Method for producing plant having enhanced secondary cell walls by nucleotide substitution in transcription factor gene

Abstract

The present invention addresses the problem of providing a plant which can serve as a material for wooden buildings or a raw material for biofuels and in which the capability of producing secondary cell walls is improved by a small-scale mutation of one nucleotide to several nucleotides. Provided is a method for increasing the production of secondary cell walls by introducing a specific nucleotide substitution of one nucleotide to several nucleotides, the nucleotide substitution being one causing the improvement of the activity, into a specific transcription factor gene derived from larch or derived from thale cress, poplar or rice, a homologue thereof or the like, or by transforming the plant with a construct which enables the expression of a transcription factor gene having the above-mentioned mutation introduced therein using a promoter capable of inducing the expression in a secondary cell wall formation cell-specific manner.

Description

Method for producing plants with enhanced secondary cell walls by base substitution in transcription factor genes

[0001] The present invention relates to a method for producing plants with enhanced secondary cell walls by either artificially inducing a base substitution in the plant body that improves the activity of a transcription factor involved in secondary cell wall (wood) production, or by transforming plant cells with a gene into which the base substitution has already been introduced.

[0002] Recently, the use of biomass, a renewable and carbon-neutral resource, has been attracting attention due to the progress of global warming caused by excessive carbon dioxide emissions. Promoting the use of wooden buildings and replacing non-wooden buildings with wooden ones is particularly expected to significantly reduce carbon dioxide emissions. However, traditional wood materials lack sufficient strength and fire resistance, necessitating the development of wood with higher density and strength, especially for use in high-rise buildings. In particular, softwood species such as larch, spruce, cedar, and cypress have excellent straightness but lower strength than hardwood species, and strengthening them is required. Furthermore, if secondary cell walls could be strengthened in not only woody plants but also herbaceous plants, the amount of biomass raw material per unit planted area could increase, potentially improving the productivity of plant-based products such as bioethanol and bioplastics.

[0003] The NST transcription factor gene, discovered by the present inventors, is well known as a gene that controls secondary cell wall production in plants (Non-Patent Document 1). The NST transcription factor was discovered in the model experimental plant Arabidopsis thaliana, and in nst1 nst3 double mutants, secondary cell walls are completely abolished except in vessels. Conversely, overexpression of the NST transcription factor gene throughout the plant body induces ectopic secondary cell wall production in various cells, resulting in growth inhibition (Non-Patent Document 2). To enhance secondary cell walls while suppressing growth inhibition, it is desirable to enhance secondary cell wall production only in xylem fibers (also called wood fibers or true fibers) in angiosperms including broad-leaved trees, tracheids in conifers, and cells with secondary cell walls in monocotyledons.

[0004] Examples of such prior art include Patent Document 1 and Non-Patent Document 3, both of which were published by the present inventors. In these documents, the rice NST2 transcription factor (OsNST2; also known as OsSWN1) was found to have high activity, and by expressing it specifically in xylem fiber cells of Arabidopsis and poplar, secondary cell wall production was successfully enhanced without growth inhibition. However, it is not known why the OsNST2 transcription factor is more active than its Arabidopsis and poplar counterparts, nor is it known what amino acid sequence differences result in its high activity. Furthermore, it is not known whether the OsNST2 transcription factor is more active than NST transcription factors of any other plant. Furthermore, in these prior arts, the OsNST2 transcription factor gene must be introduced into another plant, and the resulting plant is necessarily a genetically modified plant. Therefore, there are various restrictions on outdoor cultivation, as regulated by the Cartagena Convention and the Domestic Security Law.

[0005] If there were a method to make the NST transcription factor highly active and enhance the secondary cell wall by minimal modifications, such as substituting one to several bases, the above problems could be solved, but no reliable method has yet been established.

[0006] Patent No. 6103631

[0007] The Plant Cell,2007 19,270-280The Plant Cell,2005 17,2993-3006Scientific Reports,2016 6,19925Methods in Enzymology, 2011 498,399-406Oshima et al., 2013, Plant Cell,25: 1609Nature Plants,2018 4,777-783Frontiers in Plant Science,2013 4,383Scientific Reports 2016 6,23609Plant Biotechnology 2011 28,201-210Nature Protocols 2006 1,2796Transgenic Research 1993 2,208-218

[0008] The present invention aims to provide a technique for producing plants with enhanced secondary cell wall production ability by increasing the activity of the NST transcription factor that controls secondary cell wall production in plants through base substitution of one to several bases, and introducing such a mutation into the plant body or by introducing an NST transcription factor gene having such a mutation into the plant.

[0009] In order to selectively enhance wood composed of secondary cell walls, it is thought that it is necessary to selectively express genes that fundamentally control secondary cell wall production only in xylem fiber cells, or to introduce the mutations necessary for enhancement into secondary cell wall production control genes in the plant body. In the process of comprehensively analyzing the function of NAC family transcription factors, which are plant-specific transcription factors, in Arabidopsis thaliana, the present inventors discovered the NST transcription factor gene group (NST1 [AT2G46770] [gene names (codes) starting with AT can be identified in the Arabidopsis Information Resource Database (TAIR database, https: / / www.arabidopsis.org / )] [DNA sequence, SEQ ID NO: 16; amino acid sequence, SEQ ID NO: 3], NST2 [AT3G61910] [amino acid sequence, SEQ ID NO: 4], NST3 [AT1G32770] [amino acid sequence, SEQ ID NO: 5]) as transcription factor genes that fundamentally control secondary cell wall production (Non-Patent Documents 1 and 2).

[0010]

[0011] In the nst1 nst3 double mutant of Arabidopsis thaliana, in which NST1 and NST3 were disrupted, secondary cell walls were completely eliminated in the xylem fiber cells of the inflorescence stem and hypocotyl, resulting in a significant reduction in the strength of the inflorescence stem and failure to grow upright. However, secondary cell wall defects were not observed in the xylem vessels where secondary cell walls are also produced, and plant survival was not affected (Non-Patent Document 1). Conversely, overexpression of these genes throughout the plant resulted in ectopic secondary cell wall production in all cells, significantly inhibiting plant growth (Non-Patent Document 2).

[0012] The present inventors focused on the secondary cell wall production ability of NST transcription factor genes and, while widely examining their orthologs, found that one of the rice NST transcription factor homologs, OsNST2 (Os06g0131700) [gene names (codes) starting with Os can be identified in the RAP-DB database (https: / / rapdb.dna.affrc.go.jp / ), the NCBI HomoloGene database (https: / / www.ncbi.nlm.nih.gov / homologene / ), or the NCBI Protein database (https: / / www.ncbi.nlm.nih.gov / protein / )] (amino acid sequence, SEQ ID NO: 9), has high activity. They then expressed this gene in xylem fiber cells of Arabidopsis thaliana and poplar, and found that secondary cell wall production was enhanced, resulting in increased density and strength (Patent Document 1, Non-Patent Document 3). However, it was unclear why the OsNST2 transcription factor has higher activity than those in Arabidopsis and poplar, and small-scale mutations such as base substitutions had not been able to increase the activity of NST transcription factors in general, not just in Arabidopsis and poplar.

[0013] SEQ ID NO: 9 (Rice NST2) MSISVNGQSVVPPGFRFHPTEEELLTYYLKKKVASERIDLDVICDVDLNKLEPWDIQERCRIGSGPQNDWYFFSHKDKKYPTGTRTNRATAAGFWKATGRDKAIYSSSNRIGMRKTLVFYKGRAPHGQKSDWIMHEYRLDDPSSASASVSVNLPSYYSSSSSSSSPMHGVAGDQGAQEEGWVICRVFKKKNLVHHGGGAAAASHHAAAKLAAAAMEGSPSNCSTVTVSDHVKAQMLHSSASDDALDHILQYMGRSGCKQETKPAAMSASSAAAAAALEQHLSTPQYGKFMKLPPLEHVAGGVGLLAAAGGGGEYCSAADASGIADWDTLVRLAASYELNGALSDVASGKNMAGFFDVVDQPAGAAAFSSGDGDLWSLARSVSSSLHADLTTMNNV

[0014] Therefore, the present inventors hypothesized that by creating a large number of mutant genes by introducing artificial random mutations into the NST transcription factor gene and comprehensively measuring the activity of the resulting gene products, it might be possible to obtain mutant NST genes encoding gene products with enhanced activity. As a research subject, we focused on the NST transcription factor gene (LkNAC1) (DNA sequence, SEQ ID NO: 1; amino acid sequence, SEQ ID NO: 2) of softwood tree species, particularly larch, which is known to have excellent linearity but insufficient strength. Using methods such as error-prone PCR, we created a large number of mutant NST transcription factor genes with one to several base substitutions, and comprehensively measured their activity using a transient reporter-effector assay using Arabidopsis thaliana protoplasts. In this study, we used a reporter in which a firefly luciferase gene was linked to the promoter region of the Arabidopsis thaliana NST3 gene, and searched for mutant NST transcription factor genes with improved luminescence levels. As a result, we found that the F166Y and I133V mutations reproducibly increased the activity of the LkNAC1 transcription factor by approximately 2-fold and 1.5-fold, respectively (Fig. 1). Furthermore, we found that combining the two mutations (I133V + F166Y) produced a more additive effect (Fig. 1).F166 and I133 are widely conserved in the corresponding locations of NST homologs in other plants (Fig. 2), including Arabidopsis thaliana NST1, Arabidopsis thaliana NST2, poplar NST1 (PtrNST1) (Potri.014G104800 [gene names (codes) beginning with Potri can be identified in the Phytozome database in the Plant Comparative Genome Portal operated by the Joint Genome Institute (JGI) of the U.S. Department of Energy (https: / / phytozome-next.jgi.doe.gov / )]) (amino acid sequence, SEQ ID NO: 6), poplar NST2 (PtrNST2) (Potri.002G178700) (amino acid sequence, SEQ ID NO: 7), and poplar VNS12 (PtrVNS1 In Arabidopsis thaliana NST3 and OsNST2, the amino acid positions corresponding to F166 are F178, F173, F172, F171, F172, F175, and F187, respectively (Figure 2). However, to avoid confusion, effective mutations in any plant NST homologs will be referred to as F166Y and I133V, respectively, even if the actual amino acid positions are slightly different. When substitutions equivalent to F166Y and I133V were introduced into the NST homolog genes of Arabidopsis, poplar, and rice, experiments using Arabidopsis protoplasts confirmed that the activity of the gene product increased approximately 2-fold and 1.5-fold, respectively (Figure 1). Although no further information is available on the I133V mutation, similar desired effects to the F166Y mutation are expected, albeit to a different extent. Furthermore, in experiments using LkNAC1, no improvement in activity was observed when F166 was replaced with any other amino acid other than Y (Figure 4).

[0015]

[0016] Next, we investigated whether the F166Y mutation actually enhances secondary cell walls in plants by introducing both the unmutated LkNAC1 transcription factor gene and the LkNAC1 transcription factor gene that confers these mutations into Arabidopsis nst1 nst3 double mutants, poplar, and larch. We also conducted experiments in Arabidopsis thaliana introducing the unmutated OsNST2 transcription factor gene and the OsNST2 transcription factor gene that confers the F166Y mutation. As a result, we found that plants introduced with the LkNAC1 transcription factor gene and the OsNST2 transcription factor gene that confers these mutations had improved cell wall weight (alcohol-insoluble fraction) per fresh weight (Figures 5, 6, 9), lignification degree (Figures 7, 8), and cell wall thickness (Figure 10) compared to plants introduced with the unmutated LkNAC1 transcription factor gene and the OsNST2 transcription factor gene.

[0017] Furthermore, we measured the branch strength of poplar plants and found that plants transfected with the LkNAC1 gene carrying the F166Y mutation exhibited improved three-point bending strength compared with plants transfected with the non-mutated LkNAC1 gene (Fig. 11). In an experiment in which rice was transfected with constructs expressing both the OsNST2 transcription factor gene carrying the F166Y mutation and the non-mutated OsNST2 transcription factor gene using the OsNST2 promoter, we compared the tensile strength of flag leaf midribs and found that the rice transfected with the F166Y mutation had higher strength (Fig. 12).

