# Patent

## Title
Method for promoting the formation of secondary cell wall of plant

## Abstract

Provided is a method for increasing the thickness of the secondary cell wall of a plant. A method for increasing the thickness of the secondary cell wall of a plant by using a tracheary element differentiation (TED)-associated protein or a C-terminal fragment of the same, which comprises constructing a transgenic (Tg) plant containing, in an expressible manner, a DNA encoding protein(s) selected from the group consisting of a combination of TED6 with TED7, a C-terminal fragment of TED6, a C-terminal fragment of TED7, and a combination of a C-terminal fragment of TED6 with a C-terminal fragment of TED7, and expressing said DNA in said plant; the Tg plant; and a progeny, a cell, a tissue or a seed of the same.

## Background
This application is the national stage of international Application PCT/JP2010/055871, filed in Japan on Mar. 31, 2010, and claims priority under 35 USC §119(a)-(d) of Japanese Application 2009-086922, filed in Japan on Mar. 31, 2009.
TECHNICAL FIELD
The present invention relates to a protein having the function of promoting the formation of wood fiber secondary cell walls of plants, DNA encoding the protein, and the use thereof.
BACKGROUND ART
Human beings have long used woody biomass such as trunks, roots, leaves, and branches in various industrial fields, including paper-making, construction, feedstuff production, fuel production, and the like. Industries in which woody biomass is utilized are realized again in view of the improvement of global environmental issues because they enable the use of sustainably-usable resources in the future. Thus, such industries are expected to be recycling-oriented industries, which utilize carbon sources instead of current fossil resources. Hence, the afforestation industries mainly for fast-growing trees of the genus Eucalyptus, the genus Acacia, and the like are promoted throughout the world in order to stably and sustainably secure woody biomass.
Woody biomass is composed of vessel cells and wood fiber cells existing in the secondary xylem of plant stems. These cells are both characterized by the formation of secondary cell walls within the cells, which are composed of cellulose, hemicellulose and lignin. Wood fiber cells which account for most of the secondary xylem are used as woody biomass in industrial fields (Non-patent Document 1). The amount of secondary cell walls (the degree of thickness) of the wood fiber cells is very important because it influences the amount of biomass or the physical properties of wood fibers.
In recent years, bioenergy production and biofinery using woody biomass have become increasingly popular, as is well known. It is very beneficial to thicken the secondary cell walls to increase the amount of biomass in view of improvement of the productivity and cost reduction. Also, when woody biomass is regarded as a raw material for conventional paper-making, it is very beneficial to thicken the secondary cell walls to increase the amount of biomass in view of improvement of the productivity and cost reduction for bulky paper and the like, for which wood fibers having thick secondary cell walls and high Runkel ratio (secondary cell wall-to-lumen ratio) levels are required.
In concert with the future development of afforestation program, the formation of secondary cell walls of wood fiber cells as major source of woody biomass is promoted to change the amount of biomass (cellulose, hemicellulose, and lignin) and to change the morphology of wood fibers (e.g., the Runkel ratio). Thereby, future use for energy or expanded applications as industrial raw materials can be expected. Therefore, the development of a method for promoting the formation of secondary cell walls of wood fiber cells is very important for realizing more effective and efficient production of woody biomass on a global scale.
Several methods for promoting the secondary cell wall formation have become known to date (Patent Documents 1 to 5 and Non-patent Documents 2 to 5). However, they cannot be put to practical use because many of them relate to the promotion of the formation of secondary cell walls of plant cells other than wood fibers, and the amounts of cells and secondary cell walls resulting from the promotion are extremely low. Further, although there are techniques for promoting the formation of secondary cell walls of wood fiber cells, they cannot be practically used because adverse effects such as dwarfing are observed. As described above, it is difficult to promote the formation of secondary cell walls of wood fiber cells using the currently known techniques.
The present inventors have found many genes whose expression is specifically induced upon the formation of vessel cells (Non-patent Document 6). The group of genes found by the present inventors comprises many genes of unknown functions, and these genes are considered to play some roles in vessel formation. Vessel cells and wood fiber cells are highly analogous to each other in that both cells form secondary cell walls inside the cells, and they both are said to originate from tracheids. Therefore, it is considered that the analysis of the functions of these genes can enable the control of the secondary cell wall formation, not only for vessel cells, but also for wood fiber cells. However, there has been no such technique or finding.
PRIOR ART DOCUMENTS
Patent Documents


    [Patent Document 1] JP Patent No. 4068152
    [Patent Document 2] JP Patent Publication (Kohyo) No. 2003-509009 A
    [Patent Document 3] JP Patent Publication (Kokai) No. 2006-246852 A
    [Patent Document 4] JP Patent Publication (Kohyo) No. 2006-526990 A
    [Patent Document 5] JP Patent Publication (Kokai) No. 2008-178422 A


Non-Patent Documents


    [Non-patent Document 1] Evert, RF. Esau's Plant Anatomy, Meristems, Cells, and Tissues of the Plant Body: their Structure, Function, and Development. 3rd Ed. New Jersey: John Wiley & Sons, Inc.
    [Non-patent Document 2] Kubo et al., 2005 Genes & Dev. 19:1855-1860
    [Non-patent Document 3] Goicoechea et al., 2005 The Plant Journal 43:553-567
    [Non-patent Document 4] Mitsuda et al., 2005 The Plant Cell 17:2993-3006
    [Non-patent Document 5] Zhong et al., 2006 The Plant Cell 18:3158-3170
    [Non-patent Document 6] Demura, T. and Fukuda, H., 2007 Trends in Plant Sci. 12:64-70


SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
The present invention has been achieved in view of the aforementioned circumstances. An object of the present invention is to provide a method for using TED6 and TED7, which are genes of unknown functions, and plants in which the secondary cell wall formation is promoted.
Means for Solving the Problem
The present inventors have predicted that TED6 or TED7, which is a tracheary element differentiation (TED)-associated protein of a plant, might be involved in cellulose synthesis and pattern formation of secondary cell walls of plants. However, they have revealed that such a protein alone does not have this function. As a result of further intensive studies, they have completed the invention having the following features.
In summary, the present invention has the following features.
In a first aspect, the present invention provides a method for increasing the thickness of a secondary cell wall of a plant using a TED-associated protein or a C-terminal fragment thereof, comprising producing a transgenic plant that contains, in an expressible manner (that is, so that the genes can be expressed), DNA encoding a protein selected from the group consisting of a combination of TED6 and TED7, a C-terminal fragment of TED6, a C-terminal fragment of TED7, and a combination of a C-terminal fragment of TED6 and a C-terminal fragment of TED7, and causing the expression of the DNA within the plant.
In an embodiment of the above aspect, the above C-terminal fragment of TED6 or TED7 consists of an amino acid sequence lacking the sequence from the N-terminus to the transmembrane domain in the mature amino acid sequence of TED6 or TED7, or, an amino acid sequence containing a deletion, a substitution, or an addition of one or several amino acids on the N-terminal side and/or the C-terminal side of the amino acid sequence lacking said sequence.
Here, “several” indicates an integer of 10 or less; that is, an integer of 2 to 10.
In a second aspect, the present invention provides a transgenic plant or a progeny plant thereof characterized in that it contains, in an expressible manner, DNA encoding a protein selected from the group consisting of the combination of TED6 and TED7, the C-terminal fragment of TED6, the C-terminal fragment of TED7, and the combination of a C-terminal fragment of TED6 and a C-terminal fragment of TED7 as described above, and that the thickness of a secondary cell wall is increased as compared with that of a wild-type plant.
In a third aspect, the present invention further provides cells or tissues derived from the above transgenic plant or progeny plant thereof.
In a fourth aspect, the present invention further provides seeds of the above transgenic plant or progeny plant thereof.
A part or all of the content disclosed in the description and/or drawings of Japanese Patent Application No. 2009-086922, which is a priority document of the present application, is herein incorporated by reference.
Effects of the Invention
The method of the present invention makes it possible to promote the secondary cell wall formation in wood fiber cells of plants and thus to increase the thicknesses of secondary cell walls. Thickening of secondary cell walls is advantageous in that it can result in an increased amount of biomass.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effects of the overexpression of Ze TED6, Ze TED7, and C-terminal fragments thereof on the secondary cell wall formation of Zinnia elegans cells. The vertical axis indicates the percentage (%) of cells having visible secondary cell walls. FIG. 1A shows the overexpression of full-length Ze TED6 and the C-terminal fragment (27Leu-95Ala of SEQ ID NO: 1) of Ze TED6. FIG. 1B shows the overexpression of full-length Ze TED7-1 and the C-terminal fragment (209Trp-300Gly of SEQ ID NO: 2) of Ze TED7-1. FIG. 1C shows the co-overexpression of Ze TED6 and Ze TED7. Further, in FIG. 1, GUS (β-glucuronidase) indicates a plant control in which GUS was overexpressed instead of Ze TED6, Ze TED7, or a C-terminal fragment thereof.
FIGS. 2A-2C show: an alignment (FIG. 2A) of the amino acid sequences of Zinnia elegans TED6 and TED7 (that is, Ze TED6 and Ze TED7) and the amino acid sequences of the homologs of Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa; a dendrogram (FIG. 2B) showing the comparison of conserved regions using MEGA4 (Molecular Evolutionary Genetics Analysis software version 4.0; Tamura, K. et al., (2007) Mol. Biol. Envol. 24: 1596-1599) according to a neighbor-joining method; and the relatively conserved C-terminal regions (FIG. 2C) of the amino acid sequences used for preparation in FIG. 2B. In FIG. 2A, bold letters indicate transmembrane domains and underlines indicate the amino acid sequences of the clones Z1943 and Z16653 used in Examples. Os08g0108300 contains two repeated C-terminal domains (FIG. 2A). FIG. 2B shows the two conserved repeated C-terminal domains of Os08g0108300 being divided.
FIG. 3 shows the results of transient RNAi analysis of At TED6 and At TED7 genes in roots of Arabidopsis thaliana. FIG. 3A shows the expression of the At TED6 and At TED7 genes under conditions of transiently induced RNAi. Inverted repeat sequences corresponding to At TED6, At TED7, and both of them were transiently expressed in transgenic Arabidopsis thaliana plants under the control of a glucocorticoid-mediated induction system. Total RNAs were extracted from 1-week-old Arabidopsis thaliana seedlings after 5 hours of incubation on a growth medium supplemented with 10 μM dexamethasone. AU RT-PCR samples were prepared under the same conditions, except that 25 PCR cycles were employed for At TED6, and 30 PCR cycles were employed for At TED7 and ubiquitin. FIG. 3B to FIG. 3E show the phenotypes of the RNAi lines of the At TED6 and At TED7 genes. Three (3)-week-old transgenic Arabidopsis thaliana plants were incubated on a growth medium supplemented with 10 μM dexamethasone for 5 days, so that the inverted repeat sequences were expressed using the glucocorticoid′-mediated induction system. During induction, the effects of RNAi on vessel formation in more slowly developed roots were examined. In FIG. 3B, a bar indicates 50 μm. FIG. 3B shows wild-type Col-0. FIG. 3C shows the At TED7 RNAi line, showing linearly elongated metaxylem vessels which are scalariform. FIG. 3D shows an At TED6-TED7 chimera RNAi line and specifically shows a deletion in linearly elongated metaxylem vessels. FIG. 3E shows an YFP RNAi line and specifically shows metaxylem vessel elements with large pits. Here, “YFP” indicates a yellow fluorescent protein.



