KR101202615B1 - Method of controlling plant root hair growth by expression of auxin transporters and the plants thereof - Google Patents

Abstract

The present invention relates to a method for regulating root hair growth of plants through auxin transporter expression and to plants according to the present invention, and more particularly, to the Arabidopsis thaliana ), a recombinant plant expression vector comprising a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, a method for regulating root hair extension of a plant comprising introducing the vector into the plant, the the kidneys adjust the root hair produced by the method and the plant Arabidopsis (Arabidopsis It provides a composition for regulating root hair extension, comprising a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7 derived from thaliana ).

Description

Methods of controlling plant root hair growth by expression of auxin transporter and plant according to the method

The present invention relates to a method for regulating root hair growth of a plant through auxin transporter expression and to a plant according to the present invention, wherein the isolated PINs gene and the protein expressed from the gene are used to improve traits related to root hair growth of plants and The present invention may be usefully used for searching for a gene for regulating the growth of root hairs in another plant, and also relates to a content for generating a plant in which the root hair extension of the plant is controlled using the gene of the present invention.

Auxin is a type of plant hormone that promotes the growth of plant cells. The most widely distributed 3-indolyl acetic acid in nature and a substance exhibiting the same action are called auxin. There are several types of synthetic auxins, for example 2,4-dichlorophenoxyacetic acid (abbreviated 2.4-D). Auxins loosen the cell wall, whereby cells grow uptake. In addition, auxin enhances the permeability of cell membranes, promotes cell division, adventitious muscle formation, germ formation, and inhibits lateral growth. Synthetic auxins are used to control weeding, fall prevention, and dormancy-storage. Auxins in the renal growth of cells cause renal growth of stem and cotyledon. Optimal concentrations of auxin that have a renal effect are 10 ^-5 to 10 ^-6 M. At higher concentrations, renal growth is inhibited. This is because high concentrations of auxin promote the synthesis of ethylene, which inhibits cell growth. The cells of the root elongation also occurs in response to auxin (10 ^-10 ~ 10 ^-9 M) in the low concentration. However, when the concentration of auxin is higher than 1 μM elongation is rather suppressed.

One of the basic scientific applications of root hair research is to use the physiology and morphogenesis of root hairs to identify the mechanisms that regulate hormone action, especially auxin transport. Auxins move in polarity between cells. In the stem, auxin travels along the conduit tissue from the end of the stem to the root, from the root to the root, and again along the epidermis from the root to the root. This polar transport of auxin affects the most basic development of plants such as apical dominance, vascular bundle development, flexion, pattern formation during embryonic development. Polar transport of auxins is thought to occur because auxin transporter proteins are distributed asymmetrically in the plasma membrane.

On the other hand, the auxin releasing activity of Arabidopsis PINs has been described by the Arabidopsis and heterozygote systems as follows (Petrasek et al., 2006 Science 312: 914-918); PIN1 and PIN5 in Arabidopsis cells, PIN2, PIN3, PIN4, PIN6 and PIN7 in tobacco Bright Yellow (BY) -2 cells, PIN1, PIN2, PIN5 and PIN7 in yeast cells, and PIN1, PIN2 and PIN7 in HeLa cells. Of the eight Arabidopsis PIN members, PIN1, PIN2, PIN3, PIN4, PIN6 and PIN7 have a similar molecular structure in that they have a long central loop (hereinafter referred to as the 'long loop' PIN), which release auxin at the cellular level. Seems to promote. PIN5 and PIN8, on the other hand, have a central loop of very short estimates (hereinafter named 'short loop' PIN). Although PIN5 has recently been reported to be present in the endoplasmic reticulum (ER) and has been proposed to transport auxin metabolites to the ER lumen, the cellular function of PIN5 related to intracellular auxin transport activity has not yet been identified, PIN8 The auxin transport activity of is not yet known.

Despite the same transport direction (oxine release) and the same molecular structure, the long loops of PINs show sequence divergence not only in their central loop but also in certain residues of the transmembrane region. This structural difference in long loop PINs may be an indication of other auxin transport activity, which has not yet been quantitatively compared.

In the present invention, five long loops of PINs (PIN1, PIN2, PIN3, PIN4 and PIN7) and two short loops of PINs (PIN5 and PIN8) that are involved in auxin transport in the Arabidopsis are overexpressed at the root hairs, respectively, to give different auxin release activity. It was confirmed that having, through which it was found that by controlling the expression of PINs it is possible to control the root hair extension of the plant. On the other hand, nothing is already known about this.