[0018] In addition to the above I133V and F166Y, new mutations were investigated. In the LkNAC1 transcription factor, H135K, Y137L, K76Q, K76H, R114K, K76L, T116V, H18K, R85A, K78Y, and D69Y mutations were introduced individually, and the activity was comprehensively measured by transient reporter-effector assays using Arabidopsis protoplasts. At this time, as a reporter, the promoter region of the Arabidopsis NST3 gene, and in addition to those connected to the firefly luciferase gene, the SNBE motif to which the NST transcription factor present in the promoter of the Arabidopsis MYB46 gene binds was repeated three times to the CaMV35S core promoter, and those connected to the firefly luciferase gene. When the above mutations were not present, the reporter activity was increased compared to the LkNAC1 transcription factor (Figure 13). Furthermore, when the LkNAC1 transcription factor genes with these mutations were placed under the promoter of the Arabidopsis NST3 gene and introduced into the Arabidopsis nst1 nst3 double mutant, we found that the cell wall weight (alcohol-insoluble fraction) per fresh weight was increased compared to when an LkNAC1 transcription factor gene without any mutations was introduced (Figure 14).The above mutations are widely conserved in the corresponding positions in NST homologs of other plants ( FIG. 14 ). In Arabidopsis thaliana NST1, Arabidopsis thaliana NST2, poplar NST1 (PtrNST1) (amino acid sequence, SEQ ID NO: 6), poplar NST2 (PtrNST2) (amino acid sequence, SEQ ID NO: 7), poplar VNS12 (PtrVNS12) (amino acid sequence, SEQ ID NO: 8), Arabidopsis thaliana NST3, and OsNST2, the amino acid positions corresponding to H135 are H140, H135, H139, H139, H140, H140, and H135, the amino acid positions corresponding to Y137 are Y142, Y137, Y141, Y141, Y142, Y142, and Y137, and the amino acid positions corresponding to K76 are K81, K82, and K83. The amino acid positions corresponding to R114 are R119, R114, R118, R118, R119, R119, R114, the amino acid positions corresponding to T116 are T121, T116, T120, T120, T121, T121, T116, the amino acid positions corresponding to H18 are H23, H18, H22, The amino acid positions corresponding to R85 are R90, R85, R89, R89, R90, R90, R85, the amino acid positions corresponding to K78 are K83, K78, K82, K82, K83, K83, K78, and the amino acid positions corresponding to D69 are D74, D69, D73, D73, D74, D74, D69 (Figure 15).

[0019] Based on the above findings, the present invention has been completed.

[0020] That is, the present invention is as follows: [1] A method for producing a plant with enhanced secondary cell walls, comprising the step of enhancing the function of at least one of the following transcription factors in a cell that forms secondary cell walls: (a) a transcription factor consisting of the amino acid sequence set forth in at least one of SEQ ID NOS: 2 to 9; (b) a transcription factor in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence set forth in at least one of SEQ ID NOS: 2 to 9, and which has the activity of inducing secondary cell wall production; and (c) a transcription factor comprising the amino acid sequence set forth in at least one of SEQ ID NOS: 10 to 15, and which has the activity of inducing secondary cell wall production. [2] The production method according to [1], wherein the step of enhancing the function of the transcription factor is a step of introducing one or more base substitutions into a nucleic acid encoding the transcription factor. [3] The method of [2], wherein the base substitution is a base substitution that replaces, with tyrosine, at least one of the amino acids shown in (a) a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10, which is conserved in transcription factors consisting of the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10, which is conserved in transcription factors having a deletion, substitution, or addition of one or more amino acids in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 and having the activity of inducing secondary cell wall production; and (c) a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10, which is conserved in transcription factors comprising the amino acid sequences shown in SEQ ID NOs: 10 and 11 and having the activity of inducing secondary cell wall production.[4] The method of [2], wherein the base substitution is a base substitution that replaces, with valine, at least one of the amino acids shown in (a) an isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11, which is conserved in a transcription factor consisting of the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11, which is conserved in a transcription factor having a deletion, substitution, or addition of one or more amino acids in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 and having the activity of inducing secondary cell wall production; and (c) an isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11, which is conserved in a transcription factor comprising the amino acid sequence shown in SEQ ID NOs: 10 and 11 and having the activity of inducing secondary cell wall production. [5] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with lysine, at least one of the amino acids shown in (a) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 12, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 12, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted, or added, and which has the activity of inducing secondary cell wall production. [6] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with leucine, at least one of the amino acids shown in (a) a tyrosine corresponding to the tyrosine in the motif sequence shown in SEQ ID NO: 12, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a tyrosine corresponding to the tyrosine in the motif sequence shown in SEQ ID NO: 12, which is conserved in a transcription factor having a deletion, substitution, or addition of one or several amino acids in an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 and having the activity of inducing secondary cell wall production.[7] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with glutamine, histidine, or leucine, at least one of the amino acids shown in (a) a lysine corresponding to the first lysine in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a lysine corresponding to the first lysine in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted, or added and which has the activity of inducing secondary cell wall production. [8] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with lysine, at least one of the amino acids shown in (a) an arginine corresponding to the arginine in the motif sequence shown in SEQ ID NO: 14, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) an arginine corresponding to the arginine in the motif sequence shown in SEQ ID NO: 14, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted, or added, and which has the activity of inducing secondary cell wall production. [9] The method of [2], wherein the base substitution is a base substitution that substitutes, with valine, at least one of the amino acids shown in: (a) a threonine corresponding to the threonine in the motif sequence shown in SEQ ID NO: 14, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a threonine corresponding to the threonine in the motif sequence shown in SEQ ID NO: 14, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted, or added, and which has the activity of inducing secondary cell wall production.

[10] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with lysine, at least one of the amino acids shown in: (a) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 15, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 15, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted, or added, and which has the activity of inducing secondary cell wall production.

[11] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with alanine, at least one of the amino acids shown in: (a) an arginine corresponding to the arginine appearing first in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor consisting of the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) an arginine corresponding to the arginine appearing first in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor having the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9, in which one or more amino acids have been deleted, substituted, or added, and which has the activity of inducing secondary cell wall production.

[12] The production method according to [2], wherein the base substitution is a base substitution that substitutes, with tyrosine, at least one of the amino acids shown in: (a) a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9, in which one or more amino acids have been deleted, substituted, or added, and which has the activity of inducing secondary cell wall production.

[13] The method of [2], wherein the base substitution is a base substitution that replaces at least one of the amino acids shown in: (a) an aspartic acid corresponding to the first aspartic acid in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor consisting of the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) an aspartic acid corresponding to the first aspartic acid in the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor having the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9, with tyrosine.

[14] The method of [1], comprising a step of transforming a plant cell with a nucleic acid into which at least one of the base substitutions shown in [3] to

[13] has been introduced.

[15] The method of [1], comprising a step of ligating a nucleic acid into which at least one of the base substitutions shown in [3] to

[13] has been introduced to a promoter capable of expressing the nucleic acid in at least one type of cell that forms a secondary cell wall, and then transforming the plant cell.

[16] A method for producing the plant according to [1], comprising a step of introducing at least one base substitution selected from the base substitutions according to [3] to

[13] into a gene present in the genome of the plant by genome editing, radiation, or chemical agents.

[17] A plant or its progeny, or a part thereof, produced by the production method according to any one of

[14] to

[16] .

[18] A transcription factor in which, in at least one of (a) the amino acid sequence set forth in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequences set forth in SEQ ID NOs: 10 and 11, a phenylalanine corresponding to the phenylalanine in the motif sequence set forth in SEQ ID NO: 10, which is conserved in the amino acid sequence, is substituted with tyrosine, and which has the activity of inducing secondary cell wall production.

[19] A transcription factor having the activity of inducing secondary cell wall production, in which in at least one of: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequences shown in SEQ ID NOs: 10 and 11, the isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11, which is conserved in the amino acid sequence, is substituted with valine.

[20] A transcription factor having the activity of inducing secondary cell wall production, in which in at least one of: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 12, the histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 12, which is conserved in the amino acid sequence, is substituted with lysine.

[21] A transcription factor having the activity of inducing secondary cell wall production, in which in at least one of: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 12, a tyrosine corresponding to a tyrosine in a motif sequence shown in SEQ ID NO: 12 that is conserved in the amino acid sequence is substituted with leucine.

[22] A transcription factor having the activity of inducing secondary cell wall production, in which in at least one of: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 13, a lysine corresponding to the first lysine in the motif sequence shown in SEQ ID NO: 13 that is conserved in the amino acid sequence is substituted with glutamine, histidine, or leucine.

[23] A transcription factor having the activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 14, an arginine corresponding to the arginine in the motif sequence shown in SEQ ID NO: 14 that is conserved in the amino acid sequence is substituted with lysine.

[24] A transcription factor having the activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 14, an arginine corresponding to the arginine in the motif sequence shown in SEQ ID NO: 14 that is conserved in the amino acid sequence is substituted with valine.

[25] A transcription factor having the activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 15, a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 15 that is conserved in the amino acid sequence is substituted with lysine.

[26] A transcription factor having at least one of the following: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (c) the amino acid sequence shown in SEQ ID NO: 13, a histidine corresponding to the first arginine in the motif sequence shown in SEQ ID NO: 13 that is conserved in the amino acid sequence is substituted with alanine, and

[27] A transcription factor having the activity of inducing secondary cell wall production, in which in at least one of: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9, in which one or more amino acids have been deleted, substituted, or added; and (c) the amino acid sequence shown in SEQ ID NO: 13, a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 13, which is conserved in the amino acid sequence, is substituted with tyrosine.

[28] A transcription factor having the activity of inducing secondary cell wall production, in which in at least one of: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9, in which one or more amino acids have been deleted, substituted, or added; and (c) the amino acid sequence shown in SEQ ID NO: 12, a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 12, which is conserved in the amino acid sequence, is substituted with tyrosine.

[29] A nucleic acid encoding a transcription factor according to

[18] or

[19] or

[20] or

[21] or

[22] or

[23] or

[24] or

[25] or

[26] or

[27] or

[28] .

[0021] SEQ ID NO: 10 (NST conserved motif 1) GWV (V or I) CR (V or I or L) F (K or R or M) K SEQ ID NO: 11 (NST conserved motif 2) (K or R) (S or T or L) (D or E) WIMHEYR SEQ ID NO: 12 (NST conserved motif 3) DWIMHEYRLD SEQ ID NO: 13 (NST conserved motif 4) PQ (N or H) DWYF (Y or F) SHKDKKYP (A or T) GTRTNRA SEQ ID NO: 14 (NST conserved motif 5) G (L or M) RKTLVF SEQ ID NO: 15 (NST conserved motif 6) FRFHPT

[0022] The secondary cell wall-enhanced plant of the present invention was produced by introducing the LkNAC1 transcription factor gene, which has a mutation of one to several bases that enhances the activity of the NST transcription factor, into a plant. This allows for an increase in the density of the secondary cell wall, and therefore an increase in the amount of the secondary cell wall itself. Increasing the secondary cell wall density not only improves flame retardancy and strength, but also increases the production of bioethanol and bioplastics.

[0023] This figure shows that mutant NST proteins in which the isoleucine at position 133 of the larch NST homolog transcription factor (LkNAC1) has been replaced with valine and the phenylalanine at position 166 has been replaced with tyrosine, or both, have higher activity in Arabidopsis protoplasts than those without the replacements. This figure also shows that similar effects are obtained when the corresponding amino acid replacements are made not only in larch but also in Arabidopsis NST1, NST2, poplar NST1, NST2, and VNS12. Error bars represent standard deviation. This figure shows that the sites corresponding to I133 and F166 of LkNAC1 are conserved in NST homologs of various plants. This figure shows the effect on transcription factor activity of the Arabidopsis NST3 transcription factor and the rice NST2 transcription factor when the phenylalanine corresponding to the phenylalanine at position 166 in the LkNAC1 transcription factor is replaced with tyrosine. Error bars represent standard deviation. 1 shows the results of evaluating the activity when phenylalanine at position 166 of the LkNAC1 transcription factor is substituted with 19 other amino acids. Error bars represent standard deviation. This figure shows the results of quantifying the cell wall components (AIR / FW) per fresh weight of inflorescence stems when an unsubstituted LkNAC1 transcription factor or an LkNAC1(F166Y) transcription factor is genetically introduced into an Arabidopsis nst1 nst3 double mutant. Error bars represent standard deviation. This figure shows the results of quantifying the cell wall components (AIR / FW) per fresh weight of inflorescence stems when an unsubstituted rice NST2 transcription factor or a rice NST2 transcription factor with a substitution equivalent to F166Y in LkNAC1 is genetically introduced into an Arabidopsis nst1 nst3 double mutant. Error bars represent standard deviation.

