MODIFIED ACYLTRANSFERASE POLYNUCLEOTIDES, POLYPEPTIDES, AND METHODS OF USE

Abstract

The invention provides a method for producing a modified DGAT1 protein, comprising targeted manipulation of at least one motif selected from: a) a motif of the formula selected from RR, RXR, and RXXR, b) a motif of the formula AXXXA, c) a motif of the formula AXXXG, d) a motif of the formula GXXXG, and e) a motif of the formula GXXXA, in the N-terminal region of the protein upstream of the acyl-CoA binding site of a DGAT1 protein, where R is arginine, alanine, G is glycine and X is any amino acid. The modified DGAT1 protein can be expressed in a cell or organism, to increase the production of lipid in the cell or organism. The invention also provides the modified DGAT1 protein, polynucleotides encoding the modified DGAT1 proteins, cells and compositions comprising the polynucleotides or modified DGAT1 proteins, and methods using the modified DGAT1 proteins to produce oil.

Claims

1. A method for producing a modified DGAT1 protein, the method comprising targeted manipulation of at least one motif selected from: a) a motif of the formula selected from RR, RXR, and RXXR, b) a motif of the formula AXXXA, c) a motif of the formula AXXXG, d) a motif of the formula GXXXG, and e) a motif of the formula GXXXA, in the N-terminal region of the protein upstream of the acyl-CoA binding site of a DGAT1 protein, where R is arginine, A is alanine, G is glycine and X is any amino acid.

2. The method of claim 1 wherein the N-terminal region extends from the N-terminus of the DGAT1 protein to a position at least 1 amino acid upstream of the conserved motif ESPLSS (Glu-Ser-Pro-Leu-Ser-Ser) in the acyl-CoA binding site.

3. The method of claim 1 wherein the modified DGAT1 protein is at least 90% identical to the un-modified DGAT1 protein.

4. The method of claim 1 wherein the modified DGAT1 protein has a greater capacity to increase cellular lipid production than does the un-modified DGAT1 protein.

5. The method of claim 1 wherein when the modified DGAT1 protein is expressed in a cell, the cell produces more lipid than a suitable control cell in which modified protein is not expressed.

6. The method of claim 5 wherein when the modified DGAT1 protein is expressed in a cell, the cell produces at least 5% more than a suitable control cell in which modified protein is not expressed.

7. The method of claim 1 wherein the method includes a step of assessing the capacity of the modified DGAT1 protein to increase cellular lipid production relative to that of the un-modified DGAT1 protein.

8. The method of claim 1 wherein the method includes a step of selecting a modified DGAT1 protein with greater capacity to increase cellular lipid production than that of the un-modified DGAT1 protein.

9. The method of claim 1 wherein the modified DGAT1 protein is produced by expression from a polynucleotide encoding the modified DGAT1 protein.

10. The method of claim 9 wherein the modified DGAT1 protein is expressed in a cell or organism.

11. The method of claim 9 wherein the modified DGAT1 protein is expressed from a modified endogenous DGAT1 polynucleotide.

12. The method of claim 11 wherein the modified endogenous DGAT1 polynucleotide has been modified by a gene editing technology.

13. A modified DGAT1 protein, with an altered number or position of at least one motif selected from: a) a motif of the formula selected from RR, RXR, and RXXR, b) a motif of the formula AXXXA, c) a motif of the formula AXXXG, d) a motif of the formula GXXXG, and e) a motif of the formula GXXXA, in the N-terminal region of the protein upstream of the acyl-CoA binding site of a DGAT1 protein, where R is arginine, A is Alanine, G is Glycine and X is any amino acid.

14. The modified DGAT1 protein of claim 13 wherein the N-terminal region extends from the N-terminus of the DGAT1 protein to a position at least 1 amino acid upstream of the conserved motif ESPLSS (Glu-Ser-Pro-Leu-Ser-Ser) in the acyl-CoA binding site.

15. The modified DGAT1 protein of claim 13 that is at least 90% identical to the un-modified DGAT1 protein.

16. The modified DGAT1 protein of claim 1 that has a greater capacity to increase cellular lipid production than does the un-modified DGAT1 protein.

17. The modified DGAT1 protein of claim 13 that is produced by targeted manipulation of at least one motif selected from: a) a motif of the formula selected from RR, RXR, and RXXR, b) a motif of the formula AXXXA, c) a motif of the formula AXXXG, d) a motif of the formula GXXXG, and e) a motif of the formula GXXXA, in the N-terminal region of the protein upstream of the acyl-CoA binding site of a DGAT1 protein, where R is arginine, A is alanine, G is glycine and X is any amino acid.

18. A polynucleotide encoding a modified DGAT1 of claim 13.

19. A construct comprising the polynucleotide of claim 18.

20. A cell comprising the modified DGAT1 protein of claim 13.

21. The cell of claim 20 that produces more lipid than does a suitable control cell.

22. The cell of claim 21 that produces at least 5% more lipid than a suitable control cell in which modified protein is not expressed.

23. A plant comprising the modified DGAT1 protein of claim 13 or a polynucleotide encoding the modified DGAT1 protein.

24. The plant of claim 23 in which the polynucleotide is an endogenous DGAT1 polynucleotide that has been modified in the plant to encode the modified DGAT1 protein.

25. The plant of claim 23 that produces more lipid, in at least one of its tissues or parts, than does the equivalent tissue or part in a suitable control plant.

26. The plant of claim 23 that produces at least 5% more lipid in at least one of its tissues or parts, than does a suitable control plant.

27. The plant of claim 23 that as a whole produces at least 5% more lipid than does a suitable control plant.

28. The part, propagule or progeny of a plant of claim 23 that comprises the modified DGAT1 protein or a polynucleotide encoding the modified DGAT1 protein.

29. The part, propagule or progeny of claim 28 that produces at least 5% more lipid than does an equivalent part, propagule or progeny of a suitable control plant.

30. An animal feedstock comprising the modified DGAT1 protein of claim 13 or a polynucleotide encoding the modified DGAT1 protein.

31. A biofuel feedstock comprising the modified DGAT1 protein of claim 13 or a polynucleotide encoding the modified DGAT1 protein.

32. A method for producing oil, the method comprising extracting lipid from the cell of claim 20 or a plant, plant part, propagule or progeny comprising the cell.

