JP5019282B2 - Method for adjusting leaf water transpiration and method for improving drought tolerance of plants - Google Patents

Description

  The present invention relates to a method for adjusting moisture transpiration of leaves and a method for improving the drought resistance of plants, for example, extending the life of ornamental plants, expanding the production areas of ornamental plants and crops, drought tolerant for desert greening It can be used in fields such as the creation of sex plants.

  In land plants, about 95% or more of the water loss from the plant body is due to transpiration from the pores. Therefore, by controlling the opening and closing of the pores of the leaves, for example, moisture transpiration can be suppressed and the drought resistance of the plant can be expected to increase.

  Sphingosine 1-phosphate, a sphingolipid, is a lipid mediator involved in the abscisic acid signaling pathway that promotes stomatal closure. Sphingolipid is a membrane component widely preserved from yeast to plants and animal cells, and is a generic name for lipids having a sphingoid base (also called a long-chain base) as a structural skeleton. Initially, sphingolipids were thought to play a major role in maintaining membrane structure, but recent research has shown that sphingolipids and their metabolic intermediates are responsible for cell recognition, regulation of cell growth and differentiation, It has been found to be a highly bioactive substance that mediates intercommunication and signaling pathways (Hannun et al. 1989, Merrill et al. 1991, Hakomori 1981, Riboni et al. 1997).

The sphingolipid skeleton of animal cells is mainly sphingosine, but it is mainly phytosphingosine in plants and fungi. Naturally occurring sphingoid bases generally have 18 carbon atoms, and the stereochemical structure at the C-2,3 position is _D- erythro type. The biosynthesis pathway of major sphingolipids currently considered is explained. The biosynthesis of sphingosine begins with the formation of 3-ketodihydrosphingosine by the condensation of palmitoyl CoA and serine in the endoplasmic reticulum (ER). It starts with. 3-ketodihydrosphingosine is further converted to dihydrosphingosine by 3-ketodihydrosphingosine reductase, and dihydrosphingosine is further converted to phytosphingosine by sphingoid base C-4 hydroxylase in yeast and plants (HaaK et al. 1997, Sperling). et al. 2001). Dihydrosphingosine is also converted to sphingosine by sphingoid base C-4 desaturase in animals and plants. Dihydroceramide, phytoceramide, and ceramide are synthesized from these sphingoid bases by ceramide synthase, respectively. Ceramide is then converted into sphingomyelin or glycosphingolipid in the Golgi apparatus and transported to the cell membrane surface. In addition, sphingoid base is converted to sphingoid base 1-phosphate by sphingoid base phosphorylase and degraded by dephosphorylating enzyme or degrading enzyme.

  It has been suggested that lipids such as ceramide, sphingosine, and sphingosine 1-phosphate produced by these biosynthesis and metabolic pathways are involved in various cell functions such as cell survival, proliferation, and apoptosis. In spite of the fact that sphingosine was found to be a potent inhibitor of protein kinase C (PKC) in vitro in 1986 (Hannun 1986), sphingolipids have attracted attention as intracellular signaling agents. Therefore, research on the signal mechanism has been actively conducted. In 1993, Hannun et al. Showed that ceramide may be a signal transmitter in programmed cell death by neuronal tumor necrosis factor (TNF) -α (Obeid et al. 1993). To date, ceramide has been reported to be an important regulator of heat stress response (Dickson et al. 1997) and activation of caspase that induces apoptosis in animals and yeast (Jenkins et al. 2002). ). Sphingosine has also been shown to have a unique apoptosis-inducing function different from ceramide (Sweeney et al. 1996, Nakamura et al. 1996, Kim et al. 2001). In yeast, sphingoid bases have been reported to be involved in the regulation of endocytosis (Zanolari et al. 2000, Friant et al. 2000) and to inhibit amino acid uptake (Skrzypek et al. 2000, Chung et al. 2001).

So far, the physiological activities of ceramide and sphingoid bases have been described. In particular, sphingosine 1-phosphate (hereinafter also referred to as “S1P” for short) serves not only as an intracellular second messenger but also as an intercellular signal transmitter. It attracts attention as a new type of bioactive lipid. Intracellularly produced S1P is released outside the cell and causes various cellular responses such as cell proliferation, cell motility control, and differentiation as an intercellular signal transmitter (Pyne et al. 2000). In animal cells, the role of S1P as an intercellular signal transducer is via the G-protein-coupled EDG (endothelial differentiation growth) receptor with seven transmembrane domains (Pyne et al. 2000). Spiegel et al. 2000a, Spiegel et al. 2000b, An et al. 2000). On the other hand, S1P as an intracellular second messenger was reported to suppress calcium mobilization, activation of phospholipase D, and apoptosis and promote cell proliferation (Igarashi 1997, Spiegel 1999, Birchwood at al. 2001). In this way, the intracellular level of S1P, a signaling agent for many important events, is tightly controlled, synthesized by sphingosine kinase (SPHK), and sphingosine 1-phosphate phosphatase (sphingosine 1-phosphate). Phosphatase: Dephosphorylated by "SPP" for short). Further, it is decomposed into phosphoethanolamine and C16 aldehyde by sphingosine 1-phosphate lyase (hereinafter also referred to as “SPL” for short). For these enzymes, gene cloning and functional analysis have been performed in yeast and animals. In yeast, dihydrosphingosine-1-phosphate phosphatase (LCB3: yeast lacks sphingoid base C-4 desaturase, so there is no sphingosine, and dihydrosphingosine-1-phosphate is similar to S1P in animal cells. In the case of the deficient strain Δlcb3 (which causes the response) and in the dihydrosphingosine-1-phosphate lyase (DPL1) -deficient strain Δdpl1, several times as many as the wild type, and in the Δlcb3Δdpl1 double-deficient strain several hundred times the sphingoid base 1-phosphorus Acid accumulates in the cell (Kim et al. 2000). Further, in the growth stage, wild type strain but stops the growth in G ₁ phase reaches the stationary phase, significantly delayed growth arrest in stationary phase in Derutadpl1, about the cell concentration found in stationary phase wild-type strain 2 Doubles to stop growing (Gottlieb et al. 1999). From the above reports, it is considered that the level of intracellular sphingoid base 1-phosphate is controlled by the action of these enzymes.

