Pyrus bZIP transcription factor PbrbZIP4 and application thereof in low temperature stress response process

Abstract

The application discloses a pear bZIP type transcription factor PbrbZIP4 and application thereof in low-temperature stress response, and belongs to the technical field of plant molecular genetic engineering.The PbrbZIP4 gene in the application has a nucleotide sequence as shown in SEQ ID NO.1 and has an amino acid sequence as shown in SEQ ID NO.2.The application has the beneficial effect that a pear anti-freezing gene is obtained, a super-expression recombinant vector is constructed by using the gene, and the function of the gene in anti-freezing is proved by using molecular biology and transgenic technology.The anti-freezing property of Arabidopsis thaliana with super-expression of PbrbZIP4 is significantly stronger than that of a wild type plant.Moreover, after low-temperature treatment, the activities of anti-oxidase (SOD, POD and CAT) of the Arabidopsis thaliana with super-expression of PbrbZIP4 are significantly higher than those of the wild type plant, and the MDA content and electrolyte exosmosis rate of the Arabidopsis thaliana with super-expression of PbrbZIP4 are significantly lower than those of the wild type plant.Therefore, PbrbZIP4 can be introduced into plants as a target gene to relieve the inhibition of low-temperature stress on plant growth and significantly improve the response capacity to low-temperature stress.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the pear bZIP transcription factor PbrbZIP4 and its application in the low-temperature stress response process. Background Technology

[0002] bZIP transcription factors are conserved transcription factors widely found in plants, animals, and microorganisms. Each bZIP transcription factor possesses a conserved bZIP domain, consisting of 60-80 amino acids, including a DNA-binding base region and a leucine zipper domain (Droge-Laser et al., 2018). The base region is highly conserved, consisting of approximately 16 amino acid residues, and contains an invariant N-X7-R / K-X9 motif that binds to specific DNA sequences with an ACGT core, such as A-box (TACGTA), C-box (GACGTC), and G-box (CACGTG). Cis-elements of the ACGT sequence are frequently identified in the promoter regions of genes induced by abscisic acid or light. Furthermore, these cis-acting elements are also present in the promoter regions of genes regulating ABA-related abiotic stress and salicylic acid pathways in plants. The leucine zipper domain is relatively unconserved, consisting of heptapeptide repeats of leucine or other hydrophobic amino acids, and plays an important role in specific recognition and dimerization reactions (Wang Q et al., 2021). In recent years, the functions of an increasing number of bZIP transcription factor family genes have been revealed, including seed germination, flower development, somatic embryogenesis, vascular development, energy metabolism, cell elongation, photomorphogenesis and signal transduction, and biotic and abiotic stress.

[0003] bZIP transcription factors are widely distributed in various tissues of higher plants, participating in various signal transduction, anabolism, and responses to abiotic stresses. Some members are involved in the low-temperature stress response. In wheat, the TabZIP96 gene enhances the cold resistance of transgenic plants (Liang et al., 2022). In rice, the bZIP73 and bZIP71 genes interact to regulate the expression levels of related genes, thereby improving the rice's tolerance to low temperatures (Tian et al., 2019). Overexpression of the SlHY5 gene in tomato increases the expression of antioxidant enzyme genes, thus enhancing the plant's low-temperature tolerance (Zhang et al., 2016). The PmbZIP12 / 31 / 36 / 41 / 48 genes play an important role in plum blossom's resistance to low-temperature stress (Li et al., 2021). The above studies focus on the low-temperature tolerance of bZIP transcription factors; however, studies on the low-temperature stress response of this transcription factor in pear have not yet been reported. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to propose a pear PbrbZIP4 gene and its application in low temperature stress response.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] The first aspect of the present invention provides a pear PbrbZIP4 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides a protein encoded by the above-mentioned pear PbrbZIP4 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A third aspect of the present invention provides biological materials containing the above-mentioned pear PbrbZIP4 gene, wherein the biological materials include, but are not limited to, recombinant DNA, expression vectors, host bacteria or plant materials.