EMBODIMENTS FOR CARRYING OUT THE INVENTION
(DNA Encoding TED6 and TED7 Proteins)
TED6 and TED7 are both type I membrane proteins associated with the differentiation of tracheary elements such as vessels of plants. These proteins lead to the promotion of the formation of secondary cell walls of vessel cells or wood fiber cells when they are simultaneously overexpressed in plant cells.
In many plant species TED6 and TED7 have a characteristic structure, which is composed of, from the N-terminal side, a proline (Pro)-rich domain, a single transmembrane (TM) domain, and a C-terminal domain. The proline-rich domain is an extracellular domain, while the C-terminal domain is a cytoplasmic domain. In general, it is well known that, among plant extracellular proteins, proline-rich sequences are characteristic of hydroxyproline-rich glycoproteins (HRGPs). In particular, TED7 is assumed to be HRGP, although Ze TED7 lacks a repeat sequence which is typically observed in the HRGP family.
FIG. 2 shows the alignment of the amino acid sequences of TED6 and TED7 of exemplary plants including Zinnia elegans, Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa (FIG. 2A), and the amino acid sequences of relatively conserved C-terminal regions in the above amino acid sequences (FIG. 2C). Although the sequence identity among the TED proteins from plants of different species is low, it is understood that the above exemplary proteins belong to the tracheary element differentiation-associated protein family based on the fact that they have the above characteristic domain structures.
The amino acid sequences of TED6 and TED7 derived from these plants are listed in the Sequence Listing with the following SEQ ID NOS.
Zinnia elegans TED6, TED7-1, and TED7-2: SEQ ID NO: 1(AB377514), SEQ ID NO: 2 (AB377515), and SEQ ID NO: 3 (AB377516)
Arabidopsis thaliana TED6 and TED7: SEQ ID NO: 4 (At1g43790) and SEQ ID NO: 5 (At5g48920)
Populus trichocarpa TED6 and TED7: SEQ ID NO: 6 (eugene3. 00020671) and SEQ ID NO: 7 (eugene3. 00070382), SEQ ID NO: 8 (fgenesh1_pg. C_LG_V000008) (here, the sequences of SEQ ID NOS: 7 and 8 belong to TED7).
Oryza sativa TED: SEQ ID NO: 9 (Os08g0108300) ((note) SEQ ID NO: 10 (Os 08g0108300—1st: TERKAEVHNL SGHVHVHKAT ESGPSGAKAT VLSIDEDLKF QEVAG) and SEQ ID NO: 11 (Os08g0108300—2nd: AENKAELINV TEHIHVDEKI VSGPQGQKIE ILSEDEDIRF EEEGR) (here, the sequences of SEQ ID NOS: 10 and 11 are partial sequences prepared by dividing a duplicate domain in the sequence of SEQ ID NO: 9)).
Further, the amino acid residue positions of amino acid sequences of the transmembrane region and the C-terminal domain (FIG. 2A), as well as the relatively conserved C-terminal region (FIG. 2C) in these amino acid sequences can be determined based on the amino acid sequences of SEQ ID NOS: 1-11 and sequences listed in FIG. 2.
Regarding TED6, TED7, and C-terminal fragments thereof that can be used in the present invention, a mutation may be introduced into the wild-type amino acid sequence as long as the capability of a combination of TED6 and TED7, a C-terminal fragment of TED6 or a C-terminal fragment of TED7 to promote the formation of secondary cell walls of vessel cells or wood fiber cells is not lost. Such mutant TED6 and TED7 have 70% or more or 80% or more, preferably 90% or more or 95% or more, and further preferably 98% or more or 99% or more identity with the wild-type amino acid sequence. Amino acid mutation is, for example, deletion, substitution, or addition of 1 or a plurality of (preferably, several) amino acid(s). The substitution is desirably conservative amino acid substitution. The term “conservative amino acid substitution” refers to substitution for an amino acid having a similar property, for example, in terms of structure, electricity, polarity, or hydrophobicity. Such properties can also be classified, for example, based on the similarity of the side chains of amino acids. Amino acids having basic side chains comprise lysine, arginine, and histidine. Amino acids having acidic side chains comprise aspartic acid and glutamic acid. Amino acids having uncharged polar side chains comprise glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and the like. Amino acids having hydrophobic side chains comprise alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and the like. Amino acids having branched side chains comprise threonine, valine, and isoleucine. Amino acids having aromatic side chains comprise tyrosine, tryptophan, phenylalanine, and histidine.
DNAs encoding TED6, TED7, and C-terminal fragments thereof as described above can also contain a mutation(s). Examples of such mutation include mutation based on degeneracy of genetic code (i.e., silent mutation), splice mutation, mutation due to polymorphism. Further, examples of the above mutant DNA also include a mutant comprising a nucleotide sequence capable of hybridizing to wild-type DNA under stringent conditions. In this case, the mutant protein encoded by such DNA should have the capability to promote the formation of secondary cell walls of vessel cells or wood fiber cells, in the form of a combination of TED6 and TED7, a C-terminal fragment of TED6, or a C-terminal fragment of TED7. Such capability may be equivalent to or higher than that of the wild-type protein, or may be inferior to that of the wild-type protein. DNAs encoding mutant TED6 and mutant TED7 have 70% or more or 80% or more, preferably 90% or more or 95% or more, and more preferably 98% or more or 99% or more identity with a wild-type mature nucleotide sequence.
Here, examples of stringent conditions include conditions comprising hybridization at about 42° C. to 55° C. with 2 to 6×SSC and one or several rounds of washing at 50° C. to 65° C. with 0.1 to 1×SSC and 0.1% to 0.2% SDS. Such conditions may vary depending on the GC content of template nucleic acid, ionic strength, temperature, and the like, and thus they are not limited to the above specific conditions. Here, 1×SSC consists of 0.15 M NaCl and 0.015 M Na citrate at pH 7.0. In general, stringent conditions are determined so that the temperature is lower by about 5° C. than the melting temperature (Tm) of a specific sequence at specified ionic strength and pH. Here, the term “Tm” refers to the temperature at which 50% of probes complementary to a template sequence hybridize to the template sequence in equilibrium.