The role of S1P in plants has not been studied yet compared to yeast and animals. However, in 2000, Nishiura et al. First introduced Arabidopsis thaliana (hereinafter referred to as “A. thaliana”) to produce sphingosine kinases. The gene (AtLCBK1) has been cloned and analyzed for function (Nishiura et al. 2000, Imai et al. 2005). In 2001, Ng et al. Reported that S1P is a signaling molecule involved in the regulation of swell pressure in stomatal cells by abscisic acid (hereinafter also referred to as “ABA”), which causes calcium movement in plants. Reported (Ng et al. 2001) and that S1P-producing enzyme sphingosine kinase is activated by ABA in A. thaliana and is involved in both suppression of stomatal opening and promotion of stomatal closure by ABA (Coursol et al. 2003). In particular, it is interesting that these S1P roles have no effect on dihydrosphingosine 1-phosphate. The currently known mechanism of stomatal closure of drought stress response is as follows. Normally, ABA accumulates in the chloroplasts of mesophyll cells, and when it feels drought stress, it becomes dissociated and released to guard cells. When ABA binds to the receptor, the second messenger inositol 1, 4, 5-3 phosphate releases calcium ions (Ca ²⁺ ) from the ER calcium pool. An increase in cytosolic Ca ²⁺ concentration activates anion channels, and malate ions and chloride ions are released out of the cell, causing membrane potential depolarization. Furthermore, the potassium ion efflux channel is activated and the potassium ion evacuation is promoted to reduce the osmotic pressure of guard cells, and the swell pressure is lowered to close the pore (Schroeder et al. 2001). However, it is not well understood at what stage this mechanism and S1P are involved, or whether there is an original transmission path.

  Regarding the regulation of intracellular S1P levels in plants, only the synthetic enzyme AtLCBK1 has been identified, and the degradation system has not been investigated at all. Examples of enzymes that degrade S1P in yeast and animal cells include the SPP that is a dephosphorylating enzyme and the SPL that completely degrades S1P. In 1969, an experiment using rat liver by Stoffel et al. Confirmed that the activity of SPL was confirmed in the microsomal and mitochondrial membrane fractions, which required pyridoxal 5'-phosphate as a coenzyme, and sphingoid base 1-phosphate. It was first reported to be an enzyme that cleaves the C2-3 bond of acids (Stoffel et al. 1969). However, in 1991, Van Veldhoven et al. Reported that the activity of the mitochondrial fraction was contaminated with the microsomal fraction, and that SPL was a complete membrane protein and exposed the active site to the cytoplasmic side. (Van Veldhoven et al. 1991, Van Veldhoven 1999). Subsequently, cloning and functional analysis were performed for the first time in Saccharomyces cerevisiae (Saba et al. 1997), followed by cloning and functional analysis for mice, humans, slime molds, Drosophila, nematodes one after another (Zhou et al. 1997, Van Veldhoven et al. 2000, Li et al. 2001, Herr et al. 2003, Mendel et al. 2003). In these reports, sphingosine-1-phosphate lyase (SPL) regulates intracellular S1P levels in various organisms, and nematode SPL-deficients fail to develop embryos normally. It was semi-risal due to abnormal development.

  Regarding the SPP that is a phosphatase, the yeast Saccharomyces cerevisiae has two SPPs, LCB3p / YSR2p / Lbp1p and YSR3 / Lbp2p / YSR2-1p. These two are homologs, but LCB3p / YSR2p / Lbp1p account for the majority of their activity. These proteins are localized in the ER. In addition, mammalian SPP also has two homologs, SPP (SPP1 and SPP2), and it is thought that SPP1 is localized in the ER like yeast and accounts for most of its activity.

  Lipid phosphate phosphatases are divided into SPP (S1P phosphohydorase) family and LPP (type 2 lipid phosphate phosphohydrolase) family. These two also differ in sequence and biological properties. Among them, the most different point is that the SPP family shows high substrate specificity for S1P, dihydroS1P, and phytoS1P, but the LPP family has a wide range such as S1P, phosphatidate (PA), lysophosphatidate (LPA), ceramide-1-phosphate and diacylglucerol-pyrophosphate. It is a point that has a substrate. Both SPP and LPP proteins have the following three motifs: That is, motif 1: KXXXXXXRP, motif 2: PSGH, motif 3: SRXXXXXHXXXD. These motifs have also been found in other phosphatase superfamily (lipid phosphatases, glucose-6-phosphatases, non-bacterial acid phosphatases, chloroperoxidases, etc.). In addition, the crystal structure of chloroperoxidase showed that these three motifs are close to each other, and revealed that these three motifs form the binding pocket of the cofactor vanadate ester. . Analysis of mutants in these three conserved regions showed that the center of enzyme activity is here.

