KR20150066258A - A Mehtod for Regulating Plant Growth and Tropism Through the Control of Callose Synthesis - Google Patents

Abstract

The present invention relates to a method for controlling growth and tropism of a plant by controlling callose synthesis of a plant cell. More specifically, growth and tropism of the plant can be controlled by controlling concentration of auxin by regulating expression of GSL8 callose synthase.

Description

[0001] The present invention relates to a method for regulating plant growth and tolerance through regulation of carosynthesis,

More particularly, the present invention relates to a method for regulating plant growth or excitability by regulating carosynthesis of plant cells. More specifically, the present invention relates to a method for regulating plant growth or excitation by controlling the expression and activity of GSL8 callose synthase and functional analogue, To control plant growth and excitability.

Plants have a system to cope with and adapt to various environmental changes by controlling growth and development. Environmental stimuli and plant development programs throughout the entire life-span from seed germination to flowering are known to increase plant survival suitability through constant interactions. It is known that the regulation of the development of a plant by such environmental stimuli is mainly caused by the interaction with the external environment and the intrinsic developmental program of the plant.

Conventionally, a large amount of chemical fertilizers and pesticides were sprayed to improve the growth of plants. However, the above method causes acidosis of the soil due to the spraying of various chemical fertilizers, and as the plant growth is rather low, the crops become worse, And thus the pesticide ingredient which remains harmful to the plant is left as a result of the spraying, resulting in a serious problem that the health of the consumer is adversely affected.

In particular, plants are increasingly being used not only as food, but also as bioplants. Although many microorganisms and animal cells are used to synthesize specifically useful substances in vivo, plants are most effective considering mass production in terms of investment. Therefore, various bio-ventures and multinational companies are attempting to study plants as bio-plants.

However, since the use of plants as a source of biomaterials conflicts with the basic interests of food, the biomass of a plant stably meets the basic utility as a food resource, while the utilization as a plant for supplying biomaterials .

Therefore, it is urgently necessary to develop a method for regulating plant growth without using chemical fertilizers.

Among such methods, in particular, methods for controlling plant growth using various plant hormones and their genes have been actively researched. The present inventors intend to utilize auxin, which plays an important role in plant development, pattern formation, and excitability formation. Particularly, the function of the carnosus to control the plasmodesmata in order to function properly of the auxin hormone was investigated, and the mechanism of controlling the growth of the plant through the caloric control was utilized.

The present inventors have previously found that the GSL8 (Glucan Synthase-Like 8) protein regulates the functions of cytoplasmic breakdown, cell patterning, and platelet contact, And the ability to regulate growth and deflection was not clearly identified.

Therefore, the inventors of the present invention found that the synthesis of caros was controlled by auxin, thereby identifying a positive feedback circuit that constitutes "regulation of carosene accumulation auxin concentration gradient of callose synthase" To control the growth of plants through the accumulation of carrots, and to control the excitability of plants through the accumulation of carrots, thus completing the present invention.

Korea Patent No. 2007-0026257

The present invention relates to a method for regulating plant growth and tolerance based on the relationship between expression of GSL8 callose synthase and auxin concentration gradient. It is a primary object of the present invention to provide a method for regulating the growth of a plant by controlling the expression of GSL8 gene And a method for controlling the excitability.

Another object of the present invention is to provide a plant growth promoter or an excipient inhibitor comprising the GSL8 gene expression vector

It is another object of the present invention to provide a related mechanism of carosynthesis-auxin-plant growth and excitability and its use.

As described above, the present invention provides a mechanism for regulating the plant growth and excitability by controlling the expression level of the GSL8 carosyl synthase and thereby controlling the expression of the GSL8 gene, All included.

In order to solve the above-mentioned problems, the present invention provides a method for regulating the growth and excitability of a plant using the regulation of expression of callose synthase and a related mechanism.

The method is characterized in that it involves the regulation of the auxin concentration gradient by controlling the expression of callose synthase, and this auxin concentration gradient (slope) is essential for the growth and excitation of the plant.

Among the various methods of controlling carosyl synthesis, the method of regulating the expression of the gene for GSH8 (GLUCAN SYNTHASE-LIKE 8) may be the most efficient way to control carosynthesis.

Therefore, the present invention provides, as one embodiment, a method for enhancing plant growth and excitation by promoting expression or activity of GSL8 gene.

This method is characterized by increasing the synthesis of callus to form an oxine concentration gradient (slope) by callus accumulation (deposition).

Further, the present invention provides, as another embodiment, a method for suppressing plant growth and irritability by reducing the expression or activity of the GSL8 gene.

This method is characterized in that as the synthesis of callus is reduced, the PD permeability is increased and the auxin concentration gradient is inhibited by the increase of the symplasmic diffusion of auxin.

On the other hand, the present invention provides a plant growth promoter and a promoter containing a recombinant vector encoded so as to overexpress GSL8. In a similar aspect, a recombinant vector comprising a nucleic acid selected from the group consisting of an antisense oligonucleotide complementary to the mRNA of the GSL8 gene, a short interfering RNA, a short hairpin RNA and RNAi And a plant growth regulator and an inhibitor thereof.

In addition, plants transformed with the recombinant vectors are also provided.

At this time, the above plants can be used as rice, wheat, barley, corn, soybean, potato, red bean, oats, sorghum, Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, Peach, peach, sheep, grape, citrus, persimmon, apricot, apricot, banana, rose, peach, rose, Can be selected from the group consisting of gladiolus, gerbera, carnation, chrysanthemum, lily, tulip, raglass, red clover, orchardgrass, alfapa, tall fescue and perennial algae, It is a plant.

According to the present invention, by regulating the auxin concentration gradient by controlling the expression of carosyltransferase, plant growth and excitability can be regulated. Therefore, it is possible to control the growth rate by controlling the carosynthesis of the crop, Possibly, it can be used for various purposes.

For example, controlling the growth rate (especially, promoting growth) enables the rapid growth of the sprout grown in the growth of the sprouts, thereby increasing the yield of production, and can be utilized in the design of the utilization form of the ornamental plants utilizing the promoting properties. That is, when a plant type such as tubular water or tubular candle is designed by using light, plants with enhanced light sensitivity can be changed more economically than ordinary plants. In addition, it can be applied to the production of unfolded cucumber and eggplant using the reflex suppression technology to improve the merchantability, and it can be utilized variously such that the commercialization of the wood can be improved by the development of the unbreakable wood plant utilizing the reflex suppression technology .

1 is a graph showing the function of a carosynthesis gene related to carosis accumulation in a plasmodium (PD) and the characteristics of dsGSL8 plant characteristics.
Fig. 2 and Fig. 3 are the results of observing the effect of hypoxic luminosity and oyster neutralization according to the decrease of GSL8 activity in Arabidopsis thaliana.
FIG. 4 shows the results of observation of carosyl levels in the PD of hypocotyl cells by fluorescence microscopy and transmission electron microscopy-based immunoassay.
Fig. 5 shows the results showing that the synthesis of the novel carruth of the present invention is essential for the luminous and oyster neutral reactions.
Figs. 6 and 7 are the results showing the increase of PD permeability and auxin mobility with the decrease of GSL8 activity.
Figure 8 shows the disturbance of the auxin concentration gradient in the dsGSL8 + dex hypocotyl [not due to a PAT (polar auxin transport) system change)
Figure 9 shows the GSL8 expression pattern in the auxin response and hypocondylation during the reflex reaction.
Figure 10 shows the results showing that auxin modulates GSL8 expression through ARF7-mediated transcriptional activity.
Fig. 11 shows the results of showing a luminous bending enhanced GSL8 overexpression.
Figure 12 is a schematic diagram of a model for an auxin-carosulation regulating circuit to regulate plasma permeability (PD) permeability.

The terms used in the present invention are defined as follows.

By "gene" is meant any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term " nucleic acid " refers to nucleotide polymers of any length, including ribonucleotides as well as deoxyribonucleotides.

&Quot; Recombinant " refers to a cell expressing a protein encoded by a heterologous nucleic acid, either by replication or expression or by a heterologous nucleic acid

A "vector" is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. The vector may replicate the DNA and be independently reprogrammed in the host cell.

By "expression vector" is meant a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

&Quot; Expression " refers to the biological production of a product encoded by a coding sequence.

The term " promoter " refers to the region of DNA upstream from the structural gene and refers to the region of DNA to which the RNA polymerase binds to initiate transcription.

The improvement of the 'growth of the plant' may be caused by an increase in the growth rate of the plant, an induction of early flowering, an increase in height, an increase in seed yield or an increase in number of flowers But is not limited thereto.

The term " transformation " means that DNA is introduced into an appropriate host cell so that the DNA can replicate in the form of a chromosomal exogenous factor or a chromosomal merge.

The term "infection" refers to the introduction of a nucleic acid into an appropriate host cell by the use of a virus or viral vector.

The term 'plant morphology' refers to the external structure or shape of a plant, and it refers to the shape of a plant organs such as leaves, stems, roots, flowers, etc., Shape.

By "intracellular level" is meant the amount present in a cell, which can be controlled by a number of methods known to those skilled in the art. For example, intracellular levels may be regulated through, but not limited to, regulation at the transcription stage or post-transcription stage.

By "promoter" is meant a substance that promotes, enhances or increases the expression or activity of a gene of interest, or promotes, enhances or increases the activity of a desired function. The term " inhibitor " means a substance that inhibits, blocks or reduces the expression or activity of a target gene, or inhibits, blocks or reduces the activity of a desired function. The activation mechanism of the promoter or inhibitor is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds.

By "about" is meant a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length of 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

Auxin is a kind of plant hormone that stimulates length growth in the elongation zone where the cells generated from the apical meristem at the end of the stem and the root of the plant grow, , The generation of flowers and fruits, and the excitatory movement. In particular, endogenous auxin is present in the cotyledon (synthesis site) but confined to the hypocotyl hook region.