33. The method of claim 32 wherein the lipid is processed into at least one of: a) a fuel, b) an oleochemical, c) a nutritional oil, d) a cosmetic oil, e) a polyunsaturated fatty acid (PUFA), and f) a combination of any of a) to e).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0410] FIG. 1 shows the alignment of peptide sequences of the N-terminal cytoplasmic region of a number of plant DGAT1s including both long and short versions from the grasses as well as examples from dicotyledonous species. Left hand box represents acyl-CoA binding site (Nykiforuk et al., 2002, Biochimica et Biophysica Acta 1580:95-109). Right hand box represents first transmembrane region (McFie et al., 2010, JBC., 285:37377-37387). Left hand arrow represents boundary between exon 1 and exon 2. Right hand arrow represents boundary between exon 2 and exon 3. The sequences are AtDGAT1 (SEQ ID NO:113), BjDGAT1 (SEQ ID NO:114), BnDGAT1-AF (SEQ ID NO:115), BjDGAT1 (SEQ ID NO:116), TmajusDGAT1 (SEQ ID NO:117), EpDGAT1 (SEQ ID NO:118), VgDGAT1 (SEQ ID NO: 119), NtDGAT1 (SEQ ID NO:120), PfDGAT1 (SEQ ID NO:121), ZmL (SEQ ID NO: 122), SbDGAT1 (SEQ ID NO:123), OsL (SEQ ID NO:124), OsS (SEQ ID NO:125), SbDGAT1 (SEQ ID NO:126), ZmS (SEQ ID NO:127), PpDGAT1 (SEQ ID NO:128), SmDGAT1 (SEQ ID NO:129), EaDGAT1 (SEQ ID NO:130), VVDGAT1 (SEQ ID NO:131), GmDGAT1 (SEQ ID NO:132), GmDGAT1 (SEQ ID NO:133), LjDGAT1 (SEQ ID NO:134), MtDGAT1 (SEQ ID NO:135), JcDGAT1 (SEQ ID NO:136), VIDGAT1 (SEQ ID NO:137), RcDGAT1 (SEQ ID NO:138), PtDGAT1 (SEQ ID NO:139), Pt DGAT1 (SEQ ID NO:140).

[0411] FIG. 2 shows alignment of N-terminal peptide sequences of Arabidopsis thaliana, At (SEQ ID NO: 1); Tropolium majus, Tm (SEQ ID NO:5); Zea mays, ZmL (SEQ ID NO:10); Zea mays, ZmS (SEQ ID NO:15). Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type, while the complete AXXXA and GXXXG motifs are shown by bold face underline type.

[0412] FIG. 3 shows alignments of N-terminal peptide sequence from ZmL (SEQ ID NO:10) with versions containing modified di-argine motifs. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. Di-arginine motifs replaced by serine or glycine residues (shown by bold face and underlined S or G respectively) create ZmL.sup.R13G,R14S,R17S,R65G,R66G (SEQ ID NO:97) and ZmL.sup.R33S,R34S,R37S,R65G,R66G (SEQ ID NO:98).

[0413] FIG. 4 shows alignments of N-terminal peptide sequences from Tm (SEQ ID NO:94) with two versions having internal deletions. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Two versions of Tm with internal deletions were made; each had a separate internal section of 27 residues deleted (shown within blocks); in each case this reduced the length of the cytoplasmic N-terminus to be the same as ZmS. One removed the last 27 residues before the conserved region; this also removed the three AXXXA/GAAAG (shown by bold face and underline) motifs creating Tm.sup.68-94 (SEQ ID NO:99). The second removed a segment closer to the N-terminus; this placed the multiple AXXXA/GAAAG motifs (shown by bold face and underline) closer to the multi di-argine motif (bold face arginine resides) creating Tm.sup.35-61 (SEQ ID NO:100).

[0414] FIG. 5 shows alignments of N-terminal peptide sequences from the chimeras Tm::ZmL (SEQ ID NO: 111) and ZmS::Tm (SEQ ID NO:112) with the same chimeras that had their di-arginine motifs deleted. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. Deletion of the multi-diargine motif (shown within blocks) from the chimera Tm::ZmL created Tm.sup.R25,R26,R27::ZmL (SEQ ID NO:103). Deletion of the multi-diargine motif (shown within blocks) from the chimera ZmS::Tm created ZmS.sup.R27,L28,R29,R30::Tm (SEQ ID NO:104).

[0415] FIG. 6 shows alignments of N-terminal peptide sequences from the chimeras Tm::ZmL (SEQ ID NO:111) and ZmS::Tm (SEQ ID NO:112) with the same chimeras that had their AXXXA and GXXXG motifs perturbed by substitution. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. The AXXXA/GAAAG motifs are shown by bold face and underline. Substitution of one alanine and one glycine residue (shown by blocks) with serine residues created Tm.sup.A64S,G80S::ZmL (SEQ ID NO:101). Substitution of two alanine and two glycine residues with serine residues created ZmS.sup.A10S,G17S,A36S,G37S::Tm (SEQ ID NO:102).

[0416] FIG. 7 shows alignment of N-terminal peptide sequences from the chimera Tm::ZmL (SEQ ID NO: 111) with the same chimera that had the AXXXA and GXXXG motifs perturbed by substitution and in one case replaced with a multi di-arginine motifs. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. The AXXXA/GAAAG motifs are shown by bold face and underline. Substitution of one alanine and three glycine residue (shown by blocks) with serine residues created Tm.sup.A64S,G78R,G79R,G80R::ZmL (SEQ ID NO:105).

[0417] FIG. 8 shows alignment of N-terminal peptide sequences from the chimera ZmS::Tm (SEQ ID NO: 112) with the same chimera that had a AXXXA motif perturbed and replaced with a multi di-arginine motifs by substitution and an additional multi di-arginine motif created by substitution. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. The AXXXA/GAAAG motifs are shown by bold face and underline. Substitution of one aspartate and one alanine (shown by blocks) with arginine residues perturbed the N-terminal AXXXA motif with a multi di-arginine motif and substitution of one serine and two glycine residues (shown by blocks) generated an additional multi di-arginine motif creating ZmS.sup.D12R,A14R,S44R,G45R,G46R::Tm (SEQ ID NO:106).

[0418] FIG. 9 shows alignment of N-terminal peptide sequences from ZmS (sequence ID number 96) and Tm (SEQ ID NO:94) with the same sequences where an additional N-terminal multi di-arginine motif had been created by substitution. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. The AXXXA/GAAAG motifs are shown by bold face and underline. The substituted residues are shown by blocks; this created ZmS.sup.A9R,A10R,S11R (SEQ ID NO:107) and Tm.sup.S6R,S7R,Q8R (SEQ ID NO:108).

