WO2011047433A1 - Procédé de modification du développement et de la productivité de plantes - Google Patents

Procédé de modification du développement et de la productivité de plantes Download PDF

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WO2011047433A1
WO2011047433A1 PCT/AU2010/001402 AU2010001402W WO2011047433A1 WO 2011047433 A1 WO2011047433 A1 WO 2011047433A1 AU 2010001402 W AU2010001402 W AU 2010001402W WO 2011047433 A1 WO2011047433 A1 WO 2011047433A1
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plant
cell
arf2
seq
expression
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PCT/AU2010/001402
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English (en)
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Brian Jones
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The University Of Sydney
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development

Definitions

  • the invention relates to a method of producing plants with desirable properties by modulating the expression and/or activity of Auxin Response Factors (ARFs), and plants produced by the method. It also relates to transgenic plants having modulated expression and/or activity of Auxin Response Factors (ARFs), and seeds, plant cells, plant parts and other types of propagating materials, and uses thereof.
  • Auxin Response Factors Auxin Response Factors
  • An object of plant genetic engineering or conventional breeding is to produce novel plants with agronomically, horticulturally, or economically important traits including increased tolerance to a variety of environmental stresses, e.g., water availability, altered growth characteristics, improved plant yield, and seed quality. Plant yield and altered growth characteristics further impacts economically valuable properties of marketable plant products.
  • modified wood properties are considered desirable by the forest and pulp industries.
  • the expensive, energy-intensive, and environmentally hazardous process of turning wood into paper is attributed in part to the need to separate cellulose from lignin in wood and, as a consequence, plants having modified xylem structure or composition e.g., fast-growing, low-lignin trees, are highly desirable.
  • wood comprises layers of secondary xylem impregnated with lignin and comprises both living and dead cells.
  • Functional xylem in layers adjacent to cambium, is primarily a water and mineral-conducting tissue, whereas functional phloem, also in layers adjacent to the cambium, primarily conducts photoassimilates and signalling molecules.
  • Drought and flooding rain are defining features of the Australian landscape and are becoming increasingly prevalent globally due to climate change. It is desirable to minimize the impact that this fluctuating supply and the associated reduction in soil moisture content has on our primary production. Accordingly, it is desirable to identify genes that are implicated in a plant's ability to cope with a drying soil so that the knowledge can be used to the advantage of breeders and growers alike. Since xylem vessels are the main arteries for the transport of water and phloem is the main means for distribution of photoassimilates by plants, modification of xylem structure or function may provide desirable benefits to plants in times of environmental stresses such as water deficiency and/or mineral nutrient deficiency.
  • the time required to produce a new variety could be reduced significantly.
  • a biotechnological approach would allow closer targeting of traits considered desirable by the forest and pulp industries, in specific tree species.
  • Auxins are known to regulate many of the physiological events in a plant's life cycle.
  • the Auxin Response Factor (ARF) and Aux/IAA families are two families of transcription factor proteins important for transducing the perception of auxin through to changes in gene expression (Guilfoyle et al. (1998) Plant Physiol. 1 18:341 -347).
  • ARFs bind to auxin response cis-acting elements through an N-terminal DNA binding domain, while the carboxy-terminus contains two protein-protein interaction domains that are also found in the Aux/IAA family of early auxin-response genes.
  • ARFs are encoded by a large gene family comprising at least 23 members, of which many have unknown function.
  • a typical ARF protein comprises an N-terminal B3-like DNA-binding domain, C-terminal domains III and IV similar to those found in the C terminus of Aux/IAAs, and an intervening region that determines whether the ARF functions as a repressor or enhancer of gene expression.
  • ARFs bind to auxin-responsive cis-acting elements (AuxREs) found in the promoter regions of auxin-responsive genes.
  • Certain ARFs are known to regulate plant growth responses to blue light, and plant morphologies such as gynoecium patterning, vascular strand formation in the early embryo, and hypocotyl elongation and bending during germination. Recently, Okushima et al. (2005) The Plant Cell 17, 444-463 attempted to characterize the functions of most A.
  • thaliana ARFs by producing T-DNA insertion mutants for 18 of the 23 ARF gene family members and, notwithstanding the authors failed to demonstrate an obvious growth phenotype for most ARFs, they disclose severely-impaired lateral root formation and abnormal gravitropism in both hypocotyl and root of double ARF7/ARF19 mutants, unusual gynoecium and floral patterning defects in ARF3-deficient mutants, abnormal root meristem and cotyledon development in ARF5-deifcient mutants, impaired phototropic responses toward blue light for ARF7-deficient mutants, and pleiotropic phenotypes for ARF2-deficient mutants, including abnormal inflorescence stem, leaf and flower morphology, and flowering time.
  • auxin signalling relevant to xylem and secondary xylem development and/or structure, including effects on water use efficiency and/or stomatal conductance and/or transpiration rate and/or wood quality and/or pulpability and/or paper quality and/or coarseness.
  • the inventors sought to identify genes useful for manipulating plant properties, for example, properties of economic importance. For example, the inventors sought to identify factors that affect secondary xylem development and/or structure, including effects on water use efficiency and/or stomatal conductance and/or transpiration rate and/or wood quality and/or pulpability and/or paper quality and/or coarseness. As exemplified herein, the inventors have shown that modified expression of the Auxin Response Factor 2 (ARF2) gene in Arabidopsis thaliana produces modified or altered secondary xylem.
  • Auxin Response Factor 2 Auxin Response Factor 2
  • ARF2-deficient plants were found to have significantly enhanced stem and/or hypocotyl thickness, such as determined by measuring stem and/or hypocotyl diameter and/or the diameter of secondary xylem (wood) tissue, compared to wild-type (WT) plants.
  • ARF2-deficient plants exhibited enhanced growth level or growth rate compared to WT plants grown under identical environmental conditions.
  • the growth rate in terms of above and/or below ground biomass accumulation of ARF2-deficient plants is about 2- fold greater or about 3-fold greater or about 4-fold greater or about 5-fold greater or about 10-fold greater than WT plants.
  • the number of small vessels e.g., vessels having a cross-sectional area of about 0-20 ⁇ 2 or less
  • the number of large vessels e.g., vessels having a cross-sectional area of more than about 0-20 ⁇ 2 is reduced compared to the level of large vessels in WT plants.
  • modified expression of the Auxin Response Factor 18 (ARF18) gene in Arabidopsis thaliana produces modified xylem structure, and water use efficiency, in addition to altered leaf size, modified primary root growth and lateral root production, modified hypocotyl length and growth rate, modified flowering time, and modified seed yield. More particularly, the inventors have shown that hypocotyls of the ARF18-deficient plants have larger fibres compared to wild-type (WT) plants, indicating that ARF18 is important for determining xylem structure. Alternatively, or in addition, water usage rate is reduced in ARF18- deficient plants relative to WT plants expressing ARF18.
  • ARF18 Auxin Response Factor 18
  • stomatal conductance is reduced in ARF18-deficient plants relative to WT plants expressing ARF18.
  • transpiration rate is reduced in ARF18-deficient plants relative to WT plants expressing ARF18.
  • the rate of photosynthesis is reduced in ARF18-deficient plants relative to WT plants expressing ARF1 8.
  • leaf area is enhanced in ARF18-deficient plants relative to WT plants expressing ARF1 8.
  • ARF2 and A RF 18 exert opposing actions on one or more auxin-responsive genes affecting cell expansion in cells of the functional xylem and/or secondary xylem, including cells comprising vessels.
  • ARF2-deficient plants cell expansion is limited in vessels, whereas ARF I 8-deficient . plants exhibit larger xylem vessels than wild-type plants.
  • ARF18 acts to enhance expression of one or more genes regulating cell expansion in xylem thereby leading to xylem cell expansion.
  • ARF2 and ARF18 may exist in dynamic equilibrium in xylem cells, such that a relative level of ARF2 and ARF18 determines vessel and fibre development, including e.g., radial expansion and/or fibre length and/or vessel length and/or vessel diameter.
