ENHANCEMENT OF PRODUCTIVITY IN C3 PLANTS
20230279419 · 2023-09-07
Assignee
Inventors
Cpc classification
Y02A40/146
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
Vascular sheath tissue-specific expression of phytochrome B or variants thereof in C3 plants increases photosynthesis rate and/or introduces a carbon refixation mechanism. The heritable genetic material of a C.sub.3 plant cell is altered such that one copy of phytochrome B, or active variant or functional fragment thereof is expressed specifically in vascular sheath cells. Whole plants are regenerated from these genetically altered plant cells. Alternatively, a Crispr modification of a native phytochrome locus in a plant cell is used to insert a vascular sheath-specific regulatory element, e.g. promoter or enhancer element, so that phytochrome B is expressed in vascular sheath cells of a regenerated whole plant. Genetically altered whole plants have increased yield-related traits, e.g. increased seed yield, resulting from the enhancement of photosynthesis and/or introduction of a carbon refixation mechanism.
Claims
1. A method of increasing photosynthetic capacity of a C.sub.3 plant, the method comprising altering heritable genetic material of the C.sub.3 plant such that a gene of interest (GOI) is expressed in at least one vascular sheath cell of the C.sub.3 plant, and wherein the GOI is expressed under the control of a gene expression regulatory element active in the at least one vascular sheath cell of the C.sub.3 plant.
2. The method as claimed in claim 1, wherein the GOI encodes phytochrome B, an active variant thereof, or functional fragment thereof.
3. The method as claimed in claim 1 or claim 2, wherein the gene expression regulatory element is active specifically in the at least one vascular sheath cell of the C.sub.3 plant.
4. The method as claimed in any of claims 1 to 3, wherein the altering of the heritable genetic material comprises inserting at least one polynucleotide into the heritable genetic material of a cell of the C.sub.3 plant.
5. The method as claimed in any of claims 1 to 4, wherein the altering of the heritable genetic material comprises the use of a base editor; optionally a prime editor.
6. The method as claimed in any of claims 1 to 4, wherein the altering of the heritable genetic material comprises introducing a gene repair oligonucleobase (GRON)-mediated mutation into a target DNA sequence of the heritable genetic material of a cell of the C.sub.3 plant; optionally exposing the cell of the C.sub.3 plant to a DNA cutter and a GRON.
7. The method as claimed in claim 6, wherein the DNA cutter comprises a meganuclease, a transcription activator-like effector nuclease (TALEN), a zinc finger, an antibiotic, or a Cas protein.
8. The method as claimed in any of claims 1 to 3, wherein the altering of the heritable genetic material comprises using zinc finger nucleases (ZNFs) and/or transcription activator-like effector nucleases (TALENs) for site-specific homologous recombination of the heritable genetic material of a cell of the C.sub.3 plant.
9. The method as claimed in any of claims 1 to 3, wherein altering of the heritable genetic material comprises introducing a donor template to the heritable genetic material of a cell of the C.sub.3 plant using a viral vector.
10. The method as claimed in claim 9, wherein the viral vector comprises a protein expression vector; optionally wherein the protein expression vector comprises pQE or pET.
11. The method as claimed in any of claims 1 to 4, wherein the one or more polynucleotides comprises a polynucleotide encoding a CRISPR-Cas protein, optionally a guide RNA (gRNA), and a donor polynucleotide comprising a sequence of the gene expression regulatory element, wherein the gRNA directs the CRISPR-Cas protein to the locus of at least one copy of the GOI in the genome of a cell of the C.sub.3 plant, whereby the gene expression regulatory element is inserted so as to cause expression of the copy or copies of the GOI in the at least one vascular sheath cell of a plant regenerated from the cell.
12. The method as claimed in claim 11, wherein the CRISPR-Cas protein and the gRNA are preassembled to form ribonucleoproteins (RNPs); optionally wherein the RNPs are transfected into the cell.
13. The method as claimed in claim 11 or claim 12, wherein the RNPs are transfected into the cell using electroporation.
14. The method as claimed in any of claims 11 to 13, wherein the CRISPR-Cas protein comprises Cas9, Cas12a, or Cas 12b.
15. The method of any of claims 11 to 13, wherein the polynucleotide encoding a CRISPR-Cas protein is introduced via a plasmid.
16. The method as claimed in claim 4, wherein at least one polynucleotide comprises the expression regulatory element, a nucleotide sequence which encodes the GOI, and optionally a terminator; and a further polynucleotide encodes a CRISPR-Cas protein, and the further polynucleotide or an additional further polynucleotide optionally encodes a gRNA which directs the CRISPR-Cas protein to a desired locus in the genome of the C.sub.3 plant, such that an heterologous GOI under control of the vascular sheath regulatory element is inserted into the desired locus in the cell of the C.sub.3 plant.
17. The method as claimed in claim 16, wherein the at least one polynucleotide comprises from 5′ to 3′ the expression regulatory element, the nucleotide sequence encoding phytochrome B, or active variant thereof, or functional fragment thereof, and optionally the terminator.
18. The method as claimed in claim 4, wherein at least one polynucleotide comprises from 5′ to 3′, the expression regulatory element active specifically in at least some vascular sheath cells of a C.sub.3 plant, a nucleotide sequence which encodes a phytochrome B, or active variant thereof, or functional fragment thereof, such that the phytochrome B or active variant thereof or functional fragment thereof is inserted into the genome of the C.sub.3 plant.
19. An isolated DNA polynucleotide comprising from 5′ to 3′, an expression regulatory element active specifically in at least some vascular sheath cells of a C.sub.3 plant, a nucleotide sequence which encodes a phytochrome B or active variant thereof or a functional fragment thereof, and optionally a terminator.
20. The isolated DNA polynucleotide as claimed in claim 19, wherein the regulatory element comprises a promoter.
21. The isolated DNA polynucleotide as claimed in claim 19 or claim 20, wherein the promoter is a bundle sheath cell-specific promoter and/or a mestome sheath specific promoter or a promoter that is active throughout the vascular bundle.
22. The isolated DNA polynucleotide as claimed in any of claim 21, wherein the bundle sheath specific promoter or the mestome sheath specific promoter or the promoter that is active throughout the vascular bundle is a synthetic promoter; preferably comprised of a bundle sheath or a mestome sheath specific transcription factor binding element upstream of the promoter; optionally wherein there are two or more transcription factor binding elements.
23. The isolated DNA polynucleotide as claimed in any of claims 21 to 22, wherein the bundle sheath specific promoter or the mestome sheath specific promoter or the promoter that is active throughout the vascular bundle is selected from a minimal ZmUbi1 promoter, a NOS core promoter, a CHSA core promoter, and a minimal 35S promoter; preferably wherein the promoter has a nucleotide sequence of SEQ ID NO: 7, or SEQ ID NO: 10, or SEQ ID NO: 13 or a sequence of at least 80% identity therewith.
24. The isolated DNA polynucleotide as claimed in any of claims 21 to 234, wherein the bundle sheath specific promoter or mestome sheath specific promoter or the promoter that is active throughout the vascular bundle is derived from a bundle sheath specific gene or a mestome sheath specific gene, respectively.
25. The isolated DNA polynucleotide as claimed in any of claims 21 to 24, wherein the bundle sheath specific gene is from a plant species; including but not limited to: Arabidopsis thaliana MYB76, Flaveria trinervia GLDP, Arabidopsis thaliana SULTR2;2, Arabidopsis thaliana SCR, Arabidopsis thaliana SCRL23, Zoysia japonica PCK, Urochloa panicoides PCK1 and Hordeum vulgare PHT1;1.
