Energy sensing OsSnRK1b and its regulator OsCTK1 promote chilling-induced stomatal closure and chilling tolerance in rice

Energy sensing OsSnRK1b and its regulator OsCTK1 promote chilling-induced stomatal closure and chilling tolerance in rice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Energy sensing OsSnRK1b and its regulator OsCTK1 promote chilling-induced stomatal closure and chilling tolerance in rice Jian Hua, Jiawen Wu, Baohong Zou, Huimin Liu, Veronica Perez, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7447278/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Plants employ diverse physiological strategies for cold tolerance. Here we report an important role of low energy response and stomatal closure control in chilling tolerance through the study of SNF1-Related Kinase 1 (OsSnRK1) and its regulator Chilling Tolerance Kinase 1 (OsCTK1) in rice. OsSnRK1b, an isoform of OsSnRK1 alpha subunit, is found to have an evolutionarily conserved function in low energy response. It is positively regulated by OsCTK1 at the protein stability level through phosphorylation. Both OsCTK1 and OsSnRK1b promote chilling-induced stomatal closure and chilling tolerance, shown by slower stomatal closure and lower survival rate after chilling exposure in their respective loss of function mutants. Transcriptome analysis revealed that chilling induces low energy response which is largely dependent on OsSnRK1b. Overexpression of OsSnRK1b enhanced chilling induced stomatal closure response and promoted chilling tolerance in an OsCTK1-dependent manner. Furthermore, both OsCTK1 and OsSnRK1b promote expression of stress response gene Abscisic Acid Insensitive 5 (OsABI5) and inhibit accumulation of growth-associated ribosome protein OsRPS6 under chilling conditions. The function of CTK1 and SnRK1 in chilling tolerance, stomatal control, and growth inhibition are also observed in Arabidopsis and maize, indicating a conserved role of energy signaling in chilling tolerance and a contribution of stomatal aperture control to chilling tolerance in plants. Biological sciences/Plant sciences/Plant stress responses/Abiotic Biological sciences/Plant sciences/Plant physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Low-temperature stress poses a major challenge to plant growth and development and is a key factor limiting crop productivity and geographical distribution 1-3 . To cope with cold stress, plants employ a suite of physiological, biochemical and molecular adaptations, including changes in membrane lipid composition, accumulation of compatible solutes, activation of antioxidant systems to scavenge reactive oxygen species (ROS), and modulation of stress-responsive hormone levels 4,5 . Prior exposure to moderately low temperatures can enhance tolerance to extreme cold, a phenomenon termed cold acclimation 6-8 . Low temperature rapidly triggers calcium influx and ROS bursts, which serve as early signaling events to initiate transcriptional reprogramming. The CBF (C-repeat binding factor) transcription factors and their regulator ICE1 (Inducer of CBF expression) are key conserved players in cold signaling in diverse species including Arabidopsis, rice, and maize 9,10 . This cold-induced transcriptional response ultimately promotes expression of cold-responsive genes, leading to the accumulation of antifreeze proteins, osmoprotectants, and remodeling of the lipidome, processes essential for maintaining cellular homeostasis and enhancing low-temperature tolerance 2,11,12 . While molecular regulation of transcriptome change has been extensively studied for cold response in plants, broader physiological strategies at organismal level that contribute to cold tolerance are less well understood. Stomatal movement control is critical for adaptation to the environment, as it modulates gas exchange for photosynthesis and controls water loss for tolerance to drought and heat 13 . Stomatal movement has also been reported to occur in response to cold 14 , and it was associated with cold-induced wilting and death in Arabidopsis thaliana , Phaseolus vulgaris , and Nicotiana tabacum 15-17 . Interestingly, multiple genes critical for cold acclimation, including OST1 ( Open stomata 1 ) and CNGCs ( Cyclic nucleotide-gated channels ), are important for cold induced stomatal closure. Notably, OST1, a regulator of ABA induced stomatal closure, can phosphorylate ICE1 to enhance CBF signaling pathway under cold stress 18-20 . The rice OsCNGC14/15/16 proteins that promote cold-induced stomatal closure are positive regulators of chilling tolerance 21 . These observations suggest a role for cold induced stomatal closure in cold tolerance. Low energy response is a physiological response to energy depletion exemplified by starvation. A low energy status is also associated with some abiotic stresses including extended periods of salt and drought 3,22 , but its association with cold stress has not been investigated. In yeasts and animals, SNF1-related kinase (SnRK1/AMPK) proteins function as low energy sensors and coordinate catabolic activation and anabolic repression to maintain cellular energy homeostasis 23 . They trigger rapid cellular adaptation under low energy stress through phosphorylating key metabolic enzymes and transcription factors, thereby reprograming transcriptional network and metabolism to promote catabolism and repress anabolism 23,24 . Plant SnRK1 is also critical for adaptation to energy-deficient conditions including extended darkness and hypoxia 25,26 . It is also shown to be important for tolerance to biotic and abiotic stresses 24,27,28 . SnRK1 consists of a catalytic α-subunit and regulatory β- and γ- subunits 29 . The α-subunit consists of a N-terminal Ser/Thr kinase domain and a C-terminal regulatory domain that mediates its interaction with the β- and γ- subunits 30 . In Arabidopsis, three genes ( KIN10 , KIN11 and KIN12 ) encode the α-subunit of SnRK1 (SnRK1α). Among these, the KIN10 subunit translocates to the nucleus to de-repress metabolic stress-responsive genes under darkness, inhibited photosynthesis, or hypoxia 31 . It phosphorylates transcription factors bZIP63 and bZIP53 for starch synthesis and proline metabolism during dark-induced low-energy stress, respectively 32,33 . In rice, four isoforms of SnRK1α could be identified by sequence homology and they are named OsSnRK1a, OsSnRK1b, OsSnRK1c and OsSnRK1d (https://www.ricedata.cn/). OsSnRK1a is shown to regulate starvation response by promoting starvation-responsive genes under energy limitation but repressing these genes under energy-sufficient conditions through its interaction with the H3K27me2/3 demethylase JMJ705 34 . Whether a low energy response pathway is involved in cold tolerance remains unknown in plants. OsCTK1 ( Chilling tolerance kinase 1 ) is a positive regulator of chilling tolerance in rice as evidenced by a lower survival rate under chilling in its loss of function (LOF) mutants compared to the wild type 35 . Natural variation of the OsCTK1 gene is a major contributor to chilling tolerance variation in Indica rice. The CT (Chilling tolerant) variant of OsCTK1 , with a four amino acids insertion, exhibits a higher kinase activity and confers a greater chilling tolerance compared to the CS (Chilling sensitive) variant 35 . OsCTK1 belongs to a serine/threonine kinase family CKI (Casein kinase I) which has a broad regulatory function 36,37,38,39,40 . The Arabidopsis OsCTK1 homologous proteins, CK1.3 and CK1.4, are shown to regulate blue light signaling by phosphorylating the cryptochrome receptor CRY2 (Cryptochrome 2) 39 . OsCTK1 may target multiple phosphorylation substrates for its function, including OsP3B (Acidic ribosomal protein 3B), OsCNGC9, and OsMKP1 (Mitogen-activated protein kinase phosphatase 1) 35 . In this study, we further investigated the regulatory mechanism of chilling tolerance through the study of another substrate of OsCTK1, OsSnRK1b. We found that OsSnRK1b, like OsSnRK1a, is also a low energy sensing kinase. OsCTK1 phosphorylates and stabilizes the OsSnRK1b. Similar to OsCTK1 , OsSnRK1b promotes stomatal closure in response to chilling and enhances chilling tolerance. They both regulate the chilling-induced expression of stress-induced genes and repress the accumulation of the growth associated proteins. These findings indicate that energy metabolism and stomatal regulation are critical mechanisms for chilling tolerance in rice. Results OsCTK1 positively regulates stomatal closure in response to chilling In addition to a lower survival rate and a higher ion leakage after chilling 35 , the Osctk1-2 and Osctk1-3 LOF mutants had a rapid leaf-drooping phenotype after chilling treatment when compared to the wild type Nipponbare (NIP) (Fig. 1A). The Osctk1 mutants also had a faster water loss and a more rapid leaf curling after chilling exposure compared to the wild type (Fig. 1B and Supplementary Fig. 1A-B). These defects promoted us to measure stomatal aperture in the Osctk1 mutant after chilling treatment. Stomatal closing was observed in the wild-type plants at 1 h and continued at 3 h and 9 h, but it was not observed till 3 h in Osctk1-3 and9 h in Osctk1-2 (Fig. 1C-D). In addition, both mutants had a larger stomatal aperture at all time points measured compared to the wild type (Fig. 1C-D). This stomatal defect was supported by thermal imaging where a lower leaf temperature was detected in the Osctk1 mutants compared to the wild type (Fig. 1E). This is corroborated with a higher transpiration rate in the mutants than the wild type under normal and chilling conditions (Fig. 1F). The OsCTK1 gene is expressed in guard cells, as shown by positive signal of β ‐Glucuronidase (GUS) reporter in transgenic plants carrying the GUS reporter gene under the control of the OsCTK1 promoter (Supplementary Fig. 1C). This indicates an expression of OsCTK1 in guard cells and suggests a cell-autonomous function of OsCTK1 in guard cells. The activity of OsCTK1 in promoting stomatal closure is aligned with its activity in promoting survival rate under cold as shown by its two natural variants. The CT variant of OsCTK1 was previously shown to confer a higher survival rate under chilling stress than the CS variant 35 . Transgenic Osctk1-2 lines carrying the CT variant had a wild-type phenotype while lines carrying the CS variant partially complemented the Osctk1-2 mutant, as shown by reduced stomatal aperture and transpiration rate after a 6°C chilling treatment (Supplementary Fig. 1D-I). Therefore, the function of OsCTK1 in stomatal closure control is highly correlated with its function in promoting survival under chilling. OsCTK1 phosphorylates OsSnRK1b We conducted in vitro phosphoproteomics to identify phosphorylation targets of OsCTK1 that might mediate the function of OsCTK1 in stomatal movement control. OsSnRK1b and OsSnRK1c were among the Mass Spec identified candidate target proteins as a shared peptide of these two proteins were identified as phosphorylated by the recombinant OsCTK1. We chose these two proteins for further study because theirArabidopsis homolog KIN10 was previously implicated in stomatal movement control under normal growth temperature 41 (Supplementary Fig. 2A). OsSnRK1b was selected among the two proteins for assaying the interaction OsCTK1, as 1b and 1c are highly homologous. Direct physical interaction between OsCTK1 and OsSnRK1b was verified in planta and in vitro by Co-Immunoprecipitation (Co-IP) assays (Supplementary Fig. 2B). A direct phosphorylation of OsSnRK1b by OsCTK1 was observed by an in vitro kinase assay (Fig. 2A). The recombinant His-OsSnRK1b protein had an autophosphorylation activity, but the addition of GST-OsCTK1 in the kinase reaction resulted in a stronger phosphorylation of His-OsSnRK1b protein (Fig. 2A). In addition, natural variants of OsCTK1 exhibited a differential phosphorylation activity on OsSnRK1b in the in vitro kinase assay. The CT variant of OsCTK1 exhibited a higher phosphorylation on OsSnRK1b than the CS variant, and the critical Indel polymorphism also altered the phosphorylation level of the CT and the CS variants (Supplementary Fig. 2C). Phosphorylation of OsSnRK1b that is dependent on OsCTK1 was also detected in protoplast cells expressing a GFP-tagged OsSnRK1b by phos-tag gels. A slower migrating band of GFP-OsSnRK1b was observed in the wild type cells but not in the Osctk1-2 mutant cells (Fig. 2B). This differential band pattern was also observed from protoplast cells incubated further at 6°C for 1 h. Treatment of protein extracts by calf intestine phosphatase (CIP) abolished this slow migrating band, indicating that it was indeed a phosphorylated form of OsSnRK1b-GFP. We identified three residues, Thr 173 , Ser 174 and Ser 347 , as potential phosphorylation sites of OsCTK1 in OsSnRK1b through two approaches. Above mentioned in vitro phosphoproteomics detected phosphorylation of Thr 173 and/or Ser 174 . MassSpec analysis of GFP tagged OsSnRK1b expressed in protoplast cells revealed Ser 347 phosphorylation in the wild-type but not the Osctk1-2 mutant protoplast cells (Supplementary Fig. 2D). Mutating Thr 173 and Ser 174 of SnRK1b to Ala resulted in a 20% decrease in phosphorylation of OsSnRK1 (Supplementary Fig. 2E), while an 89% reduction of phosphorylation was observed when mutating Ser 347 , Thr 173 and Ser 1 74 of OsSnRK1bsimultaneously to Ala in vitro kinase assay with OsCTK1 (Fig. 2A). As autophosphorylation accounted for about 29% phosphorylation in the wild-type OsSnRK1b, this indicates an 85% reduction (11% versus 71%) of phosphorylation of OsSnRK1b by OsCTK1 from mutations of these three residues (Fig. 2A). OsCTK1 stabilizes OsSnRK1b through phosphorylation OsCTK1 was found to affect the abundance of OsSnRK1b when OsSnRK1b protein was detected by an antibody that targets OsSnRK1 members (Fig. 2C). This antibody primarily detects OsSnRK1b, because a similar reduction (71% and 73%) of the signal compared to the wild type was observed in the Ossnrk1b1c double mutant and a Ossnrk1b single mutant (as described in the next result section) (Fig. 2C). Strikingly, the Osctk1-2 mutant had a 46% reduction of OsSnRK1 proteins detected by this antibody compared to the wild type (Fig. 2C). This reduction of OsSnRK1 protein abundance was not due to a reduced RNA transcript of OsSnRK1 in the Osctk1-2 mutant (Supplementary Fig. 2F), indicating an effect on OsSnRK1b at the protein level by OsCTK1. A cell-free protein degradation assay indicates that a lower protein stability contributes to the low abundance of OsSnRK1b in the Osctk1-2 mutant. The recombinant His-OsSnRK1b protein had a faster degradation when incubated with protein extracts from Osctk1-2 mutant than from those of the wild type (Fig. 2D). The stability of OsSnRK1b was affected by its OsCTK1 phosphorylation sites. The His tagged mutant OsSnRK1b protein with three phosphorylation site mutation to Ala, His-OsSnRK1b TSS-AAA , had a faster degradation compared to the wild-type His-OsSnRK1b form when incubated with protein extracts from wild-type plants (Fig. 2D). In contrast, when incubated with protein extracts from the Osctk1-2 mutant, the His-OsSnRK1b TSS-AAA protein had a degradation rate similar to the wild-type His-OsSnRK1b (Fig. 2D). These findings suggest that phosphorylation of OsSnRK1b at these sites affect the protein stability in an OsCTK1-dependent manner. OsCTK1 affects the subcellular localization of OsSnRK1b in addition to its protein abundance. When expressed in protoplast cells, the GFP-OsSnRK1b fusion protein had both nuclear and cytosol distributions as assayed by nuclear fractionation. It has a lower nuclear accumulation and a higher cytosol accumulation in the Osctk1-2 mutant cells compared to the wild type (Fig. 2E). The nuclear/cytosol distribution of OsSnRK1b was likely affected by the three OsCTK1 phosphorylation residues. A lower nuclear accumulation and a higher cytosol accumulation was observed for GFP-OsSnRK1b TSS-AAA protein compared to the GFP-OsSnRK1b protein when expressed in wild-type protoplasts (Fig. 2F). These data suggests that phosphorylation of OsSnRK1b by OsCTK1 may enhance its translocation to the nucleus. OsSnRK1b promotes stomatal closure and survival under chilling The function of OsSnRK1 b and OsSnRK1c was analyzed through the mutants generated by CRISPR/Cas9 gene editing. Four LOF OsSnRK1 b mutant lines were obtained, with the -1 and -2 alleles independently created from the -3 and -4 alleles from different pairs of RNA targets (Supplementary Table 1). The Ossnrk1b 1c mutant was an Ossnrk1 b Ossnrk1 c double mutant as it contained an additional mutation in the OsSnRK1c gene (Supplementary Table 1). All four mutant lines exhibited a similar phenotype in the initial characterization, and therefore Ossnrk1 b1c double mutants and Ossnrk1b -3 ( Ossnrk1 b single mutant) were used for further analysis. Both Ossnrk1 b1c and Ossnrk1 b-3 exhibited more chilling susceptibility compared to the wild-type NIP plant. After 6°C treatment, leaf rolling and leaf drooping occurred much earlier in the mutants than the wild type (Fig. 3A, Supplementary Fig. 3A). These mutants also had a similar faster water loss, slower stomatal closure, and higher transpiration than the wild type (Fig. 3B-E, Supplementary Fig. 3B). The other two alleles, Ossnrk1 b-1 and Ossnrk1 b-4 , exhibited the same