Method for enhancing aluminum resistance of plants

Abstract

The invention discloses an application of protein kinase CPK28 (GenBank: AT5G66210) in endowing or enhancing aluminum resistance of plants, and the CPK28 is overexpressed in the plants, so that a zinc finger transcription factor STOP1 can be phosphorylated, and the protein accumulation of the STOP1 is improved, thereby enabling the plants to generate or enhance the aluminum toxicity resistance. The invention provides a theoretical basis for culturing aluminum toxicity resistant crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bioengineering and relates to a method for enhancing aluminum resistance in plants, specifically the application of a kinase CPK28 in conferring or enhancing aluminum resistance in plants. Background Technology

[0002] The zinc finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1), a key regulatory protein for biological non-stress, is crucial for enhancing plant resilience to various stresses (Sadhukhan et al., 2021). These stresses include aluminum (Al) stress, low pH, low phosphate-induced iron toxicity, low potassium, low nitrogen, low boron, and low oxygen, as well as high salinity and high ammonia. Notably, aluminum stress poses a significant obstacle to plant growth in acidic soils, which account for approximately 30% of the world's total land area and may even reach 50% of the world's potential arable land.

[0003] To combat aluminum toxicity, plants have evolved various aluminum detoxification mechanisms. The key mechanism employed by most plant species is the secretion of aluminum-induced organic acids, such as malic acid, citric acid, and oxalic acid, to facilitate aluminum detoxification. Different plant species secrete specific types of organic acids to chelate aluminum. 3+ Ions exhibit different preferences to prevent them from penetrating and damaging root tips, which are the primary targets of aluminum toxicity. Transport proteins responsible for the secretion of malic and citric acid were initially identified in crops, belonging to the aluminum-activated malate transporter (ALMT) and multidrug and toxin excretion (MATE) families, respectively. Therefore, in subsequent studies, Arabidopsis thaliana ALMT1 and MATE were identified as key players in the secretion of malic and citric acid. Arabidopsis roots release large amounts of malic acid, accompanied by a smaller proportion of citric acid, to effectively combat aluminum toxicity.

[0004] In Arabidopsis, the stress-regulating protein STOP1 primarily regulates aluminum resistance by inducing ALMT1 expression, while also inducing the expression of other aluminum resistance genes, including MATE, ALS3, GDH1 / 2, and SAUR55. Aluminum stress can trigger a MEKK1-MKK1 / 1-MPK4 cascade via an unknown mechanism. This cascade phosphorylates STOP1, increasing its accumulation by inhibiting the interaction between STOP1 and the F-box protein REGULATION OF ALMT1 EXPRESSION (RAE1). Conversely, aluminum stress can also trigger the accumulation of H2O2, which oxidizes and promotes the degradation of STOP1, enhancing the interaction between STOP1 and RAE1.

[0005] Under aluminum stress, STOP1 accumulation is induced by post-transcriptional regulatory mechanisms. However, the upstream signaling pathways responsible for aluminum-triggered STOP1 accumulation remain largely unclear. Summary of the Invention

[0006] In our research on aluminum stress in plants, our group discovered that CPK28 (GenBank: AT5G66210 or AAQ56823.1) in Arabidopsis thaliana is a calcium-dependent protein kinase (CDPK or CPK). 2+ Signaling can induce the activity of CPK28 kinase. The protein kinase CPK28 (GenBank: AT5G66210 or AAQ56823.1) can phosphorylate the Ser163 site of the zinc finger transcription factor STOP1. This Ser163 phosphorylation event inhibits the interaction between STOP1 and the F-box protein RAE1, thereby promoting STOP1 accumulation and enhancing the aluminum resistance (aluminum stress resistance, aluminum toxicity resistance, aluminum stress resistance) of Arabidopsis thaliana. The study also found that the kinase activity of CPK28 and its phosphorylation of the zinc finger transcription factor STOP1 are influenced by Ca2+. 2+ Regulation, i.e., Ca 2+ This invention positively regulates the kinase activity of protein kinase CPK28 and its phosphorylation of STOP1. Based on this research, the present invention includes the following technical solutions.

[0007] The first aspect of the present invention provides the use of a polypeptide or its encoding gene in conferring or enhancing aluminum resistance in plants, said polypeptide being selected from the group consisting of:

[0008] (a) A polypeptide with the amino acid sequence shown in SEQ ID NO:1, which is a protein kinase CPK28 (GenBank: AT5G66210 or AAQ56823.1) derived from Arabidopsis thaliana;

[0009] (b) A conserved variant polypeptide derived from (a) having the polypeptide function of (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO:1.

[0010] (c) A polypeptide derived from (a) having 90% or more homology with the polypeptide sequence defined in (a), preferably 92% or more homology, preferably 95% or more homology, preferably 97% or more homology, preferably 98% or more homology, more preferably 99% or more homology, and having the function of the polypeptide in (a); or

[0011] (d) A derivative polypeptide containing the polypeptide sequence described in (a), (b), or (c).

[0012] (SEQ ID NO:1).

[0013] The aforementioned function refers to the phosphorylation of zinc finger transcription factor STOP1 Ser163, which leads to the accumulation of STOP1 in plants, i.e., increases the abundance of STOP1 protein, thereby enabling plants to develop aluminum stress resistance, i.e., aluminum stress resistance, aluminum toxicity resistance, and aluminum resistance.

[0014] Preferably, the nucleotide sequence encoding the polypeptide CPK28 (GenBank: AT5G66210 or AAQ56823.1) shown in SEQ ID NO:1 is SEQ ID NO:2, or is a nucleotide sequence that has more than 90% homology, preferably more than 92% homology, preferably more than 95% homology, preferably more than 97% homology, preferably more than 98% homology, and more preferably more than 99% homology with SEQ ID NO:2.

[0015] As a specific application, the aforementioned polypeptides or their encoding genes are used to create aluminum-resistant plant germplasm or to cultivate aluminum-resistant plant varieties.

[0016] Preferably, the above-mentioned plants are crops selected from the group consisting of: rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, tobacco, and cruciferous plants such as Arabidopsis thaliana.

[0017] As a specific application of the above-mentioned polypeptide or its encoding gene, the present invention provides a method for improving plant aluminum stress resistance, comprising the following steps: inserting or overexpressing the encoding gene of the polypeptide described above, such as CPK28 (GenBank: AT5G66210 or AAQ56823.1), into the plant genome. The overexpressed polypeptide can be used to phosphorylate the zinc finger transcription factor STOP1 and lead to the accumulation of the zinc finger transcription factor STOP1 in the plant, thereby improving the plant's aluminum resistance.

[0018] Alternatively, the above method may be implemented, for example, in the following manner:

[0019] (i) The gene encoding the polypeptide as described above is cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, such as the CPK28 overexpression vector, and plants are transformed by Agrobacterium-mediated transformation to obtain transgenic plants that overexpress the polypeptide.

[0020] (ii) By using gene editing technology, the coding gene of the polypeptide described above is cloned into a plant chromosome to obtain a transgenic plant that overexpresses the coding gene of the polypeptide described above;

[0021] (iii) Placing CPK28 in the genome of plants such as Arabidopsis thaliana under the regulation of a functionally enhanced promoter such as UBQ10.

[0022] In one implementation, the gene editing technology described above may be selected from the group consisting of: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0023] A second aspect of the invention provides a DNA molecule comprising, for example, the nucleotide sequence SEQ ID NO:2 of the gene encoding the polypeptide CPK28 (GenBank: AT5G66210 or AAQ56823.1) as described above, or a nucleotide sequence having 90% or more homology with SEQ ID NO:2, preferably 92% or more homology, preferably 95% or more homology, preferably 97% or more homology, preferably 98% or more homology, and more preferably 99% or more homology. Preferably, the DNA molecule is, for example, an expression cassette / expression box of CPK28.

[0024] A third aspect of the invention provides a recombinant plasmid comprising the DNA molecule described above for overexpressing CPK28 (GenBank: AT5G66210 or AAQ56823.1), said recombinant plasmid being an overexpression vector formed by cloning the DNA molecule described above onto a plasmid vector suitable for expression in Agrobacterium.

[0025] In one embodiment, the plasmid vector can be selected from plant transgenic vectors or modified vectors such as pHB-YFP, pHB-FLAG, pBin19, fluorescent reporter vectors pGreenII0800-LUC, pCAMBIA-nLUC, pCAMBIA1300, pCAMBIA1301, pCAMBIA3300, pCAMBIA2301, pBI121, and pTF102.

[0026] A fourth aspect of the present invention is a microbial engineered bacterium, which is a transformant containing the recombinant plasmid described above, used to mediate the transfer of the recombinant plasmid described above into plants. Preferably, the microorganism is Agrobacterium, such as Agrobacterium tumefaciens, Agrobacterium EHA105, or Agrobacterium GV3101. For example, the above-mentioned recombinant plasmid is transferred into an Agrobacterium strain using a freeze-thaw method to form a microbial engineered bacterium.

[0027] This invention discovers that aluminum stress in Arabidopsis thaliana initiates a complex oscillatory Ca 2+ This signaling pattern rapidly induced the activity of CPK28 kinase, which interacts with the zinc finger transcription factor STOP1 and phosphorylates STOP1 at the Ser163 site. This phosphorylation event inhibits the interaction of STOP1 with the F-box protein RAE1, thereby promoting STOP1 accumulation and enhancing aluminum resistance. This study, through a simulated phosphorylation mutation at the Ser163 site of STOP1, confirmed that calcium resistance... 2+ The regulated kinase CPK28 can effectively induce STOP1 phosphorylation in Arabidopsis thaliana, thereby generating aluminum resistance. We have bred Arabidopsis thaliana with enhanced aluminum resistance, providing a theoretical basis and experimental foundation for cultivating aluminum-resistant crops. Attached Figure Description

[0028] Figure 1 It shows aluminum stress-induced root late MZ and early EZ Ca 2+ Signal generation. a, b: Pseudocolor analysis shows the Ca2+ concentration in the cytoplasm of the whole root (a) and root tip (b) under pH 4.8 treatment with 20 μM AlCl3. 2+ Concentration. A highly sensitive ratio-type Ca2+ expression model was used. 2+Imagery of seven-day-old seedlings of the reporter protein GCaMP6f-mCherry, driven by the UBQ10 promoter. The images show representative pseudocolor photographs at specific time points. The region of interest (ROI) in (b) was used for data analysis in (c). Scale bar, 100 μm. c: Cytoplasmic Ca in the ROI shown in Figure B. 2+ Quantitative analysis of the dynamic response to aluminum stress. The graphs show the Ca2+ response within 30 minutes after aluminum treatment. 2+ Concentration variation over time. Data are expressed as mean ± standard deviation (n = 4). d: Expressing ultrasensitive ratio-type Ca 2+ Seven-day-old seedlings of *Cherry*, which reported the protein GCaMP6f-mCherry, were treated with 20 μM AlCl3 at pH 4.8 for 30 min. e: The effect of CaMP on the cytoplasmic cytoplasm in the ROI shown in (d). 2+ Quantitative analysis of dynamic response to aluminum stress. The graphs show the Ca concentrations in the three ROIs within 30 minutes after treatment. 2+ Horizontal variation over time. Scale bar, 100 μm.