[0023] Figure 1 shows the results of evaluating the degree of lignification in the flower stalk when an unsubstituted LkNAC1 transcription factor or an LkNAC1(F166Y) transcription factor was genetically introduced into an Arabidopsis nst1 nst3 double mutant.

[0024] Figure 1 shows the results of evaluating the degree of lignification in the flower stalk when an unsubstituted Arabidopsis NST3 transcription factor or an Arabidopsis NST3 transcription factor with a substitution equivalent to F166Y in the LkNAC1 transcription factor LkNAC1 was genetically introduced into an Arabidopsis nst1 nst3 double mutant.

[0033] Figure 1 shows the results of quantifying cell wall components per stem fresh weight (AIR / FW) (left) and cell wall components per individual (AIR) (right) after gene transfer of unsubstituted LkNAC1 transcription factor or LkNAC1(F166Y) into poplar (Hybrid aspen). Error bars represent standard deviation.

[0034] Figure 1 shows the results of gene transfer of unsubstituted LkNAC1 transcription factor or LkNAC1(F166Y) transcription factor into larch, measuring the cell wall thickness of 50 xylem tracheids in each transverse section of the stem for eight individuals each that are thought to have different insertion positions on the genome, and averaging the results. Error bars represent standard deviation.

[0035] Figure 1 shows the results of gene transfer of unsubstituted LkNAC1 transcription factor or LkNAC1(F166Y) transcription factor into poplar (Hybrid aspen), measuring the three-point bending strength at different internodes (n = 2-5). t is a graph showing the results of measuring the tensile strength of midrib sections excised from flag leaves of rice plants transfected with either an unsubstituted OsNST2 transcription factor or an OsNST2 transcription factor with a substitution equivalent to F166Y (n=3). Error bars represent standard deviation. This figure shows that mutant NST proteins with various single amino acid substitutions in the LkNAC1 transcription factor have higher activity in Arabidopsis protoplasts than those without the substitution. Error bars represent standard deviation. This figure shows the results of quantifying the cell wall components (AIR / FW) per fresh weight of inflorescence stems when an unsubstituted LkNAC1 transcription factor or an LkNAC1 transcription factor with various single amino acid substitutions was transfected into an Arabidopsis nst1 nst3 double mutant. Error bars represent standard deviation. FIG. 1 shows that the sites corresponding to H135, Y137, D69, K76, K78, R85, R114, T116, and H18 of LkNAC1 are conserved in NST homologs of various plants.

[0024] The present invention is described in detail below. 1. Plants with Enhanced Secondary Cell Walls of the Present Invention (1) Genes for Enhancement of Secondary Cell Walls of the Present Invention Generally, the term "cell wall" refers to a strong, flexible, or rigid layer that surrounds the cells of plants, bacteria, algae, and fungi such as yeast, providing structural support and protection to the cells. Plant cell walls are characterized by being predominantly composed of polysaccharides and containing small amounts of other components (e.g., lignin, structural proteins). Cell walls are sometimes classified as "primary cell walls" and "secondary cell walls." When both types exist, the secondary cell wall is located inside the primary cell wall. Secondary cell walls are generally thickened cell walls that are produced secondarily after cell growth has nearly ceased. As is known to those skilled in the art, plants contain cellulose, hemicellulose, and lignin as the main components of their secondary cell walls.

[0025] According to the present invention, it is possible to produce plants with enhanced secondary cell walls, and the objective is, but is not limited to, the enhancement of secondary cell walls surrounding the periphery of xylem fiber cells or tracheid cells. In the present invention, an NST transcription factor gene into which a predetermined base substitution has been introduced is introduced into a host plant cell and expressed. As a result, a transformed plant into which the NST transcription factor gene has been expressed has enhanced secondary cell walls compared to a plant into which a wild-type NST transcription factor gene has been introduced. In the following explanation, this effect of enhanced secondary cell wall production in a plant into which an NST transcription factor gene into which a predetermined base substitution has been introduced is referred to as a "secondary cell wall enhancement effect."

[0026] In one embodiment, the gene that exerts the "secondary cell wall enhancement effect" in the present invention is the "LkNAC1 transcription factor gene" derived from larch and represented by SEQ ID NO: 1, and the corresponding amino acid sequence is represented by SEQ ID NO: 2. The "LkNAC1 transcription factor gene" as a gene that exerts the "secondary cell wall enhancement effect" is not limited to the larch-derived "LkNAC1 transcription factor gene" represented by SEQ ID NO: 1, but also includes its homologs such as orthologs and paralogs, and genes with partial mutations in their nucleotide sequences. That is, a "secondary cell wall enhancement effect" is exhibited in a plant engineered to express the larch-derived "LkNAC1 transcription factor gene" or its homolog, or genes with partial mutations in their nucleotide sequences, in at least one cell type that forms secondary cell walls, such as xylem fiber cells or tracheid cells. Meanwhile, in terms of amino acid sequence, the transcription factor also includes nucleic acids comprising the amino acid sequence represented by SEQ ID NO: 11, or the amino acid sequences represented by SEQ ID NOs: 10 and 11, and a nucleotide sequence encoding a polypeptide having the activity of inducing secondary cell wall formation. It is preferable that the identity to the amino acid sequence shown in SEQ ID NO: 2 is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.).

[0027] In another embodiment, the gene that exerts the "secondary cell wall-enhancing effect" is an "NST1 transcription factor gene" derived from Arabidopsis thaliana and represented by SEQ ID NO: 16, and the corresponding amino acid sequence is represented by SEQ ID NO: 3. The "NST1 transcription factor gene" as a gene that exerts the "secondary cell wall-enhancing effect" is not limited to the "NST transcription factor gene" derived from Arabidopsis thaliana and represented by SEQ ID NO: 16, but also includes homologs such as orthologs and paralogs thereof, and genes in which mutations have been introduced into part of their nucleotide sequences. In other words, a "secondary cell wall-enhancing effect" is exhibited in a plant that has been engineered so that the "NST1 transcription factor gene" derived from Arabidopsis thaliana or its homolog, or genes in which mutations have been introduced into part of their nucleotide sequences, is expressed in cells that form secondary cell walls, such as xylem fiber cells or tracheid cells.

[0028] The present inventors have demonstrated for the first time that the "NST1 transcription factor gene" is a transcription factor gene that fundamentally controls secondary cell wall production. The "NST1 transcription factor gene" is widely conserved in all plants with similar plant organelles, and has the amino acid sequence shown in SEQ ID NO: 11, or the amino acid sequences shown in SEQ ID NOs: 10 and 11. Therefore, homologous genes in other plant genomes, or genes into which partial mutations have been introduced, can also be used as long as they have the amino acid sequence shown in SEQ ID NO: 11, or the amino acid sequence shown in SEQ ID NOs: 10 and 11, and are similar genes that have the activity of inducing secondary cell wall production.

[0029] The identity of amino acid sequences or nucleotide sequences can be determined using the BLAST algorithm by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing a nucleotide sequence using BLASTN based on BLAST, parameters can be set, for example, as score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, parameters can be set, for example, as score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program can be used. Specific techniques for these analysis methods are known (http: / / www.ncbi.nlm.nih.gov).

[0030] That is, genes into which the predetermined base substitutions of the present invention are introduced to exert a "wood-enhancing effect" can generally be described as (a) to (c) below: (a) a gene encoding a transcription factor consisting of at least one of the amino acid sequences shown in SEQ ID NOs: 2 to 9, and / or (b) a gene encoding a transcription factor in which one or more amino acids have been deleted, substituted, or added in at least one of the amino acid sequences shown in SEQ ID NOs: 2 to 9, and which induces secondary cell wall production, and / or (c) a gene encoding a transcription factor having at least one of the amino acid sequences shown in SEQ ID NOs: 10 to 15, and which induces secondary cell wall production.

[0031] Here, "one or several amino acids are deleted, substituted, or added in the amino acid sequence" means that the amino acids in the amino acid sequence have been modified without significantly affecting the structure or function of the polypeptide. Furthermore, "several" refers to the presence of preferably 2 to 30, more preferably 2 to 14, and even more preferably 2 to 10 mutations, for example, 2 to 8, 2 to 6, 2 to 4, 2 to 3, 8, 7, 6, 5, 4, 3, or 2 mutations.

[0032] With respect to proteins such as transcription factors, a "conserved" amino acid or amino acid sequence means that when the amino acid sequences of two or more proteins being compared are aligned using a known program such as ClustalW so that similar portions are in the same position, the amino acid or amino acid sequence is identical at the same position or region. Alternatively, it means that one or more amino acids are several different amino acids at that position or region. "One or more" means, for example, 1 to 5, 1 to 4, 1 to 3, 5, 4, 3, 2, and 1.

[0033] The "NST transcription factor gene" of the present invention that exerts the "secondary cell wall enhancement effect" exhibits this effect by substituting bases such that specific amino acid residues in the protein encoded by the target gene are replaced with other amino acids. The specific amino acid residues are isoleucine and phenylalanine residues at specific positions that are conserved in genes encoding transcription factors that have the activity of inducing secondary cell wall production from various plants. As used herein, "induce" refers to "exerting," "inducing," "attracting," or "causing" secondary cell wall production. The degree of the secondary cell wall enhancement effect is preferably such that secondary cell wall production is increased by at least 5% or more compared to the protein without substitution at the specific amino acid residue. For example, such an increase may be 10%, 20%, 30%, 50%, or 100%.

[0034] Specifically, in proteins encoded by "NST transcription factor genes" derived from various plants, the phenylalanine residue and isoleucine residue to be replaced can be identified based on the amino acid sequences shown in SEQ ID NOs: 10 and 11, respectively. For example, by preparing a multiple alignment of the amino acid sequences of proteins encoded by transcription factor genes derived from various plants that have the activity of inducing secondary cell wall production, the motif sequences shown in SEQ ID NOs: 10 and 11 can be identified, and the amino acid sequence to be replaced can be identified. Note that the term "transcription factor" refers to a factor including a protein that positively or negatively controls (regulates) gene expression, and refers to all factors involved in transcription in a DNA sequence-dependent manner, such as transcription activators, transcription repressors, factors that neither activate nor repress transcription but bind to the upstream region of a gene, and cofactors.

[0035] Furthermore, other substitution targets, H135, Y137, D69, K76, K78, R85, R114, T116, and H18, can be identified based on the amino acid sequences shown in SEQ ID NOs: 12, 12, 13, 13, 13, 13, 14, 14, and 15, respectively.

[0036] (2) Method for Producing a Plant with Enhanced Secondary Cell Walls in the Present Invention The promoter for expressing the "NST transcription factor gene" used in the present invention is a "promoter that induces gene expression specifically in secondary cell wall-producing cells," and specifically includes a promoter of the "NST transcription factor gene" derived from the target plant or a promoter of its orthologous gene, an artificial promoter in which repeats of NST binding sequences (SNBE) are arranged upstream of a core promoter, a promoter of a secondary cell wall cellulose synthase, a promoter of enzymes involved in lignin synthesis, and a promoter of enzymes involved in xylan synthesis. In particular, a promoter of the "NST transcription factor gene" derived from the target plant is preferred. Here, "secondary cell wall-producing cells" are, for example, xylem fiber cells, tracheid cells, and cells that form secondary cell walls of plants.

[0037] A construct (recombinant vector) is constructed in which the "NST transcription factor gene" into which the above-mentioned base substitution has been introduced is placed downstream of a promoter that specifically expresses secondary cell wall-producing cells, and this construct is used to transform a wild-type strain or a plant in which the function of the NST transcription factor or its orthologous transcription factor gene is suppressed to produce a genetically modified plant. The Agrobacterium method is a typical method for introducing a foreign gene into a plant, but other methods that can be used include the PEG-calcium phosphate method, electroporation, liposome method, particle gun method, microinjection method, etc.

[0038] In addition to the method of introducing a base substitution-introduced "NST transcription factor gene" into a target plant, a method that does not result in a genetically modified plant is to directly introduce a base substitution into the "NST transcription factor gene" present in the genome of the target plant, thereby producing a plant with enhanced secondary cell wall production. Such known methods include, but are not limited to, genome editing, physical mutagenesis, methods using chemical mutagens, and methods using radiation or ultraviolet light treatment.