[0419] FIG. 10 shows alignment of N-terminal peptide sequence from At (sequence ID number 75) with the same sequences multiple AXXXA and GXXXG motifs were perturbed by substitution. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. The AXXXA/GAAAG motifs are shown by bold face and underline. The substituted residues are shown by blocks; this created At.sup.A75S,G79S,G97S,G99S (SEQ ID NO:76).

[0420] FIG. 11 shows alignment of N-terminal peptide sequence from At (SEQ ID NO:75) with the same sequences multiple AXXXA and GXXXG motifs were perturbed and an additional multi di-arginine motifs was created by substitution. Underlined type denotes the beginning of the conserved region (WO/2014/068439) which starts 13 residues upstream of the acetyl-CoA binding domain (not shown). Each arginine residue associated with a potential di-arginine motif is shown by bold face type. The AXXXA/GAAAG motifs are shown by bold face and underline. The substituted residues are shown by blocks; this created At.sup.A75S,G79S,G93R,G94R,G95R,G97S,G99S (SEQ ID NO:77).

EXAMPLES

[0421] The invention will now be described with reference to the following non-limiting examples.

Example 1: Plant DGAT1 Contain Multiple N-Terminal Di-Arginine Motifs as Well as AXXXA, GXXXG, AXXXG and GXXXA Motifs

[0422] DGAT1s from a broad range of organisms were found to contain a cluster of arginines in the first 30 residues (Siloto et al 2010). Upon examination we found most vascular plant DGAT1s from Table 1 contain multiple di-arginine motifs (RR, RXR, and RXXR) in the variable region of the N-terminus (Table 2). In comparison, the N-terminus of the mammalian DGAT1s (including: Bos Taurus, NP_777118; Mus musculus, NP_034176; Homo sapiens, NP_036211; Ovis aries, NP_0011036; Rattus norvegicus, NP_445889; Sus scrofa, NP_999216; and Mesocricetus auratus, XP_005086048) all contain the same multi motif (RRRR) close to the N-terminus and a second potential motif (RXXR) at the start of the acyl CoA binding region.

[0423] In other proteins the di-arginine motifs appear to have roles in assembly of heteromultimeric membrane proteins; retrieval of ER membrane proteins from the Golgi apparatus and the ER-Golgi intermediate; establishing the cytosolic location of the N-terminus; interaction with downstream cytosolic loops (Boulaflous et al 2009; Michelsen et al, 2005; Parks and Lamb 1993; Shikano and Li, 2003; Teasdale and Jackson 1996).

[0424] The cytosolic N-termini of the plant DGAT1s also contain varying numbers of AXXXA and GXXXG (includes AXXXG and GXXXA) motifs (Table 3); these have been shown to be involved in protein-protein interaction in transmembrane domains and in cytosolic proteins of thermophilic organisms (Teese and Langosch 2015; Kleiger et al 2002). As such the applicants postulate that they could potentially be involved in oligomerization of DGAT1.

TABLE-US-00001 TABLE 1 DGAT1 PROTEIN Species accession #s SEQ ID DNA accession #s SEQ ID Source & BAC # NO: & BAC # NO: A. thaliana NP 179535 1 NM 127503 30 B. juncea AAY40784 2 AF164434 31 B. napus AAD45536.1 3 AF164434 1 32 B. juncea AAY40785 4 DQ016107 33 T. majus AAM03340 5 AY084052 34 E. pitardii ACO55635 6 FJ226588 35 V. galamensis ABV21945 7 EF653276 36 N. tabacum AAF19345.1 8 AF129003_1 37 P. frutescens AAG23696.1 9 AF298815 1 38 Z. mays From: CHORI-201 10 From: CHORI-201 39 Maize B73 BAC Maize B73 BAC S. bicolor XP 002439419 11 XM 002439374 40 O. sativa NP_001054869 12 Os05g0196800 41 O. sativa From: AP003714.1 13 From: AP003714.1 42 S. bicolor XP 002437165 14 XM 002437120.1 43 ABV91586 15 EU039830 44 P. patens XP_001770929 16 XM_001770877.1 45 S. moellendorffii XP_002964165 17 XM_002964119 46 E. alatus AAV31083 18 AY751297 47 V. vinifera XP 002279345 19 XM 002279309 48 G. max AAS78662 20 AY496439 49 G. max BAE93461 21 AB257590 50 L. japonicus AAW51456 22 AY859489 51 M. truncatula ABN09107 23 AC174465.2 52 J. curcas ABB84383 24 DQ278448.1 53 V. fordii ABC94472 25 DQ356680.1 54 V. galamensis ABV21945 26 EF653276.1 55 R. communis XP_002514132 27 XM_002514086.1 56 P. trichocarpa XP 002308278 28 XM 002308242.1 57 P. trichocarpa XP 002330510 29 XM 002330474.1 58

TABLE-US-00002 TABLE 2 Position of Position of Position of first N- first N- first N- Number of terminally terminally terminally di-arginine located located located clusters arginine arginine arginine within the DGAT1 residue of di- residue of di- residue of di- first 65 N- Species SEQ arginine motif arginine motif arginine motif terminal Source ID NO: RR RXR RXXR residues A. thaliana 1 27, 28, 115 116 25 29, 115 1 B. juncea 2 26, 98 54, 99 23, 27, 98 2 B. napus 3 26, 98 54, 99 23, 98 2 B. juncea 4 24, 25, 98 24, 99 26, 98 1 T. majus 5 25, 26, 107 25, 108 107 1 E. pitardii 6 20, 21, 60 20 2 V. galamensis 7 21, 22, 110 21 1 N. tabacum 8 33, 34, 35, 58, 33, 34 33 2 123 P. frutescens 9 28, 29 28 1 Z. mays L 10 3, 33, 34, 65, 66 33, 35, 63 14, 17, 34 3 S. bicolor L 11 5, 35, 36. 67, 68 35, 67 16, 19 2 O. sativa L 12 6, 47, 48, 85, 86 30, 47, 49, 85 27, 48 2 O. sativa S 13 62 21, 23, 63 62 2 S. bicolor S 14 29, 47, 76 27 27 2 Z. mays S 15 29, 80 27 27 1 P. patens 16 36 34 34 1 S. moellendorffii 17 9 1 E. alatus 18 16, 17 16 1 V. vinifera 19 28 1 G. max 20 20, 21 20 1 G. max 21 20, 21 20 1 L. japonicus 22 27, 28, 98 27 1 M. truncatula 23 32, 33, 127 32 1 J. curcas 24 26, 27, 28 26, 27, 88 26, 85, 105 1 V. fordii 25 30, 31, 32 30, 31 30, 90 1 V. galamensis 26 21, 22, 110 21 1 R. communis 27 26, 27, 28 26, 27 26 1 P. trichocarpa 28 34, 35 34 84 1 P. trichocarpa 29 29, 30 29 1