  • the present invention provides a method of modifying a phenotype mediated by ARF2 in a plant, said method comprising modulating the expression of one or more Auxin Response Factor- 18 (ARF18) genes in the plant.
  • Auxin Response Factor- 18 Auxin Response Factor- 18
  • a phenotype that is enhanced by expressing ARF2 in a plant may be inhibited or reduced by expressing at least one ARF18-encoding polynucleotide in the plant.
  • a phenotype that is repressed or inhibited by expressing ARF2 in a plant may be enhanced or de-repressed by expressing at least one ARF18-encoding polynucleotide in the plant.
  • the present invention provides a method of modifying a phenotype of a plant, said method comprising reducing or inhibiting the expression of one or more Auxin Response Factor-18 (ARF18) genes in the plant, wherein the modified phenotype is selected from increased root growth, reduced leaf length, reduced petiole length, delaying flowering, enhanced flower size, increased hypocotyl length, enhanced seed yield, enhanced embryo size, enhanced seed size, enhanced tracheid fibre diameter, enhanced xylem vessel diameter, enhanced water use efficiency, enhanced drought tolerance, and enhanced or improved recovery from water stress or water deficiency.
  • Auxin Response Factor-18 Auxin Response Factor-18
  • reduced or ablated expression of ARF18 and/or activity of ARF18 enhances cellular expansion, thereby leading to increased cell size in xylem vessels and to a larger lumen size, enhanced biomass, larger seeds, enhanced root growth and/or size, and/or enhanced water usage efficiency or improved productivity during 'and/or following water stress or water deficit.
  • An enhanced water usage efficiency is a desirable property as it leads to a greater ability of the plant to cope with drying soil, when-water supply is reduced, intermittent, or during drought conditions.
  • Enhanced water usage efficiency may also be related to the root growth and/or size and/or to the increased cellular expansion in xylem.
  • Enhanced biomass is desirable for increasing plant and crop yields.
  • Woody plants according to the invention exhibiting enhanced cellular expansion in secondary xylem and/or increased coarseness are useful for production of solid wood products.
  • increased expression of ARF18 and/or activity of ARF18 decreases cellular expansion, reduced average xylem cell size, tracheid fibre diameter, xylem vessel diameter, coarseness and lignin content. Decreased cellular expansion lead to decreased cell size in secondary xylem and/or reduced coarseness and/or reduced lignin content.
  • Woody plants according to the invention exhibiting reduced cellular expansion in secondary xylem and/or low coarseness or low lignin content have utility in pulp and paper milling.
  • the present invention provides a method of modifying a phenotype of a plant, said method comprising reducing or inhibiting the expression of one or more Auxin Response Factor-2 (ARF2) genes in the plant, wherein the modified phenotype is selected from reduced average xylem cell size, reduced tracheid fibre diameter, reduced xylem vessel diameter, decreased cell size in xylem, reduced coarseness and reduced lignin content.
  • Auxin Response Factor-2 ARF2
  • increased expression of ARF2 and/or activity of ARF2 increases cellular expansion, particularly in fibre and vessels, increases root growth, reduces leaf length, reduces petiole length, delays flowering, enhances flower size, increases hypocotyl length, enhances seed size, enhances embryo size, enhances tracheid fibre diameter, enhances xylem vessel diameter, enhances water use efficiency, enhances drought tolerance, or enhances or improves recovery from water stress or water deficiency.
  • the present invention provides a method for modifying a xylem structure and/or xylem development in a plant such as a fibre-producing plant or a woody plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 and/or ARF18 are modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 expression is increased and ARF18 expression is decreased in the plant to achieve this increase in vessel and/or fibre production.
  • the present invention provides a method for improving a paper milling and/or pulp milling property of a woody plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 and/or ARF18 are Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • the present invention provides a method for reducing lignin content of a plant such as a fibre-producing plant or a woody plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 and/or ARF18 are a plant having reduced lignin content of secondary xylem.
  • ARF2 expression is decreased and ARF18 expression is increased in the plant.
  • the present invention provides a method for enhancing water use efficiency of a plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 and/or ARF18 are modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 expression is increased and ARF18 expression is decreased in the plant.
  • the present invention provides a method for enhancing drought tolerance of a plant and/or recovery from water deficit in a plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 and/or ARF18 are Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant.
  • ARF2 expression is increased and ARF18 expression is decreased in the plant.
  • Auxin Response Factors e.g.; ARF2 and/or ARF18
  • ARF2 and/or ARF18 are increased or enhanced by providing plant cells with a chimeric gene comprising the following operably linked DNA fragments:
  • a polynucleotide comprising a nucleotide sequence that encodes the Auxin Response Factor; and .
  • a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell.
  • Preferred polynucleotides that encode an auxin responsive factor will encode ARF2 or ARF18 and comprise a sequence selected from the group consisting of:
  • nucleic acid primers each comprising at least about 15 contiguous nucleotides of (i) or (ii).
  • the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 is decreased or reduced or inhibited by providing an antisense R A, co- suppression molecule, interfering RNA (iRNA) or double-stranded RNA (dsRNA) or RNAi molecule to a plant cell.
  • a chimeric gene construct comprising nucleic acid that is expressed to produce an antisense RNA, co-suppression molecule, interfering RNA (iRNA) or double-stranded RNA (dsRNA) or RNAi molecule may be expressed in the plant cell to thereby reduce expression of the Auxin Response Factor(s).
  • the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 is decreased or inhibited or reduced by providing plant cells with a chimeric gene comprising the following operably linked DNA fragments:
  • a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor as described herein and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the auxin responsive factor;
  • Exemplary dsRNA molecules for inhibiting the expression of an Auxin Response Factor will comprise a sense strand and an antisense strand that are complementary to each other, wherein the antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding an Auxin Response Factor wherein a region of complementarity with said mRNA is less than 30 nucleotides in length, and more generally 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 nucleotides in length.
  • the dsRNA upon contacting with a cell expressing the Auxin Response Factor may inhibit expression of a gene encoding the Auxin Response Factor by at least about 30% or 40% or 50% or 60% or 70% or 80%.
  • Exemplary sense strand sequences thus comprise at least about 19 contiguous nucleotides from a polynucleotide that encodes an auxin responsive factor as described herein.
  • Exemplary antisense strand sequences comprise at least about 19 contiguous nucleotides complementary to at least about 19 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor as described herein.
  • Exemplary antisense RNAs will comprise a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding an Auxin Response Factor wherein a region of complementarity with said mRNA is more than 30 nucleotides in length, and more generally 50 or 100 or 150 or 200 or 250 or 300 or 400 or 500 nucleotides in length and preferably complementary to the 3'-end of said mRNA.
  • antisense mRNA is substantially complementary to the full- length of the coding region of mRNA and the 3'-untranslated region thereof.
  • Exemplary antisense sequences thus comprise at least about 30 contiguous nucleotides complementary to at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor as described herein.
  • the promoter is a constitutive promoter e.g., CaMV 35S promoter or ubiquitin gene promoter.
  • the promoter is a fiber-specific promoter.
  • the promoter is operable in the cambium and/or xylem.
  • the transcription terminator and polyadenylation signal may be any 3-untranslated region of a plant gene.
  • the chimeric gene may be contained within an expression vector, which is transferred and stably incorporated into a plant genome by standard procedures e.g., Agrobacterium-mediated transformation as described for example by Fraley et al. ( 1983) Proc. Natl. Acad. Sci. USA. 80: 4803-4807, or by employing microparticle bombardment of plant cells by the biolistics method described by Klein et al ( 1987) Nature. 327:70-73. Cells are regenerated into whole plants according to standard procedures for regenerating plants. Transformed plants carrying the chimeric gene or an expression vector comprising same are then selected and screened to demonstrate that they exhibit the desired phenotype(s).
  • the plant according to. any example hereof may be a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.
  • a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.
  • the plant according to any example hereof may be a fiber-producing plant e.g., flax or cotton.