[0039] Genome editing is a method of modifying a target gene using a site-specific nuclease (e.g., a DNA double-strand break enzyme such as zinc finger nuclease (ZFN), transcription activation-like effector nuclease (TALEN), or CRISPR-Cas9). For example, fusion proteins such as ZFNs (U.S. Patent Nos. 6,265,196, 8,524,500, 7,888,121, and European Patent No. 1,720,995), TALENs (U.S. Patent Nos. 8,470,973 and 8,586,363), and pentatricopeptide repeat (PPR) fused with a nuclease domain (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (U.S. Patent No. 8,697,359, and International Publication No. WO 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and Examples include methods that use a complex of guide RNA and protein, such as those described in "Vertebrate Adaptive Immune Systems, Science, DOI:10.1126 / science.aaf8729,(2016)."

[0040] Physical mutagenesis methods include, for example, heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0041] Examples of methods using chemical mutagens include treating seeds with chemical mutagens (see, for example, Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). Chemical mutagens are not particularly limited, but include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0042] For plants into which mutations have been introduced by the above methods, it can be confirmed by known methods whether or not a base substitution has been introduced into the "NST transcription factor gene." Such known methods include, for example, DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, Southern blotting, and Northern blotting.

[0043] Another method for confirming whether a base substitution has been introduced into the "NST transcription factor gene" is TILLING (Targeting Induced Local Lesions IN Genomes) (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when a non-selective mutation has been introduced into the plant genome using the aforementioned heavy ion beam irradiation or chemical mutagen, the "NST transcription factor gene" or a portion thereof can be amplified by PCR, and then individuals having a mutation in the amplification product can be selected by the aforementioned TILLING or the like.

[0044] Furthermore, by crossing a plant into which a mutation has been introduced by the above-mentioned method with a wild-type plant and performing backcrossing, it is also possible to remove mutations introduced into genes other than the "NST transcription factor gene."

[0045] Plants that express an "NST transcription factor gene" into which a predetermined base substitution has been introduced are not particularly limited. That is, plants transformed with an "NST transcription factor gene" into which the above-mentioned predetermined base substitution has been introduced, or plants into which a predetermined base substitution has been introduced into the "NST transcription factor gene" on the genome by the above-mentioned method, can exert the "secondary cell wall enhancement effect" of the "NST transcription factor gene" in all plant bodies. Target plants include, but are not limited to, dicotyledonous plants and monocotyledonous plants, such as plants belonging to the Brassicaceae, Poaceae, Solanaceae, Leguminosae, and Salicaceae families, as angiosperms, and Pinaceae (see below), as gymnosperms. Furthermore, the above-mentioned plants produced by the production method of the present invention include, but are not limited to, progeny of the plants or parts of the plants (e.g., cuttings, cultured cells, callus, etc.).

[0046] Pinaceae: Cedar (Cedrus brevifolia), Fir (Abies alba), Tsuga (Tsuga sieboldii), Larch (Larix kaempferi), Todo fir (Abies sachalinensis), Picea glehnii, Norway spruce (Picea abies); Cupressaceae: Cedar (Cryptomeria japonica), Hinoki (Chamaecyparis obtuse), Hiba (Thujopsis dolabrata); Ulmaceae: Zelkova (Zelkova serrata); Fagaceae: Quercus crispula, Beech (Fagus crenata); Brassicaceae: Arabidopsis thaliana, Arabidopsis lyrata, Brassica rapa, Brassica napus, Brassica campestris; Sapindaceae: sugar maple (Acer saccharum); Euphorbiaceae: castor bean (Ricinus communis); Solanaceae: tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solanum tuberosum), tomato (Solanum lycopersicum), chili pepper (Capsicum annuum), petunia (Petunia hybrida), etc.; Fabaceae: soybean (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), peanut (Arachis hypogaea), lotus grass (Lotus japonicus), kidney bean (Phaseolus vulgaris), adzuki bean (Vigna angularis), acacia (Acacia japonica), alfalfa (Medicago sativa) truncatula), chickpea (Cicer arietinum), etc.; Asteraceae: chrysanthemum (Chrysanthemum morifolium), sunflower (Helianthus annuus), etc.;Palm family: oil palm (Elaeis guineensis, Elaeis oleifera), coconut palm (Cocos nucifera), date palm (Phoenix dactylifera), wax palm (Copernicia), etc.; Anacardiaceae: wax tree (Rhus succedanea), cashew tree (Anacardium occidentale), sumac (Toxicodendron vernicifluum), mango (Mangifera indica), pistachio (Pistacia vera), etc.; Cucurbitaceae: pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), trichosanthes cucumeroides, gourd (Lagenaria siceraria var. gourda), etc.; Rosaceae: almond (Amygdalus communis), rose (Rosa), strawberry (Fragaria), cherry (Cerasus), apple (Malus pumila var. domestica), peach (Prunus persica), etc.; Vitaceae: grape (Vitis vinifera); Caryophyllaceae: carnation (Dianthus caryophyllus), etc.; Salicaceae: poplar (Populus trichocarpa, Populus nigra, Populus tremula), etc.; Poaceae: corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), Urartu wheat (Triticum urartu), sorghum (Aegilops tauschii), wheatgrass (Brachypodium distachyon), bamboo (Phyllostachys), sugarcane (Saccharum officinarum), napier grass (Pennisetum pupureum), Erianthus (Erianthus arundinaceus), Miscanthus sinensis, Sorghum (Sorghum bicolor);Switchgrass (Panicum virgatum), etc.; Liliaceae: Tulip (Tulipa), Lilium (Lilium), etc.;

[0047] Among these, plants belonging to the Pinaceae family such as larch, Abies sachalinensis, and Norway spruce, plants belonging to the Cupressaceae family such as cedar and cypress, plants belonging to the Poaceae family such as sugarcane, corn, Erianthus, rice, sorghum, and wheat, and in particular plants belonging to the genus Saccharum, Erianthus, Sorghum, or Miscanthus are preferred.

[0048] 3. Others Other terms and concepts used in the present invention are based on the meanings of terms commonly used in the relevant technical field, and various techniques used to carry out the present invention can be easily and reliably carried out by those skilled in the art based on known literature, etc., except for techniques whose sources are specifically indicated. For example, genetic engineering techniques can be carried out by the methods described in J. Sambrook, E.F. Fritsch & T. Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); D.M. Glover et al. ed., DNA Cloning, 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995), or by methods substantially similar to or modified from these. Furthermore, various proteins and peptides used in the present invention, or DNAs encoding them, can be obtained from the existing TAIR or NCBI GenBank database (URL: http: / / www.arabidopsis.org / or http: / / www.ncbi.nlm.nih.gov / sites / entrez?db=pubmed, etc.). The contents of the technical literature, patent publications, and patent application specifications cited in this specification are to be referenced as the contents of the present invention.

[0049] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples and can be implemented in any form within the scope of the present invention.

[0050] Example 1 Screening for highly active mutant LkNAC1 transcription factors by transient reporter-effector assay (1-1) Construction of effector plasmid carrying wild-type larch LkNAC1 transcription factor gene An effector plasmid incorporating the wild-type larch LkNAC1 transcription factor gene was constructed according to the following procedure. First, the coding region of the LkNAC1 protein was amplified by polymerase chain reaction (PCR) using a larch cDNA library as a template. The following oligonucleotide pair was used as primers for the PCR reaction. 5'-ggggacaagtttgtacaaaaaagcaggcttcATGACTTTATCAGTAAATGGCC-3' (forward primer for amplifying LkNAC1, LkVNS11F, SEQ ID NO: 17) 5'-ggggaccactttgtacaagaaagctgggtcTTACTTTACGAAGTTCCATAAATCTATTTC-3' (reverse primer for amplifying LkNAC1, attB1 LkNAC1full R, SEQ ID NO: 18) In the sequences of both of the above primers, the lowercase letters represent the adapter sequence.

[0051] The amplified gene fragment was subcloned into the Gateway donor vector pDONR221 (Invitrogen) using BP Clonase (Thermo Fisher) to create an entry clone. Subsequently, the LkNAC1 gene was transferred to the vector pDEST35SHSP (Non-Patent Document 5) for transient reporter-effector assays using LR Clonase (Thermo Fisher). This resulted in the construction of a wild-type LkNAC1 effector plasmid, which serves as a benchmark for activity, in which the 35S promoter, wild-type LkNAC1 translation region, and HSP terminator sequence were linked in this order.

[0052] (1-2) Preparation of a library of random mutants of the LkNAC1 transcription factor gene To introduce random mutations into the LkNAC1 gene, error-prone PCR was performed using the GeneMorph II Random Mutagenesis kit (Agilent Technologies). Using the plasmid prepared in (1-1) as a template, mutations were introduced into the region of the LkNAC1 gene from residue 418 to just before the stop codon. The primers used here are as follows: 5'-GATTGGATTATGCATGAATATCGCCTG-3' (forward primer for random mutagenesis, random F, SEQ ID NO: 19) 5'-CACTTTGTACAAGAAAGCTGGGTCTTA-3' (reverse primer for random mutagenesis, random R, SEQ ID NO: 20)

[0053] The resulting PCR fragment was purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega), and then this DNA fragment was used to prepare a library of random mutants using the MEGAWHOP method (Non-Patent Document 4). A 50 μl PCR reaction system contained a plasmid (50 ng) containing the wild-type LkNAC1 gene prepared in (1-1) as a template, and the above PCR fragment (500 ng) as a megaprimer. A PCR reaction was performed using Phusion High-Fidelity DNA Polymerase (New England BioLabs Japan), and the original wild-type LkNAC1 gene was replaced with the mutated LkNAC1 (mutant LkNAC1). The reaction solution was then treated with the restriction enzyme DpnI at 37 ° C for 1 hour to selectively digest the template, resulting in a library of random mutants. E. coli DH5α was transformed using 2 μl of this reaction mixture, and plasmids were extracted from these E. coli using the FastGene™ Plasmid Mini Kit (Nippon Genetics Co., Ltd.). Analysis of the DNA sequence of this plasmid revealed that a good library was obtained with a low bias (bias refers to, for example, A to G or T to C substitutions alone accounting for approximately 50% of all substitutions), with a mutation frequency of 1-bp substitutions of 1 to 14 bp per 765 bp of the target portion (average of 3 bp or 6 bp). These were then used as the effector library for transient reporter-effector assays.

[0054] (1-3) Preparation of Amino Acid Substitution Clones of Arabidopsis, Rice, and Poplar NST Transcription Factors Effector plasmids incorporating the transcription factor genes of wild-type Arabidopsis NST1, 2, and 3, poplar NST1, NST2, and VNS12, and rice NST2 (OsNST2) were constructed using the following procedure. First, the protein coding regions were amplified by PCR using Arabidopsis, poplar, and rice cDNA libraries as templates. The following oligonucleotide pairs were used as primers for the PCR reaction. 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGATGTCAAAATCTATGAGCATATC-3' (entry vector construct RPF_07B02, SEQ ID NO: 21) 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATCCACTACCATTCGACACGTGACAAAA-3' (entry vector construct NST1_attB2_R, SEQ ID NO: 22) 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAACATATCAGTAAACGGACAGTCACA-3' (entry vector construct NST2_attB1_F, SEQ ID NO: 23) 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATCCACTACCGTTCAACAAGTGGCATGT-3' (entry vector construct NST2_attB2_R, SEQ ID NO: 24) 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGACAGAAAACATGAGTATATCTGTTAA-3' (Entry vector construction PtrNST1_attB1_F SEQ ID NO: 25) 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATGCACCTGTGTTTGACAACTGGCATAG-3' (Entry vector construction PtrNST1_attB2_R SEQ ID NO: 26) 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGACAGAAAACATGAGTATATCTGTGAA-3' (Entry vector construction PtrNST2_attB1_F SEQ ID NO: 27)5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATACACTAGTGTTTGGCAAGTGCCATTG-3' (Entry vector construct PtrNST2_attB2_R SEQ ID NO: 28) 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGCCTGACGATATGGTGAATCTATCAAT-3' (Entry vector construct PtxtVNS12_attB1_F SEQ ID NO: 29) 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATACTGACAAGTGGCATAGTGGGTCGGA-3' (Entry vector construct PtxtVNS12_attB2_R SEQ ID NO: 30) 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCTGATAATAAGGTCAATCTTTCGAT-3' (Entry vector construct attB1_AT1G32770_F SEQ ID NO: 31) 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACAGATAAATGAAGAAGTGGGTCTAAAGA-3' (Entry vector construct attB2_AT1G32770_R SEQ ID NO: 32) 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGCATATCGGTGAACGGGCAGTCGG-3' (Entry vector construct attB1_OsNST2_Rik SEQ ID NO: 33) 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACGTTATTCATGGTCGTCAAGTCTGCGT-3' (Entry vector construct attB2_OsNST2_Rik SEQ ID NO: 34)

[0055] The amplified gene fragment was then subcloned into the Gateway donor vector pDONR221 (Invitrogen) using Gateway BP clonase (Thermo Fisher) to create an entry clone. It was then transferred to the vector pDEST35SHSP (Non-Patent Document 5) for transient reporter-effector assays using Gateway LR clonase (Thermo Fisher). This resulted in the construction of an effector plasmid serving as a benchmark for activity, in which each NST transcription factor was linked in the order of the 35S promoter, wild-type transcription factor translation region, and HSP terminator sequence.

