Method for controlling flowering time of plant and method for producing plant with controlled flowering time

Abstract

To provide a method for controlling flowering time of a plant, and a method for producing a plant with controlled flowering time.SOLUTION: A method for controlling flowering time of a plant, the method including producing a modified plant in which a ppGpp content of the plant is regulated.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for controlling the flowering time of a plant and a method for producing a plant with a controlled flowering time. [Background technology]

[0002] In crop production, the flowering time of plants is strongly related to growth and yield, and is also a major factor in determining the harvest time. To stabilize production and optimize harvest times, there is a need to establish technology to control the flowering time.

[0003] To date, techniques for controlling flowering time have been known, such as controlling day length by light irradiation or shading (Non-Patent Document 1) or temperature control (Non-Patent Document 2).In addition, a technique for controlling flowering time by modifying the function of genes involved in flower bud formation using genetic engineering techniques is also known (Non-Patent Document 3).

[0004] In plants where the function of genes controlling flower bud formation has been modified, even if optimal day length and temperature conditions are controlled, flower bud formation can be inhibited or significantly delayed, resulting in no seed formation and requiring a long period of time for seed collection. As a result, the efficiency of genotype fixation and seed multiplication is significantly reduced, leading to problems such as delays in the development of new varieties of the resulting plants. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Guide to selecting and installing light sources for chrysanthemum cultivation, March 2014, Ministry of Agriculture, Forestry and Fisheries-commissioned project "Realizing Innovative Low Costs for Domestic Agricultural Products" compiled by the Light Flower Consortium (https: / / www.naro.go.jp / publicity_report / publication / files / light_source_guidance_201403.pdf) [Non-patent document 2] Vernalization treatment for early spring harvest of turnip "Haru no Kagayaki," Tohoku National Agricultural Research Center, FY1996 research results information (https: / / www.naro.affrc.go.jp / org / tarc / seika / jyouhou / H08 / tnaes96085.html) [Non-patent document 3] Overexpression of Arabidopsis FT gene in apple leads to perpetual flowering. Plant Biotechnology 31, 11-20 (2014), DOI: 10.5511 / plantbiotechnology.13.0912a Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for controlling the flowering time of a plant and a method for producing a plant with a controlled flowering time. [Means for solving the problem]

[0007] One aspect of the present disclosure is a method for regulating the flowering time of a plant, the method comprising producing a modified plant in which the ppGpp content of the plant is regulated. [Effects of the Invention]

[0008] According to the present disclosure, a method for controlling the flowering time of a plant and a method for producing a plant with a controlled flowering time can be provided. [Brief explanation of the drawings]

[0009] [Figure 1]The number of days after sowing at which bolting was observed in wild-type (WT), RSH3-overexpressing plants (RSH3ox2), and the RSH quadruple mutant (quadruple) is shown. Bolting time was investigated in six wild-type plants, four RSH3-overexpressing plants, and eight RSH quadruple mutant plants, and the number of days after sowing at which bolting was observed is shown as a box plot. In the figure, circles indicate outliers and crosses indicate the average. DETAILED DESCRIPTION OF THE INVENTION

[0010] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0011] <Method for controlling the flowering time of plants>

[0012] In one aspect, a method for controlling the flowering time of a plant is provided, the method comprising generating a modified plant having a regulated ppGpp content.