[0029] Figure 2 Quantitative phosphatomics analysis under different aluminum conditions is shown. a: Volcano plot of differentially phosphorylated sites in response to aluminum. Red dots (n=398) and blue dots (n=461) represent sites where phosphorylation levels of root proteins increased and decreased under aluminum induction (|fold change| ≥ 1.5, P < 0.05, two-tailed unpaired t-test). b: GO ontology analysis of root proteins with altered phosphorylation levels in response to aluminum. Left: Molecular Function; Middle: Biological Process; Right: Cellular Component.

[0030] Figure 3 Heatmaps of WRKYs and CPKs in phosphacomics are shown. a: Heatmaps showing sites where phosphorylation levels of WRKY family members change under aluminum induction. b: Heatmaps showing sites where phosphorylation levels of the three CPK members increase or decrease in response to aluminum stress.

[0031] Figure 4 The mutation in CPK28 shows that it reduces aluminum resistance in plants. a: Structural diagram of the CPK28 gene and schematic diagram of the mutation site. b: Representative photographs (left) and quantitative data (right) of aluminum resistance in WT and cpk28 mutants. Seedlings were grown for 7 days on gel medium soaked with 0, 0.75, 1, or 1.25 mM aluminum; aluminum resistance was assessed by measuring relative root length. Scale bar: 1 cm.

[0032] Figure 5The introduction of CPK28 into the cpk28-2 mutant restores the aluminum-sensitive phenotype of the cpk28-2 mutant. Representative photographs (left) and quantitative data (right) of aluminum resistance in WT, cpk28-2, and two reinjection lines (com#1 and com#2). Seedlings were grown for 7 days on solid medium soaked in 0, 0.75, 1, or 1.25 mM AlCl3; aluminum resistance was assessed by measuring relative root length. Scale bar: 1 cm.

[0033] Figure 6 The results showed that the CPK28 mutation reduced malic acid secretion, STOP1 downstream gene expression, and STOP1 accumulation. a: Compared with WT, the cpk28 mutant had reduced malic acid secretion. Seedlings were treated with solutions containing 0 (-Al) or 20 μM AlCl3 (+Al) for 12 hours, and then the malic acid concentration (pmol) in root exudates was analyzed. -1 hour -1 b: After aluminum treatment, the cpk28 mutant accumulated more aluminum than the WT. Seedlings were treated with 0.5 mM CaCl2 solution containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and then the aluminum content in the roots was measured. c: Expression of ALMT1, MATE, ALS3, STOP1, STAR1, and ALS1 in WT and cpk28 mutants. Seedlings were treated with 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and root tips (0-1 cm) were collected for RT-qPCR analysis. d: Representative protein immunoblotting data (left) and quantitative data (right) of STOP1-HA levels. WT and cpk28-2 seedlings carrying the pSTOP1:STOP1-HA transgene were treated with solution containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and root tips (0-1 cm) were collected for immunoblotting analysis. Actin was used as a control. To quantify the relative levels of STOP1-HA, STOP1-HA protein levels were first normalized to their corresponding Actin control protein levels, and then normalized relative to the protein levels of WT under the -Al condition. The data in Figure (ad) are the mean ± standard deviation of three biological replicates. Different letters indicate significant differences between means (P < 0.05, Tukey test performed after ANOVA analysis). An asterisk indicates statistically significant differences (Student's t-test). *** P<0.001).

[0034] Figure 7This study shows that CPK28 overexpression increases STOP1 accumulation and plant aluminum resistance. a: CPK28 overexpression increases the expression of STOP1-regulated genes. Seedlings of wild-type (WT) and two CPK28-overexpressing lines (CPK28-OX) were treated with solutions containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 h, and roots were then collected for RT-qPCR analysis. b: Representative photographs (left) and quantitative data (right) of aluminum resistance in WT and CPK28-overexpressing lines. Seedlings were grown for 7 days on gel media soaked in 0, 0.75, 1, or 1.25 mM AlCl3; aluminum resistance was assessed by measuring relative root length. Scale bar, 1 cm. c: Representative protein immunoblot (left) and quantitative data (right) of STOP1-HA levels in WT and CPK28-overexpressing lines. Seedlings carrying the pSTOP1:STOP1-HA transgene were treated with solutions containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and then roots were collected for immunoblotting analysis. Actin was used as a control. d: Compared with WT, malic acid release was increased in CPK28 overexpression lines. Seedlings were treated with solutions containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and the malic acid concentration (pmol) in root exudates was analyzed. -1 hour -1 The data in (a), (c), and (d) are the mean ± standard deviation of three biological replicates; the data in (b) are the mean ± standard deviation of relative root length from 24 to 26 seedlings. Different letters indicate significant differences between the means (P < 0.05, Tukey test performed after ANOVA analysis).

[0035] Figure 8The interaction between CPK28 and STOP1 and the phosphorylation of STOP1 are shown. a: CPK28 and STOP1 interact in a yeast two-hybrid assay. Yeast cells (OD600 = 1.0) co-expressed CPK28-BD and STOP1-AD, then serially diluted 10-fold and grown on Leu / Trp-deficient SD medium (SD-LT) and SD medium (SD-LTHA) for 3 days at 30°C. b: CPK28 and STOP1 interact in a luciferase-protein interaction assay (Split-LUC). The CaMV 35S promoter-driven construct shown in the figure was co-expressed in *Nicotiana benthamiana* leaves, and interaction-dependent luciferase activity was determined. c: CPK28 and STOP1 interact in a bimolecular fluorescence complementation (BiFC) assay. STOP1-nYFP or NLP8-nYFP was co-transformed with CPK28-cYFP or CPK7-cYFP in *Nicotiana benthamiana* leaves. Interaction-dependent YFP signaling and the plasma membrane localization marker BAK1-mCherry were observed using confocal microscopy. Scale bar: 40 μm. d: STOP1 and CPK28 interact in a co-immunoprecipitation (Co-IP) assay. CPK28-HA was co-expressed with STOP1-FLAG or GFP-FLAG in rae1rah1 protoplasts. Protein extracts were immunoprecipitated with anti-HA magnetic beads and then detected with an anti-FLAG antibody. e: CPK28 phosphorylates WT and mutant versions of STOP1 in vitro. His-labeled WT and mutant versions of STOP1 were co-incubated with MBP-labeled CPK28, and phosphorylated STOP1 was visualized by autoradiography following gel electrophoresis. Recombinant CPK28 and STOP1 were detected by Coomassie brilliant blue staining. f, g: Effects of aluminum (f) and / or lanthanum (g) on ​​CPK28 kinase activity. The roots of pCPK28:CPK28-FLAG transgenic seedlings were treated with 0 or 30 μM AlCl3 for 20, 60, or 180 minutes (f), or with 0 or 30 μM AlCl3 and / or 0 or 50 μM LaCl3 for 60 minutes, followed by immunoprecipitation of CPK28-FLAG (g). Phosphorylation was determined by incubation of recombinant STOP1-GFP-His with the CPK28-FLAG IP product. CPK28-FLAG was detected using anti-FLAG antibody. Data in (e) are mean ± standard deviation of three biological replicates. Different letters indicate significant differences between means (P < 0.05, Tukey test performed after ANOVA analysis).

[0036] Figure 9Phosphorylation at the Ser163 site of STOP1 was shown to enhance STOP1 protein abundance and plant aluminum resistance. a, b: Effects of S163A, S296A, or S399A mutations in STOP1 (a) and S163A or S163D mutations in STOP1 (b) on ALMT1 promoter-driven LUC activity. 35S: STOP1 WT 35S:STOP1 S163A 35S:STOP1 S163D 35S:STOP1 S296A 35S:STOP1 S399A Alternatively, 35S:GFP, along with the pALMT1:LUC reporter gene and the internal control pUBQ10:GUS, were transfected into stop1 protoplasts. The ratio of LUC to GUS activity in protoplasts was measured and compared. c: The effect of S163A or S163D mutations on STOP1 accumulation. 35S:STOP1 WT -LUC、35S:STOP1 S163A -LUC or 35S:STOP1 S163D -LUC, along with the internal control pUBQ10:GUS, is transferred into the stop1 protoplast. d: STOP1 WT and STOP1 S163A Immunoblotting (left) and quantification results (right) of representative proteins representing STOP1-HA levels in transgenic lines. STOP1 in the stop1 background. WT and STOP1 S163A The roots of the transgenic lines were treated with solutions containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and then the roots were collected for immunoblotting analysis. Actin was used as a control. e: STOP1 WT and STOP1 S163A Representative photographs (left) and quantitative data (right) of aluminum resistance in transgenic lines. Seedlings were grown for 7 days on solid media soaked in 0, 0.75, 1, or 1.25 mM aluminum; aluminum resistance was assessed by measuring relative root length. Scale bar: 1 cm. f: STOP1 WT and STOP1 S163D Immunoblotting (left) and quantification results (right) of representative proteins representing STOP1-HA levels in transgenic lines. STOP1 in the stop1 background. WT and STOP1 S163D The roots of the transgenic lines were treated with solutions containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, and then the roots were collected for immunoblotting analysis. Actin was used as an internal control. g: STOP1 WT and STOP1 S163DRepresentative photographs (left) and quantitative data (right) of aluminum resistance in transgenic lines. Seedlings were grown for 7 days on solid media soaked in 0, 0.75, 1, or 1.25 mM aluminum; aluminum resistance was assessed by measuring relative root length. Scale bar is 1 cm. h: pIMAGO was used to detect phosphorylated STOP1. pSTOP1 against the background of stop1: STOP1 WT -HA(STOP1 WT ), pSTOP1:STOP1 S163A -HA(STOP1 S163A Transgenic lines were treated for 3 hours with 0 (-Al) or 30 μM AlCl3 (+Al) and 50 μM MG132, followed by immunoprecipitation with STOP1-HA. Data in (ac, d, f, h) are the mean ± standard deviation of three biological replicates; data in (e, g) are the mean ± standard deviation of relative root length from 24 to 33 seedlings. Different letters indicate significant differences between means (P < 0.05, Tukey test performed after ANOVA analysis).