  • the plant according to any example hereof, especially those examples producing a modified pulp or paper milling property may be a wood-producing plant e.g., oak, aspen, eucalyptus, maple, pine, spruce, poplar, or larch.
  • the plant may be a species of Eucalyptus ( E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi E. camaldulensis, E.
  • Eucalyptus E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brach
  • E. glaucina E. globulus, E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis *urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora, E.
  • Sitka spruce (Picea glauca); or redwood (Sequoia sempervireris); or a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea); or a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis).
  • a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea)
  • a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis).
  • a further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF2 in a plant such as by increasing expression of ARF18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments: .
  • a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor- 18 ( ARF 18), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • ARF 18 Auxin Response Factor- 18
  • Preferred polynucleotides that encode ARF 18 comprise a sequence selected from the group consisting of:
  • nucleic acid primers each comprising at least about 15 contiguous nucleotides of (i) or (ii).
  • a further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF2 in a plant such as by down-regulating expression of ARF 18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:
  • a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor-18 (ARF 18) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF18; and
  • a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • this chimeric gene is capable of expressing antisense RNA, a co-suppression molecule, interfering RNA (iRNA), a double-stranded RNA (dsRNA) or RNAi molecule as described herein above in a plant cell, and that the sequence of the polynucleotide at (ii) supra is readily derived from a sequence encoding ARF18 as described according to any example hereof. Structures of exemplary dsRNA molecules and antisense molecules are as described herein above and said structural features apply mutatis mutandis to this example of the invention.
  • a further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF18 in a plant such as by increasing expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:
  • a promoter that is operable in the plant cell, as described according to any example hereof; ii) a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor-2 (ARF2), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • Auxin Response Factor-2 ARF2
  • Preferred polynucleotides that encode ARF2 comprise a sequence selected from the group consisting of:
  • nucleic acid primers each comprising at least about 15 contiguous nucleotides of (i) or (ii).
  • a further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF18 in a plant such as by down-regulating expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:
  • a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor-2 (ARF2) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF2; and
  • iii a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • this chimeric gene is capable of expressing antisense RNA, a co-suppression molecule, interfering RNA (iRNA), a double-stranded RNA (dsRNA) or RNAi molecule as described herein above in a plant cell, and that the sequence of the polynucleotide at (ii) supra is readily derived from a sequence encoding ARF2 as described according to any example hereof.
  • Structures of exemplary dsRNA molecules and antisense molecules are as described herein above and said structural features apply mutatis mutandis to this example of the invention.
  • a further example of the present invention provides the use of a chimeric gene as described according to any example hereof to modify a phenotype mediated by ARF2 in a plant by increasing expression of ARF18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:
  • a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor- 18 (ARF18), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • Auxin Response Factor- 18 ARF18
  • a further example of the present invention provides the use of a chimeric gene as described according to any example hereof to modify a phenotype mediated by ARF2 in a plant by down-regulating expression of ARF18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA ( fragments:
  • a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an Auxin Responsive Factor-18 (ARF18) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF18; and
  • a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • a further example of the present invention provides the use of a chimeric gene as described according , to any example hereof to modify a phenotype mediated by ARF18 in a plant by increasing expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments: i) a promoter that is operable in the plant cell, as described according to any example hereof;
  • a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor-2 (ARF2), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • Auxin Response Factor-2 ARF2
  • a further example of the present invention provides the use of a chimeric gene as described according to any example hereof to modify a phenotype mediated by ARF18 in a plant such as by down-regulating expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:
  • a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an Auxin Responsive Factor-2 (ARF2) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF 18; and
  • a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.
  • this chimeric gene is capable of expressing antisense RNA, a co-suppression molecule, interfering RNA (iRNA), a double-stranded RNA (dsRNA) or RNAi molecule as described herein above in a plant cell, and that the sequence of the polynucleotide at (ii) supra is readily derived from a sequence encoding ARF2 as described according to any example hereof. Structures of exemplary dsRNA molecules and antisense molecules are as described herein above and said structural features apply mutatis mutandis to this example of the invention.
  • a further example of the present invention provides an expression vector comprising a chimeric gene according to any example hereof and for the use of the expression vector to modify a phenotype mediated by ARF2 and/or ARF18 in a plant.
  • a further example of the present invention provides a plant cell comprising one or more chimeric genes according to any example of the invention described herein.
  • the present invention provides a plant, seed, plant cell, plant tissue, or propagating material thereof having modulated expression of ARF2 and/or ARFl 8 produced according to any example hereof.
  • Figure I provides copies of photomicrographs of transverse sections of hypocotyl of an A. thaliana ARF2-deficient mutant (panel A) and wild-type A. thaliana ecotype C24 (panel B). Data indicate small xylem cell size for the ARF2-deficient mutant plant.
  • Figure 2 provides copies of photomicrographs of transverse sections of hypocotyl of an A. thaliana ARF2-deficient mutant (panel A), an A, thaliana ARFl 1 -deficient mutant (panel B), an A. thaliana ARF13-deficient mutant (panel C), an A. thaliana ARF l 4- deficient mutant (panel D), an A. thaliana ARF15-deficient mutant (panel E), an A. thaliana ARF3-deficient mutant (panel F), an A. thaliana ARF19xARF7-double mutant deficient in both ARF19 and ARF7 (panel G), an A. thaliana ARF22-deficient mutant (panel H), an A. thaliana ARF4-deficient mutant (panel I), an A.
  • FIG. 3 provides a graphical representation showing the smaller xylem cell cross- sectional area for A. thaliana ARF2-deficient mutant (ARF2; right columns in each pair) relative to wild-type A, thaliana ecotype C24 (WT-Col; left columns in each pair).
  • A. thaliana ARF2-deficient mutant ARF2; right columns in each pair
  • WT-Col thaliana ecotype C24
  • Four size classes of xylem cell area are indicated on the x-axis. Numbers of cells in each size class are indicated on the abscissa. Sample size was 335 for each of ARF2- deficient mutant and WT-Col. Data indicate a predominance of smaller cells in the ARF2-deficient mutant.
  • Figure 4 provides copies of photomicrographs of transverse sections of hypocotyl of an
  • A. thaliana ARF18-deficient mutant (panel A, left) and wild-type A. thaliana ecotype C24 (panel A, right), and for transverse sections of flower stems of the A. thaliana ARF18-deficient mutant (panel B, left) and wild-type A. thaliana ecotype C24 (panel
  • Figure 5 provides copies of photographic representations showing A. thaliana ARF18- deficient mutant plants (N9299, left hand side) and wild-type A. thaliana ecotype C24 (C24, right hand side), after 12 days of water deficit (above) and 6-days after recommencement of watering (below). Data indicate reduced wilting of ARF18- deficient plants during the period of water deficit and more rapid recovery and enhanced survival rate of A F18-deficient plants following recommencement of watering, compared to wild-type C24.
  • Figure 6 provides graphical representations showing the rates of photosynthesis (top left panel), stomatal conductances (tope right panel), transpiration rates (lower left panel), and water use efficiencies (lower right panel) of A. thaliana ARF1 8-deficient mutant plants (N9299) and wild-type A. thaliana ecotype C24 (C24) at day 3 and day 7 after cessation of watering of plants.
  • Data indicate that ARF 18-deficient plants maintained a photosynthetic rate, stomatal conductance, transpiration rate and overall water use efficiency (WUE) during water deficit stress, whereas wild-type plants exhibit reduced rates of photosynthesis, transpiration, stomatal conductance and water use efficiency under the same conditions.
  • Figure 7 provides graphical representations showing water content of leaves at day 1 through day 12 of water deficit (top panel) and average leaf areas at day 3 of water deficit (lower panel) for A. thaliana ARF18-deficient mutant plants (diamonds in top panel; 299 in lower panel) and wild-type A. thaliana ecotype C24 (squares in top panel; C24 in lower panel). Differential water content between the lines is also indicated in the top panel (triangles). Data indicate that the A RF18-deficient plants lose less water during a period of water stress than wild-type C24 plants, as indicated by the negative value for differential water content between the lines, and marginally higher leaf area than wild-type C24 plants.