In the present invention, auxin includes natural oxines of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 4-chloroindole-3-acetic acid (4-Cl-IAA); Indole-3-butyric acid (IBA); Naphthalene acetic acid (NAA); 2,4-Dichlorophenoxyacetic acid (2,4-D); And 2,4,5-trichlorophenoxyacetic acid (2,4,5-T or a defoliant).

Complex transport systems in plants have evolved to allow the design of precise spatial and temporal auxin slopes (concentrations) that control specific developmental programs. The auxin is important in its function by regulating the hormone concentration gradient or maximum value, which is regulated by the polar auxin transport system. Studies have also been conducted on the concentration gradient mechanism of auxin via plasmodesmata, which allows for simple plasmid symplemic exchange of essential nutrients and signaling molecules for plant growth.

The present inventors have found that, in particular, the "auxin-GSL8 feedback for the regulation of the level of plasmodesmal-localized callose which locally down-regulates the symplasmic permeability during hypocotyl tropic response" Circuit ".

This system functions as a plasma contact switch to prevent the loss of gradient (tilt) formation due to simple rhemic auxin diffusion. This regulatory system represents a novel mechanism by which auxin can control the simple rustic transport of a wide variety of signaling materials.

<Mechanism>

The present invention relates to a mechanism capable of regulating plant growth and excitability by regulating the auxin concentration gradient through the regulation of GSL8 callose synthase expression, and its use.

In one aspect, the present invention relates to carosynthesis and the relationship between plant growth and oyster performance.

The polysaccharide callose, which performs a variety of functions during plant development (plant pollen production and development, stem and stem cell formation), is not only responsible for plant development, but also for signaling (plasmodermata), cell plate formation during cytokinesis, Beta-1,3-glucan, a cell wall that performs important functions in response to various stresses (high temperature, pests, wounds, etc.).

The present invention relates to a mechanism whereby the synthesis of such carrots is controlled by auxin and consequently the concentration gradient of auxin is formed depending on the amount of carrots synthesized, resulting in an influence on plant growth and excitability. A schematic diagram of such a mechanism is shown in Fig.

In particular, the mechanism of the present invention is characterized in that the regulation of the callus precipitation of plasmodesmata (PD) is essential for establishing the auxin concentration gradient.

Therefore, the present invention is based on such a mechanism, and it is based on such a mechanism that a positive feedback circuit that constitutes "synthesis of callose synthase → carosyl accumulation → regulation of auxin concentration gradient" and regulation of carosynthesis by auxin Provides the use to regulate plant growth and excitability.

Any method known in the art may be used to control the synthesis of the caros in the present invention.

For example, the carosynthesis may be regulated by using DDG (2-deoxy-Glucose; an inhibitor of carosyl synthase) or the like, but preferably a method of regulating the expression or activity of a carosynthesis gene can be used.

The callus synthesis gene may be any functional similar gene showing a carosynthesis effect, but GSL8 (GLUCAN SYNTHASE-LIKE 8) gene is preferably used.

The GSL8 gene is important for development in the early stage of growth and manifests various phenotypes, especially in embryonic development and early nutritional growth. In the present invention, GSL8 gene derived from Arabidopsis was used. As a gene coding for the GSL8 protein, oligonucleotides complementary to the terminal part of the GSL8 gene are known, and the genomic DNA or cDNA of a specific plant cell is expressed as a primer. And performing PCR as a template. The GSL8 gene according to the present invention may comprise a gene sequence of GSL8 (GLUCAN SYNTHASE-LIKE 8) described in SEQ ID NO: 2, and may further comprise a gene sequence having 70% or more homology with the gene sequence of SEQ ID NO: (GLUCAN SYNTHASE-LIKE 8) gene.

The GSL8 carosyltransferase of the present invention includes both a protein encoded by a known Arabidopsis GSL8 gene and a functional equivalent (a protein encoded by a functional similar gene). By "functional equivalent" is meant at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 70%, more preferably at least 70%, more preferably at least 70% More preferably 95% or more, and exhibits substantially the same physiological activity. &Quot; Substantially homogenous physiological activity &quot; means that the carosynthesis is promoted when overexpressed in a plant, or the carosynthesis is inhibited when the expression is suppressed in the plant.

The present invention, therefore,

In one embodiment, the expression or activity of the GSL8 gene is promoted to increase the synthesis of plasmodesmata (PD) callus to form an oxine concentration gradient (slope) by callus accumulation (deposition) Thereby inducing plant growth (growth). It also enhances the excitability in accordance with the over-accumulation of the PD callus.

At this time, according to the positive feedback circuit model, up-regulation of PD callus precipitation delays the PD-mediated spread of auxin, which is induced by auxin,

Therefore, the present invention provides a method for enhancing plant growth and excitation by promoting the expression or activity of GSL8 gene.

In another embodiment, decreasing the expression or activity of the GSL8 gene results in a decrease in the synthesis of the protoplasmic callus callus, resulting in an increase in PD permeability, thereby enhancing the symplasmic spread of auxin molecules (Ozone migration ability is enhanced) and auxin concentration gradient (slope) is not well formed, and growth and reflex reactions are reduced. That is, the PD permeability increasing effect associated with callus reduction inhibits the formation of the auxin concentration gradient required for the excitation reaction.

Therefore, the present invention provides a method for inhibiting plant growth and excitability by reducing the expression or activity of the GSL8 gene.

<Plant growth regulator>

In another aspect, the present invention relates to a plant growth regulator and an excipient regulator comprising a component that regulates the expression or activity of the GSL8 gene, based on the mechanisms and mechanisms described above. And a method for regulating the growth and the excitability of a plant by controlling the expression of the GSL8 gene.

For example, plant growth and induction promoters containing components that promote expression or activity of the GSL8 gene; Or GSL8 gene expression or activity, and a use thereof.

The plant growth and promoter of the present invention contains a component that promotes the expression or activity of the GSL8 gene, which promotes the transcription of the GSL8 gene, promotes translation of the transcribed GSL8, or promotes the function of the GSL8 protein But it is not limited thereto. For overexpression of GSL8, a suitable promoter may be operably linked to a DNA / polynucleotide encoding it and additional ' enhancer elements ' may be included if desired.

The plant growth and promoter of the present invention may be provided with a recombinant vector encoded by one of the above examples, wherein the GSL8 is overexpressed, and a plant transformed with the same. These can induce PD callus synthesis, effectively promoting plant growth and excitability.

The plant growth and inhibitor of the present invention contains a component that inhibits the expression or activity of the GSL8 gene, which inhibits transcription of the GSL8 gene, inhibits translation of the transcribed GSL8, or inhibits the function of the GSL8 protein But it is not limited thereto.

As a preferable example, a compound which is an antisense oligonucleotide which binds complementarily to the mRNA of the GSL8 gene, a short interfering RNA (short interfering RNA), a short hairpin RNA or RNAi, or a compound which binds complementarily to the GSL8 protein, , And peptide mimetics.

The plant growth and inhibitor of the present invention may, in one embodiment, be provided with a recombinant vector encoded to inhibit GSL8 expression and thus a transformed plant. By inhibiting the PD callus synthesis, they can effectively inhibit plant growth and excitation.

The above-mentioned use of the present invention also includes a method of regulating the growth and excitability of plants by controlling the expression of the GSL8 gene.

"Overexpression of a gene" and "reduction in gene expression" mean that the GSL8 gene is expressed at a level above or below that expressed in a wild-type plant, respectively. For the recombinant vector and the transformation method therefor, Can be used.

The 'plant expression vector' may be, but is not limited to, a part of itself when present in a suitable host, such as Agrobacterium tuberculosis, a Ti-plasmid vector capable of transferring the so-called T-region into a plant cell, Suitable plant expression vectors may be selected from viral vectors such as those derived from double-stranded plant viruses (e. G., CaMV) and single-stranded viruses, germinaviruses and the like, such as non-integrative plant viral vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The recombinant vector of the present invention preferably contains one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinotricin (phosphinotricin), kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, , But are not limited thereto.

The promoter that can be included in the recombinant vector of the present invention is a promoter capable of initiating transcription in a plant cell, and preferably, can overexpress a gene inserted into a plant. More preferably a constitutive promoter that is active under most environmental conditions and developmental conditions or cell differentiation. That is, a constitutive promoter is preferable because selection of the transformant can be carried out by various tissues at various stages. More preferably, the promoter that may be included in the expression vector of the invention may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter.

<Transgenic plants>

The present invention also includes cells and plants transformed with recombinant vectors which are GSL8 gene expression or activity regulators.

Any transformation method can be used to transform a nucleic acid into a plant to transform the plant. Such transformation methods include, but are not limited to, the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplast electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol (DNA or RNA-coated) particle impact method (Klein et al., 1987, Nature 327, 70) of plant elements, infiltration of plants or mature pollen or microparticles (Non-integrative) virus in the transgene-mediated gene transfer of Agrobacterium tumefaciens by transformation, and the like. A preferred method according to the present invention may be an Agrobacterium mediated transformation method

The plants that can be transformed in the present invention include, but are not limited to, food crops, vegetable crops, special crops, fruit trees, flower and feed crops, preferably rice, wheat, barley, corn, , Oats, sorghum, Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, onion, carrot, ginseng, tobacco, cotton, sesame, sugarcane, beet, Peanuts, rapeseed, apple trees, pears, jujube trees, peaches, sheep, grapes, citrus, persimmon, plum, apricot, banana, rose, gladiolus, gerbera, carnation, chrysanthemum, lily, tulip, raglass, red clover, orchard Grass, alfapa, tall fescue, and perennialla grass. In particular, in the present invention, the GSL8 gene can be most effectively expressed in plants of Arabidopsis and its closely related line, and can regulate plant growth and excitability.

The present invention also encompasses seeds produced by growing the transgenic plants.