  On the other hand, it has been studied to improve drought resistance of plants by suppressing moisture transpiration from the leaves of plants. For example, in order to maintain the freshness of cut flowers, an amine compound is used as a substance that suppresses moisture transpiration from leaves (for example, Patent Document 1). However, this method is not a method for imparting drought resistance to the plant itself.

JP 2002-53402 A

  The present invention has been made under the circumstances as described above, and the problem and purpose thereof are to study the regulation mechanism of intracellular sphingosine 1-phosphate level in plants and regulate the water transpiration of industrially useful leaves. It is to provide a method for improving the drought resistance of plants, a screening method for substances that improve drought tolerance of plants, and the like.

  In order to solve the above-mentioned problems, the present inventor has paid particular attention to the sphingosine-1-phosphate phosphatase (SPP), and SPP regulates the intracellular level of sphingosine 1-phosphate (S1P) in plants. We thought that it plays an important role and is involved in the regulation of stomatal function by S1P. Therefore, AtSPP1 (hereinafter also referred to simply as “SPP1”) gene was cloned from A. thaliana as an SPP gene, and analysis of enzyme function and the mechanism of regulation of intracellular S1P levels by SPP in plants were conducted. Furthermore, the present inventors have found that the amount of water transpiration is suppressed in the SPP1 deficient in which the SPP1 gene has been deleted, and that the suppression of SPP gene expression is involved in the water regulation of plant leaves, thereby completing the present invention.

  That is, the present invention firstly relates to a method for regulating leaf water transpiration by controlling the expression or activity of sphingosine-1-phosphate phosphatase in plant cells, particularly sphingosine-1-phosphate in plant cells. The present invention provides a method for suppressing water transpiration of leaves by suppressing the expression or activity of phosphatase.

  As shown also in the below-mentioned Example, in the SPP1-deficient body in which sphingosine-1-phosphate phosphatase (SPP1) is deleted, the amount of water transpiration is significantly reduced and suppressed as compared to the wild type. In addition, taking into account the following results that the transcription level of the SPP1 gene decreases when drought stress is applied to plants, the SPP gene is a gene that has a function to positively control leaf water transpiration. Then, it is thought that the transcription level falls.

  Therefore, by controlling the gene expression or activity of SPP, it is possible to suppress or promote water transpiration of plant leaves. In other words, SPP is thought to regulate the intracellular signal level of sphingosine 1-phosphate (S1P) in plants and regulates plant stomatal opening / closing signaling, thus controlling SPP gene expression or activity. By controlling the opening and closing of the pores, it can be used to adjust the moisture transpiration of the leaves.

  In addition, by suppressing the gene expression or activity of SPP, moisture evaporation from the pores can be suppressed, and the drought resistance of plants can be enhanced. As a result, for example, by making plants less susceptible to wilting, that is, improving the shelf life and using them to extend the life of appreciation plants, or enabling production in dry areas that were difficult to produce in the past, It can be used to expand production areas. Furthermore, since it is possible to create a plant that is resistant to a dry environment (dry stress) where the water environment is not sufficient, it can also be used to create a drought-tolerant plant for desert greening.

  Furthermore, the present inventors have found that by controlling the gene expression or activity of the sphingosine-1-phosphate lyase (SPL), it is possible to regulate water transpiration from the pores and improve drought tolerance of plants. (Japanese Patent Application No. 2005-234607). Therefore, in combination with the method described in the same specification, the method for controlling the gene expression (or activity) of SPL together with the control of gene expression (or activity) of SPP can further improve water transpiration from the pores. This is a preferable method that can be expected to improve the drought resistance of plants.

  The method for suppressing the gene expression or activity of SPP is not particularly limited. Examples of the method for suppressing the expression level of the SPP gene include a method for modifying the SPP genome (for example, a knockout method described later) and a method for suppressing after transcription (for example, a knockdown method by an RNAi method described later). Other methods may selectively inhibit or suppress transcription of the SPP gene, or selectively inhibit any of the processes of splicing, translation, and post-translational modification of SPP and specifically express SPP protein. It may be a method of restraining. “Substances that suppress the expression or activity of sphingosine-1-phosphate phosphatase” as described below include substances that inhibit and suppress the enzyme activity of SPP, as well as specifically suppress the expression of SPP protein. It may be a substance having an action to act. The present invention also includes a method of suppressing the expression or activity of SPP by administering such a substance to a plant cell, tissue or plant individual.

  The RNAi method may be used as a method for specifically suppressing the expression level of the SPP gene. For example, a method of introducing artificially produced siRNA into a plant cell, a method of producing an SPP-specific RNAi expression vector, and introducing it into a plant cell are exemplified. The SPP-specific RNAi expression vector is (1) designed to express dsRNA having a hairpin structure of an appropriate length in one cell with a single RNA, (2) sense strand and antisense strand, respectively. That are designed to be expressed and associated in the target cell. By using the RNAi expression vector, intracellular expression of the SPP gene can be continuously and stably suppressed in plant cells. The present invention is a method for specifically suppressing SPP expression by introducing such siRNA or RNAi expression vector into a plant cell, thereby enhancing the drought tolerance of plants, that is, siRNA that specifically suppresses SPP expression. And a method of using an RNAi expression vector as a drought tolerance improver for plants.

