Adapting to the projected epidemics of Fusarium head blight of wheat in Korea under climate change scenarios.

Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum Schwabe, is an emerging threat to wheat production in Korea under a changing climate. The disease occurrence and accumulation of associated trichothecene mycotoxins in wheat kernels strongly coincide with warm and wet environments during flowering. Recently, the International Panel for Climate Change released the 6th Coupled Model Intercomparison Project (CMIP6) climate change scenarios with shared socioeconomic pathways (SSPs). In this study, we adopted GIBSIM, an existing mechanistic model developed in Brazil to estimate the risk infection index of wheat FHB, to simulate the potential FHB epidemics in Korea using the SSP245 and SSP585 scenarios of CMIP6. The GIBSIM model simulates FHB infection risk from airborne inoculum density and infection frequency using temperature, precipitation, and relative humidity during the flowering period. First, wheat heading dates, during which GIBSIM runs, were predicted over suitable areas of winter wheat cultivation using a crop development rate model for wheat phenology and downscaled SSP scenarios. Second, an integrated model combining all results of wheat suitability, heading dates, and FHB infection risks from the SSP scenarios showed a gradual increase in FHB epidemics towards 2100, with different temporal and spatial patterns of varying magnitudes depending on the scenarios. These results indicate that proactive management strategies need to be seriously considered in the near future to minimize the potential impacts of the FHB epidemic under climate change in Korea. Therefore, available wheat cultivars with early or late heading dates were used in the model simulations as a realistic adaptation measure. As a result, wheat cultivars with early heading dates showed significant decreases in FHB epidemics in future periods, emphasizing the importance of effective adaptation measures against the projected increase in FHB epidemics in Korea under climate change.

Regulation of Dual Activity of Ascorbate Peroxidase 1 From Arabidopsis thaliana by Conformational Changes and Posttranslational Modifications.

Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS)-scavenging enzyme, which catalyzes the removal of hydrogen peroxide (H2O2) to prevent oxidative damage. The peroxidase activity of APX is regulated by posttranslational modifications (PTMs), such as S-nitrosylation, tyrosine nitration, and S-sulfhydration. In addition, it has been recently reported that APX functions as a molecular chaperone, protecting rice against heat stress. In this study, we attempted to identify the various functions of APX in Arabidopsis and the effects of PTMs on these functions. Cytosol type APX1 from Arabidopsis thaliana (AtAPX1) exists in multimeric forms ranging from dimeric to high-molecular-weight (HMW) complexes. Similar to the rice APX2, AtAPX1 plays a dual role behaving both as a regular peroxidase and a chaperone molecule. The dual activity of AtAPX1 was strongly related to its structural status. The main dimeric form of the AtAPX1 protein showed the highest peroxidase activity, whereas the HMW form exhibited the highest chaperone activity. Moreover, in vivo studies indicated that the structure of AtAPX1 was regulated by heat and salt stresses, with both involved in the association and dissociation of complexes, respectively. Additionally, we investigated the effects of S-nitrosylation, S-sulfhydration, and tyrosine nitration on the protein structure and functions using gel analysis and enzymatic activity assays. S-nitrosylation and S-sulfhydration positively regulated the peroxidase activity, whereas tyrosine nitration had a negative impact. However, no effects were observed on the chaperone function and the oligomeric status of AtAPX1. Our results will facilitate the understanding of the role and regulation of APX under abiotic stress and posttranslational modifications.

Machine learning, transcriptome, and genotyping chip analyses provide insights into SNP markers identifying flower color in Platycodon grandiflorus.

Bellflower is an edible ornamental gardening plant in Asia. For predicting the flower color in bellflower plants, a transcriptome-wide approach based on machine learning, transcriptome, and genotyping chip analyses was used to identify SNP markers. Six machine learning methods were deployed to explore the classification potential of the selected SNPs as features in two datasets, namely training (60 RNA-Seq samples) and validation (480 Fluidigm chip samples). SNP selection was performed in sequential order. Firstly, 96 SNPs were selected from the transcriptome-wide SNPs using the principal compound analysis (PCA). Then, 9 among 96 SNPs were later identified using the Random forest based feature selection method from the Fluidigm chip dataset. Among six machines, the random forest (RF) model produced higher classification performance than the other models. The 9 SNP marker candidates selected for classifying the flower color classification were verified using the genomic DNA PCR with Sanger sequencing. Our results suggest that this methodology could be used for future selection of breeding traits even though the plant accessions are highly heterogeneous.

