Patent Application
20220145320
This invention relates to plants with improved drought tolerance. The plants are genetically modified plants. The invention therefore also concerns isolated nucleic acids and polynucleotide constructs used in the genetic engineering of such plants. The modified plant materials and germplasm have potential application in the generation and breeding of new more drought resistant plants, including modification of existing plant varieties to improve field performance.
Water scarcity is among the main problems to be faced by many societies and the world in the 21st century. Freshwater is a limited and dwindling global resource, but water use has been growing at more than twice the rate of population increase in the last century, and, although there is no global water scarcity as such, an increasing number of regions are chronically short of water (http://www.un.org/waterforlifedecade/scarcity.shtml).
Water-limited conditions impose abiotic stresses such as dehydration and salinity on crops which limit their performance and reduce yield worldwide. Therefore, it becomes socially and economically increasingly important to achieve greater crop production under water deficient conditions.
With the aim to generate better performing plants under water scarcity, two strategies are usually pursued: marker assisted breeding programs or the more targeted approach of genetic engineering. Microarray and DNA chip technologies and more recently the advent of next-generation sequencing technology RNA-seq have identified numerous drought-responsive transcripts. Many of the genes identified in this way have been used for the generation of transgenic plants in order to test their effects on improving drought resistance in crops by overexpression and/or suppression. These genes can be classified into several groups targeting different aspects of cell function such as signalling (regulation of gene expression by protein kinases and transcription factors), protein degradation and/or modification, metabolism of phytohormones, osmotic adjustment, synthesis of protective proteins, energy regulation and signalling, transport proteins of diverse functions, amino acid metabolism and proteins involved in reactive oxygen species scavenging (see Hu, H. and Xiong L., (2014) Annual Review Plant Biology Vol. 65: 715-741, and references therein)
However, what has not been addressed in transgenic approaches hitherto is a modification of traits of plant architecture which could confer an increased survival rate in water-limited conditions. These are inherently complex traits with the contribution of many loci and are therefore difficult to target by genetic engineering. One such trait which has been shown to give an advantage to plants grown in water-limited conditions are leaves with a higher LMA (leaf dry mass per unit area in g·m−2) which is frequently used as a proxy for leaf thickness. Leaf thickness, the distance between upper (adaxial) and lower (abaxial) leaf surface, has been shown to correlate with environmental variables such as water availability, temperature and light quality and on a global scale, across habitats and land plant diversity, plants adapted to arid environments tend to have thicker leaves (see Poort, H., et al. (2009) New Phytologist 182: 565-588).
In their work on leaf thickness of the domesticated tomato Solanum lycopersicum and the desert adapted S. penneffii, Coneva, V. et al., (2017) Plant Physiology Vol 175: 376-391 attempted to elucidate the genetic mechanisms responsible for leaf thickness and identified several QTLs for this trait. In addition, they showed that increased elongation of the palisade cells was the main contributor to leaf thickness. Overexpression of the HRD gene, an AP2/ERF-like transcription factor, in Arabidopsis thaliana led to an increase in number of leaf mesophyll cells and leaf thickness and conferred drought resistance and salt tolerance to the plants (Karaba et al., (2007) Proc. Natl. Acad. Sci. USA Vol 25; 104(39):15270-15275). This increased drought resistance could be replicated in rice upon transfer of Arabidopsis HRD to rice.
Although leaf thickness is a highly functional trait, mechanistic understanding of its development during leaf ontogeny is poorly understood. This is confounded by the problem that besides the innate genetic component responsible for leaf thickness there is a prominent role for environmental cues in modulating it. Currently available data obtained in Arabidopsis thaliana suggest that increased thickness of the leaf is the result of palisade cell elongation and the formation of additional palisade cell layers and that this morphological change is regulated by perception of blue light of lower intensities through the phototropin receptor PHOT2 (Kozuka, T. et al. (2011) Plant Cell Vol 23(10): 3684-3695). On the downside however, leaf thickness is negatively correlated with yield-related traits which points towards a trade-off between investment in vegetative and reproductive biomass.
Disclosed in Steffen, A. et al., (2005) BMC Evolutionary Biology 5: 72 doc10.11186/1471-2148-5-72 is a genome-wide comparative analysis of the IQD gene families in Arabidopsis thaliana and Oryza sativa. 33 IQD1-like genes in Arabidopsis thaliana and 29 genes in Oryza sativa were identified and analyzed. The encoded IQD proteins contain a plant-specific domain of 67 conserved amino acid residues, referred to as the IQ67 domain, which is characterized by a unique and repetitive arrangement of three different calmodulin recruitment motifs, known as the IQ, 1-5-10 and 1-8-14 motifs. An IQD-like gene in bryophytes suggests that IQD proteins are an ancient family of calmodulin-binding proteins and arose during the early evolution of land plants. Comparative phylogenetic analyses indicate that the major IQD gene lineages originated before the monocot-eudicot divergence.
Disclosed in Bürstenbinder, K. et al. (2017) Plant Pysiol. 173: 1692-1708 is a scientific publication showing how the 33 members of the IQD family in Arabidopsis thaliana differentially localize, using green fluorescent protein (GFP)-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane subdomains, and nuclear compartments. The various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of calmodulin, suggesting that the diverse IQD members sequester calcium-calmodulin signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. GFP-labeled microtubule arrays were analyzed quantitatively in Nicotiana benthamiana cells transiently expressing GFP-IQD fusions. IQD-specific microtubule patterns were observed which point to a role of IQDs in MT organization and dynamics. Stable overexpression of select IQD proteins in Arabidopsis altered cellular microtubule orientation, cell shape, and organ morphology. The authors suggest that IQD families provide an assortment of platform proteins for integrating calmodulin-dependent calcium signaling at multiple cellular sites to regulate cell function, shape and growth.
To date therefore, the IQD proteins are characterised to some small degree at the level of cell structure and biochemistry. So far, only IQD1 has been assigned a putative role in glucosinolate metabolism in Arabidopsis (see Levy M et al, Plant J. 2005 July; 43(1):79-96).
Xiao, H. et al., (2008) Science Vol 319(5869): 1527-1530 describes the function of the an IQD gene known as the SUN gene in tomato. Altered expression of this gene is observed to be linked to changes in tomato fruit shape.