The present invention has been made in accordance with the above requirements, in the present invention, by overexpressing the PINs (PIN1, PIN2, PIN3, PIN4, PIN5, PIN7, and PIN8) genes involved in the auxin transport in the roots, the auxin transport activity and cells As a result of the epidemiological investigation, it was confirmed that PINox (PIN gene overexpression) each has a different auxin releasing activity, thereby completing the present invention by enabling the regulation of plant root hair extension.

In order to solve the above problems, the present invention Arabidopsis Provided is a recombinant plant expression vector comprising a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, from thaliana .

The present invention also provides a method of controlling the root hair extension of a plant comprising the step of introducing the vector into the plant.

In addition, the present invention provides a plant with controlled root hair extension produced by the above method.

In addition, the present invention Arabidopsis Arabidopsis It provides a composition for regulating root hair growth of a plant, comprising a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7 derived from thaliana .

The PINs gene isolated from the present invention and the protein expressed from the genes can be usefully used for the improvement of traits related to the root hair extension of plants and the search for genes for regulating the growth of root hairs in other plants. In addition, by using the gene of the present invention it is possible to create a plant in which the root hair extension of the plant is controlled.

1 shows the different activities of PINs in Arabidopsis root hairs. (A) is two unique PIN groups with different central hydrophilic loop sizes. The numbers above indicate the number of transmembrane helixes in each of the N- and C-terminal regions, and the numbers below show the number of amino acid residues in the central hydrophilic region. (B) shows representative root images of control (Cont; Col-0) and root hair specific PIN - overexpressing plants (PINox; ProE7 : PIN - GFP or ProE7 : PIN [-]) (bar = 100 μm) (C ) Is the root hair length of the control and PINox plants. Six to twelve independent transgenic plants (mean = 8.3), and 42-243 roots (mean = 86.8) and 336-2187 root hairs (mean = 727.8) per construct were observed for determination of root hair length. Data is mean ± standard deviation. Root hair length of the PIN5ox transformants was significantly longer than the control (P = 0.016 for PIN5ox; P <0.0001 for PIN5-GFP1ox and PIN5-GFP2ox).
2 shows the effect of root hair cell specific expression of PINs in the kidneys of root and root hair epidermal cells. (A) is seedlings of 4-day-old control (Cont; Col-0) and PINox ( ProE7 : PIN- GFP ) plants. Bar = 0.5 cm. (B) shows the root hair epidermal cell length in PINox plants. The length between two consecutive root hairs in the same root hair cell file was measured by the root hair cell length of the control and PINox plants. Five independent transgenic plants for each transformant were analyzed. Data represent mean ± standard deviation (n = 350-500).
3 is the relative retention of [ ³ H] NAA in PINox BY-2 tobacco cells. Relative accumulation of [ ³ H] NAA by PIN5ox ( ProTA: PIN5-GFP1 , [A] ) and PIN8ox ( ProTA : PIN8 - GFP , [B] ). Expression of the transgene induced by 0.01 mM Dex was induced for 24 hours. The data shows mean ± standard deviation from 8 replicates.
4 is a diagram showing that IAA restores the root hair extension of the PINox transformant. (A) is an image of representative roots of control (Cont; Col-0) and root hair specific PIN overexpression (PINox; ProE7: PIN-GFP ). Seedlings were treated with 50 nM IAA for 1 day (Bar = 100 μm). (B) shows the root hair length of the control and PINox plants. Two independent homozygous transformants were analyzed. Data represent mean ± standard deviation (n = 56-112, mean = 103).

In order to achieve the object of the present invention, the present invention provides a recombinant plant expression vector comprising a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7 derived from Arabidopsis thaliana .

The PINs gene of the present invention is characterized by auxin release transport in root hairs, and the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 are derived from Arabidopsis PIN1 , PIN2 , PIN3 , PIN4, PIN5 , PIN7 And PIN8 A nucleotide sequence representing each gene. The genes of SEQ ID NOS: 1 to 7 include both genomic DNA and cDNA. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene is a base having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with each of the base sequences of SEQ ID NOS: 1-7. Sequences may be included. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the &lt; / RTI &gt;

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the plant expression vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the plant expression vectors of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium and the terminator of the Octopine gene of Tumefaciens, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a method of controlling the root hair extension of a plant comprising the step of introducing the vector into the plant.