  • Figure 8 provides copies of photographic representations showing additional phenotypes of ARF18-deficient plants relative to wild-type C24 plants, including a rounder and wavier leaves (panels a and b), a delayed flowering (panel c), larger seeds (panel d), larger flowers (panel e), and longer primary roots (panel ⁇ ) ⁇
  • Figure 9 provides a graphical representation showing an elevated root elongation rate for ARF18-deficient plants relative to wild-type C24 plants during the first 12 days of growth.
  • the present invention provides methods and chimeric genes for modulating one or more phenotypes of a plant or a plant cell, plant organ or plant tissue.
  • the methods and chimeric genes employ polynucleotides comprising one or more sense strand sequences each of which is derived from a gene encoding an ARF2 or ARF 18 polypeptide. It will be understood by the skilled artisan that a sense strand sequence corresponds to sequence in mRNA encoding an ARF2 or ARF 18 polypeptide, however if the sense strand sequence is in DNA as opposed to mRNA it will generally comprise thymidine residues in place of uracil.
  • the chimeric gene and method may provide for ectopic expression of ARF2 and/or ARF18 polypeptides in a plant cell, tissue, organ or throughout the plant.
  • a sense strand sequence may also be employed to reduce or inhibit ARF2 and/or ARD18 expression in a plant cell, tissue, organ or whole plant, for example wherein it comprises an incomplete or partial open reading frame.
  • Polynucleotides comprising only a partial open frame of an ARF2 or ARF 18 gene may be employed as dominant negative mutants or for co-suppression of an endogenous level of expression of ARF2 and/or ARF18.
  • a sense strand sequence generally comprising only about 19-30 contiguous nucleotides of a gene encoding ARF2 or ARF18, or a combination of such sense strand sequences, may be employed in the construction of RNAi or dsRNA molecules capable of reducing or inhibiting or preventing an endogenous level of expression of ARF2 and/or ARF 18 in a plant cell, tissue, organ or throughout the plant.
  • the methods and chimeric genes of the present invention employ polynucleotides comprising one or more antisense strand sequences i.e., complementary to at least a part of mRNA encoding an ARF2 or ARF18 polypeptide and, if the antisense strand sequence is in DNA as opposed to mRNA it will generally comprise thymidine residues in place of uracil.
  • Antisense strand sequences are generally employed to reduce or inhibit or prevent expression of mRNA to which it is complementary i.e., mRNA encoding an ARF2 or ARF18 polypeptide.
  • the antisense strand sequences are complementary to a plurality of mRNAs such as because they are presented in a chimeric gene in a tandem array or linked contiguously or non- contiguously, they are employed to reduce or inhibit or prevent expression of the plurality of mRNAs.
  • Preferred methods for reducing expression of one or more mRNAs in a cell by employing antisense strand sequences include antisense technology, ribozyme technology, RNAi and dsRNA.
  • an antisense RNA may employ at least about 50 contiguous nucleotides complementary to ARF2- encoding mRNA and/or at least about 50 contiguous nucleotides complementary to ARF18-encoding mRNA, such as an antisense sequence comprising or consisting of at least 50 contiguous nucleotides of a 3'-untranslated sequence of mRNA and/or protein- encoding sequence.
  • an antisense sequence comprising or consisting of at least 50 contiguous nucleotides of a 3'-untranslated sequence of mRNA and/or protein- encoding sequence.
  • shorter regions of contiguous antisense strand sequence may be employed e.g., at least about 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 and preferably no more than 30 nucleotides in length.
  • the ARF2-derived and/or ARF18-derived sense and antisense strand sequences are employed in the manufacture of chimeric genes for modifying one or more properties and/or traits.
  • the present invention provides a method for modifying a desirable property and/or trait in a plant, which method comprises modulating the expression of an ARF2 gene and/or an ARF18 gene whose expression or transcription product is capable of directly or indirectly modifying a desirable property and/or trait in the plant or plant cell, plant part, tissue, seed or plant propagating material thereof.
  • An ARF2 gene and/or transcription product or an ARF18 gene and/or transcription product may comprise the nucleotide or amino acid sequence from Arabidopsis thaliana or an ortholog thereof from another plant species e.g., poplar or Eucalyptus spp.
  • the method of the invention may comprise the step of transforming a plant or plant propagating material with a nucleic acid molecule comprising a regulatory sequence, typically a- promoter sequence, capable of modulating expression within the plant of a nucleic acid molecule corresponding to an ARF2 gene or an ARF18 gene whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby, on expression of that sequence, the desirable property and/or trait is modified.
  • an expression cassette comprising the chimeric gene may be used to either enhance, reduce or inhibit the expression of ARF2 and/or ARF 18, or to enhance, reduce or inhibit the activity of ARF2 and/or ARF18 in the plant, seed, plant cell, tissue or plant propagating material thereof, thereby modulating the desirable property and/or desirable trait in the plant.
  • a plant-operable promoter including a plant promoter, may be operably linked to a coding region of the ARF2 and/or ARF 18 gene in the sense orientation.
  • expression of ARF2 and/or ARF18 gene(s) may be modulated by operably linking a plant promoter to a nucleic acid fragment from the gene(s) to thereby form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed.
  • the expression of ARF2 and/or ARF 18 may be modulated by introducing one or more nucleic acid fragments of the gene(s) into an appropriate vector such that double-stranded RNA is transcribed where directed by i.e., under the control of an operably-linked plant promoter, thereby producing decreased levels of mRNA and/or protein encoded by endogenous copies of the gene(s).
  • Levels of mRNA and protein encoded by orthologs or homologs of the gene(s) may also be reduced.
  • expression of ARF2 and/or ARF 18 gene(s) may be modulated by operably linking a plant promoter to a dominant-negative allele of the gene, which interferes with the function of the gene product.
  • the modulated expression of ARF2 and/or ARF 18, or the modulated activity of ARF2 and/or ARF18 in the plant, seed, plant cell, tissue or plant propagating material thereof may comprise an enhanced, reduced, or inhibited expression and/or activity relative to the expression of an otherwise isogenic plant not comprising the chimeric gene of the invention.
  • the modulated expression of ARF2 and/or ARF 18 and/or the modulated activity of ARF2 and/or ARF 18 results in an altered response of an auxin gene responsive element which drives the transcription of auxin controlled genes to thereby modulate the desirable property and/or desirable trait in the plant.
  • ARF2 and/or ARF18 may each act on a factor in the auxin signalling pathway, e.g., another Auxin Response Factor, to modulate expression of auxin- controlled genes thereby modulating the desirable property and/or desirable trait in the plant.
  • ARF2 may modulate the expression/activity of ARF18 whose expression or transcription product directly or indirectly modifies a desirable property and/or trait in the plant or plant cell, plant part, tissue, seed or plant propagating material thereof.
  • ARF18 may modulate the expression of the ARF2 gene whose expression or transcription product is capable of directly or indirectly modifying a desirable property and/or trait in the plant or plant cell, plant part, tissue, seed or plant propagating material thereof.
  • ARF2 and ARF18 may be in dynamic equilibrium to control auxin-regulated gene expression.
  • the desirable trait according to any example described herein is selected from, but not limited to altered leaf and/or vascular structure, altered secondary xylem structure including lumen size and cell wall thickness, and altered growth characteristics including cellular proliferation and/or cellular expansion.
  • a desirable, property is obtained such as increased plant size, root growth and/or root size, seed quality and/or quantity, xylem (wood) quality and water usage efficiency.
  • reduced or ablated expression of ARF2 and/or activity of ARF2 may enhance cellular expansion, leading to increased cell size in secondary xylem and to a larger lumen size, enhanced biomass, larger seeds, enhanced root growth and/or size, and/or enhanced water usage efficiency.
  • Woody plants according to the invention exhibiting enhanced cellular expansion in secondary xylem and/or reduced wall thickness and/or low density are useful for production of pulp and paper products.