stomatal closure and water retention defects as the Ossnrk1 b1c and Ossnrk1 b-3 alleles (Supplementary Fig. 3C-F). These data indicate that the loss of OsSnRK1 b function compromised chilling-induced stomatal closure. The Ossnrk1 b mutants also showed more susceptibility to chilling compared to the wild type as measured by leaf damage and survival rate (Fig. 3F). They had a higher ion leakage to a similar degree in leaf tissues after 24 h of chilling treatment compared to the wild type (Fig. 3G). The survival rates from 2 d of 6℃ treatment were 28% and 20% for the two mutants, lower than the rate of 58% for the wild type (Fig. 3H and Supplementary Fig. 3G-H). These data indicate that the loss of OsSnRK1b function leads to reduced chilling tolerance. The Ossnrk1 b Ossnrk1 c double and Ossnrk1 b-3 single mutants exhibited a similar degree of defects under chilling. This indicates that the OsSnRK1c gene had no detectable function in chilling tolerance in the absence of the OsSnRK1b function. These data indicate that the OsSnRK1 b plays a dominant role over OsSnRK1 c or an equal role as the OsSnRK1 c isoform in chilling responses (Fig. 3A-H). The relative function of OsSnRK1 b and OsSnRK1c isoforms could be further verified when single mutants of the OsSnRK1c form become available. Overexpress ing OsSnRK1 b promotes chilling tolerance and chilling induced stomatal closure in an OsCTK1 -dependent manner To further analyze the function of OsSnRK1b and its regulation by OsCTK1, we analyzed transgenic lines overexpressing OsSnRK1b in wild-type NIP (named OE- OsSnRK1b /NIP) and Osctk1-2 (named OE- OsSnRK1b / ctk1 ), respectively. Consistent with findings in vitro , the GFP-OsSnRK1b protein had a lower accumulation in the two OE- OsSnRK1b / ctk1 lines compared to the two OE- OsSnRK1b /NIP lines, despite comparable OsSnRK1b RNA expression in these four lines (Supplementary Fig. 3I-J). When transgenic plants were subject to chilling, GFP-OsSnRK1b has a more pronounced reduction in Osctk1-2 lines compared to the wild type lines (Supplementary Fig. 3J). Overexpression of OsSnRK1 b was found to promote stomatal closure and enhance chilling tolerance in the wild type. After 6°C chilling treatment, the two OE- OsSnRK1b /NIP lines showed delayed leaf rolling and drooping compared to the wild type (Fig. 3I). Water loss was lower at 3 h and 9 h of chilling treatment in the overexpression lines compared to the wild type (Supplementary Fig. 3K). The overexpression lines also had smaller stomatal apertures at 28°C and faster stomatal closure upon 6°C exposure (Supplementary Fig. 3L), resulting in higher leaf temperature and lower transpiration rate both before and after chilling compared to the wild type (Fig. 3J-K). In addition, the OE- OsSnRK1b /NIP transgenic lines suffered less chilling damage, as shown by lower ion leakage at 24 h (Fig. 3L-M) and higher survival rates (Fig. 3N) after 3.5 days at 6°C. Together, these data indicate that overexpression of OsSnRK1b promotes stomatal closure and confers a higher chilling tolerance. The enhanced chilling tolerance and stomatal closure by OsSnRK1b overexpression was not observed in the Osctk1-2 mutant background (Fig. 3I). The two OE- OsSnRK1b / ctk1 lines behaved the same as the Osctk1-2 mutant plant before and after chilling treatment, including earlier leaf dropping, higher water loss, more open stomata, and higher transpiration rate compared to the wild type (Fig. 3I-K and Supplementary Fig. 3K-L). The OE- OsSnRK1b / ctk1 lines also had higher ion leakage (Fig. 3M) and lower survival rate (Fig. 3N) compared to the wild type. These data indicate that the effects of OsSnRK1b overexpression on chilling tolerance and stomatal aperture are dependent on OsCTK1. OsSnRK1b is required for starvation response Because AtSnRK1α in Arabidopsis and OsSnRK1a in rice are shown to be low energy sensing kinases 23,34 , we assessed whether OsSnRK1b is also involved in low energy response. The Ossnrk1 b mutantsandthe OE- OsSnRK1b lines were subject to starvation with continuous darkness for 7 days (Fig. 4A). Both the Ossnrk1 b1c and the Ossnrk1b-3 mutants exhibited a larger bleached area compared to the wild type, corroborated by reduced chlorophyll levels in leaves, compared to the wild type (Fig. 4B-C). The bleaching and chlorophyll loss in the Ossnrk1b mutants was to a similar extent as the Ossnrk1a-1 mutant (Supplementary Fig. 4A-D). Conversely, the OE- OsSnRK1b lines exhibited a reduced bleached area and higher chlorophyll levels than the wild type (Fig. 4A-C). This indicates that OsSnRK1b has a conserved role in low energy stress response. In addition, the Ossnrk1a-1 mutant 42 (Supplementary Fig. 4A) which was shown to be defective in low energy response, had a significantly lower survival rate and higher transpiration rate than the wild-type after chilling treatment (Supplementary Fig. 4E-G), indicating that OsSnRK1a has a similar function to OsSnRK1b in chilling tolerance. OsSnRK1b is required for chilling induced growth inhibition We hypothesize that low energy response contributes to chilling response in plants because SnRK1b and SnRK1a are both important for low energy tolerance and chilling tolerance. To test this, we examined transcriptomic response to chilling in the wild type and the Ossnrk1b mutants by RNA sequencing (RNA-seq) analysis. The wild-type plant had 1478 cold (6℃ for 3 h) induced DEGs, and 849 (57%) of them were also induced by starvation based on a previous report (Wang et al 2021) (Fig. 4D), suggesting a contribution of low energy response to transcriptome reprogramming under chilling. The Ossnrk1b mutants had a reduced number of cold-induced genes (959) compared to the wild type, and a smaller proportion (163, 16.3%) of these genes overlapped with chilling induced and starvation induced genes compared to the wild type (Fig. 4E-F). Therefore, about half (51%) of cold-induced DEGs and 81% of the cold-induced starvation-responsive genes are dependent on OsSnRK1b (Fig. 4E-F), suggesting an important role of OsSnRK1b in inducing starvation response genes under cold. As low energy response balances plant growth and stress responses, we further investigated the regulation of growth process under chilling by OsSnRK1b and OsCTK1 . The phosphorylation of Ser 240 of the ribosome protein RPS6 (a marker for TOR regulated growth) and the abundance of RPS6 (an indicator of growth) were analyzed in the mutants before and after chilling stress treatment (Fig. 4G). Strikingly, both the Ossnrk1b1c and the Osctk1-2 mutants had a higher amount of Ser 240 phosphorylation in OsRPS6 compared to the wild type at 28℃. The difference became more pronounced after chilling treatment (Fig. 4G). The wild type had a reduced Ser 240 phosphorylation at 3 h and 12 h after chilling, reflecting the inhibition of growth at early phase of chilling stress response, but the Osctk1-2 and Ossnrk1b1c mutants had increased phosphorylation after chilling (Fig. 4G), suggesting a loss of growth inhibition under chilling. Similarly, the OsRPS6 protein amount was at a higher level in these two mutants compared to the wild type under 28℃ growth condition. In addition, the RPS6 amount decreased at 3 h, recovered at 12 h in the wild type, but it did not decrease after chilling and remained the same amount throughout the chilling treatment in the two mutants (Fig. 4H). These findings suggest that OsSnRK1b and OsCTK1 inhibit growth processes in general and more so under chilling stress. OsSnRK1 b and OsCTK1 regulate chilling-induction of the stress response gene OsABI5 Because OsSnRK1b regulates stomatal closure which is known to be associated with the ABA response pathway, we asked whether it regulates the ABA stress response pathway under cold. Chilling induced 74 ‘ABA-responsive genes’ (GO:0009737) among the 495 genes under this GO term, which was not significantly enriched among the chilling induced DEGs in the wild type. Nevertheless, 43 of them were not induced by chilling in the Ossnrk1b1c mutant, indicating a dependence on OsSnRK1b for chilling induction. We subsequently investigated the expression of OsABI5 that encodes a major transcription factor for ABA response. qRT-PCR revealed that OsABI5 expression was induced by 6°C treatment at 3 h or 24 h in the wild type but not in the Ossnrk1b1c mutants (Fig. 5A). OsCTK1 was also found to regulate the RNA expression of OsABI5 . Despite having a higher expression of OsABI5 compared to the wild type at 28°C, the Osctk1-2 mutant plants had no induction of OsABI5 gene expression by chilling and a lower OsABI5 expression compared to the wild type under chilling (Fig. 5B). The regulation of OsABI5 by OsSnRK1 b was also seen in the OE- OsSnRK1 b transgenic plants. The expression of OsABI5 was higher in the OE- OsSnRK1 b transgenic plants than in the wild type, but it was the same in the OE- OsSnRK1 b / ctk1 lines as in the Osctk1-2 mutant(Fig. 5C). These results indicated that OsSnRK1b and OsCTK1 are both required for the induction of OsABI5 in response to chilling, and OsCTK1 has an additional role in repressing OsABI5 expression under non-stress conditions. OsABI5 positively regulates chilling tolerance and stomatal closure A previous study shows that an Osabi5 mutant had reduced germination and seedling survival under cold compared to the wild type plants 62 . To further investigate the role of OsABI5 in the cold stress response, we analyzed two independent LOF mutants of OsABI5 , Osabi5-1 and Osabi5-2 , generated by CRISPR/Cas9 gene editing in the ZH11 background (Supplementary Table 1). Both Osabi5 mutants exhibited more extensive leaf drooping compared to the wild-type parent ZH11 after a 6℃ treatment (Fig. 5D). They also had higher ion leakage (Fig. 5E) and lower survival rates (Fig. 5F) compared to the wild type. In addition, the Osabi5 mutants had a higher water loss after chilling compared to the wild type (Fig. 5G and Supplementary Fig. 5A-B). The chilling susceptible defect in the Osabi5 mutants was accompanied by a larger stomatal aperture in the mutant than the wild type after chilling (Fig. 5H). This stomatal aperture defect is not chilling specific, as the Osabi5 mutants had a larger aperture without chilling treatment as well. In addition, the mutants closed stomata in response to chilling a similar dynamic to the wild type (Fig. 5H). Correlated with stomatal aperture defect, the Osabi5 mutants had a higher transpiration rate before and after chilling treatment compared to the wild type, and its leaf surface temperature was lower than the wild type at 3 h after 6°C treatment (Supplementary Fig. 5C-D). These results indicate that OsABI5 promotes stomatal closure in general, but it does not significantly affect the chilling induction of stomatal closure. OsCTK1 and OsSnRK1b do not significantly affect the expression of CBF related genes under chilling We also investigated whether OsCTK1 and OsSnRK1b regulate expression of the C-repeat binding factor ( CBF ) or dehydration-responsive element-binding ( DREB ) genes. In the wild-type plants, chilling treatment at 6°C induced the expression of eight out of the nine OsDREB genes at both 3 h and 24 h (Fig. 5I-J). All eight genes were also induced by chilling in the Ossnrk1b mutants. However, the induction of OsDREB genes in Ossnrk1b mutants were significantly lower than in the wild type at 3 h, while most OsDREB genes showed no significant difference at 24 h (Fig. 5I). In contrast, the Osctk1 mutant exhibited higher basal expression levels of seven OsDREB genes at normal growth temperature. After 24 h of 6°C treatment, the expression levels of these eight genes in the Osctk1 mutant were not significantly different from those in the wild type (Fig. 5J). These findings indicate that OsCTK1 is not a positive regulator of OsDREB expression, and its role in chilling tolerance is likely independent of the CBF pathway. Although OsSnRK1b exerts minor effects on the expression of certain OsDREB genes, whether its function in chilling tolerance is primarily mediated through the CBF pathway remains to be elucidated. Arabidopsis SnRK1 and CTK1 are regulators of chilling tolerance and chilling induced stomatal closure To determine whether or not the involvement of low energy response in chilling tolerance is conserved in plants, we analyzed homologous genes of SnRK1b and CTK1 in Arabidopsis. KIN10 (At3g10190), an apparent ortholog of three OsSnRK1 ( a, b, and c ) genes in rice (Supplementary Fig. 1A),has been shown to participate in starvation response 23 . The LOF kin10 mutant exhibited a slower stomatal closure in response to chilling. When seedlings were exposed to 6°C, stomatal closing was observed as early as 3 h of treatment in the wild type, but no closing was observed even at 24 h of chilling treatment in the kin10 mutant (Fig. 6A). Corroborated with the stomatal behavior, the kin10 mutants had a higher transpiration rate compared to the wild type throughout the 24 h of 6°C treatment (Fig. 6B-C). It was also more susceptible to freezing, as evident by the reduced survival rate following a 5 h of -8ºC treatment compared to wild-type (Fig. 6D). Most strikingly, the kin10 mutant exhibited a higher amount of growth associated molecular markers under chilling similarly to the Ossnrk1b mutant. Chilling treatment of 24 h reduced total RPS6 abundance and Ser 240 phosphorylation in wild type, and the kin10 mutant had a higher level of both total RPS6 and phosphorylation at Ser 240/244 compared to the wild type under both normal and chilling growth conditions (Fig. 6F). The closest homolog of OsCTK1 in Arabidopsis, AtCK1.3 , was also found to play a role in chilling tolerance. Compared to the wild type, the Atck1.3 mutant had a larger stomatal aperture under normal growth conditions as well as a delayed stomatal closure and increased transpiration rates throughout the 24 h period of chilling treatment (Fig. 6A-C). This mutant also had a reduced survival rate after 3 h of exposure to -8°C freezing stress compared to the wild type (Fig. 6E). In addition, the Atck1.3 mutant had a higher RPS6 phosphorylation and accumulation than the wild type after 24 h of 6℃ treatment (Fig. 6G). The ABI5 gene in Arabidopsis also promotes stomatal closure under both normal and low temperatures. The Atabi5 mutant had a larger stomatal aperture and a higher transpiration rate under normal growth conditions (Fig 6A-C). Although it responded to chilling by closing the stomata in a similar dynamic as the wild type, the mutant had a larger stomatal aperture and a higher transpiration rate throughout 36 h chilling treatment (Fig 6A-C). Maize CTK1 is a positive regulator of chilling tolerance We further investigated the function of the closest homolog of OsCTK1 in maize, ZmCTK1 (GRMZM2G156035). A LOF mutant of ZmCTK1 gene was generated by CRISPR/Cas9, and this Zmctk1-1 mutant was assayed for survival rates and stomatal closure response after chilling treatment. The wild-type maize plants exhibited stomatal closure after 1 h of 6ºC chilling treatment (Fig. 6H). While the Zmctk1 mutant had a wild-type stomatal aperture under normal growth, it displayed a reduced stomatal closure upon 6ºC treatment compared to the wild type (Fig. 6H). Additionally, the Zmctk1 mutant had a reduced survival rate (20%) than the wild type (61%) following a 3-week chilling treatment at 10ºC (Fig. 6I). Similar to the rice Osctk1 mutant, the maize Zmctk1 mutant did not exhibit chilling induced growth marker inhibition. Chilling stress caused a reduction in total RPS6 abundance and a reduction of Ser 240 phosphorylation in wild-type maize plant (Fig. 6J). The ZmCTK1 mutant had a higher level of RPS6 and phosphorylated form of RPS6 with or without 1 day of chilling treatment (Fig. 6J). These findings indicate that ZmCTK1 regulates chilling tolerance, chilling induced stomatal closure, and chilling induced RPS6 reduction. Discussion This study uncovers a new mechanism of chilling tolerance that involves low energy response for inhibiting growth and promote stress response under cold (Fig. 6K). The rice OsSnRK1b gene has an evolutionarily conserved function in starvation response, and it is also a positive regulator of chilling tolerance, as demonstrated by the altered starvation response and chilling responses in its LOF mutants and overexpression lines (Fig. 3F-H and L-N). In addition, induction of low energy responsive genes, largely carried out by OsSnRK1b , is part of transcriptome reprogramming in response to cold as revealed by RNA-seq analysis (Fig. 4D-F). Furthermore, OsSnRK1b is responsible for inhibiting growth and promoting stress response under cold, as indicated by the elevated growth status marker (OsRPS6 protein phosphorylation) and reduced expression of OsABI5 under cold in the Ossnrk1b mutant (Fig. 4G-H and 5A-C). Together, these data indicate that low energy response is critical for chilling response and chilling tolerance. While the key low energy response regulator SnRK1α activates catabolism and represses anabolism to maintain cellular energy homeostasis under starvation stress, it inhibits growth and activates stress response under chilling stress. This mechanism is likely evolutionarily conserved among plants shown by a similar role for the SnRK1α genes in Arabidopsis and maize. This study also identifies OsCTK1 as a positive regulator of OsSnRK1b . The OsCTK1 