased number of seeds, increased yield, preferably increased biomass and/or increased seed weight, relative to a control plant, if the nucleic acid is nucleic acid as defined in any one of (iv) to (vii).   
     
     
         16 . A transgenic plant
 (a) having increased yield, particularly increased biomass and/or increased seed yield, relative to a control plant, resulting from modulated expression of the nucleic acid encoding a CLC-like, OsBURP-like or AP2/ERP polypeptide as defined in  claim 1 , or   (b) having increased size or number of floral meristems, increased number of flowers, increased number of seeds, increased yield, preferably increased biomass and/or increased seed weight, relative to a control plant, resulting from modulated expression of:
 (i) a nucleic acid encoding a TPS-TPP protein fusion; 
 (ii) a first nucleic acid encoding a TPS polypeptide or an active portion thereof, and a second nucleic acid encoding a TPP polypeptide or an active portion thereof, wherein the first and second nucleic acids are comprised in a single nucleic acid molecule or in multiple, at least two, nucleic acid molecules; or 
 (iii) said one or more nucleic acids of (i) and (ii) further comprising a sequence encoding a chloroplast targeting signal; 
   or a transgenic plant cell derived from said transgenic plant.   
     
     
         17 . The transgenic plant of  claim 16 , or a transgenic plant cell derived thereof, wherein said transgenic plant is a crop plant, a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum emmer, spelt, secale, einkorn, teff, milo and oats. 
     
     
         18 . Harvestable parts of the transgenic plant of  claim 16 , wherein said harvestable parts are preferably shoot biomass and/or seeds. 
     
     
         19 . Products derived from the transgenic plant of  claim 16  and/or from harvestable parts of said plant.