Methods of increasing seed yield

Abstract

The invention relates to methods for increasing seed yield, increasing the total content of protein and/or lipid in seeds and reducing glucosinolate levels by reducing the expression or activity of UPL3. The invention also relates to genetically altered plants characterised by the above phenotypes and methods of producing such plants.

1 . A method of increasing seed yield in a plant, the method comprising reducing or abolishing the expression of at least one nucleic acid encoding a ubiquitin protein ligase 3 (UPL3) polypeptide and/or reducing the activity of a UPL3 polypeptide in said plant. 
     
     
         2 . The method of  claim 1 , wherein said increase in seed yield comprises an increase in at least one of seed weight, seed size, seed number per pod, pod length, protein and/or lipid content and weight of seed per pod. 
     
     
         3 . A method of reducing the levels of glucosinolate in a plant, the method comprising reducing the expression of a nucleic acid encoding a ubiquitin protein ligase 3 (UPL3) polypeptide and/or reducing the activity of a UPL3 polypeptide. 
     
     
         4 . A method of increasing lipid and/or protein content in a plant seed, the method comprising reducing or abolishing the expression of at least one nucleic acid encoding a ubiquitin protein ligase 3 (UPL3) polypeptide and/or reducing the activity of a UPL3 polypeptide in said plant. 
     
     
         5 . The method of any preceding claim, wherein the method comprises introducing at least one mutation into the nucleic acid sequence encoding UPL3 or the promoter of said UPL3. 
     
     
         6 . The method of  claim 5 , wherein said mutation is a loss of function mutation. 
     
     
         7 . The method of  claim 6 , wherein said mutation is an insertion, deletion or substitution. 
     
     
         8 . The method of any of  claims 5  to  7 , wherein the UPL3 nucleic acid encoding a UPL3 polypeptide comprises or consists of SEQ ID NO: 4 or 5 or a functional variant or homolog thereof, and wherein the promoter of said UPL3 nucleic acid sequence comprises or consists of SEQ ID NO: 1 or 2 or a functional variant or homolog thereof. 
     
     
         9 . The method of any preceding claim, wherein the mutation is introduced using targeted genome modification, preferably ZFNs, TALENs or CRISPR/Cas9. 
     
     
         10 . The method of any of  claims 1  to  9 , wherein the mutation is introduced using mutagenesis, preferably TILLING or T-DNA insertion. 
     
     
         11 . The method of any preceding claim, the method comprising using RNA interference to reduce or abolish the expression of a UPL3 nucleic acid. 
     
     
         12 . The method of any preceding claim, wherein said increase in seed yield is relative to a control or wild-type plant. 
     
     
         13 . The method of any preceding claim, wherein the plant is selected from a dicot plant. 
     
     
         14 . The plant of  claim 13 , wherein the plant is selected from any dicotyledonous oilseed crop, such as  Brassica  oilseed crops such as  B. juncea,  soybean, sunflower, linseed, cotton, hemp, oilpalm, coconut, peanut, safflower, Camelina and olive or wherein the plant is selected from  B.oleracea,  maize, rice, wheat or barley. 
     
     
         15 . The method of  claim 14 , wherein the plant is  Brassica napus.    
     
     
         16 . A genetically altered plant, part thereof or plant cell, wherein said plant comprises at least one mutation into at least one ubiquitin protein ligase 3 (UPL3) gene and/or at least one mutation in the UPL3 promoter. 
     
     
         17 . The plant of  claim 16 , wherein said plant is characterised by an increase in seed yield compared to a wild-type or control plant. 
     
     
         18 . The plant of  claim 17 , wherein said increase in seed yield comprises an increase in at least one of seed weight, seed size, seed number per pod, pod length, and weight of seed per pod. 
     
     
         19 . The plant of  claim 16 , wherein the plant is characterised by an increase in seed protein and/or lipid content. 
     
     
         20 . The plant of  claim 16 , wherein the plant is characterised by reduced glucosinolate levels, wherein preferably said reduction is relative to a wild-type or control plant. 
     
     
         21 . The plant of any of  claims 17  to  19 , wherein said increase is relative to a wild-type or control plant. 
     
     
         22 . The plant of any of  claims 16  to  21 , wherein said mutation is a loss of function mutation, preferably an insertion, deletion or substitution. 
     
     
         23 . The plant of any of  claims 16  to  22 , wherein the UPL3 gene comprises or consists of a nucleic acid sequence as defined in any of SEQ ID NOs: 4 to 7 or a functional variant or homolog thereof, and wherein the promoter of the UPL3 gene comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 1 or 2 or a functional variant or homolog thereof. 
     
     
         24 . The plant of any of any of  claims 16  to  21 , wherein said plant comprises an RNA interference construct that reduces the expression of a UPL3 nucleic acid. 
     
     
         25 . The plant of any of  claims 16  to  24 , wherein the plant is a dicot. 
     
     
         26 . The plant of  claim 25 , wherein the plant is selected from any dicotyledonous oilseed crop, such as  Brassica  oilseed crops such as  B. juncea,  soybean, sunflower, linseed, cotton, hemp, oilpalm, coconut, peanut, safflower, Camelina and olive or wherein the plant is selected from  B.oleracea,  maize, rice, wheat or barley. 
     