  • reduced or ablated expression of ARF18 and/or activity of ARF18 may also enhance cellular expansion leading to increased cell size in secondary xylem and to a larger lumen size, enhanced biomass, larger seeds, enhanced root growth and/or size, and/or enhanced water usage efficiency, and a greater ability of the plant to cope with drying soil when water supply is. reduced, e.g., during intermittent drought conditions.
  • Enhanced water usage efficiency may also be related to the higher root growth rate and/or increased size and/or increased cellular expansion in xylem of ARF 1 8-deficient plants.
  • Enhanced biomass is desirable for increasing plant and crop yields.
  • Woody plants according to the invention exhibiting enhanced cellular expansion in secondary xylem and/or increased coarseness are useful for production of solid wood products.
  • Increased expression of ARF18 and/or activity of ARF18 according to any example hereof may decrease cellular expansion, leading to a decreased cell size in secondary xylem and to reduced or low coarseness of xylem, and a small seed size when desirable e.g., for the production of parthenocarpic fruits.
  • Woody plants according to the invention exhibiting reduced cellular expansion in secondary xylem and/or low coarseness are useful for production of pulp and paper products.
  • the present invention extends to a plant, seed, plant cell, plant tissue, or propagating material thereof produced by the method according to any example described herein, such as a plant, seed, plant cell, plant tissue, or propagating material having modulated expression of the ARF2 and/or ARF 18 gene(s).
  • a chimeric gene of the present invention comprises a plant-operable promoter operably linked to a DNA region coding for an ARF2 and/or ARF 18 protein comprising the amino acid sequence of SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32 or a variant thereof having similar activity as the mentioned proteins, and a 3'-untranslated region involved in transcription termination and polyadenylation.
  • chimeric gene or “chimeric nucleic acid” refers to any gene or any nucleic acid, which is not normally found in a particular eukaryotic species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.
  • promoter denotes any DNA which is recognized and bound (directly or indirectly) by a DNA-dependent RNA-polymerase during initiation of transcription.
  • a promoter includes the transcription initiation site, and binding sites for transcription initiation factors and RNA polymerase, and can comprise various other sites (e.g., enhancers), at which gene expression regulatory proteins may bind.
  • regulatory region means any DNA, that is involved in driving transcription and controlling (i.e., regulating) the timing and level of transcription of a given DNA sequence, such as a DNA coding for a protein or polypeptide.
  • a 5' regulatory region is a DNA sequence located upstream (i.e., 5') of a coding sequence and which comprises the promoter and the 5'-untranslated leader sequence.
  • a 3' regulatory region is a DNA sequence located downstream (i.e., 3') of the coding sequence and which comprises suitable transcription termination (and/or regulation) signals, including one or more polyadenylation signals.
  • the promoter is a constitutive promoter.
  • the promoter activity is enhanced by external or internal stimuli (inducible promoter), such as but not limited to hormones, chemical compounds, mechanical impulses, abiotic or biotic stress conditions.
  • the activity of the promoter may also be regulated in a temporal or spatial manner (tissue-specific promoters; developmentally regulated promoters).
  • the promoter is a plant-operable promoter.
  • plant-operable promoter means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell e.g., certain promoters of viral or bacterial origin such as the CaMV 35S promoter (Hapster et al., Mol. Gen. Genet. 212, 182-190, 1988), the subterranean clover virus promoter No 4. or No. 7 (WO 1996/006932), or a T-DNA gene promoter.
  • Tissue-specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO 1989/003887), organ-primordia specific promoters (An et al., The Plant Cell 8, 15-30, 1996), stem-specific promoters (Keller et al., EMBO J. 7, 3625-3633, 1988), leaf specific promoters (Hudspeth et al., Plant Mol Biol 12, 579-589, 1989), mesophyll- specific promoters such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al., Genes Dev.
  • seed-specific promoters e.g., WO 1989/003887
  • organ-primordia specific promoters An et al., The Plant Cell 8, 15-30, 1996)
  • stem-specific promoters Kerdspeth et al., Plant Mol Biol 12, 579-589, 1989
  • tuber-specific promoters Keil et al., EMBO J. 8, 1323- 1330, 1989
  • vascular tissue specific promoters Pieris et al., Gene 84, 359-369, 1989
  • stamen-selective promoters WO 1989/010396, WO 1992/013956
  • dehiscence zone specific promoters WO 1997/013865 and the like, may also be employed.
  • the promoter may be an expansin gene promoter, a sucrose synthase (SuSy) gene promoter, an alpha-tubulin (TUB) gene promoter, an arabinogalactan protein (ARAB) gene promoter, a caffeic acid 3-O-methyltransferase (COMT) gene promoter, a cinnamyl alcohol dehydrogenase (CAD) gene promoter, a cinnamate 4-hydroxylase (C4H) gene promoter, a cinnamoyl CoA reductase (CCR) gene promoter, a ferulate-5-hydroxylase (F5H) gene promoter, a sinapyl alcohol dehydrogenase (SAD) gene promoter, a UDP-D-glucuronate carboxy lyase (UDP) gene promoter, a lipid transfer protein (LT) gene promoter, a sucrose synthase (SuSy) gene promoter, an alpha-tubulin (
  • the transcription terminator and polyadenylation signal may be any 3-untranslated region of a plant gene, preferably a gene that is expressed at a high level in a plant e.g., a CaMV 19S or CaMV 35S transcription termination and polyadenylation signal sequence, or a ubiquitin gene terminator, NOS terminator, etc.
  • the DNA region coding for ARF2 or ARF18 may comprise an open-reading frame or protein-encoding region such as the nucleotide sequence of SEQ ID No. 1 from nucleotide 502 to nucleotide 3081 , or the nucleotide sequence of SEQ ID No. 3 from nucleotide 376 to nucleotide 2955, or the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 2580, or the nucleotide sequence of SEQ ID No. 7 from nucleotide 376 to nucleotide 2955, or the nucleotide sequence of SEQ ID No.
  • nucleotide sequence of SEQ ID No. 1 from nucleotide 502 to nucleotide 3081 , or the nucleotide sequence of SEQ ID No. 13 from nucleotide 310 to nucleotide 2889, or the nucleotide sequence of SEQ ID No. 15 from nucleotide 289 to nucleotide 2868, or the nucleotide sequence of SEQ ID No. 17 from nucleotide 291 to nucleotide 2870, or the nucleotide sequence of SEQ ID No.
  • nucleotide sequence of SEQ ID No. 21 from nucleotide 339 to nucleotide 2918, or the nucleotide sequence of SEQ ID No. 23 from nucleotide 339 to nucleotide 2918, or the nucleotide sequence of SEQ ID No. 25 from nucleotide 283 to nucleotide 2091 , or the nucleotide sequence of SEQ ID No. 27 from nucleotide 1 to nucleotide 1809, or the nucleotide sequence of SEQ ID No. 29 from nucleotide 221 to nucleotide 2029, or the nucleotide sequence of SEQ ID No. 31 from nucleotide 1 to nucleotide 1809.
  • the DNA region codes for a variant of the proteins comprising the amino acid sequence of SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID o. 28, SEQ ID No. 30 or SEQ ID No. 32.
  • SEQ ID No. 4 amino acid sequence of SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID o. 28, SEQ ID No. 30 or SEQ ID No. 32.
  • SEQ ID No. l Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number N _180913.2).
  • SEQ ID No. 2 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number NM l 80913.2)
  • SEQ ID No. 3 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number NM_203251.2).
  • SEQ ID No. 4 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number NM_203251.2)
  • SEQ ID No. 5 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AY669787.1 ).
  • SEQ ID No. 6 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AY669787.1 )
  • SEQ ID No. 7 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number NM_125593.3).
  • SEQ ID No. 8 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number NM_125593.3).
  • SEQ ID No. 9 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AF378862.1 ).
  • SEQ ID No. 10 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AF378862.1 ).
  • SEQ ID No. 1 1 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number BT000784).
  • SEQ ID No. 12 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number BT000784).