protein phosphorylates and stabilizes the OsSnRK1b protein under both non-chilling and chilling conditions, as evidenced by the reduced protein abundance and nuclear accumulation of OsSnRK1b in the Osctk1 mutant (Fig. 2C-F). This regulation is also supported by a similar LOF and overexpression effects of these two genes in chilling tolerance and chilling induced stomatal movement as well as the loss of OsSnRK1b overexpression effect in the absence of the OsCTK1 . The direct involvement of starvation response for OsCTK1 is yet to be tested, but this is suggested by the elevated RPS6 phosphorylation in the Osctk1 mutant (Fig. 5H). Previous study identified a direct phosphorylation of SnRK1 by SnAK1/2 (SnRK1-activating kinases) at Thr-175 or Thr-176 residues that promotes the SnRK1α kinase activity in Arabidopsis 43,44 . These sites are distinct from the potential phosphorylation sites of OsSnRK1 by OsCTK1, suggesting different effects of these two regulators on OsSnRK1 α . Besides the effect on the protein stability of OsSnRK1b, the effects of phosphorylation by OsCTK1 on the kinase activity of OsSnRK1b has not been determined (Fig. 2D), although its effect on nuclear localization of OsSnRK1b suggests a regulation of its activity as well (Fig. 2E-F). SnRK1 was previously shown to regulate response to a number of abiotic stresses that induce low-energy status 23 . Drought stress induces stomatal closure and therefore limits CO₂ intake and ATP synthesis 45,46 . Salt stress, including Na⁺ toxicity, disrupts thylakoid membrane integrity and inhibits photosynthesis system and ATP production 47 . Hypoxia induces transition from aerobic to anaerobic respiration, causing a decline in ATP yield 48,49 . Heat stress inactivates Rubisco activate, damages chloroplast structure, and impedes photosynthesis 50 . Low temperature may also induce low energy status, like starvation, drought, salt, hypoxia, and heat. Chilling stress is known to disrupt thylakoid membrane structure, inhibit chlorophyll synthesis, and inhibit electron transport chain. It also causes phase transition of membrane lipids from the liquid crystalline state to the gel state, leading to ion leakage. The involvement of SnRK1 in chilling tolerance suggests that low energy sensing pathway could have a broad function in stress tolerance. This conserved pathway may direct the physiological reprogramming for environmental adaptation. Under chilling stress, OsSnRK1b mediates the induction of stress-responsive genes (such as OsABI5 ) and suppression of growth-related markers (OsRPS6) (Fig. 6K), suggesting a coordination of stress and growth processes according to the environment. This coordination is likely not through the regulation of DREB / CBF expression, as the expression of OsDREB genes under chilling did not have reduced expression in the Osctk1 and Ossnrk1b mutants compared to the wild type (Fig. 5I-J). Interestingly, the Osctk1 and Ossnrk1b mutants accumulate higher amount of growth marker protein RPS6 under non-chilling condition, but these mutants did not exhibit increased growth compared to the wild type (Fig. 4G-H). This suggests a broad role of OsSnRK1b and OsCTK1 genes in growth regulation other than the RPS6 associated pathway. This study also highlights that stomatal aperture regulation as a critical physiological response in chilling tolerance. OsCTK1 and OsSnRK 1b both promote stomatal closure in response to chilling as well as survival under cold (Figs. 1A-F and 3A-N). OsABI5 , a regulator of stomatal aperture regardless of chilling conditions, is also a positive regulator of chilling tolerance (Fig. 5D-H). Together with OsCNGCs in rice and OST1 in Arabidopsis, dual-function in stomatal closure and chilling tolerance for these genes strongly implicate stomatal movement control is an important part of chilling tolerance mechanism 18-21 . How SnRK1 genes in rice and Arabidopsis regulate stomatal movement under chilling still awaits further investigation. OsABI5 is a potential regulatory target, not necessarily a direct kinase target, as chilling induction of its transcript is reduced in the OsSnRK1b mutants. In addition, energy status markers such as sugar accumulation could affect stomatal movement, as starch degradation and glucose accumulation in guard cells could lead to stomatal opening 51,52 . Indeed, KIN10 ( AtSnRK1 ) overexpression was shown to promote light-induced starch degradation and thus stomatal opening 31 . This study shows that some SnRK1α members, OsSnRK1b in rice and KIN10 in Arabidopsis, regulate low-temperature response and low energy response. It is not yet known whether all members of the SnRK1α family have dual functions. KIN10 and KIN11 in Arabidopsis were shown to be low energy sensors (in starvation response) while the role of KIN12 in starvation response has not been tested 23 . KIN10 is a positive regulator of chilling tolerance, but the function of the other two Arabidopsis genes in chilling tolerance are yet to be determined. Among the four members in rice, OsSnRK1a and OsSnRK1b each have dual function in starvation response and chilling tolerance. The function of OsSnRK1c and 1d in chilling tolerance are yet to be determined by their LOF mutants. Whether or not a member has a more pronounced role in a specific stress response than other members is yet to be investigated in plants. Deciphering the shared or distinct functions of SnRK1 members in multiple stresses will provide insights into the evolution of these conserved proteins. In conclusion, this study demonstrates the involvement of low energy response in chilling tolerance and supports stomatal closure control as a critical mechanism for chilling tolerance. The knowledge of chilling tolerance at the whole plant physiology level will facilitate the development of strategies for enhancing plant resilience in a changing environment. Methods Plant materials and growth conditions The genetic backgrounds of rice mutant and transgenic plants in this study were ‘Nip’ or ‘ZH11’ as specified. Freshly harvested seeds were treated at 42℃ for a minimum of 14 days to break dormancy. Seeds were then soaked in water at room temperature for 3 to 5 days for germination. For hydroponic growth, germinated seeds with a similar development stage were transferred to a 96-well plate (with its bottom cut) that sits in water, as described previously 53 . Seedlings were grown in controlled chambers with 16 h of light and 8 h of dark at 28℃. For soil growth, germinated seeds were transferred to a mix of vermiculite: organic substrate (1:3) and cultured at 28℃ in chambers. The Arabidopsis T-DNA insertion mutants were obtained from Arabidopsis Biological Resource Center and Fuzhou Aerosa Biotechnology. Seeds were sown onto soil, stratified at 4°C for 3 days, and grown in chambers under constant light or 16 h light/8 h dark and 70% relative humidity. Maize cultivar B104 was used for this study. Seeds were placed on wet paper towels in the dark overnight at room temperature to break dormancy before being sown in soil for germination. Plants were grown under 22°C with continuous light for chilling assays or under 26°C with continuous light for stomatal measurements. Plasmid construction and plant transformation Specific targets for guide RNA spacer sequences were designed for CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 constructs by using the CRISPR-PLANT website (http://www.genome.arizona.edu/crispr/CRISPR). Two guide RNA targets were cloned into the PHUE411 vector 55 , and the resulting construct was transformed into Nip through Agrobacterium tumefaciens -mediated transformation 56 . For overexpression constructs, the coding sequence (CDS) was amplified and cloned into the pCAMBIA1300-GFP vector (Addgene). For GST (Glutathione s transferase) fusion constructs, the CDS was cloned into pGEX-2T vectors (Addgene) using a homologous recombination system (Vazyme, China). For His-fusion constructs, the CDS was cloned into the pET-30a vector. For Myc-fusion constructs, the CDS was cloned into pCAMBIA1300-221 vectors (Addgene). Vector details are listed in Supplementary Table 3. Transgenic plants were selected by hygromycin.Primes details are listed in Supplementary Table 2. The Zmctk1 mutants were generated in maize using methods as described previously 54,55 . Agrobacterium-mediated genetic transformation was performed by the Crop Bioengineering Laboratory at the Iowa State University. Chilling tolerance assays Chilling tolerance and ion leakage assays for rice plants were carried out using the method described previously 35 . For maize plants, chilling growth assay was done on seedlings grown at 26°C till 3-leaf stage. Plants were subsequently grown at 10°C for three weeks before they were transferred to 26°C for recovery for two weeks. For freezing tolerance assay in Arabidopsis, plants were germinated and grown at 22°C for two weeks. They were then subjected to -8°C for 5 h before being returned to 22°C for one week’s recovery. Plants were covered with humidity dome the first at the recovery. Stomata related assays Epidermal impressions were made using clear nail polish 63 . Dried nail polish was peeled from leaf epidermis and epidermal impression was observed under an Olympus BX53 microscope. Stomatal width and length were measured on the images using ImageJ software. Sixty stomata from four leaves were measured for each sample. Transpiration rate was measured using a steady-state porometer (METER SC-1). The second fully expanded leaf of the three-leaf stage plant was placed and stabilized within the chamber of the porometer for 30 s. Measurements were taken on the middle section of leaf. Data were collected from six leaves per genotype and time point. Leaf water loss was measured by fresh weight from ten uniformly grown seedlings per replicate, with three replicates in total to minimize individual variation. Water loss rate was expressed as the percentage of weight difference divided by the initial fresh weight. For leaf surface temperature, thermal images were captured using a thermal imager (FOTRIC 225S). Quantification of temperature was performed using AnalyzIR (FOTRIC TOOLS). RNA extraction and RT-qPCR One microgram of total RNA was reverse transcribed into cDNA using HiScript II RT SuperMix (Vazyme). OsActin ( LOC_Os03g50885 ) was used as the reference gene. Expression levels were determined using the comparative cycle threshold method (Vazyme). Primers used for RT-qPCR for each gene are detailed in Supplementary Table 2. RNA-Seq and data analysis Total RNA was isolated from the leaves of 14-day-old rice plants using Trizol reagent (TsingZol). Library construction and deep sequencing were performed by the Nanjing Jiangbei New Area Biopharmaceutical Public Service Platform Company Limited using the Illumina NovaSeq 6000 platform. DEGs were identified by DESeq2 56 , with adjusted P -values set at p 2. Protein extraction and immunoblotting Plant tissues were ground into powder using liquid nitrogen, and extracted by protein extract buffer (50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM EGTA, 1 mM MgCl 2 , 1 mM NaF, 10% glycerol, Complete TM EDTA-free protease inhibitor and PhosSTOP TM phosphatase inhibitor cocktails) at 500 μL of per 0.3 g of fresh tissue. Supernatants were collected following centrifugation at 13000 g for 15 min at 4°C as the protein extracts. Proteins were separated by SDS-PAGE on 12% gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare) in a wet tank unit (Bio-Rad). Membranes were incubated with primary anti-RPS6 antibody (rice: Solarbio, China, maize and Arabidopsis: Agrisera, Sweden) diluted to 1:1000, and polyclonal primary anti-RPS6A-P240 phosphorylated (Ser240) antibody diluted to 1:1000 (rice: Solarbio, maize and Arabidopsis: Agrisera). Antibodies were diluted in TBS-T (Tris-buffered saline with tween-20) containing 1% w/v BSA and incubated overnight at 4°C. Following six washing steps in TBS-T, membranes were incubated with a horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology) diluted 1:5000 in TBS-T containing 1% w/v BSA for 2 h. After three washing steps in TBS-T, proteins were detected by incubating the membranes in Clarity Western ECL substrate (Bio-Rad). The band intensities were quantified using ImageJ software. In vitro phosphoproteomic analysis In vitro phosphoproteomics was carried out using the method described previously 5 7 . Briefly, Total proteins extracted from chilling treated plants were dephosphorylated and then incubated with recombinant OsCTK1 in a kinase reaction containing 1 mM γ-[ 18 O 4 ] ATP. Peptides labelled with 18 O were identified by Mass Spec. Liquid chromatography tandem mass spectrometry analysis The GFP fusion protein was purified using the GFP-trap IP (KT Life). The bound proteins were eluted from the beads by heating at 95℃ for 10 min. Proteins were fractionated on 12% SDS-PAGE (Bio-Rad) and stained with Coomassie brilliant blue. Bands were excised and cut into small pieces and subjected to MS analyses (BGI). Co-immunoprecipitation assay Co-IP assay was carried out as described previously 35 . Isolation and transformation of rice protoplasts Protoplast isolation was done using method previously described 58 . Specifically, 5 grams rice leaf strips of 0.5 mm size were digested in 10 mL enzyme solution (1.5% cellulose R10, 0.75% macerozyme R10, 0.6 M mannitol, 10 mM MES) for 6 h in dark with gentle shaking (40 rpm) at 28℃. PEG-mediated transfections were carried out as described 59 . Specifically, 5-10 μg of plasmid DNA were mixed with 100 μL protoplasts. Phos-tag mobility shift assay Phos-tag reagent (AAL-107, Wako) was used for the phosphoprotein mobility-shift assay as described 60 . Protein extracts were separated on 12% (w/v) SDS-PAGE gel containing 50 μM Phos-tag and 100 μM MnCl 2 . Cell-free protein degradation assay Cell-free protein degradation assay was performed as described 61 with minor modifications. Total proteins were extracted from 14-days seedlings. Total proteins (0.3 g leaf to 500 μL extraction buffer) were incubated with purified recombinant proteins (5 mg) and 10 mM ATP. Proteins were detected with anti-His monoclonal antibody (Sangon Biotech). The Rubisco large (RbcL) subunit, detected by Ponceau S staining, was used as a loading control. Cell fractionation assay Soluble and nuclear proteins were isolated using Plant Nuclei Isolation/Extraction Kit (Sigma). PEPC protein was used as a cytosolic marker, and histone H3 was used as a nuclear marker. Declarations Acknowledgements We thank Professor Chengqiang Ding for the Ossnrk1a mutant seeds as well as Dr. Jing Li and Bioinformatics Center at Nanjing Agricultural University for RNA-seq analysis. Author contributions J.H. and B.Z. supervised the study. J.W., H.M., P.V., Y.Z., J.Y., G.X., R.Y., S.L. and S.Lu. performed the experiments, among them, J.W., B.Z. and H.M. drafted the manuscript. J.H. revised the manuscript. All authors contributed to the discussion and approved the final manuscript. Funding This study was supported by United States Department of Agriculture NIFA (2022-67013-37040), the Natural Science Foundation of China (32441064, 31971827, 32370309), Jiangsu Agricultural Science and Technology Innovation Fund (CX (23)1033) and China Postdoctoral Science Foundation (2024M761438, GZC20252664). Competing interests The authors declare no competing interests. References Ding, Y. L., Shi, Y. T. & Yang, S. H. 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Nature Protocols 2 , 1565-1572, doi:10.1038/nprot.2007.199 (2007). Mao, G. H. et al. Phosphorylation of a WRKY Transcription Factor by Two Pathogen-Responsive MAPKs Drives Phytoalexin Biosynthesis in. Plant Cell 23 , 1639-1653, doi:10.1105/tpc.111.084996 (2011). Ding, Y. L. et al. OST1 Kinase Modulates Freezing Tolerance by Enhancing ICE1 Stability in. Developmental Cell 32 , 278-289, doi:10.1016/j.devcel.2014.12.023 (2015). Li, R. Q. et al. OsNAC5 orchestrates OsABI5 to fine-tune cold tolerance in rice. J Integr Plant Biol 66 , 660-682, doi:10.1111/jipb.13585 (2024). Wu, S. and Zhao, B. Using clear nail polish to make Arabidopsis epidermal impressions for measuring the change of stomatal aperture size in immune response. Methods Mol. Biol 1578 , 243-248, doi: 10.1007/978-1-4939-6859-6_20 (2017). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementTableCTK1SnRK10820.xlsx Table S1 Characterization of the mutants used in this study. Table S2 Primers in this study. Table S3 Vectors used in this study. SupplementfigureCTK1SnRK108232025.pdf Supplementary Information The following Supporting Information is available for this manuscript: Figure S1 OsCTK1 positively regulates stomatal closure. Figure S2 OsCTK1 phosphorylates and stabilizes OsSnRK1b. Figure S3 OsSnRK1b overexpression enhances stomatal closure and chilling tolerance. Figure S4 OsSnRK1b is required for starvation response. Figure S5 OsABI5 regulates chilling tolerance. Figure S6 AtCK1.3 , KIN10 and AtABI5 regulate stomatal movement under cold in Arabidopsis. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7447278","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":523835663,"identity":"55b57388-78a5-4307-853c-8e8a02552b4e","order_by":0,"name":"Jian 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Zou","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Baohong","middleName":"","lastName":"Zou","suffix":""},{"id":523835666,"identity":"81b54363-791c-4e03-8ea9-c3eb36bfad5a","order_by":3,"name":"Huimin Liu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Liu","suffix":""},{"id":523835667,"identity":"0be778d7-c882-4c54-8759-ee5352faaaaf","order_by":4,"name":"Veronica Perez","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Veronica","middleName":"","lastName":"Perez","suffix":""},{"id":523835668,"identity":"9bc0f48e-a769-4d86-a2b9-6947c0a6b70a","order_by":5,"name":"Yao Zhang","email":"","orcid":"","institution":"College of Life Sciences, Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Zhang","suffix":""},{"id":523835669,"identity":"74605a9b-723c-4c6b-b88b-de1c293657ee","order_by":6,"name":"Pavol Vadovic","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Pavol","middleName":"","lastName":"Vadovic","suffix":""},{"id":523835670,"identity":"70fa6210-5e78-4d8d-a23c-2db54387e420","order_by":7,"name":"Yuan Jiang","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Jiang","suffix":""},{"id":523835671,"identity":"329065fc-c774-4e8f-acd4-aadc5c9a6f6e","order_by":8,"name":"Shengsong Guo","email":"","orcid":"https://orcid.org/0009-0004-6185-1308","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Shengsong","middleName":"","lastName":"Guo","suffix":""},{"id":523835672,"identity":"d67ecc0c-162e-455d-854b-9da6cb17cd42","order_by":9,"name":"Rui Yang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Yang","suffix":""},{"id":523835673,"identity":"6a6782e3-165a-4159-8da1-c4d28ff0a973","order_by":10,"name":"Shiyan Liu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shiyan","middleName":"","lastName":"Liu","suffix":""},{"id":523835674,"identity":"87e62299-b141-4f26-a09c-6cb8a0c52f04","order_by":11,"name":"Shan Lu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-08-24 16:00:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7447278/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7447278/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95702330,"identity":"5a75f726-0223-448d-a836-25dcd6d62b8d","added_by":"auto","created_at":"2025-11-12 05:45:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsCTK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promotes stomatal closure under chilling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Chilling induced leaf drooping in wild-type Nipponbare (NIP), \u003cem\u003eOsctk1-2\u003c/em\u003e and \u003cem\u003eOsctk1-3\u003c/em\u003e at 3 h of 6℃ treatments. All drooped leaves are indicated by white arrows, and bars are 5 cm, (B) Water loss (shown as percentage of fresh weight loss after chilling compared to initial weight) in leaves in NIP, \u003cem\u003eOsctk1-2\u003c/em\u003e and \u003cem\u003eOsctk1-3\u003c/em\u003e. (C-D) Stomatal phenotypes (C) and quantification of stomatal aperture (D) of NIP, \u003cem\u003eOsctk1-2\u003c/em\u003e and \u003cem\u003eOsctk1-3\u003c/em\u003e mutants before (0 h) and after a 6℃ treatment. Stomatal aperture is defined as the ratio of width and length (indicated by lines in cyan in C) of the stomata. Dot plots in C are from 60 stomata per genotype time point. Bars in C represent 100 μm. (E) Leaf surface temperature shown by the infrared thermal images of NIP, \u003cem\u003eOsctk1-2\u003c/em\u003e and \u003cem\u003eOsctk1-3\u003c/em\u003e at 3 h of 6℃ treatment (F) Transpiration rate (mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e) of NIP, \u003cem\u003eOsctk1-2\u003c/em\u003e and \u003cem\u003eOsctk1-3\u003c/em\u003e before (0 h) and after the 6℃ treatments. Shown are means and standard deviations from 60 stomata. Letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 by Duncan's multiple range test among the means via ANOVA in B, D and F.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/a68e39a230a8eaf1d86ba5a1.png"},{"id":95702323,"identity":"b828ccd6-d8e1-49c7-91fd-a24c730d8707","added_by":"auto","created_at":"2025-11-12 05:45:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":88983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsCTK1 phosphorylates and stabilizes OsSnRK1b.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phosphorylation of OsSnRK1b by OsCTK1. Shown are autoradiograph (top panel) and Coomassie Brilliant Blue (CBB) staining (bottom panel) of proteins from the kinase reaction consisting of recombinant OsCTK1 and OsSnRK1b (wild-type NIP form or the mutant form and OsSnRK1b\u003csup\u003eTSS-AAA\u003c/sup\u003eseparated on a 12% SDS-PAGE gel. Relative phosphorylation levels of OsSnRK1b (shown as black numbers) are quantified with the level of OsSnRK1b by OsCTK1 set as 1. (B) Phosphorylation of GFP-OsSnRK1b in plants carrying GFP-OsSnRK1b transgene in the NIP or \u003cem\u003eOsctk1-2\u003c/em\u003e background with or without 30 min of chilling (6℃) treatment. Total proteins were separated on the phos-tag gel (top panel) and SDS-PAGE gel (bottom panel). CIP samples were treated with calf intestine phosphatase at 37°C for 30 min. (C) Immunoblot of total proteins of NIP, \u003cem\u003eOsctk1-2\u003c/em\u003e, \u003cem\u003eOssnrk1b-3\u003c/em\u003e and \u003cem\u003eOssnrk1b1c\u003c/em\u003e detected by an anti-SnRK1b antibody. RbcL protein (Ponceau S stained) was used as the loading control of total protein. Numbers are relative amount of OsSnRK1b compared to the NIP. (D) Immunoblot of recombinant His-tagged OsSnRK1b and OsSnRK1b\u003csup\u003eTSS-AAA\u003c/sup\u003e proteins after incubation at 28℃ with total protein extracts from wild-type NIP or \u003cem\u003eOsctk1-2\u003c/em\u003e mutant plants in the presence of ATP for 0 h, 1 h or 3 h. RbcL protein stained with Ponseu S was used as the loading control of total proteins. The numbers are relative abundance of His-OsSnRK1b normalized to that of His-OsSnRK1b with WT extract at 0 h. (E) Immunoblot of GFP-OsSnRK1b in total protein extracts, nuclear fraction and cytosol fraction from protoplasts of wild-type NIP or the \u003cem\u003eOsctk1-2\u003c/em\u003e mutant transformed with GFP-OsSnRK1b. (F) Immunoblot of GFP-OsSnRK1b and GFP-OsSnRK1b\u003csup\u003eTSS-AAA\u003c/sup\u003e proteins in total protein extracts, nuclear fraction and cytosol fraction of wild-type NIP protoplasts transformed with the GFP fusion constructs. For (E) and (F), blots were probed with antibodies against GFP, PEPC (a cytosolic protein) and H3 (a nuclear protein) respectively. Loaded on the gels are 25% of the total proteins (T), 20% of nuclear proteins (N), and 50% of the cytoplasmic protein (S). LV (loading value): GFP signals quantified from blot with the first sample (WT T) set as 1. EV (equalized value): GFP signals adjusted with equalized loading of N, S and T with the first sample (WT T) set as 1.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/7c19d53a2592fcf255187b75.png"},{"id":95799153,"identity":"17a49c68-d44c-4d4a-9cf6-31d03bbc20a8","added_by":"auto","created_at":"2025-11-13 08:18:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":264927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsSnRK1b\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpression promotes stomatal closure and chilling tolerance in an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsCTK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-depdent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Leaf drooping phenotypes in NIP and the \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e mutants under chilling. Shown are three-leaf-stage seedlings grown at 28℃ for two-weeks-old before (0 h) and after a 6℃ treatment for 3 h. All drooped leaves are indicated by white arrows. Bars are 5 cm. (B) Water loss in leaves in NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e. (C) Quantification of stomatal aperture of NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e mutants before (0 h) and after a 6℃ treatment. (D) Transpiration rate (mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e) of NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e before (0 h) and after 6℃ treatments. Shown are means and standard deviations from 60 stomata. (E) Leaf surface temperature shown by the infrared thermal images of NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e at 3 h of 6℃ treatments. (F-H) Plant morphology (F), ion leakage rates (G) and survival rates (H) of NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e plants of three-leaf stage before and after 6℃ chilling treatments. Plant morphology and survival rates were taken after of 6℃ treatment for 2 days followed by 7 days of recovery at 28℃. Bars are 5 cm in F. Ion leakage were measured at 0 h and 24 h of 6℃ treatments. (I-K) Leaf drooping (I), transpiration (J) and thermal imaging (K) of NIP, \u003cem\u003eOsctk1-2\u003c/em\u003e, OE-\u003cem\u003eSnRK1b\u003c/em\u003e/NIP (#2 and #3), and OE-\u003cem\u003eSnRK1b\u003c/em\u003e/\u003cem\u003ectk1\u003c/em\u003e (#1 and #2) plants after 6℃ treatments as specified. White arrows in (I) indicate drooped leaves. Transpiration rate is mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e. A total of 60 stomata per genotype/time point were used for quantifications in (J). The bar in (I) is 5 cm. (L-N) Plant morphology (L), ion leakage rates (M) and survival rates (N) of NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOssnrk1b-3\u003c/em\u003e plants of three-leaf stage with 6℃ chilling treatments. Plant morphology and survival rates were taken after of 6℃ treatment for 3.5 days followed by 7 days of recovery at 28℃. Bars are 5 cm in L. Ion leakage were measured at 0 h and 24 h of 6℃ treatments. Letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 by Duncan's multiple range test among the means via ANOVA in B-D, G-H, J and M-N.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/ac8acbee2e9d31e5d79deda8.png"},{"id":95702325,"identity":"12501aae-387c-4339-a709-6ebe81f0a108","added_by":"auto","created_at":"2025-11-12 05:45:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsSnRK1b\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e regulates starvation response.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Starvation response phenotypes of NIP, \u003cem\u003eOssnrk1b-1\u003c/em\u003e, \u003cem\u003eOssnrk1b1c\u003c/em\u003e, and \u003cem\u003eOsSnRK1b\u003c/em\u003e overexpression lines (OE-\u003cem\u003eSnRK1b\u003c/em\u003e#1 and #2). Seedlings grown at 28℃ for 4 days followed by 7 days of growth under light (normal) or dark (starvation) before photographed. Bars are 5 cm. (B) Ratio of yellow leaf segment (flanked by white lines in A) versus total leaf length of seedlings in (A). (C) Chlorophyll content of above plants. Letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 by Duncan's multiple range test among the means via ANOVA in B-C. (D) Venn diagrams of starvation-induced up DEGs and 3 h cold-induced up DEGs in the NIP. (E) Venn diagram of 3 h cold-induced up DEGs in the NIP and the \u003cem\u003eOssnrk1b1c\u003c/em\u003e mutant. (F) Venn diagram of the cold-induced up DEGs in the \u003cem\u003eOssnrk1b1c\u003c/em\u003e mutant and starvation-induced and cold-induced up DEGs. (G) Immunoblot of Ser240/244 phosphorylation of RPS6 in NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003eand \u003cem\u003eOsctk1-2\u003c/em\u003e without (0 h) and with 3 h, 12 h, and 24 h of 6°C chilling treatment. Ponseu S stained RbcL (bottom panel) was used as the loading control. Numbers indicate relative abundance of phosphorylated RPS6 normalized to the first sample (NIP at 0 h). (H) Immunoblot of RPS6 protein in wild-type NIP, \u003cem\u003eOssnrk1b1c\u003c/em\u003e and \u003cem\u003eOsctk1-2\u003c/em\u003e seedlings before (0 h) and 3 h to 24 h after 6°C chilling treatment. Bottom panel shows stained RbcL as loading control. Numbers indicate relative abundance of RPS6 normalized to the first sample (NIP at 0 h).\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/fd8daba97ef9436b1eb2645f.png"},{"id":95702329,"identity":"01150b61-b07f-4fbe-8eaf-e844df20b197","added_by":"auto","created_at":"2025-11-12 05:45:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsABI5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e positively regulates stomatal closure and chilling tolerance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Expression of \u003cem\u003eOsABI5\u003c/em\u003eat 0 h, 3 h or 24 h after chilling (6℃) treatment in wild type and \u003cem\u003eOssnrk1b-2\u003c/em\u003e(A), \u003cem\u003eOsctk1-2\u003c/em\u003e (B) as well as OE-\u003cem\u003eSnRK1b\u003c/em\u003e/NIP and OE-\u003cem\u003eSnRK1b\u003c/em\u003e/\u003cem\u003ectk1\u003c/em\u003elines (C) at three-leaf seedling stage. Shown are relative expression with \u003cem\u003eOsActin\u003c/em\u003eas the reference gene as analyzed by qRT-PCR. (D) Leaf drooping phenotypes in wild-type ZH11, \u003cem\u003eOsabi5-1\u003c/em\u003e and \u003cem\u003eOsabi5-2\u003c/em\u003e seedlings under chilling. Shown are three-leaf-stage seedlings grown at 28℃ before (0 h) and after a 6℃ treatment for 3 h. Drooped leaves are indicated by white arrows. Bars are 5 cm. (E-F) Ion leakage rates (E) and survival rates (F) of ZH11, \u003cem\u003eOsabi5-1\u003c/em\u003e and \u003cem\u003eOsabi5-2\u003c/em\u003e plants of three-leaf stage before and after 6℃ chilling treatments. Survival rates were taken after of 6℃ treatment for 4 days followed by 7 days of recovery at 28℃. Ion leakage was measured at 0 h and 24 h of 6℃ treatments. (G) Water loss in leaves in ZH11, \u003cem\u003eOsabi5-1\u003c/em\u003eand \u003cem\u003eOsabi5-2\u003c/em\u003e plants before (0 h) and after a 6℃ treatment for 1 h, 3 h, 9 h and 24 h. (H) Quantification of stomatal aperture of ZH11, \u003cem\u003eOsabi5-1\u003c/em\u003eand \u003cem\u003eOsabi5-2\u003c/em\u003e mutants before (0 h) and after a 6℃ treatment for 1 h, 3 h and 9 h. Letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 by Duncan's multiple range test among the means via ANOVA in A-C and E-H. (I-J) Expression of \u003cem\u003eOsDREB\u003c/em\u003e genes shown as heat map before (0 h) and after 6℃ treatments (3 h or 24 h) in wild-type NIP and \u003cem\u003eOssnrk1b1c\u003c/em\u003e(I) or \u003cem\u003eOsctk1-2\u003c/em\u003e (J).\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/7e8d74dcf598da4c7dcee555.png"},{"id":95702326,"identity":"5756382d-1e36-4d41-a2fa-23b059592bee","added_by":"auto","created_at":"2025-11-12 05:45:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":104701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCTK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSnRK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eABI5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in Arabidopsis and maize promote stomatal closure under chilling stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Dot plots of stomatal aperture (width/length) of wild type Col-0, \u003cem\u003eAtck1.3\u003c/em\u003e, \u003cem\u003ekin10\u003c/em\u003e and \u003cem\u003eAtabi5-1\u003c/em\u003e before (0 h) and after treatment of 6°C for 3 h, 9 h and 24 h. (B) Quantification of transpiration rate (mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e) of wild type Col-0, \u003cem\u003eAtck1.3\u003c/em\u003e, \u003cem\u003ekin10\u003c/em\u003e and \u003cem\u003eAtabi5-1\u003c/em\u003e leaves before (0 h) and after treatment of 6°C for 3 h, 9 h, 24 h and 36 h. Shown are means and standard deviations from 50 stomata for each genotype. Letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 by Duncan's multiple range test among the means via ANOVA in A-B. (C) Infrared thermal images of wild type Col-0, \u003cem\u003eAtck1.3\u003c/em\u003e, \u003cem\u003ekin10\u003c/em\u003e and \u003cem\u003eAtabi5-1\u003c/em\u003e leaves. (D-E) Survival rate of \u003cem\u003ekin10\u003c/em\u003e (D) and \u003cem\u003eAtck1.3\u003c/em\u003e (E) plants following 3 h of freezing treatment in Arabidopsis. Asterisk indicates statistical significance, with **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, as determined by Student’s \u003cem\u003et\u003c/em\u003e-test. (F-G) Immunoblots of Phospho-RPS6 and RPS6 levels in \u003cem\u003ekin10 \u003c/em\u003e(F) and \u003cem\u003eAtck1.3 \u003c/em\u003e(G) before (0 h) and after (24 h) 6°C treatment. RbcL stained by Ponseu S in the bottom panel serves as loading control. Numbers indicate relative abundance of RPS6 or phospho-RPS6 normalized to the first sample (Col-0 at 0 h). (H) Quantification of stomatal aperture (width/length) of wild type and \u003cem\u003eZmctk1\u003c/em\u003e before (0 h) and after a 6℃ treatment for 1.5 h in maize. Letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 by Duncan's multiple range test among the means via ANOVA in H. (I) Survival rate analysis of wild type and \u003cem\u003eZmctk1\u003c/em\u003e following a 6℃ treatment in maize. Asterisk indicates statistical significance, with **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, as determined by Student’s \u003cem\u003et\u003c/em\u003e-test. (J) Immunoblots of Phospho-RPS6 and RPS6 levels in wild-type and \u003cem\u003eZmctk1 \u003c/em\u003e(G) before (0 h) and after (24 h) 10°C treatment in maize. RbcL stained by Ponseu S in the bottom panel serves as loading control. Numbers indicate relative abundance of RPS6 or phospho-RPS6 normalized to the first sample (wild-type at 0 h). (K) Proposed model for OsCTK1 and OsSnRK1b mediated chilling tolerance. Under chilling stress, OsCTK1 phosphorylates and thereby stabilizes the low-energy sensor OsSnRK1b. This post-translational modification orchestrates bidirectional regulation of downstream pathways. On the one hand, activating the stress-responsive pathway centered on \u003cem\u003eOsABI5\u003c/em\u003e, on the other hand, repressing growth-promoting pathways mediated by phosphorylated OsRPS6. Snowflakes represent chilling stress. P for phosphorylation. Arrowheads signify activation and T-bars represent inhibition.