ATL31 / 6-CPK28 molecular regulator and application thereof in regulating low-temperature stress resistance of plants

Abstract

The invention discloses a low-temperature stress molecular regulator ATL31 / 6-CPK28, the plant molecular regulator comprises ATL31 and / or ATL6 and CPK28, ATL31 / 6 and a calcium signal component CPK28 in the plant molecular regulator form a dynamic interaction network: E3 ligase activity of ATL31 is activated under a low-temperature condition, E3 ligase is promoted to be combined with CPK28, ubiquitination degradation of the CPK28 is mediated, and the ATL31 / 6 and the CPK28 are activated under a low-temperature condition. Therefore, a negative feedback regulation loop induced by environmental stimulation is constructed. The invention provides important theoretical breakthrough and molecular targets for plant adversity biology research, has obvious scientific research originality, and also has basic, wide and important application prospects.

Description

Technical Field

[0001] This invention relates to molecular biotechnology, and in particular to an ATL31 / 6-CPK28 molecular regulator and its application in regulating plant resistance to low-temperature stress. Background Technology

[0002] Low temperature is one of the key environmental factors limiting the geographical distribution of plants and agricultural production. When plants are exposed to low temperatures, a series of physiological and biochemical disorders occur within their cells, such as reduced membrane fluidity, accumulation of reactive oxygen species, protein denaturation, and metabolic imbalance, which in turn affect their normal growth and survival. To cope with cold stress, plants have evolved multi-layered adaptation mechanisms, covering a complete response chain from environmental perception and signal transduction to gene expression reprogramming and metabolic adjustment. Among these, the dynamic regulation of key regulatory proteins in signaling pathways is particularly crucial, and post-translational modification, due to its rapid and reversible characteristics, has become a core strategy for achieving precise signal transmission and timely termination.

[0003] Among numerous post-translational modifications, the ubiquitin-proteasome system plays an irreplaceable role in regulating plant stress responses. This system covalently links ubiquitin molecules to target proteins via a three-enzyme cascade, mediating their degradation or functional alteration, thereby regulating developmental processes and stress responses. E3 ubiquitin ligases, as key factors determining substrate specificity in this pathway, directly determine the targeting and biological effects of ubiquitination events. In recent years, an increasing number of E3 ubiquitin ligases have been shown to participate in the regulation of abiotic stresses such as drought, high salinity, heat stress, and low temperature in plants, suggesting their broad function in environmental adaptation.

[0004] In Arabidopsis, the Arabidopsis tóxicos en Levadura (ATL) family is a class of plant-specific RING-type E3 ubiquitin ligases involved in the regulation of various physiological processes. Studies have shown that some ATL members play important roles in plant immunity and nutrient homeostasis, but their function in temperature stress response remains poorly understood. In particular, while studies have revealed the regulatory roles of ATL31 and ATL6 in innate immunity and ion homeostasis, their roles in the low-temperature signaling network have not yet been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the various shortcomings of the prior art and provide an ATL31 / 6-CPK28 molecular regulator and its application in regulating plant resistance to low temperature stress.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0007] Firstly, this method includes a method based on the synergistic regulation of plant resistance to low-temperature stress by ubiquitin ligases ATL31 and ATL6. ATL31 and ATL6 synergistically and rapidly initiate a low-temperature response and mediate the dynamic regulation of CPK28 protein abundance, achieving precise response to cold signaling pathways simultaneously in time and space. This prevents energy loss and cell damage caused by excessive response while resisting low-temperature stress, thereby achieving regulation of plant response to low-temperature stress.

[0008] On the other hand, the present invention also includes a method for improving plant resistance to low temperature stress by reducing or inhibiting the expression of ATL31 and ATL6 in plants, constructing single mutants and / or double mutants of ATL31 and ATL6, thereby improving the cold resistance of plants.

[0009] On the other hand, the present invention also includes an industrial application of the method: increasing plant yield by improving the cold resistance of plants.

[0010] On the other hand, the present invention also includes a method for constructing low-temperature sensitive lines, improving the expression of ATL31 and ATL6 in plants, constructing single overexpression lines or double overexpression lines of ATL31 and ATL6, and the resulting overexpression lines are more sensitive to low-temperature stress than wild lines.

[0011] On the other hand, the present invention also includes an application of the method: constructing strains that are highly sensitive to low temperatures for experimental research on the regulation of plant low-temperature response.

[0012] On the other hand, the present invention also includes a low-temperature stress molecular regulator ATL31 / 6-CPK28, which includes ATL31 and / or ATL6 and CPK28. In this plant molecular regulator, ATL31 / 6 forms a dynamic interaction network with the calcium signaling component CPK28: under low temperature conditions, the E3 ubiquitin ligase activity of ATL31 is activated, which promotes its binding to CPK28 and mediates the ubiquitination and degradation of the latter, thereby constructing a negative feedback regulatory loop induced by environmental stimuli.

[0013] On the other hand, the present invention also includes a novel application of the aforementioned low-temperature stress molecular regulator ATL31 / 6-CPK28: a novel function that can both amplify the response signal and shut down the response signal in a timely manner in the plant cold signal regulation pathway.

[0014] On the other hand, the present invention also includes a novel use of the low-temperature stress molecular regulator ATL31 / 6-CPK28: using this low-temperature stress molecular regulator as a bidirectional regulatory hub to precisely regulate the positive and negative responses of plants to low temperature through phosphorylation-ubiquitination cascade reactions, to achieve rapid initiation of antifreeze response through bidirectional response, and to avoid continuous stress by dynamically regulating the abundance of CPK28 protein, thereby achieving a spatiotemporal dynamic balance between plant resistance and growth.

[0015] As a preferred embodiment of the present invention, the bidirectional response specifically includes: achieving a closed-loop regulatory mode in the plant cold signal regulation pathway of "CPK28 kinase activating E3 ubiquitin ligase, while simultaneously synergistically initiating cold E3 ubiquitin ligase to degrade CPK28 kinase", thus constructing a complete negative feedback loop; thereby achieving the following full-process signaling pathway after the low-temperature signal is initiated: low temperature induces an increase in cold Ca²⁺, which in turn induces the activation of cold CPK28, which in turn induces phosphorylation of ATL31, which in turn activates the activity of cold E3 ubiquitin ligase; at the signal termination end, the activated ATL31 / 6 induces the ubiquitination of cold CPK28, which in turn induces the degradation of cold CPK28, which in turn induces the attenuation of the cold signal; thereby enabling the plant to quickly initiate a defense response when encountering low temperatures, while preventing energy waste and / or cell damage caused by continuous signal activation.

[0016] Finally, the present invention also includes a method for improving plant survival rate, reducing electrolyte leakage rate and enhancing antioxidant capacity under low temperature stress: achieved by reducing or inhibiting the expression of ATL31 and ATL6 in plants.