  • SEQ ID No. 13 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number A 221305.1 ).
  • SEQ ID No. 14 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221305.1 ).
  • SEQ ID No. 15 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK221282.1 ).
  • SEQ ID No. 16 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number A 221282.1 ).
  • SEQ ID No. 17 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (Gen Bank; Accession number AK.221277.1 ).
  • SEQ ID No. 18 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK.221277.1 ).
  • SEQ ID No. 19 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK.221274.1 ).
  • SEQ ID No. 20 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221274.1 ).
  • SEQ ID No. 21 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK221254.1 ).
  • SEQ ID No. 22 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221254.1 ).
  • SEQ ID No. 23 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK221252.1 ).
  • SEQ ID No. 24 Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221252.1 ).
  • SEQ ID No. 25 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF 1 8 (GenBank; Accession number NM_1 16048.2);
  • SEQ ID No. 26 Predicted Arabidopsis thaliana amino acid sequence ARF 18 (GenBank; Accession number NM_1 16048.2).
  • SEQ ID No. 27 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF18 (GenBank; Accession number AF334717.1 ).
  • SEQ ID No. 28 Predicted Arabidopsis thaliana amino acid sequence ARF18 (GenBank; Accession number AF334717.1 ).
  • SEQ ID No. 29 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF 18 (GenBank; Accession number AY059746.1 ).
  • SEQ ID No. 30 Predicted Arabidopsis thaliana amino acid sequence ARF18 (GenBank; Accession number AY059746.1).
  • SEQ ID No. 31 Complete Arabidopsis thaliana mRNA nucleotide sequence ARF 18 (GenBank; Accession number AY091392.1 ).
  • SEQ ID No. 32 Predicted Arabidopsis thaliana amino acid sequence ARF18 (GenBank; Accession number AY091392.1 ).
  • variant proteins refer to proteins wherein one or more amino acids are different from the corresponding position in the proteins having the amino acid sequence of SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32, by substitution, deletion, insertion; and which have at least one of the functions of the proteins encoded by SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No.
  • SEQ ID No. 12 SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32 such as e.g. the same enzymatic or catalytic activity.
  • Methods to derive variants such a site-specific mutagenesis methods are well known in the art, as well as assays to identify the enzymatic activity encoded by the variant sequences.
  • Allelic forms of the nucleotide sequences which may encode variant proteins, including any orthologs or homologs of the exemplified sequences hereof, may be identified by hybridization of libraries, under moderate or high stringency hybridization conditions, such as to cDNA or genomic libraries of a different plant species or plant lines.
  • nucleotide sequences which hybridize under moderate or high stringency conditions to nucleotide sequences encoding the amino acid sequence of SEQ ID Nos 2, 4, 6, 8, 10; 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 or to the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29 or 31 or a sufficiently large part thereof (preferably at least about 19 contiguous nucleotides, or at least about 20 contiguous nucleotides or at least about 25 contiguous nucleotides or at least about 30 contiguous nucleotides or at least about 50 contiguous nucleotides, or at least about 100 or 200 or 300 or 400 or 500 or 1000 or 2000 contiguous nucleotides of said SEQ ID NO(s).
  • Mode stringency hybridization conditions mean that hybridization will generally occur if there is at least about 50% and preferably at least 70% sequence identity between the probe and the target sequence.
  • moderate stringency hybridization conditions includes an overnight incubation in a solution comprising 50% formamide, 2 x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 2 x SSC at approximately 55°C.
  • High stringency hybridization conditions mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence.
  • Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5 x SSC ( 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 x SSC at approximately 65°C.
  • an isothermal or polymerase chain reaction may be employed using one or more oligonucleotide primers, each comprising at least about 12 or 13 or 14 or 15 or 16 or 17or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 contiguous nucleotides, preferably at least about 30 or 35 or 40 or 45 or 50 or 60 or 70 or 80 or 90 or 100 contiguous nucleotides of a nucleotide selected from SEQ ID No. 1 , SEQ ID No. 3, SEQ ID No. 5 , SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29 and SEQ ID No. 31 , or a complementary sequence to any one of said SEQ ID NOs.
  • Preferred variants of the exemplified sequences are homologs or orthologs from plants other than A. thaliana and which encode a functional protein that can complement at least one function, but preferably all of the affected functions, in an ARF2-deficient or ARF18-deficient A. thaliana plant. Such complementation is accepted in the art as evidence of homologous or orthologous functionality.
  • variants of the exemplified sequences may be obtained from a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.
  • variants of the exemplified sequences may be obtained from a fiber-producing plant e.g., flax or cotton.
  • variants of the exemplified sequences may be obtained from a wood-producing plant e.g., oak, aspen, eucalyptus, maple, pine, spruce, poplar, or larch.
  • the plant may be a species of Eucalyptus ( E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi E. camaldulensis, E. ceracea, E. cloeziana, E. cocci/era, E. cordata, E. cornuta, E. corticosa, E. crebra, E. croajingoleisis, E. curtisii, E. dalrympleana, E.
  • Eucalyptus E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides
  • E. pachyphylla E. pauciflora
  • E. pellita E. perriniana, E. petiolaris, ⁇ . pilularis, E. piperita, E. platyphylla, E. polyanthemos, E. populnea, E. schensiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, E. risdoni, E. robertsonii E. rodwayi, E. rubida, E. rubiginosa, E. saligna, E. salmonophloia, E. scoparia, E. sieberi, E.
  • yunnanensis or a conifer as, for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), or Monterey pine (Pinus radiata); or Douglas-fir (Pseudotsuga menziesii); or Western hemlock (Tsuga canadensis); or Sitka spruce (Picea glauca); or redwood (Sequoia sempervirens); or a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea); or a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis).
  • loblolly pine Pinus taeda
  • slash pine Pin
  • the expression or activity of an ARF2 and/or ARF18 gene is decreased in a plant, plant cell, plant tissue or plant organ.
  • a method is provided to decrease expression or activity of an endogenous ARF2 and/or ARF18 gene in a plant, said method comprising the step of providing plant cells with a chimeric gene capable of reducing the expression of an endogenous ARF2 and/or ARF18 gene to the plant, wherein said endogenous gene codes for a protein comprising the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No.
  • a chimeric gene is provided to cells of the plant, wherein the chimeric gene comprises a nucleotide sequence of at least 19 contiguous nucleotides from a gene encoding an amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10. or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No.
  • nucleotide sequence is operably linked to a plant-operable promoter and a 3' region involved in transcription termination and polyadenylation (so-called "sense" RNA mediated gene silencing).
  • a chimeric gene is provided to cells of the plant, wherein the chimeric gene comprises a nucleotide sequence of at least 19 contiguous nucleotides selected from the antisense strand sequence of a gene encoding a protein comprising the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No.
  • nucleotide sequence of at least about 19 contiguous nucleotides selected from the complement of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 7 or SEQ ID No. 9 or SEQ ID No. 1 1 or SEQ ID No. 13 or SEQ ID No. 15 or SEQ ID No. 17 or SEQ ID No. 1 or SEQ ID No. 21 or SEQ ID No. 23 or SEQ ID No. 25 or SEQ ID No. 27 or SEQ ID No. 29 or SEQ ID No. 31 operably linked to a plant operable promoter and a 3' region involved in transcription termination and polyadenylation.
  • the length of the antisense or sense nucleotide sequence may vary from about 19 nucleotides in length to a length equal to a length of the target nucleic acid e.g., mRNA.
  • a length of the antisense or sense nucleotide sequence may be at least about 50 or 100 or 150 or 100 or 500 nucleotides.
  • There is really no upper limit to the total length of the antisense nucleotide or sense nucleotide sequence there is no advantage a priori in gene manipulations comprising antisense strand sequnces longer than a length of the target nucleic acid, and smaller fragments are easier to handle and may work more effectively for certain operations e.g., dsRNA.
  • a length of the antisense strand sequence should not exceed about 1 or 2 or 3 kb, and the total length of a chimeric gene for most practical applications should not exceed about 12 kb or 15 kb.