As described above, the present invention provides a mechanism for regulating the plant growth and excitability by controlling the expression level of the GSL8 carosyl synthase and thereby controlling the expression of the GSL8 gene, All included. The present invention is useful for various purposes such as controlling the growth rate by regulating the carosynthesis of crops to regulate the harvesting time or adjusting the phenotype by controlling the excitation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Plant materials and growth conditions

In the present invention, all of Arabidopsis thaliana (Arabidopsis thaliana wild type and mutant Colombia (Col-0) background were used. PGS8 :: GFP, PDR5 :: GUS, PDR5 :: RFP, P35S :: DII: VENUS (Brunoud et a., 2012), and various auxin- Dexamethasone-induced dsGSL8 was introduced in the background by transformation (Clough and Bent, 1998). And analyzed using separate F2 or F3 generation. Seeds were surface-sterilized and lx MS supplemented with 20 [mu] M dexamethasone (+ dex) And germinated on plants containing 0.6% agar medium.

The general growth conditions (Chen et al., 2009) were discussed. Sulfur seedlings were grown under continuous dark room conditions for 3 days and excitation was tested.

Experimental Preparation and Experimental Methods

Protocols used for RNA extraction, reverse transcription and RT PCR, carotid staining, export loading analysis, excitation reactions, plasmid constructs, microinjection analysis, microscopy imaging, transmission electron microscopy and carotid immunoassay, transient expression analysis, Experimental Procedures ".

One. RNA  Extraction, and real-time PCR ( Real Time PCR )

Total RNA was extracted from Arabidopsis seedlings or upper half seedlings using TRIzol (Invitrogen, CA) after cutting in the mid-dwarf region. Partial RNA (5 μg) from each sample was reverse transcribed with cDNA using Superscript II reverse transcriptase (Invitrogen) using Oligo (dT) primer.

Real-time PCR amplification using the first strand cDNA as template was performed on an ECOTM real-time PCR system (Illumina) equipped with SsoFast ™ EvaGreen® Supermix (Bio-Rad) under the following conditions: , 40 cycles of 95 ° C for 10 s, 55 ° C for 30 s, and 72 ° C for 30 s.

Expression levels of other genes were normalized to ACTIN2. All experiments were performed 3 times and the relative gene expression levels were calculated by the 2 ^-ΔΔt method (Livak and Schmittgen, 2001)

The specific primer pairs used in the real-time PCR analysis were as follows:

2. Carrot dyeing ( Callose Staining )

The Arabidopsis thaliana (Arabidopsis) Seedling cotyledon or the Carlos staining buffer (callose staining buffer, CSB) was immersed for one hour. CSB is a mixture of 0.1% w / v aniline blue and 1 M glycine (pH 9.5) in pressurized autoclaved triple-distilled water with a volume ratio of 2: 3. Samples were washed and stained in aqueous 2 mg / ml propidium iodide solution for 3-4 minutes.

3. Deflation  Loading Analysis Hypocotyl Loading Assay )

Three-day-old sulphate Arabidopsis seedlings were transferred to MS agar plates containing 0 or 20 [mu] M dex, and after 1 day of darkness the hypocotyl was cut at the hook base. A cover slip was placed between each hypocotyl cutting surface and the MS agar plate.

Individual agar blocks containing HPTS (8-hydroxypyrene-1,3,6-trisulphonic acid) (5 mg / ml) were placed on the cutback surface for dye loading and the seedlings were washed in water for 15 minutes, Probe motions were evaluated using a confocal microscope.

For auxin loading IAA (2 μM), 2,4-D (2 μM) or [14C] 2,4-D (American Radiolabeled Chemicals, specific activity 50 mCi mmol-1) . Samples were exposed to MultiSensitive Phosphor Screen (PerkinElmer, USA) for 4 days and the screen was scanned with a phosphor imager (Cyclone Plus Storage Phosphor System, PerkinElmer, USA).

4. Tyrosine Reaction

The seedlings were grown vertically on MS agar plates in a dark room at 25 ° C and exposed to unidirectional blue light of low intensity ( ² μmol · m ^-2 s ^-1 ) at 3-day old sowing seedlings. For oyster neutral experiments, the plants were rotated at 90 ° C to give a defined treatment period and the seedlings were imaged with a scanner for measuring the curvature angle

5. Chemical treatment

N-1-naphthylphthalamic acid (2 μM) and 1-NOA (1-naphthoxyacetic acid) (2 μM) as inhibitors of the bipolar auxin transfer were applied to MS agar plates and agar blocks for the auxin loading assay Respectively.

For the luminosity test, the seedlings were pretreated on MS agar plates supplemented with NPA (10 uM) or 6-fluoroindole (6-FI) (50 uM). In the auxin treatment experiments, auxin (2 μM), 2, 4-D (2 μM) or cycloheximide (CHX) (50 μM) were added to MS agar plates.

6. Plasmid construct

The preparation of the dexamethasone-inducible dsGSL8i construct was described in Chen et al. (2009). For PGSL8 :: GFP, PGSL8 :: LUC and PGSL8m :: LUC, a 1.5 kb GSL8 promoter fragment was amplified from Col-0 genomic DNA using primers 5'-caccGCAACCTATACATTGTAAAAATATG-3 'and 5'-AGAGTACGCTTGCTC-ACATACT-3' Amplified and then cloned into pENTRY-TOPO to recombine with the desired vectors GW-GAL4-5UAS-GFPer and GW-LUC.

PDR5 :: RFP-PGSL8 :: GFP construct was named PDR5 :: RFP (a gift from D. Jackson, Cold Spring Harbor Laboratory) PGSL8 :: GFP. For P35S :: GSL8, 5.7 kb GSL8 cDNA was amplified using primers 5'-caccATGGCACAGAACTTGGACCCT-3 and 5-GGTCTCAACA TTAGCTCTGTTTC- 3 ', then cloned into pENTRY-TOPO and cloned into the target vector pMDC43 Lt; / RTI &gt;

For PGSL8 :: GSL8 and PGSL8 :: GSL8, the amplified promoter fragments were cloned into pMDC99 and then recombined into pENTRY-TOPO-GSL8.

7. Microinjection analysis ( Microinjection Assays )

Microinjection was performed (Lucas et al., 1995) with reference to published literature.

Three - day - old bulbous rice seedlings were used for microinjection experiments. After the pellet was covered with 0.3 M mannitol solution, a fluorescent probe (1 mM) was injected into the epidermal cells, and cell-to-cell migration was observed with a confocal microscope (Leica model SP2).

8. Microscopic Imaging ( Microscopy Imaging )

GUS stained plants were imaged with either an Olympus dissection microscope (model SZX12) or a scanner (Epson model 1670). The curvature of the deflection angle was measured using Image J software (http://rsbweb.nih.gov/ij). For a confocal microscope, a complete plant sample was imaged on a Leica model SP2 or Olympus FV1000 confocal microscope, with the combination of laser and filter as follows: 500-550 nm band-pass for GFP and F-dextran Emission 488-nm laser line; 680 ~ 759 nm emission wavelength for chloroplast autofluorescence; 561-nm laser line with 575 to 630 nm band-pass emission for RFP; 605-651 nm band-pass emission for Texas-Red-dextran 594-nm laser line; And 405-nm laser excitation and 500-550 nm emission for aniline blue.

When imaging two or more fluorescent pigments in the same tissue, sequential laser scanning methods were used to prevent crossing between different fluorescent pigments

.

9. Transmission electron microscope ( Transmission Electron Microscopy ) And carosome immunohistochemistry Callose Immunolocalization )

Wild type, gsl8 mutants and dsGSL8 (with 20 [mu] M dexamethasone) Arabidopsis seedlings were grown vertically on MS solid medium. After the hypocotyl was cut and frozen in a Leica EM HPM100 high pressure freezer, the samples were freeze-dried in anhydrous acetone with 0.1% uranyl acetone, 0.25% glutaraldehyde and 8% dimethoxypropane for 72 h at -80 ° C, substituted. After freeze-displacement, the medium was slowly warmed from -80 ° C to -45 ° C for 35 hours. Samples were rinsed three times with anhydrous acetone and embedded in Lowicryl HM20 resin (Electron Microscopy Sciences, PA) by stepwise increasing the resin concentration from 0% to 33%, 66%, 100% for 48 hours at -45 ° C. The resin was polymerized with UV light at -45 캜 for 24 h. Freeze-displacement, resin embedding and UV polymerisation were carried out using the AFS2 automatic freeze-displacement system (Leica Microsystems, IL).

For carrots immune gold labeling, 80-120 nm-thick longitudinal sections were prepared from the hypocotyl samples and mounted on formvar coated gold-slotted grids (Electron Microscopy Sciences, PA). The instrument was floated on 0.2% Tween-20 (PBST) supplemented with 2% non-fat milk and PBS (phosphate buffered saline) for 30 minutes to inhibit non-specific antibody binding . Sections were incubated with 1/200 (v / v) diluted anti-carotid monoclonal antibody (Cat. No. 400-2, Biosupplies, Parkville, Australia) in 2% fat-free milk and PBST. At 3 hours after incubation, the instruments were thoroughly washed with PBST and probed with anti-mouse IgG secondary antibody conjugated with 15 nm gold particles (Ted Pella, CA; dilution = 1/10 [v / v] in PBST) .

Immunol Gold-labeled fragments were post-stained with 2% uranyl acetate in methanol, and Reynolds lead and observed with a Hitachi H-7000 (Pleasant, CA) transmission electron microscope operating at 100 kV. Immunogold-labeled particles were counted in 22, 19, and 21 electron micrographs showing 45, 33, 42 PD from each piece of wild type, gsl8 and dex-treated dsGSL8 transgenic plants. Immune gold particles located within 50-60 nm plasmodesmal appressed ER were counted in plasma contact-related fashion.