[0017] The beneficial effects of the above technical solution are as follows: This invention is the first to discover that 2ATL31 / 6 not only negatively regulates plant cold tolerance but also forms a dynamic interaction network with CPK28, a key component of calcium signaling: low temperature activates the E3 ubiquitin ligase activity of ATL31, promoting its binding to CPK28 and mediating the latter's ubiquitination and degradation, thereby constructing a negative feedback loop induced by environmental stimuli. This discovery reveals that E3 ubiquitin ligases have a dual function of signal amplification and timely shutdown in the cold signaling pathway, providing a new molecular perspective for understanding how plants achieve environmental adaptation through protein stability regulation. Furthermore, this invention reveals that ATL31 not only negatively regulates plant cold tolerance but also forms a dynamic interaction network with CPK28, a key component of calcium signaling in the low-temperature response: low temperature conditions activate the E3 ubiquitin ligase activity of ATL31, promoting its binding to CPK28 and mediating the latter's ubiquitination and degradation, thereby constructing a negative feedback regulatory loop induced by environmental stimuli. This mechanism reveals the unique function of E3 ligases in cold signaling pathways, enabling them to both amplify and timely shut down signals, providing a new perspective on how plants utilize ubiquitination modifications to achieve precise adaptation to environmental changes. This invention deepens our understanding of how plants finely regulate physiological processes through complex protein-protein interaction networks to cope with environmental challenges and lays the foundation for developing new strategies to improve crop cold resistance in the future.

[0018] This invention has achieved several groundbreaking advancements in the field of plant low-temperature stress response. Its core technological advancements are reflected in the following aspects: ① It is the first to reveal that ATL6 / 31-CPK28 constitutes a bidirectional regulatory hub of "phosphorylation-ubiquitination". This invention is the first to systematically elucidate the bidirectional regulatory relationship between the E3 ubiquitin ligase ATL6 / 31 and the calcium-dependent protein kinase CPK28: positive regulation: CPK28 regulates through Ca... 2+The invention utilizes a dependent phosphorylation mechanism to activate the E3 ubiquitin ligase activity of ATL31. In turn, activated ATL31 / 6 mediates the ubiquitination and proteasome degradation of CPK28. This closed-loop mechanism of "kinase activation of E3 → E3 degradation of kinase" constructs a typical negative feedback regulatory loop, a first-time report in plant cold signal transduction, providing a novel paradigm for understanding how plants achieve precise initiation and timely termination of stress signals. Secondly, it discovers a new mechanism of phosphorylation as a "molecular switch" for E3 ligase activity. Previous studies have focused on the ubiquitination regulation of substrates by E3 ligases, with little understanding of how their own activity is activated by upstream signals. This invention is the first to demonstrate that CPK28-mediated phosphorylation is a key driving force for the activation of ATL31 E3 activity at low temperatures. This discovery breaks through the traditional understanding that "E3 is only a signal endpoint," proposing that the E3 ligase itself can also act as a signal integration node, achieving functional activation through phosphorylation, thus expanding the theoretical framework of the ubiquitin system in signal transduction. ③ This invention reveals the molecular bridge between calcium signaling and the ubiquitin system. Ca²⁺ is a primary messenger in plants sensing environmental stress, while the ubiquitin-proteasome system (UPS) is responsible for protein homeostasis regulation. This invention establishes for the first time a direct link between the two in the low-temperature response: Low temperature → Ca²⁺ 2+ ↑ →CPK28 activation → ATL31 phosphorylation → CPK28 ubiquitination and degradation; Clarifying that CPK28 is both a Ca 2+The signal "decoder" is also a "regulatory target" of the ubiquitin system. This mechanism reveals how calcium signals are transformed into changes in protein stability through the kinase-E3 axis, providing key experimental evidence for the complete pathway of "signal sensing-post-translational modification-physiological response". ④ A "activation-inhibition" dynamic balance model is proposed to explain the trade-off mechanism between plant resistance and growth. The ATL31 / 6-CPK28 regulatory module proposed in this invention not only achieves rapid response but also prevents excessive signal activation through negative feedback: rapidly initiating the expression of antifreeze genes (activating the CBF pathway through CPK28); and timely terminating the signal to avoid energy waste or growth inhibition (degrading CPK28 through ATL31 / 6). This biphasic regulatory strategy provides a molecular explanation for the dynamic balance between stress resistance and normal growth in plants, and has important physiological and evolutionary significance. ⑤ Expanding the functional understanding of E3 ligases in abiotic stress. ATL31 / 6 has previously been mainly studied in immune responses. This invention, for the first time, extends the function of E3 ligases to the field of low-temperature stress response and reveals that it affects the CBF-COR pathway by regulating the key kinase CPK28, breaking the traditional view that E3 ligases only participate in protein degradation and highlighting their pivotal role in a multi-stress cross-regulatory network. In summary, this invention not only discovers a novel phosphorylation-ubiquitination cascade regulatory module in plant cold signal transduction but also proposes an innovative model at the mechanistic level in which E3 ligases can be activated by kinase phosphorylation and, in turn, regulate kinase stability. This work organically integrates calcium signaling, protein kinases, and the ubiquitin system to construct a dynamic, reversible, and self-regulating signaling network, providing an important theoretical breakthrough and molecular target for plant stress biology research. It possesses significant scientific originality and has fundamental, broad, and important application prospects. Attached Figure Description

[0019] Figure 1This diagram illustrates the negative regulation of plant frost resistance by ATL6 and ATL31. In the figure: (AC) Frozen phenotypes (A) of Col, atl6, atl31, and atl6atl31 mutants under NA and CA conditions, including survival rate (B) and ion leakage rate (C). Col and mutant plants were cultured on 1 / 2 MS medium at 22°C for 10 days before being frozen. For NA treatment, the initial temperature was 0°C, decreasing to -3°C at a rate of 1°C per hour, followed by maintenance at -4°C for 1 hour. Plants A were pretreated at 4°C for 3 days, with a freezing program starting at 0°C, decreasing to -8°C at a rate of 1°C per hour, and maintaining at -8°C for 1 hour. After treatment, plants were recovered for 12 hours in darkness at 4°C, and then transferred to normal growth conditions of 22°C, 16 hours of light / 8 hours of darkness. Representative images were taken 3 days after recovery, and survival rate and ion leakage rate were measured. Data are presented as mean ± standard error (SEM) of three independent replicates. Different lowercase letters indicate significant differences between groups as determined by one-way ANOVA (P < 0.05). (D) Expression levels of the CBF gene in Col, atl6, atl31, and atl6atl31 mutants under low-temperature stress. Plants were cultured on 1 / 2 MS plates at 22°C for 10 days, then transferred to 4°C for 6 hours. Total RNA was extracted and analyzed by qRT-PCR. The relative expression level was calculated using the expression level of the CBF gene in wild-type plants at 22°C as a baseline (set as 1.0). Data are presented as mean ± SEM of three biological replicates. Significant differences are indicated by different lowercase letters (P < 0.05, one-way ANOVA).