[0013] In an embodiment, the plant is not particularly limited and may include, for example, plants of the Malvaceae, Rubiaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Moraceae, Pedaliaceae, Araceae, Umbelliferae, Solanaceae, Papayaceae, Rosaceae, Amaryllidaceae, Leguminosae, Rutaceae, Oleaceae, Amaranthaceae, and Liliaceae families, but is not limited thereto. Brassicaceae, Asteraceae, Apiaceae, Solanaceae, Rosaceae, Amaranthaceae, and Leguminaceae are particularly preferred. More specific examples of plants according to the embodiment include kiwifruit (Actinidia deliciosa), onion (Allium cepa), leek (Allium fistulosum), garlic (Allium sativum), celery (Apium graveolens var. dulce), Arabidopsis (Arabidopsis thaliana), peanut (Arachis hypogaea), beet (Beta vulgaris subsp. Vulgaris), cauliflower (Brassica oleracea var. botrytis), cabbage (Brassica oleracea var. capitata), broccoli (Brassica oleracea var. italica), rapeseed (Brassica rapa), bok choy (Brassica rapa var. chinensis), mizuna (Brassica rapa var. nipposinica), and komatsuna (Brassica rapa var. perviridis), turnip (Brassica rapa var.rapa), tea plant (Camellia sinensis), bell pepper (Capsicum annuum), paprika (Capsicum annuum), safflower (Carthamus tinctorius), lime (Citrus aurantifolia), lemon (Citrus limon), orange (Citrus sinensis), grapefruit (Citrus x paradisi), coconut palm (Cocos nucifera), coffee plant (Coffea arabica), taro (Colocasia esculenta), mitsuba (Cryptotaenia japonica), cucumber (Cucumis sativus), pumpkin (Cucurbita maxima), persimmon (Diospyros kaki), oil palm (Elaeis spp.), buckwheat (Fagopyrum esculentum), fig (Ficus carica), strawberry (Fragaria × ananassa), garland chrysanthemum (Glebionis coronaria), soybean (Glycine max), sunflower (Helianthus annuus), barley (Hordeum vulgare), sweet potato (Ipomoea batatas), morning glory (Ipomoea nil), lettuce (Lactuca sativa), lentil (Lens culinaris), apple (Malus domestica), peppermint (Mentha x piperita), banana (Musa spp.), watercress (Nasturtium officinale), tobacco (Nicotiana tabacum), basil (Ocimum basilicum), olive (Olea europaea), rice (Oryza sativa), shiso (Perilla frutescens var.crispa), avocado (Persea americana), parsley (Petroselinum crispum), kidney beans (Phaseolus vulgaris), pepper (Piper nigrum), peas (Pisum sativum), pears (Pyrus pyrifolia), rosemary (Rosmarinus officinalis), sugarcane (Saccharum officinarum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), sorghum (Sorghum bicolor), cocoa (Theobroma cacao), thyme (Thymus vulgaris), wheat (Triticum aestivum), blueberries (Vaccinium spp.), grapes (Vitis spp.), adzuki beans (Vigna angularis), corn (Zea mays), ginger (Zingiber officinale) Examples of suitable ginseng include, but are not limited to, Arabidopsis thaliana, Zingiber officinale, and related species. Of these, Arabidopsis thaliana is preferred.

[0014] In the present disclosure, flowering time refers to the time when flowering occurs in an individual plant. As will be understood by those skilled in the art, in some plant species, flowering occurs after bolting, and therefore, a change in bolting time can be used as an indicator of a change in flowering time. Here, bolting can refer to the phenomenon in which a flower stalk is formed after the initiation of flower bud differentiation. For example, in some plant species, including Arabidopsis thaliana, an earlier bolting time leads to an earlier flowering time, and a later bolting time leads to a later flowering time.

[0015] In embodiments, controlling flowering time can be to advance (accelerate) or delay (delay) the flowering time of a plant compared to a plant not subjected to such control. In embodiments, reducing ppGpp can advance flowering time, while increasing ppGpp can delay flowering time. The advancement or delay in flowering time brought about by a decrease or increase in ppGpp can be an advancement or delay of 0.5, 1, 2, 3, 4, 5, or more days. Furthermore, advancement and delay in flowering time can be accompanied by an advancement or delay in bolting time, respectively, and the advancement or delay in bolting time can be an advancement or delay of 0.5, 1, 2, 3, 4, 5, or more days.

[0016] In an embodiment, guanosine 3',5'-bisdiphosphate refers to a compound also known as ppGpp. In the present disclosure, guanosine 4phosphate may be simply referred to as "ppGpp." The structure of ppGpp is shown below. [ka]

[0017] The compound in which another phosphate is attached to the diphosphate at the 5th position of the ribose ring of ppGpp is guanosine pentaphosphate, which is abbreviated as pppGpp.

[0018] Those skilled in the art will understand that ppGpp in the embodiments may spontaneously exist in an ionic or salt form depending on the equilibrium state in a particular aqueous environment. Therefore, ppGpp in the present disclosure should be understood to encompass both its ionic and salt forms. Salts of ppGpp may be, for example, sodium salts, potassium salts, lithium salts, ammonium salts, or other monovalent cation salts, or magnesium salts, calcium salts, strontium salts, or other divalent cation salts. Salts of ppGpp may also be, for example, fluoride ions, chloride ions, bromide ions, or other monovalent anion salts.