For example, site-directed mutagenesis, mutagenesis using PCR, or the like can be preferably employed as a technique for artificially introducing mutation (Sambrook et al., Molecular Cloning A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press; Ausubel et al., Current Protocols in Molecular Biology, 1994, John Wiley & Sons).
The term “identity” as used herein means, for example, the ratio (%) of the number of identical amino acids or nucleotides to the total number of amino acids or nucleotides observed when two amino acid sequences or nucleotide sequences are aligned with or without introduction of gaps.
Further, homologous sequence search or homology search can be performed using a known algorithm such as BLAST (e.g., BLASTN, BLASTP, and BLASTX) or FASTA.
The amino acid sequences of TED6 and TED7 proteins from plants other than those exemplified above and the nucleotide sequences of DNAs encoding such proteins can be obtained by accessing to web sites at which plant genomes are opened to the public, such as NCBI (U.S.A.), EBI (Europe), KAOS (Kazusa DNA Research Institute), IRGSP (International Rice Genome Sequencing Project), GrainGenes (U.S.A.), PGDIC (U.S.A.), ForestGEN (Forestry and Forest Products Research Institute).
According to the present invention, specifically, a combination of TED6 and TED7, a C-terminal fragment of TED6, a C-terminal fragment of TED7, and a combination of a C-terminal fragment of TED6 and a C-terminal fragment of TED7 have important functions in promotion of the formation of secondary cell walls of vessel cells or wood fiber cells.
Here, “a combination of TED6 and TED7” can be realized by substantially simultaneous expression of DNAs encoding TED6 and TED7 within a plant or a plant cell followed by the translation into the proteins. In this case, for the expression of the DNAs, the two DNAs may be incorporated into separate vectors in an expressible manner, or the two DNAs may be incorporated in tandem into the same vector in an expressible manner.
Further, “a C-terminal fragment” in “a C-terminal fragment of TED6” or “a C-terminal fragment of TED7” may be composed of a C-terminal domain on the cytoplasmic side of the TED6 or TED7 protein (i.e., a domain having an amino acid sequence that lacks the sequence from the N-terminus to the transmembrane domain in the mature amino acid sequence). Alternatively, the C-terminal fragment may have an amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids on the N-terminal side and/or the C-terminal side in the amino acid sequence of the C-terminal domain. In the latter case, for example, about 1 to 3 amino acid residues from the transmembrane region flanking the C-terminal domain may be added on the N-terminal side of the C-terminal fragment. Alternatively, for example, a portion of the sequence on the C-terminal side (e.g., 10 or less amino acid residues) may be deleted from the amino acid sequence of the C-terminal domain. Usually, it is desirable that the above C-terminal fragment in the present invention contains an amino acid sequence relatively conserved among plants as shown in FIG. 2C, and has the capability to promote the formation of secondary cell walls of vessel cells or wood fiber cells. DNA encoding such a C-terminal fragment can be obtained by performing polymerase chain reaction (PCR) using DNA encoding a TED6 or TED7 protein as a template, and 5′ and 3′ primers prepared based on a sequence to be amplified in the nucleotide sequence encoding the C-terminal domain (which may optionally contain 1 to 3 amino acid residues on the C-terminal side of the transmembrane region). Subsequently, the thus prepared DNA is incorporated into an appropriate vector.
Regarding a PCR method and conditions therefor, a usual technique is employed, which consists of an amplification cycle comprising about 20 to 40 cycles of denaturation (e.g., 94° C. for 20 seconds to 5 minutes), annealing (e.g., 55° C. for 30 seconds to 1 minute) and elongation (e.g., 72° C. for 30 seconds to 10 minutes) in a PCR buffer and in the presence of thermostable DNA polymerase (e.g., Taq polymerase), a sense primer, an antisense primer, dNTPs (N=A, T, G, C), and template DNA. A specific technique is described, for example, in Ausubel et al., Current Protocols in Molecular Biology, 1994, John Wiley & Sons. Amplification products can be isolated and purified using agarose gel or polyacrylamide gel electrophoresis.
Incidentally, the amino acid sequences of C-terminal fragments of Zinnia elegans TED6 and TED7-1 used in Examples below are the sequence ranging from 27Leu to 95Ala in SEQ ID NO: 1 and the sequence ranging from 209Trp to 300Gly in SEQ ID NO: 2, respectively.
Furthermore, “a combination of a C-terminal fragment of TED6 and a C-terminal fragment of TED7” can be realized, as in the case of “a combination of TED6 and TED7,” by substantially simultaneous expression of DNAs encoding a C-terminal fragment of the TED6 protein and DNA encoding a C-terminal fragment of the TED7 protein within a plant or a plant cell followed by translation into the proteins. In this case, for the expression of the DNAs, the two DNAs may be incorporated into separate vectors in an expressible manner, or the two DNAs may be incorporated in tandem into the same vector in an expressible manner.
The above DNAs and vectors containing them can be produced, for example, by genetic engineering techniques. As the genetic engineering techniques, for example, techniques described in Sambrook et al., Molecular Cloning a Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press; Ausubel et al., Current Protocols in Molecular Biology, 1994, John Wiley & Sons, and the like can be employed.
Briefly, for example, DNA encoding TED6 or TED7 can be amplified from a plant tissue-derived cDNA library by PCR using primers prepared based on known sequences. The DNA is purified, for example, by agarose gel or polyacrylamide gel electrophoresis and then is inserted into an appropriate expression vector in an expressible manner.