The problem therefore remains as to how to modify plant architecture of plants in a reliable enough way so as to improve the drought tolerance of such modified plants, and yet further ideally without diminution of plant growth. In the context of agricultural species and varieties, the improvement of drought tolerance ideally needs to go hand-in-hand with little or no significant diminution of yield of harvestable product.
The inventors have unexpectedly discovered that when engineering Arabidopsis thaliana and Camelina sativa plants to overexpress IQD genes, not only is there an increased leaf thickness compared to unmodified control plants, but also an increased thickness of the stem. There is a surprisingly favourable tolerance to drought in the overexpressing plants.
In accordance with the present invention there is provided a method of modifying a plant, whereby at least some of the leaves of the modified plant are: (i) thicker; and/or (ii) rounder in shape; and/or (iii) contain more chlorophyll per wet or dry weight, compared to a corresponding unmodified plant, comprising genetically modifying the plant to increase expression of a plant calmodulin-binding protein (IQD) compared to the unmodified plant grown under same or comparative conditions.
Alternatively, in accordance with the present invention is provided a method of modifying a plant to increase yield compared to a corresponding unmodified plant, comprising genetically modifying the plant so as to increase expression of a plant calmodulin-binding protein (IQD) compared to the unmodified plant grown under same or comparative conditions.
Therefore the invention provides for the use of a polynucleotide comprising a nucleic acid sequence encoding a plant calmodulin-binding protein (IQD) for increasing the expressed amount of an IQD protein or polypeptide in plant cells modified with the polynucleotide, compared to a corresponding unmodified plant cell, and wherein in a plant of such modified cells at least some of the leaves of the plant are: (i) thicker; and/or (ii) rounder in shape; and/or (iii) contain more chlorophyll per wet or dry weight, compared to a corresponding plant of unmodified cells.
The plant calmodulin-binding protein (IQD) may be a nuclear and/or a microtubule associated protein. The plant calmodulin binding protein in being associated with microtubules may be bound thereto.
In any of the aforementioned methods and uses of the invention, the IQD protein is selected from one or more of IQD22, IQD23 and IQD24. Sequence information for these IQD proteins is set forth hereinafter.
The IQD protein for use in accordance with the invention preferably comprises a conserved 67 amino acid domain (IQ67) having an amino acid sequence with at least 50% identity to FRGYLARRALRALKGLVRLQALVRG [SEQ ID NO: 10] or at least 50% identity to:
wherein “#” is a hydrophobic amino acid, and wherein “+” is a basic amino acid.
The aforementioned SEQ ID NO: 11 is referred to in Steffen, A. et al., (2005) BMC Evolutionary Biology 5: 72 doc10.11186/1471-2148-5-72. Steffen et al (2005) also refers to various motifs (1-20) of IQD proteins. Reference to numbered motifs of IQD proteins in this specification correspond to the numbered motifs described by Steffen et al (2005).
Additionally or alternatively, the polypeptide may further comprise the amino acid sequence:
or
(ii) (D/R/S/N)(K/Q/D/N/R/T)(H/R)(A/S)(I/V/M/S)(A/H)V(A/T)(A/E/F)(A/P)T(A/E)(A/H/V)(V/A) [SEQ ID NO: 16]; preferably DKHAIAVAAATAAV [SEQ ID NO: 17]. These are amino acid sequences located as at motifs 7 and/or 8 or portion thereof, of IQD proteins. The sequence DKHAIAVAAATAAV [SEQ ID NO: 18] represents an amino acid sequence portion determined by the inventors to be in common to and unique for all IQD 22, IQD 23 and IQD 24 proteins.
Additionally to the above of motif 7, the polynucleotide may further comprise an amino acid sequence portion of motif 8 being:
Additionally or alternatively to any of the above, the polypeptide may further comprise an amino acid sequence of about motif 5 being: (R/K)(R/K/T)W(S/G)F [SEQ ID NO: 33] or (R/K)(R/K/T)WSF [SEQ ID NO: 34]; preferably wherein this sequence is RRWSF[SEQ ID NO: 35] or RKWSF [SEQ ID NO: 36]. The sequence portion RRWSF represents an amino acid sequence determined by the inventors to be in common to and unique for all IQD 22, IQD 23 and IQD 24 proteins.
In any of the aforementioned aspects, the polynucleotide may comprise an amino acid sequence of motif 17 being:
Additionally or alternatively, the polynucleotide may further comprise a C-terminal portion amino acid sequence being:
In any of the aforementioned aspects, the polypeptide may have:
(1) an amino acid sequence of SEQ ID NO: 1 (IQD 22) or an amino acid sequence of at least 30% identity therewith;
(2) an amino acid sequence of SEQ ID NO: 4 (IQD 23) or an amino acid sequence of at least 30% identity therewith;
(3) an amino acid sequence of SEQ ID NO: 7 (IQD 24) or an amino acid sequence of at least 30% identity therewith.
In second separate aspect, the invention provides the use of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 (IQD 22) or an amino acid sequence of at least 30% identity therewith, for increasing (i) leaf thickness; and/or (ii) roundness of leaf shape; and/or (iii) chlorophyll content per wet or dry weight of leaf in a genetically modified plant and when compared to a corresponding unmodified plant grown under same or similar conditions, wherein the expression of the polypeptide in a modified plant is increased compared to a corresponding unmodified plant.
In other words, the invention includes the use of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 (IQD 22) or an amino acid sequence of at least 30% identity therewith, for increasing the expressed amount of the polypeptide in a plant cell modified with the polynucleotide, compared to a corresponding unmodified plant cell, and wherein in a plant of such modified cells at least some of the leaves of the plant are: (i) thicker; and/or (ii) rounder in shape; and/or (iii) contain more chlorophyll per wet or dry weight, compared to a corresponding unmodified plant; and/or the stem(s) of the plant are thicker compared to the stem(s) of a corresponding unmodified plant. In third separate aspect, the invention provides the use of a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO: 4 (IQD 23) or an amino acid sequence of at least 30% identity therewith, for increasing (i) leaf thickness; and/or (ii) roundness of leaf shape; and/or (iii) chlorophyll content per wet or dry weight of leaf in a genetically modified plant and when compared to a corresponding unmodified plant grown under same or similar conditions, wherein the expression of the polypeptide in a modified plant compared to a corresponding unmodified plant.