[0020] Figure 2This diagram illustrates the activation of E3 ligase activity of ATL31 by low-temperature treatment. In the figure: (A) Total RNA was extracted from 10-day-old Col plants after treatment at 4°C for different time periods. The transcriptional levels of ATL31 (A) and ATL6 (B) were analyzed by RT-qPCR, and Actin2 was used as an internal control for standardization. Data were calculated based on the 0-hour treatment group (set as 1). Data for each group are presented as mean ± standard deviation (SD) of three technical replicates. All experiments were repeated three times, and results are expressed as mean ± standard error (SEM). Different lowercase letters indicate significant differences (P < 0.05) as detected by one-way ANOVA. (C) Effect of low-temperature treatment on ATL31-HA protein accumulation. Seven-day-old ATL31-OE1 seedlings (10 seedlings per sample) were treated with 100 μM cyclohexylimide (CHX) to block new protein synthesis. After incubation at 4°C for a specified time, total protein was extracted and detected by Western blotting using an anti-HA antibody. Tubulin was used as a loading control. (D) MG132 inhibits low-temperature-induced degradation of ATL31 protein. Seven-day-old ATL31-OE1 seedlings were treated with CHX and 4°C for different durations with or without 50 μM proteasome inhibitor MG132, and total protein was extracted from the whole plant. Tubulin was used as a loading control. (E) In vivo ubiquitination experiments verified low-temperature-induced ubiquitination modification of ATL31. Ten-day-old Col and ATL31-OE transgenic seedlings were treated at 4°C for 1 h, and total protein was extracted and the target protein was enriched using HA magnetic beads. The total ATL31-HA protein level was confirmed by anti-HA antibody, and the polyubiquitination signal of ATL31 was detected by anti-Ub antibody. Tubulin was used as a control.

[0021] Figure 3This diagram illustrates that CPK28 is a component essential for the regulation of plant cold response by ATL6 and ATL31. In the diagram: (AC) the frozen phenotypes (A) of the Col, atl6atl31, cpk28, and atl6atl31cpk28 triple mutants, including survival rate (B) and ion leakage rate (C). Plants were cultured on 1 / 2 MS medium at 22°C for 10 days before being frozen. Under NA conditions, plants were treated at -4°C for 1 h; CA-treated plants were pretreated at 4°C for 3 days, followed by treatment at -8°C for 1 h. After treatment, the culture was restored, and relevant parameters were measured. Data are the mean ± standard error (SEM) of three independent biological replicates. Different lowercase letters indicate significant differences between groups as detected by one-way ANOVA (P < 0.05). Expression levels of the CBF gene in the (DF)Col, atl6atl31, cpk28, and atl6atl31cpk28 triple mutants under low-temperature stress. Plants were cultured on 1 / 2 MS plates at 22°C for 10 days, then transferred to 4°C for 6 hours. Total RNA was extracted and analyzed by qRT-PCR. Relative expression levels were calculated using the expression levels of CBF1 (D), CBF2 (E), and CBF3 (F) in wild-type plants at 22°C as a baseline (set as 1.0). All data are mean ± SEM from three replicates. Significant differences are indicated by different lowercase letters (P < 0.05, one-way ANOVA).

[0022] Figure 4 This diagram illustrates how low-temperature stress promotes the interaction between ATL31 and CPK28. The figure shows the Co-IP technique used to detect the interaction between ATL31 and CPK28 in Arabidopsis thaliana. HA-ATL31-overexpressing plants grown on 1 / 2 MS medium were transferred from 22°C to 4°C for 0 and 2 h. Total protein was extracted and immunoprecipitated using anti-HA agarose beads. Col was treated under the same conditions as a control. HA-ATL31 and CPK28 were detected using anti-HA and anti-CPK28 antibodies, respectively.

[0023] Figure 5 For CPK28 with Ca 2+A schematic diagram of ATL31 phosphorylation in a dependent manner. In the figure: (A) In vitro kinase assay to detect the phosphorylation of ATL31 by CPK28. Activated Flag-CPK28 was used as the kinase, and Flag-ATL31 as the substrate, and the kinase reaction was carried out in the reaction system. After separation of the reaction products by SDS-PAGE, the phosphorylation level of ATL31 was detected using an anti-phosphorylated serine / threonine (anti-pSer / pThr) antibody. (B) Phosphorylation status of ATL31 in ATL31 overexpressing plants under low-temperature response. Ten-day-old ATL31-OE transgenic seedlings were treated at 4°C for different times, or pretreated with 25mMEGTA for 45 min before low-temperature treatment, followed by low-temperature treatment for 10 s. Total protein was extracted, and ATL31-HA protein was immunoprecipitated using anti-HA agarose beads. Protein phosphorylation was detected using an anti-phosphoserine / threonine antibody (anti-ps / T). The following figure shows the expression level of ATL31-HA protein, serving as a sample control.

[0024] Figure 6 This diagram illustrates the degradation of CPK28 mediated by ATL31 / 6 under low-temperature conditions. In the figure: (A and B) the effect of low-temperature treatment on CPK28 protein accumulation. Seven-day-old Col (A) and atl6atl31 (B) seedlings (10 seedlings per sample) were treated with 100 μM CHX to block new protein synthesis. After incubation at 4°C for a specified time, total protein was extracted and detected by Western blotting using an anti-CPK28 antibody. Tubulin was used as a loading control. (C) MG132 inhibits low-temperature-induced ATL31 protein degradation. Seven-day-old Col seedlings were treated with CHX and 4°C for different times, with or without 50 μM proteasome inhibitor MG132, and total protein was extracted from the whole plant. Tubulin was used as a loading control. Detailed Implementation

[0025] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. It should be understood that, when used in this specification and appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or collections thereof. It should also be understood that the term "and / or" as used in this specification and appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes such combinations. As used in this specification and appended claims, the term "if" can be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" can be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]." Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance. References to "one embodiment" or "some embodiments" in this application mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized.

[0026] Previous Examples

[0027] 1.1 Gene Explanation

[0028] For information on the ATL31, ATL6, and CPK28 genes and their routine molecular biological procedures, please refer to the following sources: atl31 (GK_746D08, Sato et al., 2009), atl6 (Salk_149614, Sato et al., 2009), cpk28-1 (WiscDslox246D03, Monaghan et al., 2014); and: Sato et al., 2011; Yasuda et al., 2014; Chevalier et al., 2009; Obsil and Obsilova 2011; Roberts 2003; Huarancca Reyes et al., 2015; Maekawa et al., 2012; Maekawa et al., 2014; Hondo et al., 2007; Takahashi et al., 2010.

[0029] For information on the experimental materials and methods of this invention, please refer to the following materials:

[0030] 2.1 Plant materials and growing conditions

[0031] All Arabidopsis thaliana (Arabidopsis) materials used in this study were based on the Columbia (Col) ecotype. Plants were cultured on 0.8% (w / v) agar and 2% (w / v) sucrose medium (Murashige Skoog, MS) (Sigma-Aldrich) at 22°C under long-day conditions (16 h light / 8 h dark). The T-DNA insertion mutants used included cpk28, atl31, and atl6 (Salk_149614, Sato et al., 2009), and their double mutant atl31atl6. All of these materials have undergone functional identification in previous studies (Liu et al., 2022). To construct overexpression lines, the full-length coding sequence of ATL31 was obtained by PCR amplification using Col cDNA as a template and cloned into the plant binary expression vector pTF101 to construct the p35S::ATL31-HA expression vector driven by the 35S promoter and fused with the HA tag. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 strain, and introduced into Col plants using the inflorescence maceration method. Stable ATL31 overexpression transgenic lines were screened for subsequent functional analysis.