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/704a1e3f8db9afd421a77b71.png"},{"id":95804720,"identity":"c9474731-b0cb-49f1-8eaa-dcc4e6fa11f8","added_by":"auto","created_at":"2025-11-13 08:39:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2358277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/4b95c6d8-a8d6-4828-b452-652f92e7268a.pdf"},{"id":95800604,"identity":"dea03097-c3e8-4164-8944-69e0c2bd0cd2","added_by":"auto","created_at":"2025-11-13 08:22:59","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e Characterization of the mutants used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e Primers in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3\u003c/strong\u003e Vectors used in this study.\u003c/p\u003e","description":"","filename":"SupplementTableCTK1SnRK10820.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/f19a00b945eb03c423b0641b.xlsx"},{"id":95702328,"identity":"cf82075b-4294-439d-8ce4-10895ca123e2","added_by":"auto","created_at":"2025-11-12 05:45:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1208117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following Supporting Information is available for this manuscript:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e \u003cem\u003eOsCTK1\u003c/em\u003epositively regulates stomatal closure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2\u003c/strong\u003e OsCTK1 phosphorylates and stabilizes OsSnRK1b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3\u003c/strong\u003e \u003cem\u003eOsSnRK1b\u003c/em\u003eoverexpression enhances stomatal closure and chilling tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4\u003c/strong\u003e \u003cem\u003eOsSnRK1b\u003c/em\u003e is required for starvation response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S5\u003c/strong\u003e \u003cem\u003eOsABI5\u003c/em\u003eregulates chilling tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S6\u003c/strong\u003e \u003cem\u003eAtCK1.3\u003c/em\u003e, \u003cem\u003eKIN10\u003c/em\u003eand \u003cem\u003eAtABI5\u003c/em\u003e regulate stomatal movement under cold in Arabidopsis.\u003c/p\u003e","description":"","filename":"SupplementfigureCTK1SnRK108232025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7447278/v1/4cf8ce2adee91b5fa4f26b1b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Energy sensing OsSnRK1b and its regulator OsCTK1 promote chilling-induced stomatal closure and chilling tolerance in rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLow-temperature stress poses a major challenge to plant growth and development and is a key factor limiting crop productivity and geographical distribution\u003csup\u003e1-3\u003c/sup\u003e. To cope with cold stress, plants employ a suite of physiological, biochemical and molecular adaptations, including changes in membrane lipid composition, accumulation of compatible solutes, activation of antioxidant systems to scavenge reactive oxygen species (ROS), and modulation of stress-responsive hormone levels\u003csup\u003e4,5\u003c/sup\u003e. Prior exposure to moderately low temperatures can enhance tolerance to extreme cold, a phenomenon termed cold acclimation\u003csup\u003e6-8\u003c/sup\u003e.\u0026nbsp;Low temperature rapidly triggers calcium influx and ROS bursts, which serve as early signaling events to initiate transcriptional reprogramming. The CBF (C-repeat binding factor) transcription factors and their regulator ICE1 (Inducer of CBF expression) are key conserved players in cold signaling in diverse species including Arabidopsis, rice, and maize\u003csup\u003e9,10\u003c/sup\u003e.\u0026nbsp;This cold-induced transcriptional response ultimately promotes expression of cold-responsive genes, leading to the accumulation of antifreeze proteins, osmoprotectants, and remodeling of the lipidome, processes essential for maintaining cellular homeostasis and enhancing low-temperature tolerance\u003csup\u003e2,11,12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile molecular regulation of transcriptome change has been extensively studied for cold response in plants, broader physiological strategies at organismal level that contribute to cold tolerance are less well understood. Stomatal movement control is critical for adaptation to the environment, as it modulates gas exchange for photosynthesis and controls water loss for tolerance to drought and heat\u003csup\u003e13\u003c/sup\u003e. Stomatal movement has also been reported to occur in response to cold\u003csup\u003e14\u003c/sup\u003e, and it was associated with cold-induced wilting and death in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e, and \u003cem\u003eNicotiana tabacum\u003c/em\u003e\u003csup\u003e15-17\u003c/sup\u003e. Interestingly, multiple genes critical for cold acclimation, including \u003cem\u003eOST1\u003c/em\u003e (\u003cem\u003eOpen stomata 1\u003c/em\u003e) and \u003cem\u003eCNGCs\u003c/em\u003e (\u003cem\u003eCyclic nucleotide-gated channels\u003c/em\u003e), are important for cold induced stomatal closure. Notably, OST1, a regulator of ABA induced stomatal closure, can phosphorylate ICE1 to enhance CBF signaling pathway under cold stress\u003csup\u003e18-20\u003c/sup\u003e. The rice OsCNGC14/15/16 proteins that promote cold-induced stomatal closure are positive regulators of chilling tolerance\u003csup\u003e21\u003c/sup\u003e. These observations suggest a role for cold induced stomatal closure in cold tolerance.\u003c/p\u003e\n\u003cp\u003eLow energy response is a physiological response to energy depletion exemplified by starvation. A low energy status is also associated with some abiotic stresses including extended periods of salt and drought\u003csup\u003e3,22\u003c/sup\u003e, but its association with cold stress has not been investigated. In yeasts and animals, SNF1-related kinase (SnRK1/AMPK) proteins function as low energy sensors and coordinate catabolic activation and anabolic repression to maintain cellular energy homeostasis\u003csup\u003e23\u003c/sup\u003e. They trigger rapid cellular adaptation under low energy stress through phosphorylating key metabolic enzymes and transcription factors, thereby reprograming transcriptional network and metabolism to promote catabolism and repress anabolism\u003csup\u003e23,24\u003c/sup\u003e. Plant SnRK1 is also critical for adaptation to energy-deficient conditions including extended darkness and hypoxia\u003csup\u003e25,26\u003c/sup\u003e. It is also shown to be important for tolerance to biotic and abiotic stresses\u003csup\u003e24,27,28\u003c/sup\u003e. SnRK1 consists of a catalytic α-subunit and regulatory β- and γ- subunits\u003csup\u003e29\u003c/sup\u003e. The α-subunit consists of a N-terminal Ser/Thr kinase domain and a C-terminal regulatory domain that mediates its interaction with the β- and γ- subunits\u003csup\u003e30\u003c/sup\u003e. In Arabidopsis, three genes (\u003cem\u003eKIN10\u003c/em\u003e, \u003cem\u003eKIN11\u003c/em\u003e and \u003cem\u003eKIN12\u003c/em\u003e) encode the α-subunit of SnRK1 (SnRK1α). Among these, the KIN10 subunit translocates to the nucleus to de-repress metabolic stress-responsive genes under darkness, inhibited photosynthesis, or hypoxia\u003csup\u003e31\u003c/sup\u003e. It phosphorylates transcription factors bZIP63 and bZIP53 for starch synthesis and proline metabolism during dark-induced low-energy stress, respectively\u003csup\u003e32,33\u003c/sup\u003e. In rice, four isoforms of SnRK1α could be identified by sequence homology and they are named OsSnRK1a, OsSnRK1b, OsSnRK1c and OsSnRK1d (https://www.ricedata.cn/). OsSnRK1a is shown to regulate starvation response by promoting starvation-responsive genes under energy limitation but repressing these genes under energy-sufficient conditions through its interaction with the H3K27me2/3 demethylase JMJ705\u003csup\u003e34\u003c/sup\u003e. Whether a low energy response pathway is involved in cold tolerance remains unknown in plants.\u0026nbsp;\u003cem\u003eOsCTK1\u003c/em\u003e (\u003cem\u003eChilling tolerance kinase 1\u003c/em\u003e)\u0026nbsp;is\u0026nbsp;a positive regulator of chilling tolerance in rice as evidenced by a lower survival rate under chilling in its loss of function (LOF) mutants compared to the wild type\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;Natural variation of the \u003cem\u003eOsCTK1\u003c/em\u003e gene is a major contributor to chilling tolerance variation in \u003cem\u003eIndica\u003c/em\u003e rice. The CT (Chilling tolerant) variant of \u003cem\u003eOsCTK1\u003c/em\u003e, with a four amino acids insertion, exhibits a higher kinase activity and confers a greater chilling tolerance compared to the CS (Chilling sensitive) variant\u003csup\u003e35\u003c/sup\u003e. OsCTK1 belongs to a serine/threonine kinase family CKI (Casein kinase I) which has a broad regulatory function\u003csup\u003e36,37,38,39,40\u003c/sup\u003e. The Arabidopsis OsCTK1 homologous proteins, CK1.3 and CK1.4, are shown to regulate blue light signaling by phosphorylating the cryptochrome receptor CRY2 (Cryptochrome 2)\u003csup\u003e39\u003c/sup\u003e. OsCTK1 may target multiple phosphorylation substrates for its function, including OsP3B (Acidic ribosomal protein 3B), OsCNGC9, and OsMKP1 (Mitogen-activated protein kinase phosphatase 1)\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, we further investigated the regulatory mechanism of chilling tolerance through the study of another substrate of OsCTK1, OsSnRK1b. We found that OsSnRK1b, like OsSnRK1a, is also a low energy sensing kinase. OsCTK1 phosphorylates and stabilizes the OsSnRK1b. Similar to \u003cem\u003eOsCTK1\u003c/em\u003e, \u003cem\u003eOsSnRK1b\u003c/em\u003e promotes stomatal closure in response to chilling and enhances chilling tolerance. They both regulate the chilling-induced expression of stress-induced genes and repress the accumulation of the growth associated proteins. These findings indicate that energy metabolism and stomatal regulation are critical mechanisms for chilling tolerance in rice.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsCTK1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;positively regulates stomatal closure in response to chilling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to a lower survival rate and a higher ion leakage after chilling \u003csup\u003e35\u003c/sup\u003e, the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsctk1-3\u003c/em\u003e LOF mutants had a rapid leaf-drooping phenotype after chilling treatment when compared to the wild type Nipponbare (NIP) (Fig. 1A). The \u003cem\u003eOsctk1\u0026nbsp;\u003c/em\u003emutants also had a faster water loss and a more rapid leaf curling after chilling exposure compared to the wild type (Fig. 1B and Supplementary Fig. 1A-B). These defects promoted us to measure stomatal aperture in the \u003cem\u003eOsctk1\u003c/em\u003e mutant after chilling treatment. Stomatal closing was observed in the wild-type plants at 1 h and continued at 3 h and 9 h, but it was not observed till 3 h in \u003cem\u003eOsctk1-3\u003c/em\u003e and9 h in \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003e(Fig. 1C-D). In addition, both mutants had a larger stomatal aperture at all time points measured compared to the wild type (Fig. 1C-D). This stomatal defect was supported by thermal imaging where a lower leaf temperature was detected in the \u003cem\u003eOsctk1\u0026nbsp;\u003c/em\u003emutants compared to the wild type (Fig. 1E). This is corroborated with a higher transpiration rate in the mutants than the wild type under normal and chilling conditions (Fig. 1F). The\u0026nbsp;\u003cem\u003eOsCTK1\u003c/em\u003e gene is expressed in guard cells, as shown by positive signal of \u003cem\u003eβ\u003c/em\u003e‐Glucuronidase (GUS) reporter in transgenic plants carrying the GUS reporter gene under the control of the \u003cem\u003eOsCTK1\u003c/em\u003e promoter (Supplementary Fig. 1C). This indicates an expression of \u003cem\u003eOsCTK1\u0026nbsp;\u003c/em\u003ein guard cells and suggests a cell-autonomous function of \u003cem\u003eOsCTK1\u003c/em\u003e in guard cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;The activity of \u003cem\u003eOsCTK1\u0026nbsp;\u003c/em\u003ein promoting stomatal closure is aligned with its activity in promoting survival rate under cold as shown by its two natural variants. The CT variant of \u003cem\u003eOsCTK1\u0026nbsp;\u003c/em\u003ewas previously shown to confer a higher survival rate under chilling stress than the CS variant\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;Transgenic \u003cem\u003eOsctk1-2\u003c/em\u003e lines carrying the CT variant had a wild-type phenotype while lines carrying the CS variant partially complemented the \u003cem\u003eOsctk1-2\u003c/em\u003e mutant, as shown by reduced stomatal aperture and transpiration rate after a 6°C chilling treatment (Supplementary Fig. 1D-I).\u0026nbsp;Therefore, the function of OsCTK1 in stomatal closure control is highly correlated with its function in promoting survival under chilling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsCTK1 phosphorylates OsSnRK1b\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted \u003cem\u003ein vitro\u003c/em\u003e phosphoproteomics to identify phosphorylation targets of OsCTK1 that might mediate the function of OsCTK1 in stomatal movement control. OsSnRK1b and OsSnRK1c were among the Mass Spec identified candidate target proteins as a shared peptide of these two proteins were identified as phosphorylated by the recombinant OsCTK1. We chose these two proteins for further study\u0026nbsp;because theirArabidopsis homolog KIN10 was previously implicated in stomatal movement control under normal growth temperature\u003csup\u003e41\u003c/sup\u003e (Supplementary Fig. 2A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOsSnRK1b was selected among the two proteins for assaying the interaction OsCTK1, as 1b and 1c are highly homologous. Direct physical interaction between OsCTK1 and\u0026nbsp;OsSnRK1b was verified in planta and \u003cem\u003ein vitro\u003c/em\u003e by Co-Immunoprecipitation (Co-IP) assays (Supplementary Fig. 2B). A direct phosphorylation of OsSnRK1b by OsCTK1 was observed by an \u003cem\u003ein vitro\u003c/em\u003e kinase assay (Fig. 2A). The recombinant His-OsSnRK1b protein had an autophosphorylation activity, but the addition of GST-OsCTK1 in the kinase reaction resulted in a stronger phosphorylation of His-OsSnRK1b protein (Fig. 2A). In addition, natural variants of OsCTK1 exhibited a differential phosphorylation activity on OsSnRK1b in the \u003cem\u003ein vitro\u003c/em\u003e kinase assay. The CT variant of OsCTK1 exhibited a higher phosphorylation on OsSnRK1b than the CS variant, and the critical Indel polymorphism also altered the phosphorylation level of the CT and the CS variants (Supplementary Fig. 2C).\u003c/p\u003e\n\u003cp\u003ePhosphorylation of OsSnRK1b that is dependent on OsCTK1 was also detected in protoplast cells expressing a GFP-tagged OsSnRK1b by phos-tag gels. A slower migrating band of GFP-OsSnRK1b was observed in the wild type cells but not in the \u003cem\u003eOsctk1-2\u003c/em\u003e mutant cells (Fig. 2B). This differential band pattern was also observed from protoplast cells incubated further at 6°C for 1 h. Treatment of protein extracts by calf intestine phosphatase (CIP) abolished this slow migrating band, indicating that it was indeed a phosphorylated form of OsSnRK1b-GFP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe identified three residues, Thr\u003csup\u003e173\u003c/sup\u003e, Ser\u003csup\u003e174\u003c/sup\u003e and Ser\u003csup\u003e347\u003c/sup\u003e, as potential phosphorylation sites of OsCTK1 in OsSnRK1b through two approaches. Above mentioned \u003cem\u003ein vitro\u003c/em\u003e phosphoproteomics detected phosphorylation of Thr\u003csup\u003e173\u003c/sup\u003e and/or Ser\u003csup\u003e174\u003c/sup\u003e. MassSpec analysis of GFP tagged OsSnRK1b expressed in protoplast cells revealed Ser\u003csup\u003e347\u003c/sup\u003e phosphorylation in the wild-type but not the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutant protoplast cells (Supplementary Fig. 2D). Mutating Thr\u003csup\u003e173\u003c/sup\u003e and Ser\u003csup\u003e174\u003c/sup\u003e of SnRK1b to Ala resulted in a 20% decrease in phosphorylation of OsSnRK1 (Supplementary Fig. 2E), while an 89% reduction of phosphorylation was observed when mutating Ser\u003csup\u003e347\u003c/sup\u003e, Thr\u003csup\u003e173\u003c/sup\u003e and Ser\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e74\u0026nbsp;\u003c/sup\u003eof OsSnRK1bsimultaneously to Ala \u003cem\u003ein vitro\u003c/em\u003e kinase assay with OsCTK1 (Fig. 2A). As autophosphorylation accounted for about 29% phosphorylation in the wild-type OsSnRK1b, this indicates an 85% reduction (11% versus 71%) of phosphorylation of OsSnRK1b by OsCTK1 from mutations of these three residues (Fig. 2A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsCTK1 stabilizes OsSnRK1b through phosphorylation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOsCTK1 was found to affect the abundance of OsSnRK1b when OsSnRK1b protein was detected by an antibody that targets OsSnRK1 members (Fig. 2C). This antibody primarily detects OsSnRK1b, because a similar reduction (71% and 73%) of the signal compared to the wild type was observed in the \u003cem\u003eOssnrk1b1c\u003c/em\u003e double mutant and a \u003cem\u003eOssnrk1b\u0026nbsp;\u003c/em\u003esingle mutant (as described in the next result section) (Fig. 2C). Strikingly, the \u003cem\u003eOsctk1-2\u003c/em\u003e mutant had a 46% reduction of OsSnRK1 proteins detected by this antibody compared to the wild type (Fig. 2C).