In other words, the invention includes the use of a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO: 4 (IQD 23) or an amino acid sequence of at least 30% identity therewith, for increasing the expressed amount of the polypeptide in a plant cell modified with the polynucleotide, compared to a corresponding unmodified plant cell; and wherein in a plant of such modified cells at least some of the leaves of the plant are: (i) thicker; and/or (ii) rounder in shape; and/or (iii) contain more chlorophyll per wet or dry weight, compared to a corresponding unmodified plant; and/or the stem(s) of the plant are thicker compared to the stem(s) of a corresponding unmodified plant.
In fourth separate aspect, the invention provides the use of a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO: 7 (IQD 24) or an amino acid sequence of at least 30% identity therewith, for increasing (i) leaf thickness; and/or (ii) roundness of leaf shape; and/or (iii) chlorophyll content per wet or dry weight of leaf in a genetically modified plant and when compared to a corresponding unmodified plant grown under same or similar conditions, wherein the expression of the polypeptide in a modified plant compared to a corresponding unmodified plant cell.
In other words, the invention includes the use of a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO: 7 (IQD 24) or an amino acid sequence of at least 30% identity therewith, for increasing the expressed amount of the polypeptide in a plant cell modified with the polynucleotide, compared to a corresponding unmodified plant cell, and wherein in a plant of such modified cells at least some of the leaves of the plant are: (i) thicker; and/or (ii) rounder in shape; and/or (iii) contain more chlorophyll per wet or dry weight, compared to a corresponding unmodified plant; and/or the stem(s) of the plant are thicker compared to the stem(s) of a corresponding unmodified plant.
In any of the second, third or fourth aspects of the invention as herein defined, the amino acid sequence of the polypeptide may include one or more of the following amino acid sequences:
Alternatively, in any of the second, third or fourth aspects of the invention, the amino acid sequence of the polypeptide may include one or more of the following amino acid sequences:
In yet further alternative of the second, third or fourth aspects of the invention the polypeptide includes one or more of the following amino acid sequences:
The invention includes an isolated polynucleotide comprising a nucleotide sequence encoding an IQD polypeptide as described herein. In preferred aspect the nucleotide sequence is that of (a) IQD 22 as shown in
The invention also includes an isolated cDNA molecule comprising or consisting of a nucleic acid sequence complementary to any sequence of
Included in the invention is any polynucleotide construct for engineering, replication or expression, wherein the construct comprises a polynucleotide as described above. Expression constructs which may be integrated into the host cell genome at a locus other than the native IQD protein promoter will also comprise an heterologous promoter, e.g. a CMV promoter; optionally regulatory control elements such as an enhancer.
The invention also provides a genetically modified plant cell having an increased amount of an IQD polypeptide as hereinbefore defined, when compared to a corresponding unmodified plant cell.
The invention includes a genetically modified plant cell having at least one polynucleotide sequence encoding an IQD polypeptide as hereinbefore defined in addition to any naturally occurring homolog(s) of said protein(s) in a corresponding unmodified plant cell.
Genetically modified plant cells may also be defined as transgenic. Such transgenic plant cells may be modified such that they contain heterologous genetic material stably; optionally heritably, incorporated. The incorporation may be genomic, or it may be transient, e.g. extrachromosomal such as an expression plasmid.
The increased amount of polypeptide in modified plant cells may be at least 2-fold compared to the corresponding unmodified plant cell. In alternatives, the increased amount of polypeptide may be any of at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold (i.e. a log 2-fold), at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold, when compared to a corresponding unmodified plant cell. The -fold increase may correspond to the number of additional IQD gene copies which are incorporated into the modified plant cell when compared to the corresponding unmodified cell. In connection with this, each copy of the heterologous IQD gene which is present where there is a multiplicity, may be same or different. Where copies are different then their sequences may be obtained or derived from different plant species.
Where genetically modified plant cells are provided in accordance with the invention then where there is at least one additional IQD polynucleotide sequence is under the control of a native promoter for that polypeptide
In a preferred aspect, the present invention provides a genetically modified plant cell as herein defined, wherein the polynucleotide sequence is as set forth herein for IQD 22 overexpression. These embodiments of the invention are advantageous in providing the ability to regenerate and provide plants wherein the leaves of the plants have taller (i.e. more elongated) palisade leaf cells compared to a corresponding unmodified plant. Additionally or alternatively, such IQD 22 overexpression advantageously causes an increased photosynthetic activity in the leaves of plant; such photosynthetic activity increase may be at least 5% more compared to corresponding unmodified control plants subjected to and measured under the same conditions. The increase may be 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more; or 15% or more.
In some embodiments, a genetically modified plant cell of the invention may have at least one additional IQD polynucleotide sequence under the control of an heterologous promoter; optionally a constitutive promoter, such as CMV S35. Inducible promoters may be used in case it is desired to switch IQD gene overexpression on and/or off during plant growth and development.
Where there is a multiplicity of additional IQD polynucleotide sequences introduced in tot the plant cell, then they may be incorporated at the same genetic locus, or possibly into different loci.
The invention therefore includes a plant comprising or consisting of genetically modified plant cells as hereinbefore described, wherein at least some of the leaves of the plant are (i) rounder in shape; and/or (ii) thicker; and/or (iii) contain more chlorophyll per wet or dry weight, compared to a corresponding unmodified plant. In particular, when using an heterologous promoter, e.g. S35, the leaves are about 1.8-2 times thicker. In such embodiments, the leaves may be about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5 or about 3 time thicker.
In certain plants, preferably when expressed from a native IQD gene promoter, at least some leaves are thicker by at least 25% on a leaf mass (g) per unit area (m−2) basis. Other plants may have thicker leaves by at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 45%, at least 50%.
In plants with thicker leaves as described herein, this may be due to at least some leaves having a palisade layer comprising more cells and/or more elongated cells compared to a corresponding unmodified plant. As already noted, in a preferred aspect of the invention, overexpression of IQD 22 results in a modified plant with leaves comprising more elongate palisade layer cells and/or a greater level of photosynthetic efficiency, compared to leaves of an unmodified control plant.
In any of the aforementioned plants of the invention there may be an elimination of epinastic leaf curling as found in a corresponding unmodified control plant kept under same conditions for growth and development.
Additionally or alternatively in any of the aforementioned plants of the invention the stem of the plant may be thicker compared a corresponding unmodified control plant kept under same conditions for growth and development.