[0019] In an embodiment, producing a modified plant with a regulated ppGpp content may include producing a modified plant with a reduced amount of ppGpp, which may flower earlier than a plant without a reduced ppGpp content.

[0020] In the embodiment, producing a modified plant with reduced ppGpp content may include producing a modified plant with reduced ppGpp content by genetic manipulation. The genetic manipulation is not limited as long as it results in a reduction in ppGpp content. For example, the genetic manipulation may involve modifying a gene encoding an enzyme (including paralogs in various plants) with (p)ppGpp synthesis activity so as to reduce the activity of the enzyme. Alternatively, the genetic manipulation may involve modifying or introducing a gene that directly or indirectly regulates the expression of these enzymes.

[0021] The method of genetic manipulation is not limited, and any genome editing technique known to those skilled in the art, including homologous recombination, mutagenesis, or CRISPR / CAS9 (CRISPR / CAS9) technology, may be used. Alternatively, target gene expression may be post-transcriptionally silenced by expressing or introducing siRNA, dsRNA, or the like into a plant. Alternatively, a target gene may be disrupted by inserting a foreign nucleic acid sequence into the genome using transposon or knock-in technology. In particular, deleting a gene encoding an enzyme with (p)ppGpp synthesis activity (e.g., RSH, described below) using genome editing (e.g., by introducing a premature stop codon, frameshift, or aberrant splicing via point mutation, or by inactivating the enzyme) is preferred because it avoids the need to introduce a foreign recombinant sequence into the genome. While gene overexpression typically relies on the introduction of a recombinant nucleic acid sequence, this embodiment is advantageous because it allows for control of plant flowering time while minimizing genome modification.

[0022] Alternatively, the genetic manipulation may involve introducing into the plant, for example, a gene that suppresses the expression of the enzyme. Such a gene may be introduced by, for example, transformation.

[0023] In the present disclosure, transformation includes introducing a gene from an external source to increase the expression of the gene compared to a corresponding non-transformed individual. Transformation can be performed by various techniques known to those skilled in the art, including various genome editing techniques such as the Agrobacterium method, the gene gun (particle gun) method, the electroporation method, the PEG method, homologous recombination, and the CRISPR / CAS9 method, as well as combinations thereof.

[0024] In an embodiment, creating a modified plant with reduced ppGpp content includes deleting or silencing one or more, for example, all, of the plant's RSH genes. RSH stands for RelA-SpoT Homolog and refers to an enzyme family that includes members RSH1, RSH2, RSH3, and CRSH. Those skilled in the art will clearly recognize plant RSHs, including RSH1, RSH2, RSH3, and CRSH, and the genes that encode them (also referred to as RSH genes in this disclosure) (see, e.g., Masuda, 2012, The Stringent Response in Phototrophs, In book: Advances in Photosynthesis - Fundamental Aspects). For example, those skilled in the art know that plants lacking RSH2 and RSH3 have a reduced ppGpp content of approximately 80%, while plants lacking all of RSH1, RSH2, RSH3, and CRSH have a reduced ppGpp content of approximately 1 / 20 (Maekawa, M. et al. Nature Plants Vol. 1 15167, 2015). Because not only ppGpp but also RSH genes and their functions are widely conserved in plants, embodiments of the present disclosure are applicable to a variety of plants. In embodiments, when one or more RSH genes are deleted or their expression is suppressed, the one or more RSH genes may include one, two, or three of the RSH2, RSH3, and CRSH genes. Alternatively, all four genes, RSH1, RSH2, RSH3, and CRSH, may be deleted or their expression suppressed. Therefore, creating a modified plant with reduced ppGpp levels in embodiments may include deleting RSH1, RSH2, RSH3, and CRSH.

[0025] In embodiments, deleting one or more RSH genes in a plant can be achieved by various genetic manipulations described herein, but preferably involves modifying the nucleic acid sequence of the RSH gene by genome editing. In other words, in one embodiment, a plant with an earlier flowering time is produced by genome editing. The genome editing method can be any genome editing technique known to those skilled in the art, including the CRISPR / CAS9 method.

[0026] Deleting or suppressing expression of one or more RSH genes in a plant of the embodiments can be achieved by various genetic manipulations known to those skilled in the art, some of which are exemplified herein. Deficiency of the RSH gene includes a defect in the RSH gene itself that prevents it from expressing a normal gene product, and can include, for example, deletion of the gene, mutation of a transcriptional regulatory element, or mutation of the coding sequence. Suppression of expression of the RSH gene includes a state in which the RSH gene itself is not defective, but normal expression of the RSH gene is prevented by, for example, the presence of siRNA or dsRNA.