26. The isolated DNA polynucleotide as claimed in any of claims 19 to 25, wherein the promoter is derived from non-plant organisms, such as a rice tungro bacilliform virus (RTBV) promoter.
27. The isolated DNA polynucleotide as claimed in any of claims 19 to 26, wherein the nucleotide sequence which encodes a phytochrome B is any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 8, or SEQ ID NO: 11, or a sequence of at least 65% identity with any of the sequences, or a functional fragment thereof; preferably a sequence of at least 70% identity with any of the sequences, or a functional fragment thereof; more preferably a sequence of at least 80% identity with any of the sequences, or a functional fragment thereof.
28. The isolated DNA polynucleotide as claimed in any of claims 19 to 27, wherein the functional fragment of the phytochrome B has phytochrome signalling activity, but lacks light sensitivity; preferably wherein the functional fragment consists of the PAS and GAF domains.
29. The isolated DNA polynucleotide as claimed in any of claims 19 to 28, wherein the phytochrome B is light insensitive; preferably YHB and the nucleotide sequence which encodes the phytochrome B is SEQ ID NO: 1, or a sequence of at least 70% identity therewith or a functional fragment thereof.
30. A plasmid comprising a DNA polynucleotide of any of claims 17 to 29, an origin of replication, a T-DNA right border repeat of a Ti or Ri plasmid; optionally additionally a left border repeat of a Ti or Ri plasmid, and at least one bacterial selectable marker.
31. The plasmid as claimed in claim 30, further comprising an element selected from one or more of: an enhancer, a plant selectable marker, a multicloning site, or a recombination site.
32. A Ti or Ri plasmid comprising the DNA polynucleotide of any of claims 17 to 29.
33. A composition for transformation of plant cells comprising the isolated DNA polynucleotide of any of claims 19 to 29, or a plasmid of any of claims 30 to 32; optionally comprising microparticles coated with said DNA polynucleotide or said plasmid.
34. A bacterium comprising the isolated DNA polynucleotide of any of claims 19 to 29, or a plasmid of any of claims 30 to 32; optionally wherein the bacterium is E coli.
35. A bacterium comprising a plasmid of any of claims 30 to 32; preferably wherein the bacterium is Agrobacterium sp.; more preferably A. tumefaciens.
36. A plant which carries out C.sub.3 photosynthesis in at least a part thereof, the plant comprising the isolated DNA polynucleotide of any of claims 19 to 29 stably integrated into the genome thereof; preferably heritably integrated into the genome thereof.
37. A plant which carries out C.sub.3 photosynthesis in at least a part thereof, wherein the plant has an additional at least one additional copy of a phytochrome B gene or functional fragment thereof, and wherein the plant is genetically altered compared to a genetically equivalent unaltered plant, wherein an expression regulatory element(s) of at least one copy of a phytochrome B gene or functional fragment thereof in the altered plant causes an additional at least one phytochrome B gene, or functional fragment thereof, expression specifically in at least some bundle sheath cells and/or mestome sheath cells and/or vascular bundle of the plant compared to the unaltered plant.
38. The plant as claimed in claim 37, wherein the expression regulatory element is a promoter which is active specifically in the at least some vascular sheath cells of a C.sub.3 plant.
39. The plant as claimed in claim 37 or claim 38, wherein the coding sequence of the additional at least one phytochrome B gene is the same as a native phytochrome B gene or genes in the plant.
40. The plant as claimed in claim 37 or claim 38, wherein the additional at least one phytochrome B gene is different to the native phytochrome B gene or genes in the plant; optionally wherein the phytochrome B or active variant or functional fragment thereof is defined in any of claims 27 to 30.
41. The plant as claimed in any of claims 37 to 40 obtained by a process of CRISPR-Cas protein genetic modification.
42. The plant as claimed in any of claims 37 to 41, wherein the genetic modification is heritably stable.
43. The plant as claimed in any of claims 36 to 42 which is a C.sub.3 plant; preferably a crop plant, e.g. a cereal crop plant, an oilseed crop plant or a legume.
44. The plant as claimed in any of claims 37 to 43, wherein the phytochrome B has an amino acid sequence of any of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9, or SEQ ID NO: 12, or a sequence of at least 65% identity with any of the sequences or a functional fragment thereof; preferably a sequence of at least 70% identity with any of the sequences or a functional fragment thereof; more preferably a sequence of at least 80% identity with any of the sequences or a functional fragment thereof.
45. The plant as claimed in any of claims 37 to 44, wherein the functional fragment of the phytochrome B has phytochrome signalling activity, but lacks light sensitivity; preferably wherein the fragment consists of the PAS and GAF domains.
46. The plant as claimed in any of claims 37 to 45, wherein the phytochrome B is a light insensitive sequence variant or functional fragment thereof; preferably YHB with an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 12 or a sequence of at least 70% identity therewith or functional fragment thereof.
47. The plant as claimed in any of claims 37 to 46, wherein the chloroplasts present in vascular sheath cells are developmentally enhanced, in terms of size or photosynthetic capacity, compared to chloroplasts in equivalent vascular sheath cells of control unmodified plants grown under the same conditions for the same period of time.
48. The plant as claimed in any of claims 36 to 47, wherein photosynthesis is enhanced compared to a control unmodified plant grown under the same conditions.
49. The plant as claimed in any of claims 36 to 48, wherein leaf photosynthetic efficiency is greater than in the equivalent leaf or leaves of a control unmodified plant grown under the same conditions.
50. The plant as claimed in any of claims 36 to 49, wherein water use efficiency is greater than in a control unmodified plant grown under the same conditions.
51. The plant as claimed in any of claims 36 to 49, wherein the enhanced photosynthesis results in one or more of the following traits: enhanced growth rate, reduced time to flowering, faster maturation, enhanced seed yield, enhanced biomass, increased plant height, and enhanced leaf canopy area, when compared to a control unmodified plant grown under the same conditions.
52. A plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus culture, pollen grain or seed, derived or obtained from the plant of any of claims 36 to 51.
53. A processed plant product obtained from the plant of any of claims 36 to 49 or the plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus culture, pollen grain or seed of claim 52; optionally wherein the processed product comprises a detectable nucleic acid sequence of (i) a phytochrome B or active fragment thereof downstream of a gene expression regulatory element active specifically in at least some of the vascular sheath cells of a plant, or (ii) at least a portion of a polynucleotide of any of claims 19 to 29.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0103] Embodiments of the invention are further described hereinafter with reference to the Examples and accompanying Drawings, in which:
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DETAILED DESCRIPTION
[0132] In the following passages, different aspects of the invention are explained in more detail. Each aspect explained or defined may be combined with any other aspect or aspects, unless explicitly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
[0133] Conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology, and bioinformatics for use in employing the present invention are all readily known and available to a person of average skill in the art. Specific techniques are explained fully in the literature.