Where leaves of a modified plant of the invention are concerned, they are advantageously more like leaves of succulent plants in character. They tend to exhibit a reduced transpiration rate compared to an unmodified control plant kept under the same conditions. Advantageously, the IQD gene overexpressing plants of the invention may have an altered physiology, wherein wilting is delayed when water is withheld compared to unmodified plant kept under the same conditions. Further, when a wilted plant is subjected to re-watering, a wilted plant of the invention recovers more quickly compared to an unmodified plant kept under the same conditions. In some instances, the unmodified plant does not recover whilst the modified plant of the invention does revive.
Further advantages of the modified plants of the invention are found. One is that yield of the plant may be increased under drought conditions compared to an unmodified control plant kept under the same drought conditions; e.g. wherein the yield is one or more of total biomass, organ or part biomass, leaf biomass, yield of fruit or yield of seed. For example, the yield may be 2-fold compared to the corresponding unmodified plant cell. In alternatives, the increased yield of seed may be any of at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold or at least 50-fold, when compared to a corresponding unmodified plant cell.
Consequently, the invention includes any plant part, plant tissue, organ, haploid or diploid reproductive material, cell callus, fruit, seed or organ obtained from a modified plant of the invention as hereinbefore described.
The invention additionally includes a method of increasing tolerance of a plant to water stress, comprising modifying the plant so that it expresses an increased amount of an IQD polypeptide as herein defined, at least in the leaves and/or stems of the plant compared to an unmodified control plant under the same growth conditions.
The invention further includes a method of increasing leaf thickness in a plant, comprising modifying the plant so that it expresses an increased amount of an IQD polypeptide as herein defined, at least in the leaves of the plant compared to an unmodified control plant.
The invention yet further provides a method of increasing stem thickness in a plant, comprising modifying the plant so that it expresses an increased amount of an IQD polypeptide as herein described, at least in the stems of the plant compared to an unmodified control plant subjected to the same conditions for growth and development.
The invention therefore also includes any processed material of a modified plant, plant part, plant tissue, organ, cell callus, fruit, seed or organ of the invention as described herein, wherein the processed material contains on a weight basis an increased amount of an IQD polypeptide and/or of an IQD polynucleotide as defined herein, when compared to a corresponding processed material from a control unmodified plant, plant part, plant tissue, organ, cell callus, fruit, seed or organ. Such processed plant material may be selected from meal, flour, oil, biomass for energy generation, wood or wood products.
In another aspect, the invention provides an isolated polynucleotide comprising a native IQD22 gene promoter and a gene of interest (GOI) intended for expression under the control of the promoter in a plant, plant tissue or plant cell. Such polynucleotides may further include a termination sequence, e.g. nos.
The polynucleotides herein described form part of a polynucleotide expression construct. When used to transform and produce transgenic plants, the expression of a GOI driven by the native IQD22 promoter results in leaf palisade cell specific expression of the GOI, when compared to a WT, i.e. untransformed plant. The invention therefore includes such transgenic plants and transgenic plant leaves which have leaf palisade specific expression of the GOI. Examples of GOI include genes involved in photosynthesis or gene involved in resistance to foliar pathogens.
The invention also provides an IQD22 gene promoter for use in an expression construct as a leaf palisade cell specific promoter.
The IQD22 gene promoter region is set forth in SEQ ID NO: 127. The IQD22 promoter is therefore all or a functional portion of this sequence. The IQD22 promoter sequence may be a sequence of SEQ ID NO: 127 or a sequence of at least 200 contiguous nucleotides thereof, starting at any position of SEQ ID NO: 127 that provides for at least 200 contiguous nucleotides. The promoter sequence of at least 200 contiguous nucleotides may have an identity of at least 60% to the corresponding aligned nucleotides of SEQ ID NO: 127. In preferred aspects, the at least 200 contiguous nucleotides of the promoter sequence may have at least 61%, at least 62%, at least 63%, at least 64%, 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 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% sequence identity to SEQ ID NO: 127.
The invention will now be described in more detail and by way of specific examples and with reference to the drawings in which:
Camelina is a genus within the flowering plant family Brassicaceae. Camelina is an emerging biofuel crop, in particular Camelina sativa. The plant is useful as a biofuel feedstock due in part to its drought tolerance and minimal requirements for supplemental nitrogen and other agricultural inputs (Gehringer et al. (2006) Genome 49(12): 1555-63; Gugel and Falk (2006) Canadian Journal of Plant Science 86(4): 1047-1058). C. sativa is of course in the same family and so relatively close genetically and developmentally and in many ways to Arabidopsis thaliana as well as the common oilseed crop Brassica napus (canola). The inventors were looking to further increase the drought tolerance of Camelina sativa in various ways, and in doing so discovered unexpectedly that if IQD genes are overexpressed in Arabidopsis or Camelina plants, then the plants have an altered morphology which and significantly improved drought tolerance compared to a corresponding unmodified control plant allowed to grow and develop under the same regime. More particularly the inventors have found that if IQD 22, IQD 23 or IQD 24 genes are overexpressed in modified Arabidopsis or Camelina plants, then the plants have an altered morphology which and significantly improved drought tolerance compared to an unmodified control plant. The morphology and architecture of the modified overexpressing plants is altered, manifest as a significant increase in the thickness of leaves and stem. This also affects biomass in the altered plants. There are also marked differences in physiology, that is to say in terms of transpiration and drought tolerance, and in certain instances in photosynthetic efficiency. There are also advantages gained in terms of seed yield and productivity.
Comparing the IQD 22 overexpressing Arabidopsis plants in particular with control (wt) plants under conditions of drought, wilting of the IQD 22 overexpressor lines is considerably delayed. Re-watering of droughted plants leads to a much quicker and better recovery of the IQD 22 overexpressors.
Naturally, an important industrial application of this invention is the engineering of more drought tolerant crops and the making available of such engineered germplasm for use in connection with the generation of new plant varieties. The inventors consider that the enhancements of drought tolerance in plants, particularly in preferred aspects, of the Brassicaceae, may be achieved by overexpression of IQD genes; preferably IQD 22 and/or IQD 23 and/or IQD 24 genes; more preferably IQD 22.
As used herein, the terms “increase”, “improve” or “enhance” are used interchangeably. Also, the terms “reduce” or “decrease” are used interchangeably.
As described herein, when the amino acid sequences of any IQD protein are defined herein with respect to a reference sequence, then homologues (i.e. sequence variants) are also defined with reference to a percentage of sequence identity. In the broadest aspect of the invention these variant sequences are at least 30% identical to a reference sequence. The proteins of the IQD family are unusual in that there is a very wide degree of variation between plant species. However, there are motifs, as described by Steffen, A. et al., (2005) Ibid. where there is much narrower degree of variation between plant species. The inventors define certain consensus sequences of IQD 22, 23 and 24 or combinations therefore and which may be used in combination with the reference sequence and at least 30% identity variants to further define the IQD polypeptides which are overexpressed in accordance with the invention.