In a method according to an embodiment of the present invention, the gene having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 4, SEQ ID NO: 6 and SEQ ID NO: 7 corresponding to the PIN1 , PIN2 , PIN3 , PIN4 , PIN7 and PIN8 gene Overexpression in plants to reduce plant root hair extension, or PIN5 The gene having the nucleotide sequence of SEQ ID NO: 5 may be overexpressed in the plant to increase the root hair extension of the plant.

In a method for overexpressing the PIN1, PIN2, PIN3, PIN4, PIN5, PIN7 or PIN8 gene in a plant according to an embodiment of the present invention, the gene is included in a plant containing the gene or a plant not containing the gene. By introducing. "Overexpression of the gene" in the above means that the gene is expressed above the level expressed in wild-type plants. As a method of introducing the gene into a plant, there is a method of transforming a plant using an expression vector containing the gene under the control of a promoter. The above promoter is not particularly limited as long as it can overexpress an insertion gene in a plant. Examples of such promoters include, but are not limited to, 35S RNA and 19S RNA promoters of CaMV; Full length transcriptional promoters derived from Peak Water Mosaic Virus (FMV) and Coat protein promoters of TMV. In addition, ubiquitin promoters can be used to overexpress PIN1, PIN2, PIN3, PIN4, PIN5, PIN7 or PIN8 genes in monocotyledonous plants or woody plants.

In addition, the present invention provides a plant with controlled root hair extension produced by the above method.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens et al., 1982, Nature 296: 72-74; Negrutiu et al., 1987, Plant Mol. Biol. 8: 363-373), electroporation of protoplasts ( Shillito et al., 1985, Bio / Technol. 3: 1099-1102), microway injection into plant elements (Crossway et al., 1986, Mol. Gen. Genet. 202: 179-185), DNA or RNA-coated) particle bombardment (Klein et al., 1987, Nature 327: 70), in agrobacterium tumerfaciens mediated gene transfer (by non-invasion of plants or transformation of mature pollen or vesicles) Integrity) can be appropriately selected from infection by virus (EP 0 301 316) and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or whole plants.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. Plant tissue is planted in planta ) or in organ culture, tissue culture or cell culture.

Plants to which the method according to the invention can be applied may be dicotyledonous plants or monocotyledonous plants, preferably dicotyledonous plants. Examples of dicotyledonous plants include, but are not limited to, baby pole, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, There are watermelons, melons, cucumber pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, and peas. The plants are preferably Arabidopsis thaliana ).

The present invention also provides a composition for controlling root hair growth of a plant, comprising a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7 derived from Arabidopsis thaliana . In the compositions of the present invention, the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 are derived from Arabidopsis PIN1 , PIN2 , PIN3 , PIN4 , PIN5 , PIN7 and PIN8 A nucleotide sequence representing each gene. In the composition of the present invention, the PIN1, PIN2, PIN3, PIN4, PIN5, PIN7 or PIN8 gene may include the insertion, substitution, deletion of a specific base sequence in the gene sequence.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Plant and growing conditions

Columbia wild type Arabidopsis thaliana was used as a wild type plant in the present invention. All seeds were cultured in agarose plates containing 4.3 g / L Murashige and Skoog (MS) nutrient mixture (Sigma-Aldrich), 1% sucrose, 0.5 g / L MES (pH 5.7), KOH and 0.8% agarose. It was. All seeds were treated cool at 4 ° C. for 3 days and germinated in photoperiod at 23 ° C., 16h-light / 8h-cancer. Transgenic plants were selected (10 mg / mL) on plates containing hygromycin. For all pharmacological experiments, seedlings of 3 day old homozygous transformants were transferred to new plates, further grown, and root hairs were then observed.