[0032] 2.2 Experimental Reagents and Instruments

[0033] 2.2.1 The reagents used in the experiment are shown in the table below:

[0034] Table 2.1 Reagents related to plant culture

[0035]

[0036] Table 2.2 Main reagents and supplies required for protein purification and Western blotting

[0037]

[0038] Table 2.2 Main reagents and supplies required for protein purification and Western blotting (continued)

[0039]

[0040] 2.2.2 The experimental apparatus is shown in the table below:

[0041] Table 2.3 List of Experimental Instruments

[0042]

[0043] Table 2.3 List of Experimental Instruments (Continued)

[0044]

[0045] Table 2.4 RNA extraction and other reagents

[0046]

[0047] 3.1 Explanation of Abbreviations

[0048] The abbreviations used in this invention are listed in the following abbreviation table.

[0049]

[0050] Example 1: Cold Acclimation and Freezing Damage Treatment and Ion Leakage Rate Measurement

[0051] Unless otherwise specified, the Arabidopsis thaliana material used in the experiments consisted of two-week-old seedlings grown on 1 / 2 MS plates in a 22°C incubator. Prior to the frost treatment, some seedlings underwent a 3-day cold acclimation (CA) treatment at 4°C, while others were directly subjected to non-acclimated (NA) frost treatment. The frost treatment was conducted in a RuMED cryogenic incubator with the program set as follows: initial temperature 0°C, then gradually decreasing to the target temperature at a rate of 1°C per hour. After treatment, the seedlings were maintained in darkness at 4°C for 12 hours before being transferred to a 22°C incubator to recover. Survival rates were calculated after 3 days of recovery, with plants capable of sprouting new leaves considered surviving individuals. Three independent biological replicates were performed for each genotype or treatment group at each temperature gradient to ensure the reliability and reproducibility of the data.

[0052] After 3 days of recovery culture, seedlings were placed in 15 mL centrifuge tubes, and 5 mL of deionized water (denoted as S0) was added. The mixture was then extracted at 22°C with shaking for 1 h, followed by measurement of the initial conductivity (S1). The sample was then heated in a 100°C boiling water bath for 1 h to fully release intracellular ions, and then shaken at 22°C for another 1 h, with the total conductivity (S2) measured. Based on the measured data, the ion leakage rate was calculated using the formula (S1 - S0) / (S2 - S0) × 100%. This index is used to assess the degree of cell membrane damage caused by low-temperature stress and reflects the freeze resistance of plant tissues.

[0053] Example 2: RNA extraction and real-time quantitative PCR analysis

[0054] Total RNA was extracted from plant tissues using a plant total RNA extraction kit (RNAprep Pure Plant Kit, DP419, Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. A suitable amount of RNA sample was taken and reverse transcribed using a HiScript® III RT SuperMix kit (R323-01, Vazyme, Nanjing, China) to synthesize the first strand of cDNA. Real-time quantitative PCR (RT-qPCR) was performed on a LightCycler 480 II real-time quantitative PCR detection system (Roche, Basel, Switzerland) using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). Relative gene expression levels were calculated using the 2-ΔΔCt method. Data processing was performed according to the method described by Livak and Schmittgen (2001), with ACTIN2 used as an internal reference gene for standardization. Primer sequences used in this invention are detailed in Appendix S (xml sequence listing).

[0055] Table S

[0056]

[0057] Example 3, Antibody

[0058] The sources and usage information of the antibodies used in this invention are as follows: The anti-CPK28 antibody was prepared by the Animal Center of the Beijing Institute of Genetics and Developmental Biology using full-length recombinant CPK28 protein as the antigen to immunize mice. The anti-phosphorylated serine / threonine antibody (pSer / pThr, PM3801) was purchased from ECM Biosciences (Lexington, KY, USA). The anti-HA-tagged monoclonal antibody (H6533) and the anti-FLAG-tagged mouse monoclonal antibody (F1804) were both purchased from Sigma-Aldrich (USA). The anti-α-Tubulin mouse monoclonal antibody (T5168, Sigma-Aldrich, USA) was used for Western blot analysis at a working dilution of 1:4,000. The secondary antibody was goat anti-rabbit and anti-mouse IgG-HRP (M21003-S) purchased from Abmart (China) at a concentration of 1:10,000. All antibodies were validated for use in Western blot or immunoprecipitation experiments to ensure the specificity and reliability of the test results.

[0059] Example 4: Expression and purification of recombinant proteins in Escherichia coli

[0060] The constructed expression plasmid was transformed into *E. coli* BL21 competent cells. Single colonies were picked and inoculated into 5 mL of LB broth containing the appropriate antibiotic, and cultured at 37°C with shaking for 12 h for pre-culture. Then, they were transferred to 500 mL of LB broth containing the appropriate antibiotic and cultured at 37°C for another 2 to 4 h until the optical density (OD600) at 600 nm reached 0.4–0.6. Unless otherwise specified, the recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.5 mM and cultured at 16°C for another 12 h to promote the expression of soluble proteins. After induction, the bacterial cells were collected, and the protein was extracted and purified according to the instructions of the appropriate protein purification kit to obtain high-purity target recombinant protein for subsequent in vitro biochemical experiments.

[0061] Example 5: Co-immunoprecipitation assay method

[0062] Co-immunoprecipitation (co-IP) experiments were performed according to previous literature (Ding et al., 2022). Briefly, total protein was extracted from plant material expressing the Super:ATL31-HA construct. The lysis buffer composition was: 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, and 0.1% (v / v) Triton X-100. The protein extract was incubated at 4°C with anti-HA agarose beads (KTSM1363, ktsmo) for 2 hours. The extract was then washed five times with lysis buffer to remove non-specifically bound proteins, and the immunoprecipitation complex was collected. After separation by SDS-PAGE, the precipitate was detected by Western blotting using anti-HA antibody (H6533, Sigma-Aldrich, USA) and anti-CPK28 antibody to verify the interactions between the target proteins.

[0063] Example 6: In vivo phosphorylation detection experiment

[0064] To detect the in vivo phosphorylation status of ATL31, approximately 4-week-old ATL31-OE plants were cryopreserved at 4°C for 2 h, followed by total protein extraction. Protein samples were dissolved in 600 μL of extraction buffer, and 20 μL of anti-HA magnetic beads (KTSM1363, ktsmo) were added. Immunoprecipitation was performed after incubation at 4°C for 3 h. The precipitated complex was washed five times with IP buffer to remove non-specifically bound proteins. After separation by SDS-PAGE, phosphorylated ATL31 protein was detected using an anti-phosphorylated serine / threonine antibody (anti-pSer / pThr, PM3801, ECM Biosciences), and the total HA-ATL31 protein level was simultaneously detected using an anti-HA antibody to assess its expression.