Amino acid motifs which are intended to assist in defining IQD 22 and/or IQD 23 and/or IQD24 polypeptide sequences, whether or not together with SEQ ID NO: 1 or SEQ ID NO:4 or SEQ ID NO: 7, are as follows: RRWSF [SEQ ID NO: 35]; DKHAIAVAAATAAVAEAA [SEQ ID NO: 26]; WRLT; KIQS; FRGYLA [SEQ ID NO: 29]; RALRALK [SEQ ID NO: 30] or ENSPQ [SEQ ID NO: 54], whether alone or in any combination.
Amino acid motifs which are intended to assist in defining IQD22 and/or IQD 23 polypeptide sequences, whether or not together with SEQ ID NO: 1 or SEQ ID NO:4, are as follows: KRRWSF; VVRLTS or VRGHI, whether alone or in any combination.
Amino acid motifs which are intended to assist in defining IQD23 and/or IQD 24 polypeptide sequences, whether or not together with SEQ ID NO: 4 or SEQ ID NO:7, are as follows: MGFFGRLFGSKK [SEQ ID NO: 38]; DKHAIAVAAATAAVAEAAL [SEQ ID NO: 56]; AAAEVVRLT [SEQ ID NO: 39]; RSNRRW [SEQ ID NO: 40]; AAMKIQSAFRGYLARRALRALKALVKLQALV [SEQ ID NO: 41]; GHIVRKQTADMLRRMQTLVRLQ [SEQ ID NO: 42]; ARARASRSSH [SEQ ID NO: 43]; DKILEVDTWKPH [SEQ ID NO: 44]; ESPRKR [SEQ ID NO: 45]; TPFTP [SEQ ID NO: 46]; YYSGYHPNYMANTESYKAKVRSQSAP [SEQ ID NO: 47]; SGYKRS [SEQ ID NO: 48]; or QGQYYYYT [SEQ ID NO: 49].
Amino acid motifs which are intended to assist in defining IQD22 and/or IQD 24 polypeptide sequences are as follows: RRWSF [SEQ ID NO: 35]; VVRLT [SEQ ID NO: 55]; FRGYLA [SEQ ID NO: 29]; RALRALK [SEQ ID NO: 30] or ENSPQL [SEQ ID NO: 53].
Accordingly, the invention—having regard to any reference IQD sequence, i.e. SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7—also includes any variant of such reference sequence which is at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, 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 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% sequence identity thereto. 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). Other methods of sequence comparison and analysis may be used; e.g Bestfit (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711).
When using a sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably adjusted so that the percentage of identity is calculated over the entire length of the reference sequence and homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. Usually these may be default settings in commonly used software packages. Preferably therefore when a sequence has a percent identity to any one of the SEQ ID NOs as detailed herein, this refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.
The degree of sequence identity of polynucleotides of 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 reference sequence. 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.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na+ ion, typically about 0.01 to 1.0 M Na+ 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.
When employed in the invention herein, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either (a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above—becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815 both incorporated herein by reference.
Where the invention may provide a transgenic plant, the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. Thus, the plant expresses a transgene. However, as mentioned, in certain embodiments, transgenic may means that, while the nucleic acids according to the different embodiments of the invention are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified, for example by mutagenesis.
Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. According to the invention, the transgene is stably integrated into the plant and the plant is preferably homozygous for the transgene.
Modified plant cells, modified plants or parts thereof as defined herein may be stably transformed with additional genetic material. Such additional genetic material is preferably under the control of at least one regulatory sequence, but a multiplicity of control points may be built in, whether using native or modified regulatory sequences.
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 suitable ancestor cell. 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, are preferably produced via Agrobacterium tumefaciens mediated transformation.
Transformation methods are well known in the art. Thus, according to the various aspects of the invention, an heterologous nucleic acid 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” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host 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 therefrom. 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 genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner well known in the art.
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, 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, both techniques being well known in the art.
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).
The regulatory sequence may be a promoter; optionally an inducible promoter, preferably then one which may be induced by an external stress condition. In the alternative, a constitutive promoter may be employed, e.g. cauliflower mosaic 35S.
The regulatory sequence may optionally be tissue specific. The term “regulatory element” as used herein may be considered interchangeably with “control sequence” and “promoter” and all terms are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences.
The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
A “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The “plant promoter” can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed. This also applies to other “plant” regulatory signals, such as “plant” terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3′-regulatory region such as terminators or other 3′ regulatory regions which are located away from the ORF. Also possible is that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule is, as described above, preferably linked operably to or comprises a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes are known to the skilled person and include for example beta-glucuronidase or beta-galactosidase.
The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
For example, the nucleic acid sequence may be expressed using a promoter that drives overexpression. Overexpression according to the invention means that the transgene is expressed at a level that is higher than expression of endogenous counterparts driven by their endogenous promoters. For example, overexpression may be carried out using a strong promoter, such as a constitutive promoter. A “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression. Alternatively, enhanced or increased expression can be achieved by using transcription or translation enhancers or activators and may incorporate enhancers into the gene to further increase expression. Furthermore, an inducible expression system may be used, where expression is driven by a promoter induced by environmental stress conditions (for example the pepper pathogen-induced membrane protein gene CaPIMPI or promoters that comprise the dehydration-responsive element (DRE), the promoter of the sunflower HD-Zip protein genes Hahbl or Hahb4, which is inducible by water stress, high salt concentrations and ABA or a chemically inducible promoter (such as steroid- or ethanol-inducible promoter system). The promoter may also be tissue-specific. The types of promoters listed above are described in the art. Other suitable promoters and inducible systems are also known to a person of average skill.
A green tissue-specific promoter may be used. For example, a green tissue-specific promoter may be selected from the maize orthophosphate kinase promoter, maize phosphoenolpyruvate carboxylase promoter, rice phosphoenolpyruvate carboxylase promoter, rice small subunit rubisco promoter, rice beta expansin EXBO9 promoter, pigeonpea small subunit rubisco promoter or pea RBS3A promoter.