  In the present invention, the method for suppressing the expression of SPP in plant cells may be any method that substantially reduces the expression level of SPP as compared to the wild type, and completely suppresses the expression of SPP. It does not have to be.

  The sequence of the siRNA or RNAi expression vector is not particularly limited, and may be arbitrarily designed according to a known method based on the SPP gene sequence of the target plant. For example, for the SPP1 gene of Arabidopsis thaliana, its cDNA sequence is shown in SEQ ID NO: 1 and FIG. Based on this sequence, it is possible to design siRNA and RNAi expression vectors that specifically suppress the expression of the SPP1 gene of Arabidopsis thaliana.

  Examples of other methods for specifically suppressing the expression of the SPP gene include a method of administering an antisense oligonucleotide, a ribozyme, a low molecular weight compound or the like to a target plant cell, tissue or individual plant. The category of “plant” in the present invention includes individual plants, various organs (particularly leaves) such as plant roots, stems, leaves, and reproductive organs (including flower organs and seeds), various tissues, and plant cells. In addition, protoplasts, spheroplasts, induced callus, regenerated individuals and their progeny are also included. Further, the present invention is not limited to dicotyledonous plants but may be applied to monocotyledonous plants.

  Still another method for specifically suppressing the expression of the SPP gene is a knockout (gene disruption) method. In this method, a tag line (a group of gene disruption strains) is prepared using T-DNA, transposon, etc., and the SPP gene in the genome is selected by selecting a line in which the SPP gene has been disrupted by PCR or the like. Is obtained (see, for example, “Cellular Engineering Supplement, Plant Cell Engineering Series 14: Genome Research Protocol for Plants” (Shujunsha), pages 66-67, pages 82-84).

  The insertion position of T-DNA or transposon for disrupting the SPP gene may be an intron region or an exon region as long as SPP expression / activity can be significantly reduced compared to deletion or wild type. It may be a promoter region. Moreover, as long as the expression / activity of SPP can be remarkably reduced, not only SPP homo-deficient but also SPP hetero-deficient may be used. “Remarkably reduce” means that the expression or activity of SPP is 80% or less, preferably 50% or less, more preferably 30% or less, when the wild-type expression or activity is 100, for example. .

  As a method for selecting a mutant in which the SPP gene has been knocked out, a method in which T-DNA or the like is inserted into the SPP gene region is selected based on the base sequence of the T-DNA or the like and the base sequence of the SPP gene. However, the present invention is not limited to this method. Mutant plant individuals can be obtained by redifferentiating the mutant callus obtained.

  In addition to the knockout (gene disruption) method described above, the SPP gene expression in plant cells may be suppressed by artificially modifying the sequence of the SPP gene in the genome. For example, site-directed mutation (Site-Directed Mutagenesis) is used to introduce mutations such as point mutations into regions important for the enzyme activity of SPP, and to modify SPP mutant proteins that have lost their original activity. The method etc. are mentioned.

  As described above, the present invention pays attention to the function of SPP as a result of functional analysis, and is a method for regulating leaf water transpiration by controlling the expression or activity of SPP in plant cells. This control includes both suppression of expression / activity and enhancement. Examples of the method for enhancing the expression of SPP or its activity include a method of introducing an SPP expression vector into a plant cell to overexpress SPP, a method of administering a drug that activates SPP, and the like. It is possible to promote the growth and growth of plants by increasing the expression and activity of SPP and promoting moisture transpiration, and to use it to improve the productivity of ornamental plants.

  Of course, the present invention is not limited to the above-described exemplary methods, and various conventionally known methods can be applied to control the expression and activity of SPP. Further, a method newly developed after the present invention may be used.

  As described above, by suppressing the expression of SPP in plant cells or its activity, it is possible to suppress moisture transpiration from the pores and increase the drought resistance of plants, so that the expression of sphingosine-1-phosphate phosphatase or Substances that suppress activity are useful as drought tolerance improvers for plants, and screening methods thereof are also included in the present invention.

  As the screening method of the present invention, various conventionally known methods for examining gene / protein expression levels, enzyme activity changes and the like can be applied and are not particularly limited. Moreover, you may use the screening method newly developed after this invention. Any of in vitro and in vivo screening systems may be used, and screening may be performed with a cell-free system. The SPP gene / protein may be derived from other plants besides those derived from Arabidopsis thaliana. Of course, screening may be performed using information on the higher order structure of the SPP protein.

  Hereinafter, examples of the present invention will be described with reference to the drawings, but the present invention is not limited to the following examples.