2. Transgene Structure Transgene Constructs )

Arabidopsis EXPA7 promoter ProE7 was used to clone all PIN transgenes (Cho H.-T, Cosgrove DJ, 2002 Plant Cell 14: 3237-3253). Binary vector pCAMBIA1300-NOS was used as the cloning vector. ProE7 : PIN3 - GFP , ProE7 : YFP , ProE7: GFP , ProE7 : AUX1 - YFP and ProE7 : PGP4 - YFP were described above (Lee SH, Cho HT, 2006 Plant Cell 18: 1604-1616). ProE7: PIN1 - GFP, ProE7: PIN2 - GFP, ProE7: PIN4-GFP and ProE7: PIN7 - GFP construct binary vector pCAMBIA1300 - ProPIN1 in NOS (Hyg +): PIN1 - GFP (Benkova et al., 2003 Cell 115: 591-602), ProPIN2 : PIN2-GFP (Xu J, Scheres B, 2006 Genes Dev 20: 922-926), ProPIN4 : PIN4- GFP (Vieten et al., 2005 Development 132 : 4521-4531) and ProPIN7 : PIN7 - GFP (Blilou et al., 2005 Nature 433: 39-44) from the ProE7 to the PIN-GFP coding region. Made by connecting to a promoter. ProE7 : PIN2 - GFP was described in Cho et al (Cho et al., 2007a Plant Cell 19: 3930-3943), and ProE7 : PIN4 - GFP was prepared by replacing the PIN2 region in genomic PIN4 fragment in the same vector. For ProE7 : PIN1 - GFP , PIN1 - GFP fragments were prepared by PCR of genomic DNA from ProPIN1 : PIN1-GFP transformants using the following primer sets. PIN1 - for first segment of GFP 5'-TCTCCGTCGACCAAAAGATGATTACGGC-3 '(including SalI site: SEQ ID NO: 8) and 5'-TCACGGGATCCATGATGTATAGTTCATCC-3' (including BamHI site: SEQ ID NO: 9) were used and for the second segment of PIN1-GFP , 5'- CATTATCAACAAAATACTCCAATTGGCG-3 '(including BamHI site: SEQ ID NO: 10) and 5'-TTTCAGAGCTCATCATCCCACAAAAGAA-3' (including SacI site: SEQ ID NO: 11) were used and for the GFP region, 5'-TCGCCCCCGGGGTGAGCAAGGGCG -3 '(including XmaI site) : SEQ ID NO: 12) and 5'-CGGCCCCCGGGCTTGTACAGCTCGTCC-3 '(including XmaI site: SEQ ID NO: 13) were used. ProE7 : PIN7 - GFP was similarly prepared using PCR of genomic DNA from ProPIN7 : PIN7 - GFP using a primer set. 5'-ACTTTGTCGACTCCGGCGAACAACAA-3 '(including SalI site: SEQ ID NO: 14) and 5'-CCGTTGGATCCCCAAACAAACATATG-3' (including BamHI site: SEQ ID NO: 15) for the first segment of PIN7 - GFP , 5 for the second segment '-CATTATCAACAAAATACTCCAATTGGC G-3' (including BamHI site: SEQ ID NO: 16) and 5'-TCTCTGGTACCTCTTCAGCCAAGCAG-3 '(including KpnI site: SEQ ID NO: 17) were used. ProE7: PIN7 - third of the GFP fragments are ProPIN7: PIN7 - with a total area from GFP GFP was first cloned by cutting a part of the PIN7 with BamH1, BamH1 of PIN7 of the cut vector It was inserted between the first and second sections. ProE7 : PIN5 The PIN5 coding region (-6 to +1744 for ATG) was prepared by PCR of Arabidopsis genomic DNA using the following primer sets. 5'-ATTTTCTCGAGAAAACCATGATAAATTGTG-3 '(including XhoI site: SEQ ID NO: 18) and 5'-TAAAATCTAGACTTACGCTGTGCTTAGAAC-3' (including XbaI site: SEQ ID NO: 19) were used for the first fragment. ProE7: PIN8 The PIN8 coding region (-6 to +1780 for ATG) was prepared by PCR of Arabidopsis genomic DNA using the following primer sets: 5'-TACAACTCGAGAAAAACATGA TCTCCTGG-3 '(including XhoI site: SEQ ID NO: 20) and 5′-AACATTCTAGATTCATAGGTCCAATAGAAA-3 ′ (including XbaI site: SEQ ID NO: 21). ProE7 : PIN5- GFP1 The PIN5 coding region was prepared by PCR of Arabidopsis genomic DNA using the following primer set. 5'-ATTTTCTCGAGAAAACCATGATAAATTGTG-3 '(including XhoI site: SEQ ID NO: 22) and 5'-AGGAACCCGGGCTCTCCCACAACCAC-3' (including XmaI site: SEQ ID NO: 23) for the first segment and 5'-TTG TGC for the second segment CTA GGA AGT CGT TCC TTG AGG TC-3 '(including AvrII site: SEQ ID NO: 24) and 5'-TAAAATCTAGACTTACGCTGTGCTTAGAAC-3' (including XbaI site: SEQ ID NO: 25) were used. ProE7 : PIN5 - GFP2 The PIN5 coding region was prepared by PCR of Arabidopsis genomic DNA using the following primer set. 5'-ATTTTCTCGAGAAAACCATGATAAATTGTG-3 '(including XhoI site: SEQ ID NO: 26) and 5'-TATATCCCGGGGTATTCACTGTTATCTC-3' (including XmaI site: SEQ ID NO: 27) for the first segment and 5'-ACAGTCCTAGGATGCATATATACGCAGG-3 for the second segment (Including AvrII site: SEQ ID NO: 28) and 5'-TAAAATCTAGACTTACGCTGTGCTTAGAAC-3 '(including XbaI site: SEQ ID NO: 29). ProE7 : PIN8 - GFP The PIN8 coding region was prepared by PCR of Arabidopsis genomic DNA using the following primer set. 5'-TACAACTCGAGAAAAACATGATCTCCTGG-3 '(including XhoI site: SEQ ID NO: 30) and 5'-GATCTCCCGGGCACTATTGCTACTTCTT-3' (including XmaI site: SEQ ID NO: 31) for the first segment and 5'-TAGCACCTAGGAGAACAAGATCTGTTGG-3 for the second segment '(Including XmaI site: SEQ ID NO: 32) and 5'-AACATTCTAGATTCATAGGTCCAATAGAAA-3' (including AvrII site: SEQ ID NO: 33). ProE7 : PIN5 - GFP1 , ProE7 : PIN5 - GFP2 And ProE7: PIN8 - To clone the GFP area in GFP, GFP Sections were prepared using primers 5'-TCGCCCCCGGGGTGAGCAAGGGCG-3 '(including XmaI site: SEQ ID NO: 34) and 5'-CGGCCCCTAGGCTTGTACAGCTCGTCC-3' (including AvrII site: SEQ ID 35). ProTA : PIN5 - GFP1 And ProTA : For PIN8 - GFP structures, PIN - GFP The coding region was subjected to PCR amplification with the following primers from ProE7 : PIN - GFP . The modified ProTA-7001 vector (Aoyama T, Chua NH, 1997 Plant J 11: 606-612) was inserted into the Xho1-Spe1 site. PIN5 -for GFP1 5'-ATTTTCTCGAGAAAACCATGATAAATTGTG-3 '(including XhoI site: SEQ ID NO: 36) and 5'-TAAAATCTAGACTTACGCTGTGCTTAGAAC-3' (including XbaI site: SEQ ID NO: 37) were used and for PIN8 - GFP 5'-TACAACTCGAGAAAAACATGATCTCCTGG-3 '(including XhoI site: SEQ ID NO: 38) and 5'-AACATTCTAGATTCATAGGTCCAATAGAAA-3' (including XbaI site: SEQ ID NO: 39) were used. ProE7: TIR1 (TIR1ox) pCAMBIA1300 structure that includes ProE7 - NOS Designed in vector. Genomic sections of TIR1 were PCR with primers 5'-GAGATGTCGACGTTATTTGGCCGCAATG-3 '(including SalI site: SEQ ID NO: 40) and 5'-TCGTTCCCG GGTCTTATAATCCGTTAGTA-3' (XMAI site: SEQ ID 41), and the PCR product was SalI of the vector. And inserted into the XmaI site. All constructs were confirmed by nucleotide sequencing. The constructs were either Agrobacterium tumefaciens (strain C58C1) -mediated invasion (Bchtold N, Pelletier G, 1998 In JM Martinez-Zapater, J Salinas, eds, Arabidopsis Protocols) in Arabidopsis (Ecological Colombia) or tobacco BY-2 cells. , Humana, Totowa, NJ, pp. 259-266). Insertion of the transgene in Arabidopsis transformants was confirmed by PCR analysis of genomic DNA.

