Application of ZmPYL11 gene in improvement of drought resistance of plants

Abstract

The invention provides an application of a ZmPYL11 gene in improvement of drought resistance of plants, and finds that the ZmPYL11 gene can significantly improve the drought resistance of corn, and through overexpression of the ZmPYL11 gene in corn, a corn plant with higher drought resistance is successfully constructed, and the application of the ZmPYL11 gene in the corn is realized. The relative water content of the leaf of the constructed ZmPYL11 overexpression plant after drought treatment is higher than that of the wild type, the leaf stomata is more sensitive to the process of promoting stomata closing by abscisic acid (ABA) than the wild type, the water loss rate of the in-vitro leaf is reduced, the drought resistance is obviously enhanced, and the yield is increased. The gene has wide application potential and market value in the field of agricultural planting, especially in improving the drought resistance of plants and cultivating drought-resistant transgenic crops.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to an application of a ZmPYL11 gene in improving the drought resistance of plants. Background Art

[0002] With the change of global climate, drought and water shortage have become one of the main factors restricting crop growth and yield. For a long time, plants have evolved molecular mechanisms to respond to drought stress in order to cope with drought stress. The transduction of drought stress signals in plants is divided into two signal transduction pathways: ABA (abscisic acid)-dependent and ABA-independent. ABA receptor PYR1 / PYLs / RCARs is a signal regulator upstream of the ABA signaling pathway. It has the function of recognizing ABA signals and initiating signal transduction processes, and plays multiple roles in plant growth, development and stress response. In Arabidopsis, 14 PYLs have been found. PYLs have functional redundancy in perceiving ABA signals. Mutations in a single pyl often have no obvious phenotype. During the growth and development of mutants of multiple genes such as pyr1pyl1 pyl2 pyl4 pyl5 pyl8, it was found that both their growth and stomata showed an insensitive phenotype to ABA. There are 13 PYLs predicted in the rice genome. Overexpression of rice PYL3, PYL5, PYL9 or PYL11 can improve drought resistance, lead to hypersensitivity to ABA, and inhibit seed germination and seedling growth. Overexpression of wheat PYL4 can increase wheat yield under drought stress conditions. In the maize genome, 13 gene family members ZmPYL1-13 homologous to Arabidopsis PYR1 / PYLs were identified. The 13 members of the ZmPYLs gene family have different expression patterns in different developmental stages and different tissues and organs of maize. ZmPYL8 and ZmPYL9 are expressed at the highest levels in maize kernels, while the expression levels in other parts are lower. ZmPYL10 and ZmPYL11 are expressed at high levels in the whole plant, indicating that different ZmPYLs may have functional specificity.

[0003] The functions of PYLs and their responses to drought in wheat and rice have been partially reported, but the functions of ZmPYLs genes in maize have rarely been reported. Summary of the invention

[0004] The present invention provides an application of a ZmPYL11 gene in improving the drought resistance of plants. The present invention finds that the ZmPYL11 gene can significantly improve the drought resistance of corn. The present invention further overexpresses the ZmPYL11 gene in corn by using an Agrobacterium-mediated method, and successfully constructs corn plants with stronger drought resistance.

[0005] In a first aspect, the present invention provides a use of a ZmPYL11 protein in improving drought resistance of plants, wherein the amino acid sequence of the ZmPYL11 protein is at least one of the following: (1) as shown in any one of SEQ ID NOs. 3-4; (2) a protein derived from any one of SEQ ID NOs. 3-4 by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs. 3-4 and retaining the function of the amino acid sequence shown in any one of SEQ ID NOs. 3-4; (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in any one of SEQ ID NOs. 3-4 and having an equivalent functional protein; (4) An amino acid sequence obtained by connecting a tag, a restriction site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences of (1) to (3).