[0134] The inventors have generated a system comprised of a vascular sheath specific regulator of gene expression and a regulator of chloroplast activation that together increase photosynthesis and yield related traits. The inventors have demonstrated that this technology is broadly applicable to C.sub.3 plants by showing that it works in both eudicots (for example Arabidopsis thaliana) and in monocotyledons (for example wheat). The inventors have shown that this technology works irrespective of the species origin of the PHYB gene and irrespective of the vascular sheath promoter that is used. A key aspect of this invention is that PHYB, active variants, or functional fragments thereof are expressed in vascular sheath cells (which may include other cells of the vasculature or vasculature sheath extensions as hereinbefore defined) and not leaf mesophyll cells. Transgenic plants containing this system surprisingly and advantageously do not display developmental defects associated with YHB or PHYB overexpression. The transgenic plants undergo normal photomorphogenesis (no dwarfing, reduced apical dominance, delayed flowering, or decreased water use efficiency), have the same leaf thickness as control plants, and flower normally. However, these plants have higher photosynthetic rates, grow faster, have enhanced water use efficiency, mature to flowering stage sooner, produce more fruiting structures and produce significantly more seeds. The effects are dramatic, with yield increases upward of 30% in greenhouse trials.
[0135] The inventors have achieved what has not hitherto been possible, which is a manipulation of PHYB expression in planta to improve each of photosynthesis, plant growth and yield without disrupting plant development. The unexpected finding of the inventors is that combined improvements are achievable separately from the disruptive aspects of PHYB expression by expressing PHYB additionally only in the vascular bundle or component cells thereof of plants.
[0136] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
[0137] The terms “altered”, “changed” and “modified” may be used interchangeably herein. A control plant as used herein is a plant which has not been modified. Accordingly, the control plant has not been genetically modified to alter either expression of a polynucleotide of the invention as described herein. The control plant may be a wild type (WT) plant. Even if a plant were transgenic, but not in respect of the polynucleotide of the invention then it could function as a control plant. The WT or control need not be too specific, so long as it may provide a reliable reference against which the vascular bundle sheath expression of PHYB can be compared against in a modified plant material.
[0138] The terms “increase”, “improve” or “enhance” are used interchangeably herein.
[0139] The term “specific” as used herein may be considered equivalent to “exclusive” or strongly preferential.
Vascular Sheath and Vascular Sheath Cells
[0140] In C.sub.3 plants (most crops), the cells surrounding the leaf veins (i.e. the vascular sheath) are known as bundle sheath cells. In Dicotyledonous plants the bundle sheath is made up of a single layer of cells that encircle the vein, while in Monocotyledonous plants the bundle sheath can be made up of a single layer of cells or two concentric layers of cells (A. Fahn, Plant Anatomy Pergamon Press 1995). When there are two layers of cells the outer cell layer is commonly referred to as the bundle sheath and the inner cell layer is commonly referred to as the mestome sheath (A. Fahn, Plant Anatomy Pergamon press 1995). When two layers are present, both layers together make up the bundle sheath (A. Fahn, Plant Anatomy Pergamon press 1995). Thus, bundle sheath is a term used to describe either a single layer of bundle sheath cells, or a two-layer system comprised of an outer bundle sheath layer and an inner mestome sheath layer. As used throughout this specification, the terms “bundle sheath”, “bundle sheath cells”, “vascular sheath”, or “vascular sheath cells” may be used interchangeably, encompassing all types of bundle sheath cell layers unless the context clearly dictates otherwise. Bundle sheath cell layers (i.e. the single bundle sheath layer, or the outer bundle sheath and the inner mestome sheath) may contain chloroplasts. The number of chloroplasts in these bundle sheath layers may be the same or fewer than in mesophyll cells and in some cases bundle sheath cells may be devoid of chloroplasts. Furthermore, if they are present in Ca plants the size of the chloroplasts in bundle sheath cell layers is generally much smaller than in mesophyll cells (A. Fahn, Plant Anatomy: Pergamon Press (1995)). The bundle sheath cells encircle veins so they are ideally situated to ensure good water supply and for loading sugars into veins for distribution to growing plant structures.
Bundle Sheath Specific Expression
[0141] The term “specific” when used in relation to gene expression describes the biological phenomenon of enhanced gene expression within a limited subset of cell types within a plant. The term “bundle sheath specific expression” is used synonymously with “vascular sheath specific expression” to describe the phenomenon whereby the gene being expressed is expressed to a substantially higher level in bundle sheath cells than in the surrounding mesophyll cells within the leaf. This does not preclude the gene from being expressed in other non-mesophyll cells within the leaf or within the plant, just that the level of expression in the bundle sheath is high and the level of expression in the leaf mesophyll is low. The gene may also be expressed in other vascular cell types in addition to the vascular sheath cells. These cell types include some or all of the cells of the vascular bundle such as xylem and/or phloem and associated cell types. The gene may also be expressed in non-vascular cells such as guard cells, vascular sheath extension cells, bundle sheath extension cells, epidermal cells, paraveinal mesophyll cells (which are an extension of the bundle sheath and not mesophyll cells); or elsewhere in the plant not being leaf tissue, e.g. flowers, fruits, roots, stems. The key determinant is that expression is activated in the bundle sheath and not the mesophyll.
Phytochrome Proteins for Use in the Invention
[0142] “PHYB” (Phytochrome B) as hereinbefore defined is a regulatory photoreceptor. As shown in
[0143] PHYB from any plant species may be used in embodiments of the invention, whether that PHYB protein, active variant or functional fragment thereof is expressed in the same plant (homologous expression) or in a different plant (heterologous expression).
[0144] The term “active variant” and/or “functional fragment” as used herein in relation to PHYB refers to a variant or fragment of a PHYB gene or peptide sequence which retains the signal activating function of PHYB. An active variant also comprises a variant of the gene of interest encoding a peptide which has sequence alterations that do not affect the signal activating function of the resulting protein, for example in non-conserved residues.
[0145] The invention also includes functional fragments of PHYB and any variants of a PHYB protein, for use in accordance with any aspect of the invention.
Sequence Identities and Orthology
[0146] The term “variant” as used herein used in relation to a given PHYB protein from a plant species, or a functional fragment thereof, means any PHYB ortholog of differing amino acid sequence from other plant species. Such variants may be expressed in terms of a percentage identity to any of the reference nucleotide reference sequences disclosed herein (i.e. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 8 or SEQ ID NO: 11). In terms of percentage identity to an amino acid reference sequence, such as SEQ ID NO:4, a variant of PHYB may have, in increasing order of preference, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% overall sequence identity to that amino acid reference sequence.
[0147] The following table provides a non-exhaustive list of accession numbers for PHYB orthologs in 50 commercially grown plant species. Orthologs of the Arabidopsis PHYB gene were found in the NCBI publicly available sequence database. More than one PHYB accession was found for many species, indicative that PHYB has duplicated in different plant lineages; many of these paralogues arose as result of whole genome duplications. For each species, one representative, full-length, orthologous amino acid sequence was compared to Arabidopsis and wheat PHYB orthologs (AT2G18790.1 and Traes_4 AS_1F3163292.1, respectively), and percentage identity to each was quantified using multiple sequence alignments generated by Clustal Omega 2.1 with default parameters (generating alignments with mBed-like clustering guide trees and hidden Markov models using HHalign). The median PHYB percentage identity of these orthologs relative to Arabidopsis or wheat PHYB orthologs was ˜75%. There were several examples where less than 75% identity was shared between a PHYB ortholog in a given species and both the Arabidopsis and wheat orthologs. For example, Daucus carota (carrot) and Solanum lycopersicum (tomato) PHYB orthologs were >70% identical to either the Arabidopsis or wheat PHYB orthologs. Likewise, PHYB orthologs in more distantly related gymnosperm species such as Picea abies and sitchensis (spruces) were just 66-68% identical to either Arabidopsis or wheat PHYB proteins at the amino acid level. Recently duplicated PHYB paralogues fall within the PHYB similarity range indicated by the table, but more distantly related phytochromes do not. For example, a multiple sequence alignment of Arabidopsis PHYB [SEQ ID NO: 5], PHYD [SEQ ID NO: 9] and PHYA (NCBI accession NP_001322907.1), amino acids indicated that while PHYB and paralogous PHYD share 81.98% identity, PHYA shares just 52.35% identity with PHYB, and 52.20% with PHYD.