TABLE-US-00003 TABLE 3 SEQ DGAT1 ID Position of first N-terminal A or G residue located in a Species Source NO: AXXXA, AXXXG, GXXXG or GXXXA motif A. thaliana 1 71, 75, 79, 85, 89, 90, 91, 95, 97, 99 B. juncea 2 48, 61, 65, 69, 72, B. napus 3 63, 75, 76, B. juncea 4 48, 61, 65, 69, 72, T. majus 5 64, 76, 80, E. pitardii 6 25, 54 V. galamensis 7 31 N. tabacum 8 40, 63, 117 P. frutescens 9 17, 85 Z. mays L 10 19, 21, 23, 25, 27, 39, 40, 46, 49, 53, 72, 73, 77, 78, 79, 84 S. bicolor L 11 21, 23, 25, 27, 41, 42, 44, 48, 51, 55, 75, 78, 81, 82, 87 O. sativa L 12 6, 8, 11, 13, 15, 33, 37, 41, 53, 54, 56, 63, 67, 89, 96, 99, 100, 101, 103, 104, 105, 110 O. sativa S 13 8, 24, 26, 27, 29, 31, 56 S. bicolor S 14 10, 14, 36, 70 Z. mays S 15 10, 17, 36, 37, 41, 74 P. patens 16 9, 28 S. moellendorffii 17 0 E. alatus 18 44, 45 V. vinifera 19 33, 49, 53, 67, 86, 89 G. max 20 40, 44 G. max 21 25, 43, 47 L. japonicus 22 12, 85 M. truncatula 23 37, 43, 47, 50, J. curcas 24 11, 73 V. fordii 25 13, 77 V. galamensis 26 31 R. communis 27 0 P. trichocarpa 28 15 P. trichocarpa 29 0

Example 2: Generation of Recombinant Constructs for Evaluation in Saccharomyces cerevisiae

[0425] A series of construct where the di-arginine and/or AXXXA, GXXXG motifs were altered/introduced. The name of the constructs, a description of their derivation, and the corresponding peptide sequences are shown in Table 4. All DGAT1s were optimised for expression in Saccharomyces cerevisiae and had an in-frame C-terminal V5 epitope and 6 histidine tag.

[0426] Saccharomyces cerevisiae optimized DGAT1 coding sequences along with a C-terminus V5-His tag were synthesised by either GeneArt (Thermo Fisher Scientific) or GenScript and subsequently cloned into the pYES2.1/V5-His-TOPO yeast expression vector (Life Technologies, K4150-01) as per the manufacturer's instructions. This places all DGAT1s expressed in yeast were under the control of the inducible Gall promoter.

TABLE-US-00004 TABLE 4 NUCLEIC Original ACID SEQ Source of OPTIMIZED EXPRESSED DGAT FOR YEAST PROTEIN Sequence Name Description FIG. SEQ ID NO: SEQ ID NO: T. majus Tm DGAT1 from T. majus 1, 2, 59 69 Z. mays ZmL Long DGAT1 from Z. mays 1, 2, 60 70 T. majus & Tm::ZmL Chimeric DGAT1 using Tm N- 5, 6, 7 61 71 Z. mays terminus and ZmL C-terminus Z. mays N ZmL ZmL with tr ted N-terminus N/A 62 72 T. majus Tm Tm Di-arginine motifs N/A 63 73 substituted T. majus and Tm ::ZmL Tm Di-arginine motifs N/A 64 74 Z. mays substituted and chimera generated with ZmL C-terminus A. thaliana At DGAT1 from A. thaliana 1, 2, 10, 65 75 11 A. thaliana At At with multiple disrupted 10 66 76 AXXXA and GXXXG A. thaliana At At with multiple disrupted 11 67 77 AXXXA and GXXXG and new RRRR A. thaliana N At At with truncated N-terminus N/A 68 78 indicates data missing or illegible when filed

Example 3: Generation of Recombinant Constructs for Evaluation in Camelina sativa

[0427] A series of construct where the di-arginine and/or AXXXA, GXXXG motifs were altered/introduced. The name of the constructs, a description of their derivation, and the corresponding peptide sequences are shown in Table 5. All DGAT1s had an Arabidopsis thaliana DGAT1 intron 3 (Accession NC_003071, REGION: 8426117 . . . 8429853); the constructs were optimised for expression in Camelina sativa and had an in-frame C-terminal V5 epitope and 6 histidine tag. In addition, the putative serine/threonine protein kinase site in the Tropaeolum majus DGAT1 (Xu et al., 2008) was disrupted by substitution of the serine to alanine generating Tm.sup.S197A, Tm.sup.68-94,S197A, Tm.sup.35-61,S197A, ZmS.sup.A10S,G17S,A36S,G37S::Tm.sup.S170A, ZmS.sup.R27,1.28,R29,R30::Tm.sup.S170A, ZmS.sup.D12R,A14R,S44R,G45R,G46R::Tm.sup.S170A, Tm.sup.S6R,S7R,Q8R,S197A, ZmS::Tm.sup.S170A.

[0428] Brassica optimized DGAT1 coding sequences along with a C-terminus V5-His tag were synthesised by either GeneArt (Thermo Fisher Scientific) or GenScript and sub cloned into pDONR221. A cassette consisting of Not I sites flanking the Brassica napus napin seed storage promoter region and 5UTR (GenBank accession number EF627523.1)::GATEWAY cloning sequences::octopine synthase terminator was synthesised by GenScript. The cassette was digested with Not I and cloned into pRSh1 (Scott et al 2010) replacing the constitutive promoter cauliflower mosaic virus 35S (CaMV35Sp) driven GATEWAY adapted expression cassette. This created the binary vector pBR2 (ref from Somrutai) containing a seed specific expression cassette in a back-to back orientation with the CaMV35Sp driven bar gene for phosphinothricin resistant selection. DGAT1s were subsequently placed into pBR2 from pDONR221 by GATEWAY LR cloning (Thermo Fisher Scientific).