Functional properties and the oligomeric state of alkyl hydroperoxide reductase subunit F (AhpF) in Pseudomonas aeruginosa.

Alkyl hydroperoxide reductase subunit F (AhpF) is a well-known flavoprotein that transfers electrons from pyridine nucleotides to the peroxidase protein AhpC via redox-active disulfide centers to detoxify hydrogen peroxide. However, study of AhpF has historically been limited to particular eubacteria, and the connection between the functional and structural properties of AhpF remains unknown. The present study demonstrates the dual function of Pseudomonas aeruginosa AhpF (PaAhpF) as a reductase and a molecular chaperone. It was observed that the functions of PaAhpF are closely linked with its structural status. The reductase and foldase chaperone function of PaAhpF predominated for its low-molecular-weight (LMW) form, whereas the holdase chaperone function of PaAhpF was found associated with its high-molecular-weight (HMW) complex. Further, the present study also demonstrates the multiple function of PaAhpF in controlling oxidative and heat stresses in P. aeruginosa resistance to oxidative and heat stress.

LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis.

BACKGROUND: Adventitious root (AR) formation is a complex genetic trait, which is controlled by various endogenous and environmental cues. Auxin is known to play a central role in AR formation; however, the mechanisms underlying this role are not well understood. RESULTS: In this study, we showed that a previously identified auxin signaling module, AUXIN RESPONSE FACTOR(ARF)7/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN(LBD)16/LBD18 via AUXIN1(AUX1)/LIKE-AUXIN3 (LAX3) auxin influx carriers, which plays important roles in lateral root formation, is involved in AR formation in Arabidopsis. In aux1, lax3, arf7, arf19, lbd16 and lbd18 single mutants, we observed reduced numbers of ARs than in the wild type. Double and triple mutants exhibited an additional decrease in AR numbers compared with the corresponding single or double mutants, respectively, and the aux1 lax3 lbd16 lbd18 quadruple mutant was devoid of ARs. Expression of LBD16 or LBD18 under their own promoters in lbd16 or lbd18 mutants rescued the reduced number of ARs to wild-type levels. LBD16 or LBD18 fused to a dominant SRDX repressor suppressed promoter activity of the cell cycle gene, Cyclin-Dependent Kinase(CDK)A1;1, to some extent. Expression of LBD16 or LBD18 was significantly reduced in arf7 and arf19 mutants during AR formation in a light-dependent manner, but not in arf6 and arf8. GUS expression analysis of promoter-GUS reporter transgenic lines revealed overlapping expression patterns for LBD16, LBD18, ARF7, ARF19 and LAX3 in AR primordia. CONCLUSION: These results suggest that the ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in Arabidopsis.

LBD13 positively regulates lateral root formation in Arabidopsis.

MAIN CONCLUSION: Lateral Organ Boundaries Domain 13 (LBD13), which is expressed in emerged lateral roots and encodes a transcriptional activator, plays an important role in lateral root formation in Arabidopsis. Lateral roots (LRs) are major determinants of root system architecture, contributing to the survival strategies of plants. Members of the LBD gene family encode plant-specific transcription factors that play key roles in plant organ development. Several LBD genes, such as LBD14, 16, 18, 29, and 33, have been shown to play important roles in regulating LR development in Arabidopsis. In the present study, we show that LBD13 is expressed in emerged LRs and LR meristems of elongated LRs and regulates LR formation in Arabidopsis. Transient gene expression assays with Arabidopsis protoplasts showed that LBD13 is localized to the nucleus and harbors transcription-activating potential. Knock-down of LBD13 expression by RNA interference resulted in reduced LR formation, whereas overexpression of LBD13 enhanced LR formation in transgenic Arabidopsis. Analysis of ß-glucuronidase (GUS) expression under the control of the LBD13 promoter showed that GUS staining was detected in LRs emerged from the primary root, but not in LR primordia. Moreover, both the distribution of LR primordium number and developmental kinetics of LR primordia were not affected either by knock-down or by overexpression of LBD13. Taken together, these results suggest that LBD13 is a nuclear-localized transcriptional activator and controls LR formation during or after LR emergence.