[Summary of this example]
We isolated the SPP gene from Arabidopsis thaliana and analyzed the function of SPP in plants. First, a BLAST search with an amino acid sequence with yeast YSR2 was performed, and a gene presumed to be SPP1 of A. thaliana (AtSPP1) was found and cloned. In order to prove that the isolated gene is A. thaliana SPP1, it was examined whether DPL activity was complemented by introducing and expressing the A. thaliana SPP1 gene in a DPL-deficient strain (Δdpl1) of S. cerevisiae . In the presence of sphingosine, Δdpl1 is inhibited by the growth of S1P. On the other hand, Δdpl1 expressing A. thaliana SPP1 was not subject to growth inhibition. In addition, sphingosine and sphingosine 1-phosphate were extracted from these transformants and analyzed by HPLC to confirm dephosphorylation of sphingoidin 1-phosphate. From these results, it was shown that the gene isolated this time functionally encodes A. thaliana SPP1. To investigate the role of SPP1 in A. thaliana, the transcription level of each organ was analyzed using a semi-quantitative RT-PCR method. As a result, the SPP1 gene was a flower, and the mRNA accumulation level was particularly high. As a result of extracting mRNA over time from A. thaliana subjected to drought stress and analyzing by semi-quantitative RT-PCR, the accumulation level of SPP1 gene mRNA decreased as drought stress time increased. In order to investigate the role of SPP1 in the opening and closing of the pores in A. thaliana, the amount of water transpiration of A. thaliana SPP1 deficient was examined. As a result, the transpiration amount of the SPP1-deficient was reduced to about 1/3 that of the wild type. These results demonstrate in plants that regulation of SPP1 expression regulates intracellular S1P levels in A. thaliana, and can further control stomatal opening and closing via this regulation.

[Experimental materials and methods]
[I] Isolation of AtSPP1 gene and functionally active material Arabidopsis thaliana T87 cultured cells were purchased from RIKEN BioResource Center. MRNA was extracted from the cultured cells to obtain cDNA, which was cloned into a template. Yeast Saccharomyces cerevisiae BY4741 strain and Δdpl1 strain (DPL: dihydro-sphingosin-1-phosphate deficient strain) were purchased from Invitrogen.

Extraction of RNA from cultured cells and preparation of cDNA
0.1 g of T87 cultured cells cultured for 1 week were used. RNA was extracted using QIAGEN RNeasy Plant Mini Kit (50). Using this as a template, cDNA was prepared from total RNA. In addition, RNaseH (Invitrogen) treatment was performed to remove excess RNA.

Extraction of RNA from yeast and preparation of cDNA
The BY4741 strain pre-cultured in YPD medium for 16 hours was cultured overnight, and RNA was extracted in the same manner as T87 cultured cells. Using this as a template, cDNA was prepared in the same manner as described above and treated with RNaseH.

Preparation of Insert and Vector The N-terminal region and C-terminal region were subjected to PCR using the above A. thaliana and BY4741 cDNAs as templates. The primers used were AtLCB3-F, AtLCB3-R2, YSR2-F and YSR2-R2 in Table 1 below.

The PCR product was electrophoresed on a 1% agarose gel for 90 minutes, stained with ethidium bromide, and a band around 1200 bp was extracted for both. In addition, E. coli JM109 was transformed with the pYES2 vector, cultured in a liquid medium containing ampicillin, and plasmid DNA was prepared using Wizard ^™ Plus Minipreps (Promega). The two DNA solutions and plasmid DNA thus obtained were subjected to double digestion with restriction enzymes Hind III and Not I.
After restriction enzyme treatment, 10 × loading buffer was added, 5 μL of this was electrophoresed in 0.5% TBE for 90 minutes on a 1% agarose gel, and stained with ethidium bromide. The band was excised from the gel and the DNA was purified using GeneElute ^™ MINUS EtBr SPIN COLUMN (SIGMA).

Ligation reaction The prepared insert and vector were subjected to a ligation reaction at 16 ° C for 2 hours using DNA ligation Kit Ver.2 Solution I (TaKaRa).

Transformation into E. coli Using the ligation reaction solution, transformation into E. coli JM109 was performed by heat shock. Transformants were cultured in LB medium containing ampicillin at 37 ° C for 16 hours, and the resulting colonies were selected and inserted by direct PCR using the N-terminal region and C-terminal region (pYES2-F, pYES2-R). The presence or absence was confirmed. For positive clones in which the presence of the insert was confirmed, plasmid DNA was purified using Wizard ^™ Plus Minipreps (Promega).

Confirmation of Insert by Sequencing The insert sequence of purified plasmid DNA (0.20 μg / μL) was sequenced. This confirmed that the insert had the correct sequence of AtSPP1 and YSR2 and was inserted correctly. The primers used were pYES2-F, pYES2-R, AtLCB3-F, AtLCB3-R2, SEQ-AtLCB3, YSR2-F, YSR2-R2, SEQ-YSR2-F and SEQ-YSR2-R.

Transformation of pYES2-AtSPP1 and pYES2-YSR2 into yeast Δdpl1 The above pYES2-AtSPP1, pYES2-YSR2, and pYES2 (empty vector) were each introduced into the Δdpl1 strain by the lithium acetate method and transformed. The transformants thus obtained are designated as Δdpl1-pYES2-AtSPP1, Δdpl1-pYES2-YSR2, and Δdpl1-pYES2, respectively.
Using the transformants, functional complementation experiments were performed with AtSPP1 in a medium containing 10 μM sphingosine. YPD medium (-galactose, -sphingosine), minimal medium (-galactose, +10 μM sphingosine) and minimal medium (+ galactose, +10 μM sphingosine) were respectively wild strains (BY4741), Δdpl1, Δdpl1-pYES2-AtSPP1, Δdpl1- pYES2-YSR2 and Δdpl1-pYES2 were streaked and cultured at 28 ° C. Δdpl1 is a sphingoid bases lyase-deficient strain that is growth-inhibited by 10 μM sphingosine, and the pYES2 vector has a Gal1 promoter induced by galactose.