Examples of the vector include binary vectors and other vectors. A binary vector contains two about 25-bp border sequences, the right border (RB) and the left border (LB), of Agrobacterium T-DNA, between which a foreign DNA is inserted. Examples of the binary vector include pBI vectors (e.g., pBI101, pBI101.2, pBI101.3, pBI121, and pBI221 (Clontech)), pGA482, pGAH, and pBIG. Examples of other vectors include intermediate plasmids such as pLGV23Neo, pNCAT, and pMON200, and pH35GS which contains the GATEWAY cassette (Kubo et al., 2005, Genes & Dev. 19: 1855-1860). A promoter is ligated to the 5′ end of the foreign DNA. Examples of the promoter include a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthase gene promoter, a corn ubiquitin promoter, an octopine synthase gene promoter, and a rice actin promoter. Further, a terminator (e.g., a nopaline synthase gene terminator) is inserted at the 3′ end of foreign DNA. A selection marker which is required for selection of transformed cells is further inserted into the vector. Examples of the selection marker include drug resistance genes such as a kanamycin resistance gene (NPTII), a hygromycin resistance gene (htp), and a bialaphos resistance gene (bar).
(Production of Transgenic Plant)
An example of a transformation method for introducing the thus constructed vector into a plant is a method using Agrobacterium. A vector can also be introduced using other methods such as a gene gun, electroporation, a viral vector, a floral dip method, a leaf disc method. Plant transformation techniques and tissue culture techniques are described, for example, in Isao Shimamoto and Kiyotaka Okada (supervisors), Shokubutsu Saibou Kougaku Series 15, Model Shokubutsu No Jikken Protocol, Idengakuteki Shuhou Kara Genome Kaiseki Made (Plant Cell Technology Series 15, Model Plant Experimental Protocols, From Genetic Techniques to Genome Analysis), 2001, Shujunsha.
According to a method using a binary vector-Agrobacterium system, plant cells, calluses, or plant tissue sections are prepared and then infected with Agrobacterium, so as to introduce DNA encoding a protein of the present invention into plant cells. Upon transformation, a phenol compound (acetosyringone) may be added to a medium. In particular, in the case of monocotyledons, the cells can be efficiently transformed. Further, as Agrobacterium, Agrobacterium tumefaciens strains (e.g., C58, LBA4404, EHA101, EHA105, and C58C1RifR) can be used.
A transformation medium is a solid medium. For example, a plant culture medium such as an MS medium, a B5 medium, a DKN medium, or Linsmaier & Skoog medium is used as a basal medium. To the medium, 1%-5% saccharide such as maltose, sucrose, glucose or sorbitol, and 0.2%-1% polysaccharide solidifying agent such as agar, agarose, Gelrite or gellan gum can be added. Casamino acids, abscisic acid, auxin or cytokinin such as kinetin, 2,4-D, indoleacetic acid or indolebutyric acid, antibiotic such as kanamycin, hygromycin or carbenicillin, acetosyringone, and the like can be added to the medium. pH preferable for the medium ranges from 5 to 6, for example, pH 5.5 to 5.8. Further, a substance that induces transcriptional activation, such as a steroid hormone (e.g., glucocorticoid, dexamethasone, estrogen, or a derivative thereof), may be added to the medium after transformation.
Specifically, an Agrobacterium suspension is prepared by about 4 days of culture in the dark at about 25° C. Plant calluses or tissues (e.g., laminae, roots, stem sections, or growing points) are immersed in the bacterial suspension for several minutes, moisture is removed, and then the resultants are placed on a solid medium for cocultivation. A callus is a mass of plant cells, which can be induced using a callus induction medium from a plant tissue section, a fully ripened seed or the like. The transformed calluses or tissue sections are selected based on a selection marker. In case of calluses, they can subsequently be caused to redifferentiate into adventitious shoots using a redifferentiation medium. Meanwhile, in case of plant sections, calluses can be induced from the plant sections and then induced and caused to redifferentiate into adventitious shoots, or protoplasts can be prepared from the plant sections and then caused to redifferentiate into adventitious shoots after callus culture. The thus obtained adventitious shoots are transplanted into soil after rooting so that they are regenerated into plants.
Further, when a floral dip method is used, the method is performed, for example, as described by Clough and Bent et al. (Plant J. 16, 735-743 (1998)), for example, as follows: an Agrobacterium suspension is prepared by about 4 days of culture in the dark at about 25° C., floral buds of the plant host to be transformed, which have been grown until the development of immature floral buds, are immersed in the bacterial suspension for 10 seconds, and then left to stand overnight with a cover to keep humidity; the cover is removed on the next day, the plants are allowed to grow and then seeds are harvested; transformed individuals can be selected by seeding harvested seeds on a solid medium supplemented with an appropriate selection marker such as an antibiotic; the thus selected individuals are transplanted into soil and allowed to grow, so that next-generation seeds of the transgenic plants can be obtained.
Progeny plants having a novel phenotype similar to that of transgenic plants can be produced by crossing the transgenic plants with wild-type plants.
The transgenic plants or progeny plants thereof produced by the above method are characterized in that they contain, in an expressible manner, DNA encoding a protein selected from the group consisting of a combination of TED6 and TED7, a C-terminal fragment of TED6, a C-terminal fragment of TED7, and a combination of a C-terminal fragment of TED6 and a C-terminal fragment of TED7, and that the thickness of the secondary cell walls is increased as compared with wild-type plants.
Accordingly, the present invention further provides not only such a transgenic plant or a progeny plant thereof, but also a cell or tissues or a seed therefrom.
Examples of subject plants of the present invention include, but are not limited to, plants, such as dicotyledons, monocotyledons, gymnosperms, and trees. Examples of particularly useful plants include arboreous plants and herbaceous plants that are important as biomass resources such as Eucalyptus, poplar, sugarcane, rice, and the Pooideae family.
(Secondary Cell Wall Formation—Promoting Functions of TED6 and TED7)