[0037] Figure 10 This shows that CPK28 phosphorylates STOP1 at the Ser163 site to regulate its accumulation. a: The effect of the cpk28-2 mutation on STOP1 phosphorylation. Carrying pSTOP1:STOP1 WT- Roots of HA transgenic WT and cpk28-2 were treated for 3 hours with 0(-Al) or 30 μM AlCl3(+Al) and 50 μM MG132, followed by root tip (0-1 cm) immunoprecipitation for STOP1-HA and subsequent detection of STOP1 phosphorylation using pIMAGO. b: Effect of S163A mutation on CPK28-mediated STOP1 activity against pALMT1:LUC. 35S:CPK28 or 35S:GFP, 35S:STOP1 WT Or 35S:STOP1 S163A The reporter gene pALMT1:LUC was transfected into stop1 protoplasts for expression. c: The effect of the S163A mutation on CPK28-mediated 35S:STOP1WT-LUC activity. 35S:CPK28 or 35S:GFP and 35S:STOP1 WT -LUC or 35S:STOP1 S163A -LUC was transduced into stop1 protoplasts for expression. For (b, c), pUBQ10:GUS was also transduced into protoplasts as an internal control. The ratio of LUC activity to GUS activity in protoplasts was measured and compared. d: Introduction of pSTOP1:STOP1 S163D -HA(STOP1 S163DThe transgene restored the reduced STOP1 accumulation in the cpk28-2 mutant. pSTOP1:STOP1 WT -HA(STOP1 WT ), cpk28-2, pSTOP1:STOP1 S163D -HA(STOP1 S163D ) and STOP1 S163D The roots of / cpk28-2 were treated with a solution containing 0 (-Al) or 30 μM AlCl3 (+Al) for 12 hours, followed by Western blotting analysis of STOP1-HA. Actin was used as an internal control. e: Introduction of pSTOP1:STOP1 S163D -HA(STOP1 S163D The transgenic strain restored the aluminum-sensitive phenotype of the cpk28-2 mutant. (STOP 1) WT STOP1 S163D cpk28-2 and STOP1 S163D Seedlings of / cpk28-2 were grown for 7 days on solid media soaked in 0, 0.75, 1.0, or 1.25 mM AlCl3 to assess their aluminum resistance. Data in (ad) are the mean ± standard deviation of three biological replicates; data in (e) are the mean ± standard deviation of relative root length from 29 to 34 seedlings. Different letters indicate significant differences between means (P < 0.05, Tukey test performed after ANOVA analysis). Detailed Implementation

[0038] In our study of aluminum stress and aluminum toxicity resistance in Arabidopsis thaliana, we revealed that Ca 2+ The role of signaling in regulating STOP1 accumulation. Aluminum stress in Arabidopsis thaliana initiates a complex process involving Ca2+. 2+ This signaling pattern rapidly induced the activity of CPK28 kinase.

[0039] Ca 2+ As an important second messenger, calmodulin is crucial for regulating plant responses to various stresses. Three major protein families in plants—calmodulin (CAM) / calmodulin-like proteins (CMLs), calmodulin B-like proteins (CBLs), and calcium-dependent protein kinases (CDPKs / CPKs)—are key components of this process. 2+ A binding protein. Contains Ca. 2+ CaM / CMLs combined with the EF domain undergo Ca 2+ Dependent conformational changes interact with and regulate downstream target proteins. In contrast, as Ca... 2+CBLs that bind to proteins interact with specific families of CBL-interacting protein kinases (CIPKs) to phosphorylate downstream target proteins. CBL family member CBL1 confers aluminum resistance by regulating ALMT1-mediated malate secretion. CaM / CML family member CML24 enhances malate secretion and aluminum resistance by interacting with and inhibiting WRKY46-mediated transcriptional repression of ALMT1. Unlike CaM / CMLs and CBLs, which require cooperating proteins for signal transduction, calcium-dependent protein kinases (CPKs) independently phosphorylate calcium... 2+ Signal transduction is a phosphorylation event (Bender et al., 2018; Chen et al., 2021). CPKs consist of a Ser / Thr protein kinase domain and a Ca2+ domain. 2+ The combined CaM-like structural domains are connected by a self-inhibiting link (AIJ). When Ca... 2+ Upon binding to a CaM-like domain, CPKs undergo a conformational change, releasing AIJ to activate the enzyme's catalytic site (Wernimont et al., 2010; Ingram et al., 2015). CPKs play diverse roles in regulating plant responses to various stresses. For example, in Arabidopsis, CPK10, CPK30, and CPK32 are involved in sensing and resolving nitrate-triggered Ca2+. 2+ Signaling (Liu et al., 2017). CPK28 negatively regulates plant immunity by phosphorylating and activating two plant U-BOX-type E3 ligases, PUB25 and PUB26, which promote the degradation of the key immune receptor RLCK, BOTRYTIS-INDUCED KINASE 1 (BIK1) (Wang et al., 2018a). Recently, CPK28 has also been found to participate in regulating the cold response by phosphorylating and promoting the migration of the transcription factor NIN-LIKE PROTEIN7 (NLP7) to the nucleus (Ding et al., 2022). However, the role of CPKs in aluminum signaling and whether it requires Ca2+ remains to be seen. 2+ The signal used to regulate STOP1 remains uncertain.

[0040] In this study, we observed that aluminum stress specifically and rapidly induced biphasic calcium ion signaling with specific spatiotemporal patterns in the transition and elongation zones of Arabidopsis roots, while simultaneously enhancing CPK28 phosphorylation and kinase activity. Furthermore, our research revealed an interaction between CPK28 and STOP1, where CPK28 phosphorylation of STOP1 led to increased STOP1 accumulation, thereby enhancing plant aluminum resistance. Our findings highlight the role of calcium in calcium ion exchange. 2+ CPK28-dependent protein plays a key role in regulating STOP1 protein accumulation and conferring aluminum resistance.

[0041] By introducing CPK28 into other plants, such as tobacco plants, through Agrobacterium-mediated genetic transformation, these plants can express CPK28, increase the abundance of STOP1 protein, and develop aluminum resistance. Furthermore, overexpression of CPK28 in plants that already possess it, such as Arabidopsis thaliana, further enhances their aluminum resistance.

[0042] As used herein, the terms “(aluminum resistance) increase,” “enhancement,” or “enhancement” can mean an increase of at least 10% relative to a reference level (e.g., wild-type plants), such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times relative to a reference level.

[0043] Those skilled in the art will readily understand that the conserved variant polypeptide of SEQ ID NO:1, which has a high degree of homology with the structure of CPK28 (GenBank: AT5G66210 or AAQ56823.1), is expected to have similar functions and is expected to confer or enhance aluminum resistance in plants.

[0044] This invention can be implemented in various ways to overexpress or enhance CPK28 expression in plants, and these implementation methods can be combined or used in combination. For example, enhancing CPK28 gene expression can be achieved by transferring a recombinant plasmid containing the CPK28 gene into plants via Agrobacterium-mediated transformation and / or by cloning an exogenous CPK28 gene into the plant genome using gene editing technology and / or by placing the CPK28 gene in plants that already possess the CPK28 gene, such as Arabidopsis thaliana, under the regulation of a strong promoter.

[0045] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0046] When CPK28 is used for plant improvement, it can be easily transferred into plants using traditional Agrobacterium-mediated transformation methods. For this purpose, it is necessary to construct recombinant Agrobacterium-mediated strains that express CPK28.

[0047] In this article, the terms "recombinant bacteria (strain)" and "(genetically) engineered bacteria (strain)" have the same meaning, both referring to strains of wild-type Agrobacterium, such as Agrobacterium tumefaciens, that have been genetically modified and contain the CPK28 overexpression vector.

[0048] In this article, CPK28 generally refers to the protein; while italicized CPK28 refers to the gene encoding the protein.

[0049] It should be understood that the polypeptides used in the present invention are not limited to CPK28 (GenBank:AT5G66210 or AAQ56823.1) with the amino acid sequence SEQ ID NO:1, but also include calcium-dependent protein kinases CPK from other plant sources. These isoform calcium-dependent protein kinases CPK can be mutants formed by mutating one or more amino acid residues of polypeptide SEQ ID NO:1.

[0050] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions include:

[0051] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0052] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0053] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of the engineered calcium-dependent protein kinase CPK. Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0054] "Insertion" refers to a modification of a peptide by adding one or more amino acids to a reference peptide. In some embodiments, modified CPK28 includes inserting one or more amino acids into a naturally occurring calcium-dependent protein kinase CPK28 and into other modified calcium-dependent protein kinase CPK28 peptides. The insertion can be internal to the peptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions can be continuous amino acid segments or separated by one or more amino acids in a naturally occurring peptide.

[0055] To achieve overexpression of the CPK28 gene (GenBank: AT5G66210 or AAQ56823.1) in plants, the CPK28 encoding gene (SEQ ID NO:2) can be artificially synthesized or cloned by PCR using Arabidopsis thaliana genomic DNA as a template. This gene can then be used as a foreign gene to construct a gene expression cassette or expression construct. The expression cassette / expression construct can be operatively linked to a plasmid vector suitable for expression in plants and Agrobacterium tumefaciens through subcloning to obtain a recombinant plasmid. The recombinant plasmid can then be transformed into plant cells to finally obtain transgenic plants overexpressing CPK28.

[0056] As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system containing all the necessary elements required to express the target gene CPK28. Typically, it includes the following elements: a promoter such as a 35S promoter or UBQ10 promoter, a CPK28 gene sequence fragment, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc.; these elements are operatively linked.

[0057] As used herein, an "expression construct" or "expression building block" refers to a recombinant DNA molecule containing one or more intended CPK28 gene sequences, which may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector (plasmid vector).

[0058] As used herein, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein (or nucleic acid) from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, then the nucleic acid is exogenous to that host cell. A particular sequence is "exogenous" to the cell or organism in which it is inserted.

[0059] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operationally linked” to the nucleic acid sequence.

[0060] In another specific embodiment, a gene knock-in vector can also be used to integrate the polynucleotide sequence of the CPK28-encoding gene described herein, such as SEQ ID NO:2, into a region of interest in the genome. Typically, in addition to the polynucleotide sequence described herein, the gene knock-in vector may also contain a 5' homologous arm and a 3' homologous arm required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain a 5' homologous arm, the polynucleotide sequence described herein, and a 3' homologous arm. When using a gene knock-in vector, CRISPR / Cas9 technology can be used simultaneously to homologously recombine the polynucleotide sequence into the site of interest. CRISPR / Cas9 technology guides the Cas9 nuclease to modify the genome at the insertion site by designing a guide RNA targeting the target gene, resulting in increased homologous recombination efficiency in the modified gene region, thus homologously recombinating the target fragment contained in the gene knock-in vector into the target site. The steps of CRISPR / Cas9 technology and the reagents used, such as the Cas9 nuclease, are well known in the art.

[0061] We bred Arabidopsis thaliana strains with enhanced aluminum toxicity resistance by simulating phosphorylation mutation at the Ser163 site of the aluminum-resistant transcription factor STOP1. This increased STOP1 protein abundance and overall aluminum toxicity resistance in Arabidopsis. The experimental results highlight the role of calcium... 2+ The importance of CPK28-dependent signaling in aluminum-mediated responses highlights the key role of phosphorylation-mediated enhanced STOP1 accumulation in conferring aluminum resistance in Arabidopsis thaliana, providing a theoretical basis for breeding aluminum-resistant crops.