Camelina sativa Transformation

[0429] C. sativa (cf. Calena) were transformed via Agrobacterium tumefaciens (GV3101) using the floral dip method (adapted from that of Clough and Bent, 1998, Plant J. 16 (6): 735-745). Essentially seeds were sown in potting mix in 10 cm pots in a controlled environment, approximately 6 weeks after planting the flowers were dipped for 5-14 minutes under vacuum (70-80 mm Hg) in an overnight culture of appropriated Agrobacterium GV3101 cells re-suspended in a floral dip buffer. After vacuum-transformation, plants were kept for 24 h under low light conditions by partly covering with a black plastic sheet. Vacuum transformations can be repeated three times at approximately 10-12 days intervals, corresponding to the flowering duration. Plants were grown in potting mix in a controlled environment (16-h day length, 21-24 C., 65-70% relative humidity).

[0430] The T.sub.1 seeds produced can be collected and screened for transformants by germinating and growing seedlings at 22 C. with continuous light on a half-strength MS medium (pH 5.6) selection plate containing 1% (w/v) sucrose, 300 mg/L Timentin, and 25 mg/L DL-phosphinothricin to select for herbicide resistance. T.sub.2 selfed seed populations can also be screened by immuno blot for the presence of the V5 eptiope.

[0431] T.sub.2 selfed seeds may be analysed for oil content by GC. Approximately 50 individual transgenic lines (including control lines) may be selected for the next generation (10 plants/line) based on their oil content, or seed weight. T.sub.2 plants may be grown and screened by PCR for copy number and identification of null sibing lines. T.sub.2 seeds may be analysed in triplicate for oil content by NMR or GC/MS.

TABLE-US-00005 TABLE 5 Original NUCLEIC ACID SEQ Source of OPTIMIZED FOR EXPRESSED DGAT CAMELINA PROTEIN Sequence Name Description FIG. SEQ ID NO: SEQ ID: T. majus Tm DGAT1 from T. majus 1, 2, 4, 79 94 Z. mays ZmL Long DGAT1 from Z. mays 1, 2, 3, 80 95 Z. mays ZmS Short DGAT1 from Z. mays 1, 2, 9 81 96 Z. mays ZmL Long DGAT1 from Z. mays with 1 3, 82 97 and 3.sup.rd regions of di-arginines disrupted Z. mays ZmL Long DGAT1 from Z. mays with 2.sup.nd 3, 83 98 and 3.sup.rd regions of di-arginines disrupted T. majus Tm Truncated Tm disrupts AXXXA and 4 84 99 GXXXG T. majus Tm Truncated Tm disrupts AXXXA and 4 85 100 GXXXG T. majus & Z. mays Tm ::ZmL Disrupts AXXXA and GXXXG by 6 86 101 substitution Z. mays & T. majus ZmS Tm Disrupts AXXXA and GXXXG by 6 87 102 substitution T. majus & Z. mays Tm ::ZmL Tm::ZmL chimera with disrupted di- N/A 88 103 arginines Z. mays & T. majus ZmS ::Tm ZmS::Tm chimera with disrupted di- N/A 89 104 arginines T. majus & Z. mays Tm ::ZmL Tm::ZmL chimera with disrupted 7 90 105 AXXXA and additional di-arginines Z. mays & T. majus ZmS ::Tm ZmS::Tm chimera with disrupted 8 91 106 GXXXG and additional di-arginines Z. mays ZmS ZmS with additional di-arginines 9 92 107 T. majus Tm Tm with additional di-arginines 9 93 108 T. majus & Z. mays Tm::ZmL Chimera Tm N-terminus with ZmL C- 5, 6, 7 109 111 terminus (already in C. sativa) Z. mays & T. majus ZmS::Tm Chimera ZmS N-terminus with Tm C- 6 110 112 terminus (already in C. sativa) indicates data missing or illegible when filed

Example 4: Evaluation of Plant DGAT1s with Modified Di-Arginine and AXXXA, GXXXG, AXXXG and GXXXA in Saccharomyces cerevisiae

[0432] The control DGAT1s At; Tm; ZmL; N ZmL; Tm::ZmL and the DGAT1s with modified di-arginine and AXXXA, GXXXG, AXXXG and GXXXA Tm.sup.R25G,R26G,R27G; Tm.sup.R25G,R26G,R27G::ZmL; At.sup.A75S,G79S,G97S,G99S; and At.sup.A75S,G79S,G93R,G94R,G95R,G97S,G99S were over expressed in yeast and the TAG produced (as % DW) after 48 hours was determined (shown in Table 6).

TABLE-US-00006 TABLE 6 Original g FA Source Cell per L of DGAT PROTEIN DW % FA g FA (% of Sequence Name Description FIG. SEQ ID: (g/L) per cell per L control) N/A vector control vector control N/A N/A 5.36 3.22 0.173 N/A T. majus Tm DGAT1 from T. majus 1, 2, 4, 9 69 5.79 8.36 0.495 N/A Z. mays ZmL Long DGAT1 from Z. mays 1, 2, 3 70 7.26 12.6 0.915 N/A T. majus & Tm::ZmL Chimeric DGAT1 using Tm N- 5, 6, 7 71 6.33 8.6 0.544 59.5 Z. mays terminus and ZmL C-terminus (% of ZmL) Z. mays N ZmL ZmL with truncated N/A 72 6.67 12.7 0.847 92.6 N-terminus (% of ZmL) T. majus Tm Tm Di-arginine motifs N/A 73 5.79 7.86 0.455 91.9 substituted (% of Tm) T. majus & Tm ::ZmL Tm Di-arginine motifs N/A 74 5.33 7.3 0.389 71.5 Z. mays substituted, and chimera (% of Tm::ZmL) generated with ZmL C-terminus A. thaliana At DGAT1 from A. thaliana 1, 2, 10, 11 75 5.46 6.43 0.351 N/A A. thaliana At At with multiple disrupted 10 76 5.31 6.68 0.355 101.1 AXXXA and GXXXG (% of At) A. thaliana At At with maltipte disrupted 11 77 5.89 6.72 0.396 112.8 AXXXA and GXXXG and new RRRR (% of At) A. thaliana N At At with truncated N/A 78 6.02 8.68 0.523 147.3 N-terminus (% of At) indicates data missing or illegible when filed

[0433] Microsomes were extracted from the yeast cells after 48 h culture. The extracts were subjected to PAGE-immunoblot (probing with either anti-V5 antibody or anti-Kar2 antibody). The most predominant band in the in-gel-stain-free image was scanned and quantified using BioRad's ChemiDoc software. Similarly, the immunofluorescence signals indicating the V5 tag of the DGAT1 and Kar2 marker protein of the ER were also scanned and quantified. The values and relative quantifications are shown in Table 7.