3. Measurement of Root Hair and Root Hair Cell Length

Root hair length was measured according to a modified technique of Lee and Cho (Lee and Cho, 2008 In AMC Emons, T Ketelaar eds, Root hairs (Plant Cell Monographs Vol 12), Springer, Berlin, pp. 45-64). For the measurement of root hair length, digital photographs of roots were taken using a stereomicroscope (stereomicroscope, Leica MZ FLIII, Heerbrugg, Switzerland) at 40-50x magnification. Root hairs were counted in the root hair mature zone (approximately 0.78 mm from the tip). To measure root hair length, 4-5 consecutive root hairs protruding vertically from each side of the root were measured for a total of 8-10 root hairs from both sides of the root. Prominences of the short root hairs were not counted because they were thought to be stationary in the initial protruding phase. Root epidermal cell length was calculated by measuring the gap between two consecutive root hairs or protrusions in the same root hair line along the long axis of the root. Fifteen to twenty cells were observed per root.

4. Reporter Gene Expression and Microscopy

Fluorescence (green) and FM4-64 (red) from GFP were observed using confocal laser scanning microscope LSM 510 (Carl Zeiss, Gottingen, Germany). Green and red fluorescence was detected using 488 / 505-530 nm and 587/615 nm excitation / emission filter sets, respectively. The location of the transporter-reporter (PIN-GFP) fusion protein was observed from 3 or 4 day old seedlings. For observation of the intracellular location of PIN-GFP after BFA treatment, seedlings were incubated in half-strength liquid MS medium for the indicated time. The control liquid medium contained the same amount of solvent dimethylsulfoxide, which was used to dissolve BFA. The locations of PIN5-GFP1 and PIN8-GFP in transgenic tobacco BY-2 cells were observed 24 hours after treatment with 10 μM Dex. Unless otherwise noted, Arabidopsis root and tobacco BY-2 cells were treated with BFA (50 μM) for 2 hours prior to observation and FM4-64 (2 μM) treated with FM4-64 alone for 3 minutes or with BFA for 15 minutes. Treated during.

5. BY -2 Cell Culture and Transgenic Tobacco

Tobacco BY-2 cells (Nagata et al., 1992 Int Rev Cytol 13: 1-30) were cultured by shaking at 23 ° C. Liquid culture medium (pH 5.8) contains 3% (w / v) sucrose, 4.3 g / L MS salt, 100 mg / L inositol, 1 mg / L thiamine, 0.2 mg / L 2,4-D and 200 mg / L KH ₂ PO ₄ . Suspension cells were passaged weekly. Stock BY-2 calli was maintained in solid medium containing 0.8% (w / v) agar and passaged monthly. Transformed cells and callus were maintained in the same medium with 150 mg / L hygromycin added. For the transformation of tobacco BY-2 cells, 50 mL were incubated 3 days ProTA: PIN5 - GFP1 and ProTA: PIN8 - GFP 5 mL Agrobacterium (Agrobacterium containing the tumefaciens ) and Co-culture at 23 ° C. for 3 days in dark conditions. Inoculated cells were washed three times and transferred onto solid medium to which 150 mg / L hygromycin was added. After 5 days, positive callus was selected and transferred to fresh selection medium. Each callus was then maintained in solid medium or liquid medium as needed.

6. Tobacco BY -2 cells Auxin  Retention analysis ( Auxin Retention Assay )

Auxin retention analysis was performed by modified Delbarre et al. (Delbarre et al., 1996 Planta 198: 532-541). After incubation for 24 hours with or without 10 μM Dex, transformed BY-2 cells were filtered in the exponential growth phase, 1-1.5 g of cell cake was resuspended, and uptake buffer (20 Equilibration was performed by orbital shaking for 45 minutes with mM MES, 40 mM sucrose and 0.5 mM CaSO ₄ , adjusted to pH 5.7 with KOH. Equilibrated cells were collected by filtration, resuspended in fresh uptake buffer, and incubated in an orbital shaker for 1.5 hours in dark at 23 ° C. A 5 mL cell suspension was mixed with [ ³ H] NAA for a final concentration of 2 nM [ ³ H] NAA, and 0.5 mL aliquots were collected at different time intervals (0, 5, 10, 15, 20 and 25 minutes). Filtration was rapid by applying pressure on a C glass fiber filter. The cell cake was washed twice with 3 mL of cold sterile water, transferred to scintillation vials, extracted with 0.5 mL ethanol for 30 minutes, and radioactivity determined by liquid scintillation counting. Four independent experiments were performed with two iterations for each experiment.