  • the total antisense nucleotide sequence should have a sequence identity of at least about 75% with the corresponding target sequence, particularly at least about 80 %, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100% identity.
  • antisense or sense nucleotide sequences may be more effective for distantly-related species, to achieve more effective hybridization to the target mRNA where the percentage identity between the antisense strand and the target is low e.g., less than about 50%.
  • the antisense or sense nucleotide sequence always includes a sequence of at least about 19-30 nucleotides in length having at least about 90% or 95% or 99% or 100% sequence identity to a part of the target nucleic acid e.g., mRNA.
  • the number of gaps should be minimized, particularly for the shorter antisense or sense sequences.
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x l OO) divided by the number of positions compared.
  • a gap i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
  • Another example of the invention relates to a method for reducing the expression of endogenous genes of a plant, wherein said endogenous gene codes for a protein comprising an amino acid sequence having at least about 50% identity to SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No.
  • dsRNA double stranded RNA
  • a chimeric gene may be provided to a plant cell comprising a plant-operable promoter operably linked to a nucleic acid comprising a sense strand sequence comprising at least 19 consecutive nucleotides from a coding region of a nucleic acid encoding a protein having an amino acid sequence of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 and an antisense strand sequence comprising at least about 19 consecutive nucleotides having a region of complementarity or wholly complementary to the sense strand sequence.
  • the chimeric gene may comprise additional regions, such as a transcription termination and polyadenylation region > functional in plants.
  • RNA When transcribed an RNA can be produced which may form a double stranded RNA stem between the complementary parts of the sense and antisense region.
  • a spacer region may be present between the sense and antisense nucleotide sequence that forms a loop region of a hairpin loop in the expressed dsRNA.
  • the chimeric gene may further comprise an intron sequence, preferably located in the spacer region.
  • the chimeric gene used to reduce the expression of a gene endogenous to said plant, wherein said endogenous gene codes for a protein comprising the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No.
  • RNA having the nucleotide sequence of an RNA coding for a protein comprising the amino acid sequence of SEQ ID SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No.
  • the ribozyme recognizes and cleaves RNA having the nucleotide sequence of an RNA comprising the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, or 31.
  • Methods for designing and using ribozymes have been described by Haseloff and Gerlach ( 1988) and are contained e.g.,. in WO 1989/005852.
  • nucleic acids are provided which can be used to alter a plant phenotype.
  • the invention provides chimeric genes (DNA molecule) which comprise the following operably linked DNA fragments
  • a DNA region comprising a nucleotide sequence of at least 19 contiguous nucleotides selected from a nucleotide sequence coding for the protein comprising the amino acid sequence of SEQ ID SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID , No. 28 or SEQ ID No. 30 or SEQ ID No. 32 (or a variant of that protein having the same enzymatic activity), such as the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29 or 31 ; and/or
  • RNA molecules that can be obtained from the chimeric genes according any example hereof.
  • Such RNA molecules can be produced by in vivo or in vitro transcription of the chimeric genes. They can also be obtained through in vitro transcription of chimeric genes, wherein the transcribed region is under control of a promoter recognized by single subunit RNA polymerases from bacteriophages such as SP6, T3 or T7.
  • the RNA molecules may be synthesized in vitro using procedures well known in the art. Also chemical modifications in the RNA ribonucleoside backbone to make the chimeric RNA molecules more stable are well known in the art.
  • Different examples for chimeric genes or RNA molecules have been described above in relation to the methods exemplified herein for altering cellulose biosynthesis and can be applied mutatis mutandis to the examples relating to substances.
  • Chimeric genes or RNA may be provided to plant cells in a stable way, or transiently.
  • stable provision of chimeric genes or RNA molecules may be achieved by integration of the chimeric genes into the genome of the cells of a plant.
  • Methods for the introduction of chimeric genes into plants are well known in the art and include Agrobacterium-medizAed transformation, particle gun delivery, microinjection, electroporation of intact cells, polyethylene glycol-mediated protoplast transformation, electroporation of protoplasts, liposome-mediated transformation, silicon-whiskers mediated transformation etc.
  • the transformed cells obtained in this way may then be regenerated into mature fertile plants.
  • the chimeric genes or chimeric RNA molecules of the invention are provided on a DNA or RNA molecule capable of autonomously replicating in the cells of the plant, such as e.g. viral vectors.
  • the chimeric gene or the RNA molecules of the invention may be also be provided transiently to the cells of the plant.
  • the present invention also provides plant cells and plants containing the chimeric genes or the RNA molecules according to any example hereof.
  • Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants comprising the chimeric genes of the present invention, which are produced by traditional breeding methods are also included within the scope of the present invention.
  • the methods and means of the invention are particularly suited for use in a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.
  • a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.
  • the methods and means of the invention are particularly suited for use in a fiber-producing plant e.g., hemp, jute, flax or cotton.
  • exemplary cottons include Gossypium hirs tum and Gossypium barbadense, such as the varieties Coker 312, Coker310, Coker 5 Acala SJ-5, GSC251 10, FiberMax® 819, FiberMax® 832, FiberMax® 966, FiberMax® 958, FiberMax® 989, FiberMax® 5024, transgenic FiberMax® varieties exhibiting herbicide or insect-resistant traits, Siokra 1 -3, T25, GSA75, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C 1 , Acala B 1644, Acala B 1654-26, Acala BI 654-43, Acala B3991 , Acala GC356, Acala GC510, Acala GAM 1 , Acala C I , Acala
  • the methods and means of the invention are particularly suited for use in a wood-producing plant e.g., oak, aspen, eucalyptus, maple, pine, spruce, poplar, or larch.
  • the plant may be a species of Eucalyptus ( E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi E. camaldulensis, E. ceracea, E. cloeziana, E.
  • Eucalyptus E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E
  • E. globulus E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis *urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora,. E. michaeliana, E.
  • microcorys E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua, E. obtusiflora, E. occidentalis, E. optima, E. ovata, E. pachyphylla, E. pauciflora, E. pellita, E. perriniana, E. petiolaris, E. pilularis, E. piperita, E. platyphylla, E. polyanihemos, E. populnea, E. schensiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, E. risdoni, E.
  • E. willisii E. willisii subsp. falciformis
  • E. willisii subsp. willisii E. woodwardii
  • a species of poplar e.g., Populus alba, P. alba *P. grandidentata, P. alba *P. tremula
  • yunnanensis a conifer as, for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), or
  • Monterey pine Pieria
  • Douglas-fir Pseudotsuga menziesii
  • Western hemlock Tsuga canadensis
  • Sitka spruce Picea glauca
  • redwood Sequoia sempervirens
  • a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea)
  • a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis).
  • Arabidopsis thaliana was selected as a model plant for xylem development in woody plants such as poplar. All experiments were performed using Arabidopsis thaliana line N906 (wild-type syn. ecotype C24), an ARF2-deficient transgenic RNAi line, and an ARF18rdeficient transgenic mutant line.
  • the ARF18-deficient transgenic line was a GAL4-GFP enhancer trap line produced by Haseloff et al.. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom, and is publicly available from the Nottingham Arabidopsis Stock Centre.
  • the ARF2 transgenic mutant line was obtained from Okushima et al., Plant Gene Expression Center, 800 Buchanan Street, Albany, CA 94710, USA, and is publicaly available from the Arabidopsis Biological Resource Centre. Plant growth conditions
  • Arabidopsis thaliana wild-type and mutant plants were grown at 21 °C and 16 hr day period, and at 18°C during an 8hr dark period, in a controlled growth room • environment. ' Seeds were germinated on plugger soil comprising 45 g/L Osmocot general mix fertilizer. Germinated seedlings were separated and grown for 35 days until mature.
  • seeds were surface-sterilized for 10 min in 70% (v/v) ethanol/0.05% (v/v) Triton X-100, and washed three times in 95% (v/v) ethanol, sown onto the surfaces of 1 ⁇ 2MS agar plates, vernalized for 3 days at 4°C, and incubated in a growth chamber at 120- 150 ⁇ m "2 s ' ' , with a day period of 16 hr at 23°C and a dark period of 8 hr at 21°C. After 1 1 days, seedlings were transplanted to potting media and grown as describe din the preceding paragraph.