\u0026nbsp;This reduction of OsSnRK1 protein abundance was not due to a reduced RNA transcript of \u003cem\u003eOsSnRK1\u003c/em\u003e in the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutant (Supplementary Fig. 2F), indicating an effect on OsSnRK1b at the protein level by OsCTK1. A cell-free protein degradation assay indicates that a lower protein stability contributes to the low abundance of OsSnRK1b in the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutant. The recombinant His-OsSnRK1b protein had a faster degradation when incubated with protein extracts from \u003cem\u003eOsctk1-2\u003c/em\u003e mutant than from those of the wild type (Fig. 2D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe stability of OsSnRK1b was affected by its OsCTK1 phosphorylation sites. The His tagged mutant OsSnRK1b protein with three phosphorylation site mutation to Ala, His-OsSnRK1b\u003csup\u003eTSS-AAA\u003c/sup\u003e, had a faster degradation compared to the wild-type His-OsSnRK1b form when incubated with protein extracts from wild-type plants (Fig. 2D). In contrast, when incubated with protein extracts from the \u003cem\u003eOsctk1-2\u003c/em\u003e mutant, the His-OsSnRK1b\u003csup\u003eTSS-AAA\u003c/sup\u003e protein had a degradation rate similar to the wild-type His-OsSnRK1b (Fig. 2D). These findings suggest that phosphorylation of OsSnRK1b at these sites affect the protein stability in an OsCTK1-dependent manner.\u003c/p\u003e\n\u003cp\u003eOsCTK1 affects the subcellular localization of OsSnRK1b in addition to its protein abundance. When expressed in protoplast cells, the GFP-OsSnRK1b fusion protein had both nuclear and cytosol distributions as assayed by nuclear fractionation. It has a lower nuclear accumulation and a higher cytosol accumulation in the\u0026nbsp;\u003cem\u003eOsctk1-2\u003c/em\u003emutant cells compared to the wild type\u0026nbsp;(Fig. 2E). The nuclear/cytosol distribution of OsSnRK1b was likely affected by the three OsCTK1 phosphorylation residues. A lower nuclear accumulation and a higher cytosol accumulation was observed for GFP-OsSnRK1b\u003csup\u003eTSS-AAA\u003c/sup\u003e protein compared to the GFP-OsSnRK1b protein when expressed\u0026nbsp;in wild-type protoplasts\u0026nbsp;(Fig. 2F).\u0026nbsp;These data suggests that phosphorylation of OsSnRK1b by OsCTK1 may enhance its translocation to the nucleus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsSnRK1b\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;promotes stomatal closure and survival under chilling\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe function of \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsSnRK1c\u0026nbsp;\u003c/em\u003ewas analyzed through the mutants generated by CRISPR/Cas9 gene editing. Four LOF \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e mutant lines were obtained, with the \u003cem\u003e-1\u003c/em\u003e and \u003cem\u003e-2\u003c/em\u003e alleles independently created from the \u003cem\u003e-3\u003c/em\u003e and \u003cem\u003e-4\u003c/em\u003e alleles from different pairs of RNA targets (Supplementary Table 1). The \u003cem\u003eOssnrk1b\u003c/em\u003e\u003cem\u003e1c\u003c/em\u003e mutant was an \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003ec\u003c/em\u003e double mutant as it contained an additional mutation in the \u003cem\u003eOsSnRK1c\u003c/em\u003e gene (Supplementary Table 1). All four mutant lines exhibited a similar phenotype in the initial characterization, and therefore \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb1c\u0026nbsp;\u003c/em\u003edouble mutants and \u003cem\u003eOssnrk1b\u003c/em\u003e\u003cem\u003e-3\u0026nbsp;\u003c/em\u003e(\u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e single mutant) were used for further analysis.\u003c/p\u003e\n\u003cp\u003eBoth \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb1c\u0026nbsp;\u003c/em\u003eand\u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb-3\u003c/em\u003e exhibited more chilling susceptibility compared to the wild-type NIP plant. After 6°C treatment, leaf rolling and leaf drooping occurred much earlier in the mutants than the wild type (Fig. 3A, Supplementary Fig. 3A). These mutants also had a similar faster water loss, slower stomatal closure, and higher transpiration than the wild type (Fig. 3B-E,\u0026nbsp;Supplementary Fig. 3B). The other two alleles, \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb-1\u0026nbsp;\u003c/em\u003eand\u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb-4\u003c/em\u003e, exhibited the same stomatal closure and water retention defects as the \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb1c\u0026nbsp;\u003c/em\u003eand\u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb-3\u003c/em\u003e alleles (Supplementary Fig. 3C-F). These data indicate that the loss of \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e function compromised chilling-induced stomatal closure.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e mutants also showed more susceptibility to chilling compared to the wild type as measured by leaf damage and survival rate (Fig. 3F). They had a higher ion leakage to a similar degree in leaf tissues after 24 h of chilling treatment compared to the wild type (Fig. 3G). The survival rates from 2 d of 6℃ treatment were 28% and 20% for the two mutants, lower than the rate of 58% for the wild type (Fig. 3H\u0026nbsp;and Supplementary Fig. 3G-H). These data indicate that the loss of \u003cem\u003eOsSnRK1b\u003c/em\u003e function leads to reduced chilling tolerance.\u0026nbsp;The\u0026nbsp;\u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb\u0026nbsp;\u003c/em\u003e\u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003ec\u003c/em\u003e double and \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb-3\u003c/em\u003e single mutants exhibited a similar degree of defects under chilling. This indicates that the \u003cem\u003eOsSnRK1c\u003c/em\u003e gene had no detectable function in chilling tolerance in the absence of the \u003cem\u003eOsSnRK1b\u003c/em\u003e function. These data indicate that the \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e plays a dominant role over \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003ec\u003c/em\u003e or an equal role as the \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003ec\u003c/em\u003e isoform in chilling responses (Fig. 3A-H). The relative function of \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e and \u003cem\u003eOsSnRK1c\u003c/em\u003e isoforms could be further verified when single mutants of the \u003cem\u003eOsSnRK1c\u003c/em\u003e form become available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpress\u003c/strong\u003e\u003cstrong\u003eing \u003cem\u003eOsSnRK1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eb\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;promotes chilling tolerance and chilling induced stomatal closure in an\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eOsCTK1\u003c/em\u003e-dependent manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further analyze the function of OsSnRK1b and its regulation by OsCTK1, we analyzed transgenic lines overexpressing \u003cem\u003eOsSnRK1b\u0026nbsp;\u003c/em\u003ein wild-type NIP (named OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/NIP) and \u003cem\u003eOsctk1-2\u003c/em\u003e (named OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/\u003cem\u003ectk1\u003c/em\u003e), respectively. Consistent with findings \u003cem\u003ein vitro\u003c/em\u003e, the GFP-OsSnRK1b protein had a lower accumulation in the two OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/\u003cem\u003ectk1\u003c/em\u003e lines compared to the two OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/NIP lines, despite comparable \u003cem\u003eOsSnRK1b\u0026nbsp;\u003c/em\u003eRNA expression in these four lines (Supplementary Fig. 3I-J). When transgenic plants were subject to chilling, GFP-OsSnRK1b has a more pronounced reduction in \u003cem\u003eOsctk1-2\u003c/em\u003e lines compared to the wild type lines (Supplementary Fig. 3J).\u003c/p\u003e\n\u003cp\u003eOverexpression of \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e was found to promote stomatal closure and enhance chilling tolerance in the wild type. After 6°C chilling treatment, the two OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/NIP lines showed delayed leaf rolling and drooping compared to the wild type (Fig. 3I). Water loss was lower at 3 h and 9 h of chilling treatment in the overexpression lines compared to the wild type (Supplementary Fig. 3K). The overexpression lines also had smaller stomatal apertures at 28°C and faster stomatal closure upon 6°C exposure (Supplementary Fig. 3L), resulting in higher leaf temperature and lower transpiration rate both before and after chilling compared to the wild type (Fig. 3J-K). In addition, the OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/NIP transgenic lines suffered less chilling damage, as shown by lower ion leakage at 24 h (Fig. 3L-M) and higher survival rates (Fig. 3N) after 3.5 days at 6°C. Together, these data indicate that overexpression of \u003cem\u003eOsSnRK1b\u003c/em\u003e promotes stomatal closure and confers a higher chilling tolerance.\u003c/p\u003e\n\u003cp\u003eThe enhanced chilling tolerance and stomatal closure by \u003cem\u003eOsSnRK1b\u003c/em\u003e overexpression was not observed in the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutant background (Fig. 3I). The two OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/\u003cem\u003ectk1\u0026nbsp;\u003c/em\u003elines behaved the same as the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutant plant before and after chilling treatment, including earlier leaf dropping, higher water loss, more open stomata, and higher transpiration rate compared to the wild type (Fig. 3I-K and Supplementary Fig. 3K-L). The OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e/\u003cem\u003ectk1\u0026nbsp;\u003c/em\u003elines also had higher ion leakage (Fig. 3M) and lower survival rate (Fig. 3N) compared to the wild type. These data indicate that the effects of \u003cem\u003eOsSnRK1b\u003c/em\u003e overexpression on chilling tolerance and stomatal aperture are dependent on OsCTK1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsSnRK1b\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is required for starvation response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause AtSnRK1α in Arabidopsis and OsSnRK1a in rice are shown to be low energy sensing kinases \u003csup\u003e23,34\u003c/sup\u003e, we assessed whether OsSnRK1b is also involved in low energy response. The \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e mutantsandthe OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e lines were subject to starvation with continuous darkness for 7 days (Fig. 4A). Both the \u003cem\u003eOssnrk1\u003c/em\u003e\u003cem\u003eb1c\u003c/em\u003e and the \u003cem\u003eOssnrk1b-3\u0026nbsp;\u003c/em\u003emutants exhibited a larger bleached area compared to the wild type, corroborated by reduced chlorophyll levels in leaves,\u0026nbsp;compared to the wild type (Fig. 4B-C). The bleaching and chlorophyll loss in the \u003cem\u003eOssnrk1b\u0026nbsp;\u003c/em\u003emutants was to a similar extent as the \u003cem\u003eOssnrk1a-1\u003c/em\u003e mutant (Supplementary Fig. 4A-D). Conversely, the OE-\u003cem\u003eOsSnRK1b\u003c/em\u003e lines exhibited a reduced bleached area and higher chlorophyll levels than the wild type (Fig. 4A-C). This indicates that OsSnRK1b has a conserved role in low energy stress response. In addition, the \u003cem\u003eOssnrk1a-1\u003c/em\u003e mutant \u003csup\u003e42\u003c/sup\u003e (Supplementary Fig. 4A) which was shown to be defective in low energy response, had a significantly lower survival rate and higher transpiration rate than the wild-type after chilling treatment (Supplementary Fig. 4E-G), indicating that OsSnRK1a has a similar function to OsSnRK1b in chilling tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsSnRK1b\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is required for chilling induced growth inhibition\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesize that low energy response contributes to chilling response in plants because SnRK1b and SnRK1a are both important for low energy tolerance and chilling tolerance. To test this, we examined transcriptomic response to chilling in the wild type and the \u003cem\u003eOssnrk1b\u0026nbsp;\u003c/em\u003emutants by RNA sequencing (RNA-seq) analysis. The wild-type plant had 1478 cold (6℃ for 3 h) induced DEGs, and 849 (57%) of them were also induced by starvation based on a previous report (Wang et al 2021) (Fig. 4D), suggesting a contribution of low energy response to transcriptome reprogramming under chilling. The \u003cem\u003eOssnrk1b\u0026nbsp;\u003c/em\u003emutants had a reduced number of cold-induced genes (959) compared to the wild type, and a smaller proportion (163, 16.3%) of these genes overlapped with chilling induced and starvation induced genes compared to the wild type (Fig. 4E-F). Therefore, about half (51%) of cold-induced DEGs and 81% of the cold-induced starvation-responsive genes are dependent on \u003cem\u003eOsSnRK1b\u003c/em\u003e (Fig. 4E-F), suggesting an important role of \u003cem\u003eOsSnRK1b\u003c/em\u003e in inducing starvation response genes under cold.\u003c/p\u003e\n\u003cp\u003eAs low energy response balances plant growth and stress responses, we further investigated the regulation of growth process under chilling by \u003cem\u003eOsSnRK1b\u003c/em\u003e and \u003cem\u003eOsCTK1\u003c/em\u003e. The phosphorylation of Ser\u003csup\u003e240\u0026nbsp;\u003c/sup\u003eof the ribosome protein RPS6 (a marker for TOR regulated growth) and the abundance of RPS6 (an indicator of growth) were analyzed in the mutants before and after chilling stress treatment (Fig. 4G). Strikingly, both the \u003cem\u003eOssnrk1b1c\u003c/em\u003e and the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutants had a higher amount of Ser\u003csup\u003e240\u003c/sup\u003e phosphorylation in OsRPS6 compared to the wild type at 28℃. The difference became more pronounced after chilling treatment (Fig. 4G). The wild type had a reduced Ser\u003csup\u003e240\u003c/sup\u003e phosphorylation at 3 h and 12 h after chilling, reflecting the inhibition of growth at early phase of chilling stress response, but the \u003cem\u003eOsctk1-2\u003c/em\u003e and \u003cem\u003eOssnrk1b1c\u003c/em\u003e mutants had increased phosphorylation after chilling (Fig. 4G), suggesting a loss of growth inhibition under chilling. Similarly, the OsRPS6 protein amount was at a higher level in these two mutants compared to the wild type under 28℃ growth condition. In addition, the RPS6 amount decreased at 3 h, recovered at 12 h in the wild type, but it did not decrease after chilling and remained the same amount throughout the chilling treatment in the two mutants (Fig. 4H). These findings suggest that \u003cem\u003eOsSnRK1b\u003c/em\u003e and \u003cem\u003eOsCTK1\u003c/em\u003e inhibit growth processes in general and more so under chilling stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsSnRK1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eb\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and \u003cem\u003eOsCTK1\u003c/em\u003e regulate chilling-induction of the stress response gene \u003cem\u003eOsABI5\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause OsSnRK1b regulates stomatal closure which is known to be associated with the ABA response pathway, we asked whether it regulates the ABA stress response pathway under cold. Chilling induced 74 ‘ABA-responsive genes’ (GO:0009737) among the 495 genes under this GO term, which was not significantly enriched among the chilling induced DEGs in the wild type. Nevertheless, 43 of them were not induced by chilling in the \u003cem\u003eOssnrk1b1c\u003c/em\u003e mutant, indicating a dependence on \u003cem\u003eOsSnRK1b\u003c/em\u003e for chilling induction. We subsequently investigated the expression of \u003cem\u003eOsABI5\u003c/em\u003e that encodes a major transcription factor for ABA response. qRT-PCR revealed that \u003cem\u003eOsABI5\u003c/em\u003e expression was induced by 6°C treatment at 3 h or 24 h in the wild type but not in the \u003cem\u003eOssnrk1b1c\u003c/em\u003e mutants (Fig. 5A). \u003cem\u003eOsCTK1\u003c/em\u003e was also found to regulate the RNA expression of \u003cem\u003eOsABI5\u003c/em\u003e. Despite having a higher expression of \u003cem\u003eOsABI5\u003c/em\u003e compared to the wild type at 28°C, the \u003cem\u003eOsctk1-2\u0026nbsp;\u003c/em\u003emutant plants had no induction of \u003cem\u003eOsABI5\u0026nbsp;\u003c/em\u003egene expression by chilling and a lower \u003cem\u003eOsABI5\u0026nbsp;\u003c/em\u003eexpression compared to the wild type under chilling (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eThe regulation of \u003cem\u003eOsABI5\u003c/em\u003e by \u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u0026nbsp;\u003c/em\u003ewas also seen in the OE-\u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e transgenic plants. The expression of \u003cem\u003eOsABI5\u0026nbsp;\u003c/em\u003ewas higher in the OE-\u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e transgenic plants than in the wild type, but it was the same in the OE-\u003cem\u003eOsSnRK1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e/\u003cem\u003ectk1\u0026nbsp;\u003c/em\u003elines as in the \u003cem\u003eOsctk1-2\u003c/em\u003e mutant(Fig. 5C). These results indicated that \u003cem\u003eOsSnRK1b\u003c/em\u003e and \u003cem\u003eOsCTK1\u003c/em\u003e are both required for the induction of \u003cem\u003eOsABI5\u003c/em\u003e in response to chilling, and \u003cem\u003eOsCTK1\u003c/em\u003e has an additional role in repressing \u003cem\u003eOsABI5\u003c/em\u003e expression under non-stress conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsABI5\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;positively regulates chilling tolerance and stomatal closure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA previous study shows that an \u003cem\u003eOsabi5\u003c/em\u003e mutant had reduced germination and seedling survival under cold compared to the wild type plants\u003csup\u003e62\u003c/sup\u003e. To further investigate the role of OsABI5 in the cold stress response, we analyzed two independent LOF mutants of \u003cem\u003eOsABI5\u003c/em\u003e, \u003cem\u003eOsabi5-1\u003c/em\u003e and \u003cem\u003eOsabi5-2\u003c/em\u003e, generated by CRISPR/Cas9 gene editing in the ZH11 background (Supplementary Table 1). Both \u003cem\u003eOsabi5\u0026nbsp;\u003c/em\u003emutants exhibited more extensive leaf drooping compared to the wild-type parent ZH11 after a 6℃ treatment (Fig. 5D). They also had higher ion leakage (Fig. 5E) and lower survival rates (Fig. 5F) compared to the wild type. In addition, the\u003cem\u003e\u0026nbsp;Osabi5\u0026nbsp;\u003c/em\u003emutants had a higher water loss after chilling compared to the wild type (Fig. 5G and Supplementary Fig. 5A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe chilling susceptible defect in the \u003cem\u003eOsabi5\u0026nbsp;\u003c/em\u003emutants was accompanied by a larger stomatal aperture in the mutant than the wild type after chilling (Fig. 5H). This stomatal aperture defect is not chilling specific, as the \u003cem\u003eOsabi5\u0026nbsp;\u003c/em\u003emutants had a larger aperture without chilling treatment as well. In addition, the mutants closed stomata in response to chilling a similar dynamic to the wild type (Fig. 5H). Correlated with stomatal aperture defect, the \u003cem\u003eOsabi5\u003c/em\u003e mutants had a higher transpiration rate before and after chilling treatment compared to the wild type, and its leaf surface temperature was lower than the wild type at 3 h after 6°C treatment (Supplementary Fig. 5C-D).\u0026nbsp;These results indicate that \u003cem\u003eOsABI5\u003c/em\u003e promotes stomatal closure in general, but it does not significantly affect the chilling induction of stomatal closure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsCTK1\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand\u003cem\u003e\u0026nbsp;OsSnRK1b\u0026nbsp;\u003c/em\u003edo not significantly affect the expression of \u003cem\u003eCBF\u003c/em\u003e related genes under chilling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also investigated whether \u003cem\u003eOsCTK1\u003c/em\u003e and \u003cem\u003eOsSnRK1b\u003c/em\u003e regulate expression of the \u003cem\u003eC-repeat binding factor\u003c/em\u003e (\u003cem\u003eCBF\u003c/em\u003e) or \u003cem\u003edehydration-responsive element-binding\u003c/em\u003e (\u003cem\u003eDREB\u003c/em\u003e) genes. In the wild-type plants, chilling treatment at 6°C induced the expression of eight out of the nine \u003cem\u003eOsDREB\u003c/em\u003e genes at both 3 h and 24 h (Fig. 5I-J). All eight genes were also induced by chilling in the \u003cem\u003eOssnrk1b\u003c/em\u003e mutants. However, the induction of \u003cem\u003eOsDREB\u003c/em\u003e genes in \u003cem\u003eOssnrk1b\u003c/em\u003e mutants were significantly lower than in the wild type at 3 h, while most \u003cem\u003eOsDREB\u003c/em\u003e genes showed no significant difference at 24 h (Fig. 5I). In contrast, the \u003cem\u003eOsctk1\u003c/em\u003e mutant exhibited higher basal expression levels of seven \u003cem\u003eOsDREB\u003c/em\u003e genes at normal growth temperature. After 24 h of 6°C treatment, the expression levels of these eight genes in the \u003cem\u003eOsctk1\u003c/em\u003e mutant were not significantly different from those in the wild type (Fig. 5J). These findings indicate that OsCTK1 is not a positive regulator of \u003cem\u003eOsDREB\u003c/em\u003e expression, and its role in chilling tolerance is likely independent of the CBF pathway. Although OsSnRK1b exerts minor effects on the expression of certain \u003cem\u003eOsDREB\u003c/em\u003e genes, whether its function in chilling tolerance is primarily mediated through the CBF pathway remains to be elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArabidopsis \u003cem\u003eSnRK1\u003c/em\u003e and \u003cem\u003eCTK1\u003c/em\u003e are regulators of chilling tolerance and chilling induced stomatal closure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether or not the involvement of low energy response in chilling tolerance is conserved in plants, we analyzed homologous genes of \u003cem\u003eSnRK1b\u003c/em\u003e and \u003cem\u003eCTK1\u003c/em\u003e in Arabidopsis. \u003cem\u003eKIN10\u003c/em\u003e (At3g10190), an apparent ortholog of three \u003cem\u003eOsSnRK1\u0026nbsp;\u003c/em\u003e(\u003cem\u003ea, b,\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e) genes in rice (Supplementary Fig. 1A),has been shown to participate in starvation response \u003csup\u003e23\u003c/sup\u003e. The LOF \u003cem\u003ekin10\u003c/em\u003e mutant exhibited a slower stomatal closure in response to chilling. When seedlings were exposed to 6°C, stomatal closing was observed as early as 3 h of treatment in the wild type, but no closing was observed even at 24 h of chilling treatment in the \u003cem\u003ekin10\u0026nbsp;\u003c/em\u003emutant (Fig. 6A). Corroborated with the stomatal behavior, the\u0026nbsp;\u003cem\u003ekin10\u003c/em\u003e mutants had a higher transpiration rate compared to the wild type throughout the 24 h of 6°C treatment (Fig. 6B-C).\u0026nbsp;It was also more susceptible to freezing, as evident by the reduced survival rate following a 5 h of -8ºC treatment compared to wild-type (Fig. 6D). Most strikingly, the \u003cem\u003ekin10\u0026nbsp;\u003c/em\u003emutant exhibited a higher amount of growth associated molecular markers under chilling similarly to the \u003cem\u003eOssnrk1b\u0026nbsp;\u003c/em\u003emutant. Chilling treatment of 24 h reduced total RPS6 abundance and Ser\u003csup\u003e240\u003c/sup\u003e phosphorylation in wild type, and the \u003cem\u003ekin10\u003c/em\u003e mutant had a higher level of both total RPS6 and phosphorylation at Ser\u003csup\u003e240/244\u003c/sup\u003e compared to the wild type under both normal and chilling growth conditions (Fig. 6F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe closest homolog of \u003cem\u003eOsCTK1\u003c/em\u003e in Arabidopsis, \u003cem\u003eAtCK1.3\u003c/em\u003e, was also found to play a role in chilling tolerance. Compared to the wild type, the \u003cem\u003eAtck1.3\u003c/em\u003e mutant had a larger stomatal aperture under normal growth conditions as well as a delayed stomatal closure and increased transpiration rates throughout the 24 h period of chilling treatment (Fig. 6A-C). This mutant also had a reduced survival rate after 3 h of exposure to -8°C freezing stress compared to the wild type (Fig. 6E). In addition, the \u003cem\u003eAtck1.3\u003c/em\u003e mutant had a higher RPS6 phosphorylation and accumulation than the wild type after 24 h of 6℃ treatment (Fig. 6G).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eABI5\u003c/em\u003e gene in Arabidopsis also promotes stomatal closure under both normal and low temperatures. The \u003cem\u003eAtabi5\u0026nbsp;\u003c/em\u003emutant had a larger stomatal aperture and a higher transpiration rate under normal growth conditions (Fig 6A-C). Although it responded to chilling by closing the stomata in a similar dynamic as the wild type, the mutant had a larger stomatal aperture and a higher transpiration rate throughout 36 h chilling treatment (Fig 6A-C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaize \u003cem\u003eCTK1\u003c/em\u003e is a positive regulator of chilling tolerance\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;We further investigated the function of the closest homolog of \u003cem\u003eOsCTK1\u003c/em\u003e in maize, \u003cem\u003eZmCTK1\u003c/em\u003e (GRMZM2G156035). A LOF mutant of \u003cem\u003eZmCTK1\u003c/em\u003e gene was generated by CRISPR/Cas9, and this \u003cem\u003eZmctk1-1\u003c/em\u003e mutant was assayed for survival rates and stomatal closure response after chilling treatment. The wild-type maize plants exhibited stomatal closure after 1 h of 6ºC chilling treatment (Fig. 6H). While the \u003cem\u003eZmctk1\u003c/em\u003e mutant had a wild-type stomatal aperture under normal growth, it displayed a reduced stomatal closure upon 6ºC treatment compared to the wild type (Fig. 6H). Additionally, the \u003cem\u003eZmctk1\u003c/em\u003e mutant had a reduced survival rate (20%) than the wild type (61%) following a 3-week chilling treatment at 10ºC (Fig. 6I).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar to the rice \u003cem\u003eOsctk1\u0026nbsp;\u003c/em\u003emutant, the maize \u003cem\u003eZmctk1\u003c/em\u003e mutant did not exhibit chilling induced growth marker inhibition. Chilling stress caused a reduction in total RPS6 abundance and a reduction of Ser\u003csup\u003e240\u003c/sup\u003e phosphorylation in wild-type maize plant (Fig. 6J). The \u003cem\u003eZmCTK1\u003c/em\u003e mutant had a higher level of RPS6 and phosphorylated form of RPS6 with or without 1 day of chilling treatment (Fig. 6J). These findings indicate that \u003cem\u003eZmCTK1\u003c/em\u003e regulates chilling tolerance, chilling induced stomatal closure, and chilling induced RPS6 reduction.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study uncovers a new mechanism of chilling tolerance that involves low energy response for inhibiting growth and promote stress response under cold (Fig. 6K). The rice \u003cem\u003eOsSnRK1b\u003c/em\u003e gene has an evolutionarily conserved function in starvation response, and it is also a positive regulator of chilling tolerance, as demonstrated by the altered starvation response and chilling responses in its LOF mutants and overexpression lines (Fig. 3F-H and L-N). In addition, induction of low energy responsive genes, largely carried out by \u003cem\u003eOsSnRK1b\u003c/em\u003e, is part of transcriptome reprogramming in response to cold as revealed by RNA-seq analysis (Fig. 4D-F). Furthermore, \u003cem\u003eOsSnRK1b\u003c/em\u003e is responsible for inhibiting growth and promoting stress response under cold, as indicated by the elevated growth status marker (OsRPS6 protein phosphorylation) and reduced expression of \u003cem\u003eOsABI5\u003c/em\u003e under cold in the \u003cem\u003eOssnrk1b\u0026nbsp;\u003c/em\u003emutant (Fig. 4G-H and 5A-C). Together, these data indicate that low energy response is critical for chilling response and chilling tolerance. While the key low energy response regulator SnRK1α activates catabolism and represses anabolism to maintain cellular energy homeostasis under starvation stress, it inhibits growth and activates stress response under chilling stress. This mechanism is likely evolutionarily conserved among plants shown by a similar role for the \u003cem\u003eSnRK1α\u003c/em\u003e genes in Arabidopsis and maize.\u003c/p\u003e\n\u003cp\u003eThis study also identifies \u003cem\u003eOsCTK1\u003c/em\u003e as a positive regulator of \u003cem\u003eOsSnRK1b\u003c/em\u003e. The OsCTK1 protein phosphorylates and stabilizes the OsSnRK1b protein under both non-chilling and chilling conditions, as evidenced by the reduced protein abundance and nuclear accumulation of OsSnRK1b in the \u003cem\u003eOsctk1\u0026nbsp;\u003c/em\u003emutant (Fig. 2C-F). This regulation is also supported by a similar LOF and overexpression effects of these two genes in chilling tolerance and chilling induced stomatal movement as well as the loss of \u003cem\u003eOsSnRK1b\u003c/em\u003e overexpression effect in the absence of the \u003cem\u003eOsCTK1\u003c/em\u003e. The direct involvement of starvation response for \u003cem\u003eOsCTK1\u003c/em\u003e is yet to be tested, but this is suggested by the elevated RPS6 phosphorylation in the \u003cem\u003eOsctk1\u0026nbsp;\u003c/em\u003emutant (Fig. 5H). Previous study identified a direct phosphorylation of SnRK1 by SnAK1/2 (SnRK1-activating kinases) at Thr-175 or Thr-176 residues that promotes the SnRK1α kinase activity in Arabidopsis\u003csup\u003e43,44\u003c/sup\u003e. These sites are distinct from the potential phosphorylation sites of OsSnRK1 by OsCTK1, suggesting different effects of these two regulators on OsSnRK1\u003cem\u003eα\u003c/em\u003e. Besides the effect on the protein stability of OsSnRK1b, the effects of phosphorylation by OsCTK1 on the kinase activity of OsSnRK1b has not been determined (Fig. 2D), although its effect on nuclear localization of OsSnRK1b suggests a regulation of its activity as well (Fig. 2E-F).\u003c/p\u003e\n\u003cp\u003eSnRK1 was previously shown to regulate response to a number of abiotic stresses that induce low-energy status\u003csup\u003e23\u003c/sup\u003e. Drought stress induces stomatal closure and therefore limits CO₂ intake and ATP synthesis\u003csup\u003e45,46\u003c/sup\u003e. Salt stress, including Na⁺ toxicity, disrupts thylakoid membrane integrity and inhibits photosynthesis system and ATP production\u003csup\u003e47\u003c/sup\u003e. Hypoxia induces transition from aerobic to anaerobic respiration, causing a decline in ATP yield\u003csup\u003e48,49\u003c/sup\u003e. Heat stress inactivates Rubisco activate, damages chloroplast structure, and impedes photosynthesis\u003csup\u003e50\u003c/sup\u003e. Low temperature may also induce low energy status, like starvation, drought, salt, hypoxia, and heat. Chilling stress is known to disrupt thylakoid membrane structure, inhibit chlorophyll synthesis, and inhibit electron transport chain. It also causes phase transition of membrane lipids from the liquid crystalline state to the gel state, leading to ion leakage. The involvement of SnRK1 in chilling tolerance suggests that low energy sensing pathway could have a broad function in stress tolerance. This conserved pathway may direct the physiological reprogramming for environmental adaptation. Under chilling stress, OsSnRK1b mediates the induction of stress-responsive genes (such as \u003cem\u003eOsABI5\u003c/em\u003e) and suppression of growth-related markers (OsRPS6) (Fig. 6K), suggesting a coordination of stress and growth processes according to the environment. This coordination is likely not through the regulation of \u003cem\u003eDREB\u003c/em\u003e/\u003cem\u003eCBF\u003c/em\u003e expression, as the expression of \u003cem\u003eOsDREB\u003c/em\u003e genes under chilling did not have reduced expression in the \u003cem\u003eOsctk1\u003c/em\u003e and \u003cem\u003eOssnrk1b\u003c/em\u003e mutants compared to the wild type (Fig. 5I-J). Interestingly, the \u003cem\u003eOsctk1\u003c/em\u003e and \u003cem\u003eOssnrk1b\u003c/em\u003e mutants accumulate higher amount of growth marker protein RPS6 under non-chilling condition, but these mutants did not exhibit increased growth compared to the wild type (Fig. 4G-H). This suggests a broad role of \u003cem\u003eOsSnRK1b\u003c/em\u003e and \u003cem\u003eOsCTK1\u003c/em\u003e genes in growth regulation other than the RPS6 associated pathway.