10. Transient Expression Analysis Transient Expression Assays )

Agrobacterium tumefaciens containing the PGSL8 :: LUC or PGSL8m :: LUC construct [the mutation in the GSL8 promoter in the putative ARF binding site (-1102 TGTCTC → TCTGTC)] was suspended in 3 mL of YEP medium containing antibiotic and 100 μM acetosyringone in 3 mL of YEP medium. The cultured cells were harvested by centrifugation and resuspended to an OD 600 1.0 value in infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl 2 and 200 μM acetosyringone)

To minimize gene silencing effects in PGSL8 :: LUC and PGSL8m :: LUC expression, Agrobacterium cells containing the PGSL8 or PGSL8m heparase reporter gene and Tomato bushy stunt virus P19, The benthamiana was infiltrated into the leaves. Two days after Agrobacterium infiltration containing the constructs, GSL8 and GSL8m promoter activities were examined by assaying tobacco leaf discs (0.3 cm ² ). Leaf discs were treated with 2 μM IAA or water (mock control) by vacuum infiltration. After 3 hours of IAA treatment, leaf discs were sprayed with 1 mM luciferin in 0.01% v / v Triton X-100 and kept in the dark for 4 minutes to allow luciferin to penetrate all the way into the tissue. The luminescent image was obtained in 200 seconds using a pre-cooled charge-coupled device camera (-60 ° C) (ANDOR iXon Technology, CT)

Luciferase activity was quantified using ImageJ software (www.rsb.info.nih.gov/ij) and the mean activity value of the IAA- or mock-treated sample was used to calculate a multiple change relative to the time "0 & fold changes were calculated

11. Chromatin ( ChIP ) analysis

ChIP analysis was performed by Gendrel et al. (2002). 10-day-old arf7 seedlings recovered by P35S :: ARF7: GFP were treated with 1 μM IAA for 3 h. The seedlings were then harvested (~ 1 g) and 1% (w / v) formaldehyde crosslinking was carried out in vacuum for 15 minutes. The glycine was added so as to be 0.124 M and the crosslinking reaction was stopped by vacuum impregnation for 5 minutes.

The chromatin was extracted and resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, and 1 × complete protease inhibitor; Roche) and sonicated with a Bioruptor sonicator (Diagenode, Belgium).

The soluble chromatin solution was diluted 10-fold with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0 and 167 mM NaCl). The diluted chromatin solution was pre-cleared with Protein A agarose beads blocked with salmon sperm DNA (Upstate Biotechnology) and incubated overnight at 4 ° C with GFP-TRAP (ChromoTek, Germany) . After several washes, the immunocomplexes were eluted with elution buffer (1% SDS and 0.1 M NaHCO3) and reversely cross-linked overnight at 65 ° C under high-salt conditions (0.2 M NaCl). The protein was removed by proteinase K treatment, and the DNA was recovered by phenol / chloroform extraction and ethanol precipitation.

ChIP DNA products were analyzed by PCR using primers designed to amplify about 300 bp DNA fragments in the coding region or promoter region of GSL8 or IAA19 used for ChIP analysis.

Example  One : Deflation  Reflex reaction Hypocotyl Tropic Responses ) And GSL8 - Induced PD  Carlos precipitate ( deposition ) Relationship

Screening of Arabidopsis mutants deficient in PD carrots was conducted to confirm the hypothesis that the regulation of PD carrots precipitation is essential for establishing the auxin concentration gradient.

1-1 Genes encoding Arabidopsis carotene synthase

The Arabidopsis genome contains 12 genes (GSL1-12), which are presumed to encode carol synthase (Chen et al., 2009). Of the eleven gsl mutations tested, only an aniline blue-based PD carol staining of gsl8 / calS10 / massue / chorus was absent (Fig. 1A), except for the actinomatous lethal mutation gsl10. This was consistent with GSL8, a major enzyme involved in the deposition of PD carrots (Guseman et al., 2010).

The gsl8 mutant is a cytokinesis-defective mutant showing abnormal embryogenesis and seedling-lethality (Chen et al., 2009; Thiele et al., 2009), dexamethasone (dex- Knockdown transgenic plants at the GSL8 transcription level were used by expressing the double-stranded (ds) GSL8 RNAi construct in a regulated double-stranded system (Aoyama and Chua, 1997).

These lines exhibited phenotypes such as dwarf phenotype and abnormal hook opening, similar to the gsl8 mutant, and reduced GSL8 transcription levels (Chen et al., 2009) (Fig. 1 B-E).

Real-time RT-PCR analysis further confirmed that 6-h dex-treatment resulted in a 90% reduction in GSL8 transcription level compared to wild type and dex (-) plants (Fig. 1F).

Interestingly, this decrease in GSL8 transcription resulted in an increase in the transfer of other family members, most notably GSL5 and GSL7 (Fig. 1E). Significantly, the aniline blue signal was scarcely detected in dex-treated dsGSL8 RNAi (hereinafter referred to as dsGSL8 + dex) seedling plants (Fig. 1 GI). This confirms that the up-regulated GSL genes do not contribute to the PD carrots precipitation. In addition, it was confirmed that PD carrots produced by GSL8 activity were down regulated in dsGSL8 + dex plants.

1-2 concentration formation

Hypocotyls (asymmetric oxine distribution) was chosen as a model system to confirm the involvement of PD carrots in auxin concentration gradient formation. This is a precondition for the luminosity and oyster-neutral response dependent on the kidney rather than the cell division.

As a result, wild-type and non-wild-type control dsGSL8 RNAi (-dex) (hereinafter referred to as dsGSL8-dex) dominant plants showed normal luminosity (FIGS. 2A and B). However, dsGSL8 + dex based plants did not respond to either light or gravity (Figs. 2A and B). The decrease in the growth of dsGSL8 + dex hypocotyl was thought to contribute to the observed reduced light response. However, the present inventors confirmed the difference in luminous and oyster neutrality even in younger plants (Fig. 2A, B; Fig. 3A). It is believed that the non-glowing response of dsGSL8 + dex seeding is not due to a deficiency in the kidney but, on its own, rather a differential elongation between the shade and the illumination of the dysplasia due to inability .

In addition, dex-inducible GAL4: GR (control) seedlings showed similar results to wild-type plants (Fig. 3B) and other gsl mutants showed normal luminescent response (Fig. 3C).

The asymmetric distribution of the carnos was observed in the ovulatory response of dsGSL8-dex dysplasia (Fig. 2C-E, Fig. 4A). In contrast, such an asymmetric distribution did not appear in dsGSL8 + dex non-responsive depletion (Fig. 2F-H, Fig. 4A). In order to confirm whether the non-ocular properties of these dsGSL8 + dex defects are associated with PD caloric reduction, PD-related calories were visualized using anti-callose antibodies (Fig. 4B) .

Immunogold experiments revealed that the level of PD carrots was significantly reduced in dsGSL8 + dex hypocotyl compared with the wild type control (Fig. 2 I) (Fig. 2 J, Fig. 4 B, C). We also verified the efficacy of the dex-inducing system in reducing GSL8 levels and PD carrots by obtaining corresponding results in the downregulation of gsl8 mutants (FIG. 2K, FIG. 4B, C).

However, some residual PD-associated carruth was still detected in gsl8 and dsGSL8 + dex pox, suggesting that other members of the GSL family also contribute to the PD carrots precipitation.

Example  2 : Seedlings  Luminous and Oyster neutral  New Carous Synthesis Required for Reaction

GSL8 expression has been reported to occur in various organs, such as roots, leaves, stems, flower buds, and flowers (Chen et al., 2009). In addition, the light response of the plant is thought to be due to the change in the expression pattern of GSL8.

In this experiment, we have shown that the GFP signal is in agreement with the light-responsive response domain located immediately below the whitening plexus that undergoes a light response using time-lapse imaging and PGSL8 :: GFP reporter seedlings A, B).

Additional evidence on the correlation between PD caloric precipitation and plant luminous / oyster neutrality, on the other hand, was obtained through experiments that inhibited carosynthesis activity using 2-deoxy-D-glucose (DDG) (Jaffe and Leopold, 1984 ). This treatment markedly inhibited both luminous and oyster neutrality (Figures 2C and 2D). However, the dorsal elongation was not significantly affected during the period of the photopolymerization reaction.

We performed a luminous bending analysis by adding DDG at different times prior to the luminous stimulation and confirmed that the inhibition of the luminous bending occurred when both DDG and light were applied (FIG. 5E). In addition, it was confirmed that a pretreatment period of 2 hours is required to extract the maximum inhibition of the photoluminescent reaction in 20 μM dex treatment (FIG. 5 F, G). This finding strongly supports the hypothesis that new caros synthesis is essential for normal photoluminescent reactions.

Example  3: Reduced GSL8  Active, Increased PD  Permeability and Simple Rustic Auxin  Relevance of movement

First, simple raster mobility of fluorescent probes was investigated to investigate the function of GSL8 in cell and intercellular migration of fluorescent probes. The intercellular migration of HPTS (Chen et al., 2009), a simple rustic tracer, was found to occur more frequently in the dsGSL8 + dex plexus than dsGSL8-dex plexus. (Fig. 6A, and Table 1).

Microinjection experiments also showed that a 4.4-kDa fluorescein-labeled probe can radially migrate into neighboring cells in dsGSL8 + dex vacuole. However, in the control group they were trapped in the injected cells (Fig. 6B and Table 2).

These results show that reduced PD calories are consistent with increased PD permeability.

In addition, we investigated the relationship between GSL8 transcription and the migration of intercellular auxin. For this purpose, auxin shifts in the dorsiflexion were observed using [14C] -labeled 2,4-dichlorophenoxyacetic acid (2,4-D) and synthetic auxin. As a result, dsGSL8 + lt; RTI ID = 0.0 &gt; (FIG. 6C). &lt; / RTI &gt;

The treatment of the PAT inhibitor 1-N-naphthylphthalamic acid (NPA) showed that only partial inhibition of 2,4-D migration was possible in dsGSL8 + dex hypocotyls. Also, auxin loading experiments were performed on dsGSL8 plants with PDR5 :: GUS (auxin response reporter).

High type indole-3-acetic acid (IAA) and 2,4-D of oxine induced only weak GUS signals in the lower part of dsGSL8-dex hypocycles (Fig. 7A); In contrast, strong GUS signals were observed in dsGSL8 + dex defecation with or without 1-napthoxyacetic acid (1-NOA, an auxin influx transporter inhibitor) and NPA.