The present inventors have assumed in the course of the study using Arabidopsis thaliana that both TED6 and TED7 might be involved in the secondary cell wall synthesis of vessels (T. Demura, “Regulatory Mechanisms Underlying Xylogenesis in Arabidopsis, as a Model for Wood Formation,” Abstracts in FUNCFIBER 2008 International Symposium on the Biology and Biotechnology of Wood, page 12 (2008)). However, it has been revealed that TED6 or TED7 alone actually does not have a function of promoting the secondary cell wall formation.
The present inventors have found for the first time, using an RNA interference (RNAi) method, that a protein selected from the group consisting of a combination of TED6 and TED7, a C-terminal fragment of TED6, a C-terminal fragment of TED7, and a combination of a C-terminal fragment of TED6 and a C-terminal fragment of TED7 has a function of promoting the formation of secondary cell walls of vessel cells and wood fiber cells as described herein. These proteins, C-terminal fragments thereof, and examples thereof according to the present invention are as described above.
As revealed by the results of the analysis of intracellular localization, TED6 and TED7 proteins are mainly present in the plasma membranes of plant cells and are also present to some degree in cell walls. Among them, TED7 tends to be retained in cell walls at higher levels than TED6. This may be due to an interaction of the proline-rich N-terminal domain of TED7. Structurally, TED6 has a proline-rich N-terminal domain that is very shorter than that of TED7. Therefore, it is assumed that TED6 is unlikely to be retained in cell walls. Regarding TED6, the results of an experiment using Arabidopsis thaliana revealed that TED6 binds to an IRX3 subunit of cellulose synthase CesA. This is evidence demonstrating that TED6 interacts with a secondary cell wall-CesA complex.
EXAMPLES
The present invention will be described in more detail by way of Examples below, which should not be construed as limiting the scope of the present invention.
Example 1
Functional Analysis of Ze TED6 and Ze TED7 Genes in Tracheary Elements of Plants of the Genus Zinnia elegans (Zinnia)
Based on bioinformatics analysis (including SOSUI, TMHMM, and SignalP) of Ze TED6 and Ze TED7 proteins, both of these proteins were predicted to be “type I membrane proteins” (single transmembrane protein having an extracellular or lumenal N-terminus and a cytoplasmic C-terminus). The above proteins contain general single transmembrane domain-like hydrophobic regions (representing 23 out of 95 amino acids and 23 amino acids out of 300 amino acids of the Ze TED6 protein and the Ze TED7 protein, respectively). It was predicted that the C-termini thereof would be located on the cytoplasmic side. However, regarding the potential activity of these proteins, no functional domain has been predicted by ProDom, PROSITE, or Pfam. Only a Pro-rich region has been identified in the N-terminal region of the Ze TED7 protein. It is well known that, among extracellular plant proteins, Pro-rich sequences are hydroxyproline-rich glycoproteins (HRGPs). Ze TED7 might be an HRGP, although it does not have any repeated motif which is generally observed in the HRGP family.
The intracellular localization of Ze TED6 or Ze TED7 was experimentally examined by introducing into Zinnia elegans mesophylls by electroporation a plasmid encoding the protein with YFP (yellow fluorescent protein) fused to its C-terminus, which was driven by CaMV 35S promoter (Endo et al., (2008) Plant J. 53: 864-875). The fluorescence signal from each fusion protein was detected only in peripheral regions of the cells, indicating the localization of the protein in plasma membranes and/or cell walls. With plasmolyzing cells, it was clearly indicated that the proteins were localized mainly in plasma membranes and to some extent in cell walls. The Ze TED7-YFP fusion protein was retained in cell walls at higher levels than the Ze TED6-YFP fusion protein. This can be explained by the possible interaction between the Pro-rich N-terminal domain of the Ze TED7 protein and cell walls.
The effects of the overexpression of the full-length Ze TED6 or Ze TED7 protein on TE SCW (secondary cell wall) formation were examined using a dsRNA-mediated RNAi method based on the number of cells representing visible TE SCW. Moreover, a similar examination was performed for the C-terminal domains of these proteins, so that the functions of the above cytoplasmic domains in SCW formation were evaluated. As a result, when the C-terminal domain (not the full-length protein) was used alone, a slight, but a significant increase (increase by several percent as compared with the overexpressed GUS as a control) was found. Accordingly, low-density cell culture (0.5×105 cells/mL−1) was used in order to observe the positive effects on SCW formation. Such culture usually results in low-percentage SCW formation (less than 30%) in a control. It was thus confirmed that the percentage of SCW formation was increased with statistical significance with the use of the C-terminal domain (FIG. 1A and FIG. 1B). Similarly, the simultaneous overexpression of both the full-length Ze TED6 protein and the full-length Ze TED7 protein resulted in an increased percentage of SCW formation (FIG. 1C), indicating the functional interaction between the Ze TED6 protein and the Ze TED7 protein in SCW formation of TE.
Example 2
Functional Analysis of at TED6 and at TED7 in Plants Belonging to the Family Brassicaceae (Arabidopsis)
The homologs of Ze TED6 and Ze TED7 genes in Arabidopsis were found to be At1g43790 (At TED6) and At5g48920 (At TED7), respectively. The promoter activity of At TED6 and At TED7 were examined using 1 kb- and 0.5 kb-upstream sequences of At TED6 and At TED7. GUS reporter genes were ligated to these upstream sequences. Expression of the reporter transgene in adventitious shoots was limited to vessel elements undergoing differentiation for both genes, confirming the functional homology with Ze TED6 and Ze TED7. YFP reporter genes were ligated to the N-termini of coding sequences for At TED6 and At TED7, and then the genes were expressed under the control of their own promoters. At TED6 signals and TED7-YFP signals were specifically observed only beneath SCW generated in protoxylem and metaxylem vessel elements undergoing differentiation. Inconsistent localization of these signals was observed in some cases in metaxylem vessel elements, although the biological significance thereof remained unknown.