Novel functions of peroxiredoxin Q from Deinococcus radiodurans R1 as a peroxidase and a molecular chaperone.

Deinococcus radiodurans R1 is extremely resistant to ionizing radiation and oxidative stress. In this study, we characterized DR0846, a candidate peroxiredoxin in D. radiodurans. DR0846 is a peroxiredoxin Q containing two conserved cysteine residues. DR0846 exists mainly in monomeric form with an intramolecular disulfide bond between the two cysteine residues. We found that DR0846 functions as a molecular chaperone as well as a peroxidase. A mutational analysis indicates that the two cysteine residues are essential for enzymatic activity. A double-deletion mutant lacking DR0846 and catalase DR1998 exhibits decreased oxidative and heat shock stress tolerance with respect to the single mutants or the wild-type cells. These results suggest that DR0846 contributes to resistance against oxidative and heat stresses in D. radiodurans.

Functional switching of ascorbate peroxidase 2 of rice (OsAPX2) between peroxidase and molecular chaperone.

Ascorbate peroxidase (APX) is a class I haem-containing peroxidase, which catalyses the conversion of H2O2 to H2O and O2 using ascorbate as the specific electron donor. APX plays a central role in the elimination of intracellular reactive oxygen species (ROS) and protects plants from the oxidative damage that can occur as a result of biotic and abiotic stresses. At present, the only known function of APX is as a peroxidase. However, in this study, we demonstrate that Oryza sativa APX2 also operates as a molecular chaperone in rice. The different functions of OsAPX2 correlate strongly with its structural conformation. The high-molecular-weight (HMW) complexes had chaperone activity, whereas the low-molecular-weight (LMW) forms displayed predominantly APX activity. The APX activity was effectively inhibited by sodium azide, which is an inhibitor of haem-containing enzymes, but this did not affect the protein's activity as a chaperone. Additionally, the OsAPX2 conformational changes could be regulated by salt and heat stresses and these stimulated OsAPX2 dissociation and association, respectively. Our results provide new insight into the roles of APXs.

LBD18 uses a dual mode of a positive feedback loop to regulate ARF expression and transcriptional activity in Arabidopsis.

A hierarchy of transcriptional regulators controlling lateral root formation in Arabidopsis thaliana has been identified, including the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16)/LBD18 transcriptional network; however, their feedback regulation mechanisms are not known. Here we show that LBD18 controls ARF activity using the dual mode of a positive feedback loop. We showed that ARF7 and ARF19 directly bind AuxRE in the LBD18 promoter. A variety of molecular and biochemical experiments demonstrated that LBD18 binds a specific DNA motif in the ARF19 promoter to regulate its expression in vivo as well as in vitro. LBD18 interacts with ARFs including ARF7 and ARF19 via the Phox and Bem1 domain of ARF to enhance the transcriptional activity of ARF7 on AuxRE, and competes with auxin/indole-3-acetic acid (IAA) repressors for ARF binding, overriding the negative feedback loop exerted by Aux/IAA repressors. Taken together, these results show that LBD18 and ARFs form a double positive feedback loop, and that LBD18 uses the dual mode of a positive feedback loop by binding directly to the ARF19 promoter and through the protein-protein interactions with ARF7 and ARF19. This novel mechanism of feedback loops may constitute a robust feedback mechanism that ensures continued lateral root growth in response to auxin in Arabidopsis.

LBD14/ASL17 Positively Regulates Lateral Root Formation and is Involved in ABA Response for Root Architecture in Arabidopsis.