Table 7.

TABLE-US-00007 TABLE 7 Signal Intensity DGAT DGAT Total DGAT1- signal signal Protein Membrane Kar-2 V5 relative relative SEQ ID: Protein Signal Signal to MP to Kar-2 75 13072992 8318706 1334200 0.102 0.160 76 12852240 6144000 1506348 0.117 0.245 77 13031997 6906966 1993800 0.153 0.289 69 13942980 15875928 5028670 0.361 0.317 73 16206056 11252126 1585332 0.098 0.141
Summary when Expressed in Yeast [0434] Removal of di-arginine motifs in Tm decreased recombinant DGAT1 in yeast by approximately 56% (estimated by determining signal strength relative to Kar-2) and reduced FA production in yeast by approximately 10%. [0435] The same perturbation of the N-terminal di-arginine motifs in TmZmL resulted in a decrease in FA production (g FA/L) in yeast cells of approximately 30%. [0436] For the At DGAT1, disruption of AXXXA and GXXXG motifs and addition of a new RRR motif (near the C-terminus of the cytosolic variable N-terminus) increased recombinant DGAT1 in yeast microsomes by approximately 81% and increased FA production (g FA/L) in yeast cells by approximately 13%. In comparison disruption of AXXXA and GXXXG had little influence on FA production but increased accumulation of recombinant DGAT1 in yeast microsomes by approximately 53%.

Example 5: Evaluation of Plant DGAT1s with Modified Di-Arginine and AXXXA, GXXXG, AXXXG and GXXXA in Camelina Sativa

[0437] The control DGAT1s (Tm.sup.S197A, ZmL; ZmS; ZmS::Tm.sup.S170A, Tm::ZmL) and the DGAT1s with modified di-arginine and AXXXA, GXXXG, AXXXG and GXXXA (ZmL.sup.R13G,R14S,R17S,R65G,R66G; ZmL.sup.R33S,R34S,R37S,R65G,R66G; Tm.sup.68-94,S197A; Tm.sup.135-61,S197A; Tm.sup.A64S,G80S::ZmL; ZmS.sup.A10S,G17S,A36S,G37S::Tm.sup.S170A, Tm.sup.R25,R26,R27::ZmL; ZmS.sup.R27,1.28,R29,R30::Tm.sup.S170A; Tm.sup.A64S,G78R,G79R,G80R::ZmL; ZmS.sup.D12R,A14R,S44R,G45R,G46R::Tm.sup.S170A; Zm.sup.SA9R,A10R,S11R; and Tm.sup.S6R,S7R,Q8R,S197A) were over expressed in the seeds of Camelina sativa. The fatty acid content of the seed (as % DW) was determined and shown in Tables 8-13 (full data and statistics tables from each glasshouse). The data has been separated by glasshouse since the growing conditions varied within each glasshouse making comparisons between glasshouses inappropriate. However, an overall summary of the trends can be made; these are listed below.

SUMMARY when Expressed in Camelina [0438] Disruption to of the di-arginine clusters within the first 65 residues of ZmL led to a modest increase in seed FA content compared to ZmL 3.7 to 6.6% difference compared with ZmL) [0439] First internal truncation of Tm led to decreases in seed FA content (7.7% to 2.2%) compared to Tm.sup.S197A [0440] Second internal truncation of Tm led to decreases in seed FA content (7.9% to 3.3% difference compared to Tm.sup.S197A [0441] Addition of di-arginine multi-motif within first 15 residues to ZmS, resulted in an increase in seed FA content (+12% compared to ZmS) [0442] Addition of di-arginine multi-motif within first 15 residues to Tm.sup.S197A, resulted in an increase in seed FA content (+15.5% compared to Tm.sup.S197A) [0443] Disruption of first di-arginine motif in Tm::ZmL, resulted in a decrease in seed FA content (21 to 7.5% compared to Tm::ZmL) [0444] Disruption of first di-arginine motif in ZmS::Tm.sup.S170A, resulted in a decrease in seed FA content (15.6 to 14.5% compared to ZmS::Tm.sup.S170A) [0445] Disrupt AXXXA and GXXXG in ZmS of ZmS::Tm.sup.S170A resulted in a decrease in seed FA content (8.0% compared to ZmS::Tm.sup.S170A) [0446] Disrupt AXXXA and GXXXG in Tm of Tm::ZmL, resulted in an increase in seed FA content (+3.3% compared to Tm::ZmL) [0447] Disrupt AXXXA and add di-arginine motif in Tm of Tm::ZmL, resulted in an increase in seed FA content (+6.3% compared to Tm::ZmL) [0448] Disrupt AXXXA and add di-arginine motif in ZmS of ZmS::Tm.sup.S170A, resulted in an increase in seed FA content (+0.2% compared to ZmS::Tm.sup.S170A)

TABLE-US-00008 TABLE 8 (GH3 SUMMARY) TAG accumulation PROTEIN Av % A compared to DGAT1 Name Description FIG. SEQ ID: in seed control TmS197A Control Tm DGAT 1, 2, 4, 94 26.32 N/A 9 ZmL Control ZmL DGAT 1, 2, 3 95 25.78 N/A ZmS Control ZmS DGAT 1, 2, 9 96 23.46 N/A ZmLR13G, R14S, R17S, R65G, R66G disrupt di-arginines in ZmL 3 97 25.74 ns .sup.0.2 (% of ZmL) ZmLR33S, R34S, R37S, R65G, R66G disrupi di-argiranes in ZmL 3 98 27.49 ns .sup.+6.6 (% of ZmL) Tm68-94, S197A internal ation of Tm 4 99 24.24 ns 7.9 (% of Tm) Tm35-61, S197A internal ation of Tm 4 100 25.75 ns 2.2 (% of Tm) ZmSA9R, A10R, S11R creates di-arginine in ZmS 9 107 26.28 ns +12.0 (% of ZmS) indicates data missing or illegible when filed