7. Registration Number

Arabidopsis Genome Initiative locus mentioned in the present invention identifiers include Arabidopsis At1G12560 ( EXPA7 ), AT1G73590 ( PIN1 ), At5G57090 ( PIN2 ), At1G70940 ( PIN3 ), At2G01420 ( PIN4 ), At5G16530 ( PIN5 ), At1G77110 ( PIN6 ), At1G23080 ( PIN7 ), At5G15100 ( PIN8 ) At2g47000 ( PGP4 ) and At2G38120 ( AUX1 ).

Example  One. PINs Root hair cell specific expression of has different activity in inhibiting root hair extension.

In order to explain the fact that root hair specific expression of PID, PIN3 and PGPs inhibits root hair elongation by enhancing auxin release from root hair cells, the inventors of the root hair cell specific promoter EXPA7 ( Arabidopsis thaliana EXPANSIN A7 , Cho H.-T, Cosgrove DJ, 2002 Plant Cell 14: 3237-3253) (Lee SH, Cho HT, 2006 Plant Cell 18: 1604-1616). To investigate whether other PINs have a similar phenotype in root hairs, genomic sections of PIN genes ( PIN1 , PIN2 , PIN4 , PIN5 , PIN7 and PIN8 ) were linked to the reporter gene encoding the green fluorescent protein (GFP), and the fusion gene Was expressed using the EXPA7 promoter in Arabidopsis. For PIN5 , there are three different transgenes, ProE7 : PIN5 (PIN5ox, no GFP), ProE7 : PIN5 - GFP1 (PIN5-GFP1ox, 558th [from 'A' of start codon of genomic sequence] nucleotide after GFP ) And ProE7 : PIN5 - GFP2 (PIN5-GFP2ox, with GFP after 1333 nucleotides) was introduced into the Arabidopsis. For PIN8, two transgenes were prepared: ProE7: PIN8 (PIN8ox, no GFP) and ProE7 : PIN8 - GFP (PIN8-GFPox, with GFP after 675 th nucleotide). For statistical analysis of the PINox effect in root hair kidney, 6-12 independent transformants for each construct were randomly selected for determination of root hair length (FIG. 1C).

Long loop PINs (PIN1 to 4 and 7; ProE7 : PIN to GFP Root hair specific expression of [PINox]) significantly inhibited root hair elongation (FIGS. 1B and C), and it is believed that these PINs facilitated auxin release and consequently reduced internal auxin levels in root hair cells. Overexpression of short loop PIN8 significantly inhibited root hair elongation as well as long looped PINoxs (FIG. 1B). However, root hair specific overexpression of the auxin influx transporter AUX1 significantly enhanced root hair extension (FIG. 1C), presumably increasing the auxin levels of cells in root hair cells.

Although overexpression of both long loops of PINs and PIN8 significantly reduced root hair extension, the effects varied among different PIN species. Overexpression of PIN1, PIN2, PIN3 and PIN8 caused up to 90% or more inhibition of root hair extension, whereas PIN4ox and PIN7ox slightly inhibited root hair extension, on average about 60% and about 80%, respectively (FIG. 1C). Even in PINs with strong root hair inhibitory effect, somewhat different inhibitory power was observed. For example, PIN2ox almost completely inhibited root hair extension, while PIN1ox, PIN3ox and PIN8ox allowed marginal root hair extension (2-5% of control).

In contrast to other PINox plants, all three PIN5 overexpressing plants (PIN5ox, PIN5-GFP1ox and PIN5-GFP2ox) did not inhibit root hair extension but rather enhanced the kidneys (6-16%) (FIG. 1C). This PIN5ox-mediated increase was insignificant but consistent for other PIN5 overexpressing transforming constructs (P = 0.016 for PIN5ox; P <0.0001 for PIN5-GFP1ox and PIN5-GFP2ox). It can be inferred that PIN5 promotes auxin influx of cells or enhances auxin availability in the intracellular region where auxin performs molecular action.

Example  2. Root Epidermal Cell Elongation PINox Promoted by

Since high concentrations of auxins inhibit root elongation (Evans et al., 1994 Planta 194: 215-222), we found that by lowering the auxin levels of the cells, PINox promotes root epithelial cell elongation and thus root elongation. I thought I could. Root hair specific expression of PINs seems to be due to a significant increase in root elongation (FIG. 2A) and this effect at least partially enhances cell elongation. PINoxs increased the length of epidermal cells with root hairs from 8.5% (PIN4ox) to about 15% (PIN1ox, PIN2ox, PIN3ox and PIN8ox). The degree of PINox-mediated stimulation of root elongation and root hair cell elongation was somewhat proportional to the intensity of PINox-mediated root hair inhibition (2A, B). In long loop PINs, PIN1ox and PIN2ox showed the greatest effect, while PIN4ox showed the least effect in root elongation and root hair cell elongation. Epidermal cell length of the PIN5ox plant was similar to that of the control. Together with the results of PINox-mediated root hair inhibition, these data further support the hypothesis that different PINs have different auxin release activity.