[0062] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0063] Example

[0064] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0065] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0066] The molecular biology experiments in this embodiment, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments if necessary.

[0067] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0068] The primer synthesis, gene synthesis, and gene sequencing in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0069] Main experimental materials and methods

[0070] Plant materials and growing conditions

[0071] All Arabidopsis thaliana used in this study were of the Columbia (Col-0) ecotype. The following species were included: cpk28-2 (GK-061C04), cpk9-3 (GK-585B10), cpk7-1 (SALK_127223), rae1 (rae1-1), stop1 (SALK_114108), pSTOP1:STOP1-HA, and pSTOP1:STOP1. 3A -HA complementation strains have all been reported (Iuchi et al., 2007; Matschi et al., 2013; Li et al., 2015; Zhang et al., 2019c; Zhou et al., 2023). cpk28-6 is a monodeoxynucleotide insertion frameshift mutant obtained by CRISPR / Cas9 targeting CPK28 CDS. Transgenic line pSTOP1:STOP1 S163A -HA、pSTOP1:STOP1 S163D -HA and pSTOP1:STOP1 4A -HA was obtained in the context of stop1; pCPK28:CPK28 WT-FLAG was obtained in the cpk28-2 background; UBQ10:CPK28-GFP was obtained in the WT background.

[0072] Arabidopsis seeds were germinated on 1 / 2 Murashige and Skoog (MS) medium containing 1.0% agar and 1% sucrose, and cultured for 7 days in a light incubator (model CU36L4, Percival) at 22°C. Subsequently, the seedlings were transferred to soil and cultured under 14 hours of light (100 μmol / m²). 2 The cells were cultured at 22°C with a photoperiod consisting of 10 hours of darkness and 10 hours of light (using Philips TL-D 26W / 865cool daylight tubes). For Split-LUC and BiFC experiments, Nicotiana benthamiana seeds were sown in soil and grown under the same conditions as Arabidopsis thaliana.

[0073] Calcium signal imaging

[0074] Expression Ca 2+ Arabidopsis thaliana using the indicator pUBQ10:GCaMP6f-mCherry was grown on MS medium for approximately 7 days and then placed in a specially designed imaging chamber filled with imaging buffer (5 mM KCl, 10 mM MES, 0.5 mM CaCl2, pH 4.8). Arabidopsis was pretreated with the imaging buffer, followed by treatment with imaging buffer containing 0 or 20 μM AlCl3. The imaging chamber was mounted on an inverted ZEISS Axio microscope (Carl Zeiss Microimaging GmbH, Goettingen, Germany). GCaMP6f was excited by 488 nm excitation light, and the emission signal was collected in the 500–540 nm range. mCherry was excited by 5561 nm excitation light, and the emission signal was collected in the 580–640 nm range. Images were acquired at 6-second intervals for a total acquisition time of 31 minutes. To visualize Ca... 2+ During the dynamic change process, we normalized the GCaMP6f signal to the mCherry signal and used ImageJ to perform pseudo-color processing on its scale. To quantitatively analyze Ca... 2+ For dynamic changes, we normalize the scale values ​​to the baseline (R0) to calculate the relative change (ΔR / R0) at each time point. The method is similar to that used for Ca. 2+ The method using indicator YC3.672. Data analysis of high-resolution confocal laser scanning microscopy images was performed according to previously published methods (Waadt, 2022).

[0075] Identification of aluminum and lanthanum phenotypes

[0076] The method used in this study to assess aluminum resistance was an improvement on the previously established method of soaking in solid gel medium (Larsen et al., 2005). The medium (50 mL, pH 5.0) was formulated as follows: 0.25 mM (NH4)2SO4, 1 mM KNO3, 0.2 mM KH2PO4, 2 mM MgSO4, 1 mM Ca(NO3)2, 1 mM CaSO4, 1 mM K2SO4, 1 μM MnSO4, 5 μM H3BO3, 0.05 μM CuSO4, 0.2 μM ZnSO4, 0.02 μM NaMoO4, 0.1 μM CaCl2, 0.001 μM CoCl2, 1% (w / v) sucrose, and 0.4% (w / v) agar (G1910, Sigma-Aldrich). The solid culture medium was then soaked for 2 days in 40 mL of the same solution (excluding agar, pH 3.6) containing different concentrations of AlCl3 (0, 0.75, 1.0, or 1.25 mM). After 2 days, the soaking solution was discarded, the surface of the soaked solid culture medium was dried, and seeds were planted and allowed to grow for 7 days. After 7 days, the plants were photographed, and root length was measured using ImageJ. Aluminum resistance of different plant materials was assessed by calculating relative root growth, which was calculated as: root length under aluminum treatment / root length without aluminum treatment (×100) to calculate its percentage. To assess the effect of La on aluminum resistance, the solid culture medium was supplemented with 0 or 50 μM LaCl3 and then soaked in 0 or 1.0 mM AlCl3 soaking solution.

[0077] Determination of malic acid secretion and aluminum content

[0078] Arabidopsis seeds were spotted on 1 / 2 MS medium containing 1% (w / v) sucrose and allowed to grow for 7 days. They were then pretreated for 2 hours with a 2% (w / w) MGRL solution (pH 4.8) containing 1% (w / v) sucrose (Fujiwara et al., 1992), followed by treatment in 12-well plates with the same solution containing 0 or 20 μM AlCl3 at pH 4.8 for 12 hours. Root exudates were collected from the treated solutions and concentrated using a freeze dryer (Alpha 1-2LDplus; MartinChrist Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The malic acid concentration in the root exudates was determined by the NAD / NADH enzyme cycling method (HAMPP, GOLLER and FULLGRAF, 1984).

[0079] To measure the aluminum content in the roots, plants grown in a 1 / 10 concentration of Hoagland hydroponic solution for three weeks were pretreated with 0.5 mM CaCl2 solution for 6 hours, followed by treatment with the same CaCl2 solution (with or without 20 μM AlCl3) for 12 hours. The aluminum content in the roots was determined using a previously reported method (Ligaba-Osena et al., 2017). Inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmer NexION 300D) was used to measure the aluminum concentration.

[0080] RNA extraction and RT-qPCR analysis

[0081] To detect the expression of aluminum resistance genes in different materials, seeds from different materials were grown on 1 / 2 MS solid medium for 9 days. The materials were pretreated with 0.5 mM CaCl2 solution at pH 4.8 for 6 hours, followed by treatment with the same CaCl2 solution (with or without 30 μM AlCl3) for 12 hours. Root tips (0–1 cm) or whole roots were then collected for RNA extraction. Total RNA was extracted using TRIzol reagent (Beyotime) and treated with DNase I to remove DNA contamination. Approximately 1 μg of total RNA was used to synthesize cDNA using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). One-sixth of the synthesized cDNA was analyzed by RT-qPCR using a 2× Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China). UBQ10 was used as an internal control. RT-qPCR data were collected and analyzed using a CFX96 Touch real-time PCR detection system (Bio-Rad).

[0082] Yeast two-hybrid experiment

[0083] 1) To detect whether STOP1 and CPK28 interact, the CDS sequences of STOP1 and CPK28 were cloned into the pGADT7 and pGBKT7 vectors, respectively, using a common method for constructing genetically engineered bacteria that overexpress the target gene. The constructed plasmids were then co-transformed into the AH109 yeast strain.

[0084] 2) To assess protein-protein interactions, yeast cells were plated on SD-Leu-Trp, SD-Leu-Trp-His and SD-Leu-Trp-His-Ade solid media and grown for 2-4 days.

[0085] 3) Select positive single clones from SD-Leu-Trp solid medium and inoculate them into test tubes containing 5 mL of SD-Leu-Trp liquid medium. Incubate at 30°C with shaking at 200 rpm until the OD546 value of the culture medium reaches 0.6. Then, centrifuge at 2500 g for 5 minutes to collect the precipitate.

[0086] 4) Resuspend the precipitate in 1 mL of sterile deionized water and wash it. Centrifuge again at 2500 g for 5 minutes. Repeat the washing step twice, and finally resuspend the precipitate in 1 mL of sterile deionized water.

[0087] 6) After vortex mixing, take 1 μL of the suspension and measure the OD546 value. Based on the measured OD value, adjust the remaining 1 mL of suspension in all combinations to the same concentration and perform serial dilutions to achieve OD546 values ​​of 1, 0.1, 0.01 and 0.0001, respectively.

[0088] 7) Take 3 μL of suspension from each dilution and spot it onto the corresponding selective medium, then incubate at 30°C for 2 or 4 days to observe the results. After incubation, take photos to record the growth.

[0089] Luciferase-protein interaction assay (Split-LUC)

[0090] 1) To detect whether CPK28 and STOP1 interact, the commonly used transgenic plant construction method overexpressing the target gene was employed. The coding sequences of CPK28 or CPK7 were cloned into the pCAMBIA-cLUC vector, and the coding sequences of STOP1 or NLP8 were cloned into the pCAMBIA-nLUC vector. CPK7 and NLP8 were used as negative controls.

[0091] 2) Transform the constructed plasmid into Agrobacterium, plate it, and grow it at 30°C for 2 days.

[0092] 3) Select positive single clones and inoculate them into test tubes containing 5 mL of resistant LB medium. Incubate overnight at 28°C and 200 rpm.

[0093] 3) Centrifuge the overnight cultured Agrobacterium at 2500g for 5 minutes, and then adjust the OD600 to 1 using resuspension buffer (containing 10mM MgCl2, 10mM MES and 0.2mM acetylsylcholine).

[0094] 4) Mix the two bacterial cultures from each combination in a 1:1 ratio in a 1.5 mL EP tube and incubate at 28°C for 1 hour.

[0095] 5) The mixed bacterial solution was transferred into the tobacco leaves by the leaf back injection method. After the injection, the leaves were wrapped with plastic wrap and placed in the culture room for 48 hours.

[0096] 6) Two days later, spray the LUC solution evenly on the back of the leaf, darken it for 10 minutes, and then use a pre-cooled CCD imaging system to take a 5-minute exposure picture.

[0097] 7) The captured images were analyzed for fluorescence quantitative analysis using Fiji ImageJ software.

[0098] Bimolecular Fluorescence Complementary (BiFC) Experiment

[0099] 1) Construct CDS for CPK28 and CPK7 into the pCAMBIA1300-cYFP vector. Construct STOP1 and NLP8 into the pCAMBIA1300-nYFP vector.

[0100] 2) Transform the constructed plasmid into Agrobacterium, plate it, and grow it at 30°C for 2 days.

[0101] 3) Select positive single clones and inoculate them into test tubes containing 5 mL of resistant LB medium. Incubate overnight at 28°C and 200 rpm.

[0102] 4) Centrifuge the overnight cultured Agrobacterium at 2500g for 5 minutes, and then adjust the OD600 to 1 using resuspension buffer (containing 10mM MgCl2, 10mM MES and 0.2mM acetylsyleugenol).