[0006] The present invention provides an application of a ZmPYL11 gene in improving the drought resistance of plants, wherein the nucleotide sequence of the ZmPYL11 gene is at least one of the following: (1) as shown in any one of SEQ ID NOs. 1-2; (2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of any one of SEQ ID NO. 1-2 and the nucleotide sequence expresses a protein with the same function; (3) a nucleotide sequence that hybridizes with the sequence shown in any one of SEQ ID NOs. 1-2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1× SSPE solution containing 0.1% SDS or a 0.1× SSC solution containing 0.1% SDS, and the membrane is washed with the solution; (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in any one of SEQ ID NOs. 1-2 and expresses a protein with the same function.

[0007] The ZmPYL11 gene has a gene number of Zm00001eb204180 in Zea mays (B73_RefGen_v5), a gene number of Zm00001d053396 in Zeamays (B73_RefGen_v4), and a version encoding of GRMZM2G048733 in Zea mays (B73_RefGen_v3). The ZmPYL11 gene Genomic DNA consists of 3322 bases, with a total of 2 transcripts. The gene has 3 exons and 2 introns. Since the same DNA sequence in corn can produce different transcripts and translate different proteins, the different transcripts produced by the sequence are shown in SEQ ID NO.1-2, wherein the protein amino acid sequence translated from the sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.3, and the protein amino acid sequence translated from the sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.4.

[0008] In a second aspect, the present invention provides a method for improving the drought resistance of plants, specifically, increasing the expression level of the ZmPYL11 gene in plants, and the method includes but is not limited to: (1) introducing a vector that overexpresses the ZmPYL11 gene into plants through genetic engineering; or (2) Selecting and modifying the promoter that regulates the expression of the ZmPYL11 gene into a strong promoter through genetic engineering; or (3) increasing the copy number of the gene encoding ZmPYL11 in plant chromosomes by genetic engineering; or (4) Introducing enhancers through genetic engineering; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NOs. 1-2.

[0009] Preferably, the plasmid containing the ZmPYL11 gene is transformed into plants and self-fertilized. In some embodiments of the present invention, Agrobacterium-mediated transformation of plant callus is specifically used to obtain primary transgenic plants through differentiation culture, and breeding is carried out by self-fertilization.

[0010] In a third aspect, the present invention provides a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or the method described, for use in cultivating drought-tolerant plants; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

[0011] The present invention also provides a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or the method described above, for use in increasing the water content of plant seedlings in a drought environment; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

[0012] The present invention also provides a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or an application of the method described above in improving the sensitivity of plants to abscisic acid; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

[0013] The present invention finds that overexpressing the ZmPYL11 gene in a plant can increase the sensitivity of the plant to abscisic acid, reduce the stomatal aperture of the plant after abscisic acid treatment, thereby reducing water evaporation and enhancing the drought resistance of the plant.

[0014] The present invention also provides a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or the method described above, for use in reducing the water loss rate of detached plant leaves; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

[0015] The present invention found that the water loss rate of detached leaves of plants overexpressing the ZmPYL11 gene was significantly lower than that of the wild type, indicating that the ZmPYL11 gene improves the drought resistance of plants mainly due to the slowing down of the water loss rate of plants.

[0016] The present invention also provides a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or an application of the method described above in improving plant germplasm resources; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

[0017] Preferably, the plant described in the above application or method is corn, wheat, cabbage, rapeseed, broccoli, spinach, lettuce, kale, celery, water spinach, preferably corn.

[0018] The beneficial effects achieved by the present invention include: The present invention found that the ZmPYL11 gene can significantly improve the drought resistance of corn. By overexpressing the ZmPYL11 gene in corn, the present invention successfully constructed corn plants with stronger drought resistance. The relative water content of the leaves of the ZmPYL11 overexpressing plant seedlings after drought treatment was 29.25%, 29.66% and 31.03% higher than that of the wild type, respectively. The leaf stomata were more sensitive to the stomatal closure process promoted by abscisic acid (ABA) than the wild type. The average values ​​of stomatal opening after ABA treatment were 2.44 μm, 2.25 μm and 2.44 μm, respectively, which were reduced by 31.46%, 36.80% and 31.46% compared with the wild type, respectively. The water loss rate of the detached leaves was reduced, and the drought resistance was significantly enhanced. This mechanism is of great significance in the transportation and storage of various leafy vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the detection result of the expression level of the ZmPYL11 overexpressing plant in Example 1 of the present invention.