TABLE-US-00001 Arabidopsis Wheat Species name Accession identity % identity % Arachis hypogaea XP_016197860.1, XP_025694495.1 76.95 75.00 Beta vulgaris XP_010671734.1, XP_010671735.1 76.40 72.94 Brassica carinata KAG2315801.1, KAG2294593.1, 72.10 72.05 KAG2271748.1, KAG2306049.1 Brassica napus XP_013741043.1, XP_022555281.1, 90.25 71.37 CAF2100974.1, XP_022575358.1, XP_022559055.1, CAF1933771.1 Brassica oleracea XP_013585553.1, XP_013628810.1 92.53 71.30 Camelina Sativa XP_010489599 94.93 71.84 Camelina sinensis THG18270.1, THG09607.1, KAF5941548.1, 77.51 75.62 XP_028060883.1, KAF5950816.1, XP_028079860.1 Cannabis sativa XP_030506649.1, KAF4358918.1, 76.21 75.35 XP_030506648.1 Capsicum annuum XP_016581708.1, PHT94245.1 78.11 76.08 Chenopodium XP_021730340.1, XP_021774295.1 76.32 74.20 quinoa Cicer arietinum XP_004486544.1 75.09 74.26 Citrus × sinensis KDO71942.1 78.50 75.29 Coffea arabica XP_027120998.1, XP_027115886.1 77.53 75.53 Corchorus OMO53500.1 79.28 75.98 capsularis Cucumis sativus XP_004134246.2 78.48 75.91 Cucurbita pepo XP_023526818.1, XP_023540778.1, 73.74 73.07 XP_023540779.1 Daucus carota KZM85596.1 71.67 71.54 Elaeis guineensis XP_010921452.1, XP_010938231.2 75.09 72.02 Eucalyptus grandis KCW87973.1 77.76 77.67 Fragaria vesca XP_004295077.1 77.44 76.19 Glycine max NP_001240097.1, XP_006597696.1 76.19 74.80 Gossypium XP_016700852.1, XP_016677281.1 78.81 73.99 hirsutum 72.59 72.04 Helianthus annuus XP_022022035.1, XP_021987936.1 Hevea brasiliensis KAF2312734.1, XP_021668699.1 78.72 77.17 Hordeum vulgare KAE8810763.1 71.60 99.14 Jatropha curcas XP_012084068.1, XP_012084071.1, 78.68 76.53 Juglans regia XP_018805735.2 78.69 76.04 Lactuca sativa XP_023763453.1 75.24 73.48 Malus domestica XP_008368332.2, RXH80138.1 76.30 74.46 Manihot esculenta XP_021607077.1 78.48 77.17 Medicago sativa ACU21557.1, ACU21558.1 75.22 73.05 Musa acuminata AOA13605.1 71.12 77.51 Nicotiana tabacum XP_016456908.1, XP_016458771.1, 73.22 71.37 XP_016441820.1, ALN38804.1, P29130.2, XP_016454809.1 Olea europaea XP_022851738.1 77.36 75.13 Oryza sativa XP_015631282.1 74.82 93.14 Picea abies AJE63445.1 67.23 67.79 Picea sitchensis ACN40636.1 66.87 67.35 Pisum sativum AAF14344.1 75.16 73.01 Phaseolus vulgaris XP_007147366.1 76.02 75.66 Populus AAG25725.1 71.34 75.15 trichocarpa 79.25 76.65 Prunus persica XP_007227356.1 Ricinus communis XP_002519230.1 77.98 76.16 Sesamum indicum XP_011100755.1, XP_011071377.1, 75.53 74.17 XP_020555118.1, Solanum NP_001317100.1, NP_001293131.1 71.70 70.74 lycopersicum Solanum XP_006355734.1, XP_006358209.1 71.98 70.83 tuberosum Spinacia oleracea KNA10134.1, AAA17825.1, XP_021862546.1, 75.29 73.23 KNA10706.1, XP_021858666.1 Theobroma cacao EOY06733.1 79.70 76.25 Triticum aestivum KAF7042404.1, KAF7054102.1, 72.08 100.00 KAF7049239.1, AAX76779.1, KAF7054101.1 KAF7042406.1 Vigna unguiculata QCD77474.1, XP_027931104.1, QCE13780.1 76.88 76.84 Vitis vinifera CBI22877.3 78.19 77.09
[0148] The overall sequence identity may be determined using a global alignment algorithm known in the art, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys).
[0149] More examples of suitable PHYB genes can also be readily identified by a skilled person through ortholog finding programs such as OrthoFinder (Emms and Kelly. Genome Biology 2019. 20: 238). The function of such genes can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant.
[0150]
[0151] Where the bundle sheath cell specific promoter is concerned, all variants and orthologs of these are included in the invention. Where there is a reference nucleotide sequence for such a promoter, then such variants and orthologs include nucleotide sequences of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% overall sequence identity to the reference promoter sequence.
[0152] The degree of sequence identity of any polynucleotides described in connection with the invention may, instead of being expressed as a percentage identity to reference sequence, may instead be defined in terms of hybridization to a polynucleotide of any of the reference sequences [SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 8 or SEQ ID NO: 11] disclosed herein. Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
[0153] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na.sup.+ ion, typically about 0.01 to 1.0 M Na.sup.+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
[0154] PHYB is a highly conserved protein and its functions are highly conserved throughout all vascular plants. This has repeatedly been demonstrated by either increasing expression of native PHYB proteins, expressing exogenous PHYB proteins from other plant species, or knocking out native PHYB genes.
Functional Fragments
[0155] PHYB proteins are typically comprised of 7 easily recognisable protein domains. These comprise three Per-Arnt-Sim (PAS) domains (either PF08446 and/or PF00989), a GAF domain (PF01590), a PHY domain (PF00360), a His Kinase A phospho-acceptor domain (PF00512), and a GHKL domain (PF02518).
[0156] Though these protein domains are highly conserved, truncated versions of the PHYB gene can also function to initiate PHYB signalling. For example, Oka et al. (2004) “Functional Analysis of a 450-Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis” Plant Cell. 16(8): 2104-2116 showed that a 450-amino acid fragment of PHYB, which lacks the PHY domain (PF00360), the His Kinase A phospho-acceptor domain (PF00512), and the GHKL domain (PF02518), could initiate PHYB signal transduction when targeted to the nucleus. Thus, functional fragments of PHYB can provide PHYB signalling and such functional fragments are included in this invention.
Vascular Sheath (i.e. Vascular Bundle, Bundle Sheath and/or Mestome Sheath) Promoters
[0157] A person of skill in the art is well aware of many vascular bundle, vascular sheath, bundle sheath or mestome sheath specific promoters.