\u003c/p\u003e\n\u003cp\u003eThis study also highlights that stomatal aperture regulation as a critical physiological response in chilling tolerance. \u003cem\u003eOsCTK1\u003c/em\u003e and \u003cem\u003eOsSnRK\u003c/em\u003e\u003cem\u003e1b\u003c/em\u003e both promote stomatal closure in response to chilling as well as survival under cold (Figs. 1A-F and 3A-N). \u003cem\u003eOsABI5\u003c/em\u003e, a regulator of stomatal aperture regardless of chilling conditions, is also a positive regulator of chilling tolerance (Fig. 5D-H). Together with \u003cem\u003eOsCNGCs\u003c/em\u003e in rice and \u003cem\u003eOST1\u003c/em\u003e in Arabidopsis, dual-function in stomatal closure and chilling tolerance for these genes strongly implicate stomatal movement control is an important part of chilling tolerance mechanism\u003csup\u003e18-21\u003c/sup\u003e. How \u003cem\u003eSnRK1\u003c/em\u003e genes in rice and Arabidopsis regulate stomatal movement under chilling still awaits further investigation. \u003cem\u003eOsABI5\u003c/em\u003e is a potential regulatory target, not necessarily a direct kinase target, as chilling induction of its transcript is reduced in the \u003cem\u003eOsSnRK1b\u0026nbsp;\u003c/em\u003emutants. In addition, energy status markers such as sugar accumulation could affect stomatal movement, as starch degradation and glucose accumulation in guard cells could lead to stomatal opening\u003csup\u003e51,52\u003c/sup\u003e. Indeed, \u003cem\u003eKIN10\u003c/em\u003e (\u003cem\u003eAtSnRK1\u003c/em\u003e) overexpression was shown to promote light-induced starch degradation and thus stomatal opening\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study shows that some \u003cem\u003eSnRK1α\u003c/em\u003e members, \u003cem\u003eOsSnRK1b\u003c/em\u003e in rice and \u003cem\u003eKIN10\u003c/em\u003e in Arabidopsis, regulate low-temperature response and low energy response. It is not yet known whether all members of the \u003cem\u003eSnRK1α\u003c/em\u003e family have dual functions. \u003cem\u003eKIN10\u003c/em\u003e and \u003cem\u003eKIN11\u0026nbsp;\u003c/em\u003ein Arabidopsis were shown to be low energy sensors (in starvation response) while the role of \u003cem\u003eKIN12\u003c/em\u003e in starvation response has not been tested\u003csup\u003e23\u003c/sup\u003e. \u003cem\u003eKIN10\u003c/em\u003e is a positive regulator of chilling tolerance, but the function of the other two Arabidopsis genes in chilling tolerance are yet to be determined. Among the four members in rice,\u0026nbsp;\u003cem\u003eOsSnRK1a\u003c/em\u003e and \u003cem\u003eOsSnRK1b\u003c/em\u003e each have dual function in starvation response and chilling tolerance. The function of \u003cem\u003eOsSnRK1c\u003c/em\u003e and \u003cem\u003e1d\u003c/em\u003e in chilling tolerance are yet to be determined by their LOF mutants. Whether or not a member has a more pronounced role in a specific stress response than other members is yet to be investigated in plants. Deciphering the shared or distinct functions of SnRK1 members in multiple stresses will provide insights into the evolution of these conserved proteins.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study demonstrates the involvement of low energy response in chilling tolerance and supports stomatal closure control as a critical mechanism for chilling tolerance. The knowledge of chilling tolerance at the whole plant physiology level will facilitate the development of strategies for enhancing plant resilience in a changing environment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genetic backgrounds of rice mutant and transgenic plants in this study were \u0026lsquo;Nip\u0026rsquo; or \u0026lsquo;ZH11\u0026rsquo; as specified. Freshly harvested seeds were treated at 42℃ for a minimum of 14 days to break dormancy. Seeds were then soaked in water at room temperature for 3 to 5 days for germination. For hydroponic growth, germinated seeds with a similar development stage were transferred to a 96-well plate (with its bottom cut) that sits in water, as described previously\u003csup\u003e53\u003c/sup\u003e. Seedlings were grown in controlled chambers with 16 h of light and 8 h of dark at 28℃.\u0026nbsp;For soil growth, germinated seeds were transferred to a mix of vermiculite: organic substrate (1:3) and cultured at 28℃ in chambers.\u003c/p\u003e\n\u003cp\u003eThe Arabidopsis T-DNA insertion mutants were obtained from Arabidopsis Biological Resource Center and Fuzhou Aerosa Biotechnology.\u0026nbsp;Seeds were sown onto soil, stratified at 4\u0026deg;C for 3 days, and grown in chambers under constant light or 16 h light/8 h dark and 70% relative humidity.\u003c/p\u003e\n\u003cp\u003eMaize cultivar B104 was used for this study. Seeds were placed on wet paper towels in the dark overnight at room temperature to break dormancy before being sown in soil for germination. Plants were grown under 22\u0026deg;C with continuous light for chilling assays or under 26\u0026deg;C with continuous light for stomatal measurements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction and plant transformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific targets for guide RNA spacer sequences were designed for CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 constructs by using the CRISPR-PLANT website (http://www.genome.arizona.edu/crispr/CRISPR). Two guide RNA targets were cloned into the PHUE411 vector\u003csup\u003e55\u003c/sup\u003e, and the resulting construct was transformed into Nip through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation\u003csup\u003e56\u003c/sup\u003e. For overexpression constructs, the coding sequence (CDS) was amplified and cloned into the pCAMBIA1300-GFP vector (Addgene). For GST (Glutathione s transferase) fusion constructs, the CDS was cloned into pGEX-2T vectors (Addgene) using a homologous recombination system (Vazyme, China). For His-fusion\u0026nbsp;constructs,\u0026nbsp;the\u0026nbsp;CDS was cloned into the pET-30a vector. For Myc-fusion\u0026nbsp;constructs,\u0026nbsp;the CDS was cloned into pCAMBIA1300-221 vectors (Addgene).\u0026nbsp;Vector details are listed in Supplementary Table 3. Transgenic plants were selected by hygromycin.Primes details are listed in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eZmctk1\u003c/em\u003e mutants were generated in maize using methods as described previously\u003csup\u003e54,55\u003c/sup\u003e. Agrobacterium-mediated genetic transformation was performed by the Crop Bioengineering Laboratory at the Iowa State University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChilling tolerance assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChilling tolerance and ion\u0026nbsp;leakage assays for rice plants were carried out using the method described previously\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor maize plants, chilling growth assay was done on seedlings grown at 26\u0026deg;C till 3-leaf stage. Plants were subsequently grown at 10\u0026deg;C for three weeks before they were transferred to 26\u0026deg;C for recovery for two weeks.\u003c/p\u003e\n\u003cp\u003eFor freezing tolerance assay in Arabidopsis, plants were germinated and grown at 22\u0026deg;C for two weeks. They were then subjected to -8\u0026deg;C for 5 h before being returned to 22\u0026deg;C for one week\u0026rsquo;s recovery. Plants were covered with humidity dome the first at the recovery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStomata related assays\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpidermal impressions were made using clear nail polish\u003csup\u003e63\u003c/sup\u003e. Dried nail polish was peeled from leaf epidermis and epidermal impression was observed under an Olympus BX53 microscope. Stomatal width and length were measured on the images using ImageJ software. Sixty stomata from four leaves were measured for each sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranspiration rate was measured using a steady-state porometer (METER SC-1). The second fully expanded leaf of the three-leaf stage plant was placed and stabilized within the chamber of the porometer for 30 s. Measurements were taken on the middle section of leaf. Data were collected from six leaves per genotype and time point.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeaf water loss was measured by fresh weight from ten uniformly grown seedlings per replicate, with three replicates in total to minimize individual variation. Water loss rate was expressed as the percentage of weight difference divided by the initial fresh weight.\u003c/p\u003e\n\u003cp\u003eFor leaf surface temperature, thermal images were captured using a thermal imager (FOTRIC 225S). Quantification of temperature was performed using AnalyzIR (FOTRIC TOOLS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne microgram of total RNA was reverse transcribed into cDNA using HiScript II RT SuperMix (Vazyme). \u003cem\u003eOsActin\u003c/em\u003e (\u003cem\u003eLOC_Os03g50885\u003c/em\u003e) was used as the reference gene. Expression levels were determined using the comparative cycle threshold method (Vazyme). Primers used for RT-qPCR for each gene are detailed in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Seq and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from the leaves of 14-day-old rice plants using Trizol reagent (TsingZol). Library construction and deep sequencing were performed by the Nanjing Jiangbei New Area Biopharmaceutical Public Service Platform Company Limited using the Illumina NovaSeq 6000 platform. DEGs were identified by DESeq2\u003csup\u003e56\u003c/sup\u003e, with adjusted \u003cem\u003eP\u003c/em\u003e-values set at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 and a Fold Change\u0026gt;2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction and immunoblotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant tissues were ground into powder using liquid nitrogen, and extracted by protein extract buffer (50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM EGTA, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM NaF, 10% glycerol, Complete\u003csup\u003eTM\u003c/sup\u003e EDTA-free protease inhibitor and PhosSTOP\u003csup\u003eTM\u003c/sup\u003e phosphatase inhibitor cocktails) at 500 \u0026mu;L of per 0.3 g of fresh tissue.\u0026nbsp;Supernatants were collected\u0026nbsp;following centrifugation at 13000 g for 15 min at 4\u0026deg;C as the protein extracts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProteins were separated by SDS-PAGE on 12% gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare) in a wet tank unit (Bio-Rad).\u0026nbsp;Membranes were incubated with primary anti-RPS6 antibody (rice: Solarbio, China, maize and Arabidopsis: Agrisera, Sweden) diluted to 1:1000, and polyclonal primary anti-RPS6A-P240 phosphorylated (Ser240) antibody diluted to 1:1000 (rice: Solarbio, maize and Arabidopsis: Agrisera). Antibodies were diluted in TBS-T (Tris-buffered saline with tween-20) containing 1% w/v BSA and incubated overnight at 4\u0026deg;C. Following six washing steps in TBS-T, membranes were incubated with a horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology) diluted 1:5000 in TBS-T containing 1% w/v BSA for 2 h. After three washing steps in TBS-T, proteins were detected by incubating the membranes in Clarity Western ECL substrate (Bio-Rad). The band intensities were quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;phosphoproteomic analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e phosphoproteomics was carried out using the method described previously\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e7\u003c/sup\u003e. Briefly,\u0026nbsp;Total proteins extracted from chilling treated plants were dephosphorylated and then incubated with recombinant OsCTK1 in a kinase reaction containing 1 mM \u0026gamma;-[\u003csup\u003e18\u003c/sup\u003eO\u003csub\u003e4\u003c/sub\u003e] ATP. Peptides labelled with \u003csup\u003e18\u003c/sup\u003eO were identified by Mass Spec.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiquid chromatography tandem mass spectrometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GFP fusion protein was purified using the GFP-trap IP (KT Life). The bound proteins were eluted from the beads by heating at 95℃\u0026nbsp;for 10 min. Proteins were fractionated on 12% SDS-PAGE (Bio-Rad) and stained with Coomassie brilliant blue. Bands were excised and cut into small pieces and subjected to MS analyses (BGI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCo-IP assay was carried out as described previously\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and transformation of rice protoplasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtoplast isolation was done using method previously described\u003csup\u003e58\u003c/sup\u003e.\u0026nbsp;Specifically, 5 grams rice leaf strips of 0.5 mm size were digested in 10 mL enzyme solution (1.5% cellulose R10, 0.75% macerozyme R10, 0.6 M mannitol, 10 mM MES) for 6 h in dark with gentle shaking (40 rpm) at 28℃. PEG-mediated transfections were carried out as described\u003csup\u003e59\u003c/sup\u003e. Specifically, 5-10 \u0026mu;g of plasmid DNA were mixed with 100 \u0026mu;L protoplasts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhos-tag mobility shift assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhos-tag reagent (AAL-107, Wako) was used for the phosphoprotein mobility-shift assay as described\u003csup\u003e60\u003c/sup\u003e.\u0026nbsp;Protein extracts were separated on 12% (w/v) SDS-PAGE gel containing 50 \u0026mu;M Phos-tag and 100 \u0026mu;M MnCl\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell-free protein degradation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell-free protein degradation assay was performed as described\u003csup\u003e61\u003c/sup\u003e with minor modifications. Total proteins were extracted from 14-days seedlings. Total proteins (0.3 g leaf to 500 \u0026mu;L extraction buffer) were incubated with purified recombinant proteins (5 mg) and 10 mM ATP. Proteins were detected with anti-His monoclonal antibody (Sangon Biotech). The Rubisco large (RbcL) subunit, detected by Ponceau S staining, was used as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell fractionation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoluble and nuclear proteins were isolated using Plant Nuclei Isolation/Extraction Kit (Sigma). PEPC protein was used as a cytosolic marker, and histone H3 was used as a nuclear marker.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Chengqiang Ding for the \u003cem\u003eOssnrk1a\u003c/em\u003e mutant seeds as well as Dr. Jing Li and Bioinformatics Center at Nanjing Agricultural University for RNA-seq analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.H. and B.Z. supervised the study. J.W., H.M., P.V., Y.Z., J.Y., G.X., R.Y., S.L. and S.Lu. performed the experiments, among them, J.W., B.Z. and H.M. drafted the manuscript. J.H. revised the manuscript. All authors contributed to the discussion and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by United States Department of Agriculture NIFA\u0026nbsp;(2022-67013-37040),\u0026nbsp;the Natural Science Foundation of China (32441064, 31971827, 32370309), Jiangsu Agricultural Science and Technology Innovation Fund (CX (23)1033) and China Postdoctoral Science Foundation (2024M761438, GZC20252664).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDing, Y. L., Shi, Y. T. \u0026amp; Yang, S. H. 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Biol\u003c/em\u003e \u003cstrong\u003e1578\u003c/strong\u003e, 243-248, doi: 10.1007/978-1-4939-6859-6_20 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7447278/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7447278/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Plants employ diverse physiological strategies for cold tolerance. Here we report an important role of low energy response and stomatal closure control in chilling tolerance through the study of SNF1-Related Kinase 1 (OsSnRK1) and its regulator Chilling Tolerance Kinase 1 (OsCTK1) in rice. OsSnRK1b, an isoform of OsSnRK1 alpha subunit, is found to have an evolutionarily conserved function in low energy response. It is positively regulated by OsCTK1 at the protein stability level through phosphorylation. Both OsCTK1 and OsSnRK1b promote chilling-induced stomatal closure and chilling tolerance, shown by slower stomatal closure and lower survival rate after chilling exposure in their respective loss of function mutants. Transcriptome analysis revealed that chilling induces low energy response which is largely dependent on OsSnRK1b. Overexpression of OsSnRK1b enhanced chilling induced stomatal closure response and promoted chilling tolerance in an OsCTK1-dependent manner. Furthermore, both OsCTK1 and OsSnRK1b promote expression of stress response gene Abscisic Acid Insensitive 5 (OsABI5) and inhibit accumulation of growth-associated ribosome protein OsRPS6 under chilling conditions. The function of CTK1 and SnRK1 in chilling tolerance, stomatal control, and growth inhibition are also observed in Arabidopsis and maize, indicating a conserved role of energy signaling in chilling tolerance and a contribution of stomatal aperture control to chilling tolerance in plants.","manuscriptTitle":"Energy sensing OsSnRK1b and its regulator OsCTK1 promote chilling-induced stomatal closure and chilling tolerance in rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 05:45:07","doi":"10.21203/rs.3.rs-7447278/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8851ef39-edcc-4254-be40-01ca9443dbbe","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55676555,"name":"Biological sciences/Plant sciences/Plant stress responses/Abiotic"},{"id":55676556,"name":"Biological sciences/Plant sciences/Plant physiology"}],"tags":[],"updatedAt":"2025-11-12T05:45:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 05:45:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7447278","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7447278","identity":"rs-7447278","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}