Because negatively charged apoplastic 2,4-D (pKa of 2.73) can not enter the cell without the aid of an influx transporter in the 1-NOA treatment, PAT-induction through the use of two PAT inhibitors And the possibility that the migration of 2,4-D through passive apoplasmic flux could induce the expression of PDR5 :: GUS.

Hence, the enhanced auxin response observed in dsGSL8 + dex uptake treated with both 1-NOA and NPA suggests a stronger simple ruminal auxin shift than in the control dsGSL8-dex uptake (Fig. 6D-I).

And studies on seedlings that turned dsGSL8-dex PDR5 :: GUS (or PDR5 :: RFP) showed that endogenous auxin is present in the cotyledon (synthesis site), but is restricted to the backslope region 6 D).

In contrast, in the dsGSL8 + dex seedlings, GUS or RFP signals were similarly detected in the cotyledon and hook regions, but these signals were significantly extended downward (FIG. 6G, FIG. In addition, the NPA treatment did not alter the auxin reporter pattern in the dsGSL8 + dex seedlings and the control group (Fig. 6E, H).

The results obtained using the auxin input sensor DII-Venus (Brunoud et al., 2012) showed that the auxin response observed with the DR5 reporter was consistent with the auxin level (FIG. 6 J-O, FIG. 7 D).

NPA treated or untreated dsGSL8 + dex sulphated hypocotyls showed a weaker VENUS signal than the control hypersomnia (stronger auxin levels). These data show that PAT is not the cause of auxin levels increase in dsGSL8 + dex pox.

To assess the potential for increased local auxin synthesis, 6-fluoroindole (6-FI), an inhibitor of the tryptophan-mediated IAA synthesis pathway, was treated with dsGSL8 + dex sulphated hypocotyls. This treatment reduced the DR5 signal overall, but did not abolish the auxin response pattern in dsGSL8 + dex dysplasia (Fig. 6F, I; Fig. 7B, C).

The DII-Venus pattern in the 6-FI treated dsGSL8 + dex dorsiflex was similar to that of dsGSL8 + dex dorsiflexion (Fig. 6O; Fig. 7B, C). These results show that the increased auxin levels in dsGSL8 + dex uptake do not depend on the 6-FI sensitive local auxin synthesis pathway.

In addition, dsGSL8 + dex hypocomplex did not show any change in expression of the two auxin synthetic marker genes, YUCCA4 and YUCCA6. (Fig. 7E, F). In addition, these experimental results support the hypothesis that a decrease in GSL8 expression leads to an increase in PD permeability, thereby improving the ability of simple rastin auxin transport regardless of PAT and local auxin synthesis.

On the other hand, inquiries may be raised as to whether dsGSL8 + dex seedlings lead to stronger plant auxin shifts. In light-grown Arabidopsis, auxin moves downward from the cotyledons to the dorsal and roots, mostly by the PAT pathway (Lewis and Muday, 2009), and the pit length is associated with PAT (Jensen et al. , 1998). NPA treatment has been reported to significantly inhibit dorsal growth (Jensen et al., 1998; Lewis and Muday, 2009).

The present inventors have determined whether dsGSL8 induction can inhibit the effect of NPA by enhancing simple ruminal auxin transport through the PD pathway. Under NPA treatment, the dorsal growth of dsGSL8 + dex seedlings was observed to be significantly greater than in the dsGSL8-dex control (Fig. 6 P, Q). These results show that simple rumic auxin diffusion increases with decreasing levels of PD calories.

In summary, the reduction in PD carroxes achieved by downgrading GSL8 mRNA levels resulted in an increase in PD permeability, thereby confirming that enhanced simple rustic spread of small molecules such as oxine was achieved.

Example  4 : Auxin  density The gradient (not the PAT system) dsGSL8 + dex In hypocotyl  Is it disturbed?

In plants, asymmetric oxine distribution is essential for the photolytic reaction (Friml et al., 2002b); i.e., the auxin concentration needs to be high on the dorsiflexion side away from the luminous vector. Measurement of asymmetric auxin distribution and response was performed using dsGSL8-dex seedlings containing DII-Venus (PDR5 :: GUS or PDR5 :: RFP).

In the mock-treated seedlings, the auxin concentration gradient (asymmetric oxine reaction) was clearly apparent after the light-treating treatment (Fig. 8A, E, F and J, Fig. 9). Compared to the control situation, the auxin distribution and response in the dsGSL8 + dex hypocotyls were symmetric (Fig. 8B, E, H and J, Fig. 9).

These results show that the increase in PD permeability associated with the reduction of PD calories can inhibit the formation of the auxin concentration gradient required for the photoluminescence.

Alternatively, the PAT at the dorsiflexion, mediated by auxin entry and exit messengers (Ding et al., 2011; Mravec et al., 2009; Stone et al., 2008) . &Lt; / RTI &gt; This possibility was first investigated using NPA, which does not affect the growth of the axon, and effectively inhibits the luminosity of the seedlings (Friml et al., 2002b).

NPA treatment showed symmetrical auxin distribution and response at + dex conditions (Fig. 8 CE, G, I and J).

Cellular localization experiments were then performed with dsGSL8 plants containing PAUX1 :: AUX1-YFP (Stone et al., 2008), PPIN1 :: PIN1-GFP or PPIN3 :: PIN3-GFP (Ding et al., 2011) Lt; / RTI &gt;

Although PIN1-GFP signals were not detected in the dsGSL8 + dex backspace, AUX1-YFP and PIN3-GFP showed similar cellular expression patterns in the blue light-treated dsGSL8-dex and dsGSL8 + dex backs (Figure 8 KM) . Although not all of the components involved in PAT (polar auxin transport) have been tested, these results show that the lack of a photoreactive response in dsGSL8 + dex seedlings is due to an increase in PD permeability, not due to cleavage in PAT.

Example  5: Auxin - ARF7  Is regulated positively by the feedback circuit GSL8  Expression

Transgenic plants expressing PDR5 :: RFP and PGSL8 :: GFP reporters have shown a close spatial relationship between these fluorescent signals (FIG. 8 F-I and FIG. 9 N-Q).

Expression of PGSL8 :: GFP was induced by auxin application, particularly in the dorsal region (Fig. 10A, B). Quantitative RT-PCR analysis also showed that light signals and exogenously added auxins were responsible for increased GSL8 expression in nocturnal glomeruli (Fig. 10C, D).

However, GSL8 expression by these light signals and exogenous auxin was not induced in the arf7 mutation (auxin-dependent lack of photoreactive reactivity) (Tatematsu et al., 2004).

Consistent with these results, carrots accumulation in the arf7 mutant embryo pellets during the light-responsive reaction was significantly reduced compared to the control (Fig. 10E). In wild-type seedlings, the translation inhibitor cycloheximide (CHX) treatment did not block GSL8 expression by auxin (Fig. 10D), indicating that GSL8 could be the primary downstream target of auxin.

Sequence analysis of the 5 'upstream region of GSL8 confirmed the presence of one ARF binding site in the GSL8 promoter region. Therefore, we constructed PGSL8 :: LUC and mutated PGSL8m :: LUC (putative ARF binding site: TGTCTC → TCTGTC) constructs to test the auxin response of the promoter.

Using the Agrobacterium-mediated transient expression assay system, it was found that the activity of lysipyrase in the auxin-treated PGSL8 :: LUC was significantly increased compared to the control (Fig. 10F). However, this increase was not seen in samples expressing the mutant construct, PGSL8m :: LUC (lack of ARF binding site) (Fig. 10F). Chromatin immunoprecipitation (ChIP) analysis also confirmed that this ARF binding site in the GSL8 promoter region is a true target region of ARF7 (FIG. 10G).

In addition, to determine if ARF7-mediated GSL8 regulation is necessary for proper light-responsive response, the gsl8 mutation was transformed into PGSL8 :: GSL8 or PGSL8 :: GSL8.

The PGSL8 :: GSL8 construct restored both the growth / developmental phenotype and the luminosity curve of the gsl8 mutant (FIG. 10 H, I), but the PGSL8m :: GSL8 construct only partially restored the developmental phenotype and did not restore the luminous curvature 10 H, I).

These results suggest the action of the auxin-ARF7-mediated regulatory network in regulating GSL8 expression and suggest that there is a positive feedback circuit in GSL8 and auxin activity that minimizes auxin spread via PD (Figure 12) (Fig. 10J)

Example  6: PD  Over-accumulation of Carlos Over - accumulation ) &Lt; / RTI &gt; Phototropic Curvature  reinforce

According to a positive feedback circuit model (Fig. 10 J), the upregulation of auxin-induced PD carcass precipitation delays the spread of auxin mediated PD.

Therefore, in the situation where the accumulation of caros is present, the radiant bend can be predicted to occur at a shorter time than in the control condition. To confirm this hypothesis, a P35S :: GSL8 (over-accumulation) plant was constructed (FIG. 11).

Carls and accumulation plants (lines # 5 and # 6) exhibited dwarf phenotype and seedling mortality (FIG. 11A-B). In contrast, moderate carlos-accumulating plants (# 17 and # 24) expressing GSL8 transcripts slightly higher than wild-type plants showed faster growth under long-day conditions and longer growths in sulphated seedlings (Figure 11 A-D). The carlos levels in the p53 of the P35S :: GSL8- # 17 and P35S :: GSL8- # 5 were significantly higher than those present in the wild type pellet (Fig. 11 E-H). In addition, the sulfated P35S :: GSL8- # 17 seedlings were found to reduce auxin transport in the pituitary compared to wild-type seedlings (FIG. 11 I).

As predicted, these transgenic plants exhibited accelerated luminous responses (FIG. 11 J), suggesting that the hypoxic response is due to the presence of auxin-GSL8-mediated positive feedback pathways in PD, considering auxin status and PD permeability .