[0065] Example 7: Plant Protein Extraction and Immunoprecipitation Experiment

[0066] Total protein was extracted from transgenic Arabidopsis plants and lysed using pre-chilled extraction buffer (containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% Triton X-100, 0.1% NP-40, 5 mM DTT, and 1× EDTA-free protease inhibitor complex (Roche)). Samples were centrifuged twice at 13,000 g for 15 minutes each at 4°C to remove cell debris and obtain clear protein lysates. For immunoprecipitation (IP) experiments, equal volumes of total protein extract were incubated at 4°C with affinity-tagged agarose beads for 2 hours. Resins used included anti-HA agarose beads (Sigma-Aldrich, #A2095) and Ubi-trap agarose beads (Chromotek). After immunoprecipitation, the beads were washed five times with extraction buffer at 4°C for 5 minutes each time to reduce non-specific binding. The precipitated complex was eluted by adding 5× SDS loading buffer and heating at 90°C for 5 minutes. The resulting protein samples were separated by 10% or 12% (w / v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (NC membranes). Immunoblot detection was performed using the appropriate specific antibodies according to the figure captions. To ensure uniform loading volume, all membranes were validated using an anti-α-Tubulin antibody (T5168, Sigma-Aldrich, USA) as an internal control.

[0067] Example 8: Protein Accumulation Analysis Method

[0068] To detect protein accumulation levels, 10-day-old seedlings grown in the culture chamber were transferred to 1 / 2 MS medium and treated at 4°C or room temperature (22°C) for a specified time. Total protein was then extracted using 2× SDS loading buffer (containing 125 mM Tris-HCl pH 6.8, 4% SDS, 2% β-mercaptoethanol, 20% glycerol, and bromophenol blue) at 95°C for 10 min. After separation by SDS-PAGE, Western blot analysis was performed. The expression level of the target protein was detected using an anti-HA antibody (H6533, Sigma-Aldrich, USA). The signal of the internal control antibody anti-α-Tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA) on the same membrane was used as a loading control to ensure consistent protein loading and accurately assess the accumulation dynamics of the target protein under different treatment conditions.

[0069] Example 9: Ubiquitination Detection Experiment

[0070] To analyze protein ubiquitination levels, 3-day-old wild-type or ATL31-OE transgenic seedlings cultured in the dark were ground into powder in liquid nitrogen. The powder was dissolved in IP buffer (containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% Triton X-100, 0.1% NP-40, 5 mM DTT, and 1× EDTA-free protease inhibitor complex, Roche). The solution was centrifuged three times at 20,000 g for 10 min each time at 4°C, and the supernatant was collected as the total protein extract. Immunoprecipitation was then performed. An equal volume of the protein extract was incubated with anti-HA agarose beads (Sigma-Aldrich, #A2095) at 4°C for 2 h to enrich HA-labeled ATL31 protein. The precipitated complex was washed three times with washing buffer to remove non-specifically bound proteins, and then analyzed by Western blotting. The ubiquitination modification band was detected using an anti-ubiquitin antibody (Abcam, ab7254), and the expression level of ATL31 protein was confirmed using an anti-HA antibody to assess its ubiquitination status in vivo.

[0071] Example 10: ATL6 and ATL31 negatively regulate plant frost resistance

[0072] CPK28, as a core regulatory factor in plant cold stress response, integrates low-temperature-induced Ca2+... 2+ It plays a crucial role in signaling and downstream transcriptional reprogramming (Ding et al., 2022). Simultaneously, this protein also participates in the negative regulation of cytoplasmic calcium channeling mediated by Botrytis-induced kinase 1 (BIK1) triggered by pathogen-associated molecular patterns (PAMPs) in the immune pathway. 2+The outbreak of cold stress signals can finely regulate the intensity of the immune response (Monaghan et al., 2015; Wang et al., 2018). Previous studies have shown that E3 ubiquitin ligases ATL6 and ATL31 can directly mediate the ubiquitination of CPK28, promoting its degradation via the proteasome pathway and thus enhancing the plant's immune response (Liu et al., 2023). However, it remains unclear whether ATL6 and ATL31 also participate in the regulation of cold stress signals. Therefore, we systematically analyzed the freezing phenotypes of atl6 and atl31 single mutants and atl6-atl31 double mutants under NA and CA conditions. The results showed that the survival rate of both single mutants was significantly higher than that of the wild type. The atl31 mutant exhibited better cold tolerance than atl6, while the double mutant showed further enhanced cold resistance, demonstrating the strongest survival advantage. Further ion leakage measurements showed that the atl6 and atl31 mutants exhibited lower cell membrane damage and greater membrane stability after low-temperature treatment compared to the wild type, and the double mutants had the lowest ion leakage rate, consistent with the survival results.

[0073] In summary, both ATL6 and ATL31 negatively regulate plant cold tolerance, and their functions are partially redundant in regulating the cold response. The double mutants exhibit superimposed cold-resistant phenotypes, suggesting that they may participate in the negative feedback regulation of low-temperature adaptation by synergistically targeting key signaling proteins such as CPK28.

[0074] In Arabidopsis thaliana, cold stress typically induces the expression of CBF-like transcription factors, thereby activating the transcription of downstream cold-response genes and constituting a core regulatory pathway for cold adaptation. To investigate whether ATL6 and ATL31 are involved in the regulation of this pathway, we examined the CBF gene expression levels of atl6, atl31 single mutants, and atl6 atl31 double mutants under cold treatment. Real-time quantitative PCR results showed that after 6 h of treatment at 4°C, the transcription levels of CBF1, CBF2, and CBF3 in atl6 and atl31 mutants were significantly higher than those in the wild type, with the atl31 mutant showing a more pronounced induction. Furthermore, the expression of these genes was further enhanced in the atl6 atl31 double mutant, reaching the highest level. Figure 1 D). Based on the aforementioned phenotypic analysis, both atl31 and atl6 mutants exhibited higher survival rates and lower ion leakage under both non-acclimatization and cold-acclimatization conditions. Furthermore, the combined enhancement of cold resistance between the two mutants indicates that both ATL6 and ATL31 negatively regulate plant tolerance to cold stress, and that both exhibit functional redundancy in regulating the cold response, with ATL31 playing a more prominent role. In conclusion, ATL6 and ATL31 negatively regulate plant cold resistance by inhibiting the activation of the CBF pathway; their absence enhances the efficiency of cold signal transmission, thereby improving the plant's low-temperature adaptation.

[0075] Example 11: Low-temperature activation of E3 ligase activity of ATL31

[0076] To investigate the regulatory mechanisms of ATL6 and ATL31 in plant cold stress response, we first analyzed their expression patterns under low-temperature conditions. Real-time quantitative PCR results showed that after treatment at 4°C, ATL31 transcripts in seedlings began to increase at 30 min, peaked at 4 h, and then returned to basal levels at 12 h. Figure 2 A); In contrast, ATL6 transcription levels peaked after 30 min of cold treatment, then rapidly declined, returning to their initial state within 6 h. Figure 2 (B) indicates that the cold induction amplitude of ATL31 is stronger and the duration is longer. To further explore whether the stability of ATL31 protein is regulated by low temperature, we used cycloheximide (CHX) to inhibit the synthesis of new protein and examined its degradation dynamics in p35S::ATL31-HA / Col transgenic plants. The results showed that at room temperature, ATL31-HA protein gradually decreased over time, while after treatment at 4°C for 4 h, its degradation rate was significantly accelerated (B). Figure 2 C). Notably, this degradation process can be effectively blocked by the proteasome inhibitor MG132 ( Figure 2 (D) suggests that low temperature may promote the proteasome-dependent degradation of ATL31 by enhancing its own ubiquitination. Given that ATL31 is an E3 ubiquitin ligase, this phenomenon suggests that low temperature may activate its enzymatic activity. To verify this hypothesis, we conducted in vivo ubiquitination experiments using p35S::ATL31-HA transgenic seedlings. ATL31-HA protein was immunoprecipitated with and without 4°C treatment, and its ubiquitination level was detected using an anti-ubiquitin antibody. The results showed that, compared with the control, the ubiquitination modification of ATL31 was significantly enhanced after low temperature treatment (D). Figure 2 E), indicating that its E3 ligase activity was effectively activated under low temperature conditions.