[0056] Next, amino acid substitutions were introduced into each NST transcription factor. The entry clones of the Arabidopsis, poplar, and rice NST transcription factor genes prepared above were used as templates for PCR amplification using the following inverse PCR primers designed to introduce amino acid substitutions at the target site. The following oligonucleotide pairs were used as primers for the PCR reaction. 5'-CGTATTTACAAGAAGAAGAATCTTCA-3' (amino acid substitution NST1_mF166Y_F SEQ ID NO: 35) 5'-CTTCTTGTAAATACGACACACCACCC-3' (amino acid substitution NST1_mF166Y_R SEQ ID NO: 36) 5'-TGATTGGGTCATGCATGAATATAGAC-3' (amino acid substitution NST1_mI133V_F SEQ ID NO: 37) 5'-TGCATGACCCAATCAGATTTTTGGCC-3' (amino acid substitution NST1_mI133V_R SEQ ID NO: 38) 5'-CGTGTTTACAAGAAAAATAACCTTTG-3' (amino acid substitution NST2_mF166Y_F SEQ ID NO: 39) 5'-TTTCTTGTAAACACGACACACCACCC-3' (amino acid substitution NST2_mF166Y_R SEQ ID NO: 40) 5'-CGATTGGGTCATGCACGAATATAGAC-3' (amino acid substitution NST2_mI133V_F SEQ ID NO: 41) 5'-TGCATGACCCAATCGGATTTCTGACC-3' (amino acid substitution NST2_mI133V_R SEQ ID NO: 42) 5'-CGTATCTACAAGAAGAAAAACCTCAA-3' (amino acid substitution PtrNST1_mF166Y_F SEQ ID NO: 43) 5'-CTTCTTGTAGATACGGCAAACCACCC-3' (amino acid substitution PtrNST1_mF166Y_R SEQ ID NO: 44) 5'-CGATTGGGTTATGCATGAATATAGGC-3' (amino acid substitution PtrNST1_mI133V_F SEQ ID NO: 45) 5'-TGCATAACCCAATCGGATTTTTGTCC-3' (amino acid substitution PtrNST1_mI133V_R SEQ ID NO: 46) 5'-CGTATCTACAAGAAGAAAAATCTCAA-3' (amino acid substitution PtrNST2_mF166Y_F SEQ ID NO: 47)5'-CGGGTATATAGAAAGAAGAACTATCA-3' (amino acid substitution PtxtVNS12_mF166Y_F SEQ ID NO: 48) 5'-CTTTCTATATACCCGGCAAACCACCC-3' (amino acid substitution PtxtVNS12_mF166Y_R SEQ ID NO: 49) 5'-CGATTGGGTCATGCATGAATATAGGC-3' (amino acid substitution PtxtVNS12_mI133V_F SEQ ID NO: 50) 5'-TGCATGACCCAATCGGATTTCTGACC-3' (amino acid substitution PtxtVNS12_mI133V_R SEQ ID NO: 51) 5'-CGAGTGTACAGGAAGAAGAACTATCA-3' (amino acid substitution NST3_mF175Y_F SEQ ID NO: 52) 5'-CTTCCTGTACACTCGACATACCACCC-3' (amino acid substitution NST3_mF175Y_R SEQ ID NO: 53) 5'-AGGGTGTACAAGAAGAAGAACCTCGT-3' (amino acid substitution OsNST2_mF187Y_F SEQ ID NO: 54) 5'-CTTCTTGTACACCCTGCAGATCACCC-3' (amino acid substitution OsNST2_mF187Y_R SEQ ID NO: 55)

[0057] The reaction mixture was then treated with the restriction enzyme DpnI at 37°C for 1 hour to selectively digest the template, creating a library of random mutants. 2 μl of this reaction mixture was used to transform Escherichia coli DH5α, and plasmids were extracted from these Escherichia coli using the FastGene™ Plasmid Mini Kit (Nippon Genetics Co., Ltd.). The DNA sequence was then confirmed using standard Sanger sequencing to obtain NST transcription factor entry clones containing the desired substitutions. The clones were then transferred to the vector pDEST35SHSP (Non-Patent Document 5) for transient reporter-effector assays using Gateway LR Clonase (Thermo Fisher). This resulted in the construction of effector plasmids for each NST transcription factor, each containing an amino acid substitution equivalent to I133V, F166Y, or both, linked in this order: the 35S promoter, the mutant transcription factor translation region, and the HSP terminator sequence.

[0058] (1-4) Preparation of an amino acid substitution library at the F166 site in the LkNAC1 transcription factor To introduce point mutations into the LkNAC1 gene, the plasmid prepared in Example (1-1) was used as a template to introduce substitutions at residue 166 of the LkNAC1 gene. The primers used here are shown below. 5'-CGAGTTGCTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166A-F, SEQ ID NO: 56) 5'-CGAGTTTGTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166C-F, SEQ ID NO: 57) 5'-CGAGTTGATAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166D-F, SEQ ID NO: 58) 5'-CGAGTTGAAAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166E-F, SEQ ID NO: 59) 5'-CGAGTTGGAAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166G-F, SEQ ID NO: 60) 5'-CGAGTTCATAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166H-F, SEQ ID NO: 61) 5'-CGAGTTATTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166I-F, SEQ ID NO: 62) 5'-CGAGTTAAGAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166K-F, SEQ ID NO: 63) 5'-CGAGTTCTTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166L-F, SEQ ID NO: 64) 5'-CGAGTTATGAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166M-F, SEQ ID NO: 65) 5'-CGAGTTAATAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations: LkNAC1 F166N-F, SEQ ID NO: 66)5'-CGAGTTCCTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166P-F, SEQ ID NO: 67) 5'-CGAGTTCAAAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166Q-F, SEQ ID NO: 68) 5'-CGAGTTAGAAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166R-F, SEQ ID NO: 69) 5'-CGAGTTTCTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166S-F, SEQ ID NO: 70) 5'-CGAGTTACTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166T-F, SEQ ID NO: 71) 5'-CGAGTTGTTAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166V-F, SEQ ID NO: 72) 5'-CGAGTTTGGAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, LkNAC1 F166W-F, SEQ ID NO: 73) 5'-CGAGTTTACAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, F166Y F, SEQ ID NO: 74) 5'-CGAGTTTTCAAGAAGCGGAATCATCA-3' (forward primer for introducing point mutations, Y166F F 1390, SEQ ID NO: 75) 5'-CCGTTGAGATGTGATGCAACC-3' (reverse primer for introducing point mutations, LkNAC1-3-F2, SEQ ID NO: 76)

[0059] The resulting PCR fragment was purified using Wizard® SV Gel and PCR Clean-Up System (Promega Corporation) as in Example (1-2) above, and then the DNA fragment was used to prepare an amino acid substitution-introduced plasmid using the MEGAWHOP method (Non-Patent Document 4). A 50 μl PCR reaction system contained a plasmid (50 ng) containing the wild-type LkNAC1 gene prepared in (1-1) as a template and the above PCR fragment (500 ng) as a megaprimer. After amplifying the amino acid substitution plasmid by PCR using Phusion High-Fidelity DNA Polymerase (New England BioLabs Japan), the reaction solution was treated with the restriction enzyme DpnI at 37 ° C for 1 hour to selectively digest the template plasmid. 2 μl of this reaction mixture was transformed into Escherichia coli DH5α and cultured overnight in LB medium containing ampicillin to obtain transformed Escherichia coli carrying the amino acid substitution plasmid. After further culture of this transformed Escherichia coli in liquid, the plasmid was extracted using a FastGene™ Plasmid Mini Kit (Nippon Genetics Co., Ltd.).

[0060] (1-5) Construction of a reporter for transient reporter-effector assay A reporter plasmid was constructed by inserting the promoter region of the Arabidopsis thaliana NST3 transcription factor gene or an artificial promoter region in which NST binding sequences (SNBE) were repeated and placed upstream of the core promoter upstream of the firefly luciferase gene, and by placing the terminator sequence of the Arabidopsis thaliana-derived HSP18.2 gene (HSP terminator) downstream.

[0061] The promoter region of the Arabidopsis thaliana NST3 transcription factor gene was prepared by PCR amplification of the upstream 1,002 bp region, excluding the initiation codon, using the following PCR primers and inserting it upstream of the firefly luciferase gene, with an HSP terminator located downstream: 5'-GGGGACAACTTTGTATAGAAAAGTTGTAACTATTTCCGATTTAAGATGAATAG-3' (forward primer for preparing an NST3 promoter reporter construct, NST3pF1000, SEQ ID NO: 111) and 5'-GGGGACTGCTTTTTTGTACAAACTTGGTAACGAAGATAGCAATATATTTTTGGG-3' (reverse primer for preparing an NST3 promoter reporter construct, NST3pR_Asc, SEQ ID NO: 112).

[0062] The latter reporter plasmid with SNBE repeats was constructed by inserting a double-stranded DNA fragment prepared by heating and cooling the following single-stranded oligonucleotides upstream of the firefly luciferase gene of the pTATA LUC HSP vector described in Non-Patent Document 7: 5'-CGCGCCGTATACCTTGTGAATGAAGAAACTGTATACCTTGTGAATGAAGAAACTGTATACCTTGTGAATGAAGAAACTGC-3' (forward primer for artificial promoter, AscNSTx3Not F, SEQ ID NO: 77) 5'-GGCCGCAGTTTCTTCATTCACAAGGTATACAGTTTCTTCATTCACAAGGTATACAGTTTCTTCATTCACAAGGTATACGG-3' (reverse primer for artificial promoter, AscNSTx3Not R, SEQ ID NO: 78)

[0063] Equal amounts of the two oligonucleotides (100 μM) were mixed and annealed by heating at 95° C. for 5 minutes and then cooling to 25° C. over 15 minutes. This DNA fragment was diluted 1 / 1000 and inserted upstream of the above vector, which had been cleaved with the restriction enzymes AscI and NotI, using the DNA Ligation Kit <Mighty Mix> (Takara Bio Inc.).

[0064] (1-6) Preparation of Arabidopsis thaliana mesophyll cell protoplasts Arabidopsis thaliana mesophyll cell protoplasts were prepared according to the following procedure (Non-Patent Documents 3, 6, 7). Arabidopsis rosette leaves were harvested 22 to 28 days after sowing, and the epidermis was peeled off using mending tape (3M Japan). The peeled leaves were immersed in an enzyme solution (1% (W / V) Cellulase Onozuka® R-10 (Yakult Pharmaceutical Co., Ltd.), 0.25% (W / V) Macerozyme® R-10 (Yakult Pharmaceutical Co., Ltd.), 400 mM mannitol, 20 mM potassium chloride, 10 mM calcium chloride, 20 mM MES buffer (pH 5.7), 10 mM β-mercaptoethanol) while still attached to the tape, and gently shaken at 22°C in the dark. After 1 hour, the cells were diluted with an equal volume of W5 buffer (150 mM sodium chloride, 125 mM calcium chloride, 5 mM potassium chloride, 2 mM MES buffer (pH 5.7)), passed through a 70 μm nylon mesh, and then centrifuged at 100 × g for 10 minutes to remove the supernatant. The remaining precipitate was washed three times with W5 buffer and once with MMg solution (400 mM mannitol, 15 mM magnesium chloride, 4 mM MES buffer (pH 5.7)). The final cell count was 2.0-3.0 × 10 ５ The cells were suspended in MMg solution to a concentration of 100 cells / mL, and this was used as a protoplast suspension.

[0065] (1-6) Transient Gene Transfer into Protoplasts The following procedure was performed in a 96-well round-bottom plate. 35 μl of the protoplasts isolated in Example (1-4) was added to 10 μl of a mixture containing 100 ng of the effector prepared in Example 1, 500 ng of the reporter prepared in Example (1-4), and 10 ng of a reference plasmid (phRLHSP, Non-Patent Document 8) carrying the Renilla luciferase gene (hRLUC; Promega) as a reference (internal standard). An equal volume of PEG solution (40% (w / v) PEG, 200 mM mannitol, 100 mM calcium chloride) for transformation was added, and the mixture was allowed to stand at room temperature for 10 minutes. The mixture was then washed four times with W5 buffer to replace the liquid, and then allowed to stand in the dark at 22°C for 15 hours. The process from adding the PEG solution to washing with W5 was carried out using an automated liquid handling system (TECAN freedom evo 100).