The promoter may be a constitutive or strong promoter. In a preferred embodiment, the regulatory sequence is an inducible promoter or a stress inducible promoter. The stress inducible promoter is selected from the following non limiting list: the HaHB1 promoter, RD29A (which drives drought inducible expression of DREB1A), the maize rabl7 drought-inducible promoter, P5CS1 (which drives drought inducible expression of the proline biosynthetic enzyme P5CS1), ABA- and drought-inducible promoters of Arabidopsis clade A PP2Cs (ABM, ABI2, HAB1, PP2CA, HA11, HAI2 and HAI3) or their corresponding crop orthologues.
Altered plants in accordance with the invention advantageously may provide better yield characteristics. These may be designed into the alterations being made. Yield characteristics, also known as yield traits may comprise one or more of the following non-limitative list of features: biomass, seed yield, seed/grain size, starch content of grain, greenness index, increased growth rate. 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 year, 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) 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, increased biomass or grain fill. Preferably, increased yield comprises an increased number of grains/seeds/capsules/pods, increased biomass, increased growth. Yield is usually measured relative to a control plant.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest. When referred to herein, a “plant part” may be green tissue, for example a leaf.
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, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.
A dicot 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 lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine, bell pepper, chilli or citrus species. In one embodiment, the plant is oilseed rape.
Also included are biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), 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)
Other useful and preferred plants are maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
The invention also extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof.
To the extent that any experimental or practical techniques needed are not provided in the specific examples, they are available as a matter of common general knowledge in the art to a person of ordinary skill. For example, with reference to (1) J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, “Molecular Cloning: A Laboratory Manual” Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; (2) J. M. Polak and James O'D. McGee, 1990, “In Situ Hybridization: Principles and Practice” Oxford University Press; (3) B. Roe, J. Crabtree, and A. Kahn, 1996, “DNA Isolation and Sequencing: Essential Techniques”, John Wiley & Sons; (4) Ausubel, F. M. et al. (1995 and periodic supplements “Current Protocols in Molecular Biology” chapters 9, 13, and 16, John Wiley & Sons, New York, N.Y.); (5) J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: “DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology” Academic Press; (6) M. J. Gait (Editor), 1984, “Oligonucleotide Synthesis: A Practical Approach” IRL Press; and (7) E. M. Shevach and W. Strober, 1992 and periodic supplements, “Current Protocols in Immunology”, John Wiley & Sons, New York, N.Y. Each of these general texts is herein incorporated by reference.
Arabidopsis lines were generated which overexpress IQD22 under the constitutive CaMVS35 promoter.
The preparation and construction of the genetic constructs and the transformation and culturing up and reproduction of transgenic plants was as follows:
The clone containing the IQD22 cDNA was ordered from NASC. After confirmation of the full length cDNA sequence, the cDNA was amplified with Primer NcoI F: ccatggGAAAAG CGTCACGGTG GTT [SEQ ID NO: 57]; and Primer EcoRV R: gatatc tcagtacctatacccaattggcat [SEQ ID NO: 58]; and subcloned into the TOPO vector (Invitrogen). The fragment containing the IQD22 fragment was excised using the restriction enzymes NcoI and EcoRV and cloned into the vector pJD330 which had been digested with NcoI/SmaI, thereby inserting it between the S35 promoter and the nos terminator. The fragment containing S35:IQD22:nos was excised from pJD330 using BamHI/BglII and cloned into the binary transformation vector BIN19. Transformation of Arabidopsis thaliana col-0 was performed using the floral dip method (Clough S J and Bent A F, Plant J. 1998 December; 16, 735-43). Plants containing the transgene were selected on kanamycin plates (generation T1) and taken forward to homozygosity (T3). Experiments were performed with homozygous T3 plants.
Out of twelve positive transgenic plants, four plants (30%) showed a noticeable phenotype with rounder, thicker and darker green leaves (see
This alteration in growth is directly correlated with the expression level of the transgene as confirmed by Northern blotting as shown in
The photographs of
Measurements of the overall inflorescence height of 49 days old plants showed that stem height in the OE lines was reduced to 53% compared to wild type (see table 1 below). There was no change in internode number but there was a large reduction in internode length.
The stem diameter measured 1 cm above the rosette was increased by 60% in the IQD22 OE lines compared to WT (see table 1 below). Resin embedded stem cross sections shown in
The rosette phenotype obtained when IQD22 is overexpressed in Arabidopsis has significantly altered leaf shape, not being as elongated as WT and rounder than WT. As can be seen in
The IQD22 OE plant leaves are much thicker than WT. When they are bent they tend to snap and appear juicy—in a sense it is like the plant has become more of a succulent phenotype. Indeed, as shown in
The IQD22 OE lines senesce later than the WT (not measured, observed visually). Leaf shape of the IQD22 OE lines is altered. The length of the leaf blades of OE is reduced to 70-60% of WT and petioles showed a length reduction of 15-33% compared to WT. Leaf width in the OE lines is also reduced to 80% of WT at 33 days.
The height of the OE plants are 50% of WT. However, when considering the cells in the stem and in particular the interfascicular fibres (between the vein areas), these are shortened, as can be observed in
The effects on the stem of OE plants is interesting. The stem is generally shorter and shows significant increase in cross sectional diameter being 60% bigger, but this is due to an increase in the number of cells rather than size of cells.
For overexpressing plants where IQD22 is under the control of native promoter, and when grown under low white light conditions (60 μmol mm-2 sec-2)
The increased leaf thickness between wt and IQD22 overexpressing lines is reflected in an increase of leaf mass per unit area in the latter (see
In order to investigate where IQD22 is expressed in the plant, 1.6 kb of 5′ upstream sequence of the IQD22 gene, comprising the promoter sequence, were amplified and fused to the reporter gene GUS. With this construct, stable transgenic Arabidopsis lines were generated. In 7 day old seedlings GUS expression was detected in the hypocotyl and in what at first impression looked like vasculature (
The same IQD22 promoter fragment used for the GUS reporter construct was cloned in front of IQD22 which had been translationally fused at its 3′ end to the fluorescent protein mCherry (see
With this construct stable transgenic Arabidopsis lines were generated and analysed. The fluorescent fusion protein was associated with the cytoskeleton of the palisade cells (
The transgenic plants which were used for the localisation studies described in the previous section also showed an altered phenotype. Compared to the elongated leaf shape of the wildtype the leaves were much rounder and also felt thicker to the touch. In addition, the typical epinastic curling of the leaves of the wildtype is completely abolished in the lines expressing IQD22 under the control of its own native promoter (see
It is known that plants adapted to growth in arid habitats frequently are characterised by thicker leaves. As high IQD22 OE Arabidopsis lines had much thicker leaves and stems than WT, it was investigated whether the OE lines also had an advantage over the WT to cope with water stress. Two independent transgenic lines were chosen (‘fat1’ and ‘fat5’). Pots were filled with equal amounts of compost and plants grown under a normal watering regime for 4 weeks. Before starting to withhold water, pots were weighed again and brought to an equal weight by adding water. In addition, sellotape was placed over the compost in order to minimise water loss through evaporation from the compost surface. During the period of the progressive drying of the pots, pots were weighed daily and pictures were taken to record the response of the different lines. The experiment was repeated twice.