[0103] 5) Mix the two bacterial cultures from each combination in a 1:1 ratio in a 1.5 mL EP tube and incubate at 28°C for 1 hour.

[0104] 6) The mixed bacterial solution was transferred into the tobacco leaves by the leaf back injection method. After the injection, the leaves were wrapped with plastic wrap and placed in the culture room for 48 hours.

[0105] 7) Two days later, the part of the leaf injected with Agrobacterium was cut off and placed under a laser scanning confocal microscope (Zeiss LSM880 Airyscan) to detect the yellow fluorescent protein (YFP) signal.

[0106] Determination of STOP1 protein content

[0107] Seedlings grown on 1 / 2 MS medium for 10 days were first pretreated with 0.5 mM CaCl2 solution at pH 4.8 for 6 hours, and then treated with the same CaCl2 solution containing 0 or 30 μM AlCl3 for 12 hours. Root tips (0-1 cm) or whole roots were collected and frozen for protein extraction. The protein extraction buffer formulation was: 50 mM Tris-HCl (pH 7.5), 1 mM NaF, 1 mM MDT, 0.5% NP-40, 10% glycerine, 50 μM MG132, and 1× Complete Protease Inhibitor Tablets EDTA-free (5892791001, Roche). The protein extraction steps are as follows:

[0108] 1) Weigh the plant roots in a 1.5mL EP tube, freeze them quickly in liquid nitrogen, and then grind them using a grinder.

[0109] 2) Add two times the weight volume of protein extract to the ground plant tissue, vortex, and place on ice for 10 minutes.

[0110] 3) Centrifuge at 12000g (4℃) for 10 minutes, and transfer the supernatant to a new 1.5mL EP tube.

[0111] 4) Take 5 μL of the sample and add it to 1 mL of Bradford solution to measure the protein concentration. Adjust all samples to the same concentration using protein extraction liquid.

[0112] 5) Take 20 μL of sample, add 5 μL of 5× loading buffer, heat at 95℃ for 10 minutes, and use for SDS gel running.

[0113] Determination of STOP1 phosphorylation abundance

[0114] To detect STOP1 phosphorylation, seedlings grown on 1 / 2 MS medium for 10 days were first pretreated with 0.5 mM CaCl2 solution (pH 4.8) for 6 hours, and then treated with a solution containing 0 or 30 μM AlCl3, 50 μM MG132, and 0.5 mM CaCl2 (pH 4.8) for 3 hours. Root tips (0-1 cm) or whole roots were collected for subsequent experiments.

[0115] 1) First, plant protein extraction was performed. The protein extraction solution was formulated as follows: 0 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× Complete Protease Inhibitor Tablets EDTA-free, 50 μM MG132, and 1× phosphatase inhibitor.

[0116] 2) Add 20 μL of HA magnetic beads to the extracted plant protein to enrich STOP1-HA, and incubate at 4 degrees Celsius for 4 hours. After enrichment and incubation, wash the magnetic beads three times with PBS.

[0117] 3) Add 100 μL of 1× loading buffer to the magnetic beads and heat at 95°C for 10 minutes. Take 10 μL of sample for gel electrophoresis, transfer to a membrane, block, and then detect STOP1-HA with HA antibody.

[0118] 4) Quantify the developed STOP1-HA using Image J, and then adjust the protein abundance of each sample to be the same according to STOP1.

[0119] 5) After quantification according to STOP1, the samples were subjected to gel electrophoresis, transfer to a membrane, and blocked. Then, the phosphorylation of STOP1 was detected using the pIMAGO kit.

[0120] GUS staining

[0121] To construct the pCPK28:GUS transgenic line, we amplified the 2kb promoter region of the CPK28 gene and fused it with the GUS reporter gene into the pORE-R2 vector. Homozygous pCPK28:GUS transgenic lines were selected for GUS analysis. Seeds were grown on 1 / 2 MS medium for 7 days, pretreated with 0.5 mM CaCl2 solution at pH 4.8 for 6 hours, and then treated with the same CaCl2 solution containing 0 or 30 μM AlCl3 for 12 hours. Subsequently, the roots were stained with GUS staining solution. The stained roots were observed and photographed using a stereomicroscope.

[0122] Pull-down experiment

[0123] 1) Set STOP1, STOP1 S163A Or STOP1 S163D The coding sequence was amplified and constructed into the pET-29a(+) vector, and the RAE1ΔF sequence was amplified and constructed into the pGEX4T-1 vector to construct STOP1-His and STOP1. S163A -His、STOP1 S163D-His and GST-RAE1ΔF recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) strain, plated, and incubated overnight at 37°C.

[0124] 2) Pick a single colony that has transformed positively and place it in 5 mL LB broth. Incubate at 37°C (200 rpm) for 6 hours. Transfer 1 mL of the bacterial culture to 100 mL LB broth and incubate at 37°C until the OD 600 reaches 0.6. Adjust the temperature of the shaker to 16°C. When the temperature of the bacterial culture drops to 16°C, add IPTG to a final concentration of 300 μM and continue incubation for 16 hours to induce protein expression.

[0125] 3) Collect E. coli by centrifugation at 4000g for 10 min, sonicate to break down bacteria, centrifuge, collect the supernatant, and purify the protein using Ni-NTA or GST agarose beads.

[0126] 4) The purified STOP1-His and STOP1 S163A -His or STOP1 S163D Mix equal amounts of His and GST-RAE1ΔF, and take 20 μL as input. Then add 50 μL of Ni-NTA agarose beads and incubate at 4 °C for 5 h.

[0127] 5) Collect agarose beads, wash 5 times with PBS, add 100 μL of 1× loading buffer, and heat at 95°C for 10 minutes. Perform gel running, transfer, and blocking. Then, detect STOP1-His with His antibody and GST-RAE1ΔF with GST antibody.

[0128] Arabidopsis protoplast Co-IP experiment

[0129] 1) Arabidopsis protoplasts were isolated using previously reported methods. Seeds were spotted onto 1 / 2 MS (0.6% agar, 1% sucrose) plates and grown in a short-day incubator for 14 hours. The aboveground parts of the plants were collected with a blade, chopped, and incubated with enzymatic digestion buffer and TVL solution. The mixture was then placed on a shaker at 30 rpm for 12 hours. The digested liquid was filtered through a sieve into 50 mL centrifuge tubes and resuspended in 3 times the volume of W5 solution. The cells were centrifuged at 100 g for 8 minutes at room temperature, the supernatant was quickly discarded, and 50 mL of W5 solution was added to resuspend the cells. The cells were then centrifuged again at 100 g for 8 minutes and resuspended in MMG solution (approximately 2 × 10⁻⁶ cells / mL). 6 (cells / mL) for subsequent transformation.

[0130] 2) To test whether STOP1 can interact with CPK28 in vivo, 50 μg of 35S:CPK28-3HA plasmid and 50 μg of 35S:STOP1-2FLAG or 35S:GFP-2FLAG plasmid were co-transformed into 2 ml of protoplasts.

[0131] 3) For the interaction strength analysis of different versions of STOP1 and RAE1ΔF, 50 μg of 35S:STOP1-2FLAG and 35S:STOP1 were compared. S163A -2FLAG or 35S:STOP1 S163D -2FLAG plasmid was co-transferred with 50 μg of 35S:RAE1ΔF-3HA into 2 mL of rae1 rah1 mutant protoplasts.

[0132] 4) Mix the plasmid with the protoplasts resuspended in MMG, add 2 mL of PEG, mix well, and incubate in the dark for 15 min. Add 10 mL of W5 to stop the reaction. Centrifuge at 100 g for 8 minutes at room temperature and discard the supernatant. Resuspend the cells in 10 mL of W5 and incubate in the dark for 16 h. Centrifuge at 100 g for 8 minutes and discard the supernatant. Collect the protoplasts at the bottom for protein extraction.

[0133] 5) Add 300 μL of Co-IP buffer to protoplasts to extract total protein. This buffer contains 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1×Complete Protease Inhibitor Tablets EDTA-free, 50 μM MG132, and 1×phosphatase inhibitor. Take 30 μL of the protein extract as input. Add 20 μL of anti-HA or anti-FLAG M2 magnetic beads to the remaining protein extract and incubate at 4°C for 4 hours. Wash the magnetic beads 5 times with 1×PBS and add 50 μL of 1×SDS loading buffer. Detect the target protein using HA or FLAG antibodies.

[0134] Detection of LUC enzyme activity in Arabidopsis protoplasts

[0135] 1) To evaluate whether different versions of STOP1 have different activities, 5 μg of 35S:STOP1-2FLAG and 35S:STOP1 were used. S163A -2FLAG, 35S:STOP1 S296A -2FLAG, 35S:STOP1 S399A -2FLAG or 35S:STOP1 S163D -2FLAG was co-transferred to stop1 protoplasts with 8 μg of pALMT1:LUC and 2 μg of pUBQ10:GUS (as internal control).

[0136] 2) To investigate whether different versions of STOP1 affect its protein levels, 5 μg of 35S:STOP1-LUC and 35S:STOP1 were used. S163A -LUC or 35S:STOP1 S163D -LUC was co-transformed with 2 μg of pUBQ10:GUS into stop1 protoplasts.

[0137] 3) To determine whether CPK28 can activate different versions of STOP1, 8 μg of 35S:CPK28-HA or 35S:GFP-HA was mixed with 8 μg of pALMT1:LUC, 2 μg of pUBQ10:GUS, and 5 μg of 35S:STOP1-2FLAG or 35S:STOP1. S163A -2FLAG was converted into the stop1 protoplast.

[0138] 4) To investigate whether CPK28 can enhance the protein levels of different versions of STOP, 8 μg of 35S:CPK28-HA or 35S:GFP-HA was mixed with 2 μg of pUBQ10:GUS and 5 μg of 35S:STOP1-LUC or 35S:STOP1 S163A -LUC co-transfers to stop1 protoplast.

[0139] 5) Transform each plasmid group into 0.2 ml of protoplasts. Incubate in the dark for 16 h, then lyse the cells using protoplast lysis buffer. Mix the cell lysis buffer with LUC or MUG substrate, and measure the activity of LUC or GUS using a microplate reader. Calculate the LUC / GUS ratio to reflect the relative abundance or activity of the expressed protein in the protoplasts.

[0140] Detection of IP kinase activity

[0141] 1) To determine whether aluminum treatment affects CPK28 kinase activity, pCPK28:CPK28-2FLAG transgenic seedlings grown on 1 / 2 MS medium for 10 days were first pretreated with 0.5 mM CaCl2 solution at pH 4.8 for 6 hours, and then treated with solutions containing 0 or 30 μM AlCl3, 50 μM MG132 and 0.5 mM CaCl2 (pH 4.8) for 20, 60 and 180 min.