     
         27 . The plant of  claim 26 , wherein the plant is  Brassica napus.    
     
     
         28 . The plant part of any of  claims 1  to  27 , wherein the plant part is a seed. 
     
     
         29 . A method of producing a plant with increased seed yield and/or increased seed protein and/or lipid content and/or reduced glucosinolate levels, the method comprising introducing at least one mutation into at least one ubiquitin protein ligase 3 (UPL3) gene and/or at least one mutation in the UPL3 promoter. 
     
     
         30 . The method of  claim 29 , wherein the mutation is a loss of function mutation. 
     
     
         31 . The method of  claim 30 , wherein the mutation is introduced using mutagenesis or targeted genome modification. 
     
     
         32 . The method of  claim 31 , wherein the targeted genome modification is selected from ZFNs, TALENs or CRISPR/Cas9. 
     
     
         33 . A method for producing a plant with increased seed yield and/or increased seed protein and/or lipid content and/or reduced glucosinolate levels, the method comprising introducing and expressing in said plant an RNA interference construct that reduces the expression of a UPL3 nucleic acid. 
     
     
         34 . The method of any of  claims 29  to  33 , wherein the method further comprises regenerating a plant and screening for increased seed yield and/or increased seed protein and/or lipid content and/or reduced glucosinolate levels. 
     
     
         35 . The method of any of  claims 29  to  34 , wherein the plant is selected from any dicotyledonous oilseed crop, such as  Brassica  oilseed crops such as  B. juncea,  soybean, sunflower, linseed, cotton, hemp, oilpalm, coconut, peanut, safflower, Camelina and olive or wherein the plant is selected from  B.oleracea,  maize, rice, wheat or barley. 
     
     
         36 . The plant of  claim 35 , wherein the plant is  Brassica napus.    
     
     
         37 . A plant, plant part or plant cell obtained by the method of any of any of  claims 29  to 
     
     
         36 . 
     
     
         38 . A seed obtained or obtainable from the plant of any of  claims 16  to  27  or the method of any of  claims 29  to  36 . 
     
     
         39 . A method for identifying and/or selecting a plant that will have an increased seed yield phenotype, the method comprising detecting in the plant or plant germplasm at least one polymorphism in the promoter of the UPL3 gene, and/or a tandem duplication comprising SEQ ID NO: 18 and wherein said plant or progeny thereof is selected. 
     
     
         40 . The method of  claim 39 , wherein the deletion of at least one tandem duplication is indicative of a plant with a lower level of UPL3 expression than a plant comprising a different base at the above sites and/or at least two duplications of SEQ ID 
     
     
         41 . The method of  claim 39  or  40 , wherein the method further comprises introgressing the chromosomal region comprising at least one of said polymorphisms and/or deletions into a second plant or plant germplasm to produce an introgressed plant or plant germplasm. 
     
     
         42 . A nucleic acid construct comprising a nucleic acid sequence as defined in any one of SEQ ID NOs: 11 to 14 or a functional variant or homolog thereof, wherein said sequence is operably linked to a regulatory sequence, wherein preferably said regulatory sequence is a tissue-specific promoter. 
     
     
         43 . A vector comprising the nucleic acid construct of  claim 42 . 
     
     
         44 . A host cell comprising the nucleic acid construct of  claim 43 . 
     
     
         45 . A transgenic plant expressing the nucleic acid construct of  claim 44 . 
     
     
         46 . A method of increasing seed yield and/or increasing seed protein and/or lipid content and/or reducing glucosinolate levels, the method comprising introducing and expressing in said plant the nucleic acid construct of  claim 42 . 
     
     
         47 . A method for producing a plant with increased seed yield and/or increased seed protein and/or lipid content and/or reduced glucosinolate levels, the method comprising introducing and expressing in said plant the nucleic acid construct of  claim 42 . 
     
     
         48 . A method for producing a food or feed product with increased protein and/or lipid content and/or reduced glucosinolate levels, said method comprising
 a. producing a plant wherein the expression of UPL3 is reduced or abolished and/or the activity of a UPL3 polypeptide is reduced as defined in  claims 29  to  36 ;   b. obtaining a seed from said plant; and   c. producing a food or feed product from said seed.   
     
     
         49 . A method for screening a population of plants and identifying and/or selecting a plant that has or will have reduced UPL3 expression, an increased seed yield phenotype, increased seed protein and/or lipid levels and/or reduced glucosinolate levels, preferably compared to a wild-type or control plant, the method comprising detecting in the plant or plant germplasm at least one polymorphism in the UPL3 gene and/or promoter and/or at least one polymorphism in SEQ ID NO: 58, and selecting said plant or progeny thereof. 
     
     
         50 . The method of  claim 49 , wherein the polymorphism is an insertion, deletion and/or substitution. 
     
     
         51 . The method of  claim 50 , wherein the method further comprises introgressing the chromosomal region comprising at least one polymorphism in the UPL3 gene or promoter into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.