To examine the functions of At TED6 and At TED7, transgenic Arabidopsis having full length, C-terminus, or inverted repeat (RNAi) of At TED6 or At TED7 to which CaMV 35S promoter had been ligated was produced. However, no drastic morphological changes were observed expect for Arabidopsis having inverted repeat of At TED7.
The transgenic plants could not grow on germination medium, but they survived on callus induction medium. This indicated that the constitutive loss of the function of the At TED7 gene resulted in the death of adventitious shoots upon RNAi analysis.
To further examine the loss of the functions of At TED6 and At TED7. the SALK T-DNA database for insertion mutants was searched. No line could be obtained for At TED6, although T-DNA/transposon insertion lines (SALK—084115, SALK—089549 and SM—2—30444) could be obtained for At TED7. No insertion was observed in the coding region of At TED7, but an insertion was observed in the 5′ or 3′ flanking region of At TED7. These results demonstrated that the insertion did not result in a knockout mutant of At TED7.
A dexamethasone (DEX)-inducible promoter (Aoyama and Chua (1997) Plant J. 11: 605-612) was used instead of constitutive RNAi of At TED7. An inverted repeat sequence of each of At TED6, At TED7, a chimera of At TED6 and At TED7, and YFP (control), to which the inducible promoter had been ligated, was introduced into an Arabidopsis plant. Transformants were selected by selecting plants that contains inducible RNAi constructs and that had sufficiently grown under non-induction conditions. Few transformants of At TED7 RNAi and At TED6-TED7 chimera RNAi could be obtained. This is considered to be due to the remaining “leakage” expression of the At TED7 inverted repeat that causes death in adventitious shoots. The efficiency of inducible RNAi was examined via RT-PCR for each target transcript in a juvenile plant line selected after 5 hours of exposure of plants to 10 μM dexamethasone (FIG. 3A). Inducible RNAi was performed for 3-week-old Arabidopsis plants by incubating the plants on growth medium supplemented with 10 μM dexamethasone for 5 days (FIG. 3B to FIG. 3E). It was unexpectedly revealed that DEX-inducible RNAi itself has unique effects including the formation of metaxylem vessels of roots having abnormally large pits on SCW and in some cases inhibition of the formation of protoxylem vessels of roots (FIG. 3E). In addition to the aforementioned unique effects, clear defects in SCW formation of the vessel elements of roots were caused in the inducible RNAi lines of At TED7 and At TED6-TED7 chimera (FIG. 3B and FIG. 3C). In both of these lines, abnormal vessel elements having unusual scalariform SCW (FIG. 3C) were formed instead of metaxylem vessels that usually form plexiform or pitted SCW (FIG. 3B). Furthermore, the At TED6-TED7 chimera RNAi line exhibited discontinuous or deficient vessels in metaxylem (FIG. 3D). Transmission electron microscopy of the roots of the At TED6-TED7 chimera RNAi line revealed that vessels having incomplete thin SCW (which is assumed to represent scalariform SCW) were located in the metaxylem, unlike the YFP RNAi control. These results strongly indicate the involvement of At TED6 and At TED7 in SCW formation of vessel elements.
Example 3
dsRNA and Plasmid DNA
Zinnia EST clones (Demura et al., 2002, Proc. Natl. Acad. Sci. U.S.A. 99: 15794-15799) was subjected to PCR to prepare a fragment containing T7 and SP6 promoters on both ends. The PCR product was purified and then directly used as a template for in vitro synthesis of dsRNA as described by Endo et al, (Plant J. 53: 864-875, 2008). The full-length cDNAs for Ze TED6 and Ze TED7 genes (clones Z1943 and Z16653, respectively) were obtained using a SMART RACE cDNA amplification kit (Clontech). The coding sequences were inserted into pH35GY (Kubo et al., 2005, Genes Dev. 19: 1855-1860) and pY35GS (Endo et al., 2008, Plant J. 53: 864-875) and the resultant constructs were used to examine protein localization and overexpression within Zinnia cells. pY35GUS which was constructed by insertion of GUS into pY35GS was used as a control. The sequence encoding 27Leu to 95Ala of Ze TED6 (SEQ ID NO: 1) and the sequence encoding 209Trp to 300Gly of Ze TED7 (SEQ ID NO: 2) were inserted into pY35GS. The sequences (1 kb and 0.5 kb, respectively) 5′-upstream of initiation codons of At TED6 and At TED7 genes were used as promoter regions. Fragments were inserted into pBGGUS (Kubo et al., 2005 (supra)) and then their promoter activities in Arabidopsis plants were examined. A genomic fragment containing a promoter region and ORF was inserted into pHGY (which was derived from pH35GY through deletion of the CaMV 35S promoter sequence). The first intron of a FAD2 (At3g12120) gene was used as a linker for inverted repeat sequences of full-length At TED6 ORF, At TED7 ORF, and At TED6-TED7 chimera sequence. At TED7 ORF was inserted to the PshA I site of At TED6 ORF, so as to construct a chimera. The inverted repeat was inserted into pH35GS (Kubo et al., 2005 (supra)) and/or pTA7002 (Aoyama and Chua, 1997, Plant J. 11: 605-612) to construct a vector for preparing a constitutive transient RNAi line. VND7 cDNA (Yamaguchi et al., 2008, Plant J. 55: 652-664) was inserted into pER8 (Zuo et al., 2000, Plant J. 24: 265273) to construct a vector for preparing an ectopic TE-inducible line.
(RT-PCR)
RNA was prepared from the two independent lines as described by Endo et al. (2008 (supra)) and then used for cDNA synthesis. PCR was performed using primers located in 5′- and 3′-UTRs of At TED6 and At TED7. Primers used for At TED6 were 5′-AGA GCC TCA CAC ATC AAA CAC AAG-3′ (SEQ ID NO: 12) and 5′-GGT AAC ATT ATG AAT GAA GAA AGC TC-3′(SEQ ID NO: 13); primers used for At TED7 were 5′-AAC CAT TTA AGT ACA TAC ATA CTC CC-3′ (SEQ ID NO: 14) and 5′-ATG ATT GTT TAC ATT TTG AGC CTT TTG-3′ (SEQ ID NO: 15); primers used for actin 2 (At3g18780) were 5′-CCG TTT TGA ATC TTC CTC AAT C-3′ (SEQ ID NO: 16) and 5′-ATA CCG GTA CCA TTG TCA CAC A-3′ (SEQ ID NO: 17); and primers used for ubiquitin (At5g57860) were 5′-TCC AAT GTG ATC CAA CAG AGA C-3′ (SEQ ID NO: 18) and 5′-TTC AAA GTC AAA GCC ACA ACT G-3′ (SEQ ID NO: 19).
Furthermore, the following sequences were used for primers for PCR amplification of Ze TED7-1 and Ze TED7-2.