[0142] 2) For LaCl3 treatment, seedlings were pretreated in a similar manner with a 0.5 mM CaCl2 solution at pH 4.8 for 6 hours, followed by exposure to the same solution containing 0 or 50 μM LaCl3 for 30 minutes. Subsequently, seedlings were treated with a 0.5 mM CaCl2 solution (pH 4.8) containing 0 or 50 μM LaCl3 and 0 or 30 μM AlCl3 for 1 hour.

[0143] 3) Collect plant roots for total protein extraction. The protein extraction solution is: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 50μM MG132, 1×Complete Protease Inhibitor Tablets EDTA-free, and 1×phosphatase inhibitor.

[0144] 4) For immunoprecipitation, incubate 10 μL of anti-FLAG M2 magnetic beads with approximately 4 mg of total protein at 4°C for 3 hours. Enrich the magnetic beads and wash three times with PBS.

[0145] 5) For kinase activity assay, FLAG magnetic beads bound to CPK28 were mixed with 10 μg STOP1-GFP-His in 30 μL reaction buffer (25 mM Tris-HCl [pH 7.5], 12 mM MgCl2, 1 mM DTT, 10 μM ATP, and 1 mCi [γ-]). 32 The reaction was carried out in P]ATP for 1 hour at a temperature of 30°C. Subsequently, 5×SDS loading buffer was added to terminate the reaction, and the proteins were separated by SDS-PAGE. Phosphorylated proteins were detected by autoradiography.

[0146] Purification and in vitro phosphorylation experiments of recombinant proteins

[0147] 1) The CDS of CPK28 was amplified and recombined into the pMAL-c2X vector; STOP1, STOP1 S163A STOP1 S296A Or STOP1 S399A The CDS were amplified and recombined into the pET29a(+) vector. The resulting MBP-CPK28, STOP1-His, and STOP1 vectors were then used. S163A -His、STOP1 S296A -His and STOP1 S399A The His recombinant plasmid was transformed into Escherichia coli BL21(DE3) strain. The plating was plated and incubated overnight at 37°C.

[0148] 2) Select a single colony that has undergone transformation and place it in a 5 mL LB container. Incubate at 37°C (200 rpm) for 6 hours. Transfer 1 mL of the bacterial culture to a 100 mL LB container and incubate at 37°C until the OD 600 reaches 0.6. Adjust the shaker temperature to 16°C. When the bacterial culture temperature drops to 16°C, add IPTG to a final concentration of 300 μM and continue incubation for 16 hours to induce protein expression. Collect E. coli by centrifugation at 4000g for 10 min, sonicate to break down the bacteria, centrifuge again, collect the supernatant, and purify the protein using Ni-NTA or MBP agarose beads.

[0149] 3) The purified STOP1-His and STOP1 S163A -His、STOP1 S296A -His or STOP1 S399A His was reacted with MBP-CPK28 in 20 μL reaction buffer (25 mM Tris-HCl [pH 7.5], 12 mM MgCl2, 1 mM DTT, 10 μM ATP, and 1 mCi [γ-]). 32 The reaction was carried out in P]ATP for 1 hour at 30°C. The reaction was then terminated by adding 5×SDS loading buffer, and the proteins were separated by SDS-PAGE. Phosphorylated proteins were detected by autoradiography.

[0150] Mass spectrometry analysis

[0151] For the phosphorylated peptide identification experiment, seedlings grown on 1 / 2 MS medium for 14 days were first pretreated with 0.5 mM CaCl2 solution at pH 4.8 for 6 hours, and then treated with a solution containing 0 or 30 μM AlCl3, 50 μM MG132, and 0.5 mM CaCl2 (pH 4.8) for 3 hours. Whole roots were collected for subsequent experiments. Total protein was extracted using a protein extraction buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM NaF, 1 mM DTT, 1% NP-40, 2% glycerine, 50 μM MG132, 1×Complete Protease Inhibitor Tablets EDTA-free, and 1×phosphatase inhibitor. After trypsin digestion, total protein was subjected to phosphopeptide enrichment and LC-MS / MS (Thermo Fisher Scientific) analysis, according to the previously described method.

[0152] To determine which sites of STOP1 are phosphorylated by CPK28, 5 μg of recombinant GST-CPK28 protein and 10 μg of STOP1-His were incubated in kinase reaction buffer (225 mM Tris-HCl [pH 7.5], 12 mM MgCl2, 1 mM DTT, and 100 μM ATP) at 30 °C for 1 h. The protein was then trypsinized and analyzed by LC-MS / MS on a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Fisher Scientific).

[0153] Nucleocytoplasmic separation

[0154] Seedlings grown on 1 / 2 MS medium for 10 days were first pretreated for 3 hours in a 0.5 mM CaCl2 solution (pH 4.8) containing 50 μM MG132, and then treated for 3 hours in a solution containing 0 or 30 μM AlCl3, 50 μM MG132, and 0.5 mM CaCl2 (pH 4.8). Root tips (0–1 cm) or whole roots were collected for subsequent experiments. Nucleoplasmic separation was performed according to previously reported methods, as follows.

[0155] 1) Grind the plant tissue using a grinder and add the following cell lysis buffer: 300 mM sucrose, 20 mM tris-HCl (pH 8.0), 5 mM MgCl2, 5 mM KCl, 0.3% Triton X-100, 5 mM NP-40, 5 mM β-mercaptoethanol, 35% glycerol, 5 mM DTT, 1×cocktail, and 50 μM MG132. Mix thoroughly and filter through a filter membrane. Collect the filtrate as total protein.

[0156] 2) Centrifuge the filtrate at 5000g for 10 minutes at 4℃, and separate the supernatant and precipitate. Collect the supernatant as the cytoplasmic component and use the precipitate for subsequent extraction of nuclear proteins.

[0157] 3) Wash the precipitate five times with lysis buffer. After the last wash, resuspend it in a nucleoprotein extraction buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS, 5 mM DTT, 1×cocktail and 50 μM MG132 as the nuclear component.

[0158] 4) Add 5×SDS loading buffer to the total protein fraction, cytoplasmic fraction, and nuclear fraction, and separate the proteins using SDS-PAGE. Tubulin is used as an internal control in the cytoplasm, and Histone is used as an internal control in the nucleus.

[0159] Results and discussion

[0160] The results of the above study are summarized below.

[0161] 1. Aluminum stress induces biphasic Ca in the root-root transition zone and elongation zone. 2+ Signal generation.

[0162] Our initial attempt was to investigate whether exposure to physiologically relevant concentrations of aluminum would trigger calcium-related calcium metabolism in Arabidopsis roots. 2+ The signals, and whether these signals exhibit cell or tissue specificity. To this end, we used ultrasensitive Ca2+... 2+ The roots of Arabidopsis thaliana plants, containing the reporter protein GCaMP6f-mCherry, were used as the research material. Time-lapse imaging analysis was performed after replacing the imaging buffer with one containing 0 or 20 μM AlCl3. Images were acquired every 6 seconds, and GFP was calculated. em / mCherry em The proportion was calculated and converted into a pseudo-color image or graph of relative proportional change (ΔR / R0). Under control conditions, we observed a persistent slight Ca2+. 2+ Fluctuations, particularly in the late meristem zone (MZ) and elongation zone (EZ), and weak Ca2+ levels in root tip cells. 2+ These fluctuations may be associated with the normal root growth process. However, after aluminum treatment, we could repeatedly observe the appearance of Ca in specific tissues after 2 minutes. 2+ Increase, and Ca 2+ It reaches its peak after 6 minutes and returns to background levels within 30 minutes (see [link]). Figure 1 These findings reveal that root exposure to physiologically relevant concentrations of aluminum can rapidly induce calcium in specific tissues at specific time points. 2+ Increase.

[0163] To analyze Ca in more detail 2+ To elevate the level of , we defined three regions of interest (ROIs): the late meristematic zone (MZ) (ROI1), the early elongation zone (EZ) (ROI2), and the early differentiation zone (DZ) (ROI3) (see [link to relevant documentation]). Figure 1 (d). Although no obvious Ca was detected in DZ. 2+ Increased, but significant Ca was clearly observed in MZ. 2+ The increase was highest in EZ (see...) Figure 1 Quantitative evaluation and integration of multiple measurements revealed these aluminum-induced Ca2+ levels. 2+ Signal characteristics. Main Ca 2+The rise began after approximately 3 minutes (±0.5 minutes), reaching its maximum mean amplitude of 0.71ΔR / R0 (±0.10ΔR / R0) after 5.95 minutes (±0.54 minutes), lasting for 7.2 minutes (±0.38 minutes). This main signal was followed by a less significant but more persistent secondary rise, with a mean amplitude of 0.34ΔR / R0 (±0.13ΔR / R0), which gradually decreased to background levels at the end of the 30-minute measurement period (see [link to data]). Figure 1 (e). In measurements of aluminum treated for more than 3 hours, no Ca related to aluminum treatment was found. 2+ Fluctuations. For the main Ca... 2+ Signal analysis revealed that Ca in the cytoplasm 2+ The increase in concentration initially appears in the cortical and epidermal cells of the EZ, then extends to the inner cells, and longitudinally extends to the MZ and EZ, to achieve the greatest amplitude in the inner cells of the EZ (see [link to original text]). Figure 1 (e). When the Ca inside the cells of EZ 2+ When the concentration reaches its maximum, the signal intensity of the initially stimulated cortical and epidermal cells begins to decline, even though these cells exhibit the most persistent Ca2+ levels. 2+ Concentration increased. Overall, these findings establish that aluminum treatment specifically triggers specific Ca2+ concentrations in Arabidopsis roots. 2+ These cellular signals are transmitted via the cytoplasmic calcium in individual cells of the MZ and DZ cells. 2+ The increased spatiotemporal definition of the continuous formation suggests that these cells form a functional aluminum-sensing niche, participating in triggering subsequent signal transduction and anti-aluminum toxicity responses.

[0164] 2. Using phosphorylated proteomics, Ca was identified. 2+ Regulated kinase CPK28 is a key component of Arabidopsis aluminum resistance.