[0158] There are such promoters that have been isolated from several different species that a person of average skill in the art will expect to work across diverse plant species; five such examples are illustrated in
[0159] There are also bundle sheath cell promoters described in the literature for Monocots. For example, Nomura et al (2005) Plant Cell Physiology. 46(5): 754-61 which shows that Zoysia japonica PCK promoter works to drive expression in rice bundle sheath. Similarly, the Urochloa panicoides PCK1 promoter directs bundle sheath expression of reporter genes in rice and maize (Suzuki and Burnell. Plant Science. 2003 165(3):603-611). Also, Kloti et al (1999) Plant Molecular Biology 40(2): 249-266 shows rice tungro bacilliform virus promoter working in vascular bundles and other vascular cells. This promoter works in both Monocots (rice) and Dicots (tobacco) to drive expression in vascular bundles, despite these species having diverged ˜160 million years ago. Petruccelli et al 2001 PNAS 98(13) 7635-7640. Also, Schünmann et al (2004) Plant Physiol. 136(4): 4205-4214 which shows rice bundle sheath expression using the barley Pht1;1 promoter (see
Recombinant Constructs
[0160] Any suitable cloning system may be used. For example, Golden Gate modular cloning system described in Weber, E. et al (2011) PLoS ONE doi.org/10.1371/journal.pone.0016765. Otherwise genetic constructs can be fully synthesized de novo, or assembled using other molecular biology approaches.
[0161] PHYB, active variant or functional fragment sequences of the invention may be operably linked for transcription and expression, whether directly or indirectly to the vascular sheath promoter(s) employed in the invention.
Plant Transformation
[0162] Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a plant. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, can also be produced via Agrobacterium tumefaciens mediated transformation. Such routine methods are also used to introduce genome editing proteins such as CRISPR Cas nucleases, base editors and other genome editing nucleases. Collectively or in isolation these genome editing nucleases can be used to edit native PHYB gene sequences to introduce vascular sheath promoter sequences, vascular sheath regulatory elements, or convert native PHYB sequences to active variants or functional fragments.
[0163] Transformation methods are well known in the art. Thus, according to the various aspects of the invention, a polynucleotide of the invention is introduced into a plant and expressed as a transgene. The nucleic acid sequence is introduced into said plant through a process called transformation. The term “introduction” or “transformation” is used to encompass “transformation”, “transfection”, “transduction” and all such methods that result in the transfer of an exogenous polynucleotide into a host plant cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host plant genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner well known in the art.
[0164] To select transformed plants, plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis or whole genome sequencing, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis and/or RNA-Seq, each being well known in the art.
[0165] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0166] Altered plants in accordance with the invention advantageously provide better yield characteristics. Yield characteristics, also known as yield traits may comprise one or more of the following non-limitative list of features: yield, biomass, seed yield, seed/grain size, starch content of grain, early vigour, greenness index, increased growth rate, increased water use efficiency, increased resource use efficiency. The term “yield” in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and growth period, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres. The term “yield” of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds and tubers) of that plant. Thus, according to the invention, yield comprises one or more of, and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased viability/germination efficiency, increased number or size of seeds/capsules/pods, increased growth or increased branching, for example inflorescences with more branches, increased biomass, increased grain fill, increase tuber biomass. Preferably, increased yield comprises an increased number of grains/seeds/capsules/pods, increased biomass, increased growth, increased number of floral organs, increased floral branching or increased tubers. Yield is usually measured relative to a control plant.
[0167] Preferably, a plant in accordance with the invention is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal, an oilseed plant or a legume.
[0168] A plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a Monocot or a eudicot plant.
Plants and Crop Species of Interest
[0169] The term “plant” as used herein encompasses anything which is capable of undergoing photosynthesis or capable of producing structures which may undergo photosynthesis, along with parts and subcomponents thereof. Common features which undergo or are capable of undergoing photosynthesis include seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues, and organs. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores.
[0170] A Monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, oat, rye, millet, maize, or a crop such as garlic, onion, leek, yam, pineapple or banana.
[0171] A eudicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from buckwheat, lettuce, sunflower, Arabidopsis, broccoli, spinach, canola, water melon, squash, cabbage, tomato, potato, sweet potato, capsicum, cucumber, courgette, aubergine, carrot, olive, cow pea, hops, raspberry, blackberry, blueberry, almond, walnut, tobacco, cotton, cassava, peanut, sesame, rubber, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine, bell pepper, chilli, flax, camelina, cannabis/hemp, sugar beet, quinoa, citrus, cacao, tea or coffee species. In one embodiment, the plant is oilseed rape (canola).
[0172] Also included are biofuel and bioenergy crops such as rape/canola, jute, jatropha, oil palm, linseed, lupin and willow, eucalyptus, poplar, poplar hybrids, or gymnosperms, such as loblolly pine, Norway spruce or sitka spruce. Also included are crops for silage, grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. hemp, cotton, flax), building materials (e.g. pine, oak, rubber), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
EXAMPLES
Example 1: Transformation of Arabidopsis thaliana with a Genetic Construct for Bundle Sheath Expression of YHB
[0173] A genetic construct was assembled the Golden Gate cloning system and the resulting plasmid is shown in in
[0174] As noted above, six nucleotides within the YHB coding sequence [SEQ ID NO: 1] were altered to remove restriction sites prior to gene synthesis, but the amino acid sequence [SEQ ID NO: 4] was unchanged. The DHS vascular bundle specific promoter was used. The DHS promoter sequence [SEQ ID NO: 7] was cloned out of a plasmid first described in Knerova et al., (2018) “A single cis-element that controls cell-type specific expression in Arabidopsis” bioRXiv.). The level two vector contained a herbicide (Basta) resistance cassette and the domesticated YHB genetic sequence downstream of the vascular bundle promoter. Once assembled, the vector was introduced into Agrobacterium tumefasciens (strain AGL-1) cells by electroporation. Agrobacterium colonies that carried the construct were selected on LB plates and cultured on YEB media.
[0175] Arabidopsis thaliana (Columbia ecotype) plants propagated in the University of Oxford Department of Plant Sciences were selected at the point of floral emergence (approximately 4 weeks old). Some individuals were set aside and propagated to generate wildtype progeny for use as control plants. The rest were transformed by floral dipping. Once dipped, individuals were grouped into batches of plants to partition seed into independent transformation events. Seeds were sterilised using ethanol and Triton and stratified for three days in a cold room prior to germination. Following germination on soil, T1 plants were screened for transgene insertion by the application of Basta herbicide every other day for one week. T1 transformant plants were transplanted to larger pots and grown to collect T2 seed. T2 seeds were germinated on MS media containing Basta to conduct segregation analysis. Single insertion lines were identified as those that exhibited 75% survival rate on selective media, indicative of a single segregating allele. RNA was extracted from these plants to confirm expression of the YHB transgene in each line. Primers were designed and tested to confirm that they specifically amplified YHB and not native Arabidopsis thaliana Phytochrome B. Three lines representing independent transformation events were selected based on segregation and semi-quantitative PCR results and individual plants from each line were transferred onto soil at 12 days after germination. These were grown alongside wildtype plants in a greenhouse under long day conditions and watered regularly.
[0176] All phenotypic analyses in subsequent examples were conducted on all three lines unless otherwise stated, from which comparisons between one transgenic line (annotated as ‘C12’ in the figures) and control plants are displayed in subsequent plots. All error bars indicate 95% confidence intervals and t-tests were used to indicated significance (‘*’) or not (‘n.s.’) at p<0.05.