[0009] Preferably, the expression vector is pCAMBIA-1300-PbrbZIP4.

[0010] Preferably, the host bacterium is Agrobacterium.

[0011] Preferably, the plant material is Arabidopsis thaliana.

[0012] The fourth aspect of the present invention proposes the use of the above-mentioned pear PbrbZIP4 gene or biological material containing the gene in any of the following situations:

[0013] 1) Used to improve the ability of plants to resist low-temperature stress;

[0014] 2) Used to regulate plant water balance;

[0015] 3) Used to enhance the activity of antioxidant enzymes in plants under low-temperature stress;

[0016] 4) Used to enhance the frost resistance of transgenic plants;

[0017] 5) Used to improve the survival rate of plants under frost damage.

[0018] Preferably, the plant includes, but is not limited to, Arabidopsis thaliana.

[0019] A fifth aspect of the present invention provides a method for regulating the resistance of *Pyrus pyrifolia* to low-temperature stress, the method comprising:

[0020] (1) To make the pear tree contain the PbrbZIP4 gene as described in claim 1 or

[0021] (2) Overexpress the PbrbZIP4 gene as described in claim 1 in *Pyrus pyrifolia*.

[0022] Preferably, the method includes, but is not limited to, at least one of cloning the PbrbZIP4 sequence, constructing the PbrbZIP4 gene sequence into an overexpression vector, transforming the strain with the recombinant vector, transgenic material, and propagation of transgenic material.

[0023] The advantages of this invention are:

[0024] 1. This invention discloses the pear bZIP-like transcription factor gene PbrbZIP4 and its encoded protein, which is the first report of this gene in pear. Functional verification in Arabidopsis thaliana showed that overexpression of this gene significantly improved the cold tolerance of Arabidopsis plants. Therefore, it holds promise as a target gene for introduction into plants to improve their cold tolerance and thus improve plant varieties.

[0025] 2. A pear antifreeze gene was obtained, and an overexpression recombinant vector was constructed using this gene. Molecular biology and transgenic technology were used to demonstrate the gene's function in antifreeze. Arabidopsis thaliana overexpressing PbrbZIP4 exhibited significantly stronger antifreeze properties than wild-type plants. Furthermore, after low-temperature treatment, the activities of antioxidant enzymes (SOD, POD, CAT) in Arabidopsis thaliana overexpressing PbrbZIP4 were significantly higher than those in wild-type plants, while the MDA content and electrolyte leakage rate were significantly lower. Therefore, PbrbZIP4 can be used as a target gene to introduce into plants, alleviating the inhibitory effect of low-temperature stress on plant growth and significantly improving the plant's response to low-temperature stress. Attached Figure Description

[0026] Figure 1 This refers to the change in the relative expression level of the PbrbZIP4 gene under iron deficiency treatment in Example 2. Pear materials were treated with iron deficiency for 0h, 1h, 6h, and 12h, and the expression level of the PbrbZIP4 gene at each time point of iron deficiency treatment was analyzed.

[0027] Figure 2 This is a phenotypic diagram of the transgenic Arabidopsis seedlings overexpressing PbrbZIP4 in Example 4 in MS medium at normal temperature of 25°C and low temperature of 4°C. PbrbZIP4-OE-1 and PbrbZIP4-OE-2 represent two overexpressing transgenic lines, and WT is the wild type.

[0028] Figure 3 This refers to the survival rates of transgenic Arabidopsis thaliana lines (PbrbZIP4-OE-1, PbrbZIP4-OE-2) and wild-type materials (WT) in Example 4 after treatment at normal temperatures of 25°C and -20°C for 1 hour.

[0029] Figure 4The images show the phenotypic patterns and survival rates of transgenic Arabidopsis thaliana lines (PbrbZIP4-OE-1, PbrbZIP4-OE-2) and wild-type materials (WT) in Example 4 after treatment in the matrix at 25°C and -12°C for 1 hour and 20 minutes, followed by culture at 25°C for 6 days.