Measurement of AtSPP1 activity by HPLC Each yeast transformant confirmed to be complementary was treated with 50 μM sphingosine for 2 hours, washed, cells were disrupted, and sphingosine and sphingosine 1-phosphate were extracted. Reverse phase chromatographic analysis was performed on a C18 column.

[II] Analysis of AtSPP1 in A. thaliana Analysis of mRNA accumulation level in each organ A. thaliana used in this example is a Columbia strain. Cultivation was carried out on MS agar medium at 23 ° C. and 2,000 lux (National FL 20S-PG plant growing lamp) under long-day conditions of 16 hours light period and 8 hours dark period. 100 mg each of the root, stem, leaf, and flower samples grown under the above conditions for 4 weeks was used. RNA was extracted using QIAGEN RNeasy Plant Mini Kit (50). Using this as a template, cDNA was prepared and PCR was performed. First, PCR was performed using actin (Act2) gene amplification primers (At-Act2-F and At-Act2-R) expressed in the same degree in all tissues in order to align the amount of mRNA transcription. Then, PCR was performed using AtLCB3-F and AtLCB3-R2 with the template amount of each organ with the same signal intensity of At-Act2, and the signal intensity of AtSPP1 was measured, and mRNA accumulation in roots, stems, leaves, and flowers The level difference was analyzed.

Changes in AtSPP1 mRNA accumulation level due to drought stress According to the method of Yoshida et al., SRK2E, whose expression is induced by drought stress, was used as a positive control in the following manner (Yoshida et al. 2002) . Using A. thaliana grown for 4 weeks after sowing grown on MS plate medium, drought stress was applied by leaving the plate lid open in an incubator. Total RNA was extracted from leaves of A. thaliana stressed over time for 0, 1, 2, and 3 hours, and cDNAs were prepared. The control was At-Act2, and the change in the mRNA accumulation level of AtSPP1 was analyzed under the condition that the signal intensity of At-Act2 at each time when stress was applied was the same.

[III] Functional analysis using AtSPP1-deficient A. thaliana
Isolation of AtSPP1-deficient
We found several seeds with T-DNA inserted into At3g58490 (SALK_0489081, SALK_035202 and SALK_027084) from a population of Arabidopsis gene disruption strains by Salk Institute Genomic Analysis Laboratory. These seeds were sown on MS-kanamycin medium (kanamycin 25 mg / ml) plates, and genomic DNA was prepared from leaves of individuals resistant to kanamycin. Using this as a template, PCR using the following primer I (primerI) and primer II (primerII) prepared based on the sequence in the AtSPP1 genome, and primer I (primerI) and T-DNA left border primer PCR was performed using primer III (FIG. 8). Then, three types of AtSPP1-deficient bodies (Int, Ex1, Ex2) from which no amplification product was obtained by the former PCR and amplification products were obtained by the latter PCR were isolated. Furthermore, total RNA was extracted from the leaves of these AtSPP1-deficient bodies, and RT-PCR was performed to confirm that there was no mRNA expression.
primerI: 5'-ATGAAGAAGATTCTAGAAAGATC-3 '(14) Int, Ex1
For 5'-CTACAAGTTAACGTAAGAGAATAGT-3 '(15) Ex2
primerII: 5'-CTACAAGTTAACGTAAGAGAATAGT-3 '(15) Int, Ex1
5'-ATGAAGAAGATTCTAGAAAGATC-3 '(14) For Ex2
primerIII: 5'-TGGTTCACGTAGTGGGCCATCG-3 '(16)
The numbers in parentheses indicate the sequence numbers in the sequence listing.

Measurement of transpiration rate in AtSPP1-deficient body The transpiration rate of wild strain and AtSPP1-deficient body was measured as follows. In order to arrange the germination date, it was seeded in MS medium. Three weeks after germination, the plants were replanted into vermiculite, and the humidity was kept high for 3 days so that drought stress was not applied. Thereafter, the plant was returned to normal growth conditions, and the roots of the plant body 4 weeks after germination were cut off, and the transpiration amount of the above-ground part was measured over time in an incubator so as not to be affected by light.