[0165] After determining that aluminum treatment induces specific Ca in the roots 2+ Following the signal pattern, we then sought to identify components that might decode this signal into downstream responses. To this end, we performed mass spectrometry-based quantitative phosphoproteomics analysis on Arabidopsis roots treated with 0 and 30 μM aluminum for 3 h. A total of 7521 distinct phosphopeptides were identified from 2700 proteins. Compared to control conditions, we observed changes in phosphorylation levels, with 398 phosphopeptides from 288 proteins showing increased phosphorylation under aluminum stress, while 461 phosphopeptides from 303 proteins showed decreased phosphorylation under aluminum stress (fold change >1.5, p <0.05, two-tailed unpaired t-test) (see [link to relevant documentation]). Figure 2(a) Notably, 41 proteins showed both increased and decreased phosphorylation levels at different phosphorylated peptides. GO (Gene Ontology) analysis revealed that 632 proteins with altered phosphorylation levels after aluminum treatment were primarily enriched in areas including "protein binding," "calmodulin binding," "cortical microtubules," "development and growth," and "nuclear tissue" (see [reference]). Figure 2 (b)

[0166] The phosphorylation levels of 53 transcription factors were affected by aluminum treatment. Among these transcription factors are several WRKY family proteins (see [link to relevant documentation]). Figure 3 (a) This reveals their important roles in regulating aluminum resistance. Among them, WRKY46, previously identified as a negative regulator of aluminum resistance, exhibits aluminum-induced phosphorylation at the Ser168 site. Due to the known role of Ca... 2+ The activity of decoding components such as CAMs / CMLs, CBLs, CIPKs, and CPKs is regulated by their phosphorylation state; we focus on the regulation of the abundance of phosphorylation in these proteins induced by aluminum. This method identifies several Ca2+-dependent phosphorylation processes under aluminum stress. 2+ The phosphorylation abundance of sensory proteins, including kinases CPK28, CPK7, and CPK9, as well as CAM4 and CML41, was altered (see [link to relevant documentation]). Figure 3 (b)

[0167] 3. CPK28 positively modulates aluminum resistance and STOP1 accumulation.

[0168] To confirm the importance of CPK28 in the regulation of aluminum resistance, we ordered the cpk28 TDNA insertion mutant cpk28-2 and constructed a second line of the cpk28 mutant using CRISPR / Cas9 gene editing technology. This line contained a frameshift mutation in the fourth exon of CPK28, which we named cpk28-6. Figure 4 (a). Compared to the wild type (WT), cpk28-6 also showed increased sensitivity to aluminum. Figure 4 (b) To further confirm the role of CPK28 in aluminum resistance, we constructed pCPK28:CPK28-FLAG into a plant-stable vector and used it to reintroduce cpk28-2. The results showed that in both reintroduced lines, aluminum resistance in cpk28-2 was completely restored to wild-type levels (see [link]). Figure 5 ).

[0169] To further elucidate the mechanism by which the cpk28 mutant increases aluminum sensitivity, we compared malate secretion between the wild-type and mutant strains. Malate secretion is crucial for aluminum resistance in Arabidopsis thaliana, and both cpk28 mutants exhibited lower levels of malate secretion (see [link to cpk28 mutant]). Figure 6 (a) Consistent with the reduced malate secretion in cpk28, the mutant accumulated more aluminum than the wild type under aluminum stress (see [reference]). Figure 6 (b) Then, we compared the expression levels of ALMT1 and other aluminum resistance genes in the root tip region of wild-type and mutants. Consistent with reduced malate secretion, ALMT1 expression in mutants was lower than in wild-type (see [reference]). Figure 6 (c). Furthermore, we found that other genes regulated by STOP1, including MATE and ALS3, were also expressed lower in the mutant than in the wild type (see [link]). Figure 6 In c), the expression levels of STAR1, ALS1, and STOP1, genes not regulated by STOP1, were similar between wild-type and mutant. The reduced expression of STOP1-regulated genes in cpk28-2 was restored in both complement lines. These results suggest that loss of CPK28 function appears to suppress STOP1 function. Therefore, to investigate whether STOP1 accumulation in cpk28 is reduced, we crossed the previously constructed pSTOP1:STOP1-HA transgenic line with cpk28-2, introducing the transgene into a mutant background. Immunoblotting analysis using an anti-HA antibody showed that, under -Al and +Al conditions, the STOP1-HA protein level in the root tip region of the mutant was significantly lower than that of the wild type (see [link to original text]). Figure 6 (d). To investigate whether elevated CPK28 expression levels also regulate plant resistance to aluminum stress, we overexpressed CPK28 fused to the GFP gene using the UBQ10 promoter (see d). Figure 7 Phenotypic analysis showed that increased CPK28 expression levels enhanced aluminum resistance in plants at different aluminum concentrations (see [reference]). Figure 7 (See b). To explore the mechanism of this enhanced resistance, we compared the expression levels of STOP1 regulatory genes in wild-type and CPK28 overexpression lines. The results showed that under -Al and +Al conditions, the expression levels of STOP1 regulatory genes, including ALMT1, MATE, and ALS3, were higher in the overexpression lines than in the wild-type (see b). Figure 7 (a) Correspondingly, compared with the wild type, the accumulation of STOP1 protein was increased in the CPK28 overexpression lines (see [reference]). Figure 7 (c) Consistent with increased ALMT1 expression, CPK28 overexpressing lines exhibited greater malate secretion than wild-type (see c). Figure 7 (d). In summary, these findings establish that CPK28 positively regulates aluminum toxicity resistance in Arabidopsis thaliana by modulating STOP1 accumulation.

[0170] 4. CPK28 can interact with STOP1 and phosphorylate STOP1.

[0171] To determine whether CPK28 interacts with STOP1, we first performed a yeast two-hybrid experiment and found that CPK28 can directly interact with STOP1 (see [link to experiment]). Figure 8 (a) Then, we tested their interactions in tobacco leaves using a luciferase activity complementation assay (split-LUC) and a bimolecular fluorescence complementation assay (BiFC). Only the combination of CPK28-cLUC and STOP1-nLUC produced a LUC signal indicating a direct protein-protein interaction (see [reference]). Figure 8 (b) , while no signal was observed in combinations of CPK7-cLUC and STOP1-nLUC or CPK28-cLUC and NLP8-nLUC, indicating the plant-specific interaction between CPK28 and STOP1. BiFC experiments also revealed interaction-dependent fluorescence signals between STOP1-nYFP and CPK28-cYFP (see [b]). Figure 8 (See c). In addition, we performed co-immunoprecipitation (Co-IP) experiments using Arabidopsis protoplasts to investigate whether there is an interaction between CPK28 and STOP1 in Arabidopsis. The CPK28-HA fusion protein co-precipitated with STOP1-FLAG, but not with the GFP-FLAG control protein (see c). Figure 8 (d). In summary, these data support the conclusion that CPK28 interacts with STOP1 in vivo.

[0172] To assess the potential phosphorylation of STOP1 by CPK28, we performed in vitro kinase activity assays by incubating MBP-tagged CPK28 with His-tagged STOP1 and detecting phosphorylation of STOP1 by CPK28 (see [link to assay]). Figure 8 (See also e). Furthermore, by immunoprecipitating CPK28-FLAG from the roots of pCPK28:CPK28-FLAG / cpk28-2 recombinant lines subjected to aluminum stress for different durations, we discovered the ability of CPK28 to phosphorylate STOP1 in vivo. Notably, CPK28 activity significantly increased after 20 minutes or longer of aluminum stress (see [reference e]). Figure 8 (f). The application of the calcium channel blocker La inhibited aluminum-induced CPK28 activity (see f). Figure 8 (g), indicating that aluminum stress is mediated by Ca. 2+ The signal enhanced the activity of CPK28.

[0173] To determine the phosphorylation sites targeted by CPK28 in STOP1, we performed liquid chromatography-tandem mass spectrometry (LC-MS / MS) experiments on CPK28-incubated STOP1 samples. The results revealed that three phosphorylation sites in STOP1 showed the highest scores after reaction with CPK28: Ser163, Ser296, and Ser399. We replaced Ser163, Ser296, and Ser399 with Ala to examine whether individual mutations at these sites would affect phosphorylation. The results showed that individual mutations at all three sites significantly reduced CPK28 phosphorylation in STOP1 (see [link to relevant documentation]). Figure 8 (e).

[0174] 5. Phosphorylation of STOP1 at the Ser163 site increases STOP1 accumulation and plant resistance to aluminum stress.

[0175] To investigate the functional importance of three phosphorylation sites (Ser163, Ser296, and Ser399) in STOP1, we mutated these sites from Ser to mimic the non-phosphorylated form, Ala. Wild-type or mutant STOP1 was co-transformed with the reporter gene pALMT1:LUC into stop1-2 protoplasts. The results showed that, along with STOP1… WT Compared to STOP1 S163A Significantly reduced reporter gene induction, while S296A or S399A mutations had no effect on LUC activity (see [link]). Figure 9 (a) This study revealed the crucial role of the Ser163 site in STOP1 function. Furthermore, replacing Ser163 with phosphorylated Asp enhanced reporter gene induction (see [reference]). Figure 9 (b) This indicates that phosphorylation at Ser163 enhances STOP1 function. To investigate whether Ser163 phosphorylation regulates STOP1 protein accumulation, we constructed different versions of STOP1-LUC and transformed them into stop1-2 protoplasts. 35S:STOP1 S163A -LUC and 35S:STOP1 S163D- LUC-transformed cells and 35S:STOP1 WT Compared to -LUC, it showed reduced and increased STOP1-dependent LUC activity, respectively (see [link to relevant documentation]). Figure 9 c), which indicates that phosphorylation at Ser163 positively regulates the accumulation of STOP1 protein.

[0176] To further explore the role of Ser163 phosphorylation in the accumulation of STOP1 in Arabidopsis thaliana, we constructed pSTOP1:STOP1 in a stop1-2 mutant background. S163A -HA(STOP1S163A Transgenic lines. We selected two independent STOP1 lines. S163A Further investigation was conducted using transgenic lines to study the STOP1 expression levels and pSTOP1:STOP1-HA(STOP1) expression levels in these two lines. WT Similar to STOP1. We observed that STOP1 S163A The STOP1 protein content in the strain was higher than that of STOP1 under both -Al and +Al conditions. WT Medium and low (see Figure 9 (d). Therefore, with STOP1 WT Compared to other strains, STOP1 S163A The strain is more sensitive to aluminum toxicity (see Figure 9 e) We also constructed pSTOP1:STOP1 in the context of the stop1-2 mutant. S163D -HA(STOP1 S163D Transgenic lines. STOP1 S163D The strains were better than STOP1 under both -Al and +Al conditions. WT The strains accumulate more STOP1 protein (see Figure 9 (f) Consistent with this, STOP1 S163D The strain's resistance to aluminum toxicity is higher than that of STOP1 WT Stronger (see also) Figure 9 (g). These results indicate that phosphorylation of STOP1 Ser163 promotes protein accumulation and enhances the plant's resistance to aluminum toxicity.

[0177] To verify in vivo phosphorylation of STOP1 at Ser163, we used pIMAGO to detect protein phosphorylation and compare STOP1 levels. WT and STOP1 S163A STOP1 phosphorylation levels among strains (see) Figure 9 (h). The results showed that, under both aluminum-free and aluminum-containing conditions, STOP1 S163A The phosphorylation level of STOP1 in the strain was lower than that of STOP1 WT This indicates that STOP1 does indeed undergo phosphorylation at the Ser163 site in vivo.

[0178] 6. CPK28 regulates STOP1 accumulation via Ser163 phosphorylation.

[0179] To illustrate in more detail the role of CPK28 in regulating STOP1 accumulation through phosphorylation, we compared the STOP1 phosphorylation levels in WT and cpk28-2 cells carrying the pSTOP1:STOP1-HA transgene (see [link to original text]). Figure 10(a) Notably, the cpk28-2 mutation resulted in reduced phosphorylation levels of STOP1 under both aluminum-free and aluminum-containing conditions, suggesting that CPK28 may regulate STOP1 accumulation through phosphorylation.