  • Transverse sections of stem and hypocotyls were harvested, cleaned to remove excessive plant material, including lateral roots, trimmed to a maximum length of 2mm, and immersed in fixative according to standard procedures for 4 hr. The fixative was replaced and plant tissue fixed for a further 4 hr period. Plant material was stored at 5°C in fixative. Fixed plant material was wax-embedded according to standard procedures. A Leica RM2235 rotary microtome was employed to slice tissue sections to an appropriate thickness e.g., 2 ⁇ .
  • Wax -embedded blocks were pre-cooled on ice, and transverse sections of stem and hypocotyl were obtained, wherein the wrinkle effect was reduced by incubating tissue ribbons in 1% (w/v) horse serum for 2min, and sections were transferred onto glass slides for staining. Multiple sections were produced from each specimen to ensure a complete representation of the specimen structure.
  • hypocotyls and flower stems were incubated in a fixative solution (5 mL of 16% paraformaldehyde, 2 mL of 25% gluteraldehyde, 10 mL of 0.2 M phosphate- buffered saline (PBS), 3 mL of distilled water) for 2 hours, washed twice for 5 min per wash in 0.2 M PBS, and washed successively in 30% ethanol for 15 min, , then in 70% ethanol for 15 min, then in 00% ethanol for 15 min then in 99% ethanol for 15 min, and finally in absolute ethanol for 60 minutes.
  • a fixative solution 5 mL of 16% paraformaldehyde, 2 mL of 25% gluteraldehyde, 10 mL of 0.2 M phosphate- buffered saline (PBS), 3 mL of distilled water
  • Digital images of secondary xylem and vascular bundles were obtained using a light microscope with in-built camera comprising a 1 OX or 40X objective and an 8X eye piece.
  • Root length and root elongation rate were determined for seedlings grown on 1 ⁇ 2MS agar plates. Root tips of seedlings were marked from day 6 post-germination to day 12 post-germination and root lengths determined at day 12 by imaging using ImageJ software. Elongation rates of roots were then calculated. Hypocotyl lengths were determined for plants grown in potting medium, or for etiolated seedlings grown on 1 ⁇ 2MS agar plates. Leaf areas were determined using EZ-Rhizo software.
  • Figure 1 provides representative transverse sections of hypocotyl of the ARF2-deficient mutant ( Figure l a) and otherwise isogenic wild-type (WT) plants ( Figure l b). Data indicate that that the number of small vessels is increased in xylem of the ARF2- deficient A. thaliana mutant. Tears in secondary xylem were apparent in all sections of ARF2-deficient secondary xylem, suggesting possible reduced lignification, because only non-lignified structures of wild-type plants showed tearing.
  • Data presented in Figure 2 show that, in contrast to ARF2-deficient (and ARF18- deficient) mutant plants, the vasculatures of ARF 1 1 -deficient mutant, ARF 13-deficient mutant, ARFI4-deficient mutant, ARF15-deficient mutant, ARF3-deficient mutant, ARF22-deficient mutant, ARF4-deficient mutant, ARF5-deficient mutant, and the ARF19-deficient and ARF7-deficient ARF19xARF7 double-mutant, more closely resembled the vasculature of wild-type plants.
  • vessels of the ARF2-deficient mutant have a cross-sectional area in the range 0-7 ⁇ 2
  • about 27% have a cross-sectional area in the range 8-20 ⁇ 2 .
  • the small cell area in secondary xylem of ARF2-deficient plants indicates reduced cell expansion and vessel formation.
  • the ARF2-deficient mutant has enhanced lignification relative to wild-type plants, as evidenced by increased thickening of cell walls (not shown). Hypocotyl diameter of the ARF2-deficient mutant was also larger than in wild-type plants, the increase in size being correlated with increased cell number and density.
  • the mutant exhibited enhanced drought tolerance or reduced susceptibility to water deficit stress than wild-type plants, as determined by reducing wilting after water supply was reduced (Figure 5). Following water-deficit, the mutant line also recovered better than wild-type plants, as determined by a higher percentage of plants that survived when watering was resumed after a period of water deficit. As shown in Figure 5, about 60% of ARF 18-deficient plants survive a period of water deficit compared to only about 10% of wild-type plants.
  • Data in Figure 6 also indicate that ARF 18-deficient plants maintained a photosynthetic rate, stomatal conductance and transpiration rate during and following water stress, whereas wild-type plants exhibit reduced rates of photosynthesis, transpiration, and stomatal conductance under the same conditions.
  • the weights of potted plants and average total leaf areas of plants were determined for 39-day old plants e.g., after 3 days of water deficit.
  • ARF18-deficient plants had higher average total leaf areas, and reduced water content (p ⁇ 0.001 ) than wild-type plants.
  • the roots of 12-day-old ARF 18-deficient plants were also found to be significantly longer (p ⁇ 0.001 ) than the roots of 12-day-old wild-type C24 seedlings, and this was correlated with a higher rate of root elongation (pO.001 ) i.e., about 5.73 mm/day for the ARF 18-deficient mutant compared to 3.82 mm/day for wild-type plants (Table 1 ; Figure 8f, Figure 9).
  • the hypocotyl length of the ARF18-deficient mutant was also found to be significantly higher than that of the wild- type seedlings (p ⁇ 0.001 ; Table 1 ). Seeds of ARF 18-deficient plants were also heavier than seeds of wild-type plants (p ⁇ 0.001 ; Table 1 ).
  • the ARF 18 gene affects secondary xylem structure and water use efficiency, and also hypocotyl elongation in the light and in the dark, root elongation, cell size, cotyledon size, leaf size, flowering time and seed yield.

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Abstract

La présente invention concerne des constructions de gènes chimériques permettant de moduler l'expression et l'activité de facteurs de réponse à l'auxine (ARF) endogènes dans des plantes, en particulier ARF2 et/ou ARF18. Elle concerne également un procédé d'utilisation des gènes chimériques pour moduler un ou plusieurs phénotypes végétaux comprenant des modifications de la structure et de la fonction du xylème. L'invention convient particulièrement à la production de plantes présentant des propriétés améliorées de fabrication de pâte à papier et une meilleure efficacité de l'utilisation de l'eau, une meilleure résistance au stress de déficit hydrique et une meilleure aptitude à la réparation après un stress de déficit hydrique.
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WO2013087833A1 (fr) * 2011-12-14 2013-06-20 Biogemma Procédé pour l'amélioration de plantes
CN105936908A (zh) * 2016-06-22 2016-09-14 中国农业科学院生物技术研究所 玉米生长素应答因子ZmARF21基因及其应用
CN105936908B (zh) * 2016-06-22 2019-12-27 中国农业科学院生物技术研究所 玉米生长素应答因子ZmARF21基因及其应用
CN108864267A (zh) * 2018-08-01 2018-11-23 中国农业大学 甘薯类胡萝卜素合成和耐盐抗旱相关蛋白IbARF5及其编码基因与应用
CN108864267B (zh) * 2018-08-01 2020-09-04 中国农业大学 甘薯类胡萝卜素合成和耐盐抗旱相关蛋白IbARF5及其编码基因与应用
CN110092819A (zh) * 2018-11-13 2019-08-06 中国农业大学 玉米苞叶宽度调控蛋白arf2及其编码基因与应用
CN110092819B (zh) * 2018-11-13 2021-07-16 中国农业大学 玉米苞叶宽度调控蛋白arf2及其编码基因与应用
CN112358534A (zh) * 2020-10-27 2021-02-12 中国林业科学研究院林业研究所 一种调控杨树纤维长度的生长素响应因子基因及其应用
CN112358534B (zh) * 2020-10-27 2021-07-30 中国林业科学研究院林业研究所 一种调控杨树纤维长度的生长素响应因子基因及其应用

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