[Experimental results and discussion]
[I] Isolation and structural analysis of Arabidopsis thaliana SPP1 (AtSPP1) gene A BLAST search was performed with the yeast Saccharomyces cerevisiae YSR2 (accession number NC_001142), and sphingosine-1 was obtained from Arabidopsis thaliana -The phosphate phosphatase gene (AtSPP1) was cloned.
The base sequence of the cloned AtSPP1 cDNA and the amino acid sequence encoded by it are shown in FIG. 10 (and SEQ ID Nos. 1 and 2 in the sequence listing). The ORS of AtSPP1 is 1251 bp, consists of 416 amino acid residues, and encodes a protein with an isoelectric point of 8.23. The AtSPP1 genome is located on chromosome 3 and consists of 8 exons.
FIG. 1 shows the result of alignment of this AtSPP1 amino acid sequence with the yeast and human SPP1 amino acid sequences that have already been cloned and analyzed for functions. From the alignment results, AtSPP1 had three important motifs (motif1: KXXXXXXRD, motif2: PSGH, motif3: SRXXXXHXXXD) as well as yeast and human SPP1. These motifs are vanadate binding motifs. AtSPP1 homology at the amino acid level with other SPPs was 26.7% for Identity and 38.8% for Similarities. Although the homology was not so high, there is a similar tendency when yeast and human or yeast and mouse are compared.
Moreover, the result of the hydropathy analysis of AtSPP1 is shown in FIG. As shown in the figure, a vanadate binding motif was confirmed in AtSPP1 as well as YSR2 in yeast, and it is thought to be a transmembrane enzyme. In AtSPP1, about 75 amino acids on the N-terminal side were specific to AtSPP1. Furthermore, as a result of the phylogenetic analysis of AtSPP1, as shown in FIG. 3, AtSPP1 was determined to belong to the SPP Family using only SIP as a substrate.

[II] Expression and functional analysis of AtSPP1 in yeast
Functional complementarity experiments with AtSPP1
We cloned the cDNA of the AtSPP1 gene, which is expected to encode A. thaliana SPP1, and confirmed that the protein encoded by this gene has functional SPP activity using yeast lyase mutant (Δdpl1). confirmed. Since Δdpl1 has no lyase activity, growth is inhibited in a medium containing sphingosine because S1P accumulates without being decomposed. Therefore, an experiment was conducted to investigate whether the cloned DPL activity could be compensated by introducing the cloned cDNA into Δdpl1 and expressing the protein.

  Δdpl1 was inhibited by 10 μM sphingosine. However, the pYES2-AtSPP1 and pYES2-YSR2 transformants grew on a medium containing 10 μM sphingosine when the expression of the GAL1 promoter was induced by galactose. On the other hand, an empty vector containing only pYES2 and Δdpl1 did not grow (FIG. 4). From this, it can be said that the transformants of pYES2-AtSPP1 and pYES2-YSR2 complemented the S1P-sensitive traits. The fact that the SPL trait that degrades S1P is complemented indicates that AtSPP1 has an enzyme activity that degrades S1P. Furthermore, cloned AtSPP1 was determined to be an S1P phosphatase gene from the viewpoint of sequence homology.

AtSPP1 activity measurement by HPLC. AtSPP1 was expressed in Δdpl1 and functional complementation was confirmed by giving sphingosine resistance, but activity measurement using HPLC to confirm that S1P was actually dephosphorylated Went. Before extracting sphingolipids from Δdpl1-pYES2, Δdpl1-pYES2-YSR2 (Δdpl1-YSR2) and Δdpl1-pYES2-AtSPP1 (Δdpl1-AtSPP1), sphingosine was absorbed by culturing in 50 mM sphingosine medium for 2 hours . Since yeast does not have a sphingosine type sphingoid base, S1P was accumulated by absorbing sphingosine, and this operation was performed to confirm the dephosphorylation of S1P by SPP. The result is shown in FIG. As shown in the figure, Δdpl1-AtSPP1 was able to dephosphorylate S1P. The above functional complementation test in AtSPP1 and in vivo analysis using HPLC revealed that AtSPP1 dephosphorylates sphingoid base 1-phosphate and further uses S1P as a substrate.

[III] Functional analysis of AtSPP1 in A. thaliana Analysis of mRNA accumulation level in each organ
For the expression analysis of AtSPP1 in A. thaliana, the mRNA accumulation level of each organ was analyzed by the semi-quantitative RT-PCR method. AtSPP1 was analyzed using root, stem, leaf, and flower template amounts that matched the signal intensity of At-Act2. The result is shown in FIG. As shown in the figure, mRNA expression was observed in all organs, but there was no significant difference in AtSPP1 mRNA accumulation level in roots, stems, and leaves. However, in flowers, AtSPP1 mRNA accumulation was about 5 times that of roots.

Changes in AtSPP1 mRNA accumulation level due to drought stress Next, we examined AtSPP1 by applying drought stress to investigate how AtSPP1 is involved in the process of stomatal closure by regulating intracellular S1P levels. The accumulation level of mRNA was examined by the same method as described above. The result is shown in FIG. As shown in the figure, in AtSPP1, the mRNA accumulation level decreased with increasing time for applying drought stress. From this result, it is considered that AtSPP1 is involved in the stomatal closure mechanism in which S1P acts as a signal transmitter, and regulates the amount of intracellular S1P.

[IV] Functional analysis using AtSPP1-deficient A. thaliana
Isolation of AtSPP1-deficient plants Based on the results of examining changes in mRNA accumulation level due to drought stress, plants lacking AtSPP1 are thought to accumulate more S1P than normal, so we believe that they will become plants that are resistant to drought stress. It was. Therefore, individuals lacking AtSPP1 were screened using SALK seeds. As a result, three types of AtSPP1-deficient bodies (Int, Ex1, Ex2) were obtained (FIG. 8).
That is, PCR was carried out using primers extracted from WT and each AtSPP1 deficient as a template with the positions shown in FIG. As a result, in PCR performed with primer I (primerI) and primer II (primerII), a signal of about 2,027 bp was confirmed only in WT. In addition, in PCR performed with primer I (primerI) and T-DNA LB primer (primerIII), amplification bands were confirmed only in each AtSPP1-deficient (Int, Ex1, Ex2). From this result, it can be said that each AtSPP1-deficient body is homozygous and the AtSPL1 gene is disrupted. As a result of sequence analysis, it was found that T-DNA was inserted into the first intron in Int, the second exon in Ex1, and the third exon in Ex2.