(SEQ ID NO: 20)



5′-TTC CCT CAT TTT CCA CCG CCA TC-3′



and



 



(SEQ ID NO: 21)



5′-TGT TGT GGA ATG GTT GCT TGG AGA-3′






INDUSTRIAL APPLICABILITY
The present invention promotes the formation of secondary cell walls of vessel cells and wood fiber cells in plants and thus increases the thickness of secondary cell walls. The present invention enables increased cellulose content in arboreous plants and herbaceous plants which are biomass resources and therefore has a high degree of industrial usefulness.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
Sequence Listing Free Text


    SEQ ID NOS: 12-21: primers
    Sequencing Listing

## Claims

1. A method for increasing the thickness of a secondary cell wall of a plant, said method comprising producing a transgenic plant that contains expressible DNA encoding a protein consisting of a C-terminal domain of a tracheary element differentiation (TED) protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO: 1, amino acids 44-116 of SEQ ID NO: 4, amino acids 79-173 of SEQ ID NO: 6, amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-273 of SEQ ID NO: 8, amino acids 205-342 of SEQ ID NO: 9, and an amino acid sequence having at least 95% identity thereto; wherein the expressible DNA is expressed.

2. The method according to claim 1, wherein the expressible DNA encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO:1, amino acids 44-116 of SEQ ID NO: 4, amino acids 79-173 of SEQ ID NO: 6, and an amino acid sequence having at least 95% identity thereto; and
also encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-271 of SEQ ID NO: 8, amino acids 205-342 of SEQ ID NO: 9, and an amino acid sequence having at least 95% identity thereto.

3. A method for increasing the thickness of a secondary cell wall of a plant, said method comprising producing a transgenic plant that contains expressible DNA encoding a protein consisting of a C-terminal domain of a tracheary element differentiation (TED) protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO: 1, amino acids 44-116 of SEQ ID NO: 4, amino acids 79-173 of SEQ ID NO: 6, amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-273 of SEQ ID NO: 8, amino acids 205-342 of SEQ ID NO: 9, and an amino acid sequence having at least 95% identity thereto; wherein the expressible DNA encodes a protein that further comprises an additional 1 to 3 amino acids on the N-terminal side of the C-terminal domain of the TED protein.

4. A transgenic plant or a progeny plant thereof produced by the method of claim 1, wherein the thickness of the secondary cell wall is increased as compared to that of a wild-type plant; and wherein the progeny plant contains the expressible DNA.

5. The transgenic plant or progeny plant thereof according to claim 4, wherein the expressible DNA encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO: 1, amino acids 44-116 of SEQ ID NO: 4, amino acids 79-173 of SEQ ID NO: 6, and an amino acid sequence having at least 95% identity thereto;
and also encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-271 of SEQ ID NO: 8, amino acids 205-342 of SEQ ID NO: 9, and an amino acid sequence having at least 95% identity thereto.

6. A cell or tissue of the transgenic plant or the progeny plant thereof according to claim 4, wherein the cell or tissue contains the expressible DNA.

7. A cell or tissue of the transgenic plant or the progeny plant thereof according to claim 5, wherein the cell or tissue contains the expressible DNA.

8. A seed of the transgenic plant or the progeny plant thereof according to claim 4, wherein the seed contains the expressible DNA.

9. A seed of the transgenic plant or the progeny plant thereof according to claim 5, wherein the seed contains the expressible DNA.

10. The method of claim 1, in which the expressible DNA encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO: 1, amino acids 44-116 of SEQ ID NO: 4, amino acids 79-173 of SEQ ID NO: 6, amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-273 of SEQ ID NO: 8, and amino acids 205-342 of SEQ ID NO: 9.

11. The method according to claim 1, wherein the expressible DNA encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO: 1, amino acids 44-116 of SEQ ID NO: 4, and amino acids 79-173 of SEQ ID NO: 6;
and also encodes a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-273 of SEQ ID NO: 8, and amino acids 205-342 of SEQ ID NO: 9.

12. The transgenic plant or a progeny plant thereof according to claim 4, that comprises an expressible DNA encoding a protein consisting of a C-terminal domain of a TED protein, wherein the C-terminal domain is selected from the group consisting of: amino acids 29-95 of SEQ ID NO: 1, amino acids 44-116 of SEQ ID NO: 4, amino acids 79-173 of SEQ ID NO: 6, amino acids 211-300 of SEQ ID NO: 2, amino acids 194-283 of SEQ ID NO: 3, amino acids 120-205 of SEQ ID NO: 5, amino acids 127-210 of SEQ ID NO: 7, amino acids 174-273 of SEQ ID NO: 8, and amino acids 205-342 of SEQ ID NO: 9.