[0030] Figure 5 The SOD activity of transgenic Arabidopsis thaliana in Example 5 after treatment at 25°C and -12°C is shown. PbrbZIP4-OE-1 and PbrbZIP4-OE-2 represent two overexpressed transgenic lines, and WT is the wild type.

[0031] Figure 6 The POD activity of transgenic Arabidopsis thaliana (PbrbZIP4-OE-1, PbrbZIP4-OE-2) and wild-type Arabidopsis thaliana (WT) in Example 5 was measured at 25°C and -12°C.

[0032] Figure 7 The CAT activity of transgenic Arabidopsis thaliana (PbrbZIP4-OE-1, PbrbZIP4-OE-2) and wild-type Arabidopsis thaliana (WT) in Example 5 was measured at 25°C and -12°C.

[0033] Figure 8 This refers to the MDA content of Arabidopsis thaliana seedlings overexpressing PbrbZIP4 after treatment at 25℃ and -12℃ in Example 5. PbrbZIP4-OE-1 and PbrbZIP4-OE-2 represent two overexpressing transgenic lines, and WT represents the wild type.

[0034] Figure 9 This refers to the electrolyte leakage rate of transgenic Arabidopsis seedlings overexpressing PbrbZIP4 in Example 5, cultured at 25℃ and -12℃. PbrbZIP4-OE-1 and PbrbZIP4-OE-2 represent two overexpressing transgenic lines, and WT represents the wild type. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0037] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0038] Example 1: Obtaining the PbrbZIP4 gene

[0039] Young leaves of Dangshan pear were ground into powder in liquid nitrogen and processed according to the instructions of the RNA extraction kit. After obtaining RNA, cDNA was synthesized using a cDNA reverse transcription kit and stored at -20°C.

[0040] The applicant identified PbrbZIP4 in pears and located the coding sequence of this gene. Specific primers were designed using Primer Premier5 software.

[0041] PbrbZIP4-F: ATGGCTTCGTCGAAGCTAATGT; (SEQ ID NO: 3)

[0042] PbrbZIP4-R: CCACTGCAATGAGTGAACTCTTCG. (SEQ ID NO: 4)

[0043] Using pear cDNA as a template, PCR amplification was performed using DNA polymerase. The amplification conditions were: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 40 s, for 35 cycles; followed by a final extension at 72℃ for 1 min. The amplified products were detected by agarose gel electrophoresis. After detection, the gel was excised and recovered. The recovered product was sequenced by a sequencing company to obtain the complete sequence, and the gene was named PbrbZIP4, with its nucleotide sequence shown in SEQ ID NO: 1.

[0044] ATGGCTTCGT CGAAGCTAAT GTCGTCGTCG TCGACATCAC AAAACTCGGA TCTCTCCCGA60CGCCCTTCCA CTTTCTCCTC CGCTACAAAG CCCAAGCCCA CCACCACCAC ACCACACCCG120TCTTCCTCCT CTGCGCACAC CCAAACCCTA AACAACTACC AGAACAGCAT CGTTAACACG180ACGCCCTCGC TTCCAATCGG CTCCATGACC GCCGACGGCC TCCTCTACGA CCCCACCCCC240ATGACCGACG CCACCCTCCT CGACACCCAA ATAACCCTAC TCGACGCCGC TGCAAATGTT300AATGGACCAT CAGCGCCGCC GCCTAAGACC GTCGACGAAG TTTGGCGGGA AATTGTGTCC360GGCGACAGTC GGAAGGAGTG TAAAGCGGAG GTTCCGGATG AGATGATGAC ACTGGAGGAC420TTTTTGGCTC GGGCCGGGGC CGTGGAAGAG AACGACATTA AGGATTTTCC GCTTGCGCCA480CCGCCGGAGA TGGAGAGATT GAGGAGGGA GTGTTTTCCT TCGATCAGAT CCCGCTCAGC540CCGTTCGGGT CGATTGATAA GGTCGAAGGG TCGATTGTTG GGTTTGGAAA TGGGGTGGAC600CCTGCTGGGA GTGGAGGTAG GGTGGGGAGG GGGAAGAGAG GGCGCGCCGT CATGGAGCCG660ATGGATAAGG CGGCGCAGCA GAGACAGAAA AGAATGATCA AGAACCGGGA ATCCGCGGCC720AGGTCCAGAG AGAGAAAGCA GGCTTACCAA GTTGAATTGG AGTCATTGGC GGTCAGGTTA780GAGGAGGAAA AAGAGCAGCT TTTGAAAGAA CAGGCCGAGA GGACTCGGGC AAGACTTAAG840CAGCTAATGG AAAACATCAT TCCGGTTGTG GAGAAGCGAAGACCACCTCG TGTGCTCCGA900AGAGTTCACT CATTGCAGTG GTAA 924