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> A Mehtod for Regulating Plant Growth and Tropism Through the          Control of Callose Synthesis <130> PN1310-359 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1904 <212> PRT <213> GSL8 protein sequence <400> 1 Met Ala Arg Val Tyr Ser Asn Trp Asp Arg Leu Val Arg Ala Thr Leu   1 5 10 15 Arg Arg Glu Gln Leu Arg Asn Thr Gly Gln Gly His Glu Arg Val Ser              20 25 30 Ser Gly Leu Ala Gly Ala Val Pro Pro Ser Leu Gly Arg Ala Thr Asn          35 40 45 Ile Asp Ala Ile Leu Gln Ala Ala Asp Glu Ile Gln Ser Glu Asp Pro      50 55 60 Ser Val Ala Arg Ile Leu Cys Glu Gln Ala Tyr Ser Met Ala Gln Asn  65 70 75 80 Leu Asp Pro Asn Ser Asp Gly Arg Gly Val Leu Gln Phe Lys Thr Gly                  85 90 95 Leu Met Ser Val Ile Lys Gln Lys Leu Ala Lys Arg Asp Gly Ala Ser             100 105 110 Ile Asp Arg Asp Arg Asp Ile Glu Arg Leu Trp Glu Phe Tyr Lys Leu         115 120 125 Tyr Lys Arg Arg His Arg Val Asp Asp Ile Gln Lys Glu Glu Gln Lys     130 135 140 Trp Arg Glu Ser Gly Thr Thr Phe Ser Ser Asn Val Gly Glu Ile Leu 145 150 155 160 Lys Met Arg Lys Val Phe Ala Thr Leu Arg Ala Leu Ile Glu Val Leu                 165 170 175 Glu Val Leu Ser Arg Asp Ala Asp Pro Asn Gly Val Gly Arg Ser Ile             180 185 190 Arg Asp Glu Leu Gly Arg Ile Lys Lys Ala Asp Ala Thr Leu Ser Ala         195 200 205 Glu Leu Thr Pro Tyr Asn Ile Val Pro Leu Glu Ala Gln Ser Met Thr     210 215 220 Asn Ala Ile Gly Val Phe Pro Glu Val Gly Ala Val Gln Ala Ile 225 230 235 240 Arg Tyr Thr Glu His Phe Pro Arg Leu Pro Val Asp Phe Glu Ile Ser                 245 250 255 Gly Gln Arg Asp Ala Asp Met Phe Asp Leu Leu Glu Tyr Ile Phe Gly             260 265 270 Phe Gln Arg Asp Asn Val Arg Asn Gln Arg Glu His Leu Val Leu Thr         275 280 285 Leu Ser Asn Ala Gln Ser Gln Leu Ser Ile Pro Gly Gln Asn Asp Pro     290 295 300 Lys Ile Asp Glu Asn Ala Val Asn Glu Val Phe Leu Lys Val Leu Asp 305 310 315 320 Asn Tyr Ile Lys Trp Cys Lys Tyr Leu Arg Ile Arg Val Val Tyr Asn                 325 330 335 Lys Leu Glu Ala Ile Asp Arg Asp Arg Lys Leu Phe Leu Val Ser Leu             340 345 350 Tyr Phe Leu Ile Trp Gly Glu Ala Ala Asn Val Arg Phe Leu Pro Glu         355 360 365 Cys Ile Cys Tyr Ile Phe His Asn Met Ala Lys Glu Leu Asp Ala Lys     370 375 380 Leu Asp His Gly Glu Ala Val Arg Ala Asp Ser Cys Leu Thr Gly Thr 385 390 395 400 Asp Thr Gly Ser Val Ser Phe Leu Glu Arg Ile Ile Cys Pro Ile Tyr                 405 410 415 Glu Thr Ile Ser Ala Glu Thr Val Arg Asn Asn Gly Gly Lys Ala Ala             420 425 430 His Ser Glu Trp Arg Asn Tyr Asp Asp Phe Asn Glu Tyr Phe Trp Thr         435 440 445 Pro Ala Cys Phe Glu Leu Ser Trp Pro Met Lys Thr Glu Ser Arg Phe     450 455 460 Leu Ser Lys Pro Lys Gly Arg Lys Arg Thr Ala Lys Ser Ser Phe Val 465 470 475 480 Glu His Arg Thr Tyr Leu His Leu Phe Arg Ser Phe Ile Arg Leu Trp                 485 490 495 Ile Phe Met Phe Ile Met Phe Gln Ser Leu Thr Ile Ile Ala Phe Arg             500 505 510 Asn Glu His Leu Asn Ile Glu Thr Phe Lys Ile Leu Leu Ser Ala Gly         515 520 525 Pro Thr Tyr Ala Ile Met Asn Phe Ile Glu Cys Leu Leu Asp Val Val     530 535 540 Leu Met Tyr Gly Ala Tyr Ser Met Ala Arg Gly Met Ala Ile Ser Arg 545 550 555 560 Leu Val Ile Arg Phe Leu Trp Trp Gly Leu Gly Ser Ala Phe Val Val                 565 570 575 Tyr Tyr Tyr Val Lys Val Leu Asp Glu Arg Asn Lys Pro Asn Gln Asn             580 585 590 Glu Phe Phe Phe His Leu Tyr Ile Leu Val Leu Gly Cys Tyr Ala Ala         595 600 605 Val Arg Leu Ile Phe Gly Leu Leu Val Lys Leu Pro Ala Cys His Ala     610 615 620 Leu Ser Glu Met Ser Asp Gln Ser Phe Phe Gln Phe Phe Lys Trp Ile 625 630 635 640 Tyr Gln Glu Arg Tyr Phe Val Gly Arg Gly Leu Phe Glu Asn Leu Ser                 645 650 655 Asp Tyr Cys Arg Tyr Val Ala Phe Trp Leu Val Val Leu Ala Ser Lys             660 665 670 Phe Thr Phe Ala Tyr Phe Leu Gln Ile Lys Pro Leu Val Lys Pro Thr         675 680 685 Asn Thr Ile Ile His Leu Pro Pro Phe Gln Tyr Ser Trp His Asp Ile     690 695 700 Val Ser Lys Ser Asn Asp His Ala Leu Thr Ile Val Ser Leu Trp Ala 705 710 715 720 Pro Val Leu Ala Ile Tyr Leu Met Asp Ile His Ile Trp Tyr Thr Leu                 725 730 735 Leu Ser Ala Ile Ile Gly             740 745 750 Glu Ile Arg Thr Ile Glu Met Val His Lys Arg Phe Glu Ser Phe Pro         755 760 765 Glu Ala Phe Ala Gln Asn Leu Val Ser Pro Val Val Lys Arg Val Pro     770 775 780 Leu Gly Gln His Ala Ser Gln Asp Gly Gln Asp Met Asn Lys Ala Tyr 785 790 795 800 Ala Ala Met Phe Ser Pro Phe Trp Asn Glu Ile Ile Lys Ser Leu Arg                 805 810 815 Glu Glu Asp Tyr Leu Ser Asn Arg Glu Met Asp Leu Leu Ser Ile Pro             820 825 830 Ser Asn Thr Gly Ser Leu Arg Leu Val Gln Trp Pro Leu Phe Leu Leu         835 840 845 Cys Ser Lys Ile Leu Val Ala Ile Asp Leu Ala Met Glu Cys Lys Glu     850 855 860 Thr Gln Glu Val Leu Trp Arg Gln Ile Cys Asp Asp Glu Tyr Met Ala 865 870 875 880 Tyr Ala Val Gln Glu Cys Tyr Tyr Ser Val Glu Lys Ile Leu Asn Ser                 885 890 895 Met Val Asn Asp Glu Gly Arg Arg Trp Val Glu Arg Ile Phe Leu Glu             900 905 910 Ile Ser Asn Ser Ile Glu Gln Gly Ser Leu Ala Ile Thr Leu Asn Leu         915 920 925 Lys Lys Leu Gln Leu Val Val Ser Arg Phe Thr Ala Leu Thr Gly Leu     930 935 940 Leu Ile Arg Asn Glu Thr Pro Asp Leu Ala Lys Gly Ala Ala Lys Ala 945 950 955 960 Met Phe Asp Phe Tyr Glu Val Val Thr His Asp Leu Leu Ser His Asp                 965 970 975 Leu Arg Glu Gln Leu Asp Thr Trp Asn Ile Leu Ala Arg Ala Arg Asn             980 985 990 Glu Gly Arg Leu Phe Ser Arg Ile Ala Trp Pro Arg Asp Pro Glu Ile         995 1000 1005 Ile Glu Gln Val Lys Arg Leu His Leu Leu Leu Thr Val Lys Asp Ala    1010 1015 1020 Ala Ala Asn Val Pro Lys Asn Leu Glu Ala Arg Arg Arg Leu Glu Phe 1025 1030 1035 1040 Phe Thr Asn Ser Leu Phe Met Asp Met Pro Gln Ala Arg Pro Val Ala                1045 1050 1055 Glu Met Val Pro Phe Ser Val Phe Thr Pro Tyr Tyr Ser Glu Thr Val            1060 1065 1070 Leu Tyr Ser Ser Ser Glu Leu Arg Ser Glu Asn Glu Asp Gly Ile Ser        1075 1080 1085 Ile Leu Phe Tyr Leu Gln Lys Ile Phe Pro Asp Glu Trp Glu Asn Phe    1090 1095 1100 Leu Glu Arg Ile Gly Arg Ser Glu Ser Thr Gly Asp Ala Asp Leu Gln 1105 1110 1115 1120 Ala Ser Ser Thr Asp Ala Leu Glu Leu Arg Phe Trp Val Ser Tyr Arg                1125 1130 1135 Gly Gln Thr Leu Ala Arg Thr Val Arg Gly Met Met Tyr Tyr Arg Arg            1140 1145 1150 Ala Leu Met Leu Gln Ser Phe Leu Glu Arg Arg Gly Leu Gly Val Asp        1155 1160 1165 Asp Ala Ser Leu Thr Asn Met Pro Arg Gly Phe Glu Ser Ser Ile Glu    1170 1175 1180 Ala Arg Ala Gln Ala Asp Leu Lys Phe Thr Tyr Val Val Ser Cys Gln 1185 1190 1195 1200 Ile Tyr Gly Gln Gln Lys Gln Gln Lys Lys Pro Glu Ala Thr Asp Ile                1205 1210 1215 Gly Leu Leu Leu Gln Arg Tyr Glu Ala Leu Arg Val Ala Phe Ile His            1220 1225 1230 Ser Glu Asp Val Gly Asn Gly Asp Gly Gly Ser Gly Gly Lys Lys Glu        1235 1240 1245 Phe Tyr Ser Lys Leu Val Lys Ala Asp Ile His Gly Lys Asp Glu Glu    1250 1255 1260 Ile Tyr Ser Ile Lys Leu Pro Gly Asp Pro Lys Leu Gly Glu Gly Lys 1265 1270 1275 1280 Pro Glu Asn Gln Asn His Ala Ile Val Phe Thr Arg Gly Glu Ala Ile                1285 1290 1295 Gln Thr Ile Asp Met Asn Gln Asp Asn Tyr Leu Glu Glu Ala Ile Lys            1300 1305 1310 Met Arg Asn Leu Leu Glu Glu Phe His Gly Lys His Gly Ile Arg Arg        1315 1320 1325 Pro Thr Ile Leu Gly Val Arg Glu His Val Phe Thr Gly Ser Val Ser    1330 1335 1340 Ser Leu Ala Trp Phe Met Ser Asn Gln Glu Thr Ser Phe Val Thr Leu 1345 1350 1355 1360 Gly Gln Arg Val Leu Ala Tyr Pro Leu Lys Val Arg Met Met Tyr Gly                1365 1370 1375 His Pro Asp Val Phe Asp Arg Ile Phe His Ile Thr Arg Gly Gly Ile            1380 1385 1390 Ser Lys Ala Ser Arg Val Ile Asn Ile Ser Glu Asp Ile Tyr Ala Gly        1395 1400 1405 Phe Asn Ser Thr Leu Arg Gln Gly Asn Ile Thr His His Glu Tyr Ile    1410 1415 1420 Gln Val Gly Lys Gly Arg Asp Val Gly Leu Asn Gln Ile Ala Leu Phe 1425 1430 1435 1440 Glu Gly Lys Val Ala Gly Gly Asn Gly Glu Gln Val Leu Ser Arg Asp                1445 1450 1455 Val Tyr Arg Ile Gly Gln Leu Phe Asp Phe Phe Arg Met Met Ser Phe            1460 1465 1470 Tyr Phe Thr Thr Val Gly Phe Tyr Val Cys Thr Met Met Thr Val Leu        1475 1480 1485 Thr Val Tyr Val Phe Leu Tyr Gly Arg Val Tyr Leu Ala Phe Ser Gly    1490 1495 1500 Ala Asp Arg Ala Ile Ser Arg Val Ala Lys Leu Ser Gly Asn Thr Ala 1505 1510 1515 1520 Leu Asp Ala Leu Asn Ale Gln Phe Leu Val Gln Ile Gly Ile Phe                1525 1530 1535 Thr Ala Val Pro Met Val Met Gly Phe Ile Leu Gly Leu Gly Leu Leu            1540 1545 1550 Lys Ala Ile Phe Ser Phe Ile Thr Met Gln Phe Gln Leu Cys Ser Val        1555 1560 1565 Phe Phe