Recording the daily weight loss of the pots, the first observation made was that water loss of the pots containing the IQD22 OE lines was much reduced when compared to WT as they consistently weighed in heavier than the pots containing the WT plants (see
Secondly, as the series of pictures in
After 11 days of no watering, pots were thoroughly saturated with water and a normal watering regime was resumed for a couple of days. As can be seen in
The varying ability of the WT and IQD22 OE plants to cope with progressive drought was investigated by looking at gene expression. RNAseq was performed with RNA obtained from leaf samples at different time points during the imposition of the stress.
As can be seen in
As the PCA plot in
Looking at
To test whether differences in gas exchange between the WT and IQD22 OE lines could be detected measurements with the LI-6400XT Portable Photosynthesis System were performed. Data was collected at PAR (photosynthetic active radiation) levels of 500, 250, 125, 60 and 30 (μmol (photons) m−2 s−1) with 500 μmol m−2 s−1 representing saturating levels of PAR. Conductance and photosynthesis rate was measured at each light level and plotted against each other. The method of residual maximum likelihood (REML) was used to fit a multivariate linear mixed model to the photosynthesis rate and conductance data combined, testing (F-tests) for the main effects and interactions between the factors of light level and genotype, and accounting for the structure of the variance-covariance matrix underlying the data from the two variables. The plots thus obtained for IQD22 OE lines ‘fat1’ ‘fat5’ and ‘fat4’ are significantly different to WT with regard to conductance and show at the same photosynthetic rate a reduced conductance (see
In order to investigate whether a reduced number of stomata could explain the observed decrease in water loss of the IQD22 overexpressing lines, stomata numbers per mm−2 and stomatal index were determined. Although a higher number of stomata were counted per mm−2 leaf area for the IQD22 overexpressing lines, when stomatal index was measured no differences for those lines compared to wt were observed (
In Arabidopsis thaliana the two closest homologues of IQD22 are IQD23 (At5g62070) and IQD24 (At5g07240) (
Both IQD23 and IQD24 were cloned and tested whether they confer similar traits to Arabidopsis as IQD22 when overexpressed.
As is the case for IQD22, transgenic plants with thicker leaves were obtained for the S35:IQD23 and S35:IQD24 OE constructs. In both cases, not all the transformed plants displayed the phenotype of increased leaf thickness. This trait was shown to be related to expression levels of the transgenes. IQD23 and IQD24 OE also resulted in increases in leaf thickness and gave drought resistance compared to WT plants.
The drought experiment was set up as already described in Example 6 for IQD22 OE lines. After 4 weeks of growth under well-watered conditions in a long day cabinet (16 hours of daylight) watering was stopped and the response of the plants to progressive drought recorded.
Transgenic IQD24 OE were generated as described. As the case for IQD22 and IQD23, the IQD24 OE plants show increased leaf and stem thickness which is dependent on the expression level of the IQD24 transgene. The percentage of plants with a thicker phenotype obtained after the transformation was however lower than that for IQD23 and IQD22. The increase in thickness of leaves and stem is the trait that confers the ability to cope with water stress and so it is fully expected that the IQD24 OE lines also to do better under water scarce conditions compared to WT plants.
In order to establish whether IQD22 overexpression has an effect on seed yield, 12 plants each for wt and IQD22 OE lines ‘fat1’, ‘fat4’ and ‘fat5’ were grown to maturity and traits affecting seed yield recorded. Measurements of the length of the siliques revealed that in the IQD22 overexpressing lines silique length was in average ca 40% shorter than in wt (
As a consequence of the reduced silique length, the number of seeds obtained per silique is also reduced in the IQD22 overexpressing lines (see
Total seed yield was reduced in the IQD22 overexpressing lines (see
Where the WT and IQD22 OE lines were subjected to a severe drought stress during their growing period however, the seed yield obtained from transgenic OE lines was much higher than the one obtained from WT (see
The same construct as used in Arabidopsis Example 1 was used in this example Camelina sativa ‘Celine’. This is shown schematically in
Transformants were grouped into ‘strong’ or ‘intermediate’ transgenic lines depending on the strength of the phenotype shown. Both are fertile. As shown in
The ‘intermediate’ type plants are between WT and the ‘strong’ OE phenotype, having longer, thinner (i.e. more lanceolate) leaves than the strong ones. The plants are of normal height but slightly later flowering than WT. The seed pod shape was the same as that observed for the ‘strong’ lines.
This experiment was carried out in order to establish whether IQD22 OE confers the same enhanced ability to cope with water stress to Camelina as was observed for A. thaliana. Five plants displaying either the ‘strong’ or the ‘intermediate’ phenotype were planted out together with five wildtype plants. Before planting, it was ensured that all pots contained equal amounts of soil and had the same weight. After 4 weeks of growth, when flowers were about to open, the pot weights were brought again to equal weights with water and subsequently watering was stopped and the response of the plants to decreasing water availability was observed.
As reported for Arabidopsis, soil-drying occurred at a much slower speed in the case of the transgenic lines, suggesting a reduced transpiration rate for these plants. As is shown in
After 11 days without watering, WT Camelina showed distinctive stress symptoms such as severely wilted and yellowing leaves and drying of the inflorescences with shriveled flowers whereas the two transgenic lines still had green leaves (although they started to lose their turgor) and upright inflorescences. However, the ‘intermediate’ OE line showed stronger water-stress induced symptoms than the ‘strong’ line.