TABLE-US-00009 TABLE 9 (GH3 stats) DNA Seed Size % Lipid Lipid Seq (mg/seed) Null Seed Size (% DW) Null % Lipid (mg/seed) Null Lipid Line ID DGAT1 ID# n = 6 (p-Value) n = 6 (p-Value) n = 6 (p-Value) WT Wild-Type 1.22 24.33 0.30 pBR2 Vector Control 1.16 24.99 0.29 Tm#2 Tm 1.25 1.23 (0.6355) 26.32 26.53 (0.6279) 0.33 0.33 (0.8333) ZmS#1 ZmS 1.16 1.24 (0.0803) 23.46 25.41 (0.3489) 0.27 0.31 (0.2027) ZmL#9 ZmL 1.17 1.15 (0.6847) 25.78 27.50 (0.3268) 0.30 0.32 (0.6291) 1-E ZmL R13G R14S R17S R65G R65G 82 1.21 1.17 (0.2047) 27.09 26.58 (0.7733) 0.33 0.31 (0.4663) 1-D 1.25 1.19 (0.1056) 24.39 24.47 (0.9232) 0.31 0.29 (0.2429) 2-29 ZmL R33S R34S R37S R65G R66 83 1.18 1.17 (0.7351) 28.11 26.32 (0.1236) 0.33 0.31 (0.2251) 2-H11 1.19 1.15 (0.3879) 24.91 25.22 (0.8865) 0.30 0.29 (0.8381) 2-H13 1.24 1.22 (0.5211) 28.80 28.32 (0.7381) 0.36 0.35 (0.5074) 2-H14 1.28# 1.23 (0.2203) 28.12 28.15 (0.9815) 0.36 0.35 (0.5713) 3-A Tm Delta 68-94 84 1.31 1.24 (0.1255) 23.88 23.35 (0.7251) 0.31 0.29 (0.3536) 3-H 1.27 1.22 (0.4819) 24.59 23.68 (0.4413) 0.31 0.29 (0.3886) 4-48 Tm Delta 35-61 85 1.27 1.25 (0.6464) 24.70 23.93 (0.6589) 0.31 0.30 (0.5273) 4-50 1.25 1.22 (0.6047) 26.80 24.67 (0.3536) 0.34 0.30 (0.3494) 11-6 ZmS ASR A10R S11R 92 1.27## 1.15 (0.0003) 25.93 26.02 (0.9319) 0.33 0.30 (0.0402) 11-7 1.25# 1.21 (0.2503) 27.10 26.81 (0.8333) 0.34 0.32 (0.5236) 11-13 1.29## 1.18 (0.0019) 25.58 23.36 (0.0899) 0.33 0.28 (0.0163) 11-15 1.25# 1.19 (0.0335) 24.26 25.25 (0.4124) 0.31 0.30 (0.8027) 11-H14 1.29# 1.22 (0.1419) 28.52 27.32 (0.6397) 0.37# 0.33 (0.3982) Glasshouse 3 Plants (* = p-Value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, compared to WT and VC) (# = p-value < 0.05, ## = p-value < 0.01, ### = p-value < 0.001, compared to the parent)

TABLE-US-00010 TABLE 10 (GH5 BATCH 1 SUMMARY) PROTEIN % FA in TAG accumulation DGAT1 Name Description FIG. SEQ ID: seed compared to control TmS197A Control Tm DGAT 1, 2, 4, 9 94 27.24 N/A ZmL Control ZmL DGAT 1, 2, 3 95 26.06 N/A ZmS Control ZmS DGAT 1, 2, 9 96 27.68 N/A ZmLR13G, R14S, R17S, disrupt di-arginines in 3 97 27.03 .sup.+3.7 (% of ZmL) R65G, R66G ZmL Tm68-94, S197A internal truncation of Tm 4 99 26.34 3.3 (% of Tm) Tm35-61, S197A internal truncation of Tm 4 100 25.15 7.7 (% of Tm) ZmS A10S, G17S, A36S, disrupt AXXXA & GXXXG 6 102 27.65 8.0 (% of ZmS::Tm) G37S::TmS170A TmR25, R26, R27::ZmL disrupt first di-arginine 5 103 25.46 21.0 (% of Tm::ZmL) motif in Tm::ZmL mS R17, L28, disrupt first di-arginine 5 104 25.36 15.6 (% of ZmS::Tm) R29, R30::TmS170A motif in ZmS::Tm Tm::ZmL Control Tm::ZmL Chimera 6, 7 111 32.21 N/A ZmS::TmS170A Control ZmS::Tm Chimera 6 112 30.04 N/A indicates data missing or illegible when filed

TABLE-US-00011 TABLE 11 (GH5 BATCH 1 STATS) DNA Seed Size Null % Lipid Null Lipid Null Seq mg/seed Seed Size % DW % Lipid mg/seed Lipid Line ID DGAT1 ID # n = 6 p-Value n = 6 p-Value n = 6 p-Value WT Wild type 1.24 26.46 0.33 pBR2 Vector Control 1.18 25.44 0.30 TM Tm 1.38** 1.29 (0.0056) 27.24 26.48 (0.3299) 0.38 0.34 (0.0099) Zm ZmS 1.29 1.22 (0.1073) 27.68 26.95 (0.2524) 0.36 0.32 (0.0382) Zm ZmL 1.20 1.17 (0.6170) 26.06 26.47 (0.7288) 0.31 0.31 (0.8847) TM::Zm Tm::ZmL 1.16 1.20 (0.6159) 31.71** 27.42 (0.01025) 0.36 0.33 (0.2458) TM::Zm Tm::ZmL 1.25 1.16 (0.1419) 32.71** 26.37 (0.0029) 0.41** 0.31 (0.0057) ZmS::Tm ZmS::Tm 1.42* 1.23 (0.0325) 30.04** 26.09 (0.0006) 0.43*** 0.32 (0.0006) 1-23 ZmL R136 R145 R175 R65G 82 1.29 1.17 (0.0274) 27.03 26.65 (0.7625) 0.35 0.31 (0.1463) R66G 3-10 Tm Delta 68-94 84 1.15### 1.19 (0.4990) 25.73 25.92 (0.9449) 0.30 0.31 (0.7562) 3-17 1.21### 1.05 (0.0126) 26.95 22.85 (0.0883) 0.33*# 0.24 (0.0168) 4- Tm Delta 35-61 85 1.27## 1.14 (0.0159) 25.15# 25.20 (0.9724) 0.32## 0.28 (0.1746) 5-1 Tm Delta R25R26R27::ZmL 88 1.16 1.21 (0.3930) 25.52## 25.78 (0.8661) 0.30## 0.31 (0.5845) 5-2 1.32 1.22 (0.3007) 25.92## 26.69 (0.5484) 0.34# 0.33 (0.5523) 5-11 1.28 1.33 (0.4643) 24.94### 26.01 (0.3461) 0.32## 0.35 (0.2959) 6-14 ZmS Delta R27L28R29R30::Tm 89 1.33 1.12 (0.0038) 24.48### 20.59 (0.1847) 0.33## 0.24 (0.0506) 6-15 1.29# 1.25 (0.1860) 26.23## 23.89 (0.0366) 0.34### 0.30 (0.0117) 8-14 ZmS A10S G17S A36S G37S::Tm 87 1.31*# 1.20 (0.0703) 30.63** 26.01 (0.0010) 0.40*** 0.31 (0.0001) 8- 1.34* 1.15 (0.0022) 24.66### 25.93 (0.2769) 0.33### 0.30 (0.1115) Glasshouse 5-Batch 1 Plants (*= p-Value < 0.05, **=p-value <0.01, ***= p-value < 0.001, compared to WT and VC) (#= p-value < 0.05, ##= p-value < 0.01, compared to the parent) indicates data missing or illegible when filed