[0045] (SEQ ID NO: 1)

[0046] The encoded amino acid sequence is shown in SEQ ID NO: 2:

[0047] MASSKLMSSSSTSQNSDLSRRPSTFSSATKPKPTTTTPHPSSSSAHTQTLNNYQNSIVNTTPSLPIGSMTADGLLYDPTPMTDATLLDTQITLLDAAANVNGPSAPPPKTVDEVWREIVSGDSRKECKAEVPDEMMTLEDFLARAGAVEENDIKD FPLAPPPEMERLSSGVFSFDQIPLSPFGSIDKVEGSIVGFGNGVDPAGSGGRVGRGKRGRAVMEPMDKAAQQRQKRMIKNRESAARSRERKQAYQVELESLAVRLEEEKEQLLKEQAERTRARLKQLMENIIPVVEKRRPPRVLRRVHSLQW(SEQ ID NO:2)

[0048] Example 2: Expression analysis of the PbrbZIP4 gene

[0049] Rooted pear tissue culture seedlings were selected, and after their growth stabilized, the roots were subjected to low-temperature treatment. Samples were taken at 0h, 1h, 6h, and 12h after treatment, and quickly frozen in liquid nitrogen and stored at -80℃. Total RNA was then extracted from the samples using an RNA extraction kit and reverse transcribed into cDNA.

[0050] Design quantitative primers based on the PbrbZIP4 sequence:

[0051] PbrbZIP4-qPCR-F:GCCTCCTCTACGACCCCAC; (SEQ ID NO: 5)

[0052] PbrbZIP4-qPCR-R:CCGCTTTACACTCCTTTCG. (SEQ ID NO: 6)

[0053] Actin, a pear internal reference gene, was used as a control. Reaction systems were prepared according to the kit instructions, with each reaction repeated three times. The reaction program on the quantitative PCR instrument was set as follows: pre-denaturation, 95℃, 2 min; denaturation, 95℃, 15 s; annealing, 58℃, 30 s, for 40 cycles. Melting curve analysis was performed at 72℃. Three biological replicates were set up for each time point. The final value for each sample was expressed as 2. -ΔΔCT The method was used for computational analysis. Results showed that PbrbZIP4 expression significantly increased at 12 hours after low-temperature treatment (e.g., ...). Figure 1 (As shown).

[0054] Example 3: Construction of an overexpression vector for the PbrbZIP4 gene

[0055] PbrbZIP4 was constructed into an overexpression vector, and two restriction enzyme sites, Xba I and BamHI, were selected on the pCAMBIA-1300 overexpression vector. PbrbZIP4 primers were then designed, with corresponding vector homologous sequences added before and after the primers. The primers are as follows:

[0056] P5: gagaacacgggggactctagaATGGCTTCGTCGAAGCTAATGT; (SEQ ID NO: 7) P6: gcccttgctcaccatggatccCCACTGCAATGAGTGAACTCTTCG. (SEQ ID NO: 8)

[0057] The pCAMBIA-1300 vector was double-digested using two restriction endonucleases, Xba I and BamHI, to obtain a linearized vector.