[0066] (1-7) Screening for highly active mutant LkNAC1 transcription factors by measuring luciferase activity To measure the luciferase activity of protoplasts, Picagene (registered trademark) Dual Sea Pansy Luminescence Kit (Fujifilm Wako Pure Chemical Industries, Ltd.) or Dual-Luciferase (registered trademark) Reporter Assay System (Promega Corporation) was used. First, 20 μl of a 5x concentration cell lysing agent was added to 80 μl of protoplast solution, and the mixture was stirred at 900 rpm at room temperature for 15 minutes to lyse the cells. 4 μl of the lysed solution was dispensed into a 96-well plate, and 20 μl of firefly luciferase luminescent substrate solution was added, and luminescence was measured. Subsequently, 20 μl of Renilla luciferase luminescent substrate solution was added, and luminescence was measured. A microplate reader (Infinite 200 PRO, Tecan Japan) was used for the entire series of operations, from dispensing the detection reagent to measuring luminescence. The obtained firefly luciferase measurements were corrected with the Renilla luciferase measurements to determine the activity value. From approximately 5,000 clones subjected to transient reporter-effector assay tests, I133V and F166Y mutations were identified as mutant LkNAC1 with higher reporter activity than wild-type LkNAC1. Next, to verify the effect of substituting F166Y, which had a particularly significant effect, with other amino acids, a test was conducted in which 19 other amino acids were substituted, including back-substitutions. The plasmid library prepared in Example (1-3) in which the F166 amino acid had been substituted with another amino acid was used as an effector to examine the effect on transcriptional control ability. As a result, various activities were exhibited depending on the amino acid substituted, but only F166Y was substituted to significantly improve activity (Figure 4). Furthermore, we investigated whether a substitution equivalent to F166Y was effective in enhancing function in the NST transcription factors of plants other than larch, such as Arabidopsis, rice, and poplar. As a result, we found that the F166Y substitution was effective in enhancing activation ability in the NST transcription factors of Arabidopsis and rice (Figure 3), and that the I133V substitution was also effective in enhancing activation ability in the NST transcription factors of Arabidopsis, rice, and poplar (Figure 1).

[0067] Example 2 Evaluation of the "Secondary Cell Wall Enhancement Effect" of Highly Active Mutant LkNAC1 Transcription Factor in Plants (2-1) Construction of Transformation Vector ProNST3:mLkNAC1 A transformation vector was constructed as follows: The LkNAC1(F166Y) sequence prepared in Example 1-3 above was cloned into pDONR207 using site-specific recombination with BP clonase, and then inserted into the pDEST NST3p HSP GWB5 vector described in Non-Patent Document 6 via recombination with LR clonase to obtain the pDEST NST3p:LkNAC1(F166Y):HSP GWB5 plasmid.

[0068] (2-2) Transformation of Arabidopsis thaliana nst1 nst3 double mutant with hyperactive mutant LkNAC1 transcription factor using floral dip method The plasmid obtained in Example (2-1) above was introduced into a soil bacterium (Agrobacterium tumefaciens strain GV3101(C58C1Rifr)pMP90(Gmr) (Koncz and Schell, Molecular and General Genetics, 204: 383-396, 1986)) by electroporation, and the resulting agrobacterium was plated on LB agar medium containing gentamicin, spectinomycin, and rifampicin and cultured at 28°C for 2 days to obtain transformed agrobacteria. The resulting agrobacteria were cultured in 50 ml of LB medium for 1 day. Next, the fungal cells were collected from the culture medium and suspended in 500 ml of infiltration medium. The Arabidopsis thaliana nst1 nst3 double mutant was immersed in this for 2 minutes to infect it, and then grown as usual to harvest T1 seeds. The T1 seeds were then sterilized in a hypochlorous acid solution for 7 minutes, rinsed three times with sterile water, and sown on MS selection medium containing 30 mg / L hygromycin. Transformed plants (proNST3:LkNAC1 (F166Y):HSP nst1 nst3) exhibiting antibiotic resistance were selected by growing on hygromycin plates for about 2 weeks, and then replanted in soil and grown for about 6 weeks.

[0069] (2-3) Quantification of cell wall components of Arabidopsis plants into which highly active mutant NST transcription factors were introduced proNST3 obtained in Example (2-2): LkNAC1 (F166Y): HSP nst1 nst3 Arabidopsis thaliana T1 plants were collected from the main stems that had grown to more than 25 cm about 8 weeks after sowing, and then chlorophyll was removed and defatted by changing the solution three times in methanol, acetone, methanol: chloroform solution (1:1), for 30 minutes or more, and then dried overnight at 65 ° C. to measure the dry weight. As a result, it was found that the dry weight ratio per fresh weight of proNST3: LkNAC1 (F166Y): HSP nst1 nst3 was increased by about 20% compared to the wild-type strain (Figure 5). Similarly, we found that the dry weight ratio per fresh weight of rice proNST3:OsNST2(F166Y):HSP nst1 nst3 plants, in which an amino acid substitution corresponding to F166Y was introduced into the rice NST gene, was approximately 30% higher than that of the wild-type strain (Figure 6).

[0070] (2-4) Observation of sections of inflorescence stems of Arabidopsis plants into which highly active mutant NST transcription factors were introduced In the proNST3:LkNAC1(F166Y):HSP nst1 nst3 Arabidopsis thaliana T1 plants obtained in Example (2-2) above, inflorescence stems were collected approximately 8 weeks after sowing, and transverse manual sections were prepared at a height of approximately 3 cm above ground level, and autofluorescence images were observed under UV irradiation. When the transcription activation ability of the introduced gene is high, lignin autofluorescence, i.e., lignification, is observed even in the central parenchyma cells (Non-Patent Document 3), and it has been found that the frequency of individuals with lignified parenchyma cells increases. A comparison was made between proNST3:LkNAC1:HSP nst1 nst3, an LkNAC1 expression strain without amino acid substitutions, and proNST3:LkNAC1(F166Y):HSP nst1 nst3 into which amino acid substitutions were introduced. As a result, the incidence of individuals emitting lignin autofluorescence in parenchyma cells was about 40% in proNST3:LkNAC1:HSP nst1 nst3, whereas proNST3:LkNAC1(F166Y):HSP nst1 nst3 was about 75%, and it was found that the incidence of individuals lignifying in parenchyma cells by introducing amino acid substitutions is significantly increased (Figure 7). In addition, proNST3:NST3:HSP nst1 nst3, an Arabidopsis NST expression strain without amino acid substitutions, was compared with proNST3:NST3(F166Y):HSP nst1 nst3, which introduced amino acid substitutions. As a result, the incidence of individuals emitting lignin autofluorescence in parenchyma cells was increased from about 50% to about 75% (Figure 8).

[0071] (2-5) Preparation of poplar plants into which highly active mutant NST transcription factors have been introduced The plasmid obtained in Example (2-1) above was introduced into a soil bacterium (Agrobacterium tumefaciens strain GV3101(C58C1Rifr)pMP90(Gmr) (Koncz and Schell, Molecular and General Genetics, 204: 383-396, 1986)) by electroporation, and the resulting mixture was spread on an LB agar medium containing gentamicin, spectinomycin, and rifampicin and cultured at 28°C for 2 days to obtain transformed Agrobacterium. The resulting Agrobacterium was precultured for 1 day in 2 ml of LB liquid medium containing gentamicin, spectinomycin, and rifampicin, and then 500 μL of the preculture was cultured for 1 day in 100 ml of LB liquid medium containing gentamicin, spectinomycin, and rifampicin. The resulting Agrobacterium suspension was centrifuged at 1750 × g for 15 minutes in a swing-out rotor to collect the cells, which were then resuspended to an OD value of 0.4-0.6 in MS liquid medium (Shiotani MS, prepared with adjusted mixed salts) supplemented with acetosyringone to a final concentration of 20 μM, and then shaken at 25°C and 50 rpm for 1 hour to prepare an Agrobacterium infection solution. A small piece (approximately 2 cm long; hereafter referred to as "explant") of sterilely cultured poplar stem (subcultured approximately 1 month) was immersed in the resulting infection solution for approximately 5 minutes. After thoroughly removing the infection solution with sterilized filter paper, the explant was approximately half-buried in MS1 ​​coculture medium (0.1 mg / L IBA, 0.01 mg / L thidiazuron) and cocultured at 25°C for 2 days in the dark. After coculture, the explant was washed twice with MS liquid medium containing 10 mg / L claforan and then cultured in MS1 ​​medium (containing 0.1 mg / L IBA, 0.01 mg / L thidiazuron, 500 mg / L claforan, and 30 mg / L hygromycin) so that the explant was approximately half-buried. After changing to fresh MS1 medium every two weeks, when the selected multiple shoots began to grow, they were subcultured onto MS1 medium with a reduced amount of thidiazuron of 0.001 mg / L to promote their growth, and then transplanted onto MS2 medium (containing 0.1 mg / L IBA and 30 mg / L hygromycin) to induce shoot formation and growth.The shoots were then excised and rooted on 1 / 2 MS medium, after which the presence or absence of the introduced gene was confirmed to produce transformed poplars. The resulting poplars were maintained by shoot apex culture, and those approximately three weeks after transplantation were potted and grown for approximately two months under a 16-hour photoperiod (60-80 μmol m ). －２ ・s －１ ) / 8-hour dark period, and 22°C until the stem length reached approximately 80 cm and the number of nodes reached approximately 40, and the amount of cell wall components in the stems was measured using the method in Example (2-3). As a result, the amount of cell wall components per fresh weight of the proNST3:LkNAC1(F166Y):HSP recombinant poplar was increased by approximately 20% compared to the proNST3:LkNAC1:HSP recombinant poplar without the amino acid substitution, and the amount of cell wall components in the stem per individual was also increased by approximately 30% (Figure 9).

[0072] (2-6) Construction of larch transformation vector ProLkNST1:LkNAC1(F166Y) A transformation vector was constructed as follows. The larch NST promoter (LkNAC1p) was amplified by PCR using genomic DNA extracted from larch leaves as a template. The Arabidopsis thaliana NST3 promoter (NST3p) was removed from the pDEST NST3p:LkNAC1(F166Y):HSP GWB5 plasmid of Example 2-1 by inverse PCR to linearize the plasmid, and LkNST1p was inserted into this plasmid using an in-fusion enzyme to obtain the pDEST LkNAC1p:LkNAC1(F166Y):HSP GWB5 plasmid.

[0073] (2-7) Transformation of larch with a highly active mutant LkNAC1 transcription factor gene using the Agrobacterium method The plasmid obtained in Example (2-6) above was introduced into the soil bacterium Agrobacterium (Agrobacterium tumefaciens strain EHA105 (C58C1Rifr) pEHA105) by electroporation, and the resulting mixture was spread on an LB agar medium containing spectinomycin and rifampicin and cultured at 28°C for 2 days to obtain transformed Agrobacterium. The resulting Agrobacterium was cultured in 50 ml of LB medium for 1 day, after which the cells were harvested from the culture and suspended in 20 ml of infiltration medium. Larch somatic embryogenic cells (PEMs) were immersed in the suspension for 20 minutes to infect them. The PEMs were then harvested on filter paper and co-cultured with Agrobacterium in a sealed Petri dish for 2 days. The PEMs were washed with infiltration medium, harvested again on filter paper, and then cultured in modified Campbell and Durzan medium (CD) containing 10 mg / L meropenem and 20 mg / L hygromycin. While eradicating the Agrobacterium, PEMs that grew with antibiotic resistance were selected. Next, the selected PEMs were cultured in LM medium (lobollly pine medium (Litvay et al. Plant Cell Rep. 4: 325-328, 1985)) for approximately 40 days to induce somatic embryos. The somatic embryos were placed on modified CD medium lacking plant hormones and allowed to germinate, yielding transformed larch plantlets. After approximately one month, the plantlets were transplanted into soil and grown for a further six months. Transformed larch seedlings 20-30 cm tall were used to evaluate secondary cell walls.