[0077] Example 12: CPK28 is an essential component for ATL6 and ATL31 to regulate plant cold response.

[0078] To elucidate the genetic relationship between ATL6 / 31 and CPK28 in the cold stress signaling pathway, we constructed the atl31 atl6 cpk28 triple mutant through genetic hybridization. Freezing tolerance analysis showed that the atl31 atl6 double mutant exhibited significantly enhanced freezing resistance compared to Col, while the atl31 atl6 cpk28 triple mutant showed freezing sensitivity comparable to the cpk28 single mutant. Figure 3(A and B) indicates that the deletion of CPK28 completely suppressed the enhanced cold resistance phenotype in the atl31 atl6 double mutant. Ion leakage assays further confirmed that the cell membrane damage after low-temperature treatment in the atl31 atl6 double mutant was significantly lower than that in Col, while the ion leakage level of the triple mutant was not significantly different from that of the CPK28 single mutant. Figure 3 C). Furthermore, real-time quantitative PCR analysis showed that after 6 h of treatment at 4°C, the expression of CBF1, CBF2, and CBF3 was significantly upregulated in the atl6 atl31 double mutant; while the induction of these key cold-response transcription factors was significantly inhibited in the cpk28 mutant; in the atl6 atl31 cpk28 triple mutant, the expression pattern of the CBF gene was consistent with that of the cpk28 single mutant, both being in a low-induction state (C). Figure 3 (D–F). The above results indicate that the functions of ATL6 and ATL31 in regulating plant cold resistance depend on the presence of CPK28. CPK28 is located downstream of ATL6 / 31 genetically and is an indispensable signaling component for mediating cold response regulation.

[0079] Example 13: Low temperature stress promotes the interaction between ATL31 and CPK28

[0080] To verify whether there is a physical interaction between ATL31 and CPK28, we conducted a co-IP experiment in p35S::ATL31-HA / Col transgenic seedlings. The results showed that ATL31-HA could effectively co-precipitate endogenous CPK28 protein, and the interaction between the two was significantly enhanced after treatment at 4°C. Figure 4 The results suggest that low temperature conditions can promote the binding of ATL31 and CPK28.

[0081] Example 14, CPK28 with Ca 2+ Dependent phosphorylation of ATL31

[0082] Given that CPK28 is a functional Ca 2+ Dependent protein kinase, and Ca 2+ Since CPK28 can regulate its interaction with ATL31, we further investigated whether it can directly phosphorylate ATL31. Through in vitro kinase experiments, using His-Flag-CPK28 as the kinase and His-Flag-ATL31... △TM Using the transmembrane domain-depleted mutant as a substrate, His-Flag-CPK28 exhibited significant autophosphorylation activity under ATP-added conditions and could effectively phosphorylate ATL31. △TM ( Figure 5A) indicates that CPK28 possesses the ability to phosphorylate ATL31. To confirm this result, we performed liquid chromatography-tandem mass spectrometry analysis on the kinase reaction products to identify phosphorylation sites on ATL31. The results showed that when His-Flag-CPK28 with kinase activity was used as the kinase, ATL31 was identified. △TM There are 17 potential phosphorylation sites; while when using the kinase-inactivating mutant His-Flag-CPK28 D188A As a control, no ATL31 was detected. △TM The phosphorylation signal further confirmed that ATL31 phosphorylation depends on the kinase activity of CPK28 (Matschi et al., 2013). Furthermore, in p35S::ATL31-HA / Col transgenic seedlings, the phosphorylation level of ATL31-HA significantly increased after treatment at 4°C for 45 min; however, the phosphorylation level decreased after the addition of Ca... 2+ After chelation with EGTA, the phosphorylation level returned to the baseline state at 0 min of low-temperature treatment. Figure 5 B). The above results indicate that CPK28 can phosphorylate ATL31 under low-temperature conditions, and this process is strictly dependent on Ca. 2+ Signal activation. In summary, ATL31 is a direct phosphorylation substrate of CPK28, and a functional kinase-substrate relationship exists between the two. Furthermore, this phosphorylation event is induced by low-temperature Ca2+. 2+ The concentration change regulation suggests that CPK28 regulates ATL31 activity through phosphorylation modification, thereby participating in the transmission and feedback regulation of cold signals.

[0083] Example 15: ATL31 / 6-mediated degradation of CPK28 under low-temperature conditions

[0084] Previous research results indicate that low-temperature treatment can activate the E3 ubiquitin ligase activity of ATL31 ( Figure 2 E), and enhance its interaction with CPK28 ( Figure 4 To further verify the regulatory effects of ATL31 and ATL6 on CPK28 protein abundance under low temperature conditions, we used an anti-CPK28 antibody to detect the dynamic accumulation of CPK28 protein in the atl31 atl6 double mutant. Under CHX treatment to block the synthesis of new protein, the CPK28 protein level in Col plants decreased significantly within several hours after low temperature treatment. Figure 6 A); however, in the atl31 atl6 double mutant, this degradation process was significantly inhibited, and the CPK28 protein remained relatively stable ( Figure 6B). Furthermore, we compared the changes in CPK28 ubiquitination levels in the Col and atl31 atl6 mutants after low-temperature treatment. Immunoblotting results showed that, after treatment at 4°C, the ubiquitination modification of CPK28 in the atl31 atl6 double mutant was significantly lower than that in the wild-type plant (B). Figure 6 C). The above results indicate that, under low-temperature conditions, ATL31 and ATL6 promote the proteasome-dependent degradation of CPK28 by mediating ubiquitination modification, thereby regulating the protein stability of CPK28.

[0085] In summary, ATL31 and ATL6, as key negative regulators, are activated after cold signal sensing, and promote the degradation of CPK28 by enhancing ubiquitination modification, thereby finely regulating the activation level of downstream cold response pathways.

[0086] Example 16: Activation of the CBF-COR pathway and negative regulatory function of ATL6 / 31

[0087] In Arabidopsis, low-temperature stress rapidly induces the expression of CBF (a transcription factor-like gene, CBF1 / 2 / 3), which in turn activates downstream cold-response genes (COR genes), constituting a core signaling pathway for plant cold adaptation. The degree of activation of this pathway is positively correlated with the plant's freeze resistance and is an important molecular basis for plant responses to abiotic stresses (Lissarre et al., 2010; Thomashow, 2010). This invention, through systematic analysis of the freezing phenotypes of atl6, atl31, and atl6 atl31 double mutants under NA and cold acclimatization CA conditions, found that these mutants all exhibited significantly enhanced freeze resistance. Figure 1 (A and B). Ion leakage experiments further confirmed that the mutant showed significantly less cell membrane damage after low-temperature treatment than the wild-type plant (Figure 1 C), indicating that its cell protection mechanism was more effective.