[0058] Using pear leaf cDNA as a template, the complete coding sequence of PbrbZIP4 was amplified using primers. The cDNA was recovered using a kit, ligated into a vector, and transformed into *E. coli* (DH5α) competent cells. The ligation product was then placed on ice for 5 minutes, followed by incubation in a 42°C metal bath for 1 minute, and then back on ice for 10 minutes. Antibiotic-free LB medium was then added to the tubes, and the tubes were incubated at 37°C with shaking for 1 hour. Bacterial cells were then collected and plated onto plates containing the corresponding antibiotic. After 12 hours, single colonies were picked and propagated in liquid LB medium containing the same concentration of kanamycin sulfate. PCR and agarose gel electrophoresis were used to detect the presence of the target band for further sequencing. After successful sequencing alignment, 50% glycerol was added as needed, and the cells were stored at -80°C.

[0059] Example 4: Arabidopsis thaliana transformation and screening

[0060] Agrobacterium, after being transformed into the PbrbZIP4 overexpression vector and activated, was used to infect Arabidopsis thaliana using the flower immersion method. An Arabidopsis thaliana infection suspension was prepared, and Agrobacterium colonies were mixed by pipetting with the infection solution. Wild-type Arabidopsis thaliana plants that had bolted one week prior were then subjected to flower immersion infection when the OD600 was measured to be 0.6. The infected Arabidopsis thaliana plants were then treated in darkness for 1-2 days, and reinfected after one week. Finally, seeds were collected. To obtain transgenic Arabidopsis thaliana plants with stable overexpression of PbrbZIP4, selection was required from generation T0 to T2. The collected seeds were washed, dried, and evenly sown on a selection medium containing hygromycin. After approximately one week of cultivation in a light incubator, leaf DNA was extracted from healthy seedlings for identification; positive plants were designated as T0 plants. Further self-pollination and selection were then performed to obtain the T2 generation of transgenic Arabidopsis thaliana.

[0061] Example 5: Verification of the freeze resistance of transgenic plants

[0062] (1) Frost Resistance Identification (MS Medium Method): Appropriate amounts of transgenic and wild-type Arabidopsis seeds were washed, dried, and sown separately on normal MS medium. One group was cultured at 25℃, and the other at 4℃. Phenotypic differences were observed. After two weeks of growth on the medium, significant differences in root length were observed under different temperature treatments. At 25℃, there were no significant differences in growth and root length between wild-type and transgenic plants; however, at 4℃, the root length of Arabidopsis plants overexpressing PbrbZIP4 was significantly longer than that of wild-type plants (e.g., ...). Figure 2 (As shown).

[0063] Appropriate amounts of transgenic and wild-type Arabidopsis seeds were washed, dried, and sown separately on normal MS medium. They were cultured normally for one week in a 25℃ light incubator, followed by freeze-thaw treatment. One group remained at 25℃, while the other group was treated at -20℃ for 1 hour. Phenotypic differences were observed. As shown in the figure, there was no significant difference in growth and survival rate between wild-type and transgenic plants at 25℃. However, the survival rate of Arabidopsis plants overexpressing PbrbZIP4 treated at -20℃ for 1 hour was significantly higher than that of wild-type plants (e.g., ...). Figure 3 (As shown).

[0064] Appropriate amounts of transgenic and wild-type Arabidopsis seeds were washed, dried, and sown separately on normal MS medium. After one week of normal culture in a 25℃ light incubator, they were transplanted into soil for greenhouse growth. Twenty days later, they were subjected to a -12℃ low-temperature treatment for 1 hour and 20 minutes, followed by 6 days of culture at 25℃. Phenotypic differences in the plants were compared. At 25℃, there was no significant difference in leaf condition and survival rate between wild-type and transgenic plants. Plants that had undergone low-temperature treatment and then returned to normal temperature showed yellowing leaves and a significantly decreased survival rate; however, the survival rate of Arabidopsis plants overexpressing PbrbZIP4 was significantly higher than that of wild-type plants (e.g., ...). Figure 4 (As shown).