Thr Phe Ser Leu Gly Thr Arg Thr His Tyr Phe Gly Arg Thr    1570 1575 1580 Ile Leu His Gly Gly Ala Lys Tyr Arg Ala Thr Gly Arg Gly Phe Val 1585 1590 1595 1600 Val Gln His Ile Lys Phe Ala Asp Asn Tyr Arg Leu Tyr Ser Arg Ser                1605 1610 1615 His Phe Val Lys Ala Phe Glu Val Ala Leu Leu Leu Ile Ile Tyr Ile            1620 1625 1630 Ala Tyr Gly Tyr Thr Asp Gly Aly Ser Ser Phe Val Leu Leu Thr        1635 1640 1645 Ile Ser Ser Trp Phe Leu Val Ile Ser Trp Leu Phe Ala Pro Tyr Ile    1650 1655 1660 Phe Asn Pro Ser Gly Phe Glu Trp Gln Lys Thr Val Glu Asp Phe Glu 1665 1670 1675 1680 Asp Trp Val Ser Trp Leu Met Tyr Lys Gly Gly Val Gly Val Lys Gly                1685 1690 1695 Glu Leu Ser Trp Glu Ser Trp Trp Glu Glu Glu Gln Ala His Ile Gln            1700 1705 1710 Thr Leu Arg Gly Arg Ile Leu Glu Thr Ile Leu Ser Leu Arg Phe Phe        1715 1720 1725 Met Phe Gln Tyr Gly Ile Val Tyr Lys Leu Asp Leu Thr Arg Lys Asn    1730 1735 1740 Thr Ser Leu Ala Leu Tyr Gly Tyr Ser Trp Val Val Leu Val Val Ile 1745 1750 1755 1760 Val Phe Leu Phe Lys Leu Phe Trp Tyr Ser Pro Arg Lys Ser Ser Asn                1765 1770 1775 Ile Leu Ala Leu Arg Phe Leu Gln Gly Val Ala Ser Ile Thr Phe            1780 1785 1790 Ile Ala Leu Ile Val Ala Ile Ala Met Thr Asp Leu Ser Ile Pro        1795 1800 1805 Asp Met Phe Ala Cys Val Leu Gly Phe Ile Pro Thr Gly Trp Ala Leu    1810 1815 1820 Leu Ser Leu Ala Ile Thr Trp Lys Gln Val Leu Arg Val Leu Gly Leu 1825 1830 1835 1840 Trp Glu Thr Val Arg Glu Phe Gly Arg Ile Tyr Asp Ala Ala Met Gly                1845 1850 1855 Met Leu Ile Phe Ser Pro Ile Ala Leu Leu Ser Trp Phe Pro Phe Ile            1860 1865 1870 Ser Thr Phe Gln Ser Arg Leu Leu Phe Asn Gln Ala Phe Ser Arg Gly        1875 1880 1885 Leu Glu Ile Ser Ile Leu Ala Gly Asn Arg Ala Asn Val Glu Thr    1890 1895 1900 <210> 2 <211> 5715 <212> DNA <213> GSL8 cDNA ORF sequence <400> 2 atggctaggg tttatagtaa ttgggatagg ctggttcgag ccactttaag aagagagcag 60 ctgagaaata caggtcaagg tcatgagcgt gttagtagcg gacttgctgg agcagttccg 120 ccatcacttg gtcgagctac aaatatcgat gctatcttac aagctgctga tgagattcag 180 tctgaggatc cttctgtagc tagaattcta tgtgagcaag cgtactctat ggcacagaac 240 ttggacccta acagtgatgg tagaggtgtt cttcaattca aaactggttt gatgtccgtg 300 attaagcaaa aacttgccaa gagagatggt gcttcaatag accgcgatcg tgatattgaa 360 cgactatggg aattttacaa actttataaa agacggcata gagtggatga tattcagaag 420 gaagagcaaa agtggaggga atcaggaaca actttttctt ctaacgtggg ggagatcttg 480 aagatgagga aggtgtttgc cacattgagg gccctaattg aagtgttaga ggtgctaagc 540 agagatgctg atccaaatgg tgttgggagg tctatcaggg acgagcttgg gcggattaaa 600 aaagctgatg caactttgtc tgcggaacta actccttaca atattgtccc tctagaagcg 660 cagtccatga ccaatgcaat tggggttttc cctgaagtac gtggtgcagt ccaagctatc 720 agatacactg aacatttccc taggcttccc gttgattttg agatttccgg acaacgtgat 780 gcagatatgt ttgatttatt ggagtatatc tttgggtttc agagagacaa tgtaaggaac 840 caacgggagc atttggttct cacactttca aatgcacagt cccagcttag tatacctggg 900 cagaatgacc caaaaattga tgagaatgca gtcaatgagg ttttcttaaa ggttttggac 960 aactacatca agtggtgcaa atacttgagg atacgcgttg tgtacaacaa gttagaagcc 1020 attgaccggg acaggaagct gtttcttgtt tctttgtact tcctaatctg gggtgaagct 1080 gctaatgtcc gctttcttcc agagtgtatc tgctatatat ttcacaacat ggccaaggaa 1140 ttggatgcaa aattggatca tggagaagct gttcgtgctg acagttgctt aactggaacg 1200 gataccggtt ctgtatcatt tcttgagcga attatctgcc ctatatatga aacaatctca 1260 gcggaaactg ttcgaaacaa tggtgggaaa gctgcacatt ctgaatggcg gaattatgat 1320 gacttcaatg aatacttttg gacacctgct tgctttgagt taagctggcc tatgaaaaca 1380 gagtcacgct ttttaagcaa gccgaaaggg cggaaaagga ctgctaaaag tagctttgtt 1440 gagcatcgga cataccttca ccttttccga agttttattc gactttggat cttcatgttt 1500 attatgttcc agtctttgac gattatagcc ttcaggaatg aacatctcaa tatcgaaact 1560 ttcaagatct tactcagtgc tgggcctaca tatgctatta tgaacttcat tgaatgtctt 1620 cttgatgttg tacttatgta tggcgcctac agcatggcaa gaggaatggc tatatccagg 1680 ctggttatca ggtttctttg gtggggcttg ggatcggcat ttgtggtgta ttattatgtg 1740 aaggttctgg atgaaagaaa taaaccaaac caaaatgagt ttttcttcca tttatacatt 1800 cttgtattgg ggtgttatgc cgctgttcgc ctcatctttg gacttttagt caaacttcca 1860 gcatgccatg ctctgtcaga aatgtcagat caatctttct tccagttttt taagtggata 1920 tatcaggaac gatacttcgt gggacgtggc ctctttgaga atttaagtga ttattgcagg 1980 tatgtggcgt tttggttggt tgtcctggcc tccaaattca catttgcata cttcctccag 2040 atcaaaccac tcgttaaacc caccaacact atcattcatc ttcctccatt tcaatattca 2100 tggcatgata ttgtctcaaa atctaacgat catgcgttga ccattgttag cttgtgggct 2160 ccagttttgg cgatttatct tatggatatt catatatggt acacactctt gtctgcaatt 2220 attggtggtg tgatgggtgc aaaagctcgc ctaggcgaga tacgcaccat cgagatggtt 2280 cataagcgtt ttgagagttt tccagaggct tttgctcaga accttgtctc tcctgttgtg 2340 aaaagagtac cacttggcca acatgcttct caggatggtc aggatatgaa caaggcatat 2400 gctgcgatgt tttcaccttt ttggaatgag ataataaaga gcttgagaga ggaagattat 2460 ctcagtaaca gggagatgga tttgctttct attcccagta acacggggag ccttagacta 2520 gtccagtggc ccttgtttct tctctgcagt aagattctgg tagccattga tttagccatg 2580 gagtgcaaag aaacacaaga agtactatgg agacaaatat gcgatgatga atacatggcg 2640 tatgcagttc aggagtgcta ttacagtgtt gaaaaaatat tgaattccat ggttaatgat 2700 gaagggcgac gttgggtgga aagaattttt ctggagatca gcaacagtat agaacagggt 2760 tcccttgcga taactctaaa tcttaagaag cttcagttag tggtttccag atttactgca 2820 ttaaccgggc ttttgattcg aaatgaaacc ccagatcttg caaaaggtgc tgcaaaagct 2880 atgtttgatt tttacgaggt ggtcacgcat gaccttcttt cacatgattt aagggagcag 2940 cttgatacat ggaatatttt agcacgggct cgaaatgaag ggcgtctctt ttccagaata 3000 gcttggccta gggacccaga aattatagag caggtcaagc ggctacatct tcttctcact 3060 gtgaaggatg ccgctgctaa cgtcccaaaa aatctagaag caagaagaag attggagttt 3120 ttcacaaatt cgttatttat ggatatgccc caagcaaggc ctgttgctga aatggttcca 3180 ttcagtgtct tcaccccata ctatagtgag acagtgctct atagctcctc agaactgcga 3240 agtgagaatg aggatggaat atctattctt ttctatcttc aaaagatatt tcctgatgaa 3300 tgggagaact tcttggagag gattggcagg tctgaatcaa caggggatgc agatcttcaa 3360 gcaagttcaa ctgatgcttt ggagcttcgg ttttgggtat cttatcgagg acagacttta 3420 gcgaggactg tgcgaggtat gatgtattac cgaagggctt tgatgctcca gagtttctta 3480 gaaagacgag gcttgggagt ggatgatgca tctctgacca acatgccacg aggatttgaa 3540 tcgtcaattg aagctcgagc tcaagcggac cttaagttta cgtatgttgt gtcgtgccaa 3600 atttatgggc aacagaaaca gcaaaagaaa ccagaggcta ctgatatcgg acttttattg 3660 caaagatacg aggcgctacg agttgctttc atacattcgg aggatgttgg aaatggagat 3720 ggtggaagtg gagggaaaaa ggaattctat tctaaattag taaaagctga cattcacgga 3780 aaagatgagg aaatttattc aattaaactt cccggagatc ctaaacttgg tgaagggaag 3840 cctgagaatc aaaatcatgc tatcgtgttt acgcggggag aagctatcca gaccatagat 3900 atgaatcagg acaattatct ggaggaagca atcaaaatga gaaatctact tgaggagttt 3960 catggaaaac atggaattcg acgtcctact attttgggag ttagagagca tgttttcacg 4020 ggaagtgttt catcactggc gtggttcatg tcgaatcaag agaccagttt tgtaacgttg 4080 ggtcaacggg ttctagcata tccacttaaa gttcgaatgc actatgggca tccagatgtg 4140 ttcgatagaa ttttcatat aactcgcgga ggaatcagca aagcatcccg tgttattaac 4200 atcagtgaag atatatatgc tggatttaac tcgacactca ggcaggggaa tatcacccac 4260 catgagtata ttcaggttgg taaaggaaga gatgtaggac ttaaccagat tgccctattt 4320 gaagggaagg tagcaggtgg aaatggagaa caagttctta gcagggatgt ctacagaatt 4380 gggcagctgt ttgacttttt tagaatgatg tcattctact tcacaacagt tggattctat 4440 gtctgcacaa tgatgaccgt ccttactgtg tacgtctttt tgtatggaag agtttatctg 4500 gccttttctg gtgctgatcg tgcaatatca agagtagcca aactgtcagg taacactgca 4560 ttggatgctg cactaaacgc acagttttta gtccagattg gaatctttac tgccgtccct 4620 atggtgatgg gtttcatact tgaacttggg ttgctaaagg ctattttcag cttcattaca 4680 atgcaattcc agttgtgttc tgtctttttc acattttctc tcgggacaag aactcattac 4740 tttggacgta ctattcttca cggtggtgca aagtatcgag caactggcag aggttttgtg 4800 gttcagcaca taaagtttgc cgataattac agactttatt ctagaagtca ttttgttaaa 4860 gcttttgaag tagctctact gttgatcatt tacattgcct atgggtatac ggacgggggt 4920 gcctcttcgt tcgttttgct aactatcagc agttggttcc ttgtaatatc ctggttgttt 4980 gcaccctata tcttcaatcc ctctggattt gaatggcaaa agactgtcga ggattttgaa 5040 gactgggtca gttggcttat gtacaaaggt ggagtgggag taaagggaga gctcagttgg 5100 gagtcgtggt gggaggaaga acaggcgcat atccaaacat taagaggaag gatattggag 5160 actattttga gcctgagatt tttcatgttt caatatggca ttgtgtacaa gcttgatctc 5220 actcggaaaa acacttcact tgcgctctat ggctactcgt gggttgtttt ggttgttatt 5280 gtgttcttgt ttaagctttt ctggtatagt cccagaaaat caagcaacat tctgctggca 5340 ctgcgttttc tacagggagt ggcttcgatc acattcatcg ccctcatcgt tgtagccatt 5400 gcaatgacag atctatcgat tccagacatg tttgcatgtg tccttggttt tatcccgacc 5460 ggctgggctc tactctcttt agccatcaca tggaaacagg tgcttagggt cctgggcttg 5520 tgggaaacag tccgtgagtt tgggcgtata tatgatgctg caatgggtat gctcatattt 5580 tctccgatcg cccttctttc ctggttccct ttcatctcca cgttccagtc tcggctcctc 5640 ttcaaccaag ctttcagtcg cggacttgaa atctccatta tccttgccgg aaacagagct 5700 aatgttgaga cctga 5715