[0088] Real-time quantitative PCR analysis showed that after treatment at 4°C for 6 h, the transcriptional levels of CBF1, CBF2, and CBF3 were significantly upregulated in both atl6 and atl31 single mutants, with the atl31 mutant showing a higher induction amplitude than atl6; while the expression of these genes was further enhanced in the atl6 atl31 double mutant. Figure 1 D), exhibiting a significant additive effect. This result not only confirms the functional redundancy of ATL6 and ATL31 in regulating the cold response, but also reveals their negative regulatory role in the CBF pathway at the physiological level. Notably, the E3 ubiquitin ligase activity of ATL31 is significantly enhanced under low temperature conditions (D). Figure 2(E), suggesting that it may target and degrade positive regulators through the ubiquitin-proteasome system, thereby inhibiting the overactivation of cold signals. This finding positions ATL6 / 31 as a key "brake" element in the cold response pathway, the absence of which leads to the continuous activation of the signaling pathway, thereby enhancing the plant's frost resistance.

[0089] Example 17: CPK28 is a necessary downstream actuator for the negative regulation function of ATL6 / 31.

[0090] Although ATL6 and ATL31 belong to the RING-type E3 ubiquitin ligase family, their specific target proteins and mechanisms of action in cold responses remain unclear. Previous studies have shown that ATL31 can interact with the calcium-dependent protein kinase CPK28 and promote its degradation in immune responses (Liu et al., 2022). To investigate the genetic relationship between these two in the cold signaling pathway, we constructed an atl31 atl6 cpk28 triple mutant and performed cryotolerance analysis. The results showed that the atl31 atl6 double mutant exhibited a very strong antifreeze phenotype; however, when the cpk28 mutation was introduced, this phenotype was completely suppressed, and its cryosensitivity was comparable to that of the cpk28 single mutant. Figure 3 A and B). Ion leakage and CBF gene expression analyses also consistently showed that the physiological and molecular phenotypes of the triple mutant were not significantly different from those of the cpk28 single mutant. Figure 3 CF).

[0091] The aforementioned genetic evidence strongly suggests that CPK28 is an indispensable downstream component for the negative regulatory function of ATL6 / 31. In other words, the "negative regulatory" effect of ATL6 / 31 is not a direct inhibition of the CBF pathway, but rather an indirect effect achieved by regulating the stability of CPK28. This finding challenges the traditional view of E3 ligases as mere "degradation machines," revealing their complex role as a "regulatory hub" in signal transduction. Furthermore, the contrast between CPK28's function as a positive regulator in the immune response (Monaghan et al., 2013) and its regulatory role in the cold response suggests that this kinase may play a multifunctional role in different stress pathways, with its functional output depending on upstream signal input and downstream target status.

[0092] Example 18, Low-temperature induced Ca 2+ Signal-driven interaction between ATL31 and CPK28

[0093] Upon sensing low temperatures, plant cytoplasmic Ca²⁺ concentration rapidly increases, forming a specific "calcium fingerprint" that serves as a primary signal to initiate downstream responses (Knight et al., 1996; Misbah et al., 2024). CPK28, a Ca²⁺-dependent protein kinase, can sense this signal and undergo a conformational change, thereby activating its kinase activity. This invention reveals that low-temperature treatment significantly enhances the physical interaction between ATL31 and CPK28 (…). Figure 4 This interaction can be significantly weakened in vitro by the Ca²⁺ chelator EGTA. Figure 4 The results indicate that it depends on the presence of Ca²⁺. This suggests that low-temperature-induced Ca²⁺ influx not only activates CPK28 but may also promote its binding with ATL31 by altering its affinity, thus providing a structural basis for subsequent phosphorylation and ubiquitination events. This "signal sensing-protein interaction enhancement" mechanism embodies how plants transform environmental stimuli into the assembly process of specific protein complexes, and is a key guarantee for the precision of signal transduction.

[0094] Example 19: CPK28-mediated phosphorylation of ATL31: A molecular switch activating E3 activity

[0095] Building upon the confirmed interaction between ATL31 and CPK28, we further explored their functional relationship. In vitro kinase assays showed that CPK28 can directly phosphorylate ATL31. △TM (Transmembrane domain deletion), and this process depends on ATP and Ca. 2+ ( Figure 5 A). LC-MS / MS identification revealed 17 potential phosphorylation sites, primarily located in the N-terminal regulatory region of ATL31, suggesting that these modifications may affect its stability, subcellular localization, or ubiquitination activity (Matschi et al., 2013). More importantly, in vivo experiments showed that phosphorylation levels of ATL31-HA significantly increased after 45 min of cryotherapy, while pretreatment with EGTA completely blocked this phenomenon. Figure 5 B). This indicates that the phosphorylation modification of ATL31 by CPK28 is a key event in the low-temperature response. The result shows that the E3 activity of ATL31 is activated at low temperatures (…). Figure 2 (E) We propose that CPK28 phosphorylation of ATL31 acts as a "molecular switch" for activating its E3 ubiquitin ligase function. This phosphorylation may enhance ubiquitin transfer efficiency by altering the conformation of ATL31, promoting its oligomerization, or strengthening its binding affinity to E2 enzymes. This mechanism provides a new paradigm for understanding how E3 ligases are dynamically regulated by upstream kinases.

[0096] Example 20: ATL31 / 6-mediated ubiquitination and degradation of CPK28: Construction of a negative feedback control loop

[0097] The most innovative finding of this invention is the elucidation of the ubiquitination and degradation mechanism of CPK28 by ATL31 / 6. Under CHX treatment to block the synthesis of new proteins, the CPK28 protein level in wild-type plants decreased significantly within hours after low-temperature treatment, while this degradation process was completely inhibited in the atl31 atl6 double mutant. Figure 6 A). Further immunoblotting analysis showed that the ubiquitination level of CPK28 in the atl31 atl6 mutant was significantly lower than that in the wild type ( Figure 6 (B) This directly proves that ATL31 / 6 is the key E3 ligase mediating CPK28 degradation. This closed-loop regulatory model of "kinase activates E3 → E3 degrades kinase" constitutes a typical negative feedback loop. Its biological significance lies in the signal initiation: low temperature → Ca2+ 2+ Up → CPK28 activation → phosphorylation of ATL31 → activation of its E3 activity; signal termination: activated ATL31 / 6 → ubiquitination of CPK28 → CPK28 degradation → signal attenuation. This circuit ensures that plants can quickly initiate a defense response when exposed to low temperatures, while preventing energy waste or cell damage caused by continuous signal activation. This dynamic balance mechanism of "activation-inhibition" is a high-efficiency stimulation strategy developed by plants over a long period of evolution, reflecting the tunability of the signaling network.