[0177]
[0178] Leaf thickness was measured magnetically using a Multispeq V1.0 device. The same leaf (9) was identified for n=10, 5.5 week old plants, measured in three spots near the centre of the leaf and the median value was used for each replicate. As shown in
Example 2: Expression of YHB in Bundle Sheath Cells Enhances Photosynthetic Capacity in Arabidopsis thaliana
[0179] To demonstrate photosynthetic enhancement in transgenic plants compared to non-transformed controls, the plants generated in Example 1 were analysed by gas exchange measurements using a LICOR 6800 device equipped with a multiphase fluorometer head. What is being measured is the amount of carbon that control plants and transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB could fix given a determined level of ambient carbon dioxide around the leaf (i.e., their photosynthetic rate). Arabidopsis plants growing in a greenhouse were analysed by clamping a leaf in the gas exchange chamber and controlling environmental conditions at 23° C., 65% relative humidity, flow was set to 500 μmol s.sup.−1 and fan speed to 10,000 rpm. The same leaf was used for each plant and all plants were measured between 32 to 35 days old, with a mixture of transgenic lines and control plants tested each day between 10 am to 3 pm. Plants were adapted to 400 μmol mol.sup.−1 CO.sub.2 and 1500 μmol m.sup.−1 s.sup.−1 light (with a mixture of 90% red and 10% blue) for 15 minutes, then the carbon dioxide concentration was decreased stepwise from 400 μmol mol.sup.−1 to 10 μmol mol.sup.−1 then raised back up to 400 μmol mol.sup.−1 before increasing to a maximum of 2000 μmol mol.sup.−1. Plants were given 5 minutes to acclimate to each new CO.sub.2 concentration then carbon assimilation was measured. Plant leaf area was measured to adjust for slight differences in leaf sizes. The resulting A/Ci curves (
[0180] Transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB consistently outperformed controls until carbon dioxide was too low to facilitate photosynthesis in either genotype. The initial slope of these curves show that these transgenic plants have a greater carboxylation efficiency, and the plateauing phase (towards the highest values of carbon dioxide concentrations tested) demonstrate that the maximum photosynthetic rate of these transgenic plants was also increased. Just considering ambient carbon dioxide levels (as would be encountered by crops in fields), what the experiment shows is that these transgenic plants fix significantly more carbon out of the surrounding air than control plants. Thus, unlike previous studies that have manipulated the expression of PHYB, the invention described here substantially improves leaf level photosynthetic rate.
Example 3: Expression of YHB in Bundle Sheath Cells Enhances Water Use Efficiency in Arabidopsis thaliana
[0181] To demonstrate that transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB showed no negative effects on water use efficiency when compared to control plants, stomatal conductance was measured. This is important, because previous attempts by others to modulate PHYB/YHB expression (e.g., Rao et al., (2011)), have resulted in large increases in water consumption. Stomatal conductance was measured at 400 μmol mol.sup.−1 CO.sub.2, 65% relative humidity, 23° C. temperature with flow set to 500 μmol s.sup.−1 and a fan speed of 10000 rpm. Importantly, there was no increase in stomatal conductance in transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB when compared to controls (
[0182] By dividing carbon assimilation rate by stomatal conductance, instantaneous water use efficiency was calculated (carbon captured per water flux). This demonstrated that while photosynthetic rate was at a maximum (as shown in
Example 4: Expression of YHB in Bundle Sheath Cells Enhances Chloroplast Development in Bundle Sheath Cells but not Mesophyll Cells in Arabidopsis thaliana
[0183] In Arabidopsis leaves, mesophyll cells contain fully developed, photosynthetically active chloroplasts whilst bundle sheath cells contain smaller chloroplasts with reduced photosynthetic capacity. To demonstrate that chloroplasts in bundle sheath cells of transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB were enhanced compared to control plants, the plants were subject to confocal microscopy and electron microscopy analysis. Equivalent leaves (leaf 6) were harvested from transgenic and control Arabidopsis plants 25 days after germination (as generated in Example 1). The lower epidermis was peeled away, and leaves were fixed in formaldehyde. Once fixed, paradermal sections were placed on a slide and imaged using a confocal microscope. To allocate chloroplasts to particular cell types, both chlorophyll and lignin autofluorescence were imaged in cells surrounding veins. Lignin and chlorophyll autofluorescence were detected by excitation with 458 nm and 633 nm lasers and emission spectra recorded between 465-599 nm and 650-750 nm, respectively. Z stacks were taken around veins to capture mesophyll and bundle sheath cells from a total of five leaves per genotype. For each leaf, five mesophyll and five bundle sheath cells were identified from at least two different images and the chloroplast area plans of the five largest chloroplasts in each cell (i.e. positioned parallel to the Z plane) were calculated using ImageJ. Hence the average chloroplast size per genotype was calculated by measuring a total of 125 chloroplasts across 25 cells distributed between five different plants. Additionally, transmission electron micrographs were obtained by sampling plants at the same time of day (11 am). Tissues were stained and embedded in resin, then thin sections were cut using an ultramicrotome diamond knife. Images were taken on a Siemens transmission electron microscope.
[0184] As shown in
[0185] Electron microscopy analysis of transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB revealed that bundle sheath chloroplasts were equivalent to mesophyll cell chloroplasts in terms of size and organisation of photosynthetic apparatus (
Example 5: Expression of YHB in Bundle Sheath Cells Enhances Refixation of Respired Carbon Dioxide in Arabidopsis thaliana
[0186] Whilst it is the photosynthetic cells that fix CO.sub.2 into sugars, every single plant cell respires, consuming sugars and releasing CO.sub.2. Respiration by cells in the veins releases CO.sub.2, which normally diffuses out of the veins, through the encircling bundle sheath cells, and into the intercellular space where it is either taken back up by the mesophyll or lost from the leaf through stomata. Since transgenic plants showed increased capacity to fix carbon, measurements were made to see if this was in part due to refixation of respired CO.sub.2 by the veins, back into sugars to fuel more growth.
[0187] Whilst the CO.sub.2 in the air is comprised of a mixture of carbon-12 and carbon-13 isotopes, the carbon in plant tissues have a signature of less carbon-13 relative to carbon-12 than the air. This is because the enzyme that fixes carbon out of the air, rubisco for C.sub.3 species, discriminates against the heavier carbon-13 isotope, resulting in a negative δ13C ratio as measured by dry matter carbon isotope analysis. If the transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB (Example 4) were refixing respired carbon (i.e. carbon that had already been fixed once before), then the carbon that ended up in the leaves would be subject to multiple rounds of rubisco mediated fixation and thus multiple rounds of discrimination. Thus, if enhanced refixation of respired CO.sub.2 was occurring in the transgenic plants containing the genetic vector for bundle sheath expression of YHB then one would expect to see a signature of this in carbon isotope analysis. Specifically, one would expect to see a more negative 613C than in equivalent tissues from control plants.
[0188] At 36 days old (plants as generated in Example 1) equivalent leaves (leaf 9) were flash frozen in liquid Nitrogen and freeze dried in a lyophiliser for 4 days. Approximately 1 mg of dry leaf powder was weighed out per 6 samples per genotype (two genotypes were tested, one transgenic line and one control group) and subject to stable isotope analysis. This demonstrated that transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB had a significantly more negative 613C, indicating that respired CO.sub.2 was a significant carbon source in these plants (see
[0189] Normal C.sub.3 plants fix the carbon that diffuses into the leaf intercellular space into sugars. Rubisco in photoactivated mesophyll cells fixes the carbon, which is then exported to the vasculature as sugar. These sugars are respired to fuel plant growth throughout the plant. This releases carbon dioxide, which diffuses back out of the veins, around/through bundle sheath cells and out of the leaf.