[0180] To further elucidate the mechanism by which CPK28 regulates STOP1 abundance, particularly focusing on the Ser163 phosphorylation site of STOP1, we co-expressed 35S:CPK28 and 35S:STOP1 in stop1-2 protoplasts. WT Or 35S:STOP1 S163A The construct, and the pALMT1:LUC reporter gene. Compared with the GFP control, CPK28 significantly enhanced LUC activity via STOP1 (see [link to documentation]). Figure 10 (b). However, CPK28-induced increased LUC activity was observed in the expression of 35S:STOP1. S163A Cells expressing 35S:STOP1 WT The levels were significantly reduced in cells, highlighting the key role of the Ser163 site in the CPK28-targeted STOP1 function.

[0181] In addition, 35S:STOP1 is expressed in protoplasts. WT- LUC or 35S:STOP1 S163A -LUC and 35S:CPK28, the results showed that the S163A mutation significantly impaired CPK28-induced LUC activity (see...). Figure 10 (c) In addition, STOP1 was hybridized. S163D Introducing the gene into the cpk28-2 mutant background significantly restored the observed reduction in STOP1 levels and decreased expression of STOP1-regulated genes in the cpk28-2 mutant (see [link to article]). Figure 10 (d). Therefore, in STOP1 S163D In the / cpk28-2 strain, the damaged aluminum resistance was largely restored (see...). Figure 10 (e). In summary, these findings highlight the role of CPK28-mediated phosphorylation of STOP1 at Ser163 in regulating STOP1 accumulation and conferring aluminum resistance.

[0182] 7. Phosphorylation of STOP1 at Ser163 inhibits STOP1 degradation mediated by the F-box protein RAE1.

[0183] To illustrate in more detail how CPK28 regulates STOP1 protein accumulation through phosphorylation, we compared the phosphorylation levels of STOP1 in WT and cpk28-2 cells carrying the pSTOP1:STOP1-HA transgene. Figure 10(a) Notably, the cpk28-2 mutation resulted in decreased STOP1 phosphorylation levels under both -Al and +Al conditions, suggesting that CPK28 may regulate STOP1 protein accumulation by phosphorylating it. To further elucidate the role of STOP1 Ser163 in this process, we co-expressed 35S:CPK28 and 35S:STOP1 in stop1-2 protoplasts. WT Or 35S:STOP1 S163A The construct, and the pALMT1:LUC reporter gene. Compared with the GFP control, CPK28 significantly enhanced LUC activity via STOP1. Figure 10 (b). However, the extent of the CPK28-induced increase in LUC activity was observed in the expression of 35S:STOP1. S163A Cells expressing 35S:STOP1 WT Significantly reduced in cells ( Figure 10 (b) This reveals that the STOP1 Ser163 site plays a crucial role in the phosphorylation of STOP1 by CPK28. Furthermore, 35S:STOP1 is co-expressed in protoplasts. WT -LUC or 35S:STOP1 S163A -LUC and 35S:CPK28, the results showed that the S163A mutation significantly reduced CPK28-induced LUC activity ( Figure 10 (c)

[0184] Through hybridization, STOP1 S163D Introducing the gene into the cpk28-2 mutant background significantly restored the reduced STOP1 content observed in the cpk28-2 mutant. Figure 10 (d). Therefore, in STOP1 S163D In the / cpk28-2 strain, the weakened aluminum resistance of cpk28-2 was largely restored. Figure 10 (e). In summary, these results demonstrate the crucial role of CPK28-mediated phosphorylation of STOP1 at the Ser163 site in regulating STOP1 accumulation and plant resistance to aluminum toxicity. To further elucidate the mechanism by which the S163A mutation mediates STOP1 degradation, we investigated STOP1 degradation using pull-down experiments. WT -His、STOP1 S163A -His or STOP1 S163D - Interaction between His and GST-fused RAE1 (RAE1ΔF) fusion proteins with the F-box domain removed. Results showed that GST-RAE1ΔF interacts with STOP1. S163D -His interaction ratio and STOP1 WT -His or STOP1S163A -His interaction was weak. Consistent with this, Co-IP experiments in Arabidopsis protoplasts further confirmed that the phosphorylated S163D mutation suppressed the interaction between STOP1 and RAE1ΔF. Conversely, the non-phosphorylated S163A mutation enhanced the interaction between STOP1 and RAE1ΔF. This result suggests that WT STOP1 may be phosphorylated to some extent in the protoplast, inhibiting its interaction with RAE1ΔF. In summary, these results demonstrate that phosphorylation of STOP1 at the Ser163 site inhibits its interaction with RAE1.

[0185] 8. CPK28-mediated phosphorylation at Ser163 enhances the nuclear localization of the STOP1 protein.

[0186] To assess the impact of the cpk28 mutation on STOP1 subcellular localization, we isolated nuclear and cytoplasmic components from WT and cpk28-2 mutants carrying the pSTOP1:STOP1-HA transgene. These plant materials were treated with the 26S proteasome inhibitor MG132 under both aluminum- and aluminum-free conditions to prevent STOP1 degradation. Immunoblot analysis showed that in WT plants, aluminum treatment increased the proportion of STOP1 in the nucleus while decreasing its proportion in the cytoplasm. In contrast, under control and aluminum stress conditions, the proportion of STOP1 in the nucleus was significantly lower in the cpk28-2 mutant compared to WT. This result indicates that CPK28-mediated STOP1 phosphorylation is crucial for promoting its nuclear localization. To further investigate whether the CPK28-mediated altered STOP1 localization is caused by phosphorylation at the Ser163 site, we isolated nuclear and cytoplasmic components from STOP1… S163A and STOP1 S163D Nuclear and cytoplasmic proteins were isolated from the strain. Results showed that under -Al and +Al conditions, in STOP1... S163A In the strains, the proportion of STOP1 in the cell nucleus was significantly reduced; while in STOP1 S163D In the strains, this proportion increased significantly. These results indicate that phosphorylation of STOP1 by CPK28 at the Ser163 position is an important mechanism for enhancing its nuclear localization.

[0187] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

[0188] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are prior art or common general knowledge.

[0189] References

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[0191] Chen XX, et al. Protein kinases in plant responses to drought, salt, and cold stress. J Integr Plant Biol 63, 53 - 78 (2021).

[0192] Wernimont AK, et al. Structures of apicomplexan calcium - dependent protein kinases reveal mechanism of activation by calcium. Nat Struct Mol Biol 17, 596 - U595 (2010).

[0193] Ingram JR, et al. Allosteric activation of apicomplexan calcium - dependent protein kinases. Proc Natl Acad Sci USA 112, E4975 - E4984 (2015).

[0194] Liu KH, et al. Discovery of nitrate - CPK - NLP signalling in central nutrient - growth networks. Nature 545, 311 - +(2017).

[0195] Wang JL, et al. A regulatory module controlling homeostasis of a plant immune kinase. Mol Cell 69, 493 - +(2018).

[0196] Ding YL,et al.CPK28-NLP7 module integrates cold-induced Ca 2+ signal andtranscriptional reprogramming in Arabidopsis.Sci Adv 8,(2022).

[0197] Iuchi S,et al.Zinc finger protein STOP1 is critical for protontolerance in Arabidopsis and coregulates a key gene in aluminumtolerance.Proc Natl Acad Sci USA 104,9900-9905(2007).

[0198] Matschi S,Werner S,Schulze WX,Legen J,Hilger HH,Romeis T.Function ofcalcium-dependent protein kinase CPK28 of Arabidopsis thaliana in plant stemelongation and vascular development.Plant J 73,883-896(2013).

[0199] Li G,et al.The calcium-dependent protein kinase CPK7 acts on roothydraulic conductivity.Plant Cell Environ 38,1312-1320(2015).

[0200] Zhang Y,et al.F-box protein RAE1 regulates the stability of thealuminum-resistance transcription factor STOP1 in Arabidopsis.Proc Natl AcadSci USA 116,319-327(2019).

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Claims

1. The use of a polypeptide or its encoding gene in conferring or enhancing aluminum resistance in plants, wherein the polypeptide is selected from the group consisting of: (a) A polypeptide with the amino acid sequence shown in SEQ ID NO:1, which is a protein kinase CPK28 (GenBank: AT5G66210 or AAQ56823.1) derived from Arabidopsis thaliana; (b) A conserved variant polypeptide derived from (a) having the polypeptide function of (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO:

1. (c) A polypeptide derived from (a) that has more than 90% homology to the polypeptide sequence defined in (a) and possesses the function of the polypeptide in (a); or (d) A derivative polypeptide containing the polypeptide sequence described in (a), (b), or (c).

2. The application as described in claim 1, characterized in that, The nucleotide sequence encoding the polypeptide CPK28 (GenBank:AT5G66210) with the amino acid sequence shown in SEQ ID NO:1 is SEQ ID NO:2, or a nucleotide sequence that has more than 90% homology with SEQ ID NO:

2.

3. The application as described in claim 1, characterized in that, Used to create aluminum-resistant plant germplasm or to cultivate aluminum-resistant plant varieties.

4. The application as described in claim 1 or 3, characterized in that, The plants mentioned are crops selected from the group consisting of: rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, tobacco, and cruciferous plants such as Arabidopsis thaliana.

5. A method for improving the aluminum stress resistance of plants, characterized in that, It includes the following steps: inserting or overexpressing the gene encoding the polypeptide as described in claim 1 into the genome of a plant.

6. The method as described in claim 5, characterized in that, Implemented in the following manner: (i) Cloning the encoding gene of the polypeptide as described in claim 1 or 2 into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, and transforming plants by Agrobacterium-mediated transformation to obtain transgenic plants overexpressing the polypeptide; (ii) Using gene editing technology to... The gene encoding the polypeptide as described in claim 1 or 2 is cloned onto a plant chromosome to obtain a transgenic plant overexpressing the gene encoding the polypeptide as described in claim 1 or 2; and / or (iii) Place CPK28 in the plant genome under the regulation of a functionally enhanced promoter.

7. A DNA molecule, characterized in that, It contains the gene encoding the polypeptide CPK28 (GenBank: AT5G66210 or AAQ56823.1) as described in claim 2.

8. A recombinant plasmid, characterized in that, It contains the DNA molecule as described in claim 7.

9. The recombinant plasmid as described in claim 8, characterized in that, The plasmid vectors are selected from pHB-YFP, pHB-FLAG, pBin19, fluorescent reporter vectors pGreenII0800-LUC, pCAMBIA-nLUC, pCAMBIA1300, pCAMBIA1301, pCAMBIA3300, pCAMBIA2301, pBI121, and pTF102.

10. A type of engineered microbial bacterium, characterized in that, It is a transformant containing the recombinant plasmid as described in claim 8 or 9.

Images