[0098] Example 21: Bidirectional Regulation of the ATL31 / 6-CPK28 Module: Constructing a Signal Hub and Its Physiological Significance

[0099] Based on all the data, we propose that ATL31 / 6-CPK28 constitutes a bidirectional regulatory hub, precisely regulating the plant's low-temperature response through a phosphorylation-ubiquitination cascade: Positive pathway: Low-temperature signaling is transmitted via Ca2+... 2+Activation of CPK28 leads to phosphorylation and activation of ATL31, initiating an early antifreeze response. A negative pathway involves activated ATL31 / 6 ubiquitinizing and degrading CPK28, terminating signal output and achieving self-regulation. This bidirectional regulation not only ensures the rapid initiation of the antifreeze response but also avoids sustained stress by dynamically regulating CPK28 protein abundance, thus achieving a balance between resistance and growth. Similar mechanisms have been reported in animal systems (e.g., IκBα negative feedback in the NF-κB pathway), but this is the first systematic elucidation of the cold response in plants (Hay et al., 1999). From an evolutionary perspective, the interaction between ATL31 / 6 and CPK28 may originate from the cross-regulation of immune and stress responses, subsequently being recruited to the cold signaling pathway, demonstrating the modularity and reusability of plant signaling networks. Future research could further explore: whether other CPK family members participate in the cold response; functional verification of the phosphorylation site of ATL31; the conservation of this module in different plant species; and the application prospects of using gene editing technology to improve crop cold resistance.

[0100] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0101] As demonstrated in the above examples, this invention explores the negative regulatory functions of ATL31 and ATL6 in plant cold stress responses: the atl31atl6 double mutant exhibits significantly enhanced cold tolerance under low-temperature conditions, including higher survival rates, lower electrolyte leakage rates, and stronger antioxidant capacity, while overexpression lines are more sensitive. Further research shows that low-temperature stress can activate the E3 ubiquitin ligase activity of ATL31 and promote its physical interaction with the calcium-dependent protein kinase CPK28; CPK28 in Ca… 2+ Phosphorylation of ATL31 under cold stress enhances its protein stability and functional activity. Notably, under cold stress, ATL31 and ATL6, acting as E3 ligases, mediate K48-linked polyubiquitination of CPK28, thereby promoting its degradation via the 26S proteasome pathway, forming a low-temperature-induced negative feedback regulatory loop. This mechanism achieves precise temporal and genomic control of the cold signaling pathway by dynamically regulating CPK28 protein abundance, preventing energy loss and cell damage caused by excessive response. This invention reveals the molecular mechanism by which the ATL31 / 6–CPK28 module finely regulates plant low-temperature adaptation through the synergistic action of phosphorylation and ubiquitination, elucidating the dual role of E3 ligases as both "signal transducers" and "signal terminators" in the stress signaling network. This invention not only elucidates the negative regulatory mechanism of ATL6 and ATL31 in plant cold resistance but also reveals a mechanism by which Ca2+... 2+The sophisticated signal regulatory network composed of kinases and E3 ligases provides a new theoretical framework for understanding the molecular basis of plant environmental adaptation. Specifically, this invention systematically elucidates the molecular mechanism of the E3 ubiquitin ligases ATL6 and ATL31 in the low-temperature response of Arabidopsis thaliana, revealing a new mechanism by which they finely regulate plant cold resistance through a bidirectional regulatory module formed with the calcium-dependent protein kinase CPK28. The main experimental results are summarized as follows: ATL6 and ATL31 negatively regulate plant cold resistance; CPK28 is a key downstream component for the function of ATL6 / 31; CPK28 phosphorylates ATL31 and activates its E3 ligase activity. In summary, this invention, through genetic, biochemical, and molecular biological methods, fully reveals the bidirectional regulatory network of the ATL6 / 31-CPK28 module in the plant cold response: low temperature induces Ca2+... 2+ Influx of nutrients activates CPK28; CPK28 phosphorylates and activates ATL31 / 6; the activated ATL31 / 6, in turn, ubiquitinates and degrades CPK28, thereby achieving precise initiation and timely termination of antifreeze signals. This discovery not only expands our understanding of plant hypothermia sensing and signal transduction mechanisms, but also provides potential molecular targets for genetic improvement of crop cold resistance.

[0102] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for synergistically regulating plant resistance to low temperature stress based on ubiquitin ligase ATL31 and ATL6, characterized in that: ATL31 and ATL6 synergistically initiate the low temperature response rapidly and mediate the dynamic regulation of CPK28 protein abundance, which realizes the precise response to the cold signal pathway in time and space, prevents the energy loss and cell damage caused by over-response while resisting low temperature stress, and thus regulates the plant response to low temperature stress.

2. A method for increasing the resistance of a plant to low temperature stress, characterized in that: Decreasing or inhibiting the expression of ATL31 and ATL6 in plants, constructing single mutant and / or double mutant of ATL31 and ATL6, thereby improving the cold resistance of plants.

3. The industrial application of the method of claim 2: improving the yield of plants by improving the cold resistance of plants.

4. A method for constructing a low temperature sensitive strain, characterized in that: Increasing the expression of ATL31 and ATL6 in plants, constructing single overexpression line or double overexpression line of ATL31 and ATL6, and the obtained overexpression line has higher sensitivity to low temperature stress than the wild type line.

5. The application of the method of claim 4: constructing a line highly sensitive to low temperature for experimental study on the regulation of plant response to low temperature.

6. A cold stress molecular regulator ATL31 / 6-CPK28, characterized by: The plant molecular regulator comprises ATL31 and / or ATL6 and CPK28, and ATL31 / 6 and CPK28 form a dynamic interaction network in the plant molecular regulator: under low temperature conditions, the E3 ubiquitin ligase activity of ATL31 is activated, which promotes the combination of ATL31 and CPK28 and mediates the ubiquitination and degradation of CPK28, thereby constructing a negative feedback regulation loop induced by environmental stimulation.

7. The new use of the low temperature stress molecular regulator ATL31 / 6-CPK28 of claim 6: a brand-new function of amplifying the response signal and timely closing the response signal in the plant cold signal regulation pathway.

8. The new use of the low temperature stress molecular regulator ATL31 / 6-CPK28 of claim 6: using the low temperature stress molecular regulator as a two-way regulation hub to accurately regulate the positive and negative responses of plants to low temperature through the phosphorylation-ubiquitination cascade reaction, realizing the rapid initiation of the anti-freezing response through the two-way response, avoiding continuous stress through the dynamic regulation of CPK28 protein abundance, and realizing the time and space dynamic balance between resistance and growth of plants.

9. The new use according to claim 8: said bidirectional response specifically comprises: In the plant cold signal regulation pathway, the "CPK28 kinase activates E3 ubiquitin ligase, while cooperatively starting the cold E3 ubiquitin ligase to degrade CPK28 kinase" closed-loop regulation mode is realized, and a complete negative feedback loop is constructed. Thus, the following whole signal pathway after the low temperature signal is started is realized: low temperature induces cold Ca 2+ CPK28 activation, in turn induces phosphorylated ATL31, in turn cold activates cold E3 cold ubiquitin ligase activity; at the same time, at the signal termination end, activated ATL31 / 6 cold induces cold CPK28 ubiquitination, in turn cold induces cold CPK28 degradation, in turn cold induces cold signal attenuation; thus, the plant can quickly start the defense response when encountering low temperature, while preventing energy waste and / or cell damage caused by persistent signal activation.

10. A method for improving the survival rate of plants, reducing the electrolyte leakage rate and improving the antioxidant capacity of plants under low temperature stress: by decreasing or inhibiting the expression of ATL31 and ATL6 in plants.

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