[0027] In the present disclosure, the RSH gene may be a gene encoding an RSH protein having (p)ppGpp synthase activity. In the present disclosure, (p)ppGpp synthase activity refers to the enzyme activity of synthesizing guanosine tetraphosphate (ppGpp). Since enzymes with the activity of synthesizing ppGpp can also synthesize pppGpp, the term "(p)ppGpp" synthase is used by those skilled in the art. For example, the same enzyme can synthesize ppGpp using GDP as a substrate, or pppGpp using GTP as a substrate. Such enzyme activity can be detected and measured by methods known to those skilled in the art (see, for example, Tozawa, Y. et al., The Journal of Biological Chemistry, Vol. 282, 49 (2007): 35536-45). Creating a modified plant with reduced ppGpp levels in the present embodiment may involve deleting all RSH genes having (p)ppGpp synthase activity in the plant.

[0028] Producing an engineered plant with reduced ppGpp levels according to embodiments can include producing an engineered plant with reduced ppGpp content through environmental manipulation, such as by inhibiting the activity of an enzyme involved in ppGpp synthesis with a drug, or any other non-genetic manipulation that reduces ppGpp content.

[0029] In an embodiment, producing a modified plant with a regulated ppGpp content can be producing a modified plant with an increased amount of ppGpp, in which case the flowering time of the plant can be delayed compared to a plant without the increased ppGpp content.

[0030] In the present embodiment, creating a modified plant with an increased amount of ppGpp can include creating a modified plant that overexpresses the RSH gene. Creating a modified plant that overexpresses the RSH gene can involve increasing the expression level of the RSH gene in a plant through genetic manipulation, and such genetic manipulation can be plant transformation using the RSH gene. The RSH gene used for transformation can be one or more RSH genes selected from the group consisting of RSH1, RSH2, RSH3, and CRSH derived from the plant to be transformed. Alternatively, the RSH gene can be one or more RSH genes derived from a heterologous species. The RSH gene used for transformation can be a gene operably linked to a functional promoter. The functional promoter is not particularly limited as long as it can induce expression of the RSH gene in plant individuals, tissues, or cells. Preferred examples include the cauliflower mosaic virus (CaMV) 35S promoter and its variants. The RSH gene linked to a functional promoter can be contained in any vector suitable for transformation. The RSH gene can also include untranslated regions (UTRs) in addition to the coding region. The plant used for transformation may be a wild-type plant or a mutant lacking one or more of the RSH genes. Creating a modified plant with increased ppGpp levels according to an embodiment can involve creating a double mutant lacking the rsh2 and rsh3 genes and overexpressing RSH3 (Maekawa, M. et al., Nature Plants, Vol. 1, pp. 15167, 2015).

[0031] Producing a modified plant with increased ppGpp levels according to the embodiments may include producing a modified plant with increased ppGpp content through environmental manipulation, such as drug inhibition of a protein that negatively regulates the activity of an enzyme involved in ppGpp synthesis, or any other non-genetic manipulation that increases ppGpp content.

[0032] <Method for producing plants with controlled flowering time>

[0033] In another aspect, a method for producing a plant with a controlled flowering time is provided. The method for producing a plant with a controlled flowering time of the embodiment may include controlling the flowering time of a target plant using the method described in the section <Method for controlling the flowering time of a plant>. It will also be understood by those skilled in the art that the method described in the section <Method for controlling the flowering time of a plant> may also include producing a plant with a controlled flowering time.

[0034] The method for producing a plant with controlled flowering time according to an embodiment can be characterized by breeding at least one more generation of plants from the parent plant whose flowering time is genetically controlled. In this way, a plant line with controlled flowering time can be established. The bred next generation plants are plants in which the reduced or increased ppGpp content is at least partially maintained. The bred next generation plants may be pure lines having the same genotype as the parent plant with respect to the genetic modification that reduces or increases the ppGpp content, or they may be hybrids that inherit the modified gene. [Example]

[0035] Examples of the present disclosure will be described below, but the present disclosure is not limited to the examples described below.

[0036] Arabidopsis thaliana contains four ppGpp synthases, designated RSH1, RSH2, RSH3, and CRSH, which belong to the well-established RSH family. It is a protein.