[0021] Figure 2 This is a diagram showing the phenotypic results of the ZmPYL11 overexpressing plants and the wild type under drought treatment in Example 2 of the present invention.

[0022] Figure 3 This is a graph showing the relative water content of leaves of ZmPYL11 overexpressing plants and wild type plants under drought treatment in Example 2 of the present invention.

[0023] Figure 4 This is a graph showing the aboveground dry weight results of ZmPYL11 overexpressing plants and wild type plants under drought treatment in Example 2 of the present invention.

[0024] Figure 5 This is a graph showing the stomatal aperture changes of ZmPYL11 overexpressing plants and wild type under ABA treatment in Example 3 of the present invention.

[0025] Figure 6 This is a graph showing the water loss rate of detached leaves of ZmPYL11 overexpressing plants and wild type plants under drought treatment in Example 4 of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The pBCXUN vector recombinant vector used in the present invention is provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0027] Example 1 Preparation of ZmPYL11 overexpressing corn plants 1. Construction and detection of ZmPYL11 gene overexpression vector Total RNA was extracted from B73 maize (Zea mays L.), and cDNA was obtained by reverse transcription. The CDS region of the target gene ZmPYL11 was amplified using upstream and downstream primers and high-fidelity enzymes using cDNA as a template. In order to facilitate subsequent protein experiments, the MYC tag DNA sequence (SEQ ID No.5) was fused before the CDS region of ZmPYL11. The ZmPYL11 gene DNA sequence with the MYC tag was inserted into the pBCXUN vector using restriction endonuclease XcmⅠ. The recombinant vector can express the protein shown in (Seq ID No.6).

[0028] 2. Identification of Overexpressing Transgenic Maize Plants The constructed pBCXUN recombinant expression vector (promoter is maize Ubiquitin promoter) containing the target gene ZmPYL11 was transformed into Agrobacterium EHA105 strain. Transgenic operation was carried out through Agrobacterium-mediated transgenic operation into the immature embryos of the maize inbred line ND101 (ZmCRK1 negatively regulates maize's response to drought stress byphosphorylating plasma membrane H+-ATPase ZmMHA2. New Phytologist, 2024.8), dedifferentiated to form callus tissue, and then differentiated to form tissue organs to obtain T0 generation transgenic plants.

[0029] The T0 transgenic seedlings were screened using Basta resistance medium, and the T0 plants with a copy number of 1 were transplanted to the greenhouse of the West Campus of China Agricultural University for self-pollination to obtain T1 transgenic seeds; T1 transgenic plants were self-pollinated to obtain T2 transgenic seeds; T2 transgenic plants were self-pollinated again to obtain T3 transgenic seeds. In each generation, positive transgenic plants were identified by PCR amplification, and strict self-pollination was performed to obtain the next generation of seeds.

[0030] In order to detect the expression of ZmPYL11 gene in transgenic lines, RNA was extracted from the leaves of the recipient ND101 and three transgenic lines OE#1, OE#2, and OE#3 at the seedling stage, reverse transcribed into corresponding cDNA, and fluorescence quantitative PCR experiments were performed to detect its expression level (primers are as follows, results are shown in Figure 1 Seeds of T3 transgenic homozygous lines were used for subsequent experiments.

[0031] Primer-F: 5'-GGAACATCGAGATCGGTAGCG-3' (Seq ID No. 7).

[0032] Primer-R: 5'-ATGGAGGAGTAGTTCTGCAGCC-3' (Seq ID No. 8).

[0033] Result analysis: Depend on Figure 1 It can be seen that the ZmPYL11 gene was successfully transferred into the corn plant and integrated into the genomic DNA, and can be stably propagated under self-pollination.