TABLE-US-00012 TABLE 12 (GH7 SUMMARY) TAG accumulation PROTEIN % FA compared to DGAT1 Name Description FIG. SEQ ID: in seed control TmS197A Control Tm DGAT 1, 2, 4, 94 25.08 N/A 9 ZmL Control ZmL DGAT 1, 2, 3 95 28.56 N/A ZmS Control ZmS DGAT 1, 2, 9 96 27.19 N/A TmA64S, G80S::ZmL disrupt AXXXA & GXXXG 6 101 30.59 +3.3 (% of Tm::ZmL) TmR25, R26, R27::ZmL disrupt di-arginines in Tm portion 5 103 27.40 7.5 (% of Tm::ZmL) ZmS R27, L28, R29, R30::TmS170A disrupt di-arginines in ZmS::Tm 5 104 27.83 14.5 (% of ZmS::Tm) TmA64S, G78R, G79R, G80R::ZmL disrupt AXXXA: added di-arginine in 7 105 31.48 +6.3 (% of Tm::ZmL Tm::ZmL) ZmSD12R, A14R, S44R, G45R, G46R::TmS170A disrupt AXXXA; added di-arginine in 8 106 32.62 +0.2 (% of ZmS::Tm ZmS::Tm) TmS6R, S7R, Q8R, S197A creates di-arginine in Tm 9 108 28.97 +15.5 (% of Tm) Tm::ZmL Control Tm::ZmL Chimera 6, 7 111 29.62 N/A ZmS::TmS170A Control ZmS::Tm Chimera 6 112 32.54 N/A

TABLE-US-00013 TABLE 13 (GH7 STATS) DNA Seed Size Null % Lipid Lipid Seq (mg/seed) Seed Size (% DW) Null % Lipid (mg/seed) Null Lipid Line ID DGAT1 ID# n = 6 (p-Value) n = 6 (p-Value) n = 6 (p-Value) None Wild-Type 1.17 27.16 0.32 pBR2 Vector Control 1.17 25.96 0.30 Tm#2 Tm 1.15 1.15 (0.8877) 25.08 23.91 (0.2692) 0.29 0.27 (0.4350) ZmS#1 ZmS 1.16 1.14 (0.7798) 27.19 27.28 (0.9549) 0.31 0.31 (0.8899) ZmL#9 ZmL 1.18 1.16 (0.7606) 28.56 26.17 (0.1805) 0.34 0.30 (0.1409) Tm::ZmL#5 Tm::ZmL 1.21 1.20 (0.7038) 27.57 21.22 (5.7E01) 0.33 0.25 (5.1E04) Tm::ZmL#13 Tm::ZmL 1.29** 1.12 (7.8E04) 31.66*** 25.43 (1.2E04)) 0.41*** 0.29 (9.0E05) ZmS::Tm#3 ZmS::Tm 1.32** 1.17 (0.0222) 32.93*** 27.92 (4.1E04) 0.44*** 0.33 (6.9E04) ZmS::Tm#4 ZmS::Tm 1.46*** 1.14 (1.0E05) 32.14*** 27.96 (6.3E04) 0.47*** 0.32 (2.0E05) 5-15 Tm Delta 88 1.18## 1.16 (0.5842) 28.10# 26.87 (0.3537) 0.33## 0.31 (0.1856) R25R26R27::ZmL 5-21 1.24 1.19 (0.4706) 26.70## 25.30 (0.4377) 0.33## 0.30 (0.1811) 6-23 ZmS Delta 89 1.20## 1.18 (0.5964) 27.79## 27.09 (0.5961) 0.33## 0.32 (0.5658) R27L28R29R30::Tm 6-27 1.29## 1.19 (0.0609) 27.86## 25.34 (0.0791) 0.36*## 0.30 (0.0169) 7 Tm A64S G80S::ZmL 86 1.23 1.18 (0.2181) 30.13**# 28.15 (0.0255) 0.37**# 0.33 (0.0419) 7 1.26* 1.16 (0.0537) 30.35 27.34 (0.1794) 0.38* 0.32 (0.0289) 7 1.29 1.18 (0.1179) 32.42** 26.93 (0.0062) 0.42*** 0.32 (1.3E04) 7 1.32** 1.21 (0.0440) 29.47 27.27 (0.1677) 0.39** 0.33 (0.0262) 9-20 Tm A64S G78R G79R 90 1.15# 1.16 (0.8637) 31.55** 27.49 (5.9E04) 0.36**# 0.32 (0.0113) G80R::ZmL 9-21 1.14# 1.18 (0.1567) 31.41* 27.30 (0.0323) 0.35 0.32 (0.1367) 10-3 ZmS D12R A14R S44R 91 1.20## 1.19 (0.7306) 31.28*** 26.60 (3.1E04) 0.38***## 0.32 (0.0170) G45R G45R::Tm 10-13 1.22## 1.21 (0.8347) 35.17** 28.13 (0.0016) 0.43** 0.34 (0.0265) 10-15 1.26## 1.09 (0.0354) 31.41** 27.44 (0.0020) 0.40**# 0.30 (0.0027) 12-8 Tm S6R S7R Q8R 93 1.32**# 1.16 (0.0053) 28.97# 24.39 (0.0151) 0.38**## 0.28 (0.0013) Glasshouse 7 Plants (*= p-Value < 0.05, **= p-value < 0.01, ***= p-value < 0.001, compared to WT and VC) (#=pvalue < 0.05, ##= p-value < 0.01, compared to the parent) indicates data missing or illegible when filed

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