[0037] In a previous study, a quadruple mutant lacking all four RSHs was generated by further deleting CRSH from the triple mutant rsh1rsh2rsh3, which lacks RSH1, RSH2, and RSH3, using genome editing technology. In this quadruple mutant, the basal level of ppGpp was reduced to approximately one-twentieth of that of the wild type (Inazu, M. et al. Plant & cell physiology, pcad136., 2023).

[0038] In this example, we investigated the flowering time of this quadruple mutant as well as RSH3ox2 (Maekawa, M. et al. Nature plants vol. 1 15167, 2015), a strain in which RSH3 is overexpressed in the rsh2rsh3 strain, which is deficient in RSH2 and RSH3, and accumulates approximately three times as much ppGpp as the wild type.

[0039] The wild-type (WT), overexpression (RSH3ox2), and quadruple mutants were grown in soil under short-day conditions, and the number of days after sowing at which flower bolting occurred was investigated. Compared to the wild-type, the number of days after sowing at which flower bolting occurred was approximately three days later in the overexpression lines, while it was approximately four days earlier in the quadruple mutant (Fig. 1). Furthermore, normal flowering was observed after flower bolting in all lines.

[0040] These results demonstrate that increasing or decreasing ppGpp accumulation in the model plant Arabidopsis thaliana can delay or advance bolting, respectively. Furthermore, they demonstrate that altering ppGpp accumulation can control flowering time while maintaining normal flowering. Furthermore, they suggest that a similar method may be used to control flowering time in plants other than Arabidopsis.

[0041] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the examples in the above embodiments. Various modifications can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0042] The present disclosure includes the following embodiments. (Section 1) A method for controlling the flowering time of a plant, comprising: and producing a modified plant in which the ppGpp content of the plant is regulated. method. (Section 2) Item 1. The method according to Item 1, wherein producing a modified plant with regulated ppGpp content comprises producing a modified plant with reduced ppGpp amount, and the flowering time of the plant is earlier than that of the plant without reduced ppGpp content. (Section 3) Item 3. The method according to Item 2, wherein producing a modified plant with a reduced amount of ppGpp comprises deleting one or more of the RSH genes of the plant. (Section 4) Item 4. The method according to Item 2 or 3, wherein producing a modified plant with reduced ppGpp amount comprises deleting all RSH genes having (p)ppGpp synthase activity in the plant. (Section 5) Item 5. The method according to any one of Items 2 to 4, wherein the producing a modified plant having a reduced amount of ppGpp comprises deleting RSH1, RSH2, RSH3, and CRSH. (Section 6) Item 1. The method according to Item 1, wherein producing a modified plant with regulated ppGpp content comprises producing a modified plant with increased ppGpp amount, and the flowering time of the plant is delayed compared to the plant without increased ppGpp content. (Section 7) Item 7. The method according to Item 6, wherein producing a modified plant with an increased amount of ppGpp comprises producing a modified plant that overexpresses the RSH gene. (Section 8) Item 8. A method for producing a plant whose flowering time is controlled, comprising controlling the flowering time of a target plant using the method according to any one of Items 1 to 7.

Claims

1. A method for controlling the flowering time of a plant, comprising: and producing a modified plant in which the ppGpp content of the plant is regulated. method.

2. The method of claim 1, wherein producing a modified plant with regulated ppGpp content comprises producing a modified plant with reduced ppGpp amount, and the flowering time of the plant is earlier than that of a plant without reduced ppGpp content.

3. The method according to claim 2, wherein producing a modified plant with a reduced amount of ppGpp comprises deleting one or more of the RSH genes of the plant.

4. The method according to claim 2, wherein producing a modified plant with a reduced amount of ppGpp comprises deleting all RSH genes having (p)ppGpp synthase activity that are present in the plant.

5. The method of claim 2, wherein producing a modified plant with a reduced amount of ppGpp comprises deleting RSH1, RSH2, RSH3, and CRSH.

6. The method of claim 1, wherein producing a modified plant with regulated ppGpp content comprises producing a modified plant with increased ppGpp content, and the flowering time of the plant is delayed compared to a plant that does not have increased ppGpp content.

7. The method of claim 6, wherein producing a modified plant with increased amounts of ppGpp comprises producing a modified plant that overexpresses an RSH gene.

8. A method for producing a plant whose flowering time is controlled, comprising controlling the flowering time of a target plant using the method according to any one of claims 1 to 7.

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