Example  3. PIN8 Silver cigarette BY -2 suspension  In a cell Auxin  Release activity.

We previously described that auxin transporters, such as PIN3 and PGP4, are present in the plasma membrane and act as release transporters for 1-naphthalene acetic acid (NAA) in tobacco Bright Yellow-2 (BY-2) cells (Lee). SH, Cho HT, 2006 Plant Cell 18: 1604-1616. In addition, PIN4, PIN6, PIN7 and PGP19 have been shown to promote auxin release in tobacco BY-2 cells (Petrasek et al., 2006 Science 312: 914-918). Recently, PIN5 has been reported to have auxin releasing activity in yeast plasma membrane and possibly intracellular transport activity in Arabidopsis cells (Mravec et al., 2009 Nature 459: 1136-1142). In the present invention, in order to directly measure the intracellular auxin transport activity of short loop PINs (PIN5 and PIN8) in plant cells, the present inventors have determined that dexamethasone (Dex) -inducible promoter ( ProTA ; Aoyama T, Chua NH, 1997 Plant J 11: 606-612) was used to express GFP-tagged versions.

Auxin transport for analysis of cells, transgenic tobacco cells were processed for ^{[3 H] NAA, [3} H] retention of NAA in cells was measured at specific time intervals. NAA can easily enter cells by dispersion, requiring an active auxin release transporter to be released into the apoplast (Delbarre et al., 1996 Planta 198: 532-541). Induction of PIN8 significantly reduced the retention of [ ³ H] NAA compared to control cells that were not induced (FIG. 3B). After 20 minutes of incubation with [ ³ H] NAA, PIN8ox cells retained about 60% [ ³ H] NAA compared to control cells, indicating that PIN8 mediates auxin net flow from tobacco cells. In contrast, PIN5 induction in tobacco cells did not significantly alter [ ³ H] NAA retention levels compared to control cells (FIG. 3A). The impossibility to detect net flow of auxin in PIN5-expressing cells may be due to intracellular auxin transport activity rather than its intercellular auxin transport.

Example  4. external Auxins PINox  Restore the root hair extension of the transformants.

If PINoxs inhibit root follicle elongation by reducing auxin levels in root hair cells, external auxin application may restore root hair cell elongation in these transformants. 50 nM indole-3-acetic acid (IAA) increases root hair elongation of control plants by 30% and PIN5ox transformants by 20%, while compared to other PINox when compared to root hair elongation of untreated transformants. Root hair extension of the transformants was stimulated by 5 to 44 folds (average 15 folds) (FIG. 4). Auxin-mediated root hair recovery levels in PINoxs averaged 82% of root hair length (100%) of untreated control plants. These results indicate that the diffuse AUX1-utilized auxin intake rate exceeds the PINox-mediated auxin release rate until certain equivalences are reached. Although external IAA significantly increases the root hair elongation of PINox transformants overall, the extent of auxin-mediated root hair recovery varies between PINoxs. The PIN2ox plant exhibits some root hair recovery by IAA, suggesting that PIN2 has the strongest auxin releasing activity in root hair cells.

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Claims (7)

delete Arabidopsis (Arabidopsis thaliana) derived from the SEQ ID NO: 1 to 7 groups stage root hair elongation of Arabidopsis plants comprising a for introducing a recombinant plant expression vector containing the gene having the nucleotide sequence in Arabidopsis plant is selected from the consisting of How to adjust. The method according to claim 2, wherein the gene having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 4, SEQ ID NO: 6 and SEQ ID NO: 7 is overexpressed in Arabidopsis plants, characterized in that to reduce the root hair growth of Arabidopsis plants Way. The method according to claim 2, wherein the gene having the nucleotide sequence of SEQ ID NO: 5 is overexpressed in Arabidopsis plants to increase root hair growth of Arabidopsis plants. A Arabidopsis plant with controlled root hair elongation produced by the method of any one of claims 2 to 4. delete delete

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