Measurement of transpiration rate in AtSPL1-deficient body
The results of examining the transpiration rate of WT and each AtSPP1-deficient are shown in FIG. There was no difference in morphology between WT and each AtSPP1-deficient. However, as shown in FIG. 9, WT lost about 70% of the water by transpiration after 2 hours. On the other hand, in each AtSPP1-deficient body, the amount of transpiration was clearly lower than that of WT immediately after the start of measurement. For example, Ex1 had a transpiration rate of about 20% even after 2 hours. For other AtSPP1-deficient bodies (Ex2 and Int), the transpiration rate remained low.
Since SPP1 is knocked out in the AtSPP1-deficient body, it is considered that S1P is accumulated in the plant rather than WT. Therefore, it is considered that there was a clear difference immediately after the start of measurement. In addition, since there were many S1Ps from the beginning of the pores that were not closed, it was thought that as a result, more pores could be closed earlier than WT. From these facts, for example, it is considered that the transpiration rate of AtSPP1-deficient Ex1 finally decreased by about one third as compared with WT.

  Based on the above results, AtSPP1 was isolated from A. thaliana for the first time in plants, and SPP enzyme activity was confirmed by functional complementation experiments using Δdpl1 and in vivo analysis by HPLC. In plants, the mRNA accumulation level of AtSPP1 was high in flowers. In addition, drought stress reduces AtSPP1 mRNA accumulation. In the analysis using AtSPP1-deficient, the transpiration rate of AtSPP1-deficient decreased by about 1/3 compared with WT. These results suggest that SPP regulates intracellular S1P levels in plants as well as animal cells and yeast, and regulates stomatal opening and closing.

  As described above, in the sphingosine-1-phosphate phosphatase (SPP) homo-deficient body, a large amount of S1P is accumulated, so that it is considered that more pores are closed than WT. From this result, SPP is involved in the regulation mechanism of stomatal opening and closing, and is used to promote photosynthesis by opening the pore as much as possible in an environment with sufficient water content by changing the expression level and activity of SPP. On the other hand, in an environment where the water environment is not sufficient, it can be used to create plants that are resistant to drought stress by promoting pore closure.

  As described above, the present invention relates to a method for regulating leaf water transpiration by controlling the expression or activity of sphingosine-1-phosphate phosphatase in plant cells, and a method for improving drought tolerance of plants. As mentioned above, it can be used in fields such as extending the life of ornamental plants, expanding the production area of ornamental plants and crops, creating drought-tolerant plants for greening deserts, and in various industrial fields. Is possible.

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It is a figure which shows alignment with AtSPP1 and another phosphatase. It is a figure which shows the comparison of the hydropathy of AtSPP1 and yeast YSR2. It is a figure which shows the Lipid phosphate phosphatase system | strain analysis result. It is a figure which shows the result of the functional complementation test in yeast (DELTA) dpl1 strain | stump | stock. It is a graph which shows the result of having measured the activity of AtSPP1. It is a graph which shows the accumulation amount of mRNA of AtSPP1 in each organ of Arabidopsis thaliana. It is a graph which shows the accumulation amount of mRNA of AtSPP1 under drought stress conditions. It is a figure which shows isolation of an AtSPP1 deletion body. It is a figure which shows the change of the transpiration rate in an AtSPP1-deficient body and a wild type. It is a figure which shows the base sequence and amino acid sequence of AtSPP1. The underlined portion indicates a vanadate binding motif.

Claims (8)

  A method for inhibiting leaf water transpiration by inhibiting the expression or activity of sphingosine-1-phosphate phosphatase in plant cells.   A method for improving drought tolerance of plants by suppressing the expression or activity of sphingosine-1-phosphate phosphatase in plant cells.   A method of inhibiting leaf water transpiration by inhibiting the expression or activity of sphingosine-1-phosphate phosphatase in plant cells and inhibiting the expression or activity of sphingosine-1-phosphate lyase.   A method for improving drought tolerance of plants by suppressing the expression or activity of sphingosine-1-phosphate phosphatase in plant cells and suppressing the expression or activity of sphingosine-1-phosphate lyase. The expression of sphingosine-1-phosphate phosphatase is suppressed by knocking out the sphingosine-1-phosphate phosphatase gene in the genome or by modifying the gene sequence by other methods . The method according to any one of the above. By specifically inhibiting RNA expression of sphingosine-1-phosphate phosphatase is introduced into a plant cell, suppress the expression of a sphingosine-1-phosphate phosphatase, according to any one of claims 1 to 4 the method of. Sphingosine-1-specific RNA that suppresses expression of phosphoric acid phosphatase, or the RNA containing base compactors constructed to express in plant cells, drying resistance improver of plants.   A screening method for a substance that improves drought tolerance of a plant, comprising searching for a substance that suppresses the expression or activity of sphingosine-1-phosphate phosphatase.