[0074] (2-8) Observation of Stem Sections of Larch Plants Introduced with a Highly Active Mutant LkNAC1 Transcription Factor Gene. A section 20 cm below the apex of the stem of the LkNAC1p:LkNAC1(F166Y):HSP larch plant obtained in Example (2-7) above was collected, and a 50-micrometer-thick cross-section of the stem was prepared using a microtome. The cross-section was stained with safranin, and fluorescence images (observed at wavelengths of 575 nm or longer) were observed under excitation light of 530-550 nm. Cell walls of tracheids in the secondary xylem were randomly selected, and the cell wall thickness was measured at 50 locations. When the LkNAC1 expression strain LkNAC1p:LkNAC1:HSP, which does not have an amino acid substitution, was compared with the LkNAC1p:LkNAC1(F166Y):HSP strain, which has an amino acid substitution, the average cell wall thickness was 3.3 micrometers in the former strain, while it was 4.2 micrometers in the latter strain, approximately 1.3 times thicker (Figure 10). This indicates that the LkNAC1 transcription factor, highly activated by the amino acid substitution (F166Y), enhances the secondary cell wall of larch.

[0075] (2-9) Strength test of poplar plant stems into which highly active mutant LkNAC1 transcription factor was introduced The bending modulus was measured as an indicator of strength for the proNST3:LkNAC1(F166Y):HSP recombinant poplar obtained in Example (2-5) above, and the proNST3:LkNAC1:HSP recombinant poplar without amino acid substitutions. Samples were obtained in Example (2-5) above. For the recombinant poplar with a stem length of approximately 80 cm and a node count of approximately 40, the 14th-16th, 19th-21st, and 24th-26th internodes counted from the shoot apex were excised, and the tissue was fixed by immersion in FAA solution [45% (w / w) ethanol, 2.5% (w / w) acetic acid, approximately 1% (w / w) formaldehyde]. The tissue was then stored at room temperature until measurement. After storage, samples were washed three times with approximately 10 mL of ultrapure water before measurement, and then the flexural modulus was measured using a strength tester described below. Measurements were performed using a plant stem tensile / bending tester (AC-500N-CM, manufactured by TSE Corporation) using a three-point bending test. Measurement conditions were a support distance of 20 mm, a loading rate of 10 mm / min, and a loading distance of 20 mm. The flexural modulus (MPa) was measured by measuring the sample diameter before measurement and calculating the loading pressure relative to the diameter. As a result, the flexural modulus of the proNST3:LkNAC1:HSP recombinant poplar with the amino acid substitution introduced was higher at internodes 14-16 (6.3%), internodes 19-21 (13.5%), and internodes 24-26 (9.1%) than that of the proNST3:LkNAC1:HSP recombinant poplar without the amino acid substitution introduced (Figure 11).

[0076] (2-10) Preparation of rice plants into which highly active mutant rice NST2 transcription factor has been introduced (a) Construction of cloning vector In order to construct a cloning vector into which a target gene is introduced, an entry vector was prepared in which the terminator portion (NOS terminator, SEQ ID NO: 79) of the pNOS Entry vector described in Non-Patent Document 3 was replaced with an Arabidopsis-derived HSP terminator (SEQ ID NO: 80). The HSP terminator was amplified by PCR using primers of SEQ ID NOs: 81 and 82, and p35SHSPG described in Non-Patent Document 9 as a template. The resulting PCR product was cleaved with SalI and EcoRI at 37 ° C. for 3 hours, then electrophoresed on a 0.9% agarose gel and purified using a gel extraction kit (FastGene™ Gel / PCR Extraction Kit, manufactured by Nippon Genetics Co., Ltd.) according to the kit's protocol. The pNOS Entry plasmid was then subjected to the same restriction enzyme and purification procedures, followed by ligation according to standard methods. The resulting reaction product was then introduced into E. coli, and the plasmid containing the target sequence was confirmed by standard sequencing to obtain the target cloning vector (hereinafter referred to as pHSP entry).

[0077] SEQ ID NO: 79 (NOS terminator) GTCGACGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTC SEQ ID NO: 80 (HSP terminator) GTCGACGAGCTCATATGAAGATGAAGATGAAATATTTGGTGTGTCAAATAAAAAGCTTGTGTGCTTAAGTTTGTGTTTTTTTCTTGGCTTGTTGTGTTATGAATTTGTGGCTTTTTCTAATATTAAATGAATGTAAGATCTCATTATAATGAATAAACAAATGTTTCTATAATCCATTGTGAATGTTTTGTTGGATCTCTTCTGCAGCATATAACTACTGTATGTGCTATGGTATGGACTATGGAATATGATTAAAGATAAGGAATTC SEQ ID NO: 81 (Sal-HSP-s) GGTACCGTCGACGAGCTCATATGAAGATGA SEQ ID NO: 82 (EcoRI-HSP-as) CGCGAATTCCTTATCTTTAATCATATTCCATAGTC

[0078] (b) Construction of rice transformation vectors Construction of rice transformation vectors was carried out as follows. First, DNA fragments of the promoter region (SEQ ID NO: 83) and gene coding region (SEQ ID NO: 84) of the rice NST transcription factor gene (OsNST2) were obtained. For the promoter region, genomic DNA extracted from leaves of rice (Nipponbare) 2 weeks after sowing was used as a template, and the OsNST2 promoter region (SEQ ID NO: 83) 3631 bp was amplified by PCR using primers with SEQ ID NOs: 85 and 86. The obtained DNA was electrophoresed on a 0.9% agarose gel, and a band around 3 kbp was excised and extracted and purified using a gel extraction kit (FastGene™ Gel / PCR Extraction Kit, manufactured by Nippon Genetics Co., Ltd.).

[0079] The gene coding region (SEQ ID NO: 84) was amplified by PCR using an entry clone containing rice OsNST2 or a mutant thereof prepared by the method described in (1-3) above as a template and primers of SEQ ID NOs: 87 and 88. The resulting DNA was electrophoresed on a 0.9% agarose gel, as described above, and then extracted and purified using a gel extraction kit (FastGene™ Gel / PCR Extraction Kit, manufactured by Nippon Genetics Co., Ltd.). The resulting promoter region, gene coding region, and the cloning vector prepared in (2-10)(a) were treated with the restriction enzymes AscI and SmaI and then assembled using In-Fusion® Snap Assembly Master Mix (manufactured by Takara Bio Inc.). This resulted in the pOsNST2:OsNST2:HSP intermediate clone and the pOsNST2:OsNST2(F166Y equivalent):HSP intermediate clone. Subsequently, the resulting intermediate clones were introduced into the vector pBCSH described in Non-Patent Document 7 by the Gateway LR reaction, which is a sequence-dependent recombination reaction, to obtain the pOsNST2:OsNST2:HSP rice transformation vector and the pOsNST2:OsNST2(F166Y equivalent):HSP rice transformation vector.

[0080]

[0081] (c) Preparation of Transgenic Rice Plants Transformation of rice callus was performed according to the method described in Non-Patent Document 10. The rice transformation vector prepared in (2-10)(b) above was introduced into the soil bacterium (Agrobacterium tumefaciens) strain EHA105 (Non-Patent Document 11) by electroporation. The cells were then collected and suspended in 30 mL of infection medium (AAM containing acetosyringone). Embryogenic callus induced from rice seeds for 3-4 weeks was immersed in this solution for 1.5 minutes and then co-cultured for 48-60 hours on co-culture medium (2N6-AS medium). After washing, the callus was cultured for 3-4 weeks on selective medium (N6D-S medium) containing hygromycin. Calli successfully transformed with rice transformation vectors acquire hygromycin resistance. Transformed calli that grow on the hygromycin medium were selected and cultured on a regeneration medium containing hygromycin (MS-NK medium) for 3 to 4 weeks, followed by culturing on a plant growth medium containing hygromycin (MS-HF medium) for 3 to 4 weeks to regenerate transformed plants. The resulting transformed plants were transplanted into soil and incubated in 100 to 200 μmol m －２ ・s －１ The transgenic rice plants were grown under a 14-hour light / 10-hour dark cycle at 27°C. The transgenic plants obtained by introducing the pOsNST2:OsNST2:HSP rice transformation vector are referred to as OsNST2 recombinant rice plants, and those obtained by introducing the pOsNST2:OsNST2(equivalent to F166Y):HSP rice transformation vector are referred to as mOsNST2(F166Y) recombinant rice plants. Transgenic rice seeds were obtained by continuously cultivating the transgenic rice under the above conditions. The transgenic rice seeds obtained by this process are referred to as T1 generation recombinant seeds. Transgenic rice was selected from the T1 generation recombinant seeds on a medium containing 50 mg / L hygromycin, ½ MS, 2% (w / v) sucrose, and 0.8% agar. The transgenic rice plants obtained by this process are referred to as T1 generation recombinant rice plants. The T1 generation recombinant rice plants were transplanted into soil and exposed to 100 to 200 μmol m －２ ・s －１) The plants were grown under a 14-hour light / 10-hour dark cycle at 27°C for approximately two months to obtain OsNST2 T1 generation recombinant rice plants or mOsNST2(F166Y) T1 generation recombinant rice plants.

[0082] (d) Measurement of tensile strength of recombinant rice. The flag leaf of each T1 generation recombinant rice plant obtained in (c) above was sampled, and only the midrib was excised and used for tensile strength testing. Measurements were performed using a plant stem tensile / bending tester (AC-500N-CM, manufactured by TSE Corporation) with a 100 N load cell and a tensile test fixture. The measurement conditions were a sample distance of 10 mm, a loading rate of 1 mm / min, and a loading distance of 1 mm. The sample diameter was measured before measurement, and the tensile modulus (MPa) was calculated by calculating the loading pressure relative to the diameter. The tensile modulus of the OsNST2 recombinant rice without amino acid substitutions was 65.0 ± 1.8 MPa, whereas the bending modulus of the mOsNST2(F166Y) recombinant rice with amino acid substitutions was significantly increased to 99.2 ± 3.7 MPa (Figure 12).

[0083] (2-11) Verification of the wood-enhancing effect of highly active mutant LkNAC1 transcription factors other than F166Y (a) Preparation of new substitution clones To introduce new point mutations into the LkNAC1 gene, targeted amino acid substitutions were introduced by PCR using the wild-type LkNAC1 effector plasmid prepared in Example (1-1) as a template and primers for substitution introduction. The primers used here are shown below. 5'-ATTATGAAAGAATATCGCCTGGATGAC-3' (forward primer for point mutation introduction H135K-F, SEQ ID NO: 89) 5'-ATATTCTTTCATAATCCAATCTGTTTTCTG-3' (reverse primer for point mutation introduction H135K-R, SEQ ID NO: 90) 5'-CATGAACTCCGCCTGGATGACGCTGAA-3' (forward primer for point mutation introduction Y137L-F, SEQ ID NO: 91) 5'-CAGGCGGAGTTCATGCATAATCCAATC-3' (reverse primer for point mutation introduction Y137L-R, SEQ ID NO: 92) 5'-AGCCACCAAGACAAGAAGTACCCCACC-3' (forward primer for point mutation introduction K76Q-F, SEQ ID NO: 93) 5'-CTTGTCTTGGTGGCTAAAGAAATACCA-3' (reverse primer for point mutation introduction K76Q-R, SEQ ID NO: 94) 5'-AGCCACCATGACAAGAAGTACCCCACC-3' (forward primer for point mutation introduction K76H-F, SEQ ID NO: 95) 5'-CTTGTCATGGTGGCTAAAGAAATACCA-3' (reverse primer for point mutation introduction K76H-R, SEQ ID NO: 96) 5'-GGAATGAAAAAGACTCTCGTGTTCTAC-3' (forward primer for point mutation introduction R114K-F, SEQ ID NO: 97) 5'-AGTCTTTTTCATTCCTATTTTCTTGAA-3' (reverse primer for point mutation introduction R114K-R, SEQ ID NO: 98) 5'-AGCCACCTTGACAAGAAGTACCCCACC-3' (forward primer for point mutation introduction K76L-F, SEQ ID NO: 99) 5'-CTTGTCAAGGTGGCTAAAGAAATACCA-3' (reverse primer for point mutation introduction K76L-R, SEQ ID NO: 100)5'-CGAAAGGTGCTCGTGTTCTACAAAGGC-3' (forward primer for introducing point mutation T116V-F, SEQ ID NO: 101) 5'-CACGAGCACCTTTCGCATTCCTATTTT-3' (reverse primer for introducing point mutation T116V-R, SEQ ID NO: 102) 5'-CGGTTCAAACCAACTGAGGAGGAGCTC-3' (forward primer for introducing point mutation H18K-F, SEQ ID NO: 103) 5'-AGTTGGTTTGAACCGAAACCCCGGCGG-3' (reverse primer for introducing point mutation H18K-R, SEQ ID NO: 104) 5'-GGGACTGCGACTAATAGGGCAACAGCC-3' (forward primer for introducing point mutation R85A-F, SEQ ID NO: 105) 5'-ATTAGTCGCAGTCCCGGTGGGGTACTT-3' (reverse primer for introducing point mutation R85A-R, SEQ ID NO: 106) 5'-AAGGACTATAAGTACCCCACCGGGACT-3' (forward primer for point mutation introduction K78Y-F, SEQ ID NO: 107) 5'-GTACTTATAGTCCTTGTGGCTAAAGAA-3' (reverse primer for point mutation introduction K78Y-R, SEQ ID NO: 108) 5'-CAGCACTATTGGTATTTCTTTAGCCAC-3' (forward primer for point mutation introduction D69Y-F, SEQ ID NO: 109) 5'-ATACCAATAGTGCTGTGGCGTGGATCC-3' (reverse primer for point mutation introduction D69Y-R, SEQ ID NO: 110)