[0034] Example 2 Application of ZmPYL11 overexpressing plants in drought stress 1. Soil drought treatment and phenotypic detection of ZmPYL11 overexpressing plants at the seedling stage Imported soil: peat soil: vermiculite were mixed in a ratio of 1:1:1 and placed in small pots, with 170 g of soil in each small pot. Seeds of wild-type and ZmPYL11 overexpressing plants were sown in the soil, 4 seeds per pot, evenly distributed. When the seedlings grew to the one-leaf and one-heart stage, thinning was performed, and 3 plants with relatively consistent growth size and status were retained in each pot. When the seedlings grew to the two-leaf and one-heart stage, water was added to the tray, and after the water was fully absorbed, all small pots were transferred to another dry tray. The wild-type and overexpressing plants were divided into two groups, respectively. At this time, the planted wild-type and overexpressing plants were divided into two treatments (normal watering treatment and drought treatment), and the normal watering treatment continued to be cultivated normally, watered on time, and water was added to the tray once every 3 days. After the water was absorbed to saturation, all small pots were transferred to another dry tray. The plants in the drought treatment were no longer watered, and drought stress treatment was carried out, and observation was performed regularly every day.

[0035] The drought treatment lasted for about 7 days (at this time, the three leaves and one heart stage) and the photos were recorded. Figure 2 As shown, Figure 2 The left side shows the normal watering condition, and the right side shows the drought treatment condition.

[0036] 2. Detection of relative water content of leaves during drought treatment at the seedling stage Observe the state of the seedlings in the pots. When drought phenotypes appear, take 5 pots of overexpression plants and control plants under normal watering and drought conditions, take the third fully expanded leaf, and the leaves of 3 plants in each pot are 1 replicate, with a total of 5 replicates. Weigh the weight of the leaves and record it as W1; then put the leaf segments into a 50 mL centrifuge tube, add distilled water, and let the leaves absorb water to saturation (about 24 hours); then wipe the water on the leaves with absorbent paper, weigh the saturated weight and record it as W2; after weighing, dry the leaves (80℃, 3 days), and measure the dry weight and record it as W3. Calculate the relative water content, relative water content = (W1-W3) / (W2-W3)×100%, the results are as follows Figure 3 shown.

[0037] 3. Measurement of aboveground dry weight of seedlings during drought treatment Observe the status of the seedlings in the pots. When drought phenotypes appear, take 5 pots of overexpression plants and control plants under normal watering and drought conditions, and 3 corn plants in each pot as 1 replicate, for a total of 5 replicates. Take the aboveground part of the corn plant in a kraft paper bag, dry it in an oven at 80 degrees (about 3 days), and then weigh the dry weight of the aboveground part.

[0038] Result analysis: Depend on Figure 2 It can be seen that after drought treatment, the leaves of plants overexpressing the ZmPYL11 gene were more stretched than those of the wild type, showing an obvious drought-resistant phenotype.

[0039] Depend on Figure 3 It can be seen that under normal watering conditions, there was no significant difference in the relative water content of leaves between the ZmPYL11 gene overexpressing plants and the wild type. The relative water content of leaves of the ZmPYL11 gene overexpressing plants under drought treatment was significantly higher than that of the wild type, among which the relative water content of leaves of PYL11OE#1 was 94.56%, the relative water content of leaves of PYL11OE#2 was 94.80%, the relative water content of leaves of PYL11OE#3 was 95.76%, and the relative water content of leaves of the wild type was 73.12%.

[0040] Depend on Figure 4 It can be seen that drought treatment will inhibit plant growth, and the degree of inhibition on the wild type after drought treatment is greater than that on the ZmPYL11 overexpressing plants.

[0041] Example 3 Detection of the effect of exogenous ABA on stomatal aperture in ZmPYL11 overexpressing plants The leaves of wild-type and ZmPYL11 overexpressing plants were used as the operation objects, and the control group and experimental group (10μM ABA treatment) were set up respectively. Transparent nail polish was applied on the back of the corn leaves, and the lower epidermis was torn off. The photos were taken under a microscope, and the stomatal aperture (expressed by the horizontal diameter of the stomatal opening) was counted using ImageJ software.