Claims (13)

A method for regulating the growth and excitability of a plant using expression or activity regulation of callose synthase. The method according to claim 1,
Wherein said method comprises the modulation of an auxin concentration gradient upon modulation of expression or activity of callose synthase. The method according to claim 1,
The activity of the carosyl synthase may be regulated by GSL8 (GLUCAN SYNTHASE-LIKE 8) polypeptide described in SEQ ID NO: 1 or GSL8 (GLUCAN SYNTHASE-LIKE 8) containing an amino acid sequence having 70 or more homology with the polypeptide of SEQ ID NO: Lt; RTI ID = 0.0 &gt; polypeptide. &Lt; / RTI &gt; The method according to claim 1,
The expression of the carosyltransferase may be regulated by a gene sequence of GSL8 (GLUCAN SYNTHASE-LIKE 8) described in SEQ ID NO: 2 or a gene sequence of GSL8 (GLUCAN SYNTHASE- LIKE 8) gene. &Lt; / RTI &gt; 5. The method of claim 4,
The GSL8 gene may be selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, sorghum, Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, Peach, pear, grape, citrus, persimmon, plum, apricot, banana, rose, gladiolus, peanut, peanut, Selected from the group consisting of gerbera, carnation, chrysanthemum, lily, tulip, raglass, red clover, orchardgrass, alfapa, tall fescue, bamboo, buckwheat and perennialla grass and broccoli, red cabbage, &Lt; / RTI &gt; The method according to claim 1,
Wherein said method promotes plant GSL8 gene expression or GSL8 protein activity thereby enhancing plant growth and excitation. The method according to claim 6,
Wherein the method increases the synthesis of callus to form an oxine concentration gradient (slope) by callus accumulation (deposition). The method according to claim 1,
Wherein said method inhibits plant growth and irritability by reducing the expression of GSL8 gene or the activity of GSL8 protein. 9. The method of claim 8,
The method is characterized in that as the synthesis of callus is reduced, an increase in PD permeability and a symplasmic diffusion enhancement of auxin occur, thereby inhibiting the formation of an oxine concentration gradient. Plant growth and excitatory promoters by increased carosyl synthesis, containing a recombinant vector encoded to overexpress GSL8. Which comprises a recombinant vector comprising a nucleic acid selected from the group consisting of an antisense oligonucleotide complementary to the mRNA of the GSL8 gene, a short interfering RNA, a short hairpin RNA and RNAi, Plant growth and inhibition by synthetic reduction. A plant transformed with a recombinant vector encoded so that GSL8 is overexpressed. 13. The method of claim 12,
The plant may be selected from the group consisting of rice, wheat, barley, corn, beans, potatoes, red beans, oats, sorghum, Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, Ginseng, tobacco, cotton, sesame seeds, sugar beet, perilla, peanut, rapeseed, apple tree, pear, jujube tree, peach, sheep, grape, citrus, persimmon, apricot, banana, rose, gladiolus, gerbera Wherein the plant is selected from the group consisting of carnations, chrysanthemums, lilies, tulips, ragras, red clover, orchardgrass, alfapa, tall fescue and perennialla grass, bamboo, broccoli and other sprouts.

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