Method for regulating photosynthetic response of plant, method for producing plant with accelerated photosynthetic response, and system for cultivating plant with accelerated photosynthetic response

Abstract

To provide a method for regulating photosynthetic response of a plant, a method for producing a plant with accelerated photosynthetic response, and a system for cultivating a plant with accelerated photosynthetic response.SOLUTION: A method for regulating photosynthetic response of a plant, the method including producing a modified plant in which a ppGpp content of the plant is regulated.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to methods for modulating the photosynthetic response of plants, methods for producing plants with an accelerated photosynthetic response, and systems for growing plants with an accelerated photosynthetic response. [Background technology]

[0002] In the field, light intensity constantly fluctuates on the scale of fractions of a second to minutes, and it is known that the speed of the photosynthetic response to fluctuating light has a significant effect on the amount of carbon fixation and growth of plants. Therefore, accelerating the photosynthetic response to fluctuating light is expected to be an effective approach to achieving higher crop yields and promoting tree growth and carbon fixation capacity.

[0003] To date, there are known techniques for speeding up the photosynthetic response to fluctuating light by modifying the function of genes involved in the control of stomatal opening and closing (Non-Patent Document 1, Non-Patent Document 2), and a technique for speeding up the photosynthetic response to fluctuating light by modifying the function of genes involved in the biosynthesis and activity control of photosynthetic proteins (Non-Patent Document 3). These previous studies have reported that it is possible to speed up the photosynthetic response to fluctuating light at the individual leaf level by modifying the function of genes involved in the opening and closing and formation of stomata and the biosynthesis and activity control of photosynthetic proteins.

[0004] However, even if these genes are modified, it is possible that the photosynthetic response will not be accelerated at the individual plant level. Because the amount of carbon fixation and growth of a plant is determined by the amount of photosynthesis in the individual plant, including all individual leaves, there is a need for a technology to accelerate the photosynthetic response at the individual plant level, but such a technology has not yet been established. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kimura, H. et al. “Improved stomatal opening enhances photosynthetic rate and biomass production in fluctuating light.” Journal of experimental botany vol. 71,7 (2020): 2339-2350. doi:10.1093 / jxb / eraa090 [Non-patent document 2] Sakoda, K. et al. “Higher Stomatal Density Improves Photosynthetic Induction and Biomass Production in Arabidopsis Under Fluctuating Light.”Frontiers in plant science vol. 11 589603. 21 Oct. 2020, doi:10.3389 / fpls.2020.589603 [Non-patent document 3] Yamori, W. et al. “Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.” The Plant journal : for cell and molecular biology vol. 71,6 (2012): 871-80. doi:10.1111 / j.1365-313X.2012.05041.x 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 regulating the photosynthetic response of plants, a method for producing plants with an accelerated photosynthetic response, and a system for cultivating plants with an accelerated photosynthetic response. [Means for solving the problem]

[0007] One aspect of the present disclosure is a method of modulating a photosynthetic response in a plant, the method comprising generating a modified plant wherein the ppGpp content of the plant is modulated. [Effects of the Invention]

[0008] According to the present disclosure, it is possible to provide a method for regulating the photosynthetic response of a plant, a method for producing a plant with an accelerated photosynthetic response, and a system for cultivating a plant with an accelerated photosynthetic response. [Brief explanation of the drawings]

[0009] [Figure 1] The time course of the relative photosynthetic rate (Aind) when exposed to approximately 100 μmol m-2 s-1 of light (weak light) converted to photosynthetic photon flux density (PPFD), followed by approximately 500 μmol m-2 s-1 of light (strong light) for 30 minutes. Average values ​​(n=5) are shown for the wild type (dotted line) and the RSH gene quadruple mutant (solid line). The time at which Aind reaches 0.95 (t0.95) for the wild type and quadruple mutant is indicated by black circles. [Figure 2] For five wild-type (WT) and four-mutant (KO) plants, the times at which the relative photosynthetic rate (Aind) reached 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95 (t0.5, t0.6, t0.7, t0.8, t0.9, and t0.95) were calculated and shown as boxplots. The ×s in the figures indicate the mean values. 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 regulating photosynthetic responses in plants>

[0012] In one aspect, a method for modulating a photosynthetic response in a plant is provided, the method comprising generating a modified plant having a modulated ppGpp content in the plant.