[0084] For the PCR reaction, the effector plasmid (1 ng) containing the wild-type LkNAC1 gene prepared in (1-1) was used as a template, and the amino acid substitution plasmid was amplified by PCR using PrimeSTAR GXL (Takara Bio Inc.). The reaction solution was then treated with the restriction enzyme DpnI at 37°C for 1 hour to selectively digest the template plasmid. 2 μl of this reaction solution was transformed into Escherichia coli DH5α and cultured overnight in LB medium containing ampicillin to obtain transformed Escherichia coli containing the amino acid substitution plasmid. This transformed Escherichia coli was further cultured in liquid culture, and the plasmid was extracted using a FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.). The sequence of the extracted plasmid was confirmed by conventional sequencing, and an effector plasmid containing the LkNAC1 gene into which the desired amino acid substitution had been introduced was obtained. Hereinafter, these are referred to as amino acid substitution-introduced LkNAC1 effector plasmids. The amino acid substitution-introduced LkNAC1 effector plasmid was used in protoplasts prepared according to Example (1-5), and the transcriptional activation ability was measured by transient reporter-effector assay using the method described in Examples (1-6) and (1-7). As a result, it was found that these had significantly higher activity than the effector plasmid containing the wild-type LkNAC1 gene, so the wood enhancement effect in plants was verified. First, a construct for producing recombinant plants was prepared. The above-mentioned amino acid substitution-introduced LkNAC1 effector plasmid was subcloned into the pDONR221 vector (Thermo Fisher) by Gateway BP reaction to prepare an entry clone. Subsequently, by Gateway LR reaction, as in Example (2-1), it was inserted into the pDEST NST3p HSP GWB5 vector described in Non-Patent Document 5, to obtain the amino acid substitution-type pDEST NST3p:mLkNAC1:HSP GWB5 vector. The obtained amino acid substituted pDEST NST3p:mLkNAC1:HSP GWB5 vector was introduced into the Arabidopsis thaliana nst1 nst3 double mutant by the method described in Example (2-2) above, to obtain an amino acid substituted proNST3:mLkNAC1:HSP nst1 nst3 transformed plant.The recombinant Arabidopsis plants into which each amino acid substitution was introduced are collectively referred to as "amino acid substituted proNST3:mLkNAC1:HSP nst1 nst3 transformants," and when referring to each amino acid substituted transformant, the amino acid substitution will be indicated in parentheses after "mLkNAC1." The resulting amino acid substituted proNST3:mLkNAC1:HSP nst1 nst3 transformed plants were subjected to quantification of cell wall components (AIR / FW [%]) using the method described in Example (2-3) above. When the amount of cell wall components was compared with that of wild-type proNST3:mLkNAC1:HSP nst1 nst3 transformed plants for each experimental lot, in Experiment 1, the amount of cell wall components in the amino acid substituted proNST3:mLkNAC1(H135K):HSP nst1 nst3 transformants was increased by 16%. In Experiment 2, the amino acid substituted proNST3:mLkNAC1(Y137L):HSP nst1 nst3 transformant and the amino acid substituted proNST3:mLkNAC1(Y137L):HSP nst1 nst3 transformant increased by 22% and 17%, respectively. In Experiment 3, the amino acid substituted proNST3:mLkNAC1(K76H):HSP nst1 nst3 transformant, amino acid substituted proNST3:mLkNAC1(R144K):HSP nst1 nst3 transformant, amino acid substituted proNST3:mLkNAC1(K76L):HSP nst1 nst3 transformant, and amino acid substituted proNST3:mLkNAC1(T116V):HSP nst1 nst3 transformant increased by 39%, 34%, 30%, and 29%, respectively. In Experiment 4, the amino acid-substituted proNST3:mLkNAC1(H18K):HSP nst1 nst3 transformant, the amino acid-substituted proNST3:mLkNAC1(R85A):HSP nst1 nst3 transformant, and the amino acid-substituted proNST3:mLkNAC1(K78Y):HSP nst1 nst3 transformant increased by 27%, 26%, and 16%, respectively. In Experiment 5, the amino acid-substituted proNST3:mLkNAC1(D69Y):HSP nst1 nst3 transformant increased by 41% ( FIG. 14 ).

Claims

1. A method for producing a plant with enhanced secondary cell walls, comprising the step of enhancing the function of at least one of the following transcription factors in a cell that forms secondary cell walls: (a) a transcription factor consisting of at least one of the amino acid sequences shown in SEQ ID NOs: 2 to 9; (b) a transcription factor in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 and which has the activity of inducing secondary cell wall production; and (c) a transcription factor comprising at least one of the amino acid sequences shown in SEQ ID NOs: 10 to 15 and which has the activity of inducing secondary cell wall production.

2. The method according to claim 1, wherein the step of enhancing the function of the transcription factor is a step of introducing one or several base substitutions into a nucleic acid encoding the transcription factor.

3. The method according to claim 2, wherein the base substitution is a base substitution that replaces, with tyrosine, at least one of the amino acids shown in: (a) a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production; and (c) a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10, which is conserved in a transcription factor comprising the amino acid sequences shown in SEQ ID NOs: 10 and 11 and which has the activity of inducing secondary cell wall production.

4. The method according to claim 2, wherein the base substitution is a base substitution that replaces at least one of the amino acids shown in: (a) an isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production; and (c) an isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11 conserved in a transcription factor comprising the amino acid sequences shown in SEQ ID NOs: 10 and 11 and which has the activity of inducing secondary cell wall production, with valine.

5. The method according to claim 2, wherein the base substitution is a base substitution that substitutes, with lysine, at least one of the amino acids shown in: (a) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 12 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 12 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

6. The method according to claim 2, wherein the base substitution is a base substitution that replaces, with leucine, at least one of the amino acids shown in: (a) a tyrosine corresponding to the tyrosine in the motif sequence shown in SEQ ID NO: 12 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a tyrosine corresponding to the tyrosine in the motif sequence shown in SEQ ID NO: 12 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

7. The method according to claim 2, wherein the base substitution is a base substitution that substitutes, with glutamine, histidine or leucine, at least one of the amino acids shown in: (a) a lysine corresponding to the first lysine in the motif sequence shown in SEQ ID NO: 13 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a lysine corresponding to the first lysine in the motif sequence shown in SEQ ID NO: 13 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

8. The method according to claim 2, wherein the base substitution is a base substitution that substitutes, with lysine, at least one of the amino acids shown in: (a) an arginine corresponding to the arginine in the motif sequence shown in SEQ ID NO: 14 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) an arginine corresponding to the arginine in the motif sequence shown in SEQ ID NO: 14 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

9. The method according to claim 2, wherein the base substitution is a base substitution that replaces, with valine, at least one of the amino acids shown in: (a) a threonine corresponding to the threonine in the motif sequence shown in SEQ ID NO: 14 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a threonine corresponding to the threonine in the motif sequence shown in SEQ ID NO: 14 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

10. The method according to claim 2, wherein the base substitution is a base substitution that replaces at least one of the amino acids shown in: (a) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 15 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a histidine corresponding to the histidine in the motif sequence shown in SEQ ID NO: 15 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production, with lysine.

11. The method according to claim 2, wherein the base substitution is a base substitution that replaces, with alanine, at least one of the amino acids shown in: (a) an arginine corresponding to the first arginine in the motif sequence shown in SEQ ID NO: 13 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) an arginine corresponding to the first arginine in the motif sequence shown in SEQ ID NO: 13 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

12. The method according to claim 2, wherein the base substitution is a base substitution that substitutes, with tyrosine, at least one of the amino acids shown in: (a) a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 13 conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 13 conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

13. The method according to claim 2, wherein the base substitution is a base substitution that replaces, with tyrosine, at least one of the amino acids shown in: (a) an aspartic acid corresponding to the aspartic acid appearing at the beginning of the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor consisting of an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; and (b) an aspartic acid corresponding to the aspartic acid appearing at the beginning of the motif sequence shown in SEQ ID NO: 13, which is conserved in a transcription factor having an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added and which has the activity of inducing secondary cell wall production.

14. A method for producing the plant according to claim 1, comprising the step of transforming a plant cell with a nucleic acid into which at least one of the base substitutions according to claims 3 to 13 has been introduced.

15. A method for producing the plant according to claim 1, comprising the step of transforming a plant cell with a nucleic acid into which at least one of the base substitutions according to claims 3 to 13 has been introduced, linked to a promoter capable of expressing the nucleic acid in at least one type of cell that forms a secondary cell wall.

16. A method for producing the plant according to claim 1, comprising a step of introducing at least one of the base substitutions according to claims 3 to 13 into a gene present in the genome of the plant by genome editing, radiation, or chemical agents.

17. A plant or its progeny, or a part thereof, produced by the production method according to claims 14 to 16.

18. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following amino acid sequences is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NOs: 10 and 11, a phenylalanine corresponding to the phenylalanine in the motif sequence shown in SEQ ID NO: 10 conserved in the amino acid sequence is replaced with tyrosine.

19. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NOs: 10 and 11, in which the isoleucine corresponding to the isoleucine in the motif sequence shown in SEQ ID NO: 11 conserved in the amino acid sequence is replaced with valine.

20. A transcription factor having an activity of inducing secondary cell wall production in at least one of the following: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 12 in which a histidine corresponding to a histidine in the motif sequence shown in SEQ ID NO: 12 conserved in said amino acid sequence has been replaced with a lysine.

21. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 12 in which a tyrosine corresponding to a tyrosine in the motif sequence shown in SEQ ID NO: 12 conserved in said amino acid sequence is replaced with a leucine.

22. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 13, in which the lysine corresponding to the first lysine in the motif sequence shown in SEQ ID NO: 13 conserved in the amino acid sequence is replaced with glutamine, histidine or leucine.

23. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 14 in which an arginine corresponding to an arginine in the motif sequence shown in SEQ ID NO: 14 conserved in the amino acid sequence is replaced with a lysine.

24. A transcription factor having an activity of inducing secondary cell wall production in at least one of the following: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 14 in which a threonine corresponding to a threonine in the motif sequence shown in SEQ ID NO: 14 conserved in the amino acid sequence has been replaced with a valine.

25. A transcription factor having an activity of inducing secondary cell wall production in at least one of the following: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 15 in which a histidine corresponding to a histidine in the motif sequence shown in SEQ ID NO: 15 conserved in the amino acid sequence has been replaced with a lysine.

26. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 13 in which the arginine corresponding to the first arginine in the motif sequence shown in SEQ ID NO: 13 conserved in the amino acid sequence is replaced with alanine.

27. A transcription factor having an activity of inducing secondary cell wall production in at least one of the following: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 13 in which a lysine corresponding to the second lysine in the motif sequence shown in SEQ ID NO: 13 conserved in the amino acid sequence has been replaced with tyrosine.

28. A transcription factor having an activity of inducing secondary cell wall production, in which at least one of the following is present: (a) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9; (b) an amino acid sequence shown in at least one of SEQ ID NOs: 2 to 9 in which one or more amino acids have been deleted, substituted or added; and (c) an amino acid sequence shown in SEQ ID NO: 12 in which an aspartic acid corresponding to an aspartic acid in the motif sequence shown in SEQ ID NO: 12 conserved in the amino acid sequence is replaced with tyrosine.

29. Claim 18 or claim 19 or claim 20 or claim 21 relates to a nucleic acid encoding a transcription factor according to claim 22 or claim 23 or claim 24 or claim 25 or claim 26 or claim 27 or claim 28.