Example 6: Expression of YHB in Bundle Sheath Cells Enhances Plant Growth in Arabidopsis thaliana
[0190] Given that transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB had a higher photosynthetic capacity than control plants (Examples 2-5) it was determined how this increase in net carbon uptake might fuel increased plant growth. Photographs were taken from above of trays of 15 plants (as generated in Example 1) at days 14 and 21 after germination. Images were analysed in ImageJ to calculate total rosette area per plant. This showed that, consistent with the increase in photosynthetic rate, the transgenic plants of the invention grew faster than control plants over this time window (
[0191] Bolts are the flowering structures of Arabidopsis. Once plants have obtained enough resources during vegetative growth they mature to flowering and invest resources into reproductive structures. Bolting time was measured as the number of days after germination for the plant to grow a bolt that was greater than 3 mm in height. This demonstrated that bolting time is reduced in transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB compared to wildtype controls (
[0192] In addition to measuring flowering (bolting) time above, the size of the flowering structure (bolt) was also measured. For each of n=12 plants the tallest bolt was measured at 12 pm using a ruler. Bolts from transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB were taller than those of control plants 35 days after germination. Rather than being dwarfed, as would be expected given previous work by others in PHYB and YHB overexpression, the inventors found these transgenic plants taller at the same time point (see
[0193] Thus, the invention of driving precise expression of YHB in bundle sheath cells resulted in faster growth, earlier flowering and larger flowering structures. These are all advantageous traits for agriculture as they mean a shorter growing season, reducing risk of crop lost from adverse weather or pests/pathogens and potentially allowing more harvest cycles per year, something which has added additional value.
Example 7: Expression of YHB in Bundle Sheath Cells Enhances Yield in Arabidopsis thaliana
[0194] Given that the plants of the invention, had higher photosynthetic rates, grew faster, flowered earlier and produced larger flowering structures (
[0195]
[0196] Previous experiments on PHYB/YHB overexpression in plants often reports yield enhancement, but this is misleading and would not translate to crop harvests due pleiotropic delays in flowering. For example, Thiele et al., (1999) overexpress PHYB in potato and produce fewer, but more numerous tubers resulting in a reported yield increase. However, they also clarify that this does not occur in the same time frame as conventional potato harvests; and when harvested at the same time as normal potato harvesting, the yield of conventional PHYB overexpressors is lower than controls. Indeed, in Hu et al., (2019) supra, YHB (either derived from Arabidopsis or rice) was overexpressed in a range of diverse species (Arabidopsis, rice, tobacco, tomato and Brachypodium) and YHB overexpression consistently had a negative impact on seed yield. In distinct contrast, the transgenic plants of the inventors show surprisingly a much greater seed yield than controls when harvested at the same time, regardless of the stage at which they are harvested.
[0197] Ultimately, transgenic Arabidopsis plants containing the genetic vector for bundle sheath expression of YHB filled these siliques to produce significantly more seed than controls (see
[0198] Thus, the invention results in higher photosynthetic rate, enhanced water use efficiency, enhanced CO.sub.2 refixation, faster growth, early flowering, larger flowering structures, and more yield when compared to control plants.
Example 8: Transformation of Triticum aestivum with a Genetic Construct for Bundle Sheath Expression of YHB
[0199] To demonstrate the broad general applicability of this invention and validate the crop enhancement potential of bundle sheath expressed YHB, a monocot optimized plasmid was designed and tested in the monocot crop plant wheat, Triticum aestivum variety Cadenza. Unlike Example 1 which used a synthetic bundle sheath promoter, here the Zoysia japonica phosphoenolpyruvate carboxykinase promoter was used (previously described to provide bundle sheath specific gene expression in monocots (Nomura et al., (2005), Plant Cell Physiol. And
[0200] The full-length promoter-gene-Nos terminator sequence was fully synthesized de novo. This sequence was integrated into a binary vector containing an nptII selection cassette, transferred into agrobacterium, and used to transform cultured wheat calli using standard plant tissue culture and transformation methods. Transformants were screened to confirm successful genomic insertion and to identify single insertion transgenic plants by qPCR.
[0201] Transformants were potted and grown in growth chambers alongside control plants which had been through callus regeneration, but not received the construct for bundle sheath expression of YHB.
Example 9: Expression of YHB in Bundle Sheath Cells Enhances Photosynthetic Rates in Triticum aestivum
[0202] After seven weeks of growth in a growth chamber, photosynthetic rates of transformant wheat plants generated in Example 8 were quantified and compared to control plants. As in Example 2, LICOR 6800 devices were used to accurately measure photosynthetic rate. Environmental constants were as follows: flow 500 μmol s.sup.−1, fan speed 10,000 rpm, leaf temperature 25° C., 65% relative humidity. To measure the ambient photosynthetic rate in the growth chambers, PAR (photosynthetically active radiation, i.e. the amount of light available for photosynthesis) was set to 350 μmol m.sup.−1 s.sup.−1 (which was the measured light intensity at canopy height in the growth chamber) and carbon dioxide to 400 μmol mol.sup.−1. For each plant, the leaf below the flag leaf was selected and clamped ˜⅓ from the leaf tip. Following 10 minutes of acclimation (confirmed by observing no change in assimilation rate, fluorescence or stomatal conductance following this acclimation), an ambient photosynthetic measurement was recorded. Four controls and eight single insert wheat plants were screened between 12:00-14:00 on the same day.
Example 10: Expression of YHB in Bundle Sheath Cells Enhances Growth Rates in Triticum aestivum
[0203] As demonstrated in Example 6, enhanced Phytochrome B signalling in the vascular bundles of Arabidopsis was associated with faster growth, indicated by increase biomass accumulation compared to controls in the same time window, but not with changes to overall development of plant architecture. Likewise, wheat plants containing the genetic vector for bundle sheath expression of YHB showed no changes in development (such as dwarfing) and normal flowering was observed. As indicated by
[0204] Indeed, plant height (as measured as maximum canopy height, from soil surface to tip of tallest point) was significantly higher (by t-test, at p<0.05) in transformants (n=8) than controls (n=4) (
[0205] A person of average skill in the art could therefore combine any promoter sequence known to activate vascular bundle expression (either those known in literature or by designing a new promoter), and over express either an endogenous Phytochrome B gene or an exogenous Phytochrome B gene or YHB variant or functional fragment thereof to apply this invention to any desired crop. Likewise, various transformation methods can be used (whether floral dipping as in Example 1 or callus transformation as in this example) depending on species of interest.
Example 11: Gene Editing Brassica napus for Bundle Sheath Expression of PHYB and or YHB
[0206] As noted in
[0207] Brassica napus provides an example species where genome editing may be used to achieve bundle sheath expression of YHB using standard genome editing technologies known to a person of average skill in the art.