[0042] The results of the stomatal movement experiment are as follows Figure 5 Under the control condition, there was no difference in stomatal aperture among the plants, all around 6.00 μm; under ABA treatment, the stomatal aperture of the three groups of ZmPYL11 overexpressing plants was smaller than that of the wild type, 2.44 μm, 2.25 μm and 2.44 μm respectively, while that of the wild type was 3.56 μm, indicating that ZmPYL11 overexpressing plants were more sensitive to ABA, and the ZmPYL11 gene responded to the regulation of plant stomatal movement and played an important role in plant response to drought stress.

[0043] Example 4 Detection of water loss rate of detached leaves of ZmPYL11 overexpressing plants The second fully expanded leaf of corn seedlings that have grown normally to the V2 stage was removed, and the initial fresh weight was immediately weighed with an electronic balance, and the fresh weight was weighed with a 1 / 10,000 electronic balance; the weight was weighed after being placed for 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 hours under normal light conditions at a temperature of 27°C and a humidity of 30%-45%. The water loss rate was calculated as follows: water loss rate = (initial weight - weight after water loss) / initial weight × 100%.

[0044] The results of the water loss rate test of detached leaves are as follows Figure 6 As shown, the water loss rate of detached leaves of the overexpressing plants was significantly lower than that of the wild type, indicating that the drought resistance phenotype of the ZmPYL11 overexpressing material was mainly caused by the slower water loss rate of the plants.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A use of ZmPYL11 protein in improving plant drought resistance, characterized in that: The amino acid sequence of the ZmPYL11 protein is at least one of the following: (1) as shown in any one of SEQ ID NOs. 3-4; (2) a protein derived from any one of SEQ ID NOs. 3-4 by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs. 3-4 and retaining the function of the amino acid sequence shown in any one of SEQ ID NOs. 3-4; (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in any one of SEQ ID NOs. 3-4 and having an equivalent functional protein; (4) An amino acid sequence obtained by connecting a tag, a restriction site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences of (1) to (3).

2. An application of ZmPYL11 gene in improving drought resistance of plants, characterized in that: The nucleotide sequence of the ZmPYL11 gene is at least one of the following: (1) as shown in any one of SEQ ID NOs. 1-2; (2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of any one of SEQ ID NO. 1-2 and the nucleotide sequence expresses a protein with the same function; (3) a nucleotide sequence that hybridizes with the sequence shown in any one of SEQ ID NOs. 1-2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1× SSPE solution containing 0.1% SDS or a 0.1× SSC solution containing 0.1% SDS, and the membrane is washed with the solution; (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in any one of SEQ ID NOs. 1-2 and expresses a protein with the same function.

3. A method for improving the drought resistance of plants, characterized in that: The method for increasing the expression level of the ZmPYL11 gene in a plant includes but is not limited to: (1) Using genetic engineering methods to transfer a vector that overexpresses the ZmPYL11 gene into plants; or (2) Selecting and modifying the promoter that regulates the expression of the ZmPYL11 gene into a strong promoter through genetic engineering; or (3) increasing the copy number of the gene encoding ZmPYL11 in plant chromosomes by genetic engineering; or (4) Introducing enhancers through genetic engineering; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NOs. 1-2.

4. The method according to claim 3, characterized in that: The plasmid containing the ZmPYL11 gene was transformed into plants and self-bred.

5. Use of a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or a method according to any one of claims 3 to 4 in cultivating drought-tolerant plants; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

6. Use of a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or a method according to any one of claims 3 to 4 in increasing the water content of plant seedlings under drought conditions; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

7. Use of a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or a method according to any one of claims 3 to 4 in improving the sensitivity of plants to abscisic acid; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

8. Use of a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or a method according to any one of claims 3 to 4 in reducing the water loss rate of detached plant leaves; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

9. Use of a ZmPYL11 gene, or a protein encoded by it, or a biological material containing the gene, or a method according to any one of claims 3 to 4 in improving plant germplasm resources; The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2; or The amino acid sequence of the protein is shown in any one of SEQ ID NO.3-4.

10. The use according to any one of claims 1-2, 5-9 or the method according to any one of claims 3-4, characterized in that: The plants are corn, wheat, cabbage, rapeseed, broccoli, spinach, lettuce, kale, celery and water spinach.

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