[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] As will be understood by those skilled in the art, photosynthetic response may refer to a change in physiological activity and / or morphology associated with photosynthesis in response to changes in the environment. In particular, in the present disclosure, photosynthetic response may refer to the regulation of photosynthetic rate in response to light intensity.

[0015] Regulating the photosynthetic response of an embodiment generally refers to, but is not limited to, accelerating the regulation of the photosynthetic rate of a plant in response to changes in light intensity. Here, the photosynthetic rate can be measured by methods known to those skilled in the art, including measuring the carbon dioxide absorption rate. Furthermore, light intensity can be measured by photosynthetic photon flux density (PPFD), which is defined as the number of photons in the wavelength range of 400 to 700 nm that are irradiated to a plant per unit time and unit area and that can be used for photosynthesis by the plant's chlorophyll. Regulating the photosynthetic response of a plant or modifying its regulatory ability in an embodiment can result in better photosynthetic performance under varying light intensities (e.g., increasing the irradiated light intensity to 100 μmol m s) compared to a plant without such regulation or modification of its regulatory ability. -2 s -1 to 500 μmol m -2 s -1 The relative photosynthetic rate (A ind It will be understood by those skilled in the art that this may include having the ability to make the plant have the ability to achieve a phototrophic ratio (phototrophic ratio) of 0.95, or may include having a greater carbon dioxide absorption. If the plant's photosynthetic rate can be adjusted more quickly in response to light intensity, the plant may be able to grow in areas where light intensity fluctuates greatly over time, which was previously unsuitable for growth, and this may lead to an expansion of the areas where the plant can be cultivated as an agricultural plant.

[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] Producing a modified plant with regulated ppGpp content according to an embodiment may include producing a modified plant with reduced ppGpp levels, which may result in a more rapid photosynthetic response in the plant compared to a plant without 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 the modulation of plant photosynthetic responses with minimal 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 in plants lacking RSH2 and RSH3, ppGpp content is reduced by approximately 80%, while in plants lacking all of RSH1, RSH2, RSH3, and CRSH, ppGpp content is reduced by approximately 1 / 20 (Maekawa, M. et al. Nature Plants Vol. 1 15167, 2015). The reduction in ppGpp content may correlate with the degree of rapid photosynthetic response. 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 in which 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, producing a modified plant with reduced ppGpp levels according to the embodiments may involve 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 a regulated photosynthetic response is produced by genome editing. The genome editing method can be any genome editing technology 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] <Plant production method>

[0030] In another aspect, a method for producing a plant is provided. The method for producing a plant with an accelerated photosynthetic response of the embodiment may include accelerating the photosynthetic response of a target plant using the method described in the section <Method for regulating the photosynthetic response of a plant>. It will be understood by those skilled in the art that the method described in the section <Method for regulating the photosynthetic response of a plant> may also include producing a plant with an accelerated photosynthetic response.

[0031] In one embodiment, the method for producing a plant with an accelerated photosynthetic response can be characterized by breeding at least one more generation of plants from a parent plant whose photosynthetic response has been genetically improved. In this way, a plant line with an accelerated photosynthetic response can be established. The bred next generation plants are plants in which the reduced 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 the ppGpp content, or they may be hybrids that inherit the modified gene.

[0032] <System for growing plants with accelerated photosynthetic response>

[0033] In yet another aspect, a system for cultivating a plant with an accelerated photosynthetic response is provided, which includes the plant described in the section <Method for regulating the photosynthetic response of a plant>.

[0034] In an embodiment, a system for cultivating a plant with an accelerated photosynthetic response may include, in addition to the plant, soil for the plant to take root in, and one or more containment elements selected from the group consisting of a hydroponic system, a greenhouse, and a field plot to contain and cultivate the plant in. The system may cultivate the plant under fluctuating light, for example, natural light. [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 of proteins.