In both transgenic lines the water stress resulted in stunted inflorescences as the distance between seed pods was reduced when compared the growth habit of well-watered plants, as can be seen in
Watering of the plants was resumed after 11 days without water (after the pictures in
The results here are based on data obtained from 5 plants for wt, ‘intermediate’ and ‘strong’ phenotype plants respectively. Fully ripened seeds obtained from each plant were harvested and weighed. This was done for plants grown under optimal water conditions and also for the droughted plants described in Example 17.
As can be seen in
However, when plants were subjected to the severe water stress of withholding water for 11 days, after which normal watering was resumed, the IQD22-overexpressing lines performed much better than the wt plants (see
The reduction of seed yield observed in the well-watered transgenic lines compared to wt was not due to a reduction of seed number per seed pod (see
Transgenic A. thaliana plants are produced according to procedures described in Example 1, but the expression construct use comprises a native IQD22 promoter driving expression of a GUS reporter gene. The construct is shown in
Wild type hybrid poplar (Populus tremula X Populus alba Institut National de la Recherche Agronomique (INRA) clone 7171-B) was used. Transgenic poplar plants were produced according to the procedures described in Example 1 using an expression construct comprising an S35 constitutive promoter, the Arabidopsis IQD22 gene and the nos termination sequence. The expression construct is shown in
What the inventors have found with the transgenic plants made so far, is that when IQD22 is expressed constitutively (e.g. with a S35 promoter) the particular phenotype is related to the relative level of overexpression of the transgene. In practice, this means that some transgenic lines with high levels of expression show what is described herein as the ‘fat’ phenotype. However, when IQD22 is overexpressed using a native promoter, all of the lines analysed so far have leaves with increased cell layers in the leaf mesophyll and elongated palisade cells.
The following genetic resources were used in the making of the invention:
Seeds of Arabidopsis thaliana col-0 were obtained from the seed collection at Rothamsted Research, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom.
Seeds of Camelina sativa ‘Celine’ were obtained the seed collection at Rothamsted Research, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom.
Wild type hybrid poplar (Populus tremula X Populus alba) Institut National de la Recherche Agronomique (INRA) clone 7171-B was obtained from the University of Malaga, Spain.
Arabidopsis thaliana IQD22 protein (AT4G23060.1)
Arabidopsis thaliana IQD22 full length genomic DNA. Underlined sequence portion is the coding region. The start codon is marked in bold:
ATGGG AAAAGCGTCA
CGGTGGTTTA GGAGTCTATT CGGAGTTAAG AAACCCGACC
CGGGTTATCC
GGATCTATCC GTCGAGACGC CTTCTCGGTC AACTTCCTCT
AATCTCAAAC
GCCGATGGAG TTTCGTCAAA TCCAAACGAG AAAAAGAAAG
TACACCGATT
AATCAAGTTC CTCATACTCC ATCGCTACCG AATTCGACGC
CTCCACCGCC
GTCTCACCAC CAATCGTCGC CGAGACGACG GAGAAAACAA
AAGCCGATGT
GGGAGGATGA GGGAAGTGAA GATTCGGACA AGCATGCTAT
TGCGGTGGCT
GCCGCGACTG CTGCGGTTGC TGAAGCTGCA GTCGCCGCCG
CTAATGCTGC
TGCTGCGGTC GTCAGGCTGA CGAGCACAAG TGGGAGGTCG
ACTCGAAGTC
CTGTTAAGGC ACGGTTTAGC GACGGATTCG ACGACGTGGT
GGCGCATGGT
AGCAAGTTTT ATGGACACGG CCGTGACAGT TGTGAACTTG
CGGTGATTAA
GATACAATCT ATATTTCGCG GATACTTG GT AATTTTCTTT
GCA AAGAGAGCGT
TAAGGGCACT CAAGGGTTTG GTTAGGCTTC AAGCGATAGT
TAGAGGCCAT
ATCGAAAGAA AGAGAATGTC AGTCCATCTG CGCAGGATGC
ACGCTTTGGT
TCGAGCTCAG GCTCGTGTGC GTGCCACTCG GGTTATTGTC
ACGCCTGAAT
CTTCTTCTTC TCAATCCAAC AATACCAAAT CTTCTCACTT
CCAAAACCCT
TCCACCAACT CCGGAAAAAC TCGAGCATTC GATCTCTTCT
CGCAGCTCCA
AACTCGCTCA TTCTCATCTT TTCAAG GTAT TACATTTTCA
AGGAATGGTT
CGAAGGCAAG CGACAACAAC AGACTGTACC CTGCTCACAG
GGAGACATTC
TCAGCCACAG ACGAAGAAGA AAAGATTCTT CAAATCGACA
GGAAACACAT
CAGTTCTTAC ACAAGACGCA ACAGACCAGA CATGTTCTAC
TCATCCCACC
TCATCCTAGA CAATGCTGGC CTGTCTGAAC CAGTTTTCGC
CACGCCTTTT
AGCCCGTCCT CGTCGCATGA AGAGATTACA AGCCAGTTTT
GCACTGCAGA
GAACAGTCCT CAGTTATACT CAGCTACTTC TAGAAGCAAA
CGCAGTGCTT
TCACCGCTAG TTCTATAGCA CCGAGCGATT GCACAAAAAG
CTGCTGTGAT
GGTGACCATC CAAGCTACAT GGCTTGTACA GAGTCCTCTA
GGGCTAAGGC
TAGGTCCGCT AGTGCCCCGA AGTCTCGACC ACAGTTATTT
TACGAGCGGC
CTTCATCAAA ACGGTTTGGA TTTGTTGATT TGCCGTACTG
TGGTGATACA
AAGTCCGGTC CCCAGAAAGG CTCTGCTCTG CATACTAGTT
TTATGAACAA
GGCTTATCCC GGTTCAGGTC GGTTGGACCG TCTCGGGATG
CCAATTGGGT
ATAGGTACTG A GAACGTTGT TGGACCCTTT AACCTGGTTT
Arabidopsis thaliana IQD22 Full Length cDNA:
Arabidopsis thaliana IQD23 Protein (AT5G62070.1)
Arabidopsis thaliana IQD23 Full Length Genomic DNA:
Arabidopsis thaliana IQD23 Full Length cDNA
Arabidopsis thaliana IQD24 Protein (AT5G07240.1)
Arabidopsis thaliana IQD24 Full Length DNA
Arabidopsis thaliana IQD24 cDNA
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.
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.
The readers 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1903485.9 | Mar 2019 | GB | national |
| 1905793.4 | Apr 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2020/050639 | 3/13/2020 | WO | 00 |