[0065] (2) Determination of physiological indicators at low temperature: The antioxidant enzyme activity, MDA content and electrolyte extravasation rate of wild-type and PbrbZIP4 overexpressing Arabidopsis thaliana were determined at normal temperatures of 25℃ and -12℃, respectively.

[0066] Superoxide dismutase (SOD) scavenges superoxide free radicals generated under abiotic stress, thereby enabling plants to resist the damage to cells caused by harmful substances produced during metabolic processes under abiotic stress, exhibiting a certain degree of stress resistance. Under low-temperature stress, SOD synthesis is inhibited, and its activity begins to decline. At 25℃, the SOD activity of PbrbZIP4-overexpressing plants and wild-type plants is almost identical, with no significant difference. At -12℃, the SOD activity of PbrbZIP4-overexpressing Arabidopsis thaliana is significantly higher than that of wild-type Arabidopsis thaliana (e.g., ...). Figure 5 As shown in the figure, overexpression of PbrbZIP4 increases the cold resistance of plants. POD is mainly located at the plasmolysis boundary of plant cells and can scavenge intracellular peroxides, mitigating oxidative damage caused by environmental stress. At normal temperature (25℃), there is no significant difference in POD activity between transgenic and wild-type plants. We found that POD activity increases under low-temperature stress, but the POD activity of transgenic plants is significantly higher than that of wild-type plants (e.g., ...). Figure 6 (As shown). At 25℃, there was no significant difference in CAT activity between plants overexpressing PbrbZIP4 and wild-type plants. At -12℃, the CAT activity of plants overexpressing PbrbZIP4 was significantly higher than that of wild-type plants, showing a statistically significant difference (e.g., ...). Figure 7 (As shown).

[0067] Low-temperature stress disrupts the balance between free radical production and elimination in plant cells. Therefore, cell membrane lipid peroxidation occurs under low-temperature stress, generating MDA. As shown in the figure, at 25℃, there was no significant difference in MDA content between leaves of PbrbZIP4-overexpressing and wild-type plants. At -12℃, the MDA content in leaves of PbrbZIP4-overexpressing Arabidopsis thaliana was significantly lower than that in wild-type Arabidopsis thaliana (e.g., ...). Figure 8 As shown in the figure, overexpression of PbrbZIP4 increases the plant's cold resistance.

[0068] Low-temperature stress can cause changes in the structure of plant cell membranes, increasing membrane permeability and leading to the leakage of intracellular solutes. The amount of electrolyte leakage from plant cells often reflects the severity of damage caused by low temperatures. At 25℃, there was no significant difference in electrolyte leakage rates between PbrbZIP4-overexpressing plants and wild-type plants. At -12℃, under low-temperature stress, the electrolyte leakage rates of both PbrbZIP4-overexpressing and wild-type plants increased significantly; however, the electrolyte leakage rate of PbrbZIP4-overexpressing Arabidopsis thaliana was significantly lower than that of wild-type Arabidopsis thaliana (e.g., ...). Figure 9 (As shown).

[0069] All of the above physiological indicators suggest that overexpression of PbrbZIP4 is beneficial in resisting the negative effects of low-temperature stress.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of pear PbrbZIP4 Genes, characterized by, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The pear according to claim 1 PbrbZIP4 Gene-encoded proteins are characterized by, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A pear containing the pear as described in claim 1 PbrbZIP4 Gene-based biomaterials, characterized in that, The biological materials are expression vectors and host bacteria.

4. The biomaterial according to claim 3, characterized in that, The expression vector is pCAMBIA-1300-PbrbZIP4.

5. The biomaterial according to claim 3, characterized in that, The host bacterium is Agrobacterium.

6. The pear according to claim 1 PbrbZIP4 The use of the gene or the biological material according to any one of claims 3-5 in any of the following situations: (1) Used to improve the ability of plants to resist low temperature stress; (2) Used to improve the activity of antioxidant enzymes in plants under low temperature stress; (3) Used to enhance the frost resistance of transgenic plants; (4) Used to improve the survival rate of plants under frost damage; The plant in question is Arabidopsis thaliana.

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