[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] The wild-type line (WT) and the quadruple mutant (KO) were grown in soil under short-day conditions, and the photosynthetic response to fluctuating light was evaluated at the individual plant level. The photosynthetic rate of Arabidopsis plants 48-58 days after sowing was measured using an individual measurement chamber. Individual measurements were carried out in an artificial climate chamber maintained at a temperature of 22°C and humidity of 50%. The environmental conditions in the measurement chamber were controlled by an open photosynthesis measurement device, with a temperature of 26°C and a flow rate of 1600 μmol s -1 The CO2 concentration was set to 400 ppm and humidity to 55-80%.

[0039] Using LEDs installed in the artificial weather chamber, the photosynthetic photon flux density (PPFD) was calculated to be approximately 100 μmol m -2 s -1 The photosynthetic rate was then stabilised by irradiating the plants with weak light, and the PPFD was then converted to approximately 500 μmol m -2 s -1 The cells were exposed to strong light for 30 minutes.

[0040] Since the measured values ​​were affected by soil and root respiration, after individual measurements, the above-ground parts of the plants were removed and similar measurements were performed to measure the soil and root respiration rates, and the individual photosynthetic rate (A) under weak and strong light exposure was calculated by subtracting these values.

[0041] To facilitate comparison of individual photosynthetic responses between the wild type and the quadruple mutant, the relative photosynthetic rate A ind was calculated based on the following formula: A ind =(A t -A min ) / (A max -A min ) In this formula, A t is the photosynthetic rate at a given time, A min is the steady-state photosynthetic rate under low light conditions, A max is defined as the steady-state photosynthetic rate under strong light conditions. ind The times when the 0.5 , t 0.6 , t 0.7 , t 0.8 , t 0.9 , t 0.95 The smaller these values, the more rapid the photosynthetic response (Fig. 1).

[0042] Wild-type and quadruple mutant t 0.5 , t 0.6 , t 0.7 , t 0.8 , t 0.9 , and t 0.95 The results are shown in Figure 2.0.5 , t 0.6 , t 0.7 , t 0.8 The t of the quadruple mutant was similar. 0.9 , t 0.95 was smaller than that of the wild type (Fig. 2). This result indicates that the photosynthetic response of the quadruple mutant is accelerated compared to that of the wild type.

[0043] The above examples demonstrate that artificially reducing ppGpp accumulation in the model plant Arabidopsis thaliana can accelerate the photosynthetic response to fluctuating light at the individual level. Furthermore, similar methods may be used to accelerate the photosynthetic response to fluctuating light in individual plants other than Arabidopsis thaliana.

[0044] 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.

[0045] The present disclosure includes the following embodiments. (Section 1) 1. A method for modulating a photosynthetic response in 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 photosynthetic response of the plant is accelerated compared to the plant in which the ppGpp content is not reduced. (Section 3) Item 3. The method according to Item 1 or 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 any one of Items 1 to 3, 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. (Section 5) Item 5. The method according to any one of Items 1 to 4, wherein the step of producing a modified plant having a reduced amount of ppGpp comprises deleting RSH1, RSH2, RSH3, and CRSH. (Section 6) A method for producing a plant with an accelerated photosynthetic response, comprising accelerating the photosynthetic response of a target plant using the method according to any one of Items 1 to 5. (Section 7) 1. A system for growing plants with accelerated photosynthetic response, comprising: Item 6. Plants produced by the method described in Item 6. system. (Section 8) Item 8. The system according to item 7, comprising one or more selected from the group consisting of a hydroponic cultivation facility, a greenhouse, and a field plot.

Claims

1. 1. A method for modulating a photosynthetic response in 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 photosynthetic response of the plant is accelerated compared to the plant in which the ppGpp content is not reduced.

3. The method of claim 1, wherein producing a modified plant with a reduced amount of ppGpp comprises deleting one or more RSH genes in the plant.

4. The method according to claim 1, 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 according to claim 1, wherein creating a modified plant with a reduced amount of ppGpp comprises deleting RSH1, RSH2, RSH3, and CRSH.

6. A method for producing a plant with an accelerated photosynthetic response, comprising accelerating the photosynthetic response of a target plant using the method according to any one of claims 1 to 5.

7. 1. A system for growing plants with accelerated photosynthetic response, comprising: Plants produced by the method of claim 6 are included. system.

8. 8. The system of claim 7, comprising one or more selected from the group consisting of a hydroponic facility, a greenhouse, and a field plot.

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