[0208] Initially, the gene expression domain of a native PHYB gene would be changed such that it was expressed in the vascular bundle. This would be achieved through a knock in of a short promoter sequence (e.g. SEQ ID: 7) or any vascular bundle or bundle sheath promoter or bundle sheath enhancer element known to a person of average skill in the art into the 5′ upstream region of a native PHYB gene (e.g. BnaA05). The bundle sheath promoters hereinbefore described and also illustrated in
[0209] In accordance with the invention, the editing of the native PHYB gene which inserts a vascular sheath promoter results in expected expression of PHYB in the requisite tissue. A stably inherited PHYB sequence is functionally equivalent to the polynucleotide integrated into the Arabidopsis or wheat genomes as described in Example 1 and Example 8. Any region in the 5′ upstream region may be a suitable target site for knocking in these promoter sequences. An endonuclease would be directed to a specific site to induce a double strand DNA break, and homology arms would direct the promoter polynucleotide to this area, to be incorporated into the DNA by homology directed repair. This has already been demonstrated in plants with suitable efficiency. For example, CRISPR-Cpf1 has been used to knock in >3,000 bp pieces of DNA into the rice genome with 8% efficiency (Begemann et al., (2017) Sci. Reps. 7:11606). Given that the vascular bundle promoter element is much shorter than this example, and shorter sequences result in higher knock in efficiency, this knock in will be feasible without further inventive steps. B. napus can be transformed using Agrobacterium (as Example 1) and independent transformation events screened by PCR to find individuals in which the promoter element has successfully been incorporated upstream of PHYB. Plants descended from these individuals would have enhanced PHYB expression in the vascular bundles, which can be tested by gene expression analysis, and would be expected to show some enhanced chloroplast development, photosynthetic rates and productivity, without the developmental defects associated with altering PHYB expression at the whole plant level (such as the semi-dwarfing phenotype that results from ubiquitous overexpression of PHYB). The phenotype would be expected to be analogous to that described in this document from introduction of vascular bundle expressed PHYB using conventional genetic modification approaches.
[0210] To further amplify PHYB signalling activity in the vasculature of B. napus, it may also be necessary to make a second edit, to convert the vascular-bundle driven PHYB into YHB. This too could be delivered with gene editing, but only requires a point mutation rather than a double strand DNA break. In Arabidopsis PHYB, a TAT′ codon is changed into ‘CAT’ to convert residue 276 from tyrosine to histidine and change PHYB into YHB. For BnaA05, a ‘TAO’ codon encodes the equivalent tyrosine residue, which can be changed into ‘CAC’ to make the equivalent modification to histidine by the introduction of a single nucleotide change.
[0211] As indicated by
[0212] Both of the genome edits proposed here have been demonstrated in planta to high levels of efficiency, even in species that are hard to transform and require methods other than floral dip, such as callus regeneration or particle bombardment. Thus, this B. napus example provides a general methodology for introducing vascular bundle expressed YHB through genome editing, in any species containing more than one copy of PHYB. Moreover, this approach can also be taken in any diploid plant so long as the transformants were maintained as heterozygous plants containing one unaltered copy of the PHYB allele and one altered copy of the PHYB allele. In summary, a single copy of PHYB is targeted for editing using nucleotide variation that is specific to that copy. In the first instance, PHYB expression is enhanced in the vascular bundles by knocking in a vascular sheath or vascular bundle specific promoter into the 5′ upstream region. This same gene is then subsequently targeted for a single nucleotide mutation in the CDS (coding sequence); the codon encoding a tyrosine residue that gives native PHYB the ability to revert from its photoactive form is mutated into histidine. This converts the native PHYB into constitutively active YHB, which further enhances PHYB signalling cascades in the vascular bundle. It is worth noting that even in species that lack a redundant copy of PHYB, it would even be possible to knock in a full length PHYB copy first, thereby creating a copy that can be gene edited further. Notably, all of these gene editing proposals achieve the same end result that was demonstrated in Example 1 and Example 8 by genetic modification methodology: A PHYB homolog that is expressed in vascular sheath cells.
[0213] Finally, the effects of altering PHYB expression (by knock out or overexpression) are highly conserved between distantly related species (
Example 12: Generalised Gene Editing Protocol for Activating Bundle Sheath Expression of PHYB and or YHB in any Plant Species
[0214] In addition to the full promoter knock-in example of Example 11, it is also possible to restrict the size of the gene edit to a few base pairs by only introducing a small vascular sheath or vascular bundle motif or enhancer element into the promoter region of endogenous PHYB genes.
[0215] The MYB76 promoter used in Example 1 has also been shown to drive tissue specific gene expression through the action of a small minimal enhancer motif (Dickinson et al., (2020) Nature Plants. 6:1468-1479). Such an enhancer motif sequence could be introduced in close proximity to the transcription start site of soybean's PHYB gene to confer the desired expression pattern. Unlike the tomato design described above, this approach would leave the endogenous core promoter intact, as indicated in
Genetic Resources
[0216] Seeds of Arabidopsis thaliana (Columbia ecotype) were obtained in September 2018 from the University of Oxford Department of Plant Sciences greenhouses.
[0217] Golden gate cloning parts were provided by Sylvestre Marillonnet (Liebnitz Institute of Plant Biochemistry: Weber et al., (2011) PLOS ONE). The DHS vascular bundle promoter was provided by Patrick Dickinson from Julian Hibberd's lab, Cambridge University (Kneřová et al., (2018) bioRxiv).
[0218] Cadenza wheat plants and wheat transformation was provided by NIAB Crop Transformation Services.
Nucleotide and Amino Acid Sequences
[0219] [SEQ ID NO: 1] The domesticated Arabidopsis thaliana PHYB coding sequence, containing the YHB mutation.
[0220] [SEQ ID NO: 2] The domesticated Arabidopsis thaliana PHYB coding sequence (Arabidopsis_PHYB_AT2G18790.1).
[0221] [SEQ ID NO: 3] The rice PHYB coding sequence (Rice_PHYB_LOC_Os03g19590.1).
[0222] [SEQ ID NO: 4] The Arabidopsis thaliana YHB amino acid sequence.
[0223] [SEQ ID NO: 5] The Arabidopsis thaliana PHYB amino acid sequence (Arabidopsis_PHYB_AT2G18790.1).
[0224] [SEQ ID NO: 6] The rice PHYB amino acid sequence (Rice_PHYB_LOC_Os03g19590.1).
[0225] [SEQ ID NO: 7] The nucleotide sequence of the Arabidopsis derived MYB76 vascular bundle promoter. This is a synthetic promoter comprised of an oligomerised MYB76 sequence containing a minimal enhancer element, and a 35S minimal core promoter element.
[0226] [SEQ ID NO: 8] Arabidopsis thaliana phytochrome D nucleotide coding DNA sequence (Arabidopsis_PHYD_AT4G16250.1).
[0227] [SEQ ID NO: 9] Arabidopsis thaliana phytochrome D amino acid sequence (Arabidopsis_PHYD_AT4G16250.1).
[0228] [SEQ ID NO:10] The Zoysia japonica PCK promoter sequence.
[0229] [SEQ ID NO:11] The wheat PHYB coding sequence containing the YHB mutation (derived from Traes_4 AS_1F3163292).
[0230] [SEQ ID NO:12] The wheat PHYB amino acid sequence containing the YHB mutation (derived from Traes_4 AS_1F3163292).
[0231] [SEQ ID NO:13] The GLDP1 V-box containing promoter DNA sequence.
[0232] [SEQ ID NO: 14] Brassica napus PHYB coding sequence excerpt (BnaC03g39830D).
[0233] [SEQ ID NO: 15] Brassica napus PHYB coding sequence excerpt (BnaA05g22950D).
[0234] [SEQ ID NO: 16] Brassica napus PHYB coding sequence excerpt (BnaC05g36390D).
[0235] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0236] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0237] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.