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family members play key roles in diverse aspects of plant development. Previous studies have shown that LBD16, 18, 29 and 33 are critical for integrating the plant hormone auxin to control lateral root development in Arabidopsis thaliana. In the present study, we show that LBD14 is expressed exclusively in the root where it promotes lateral root (LR) emergence. Repression of LBD14 expression by ABA correlates with the inhibitory effects of ABA on LR emergence. Transient gene expression assays with Arabidopsis protoplasts demonstrated that LBD14 is a nuclear-localized transcriptional activator. The knock-down of LBD14 expression by RNA interference (RNAi) resulted in reduced LR formation by delaying both LR primordium development and LR emergence, whereas overexpression of LBD14 in Arabidopsis enhances LR formation. We show that ABA (but not other plant hormones such as auxin, brassinosteroids and cytokinin) specifically down-regulated ß-glucuronidase (GUS) expression under the control of the LBD14 promoter in transgenic Arabidopsis during LR development from initiation to emergence and endogenous LBD14 transcript levels in the root. Moreover, RNAi of LBD14 enhanced the LR suppression in response to ABA, whereas LBD14 overexpression did not alter the ABA-mediated suppression of LR formation. Taken together, these results suggest that LBD14 promoting LR formation is one of the critical factors regulated by ABA to inhibit LR growth, contributing to the regulation of the Arabidopsis root system architecture in response to ABA.

Dimerization in LBD16 and LBD18 Transcription Factors Is Critical for Lateral Root Formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (hereafter referred to as LBD) are plant-specific transcription factors that play important roles in a plethora of plant growth and development. The leucine (Leu) zipper-like coiled-coil motif in the lateral organ boundaries domain of the class I LBD proteins has been proposed to mediate protein dimerization, but it has not been experimentally assessed yet. LBD16 and LBD18 have been well characterized to play important roles in lateral root development in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of the coiled-coil motif in the dimerization of LBD16 and LBD18 and in transcriptional regulation and biological function. We built the molecular models of the coiled coil of LBD16 and LBD18, providing the probable Leu zipper models of the helix dimer. Using a variety of molecular techniques, such as bimolecular fluorescence complementation, luciferase complementation imaging, GST pull down, and coimmunoprecipitation assays, we showed that the conserved Leu or valine residues in the coiled-coil motif are critical for the dimerization of LBD16 or LBD18. Using transgenic Arabidopsis plants that overexpress HA:LBD16 or HA:LBD16Q in lbd16 or HA:LBD18 or HA:LBD18Q in lbd18, we demonstrated that the homodimerization of LBD18 mediated by the coiled-coil motif is crucial for transcriptional regulation via promoter binding and for lateral root formation. In addition, we found that the carboxyl-terminal region beyond the coiled-coil motif in LBD18 acts as an additional dimerization domain. These results provide a molecular basis for homodimerization and heterodimerization among the 42 Arabidopsis LBD family members for displaying their biological functions.

CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3 Regulate Lateral Root Development in Response to Cold Stress in Arabidopsis.

Lateral roots (LRs) are a major determinant of the root system architecture in plants, and developmental plasticity of LR formation is critical for the survival of plants in changing environmental conditions. In Arabidopsis thaliana, genetic pathways have been identified that regulate LR branching in response to numerous environmental cues, including some nutrients, salt, and gravity. However, it is not known how genetic components are involved in the LR adaptation response to cold. Here, we demonstrate that CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3, encoding APETALA2 transcription factors, play an important role in regulating Arabidopsis LR initiation under cold stress. Analysis of LR developmental kinetics demonstrated that both CRF2 and CRF3 regulate LR initiation. crf2 and crf3 single mutants exhibited decreased LR initiation under cold stress compared with the wild type, and the crf2 crf3 double mutants showed additively decreased LR densities compared with the single mutants. Conversely, CRF2 or CRF3 overexpression caused increased LR densities. CRF2 was induced by cold via a subset of the cytokinin two-component signaling (TCS) pathway, whereas CRF3 was upregulated by cold via TCS-independent pathways. Our results suggest that CRF2 and CRF3 respond to cold via TCS-dependent and TCS-independent pathways and control LR initiation and development, contributing to LR adaptation to cold stress.

Inhibitory effect of gamma irradiation and its application for control of postharvest green mold decay of Satsuma mandarins.

Gamma irradiation has been shown to be effective for the control of postharvest fungi in vitro, but little is known regarding antifungal action, responses to gamma irradiation, and its application to fresh produce. Gamma irradiation was evaluated for its in vitro and in vivo antifungal activity against Penicillium digitatum on Satsuma mandarin fruits. Green mold was inhibited in a dose-dependent manner. Gamma irradiation showed a complete inhibition of spore germination, germ tube elongation, and mycelial growth of P. digitatum, particularly at 1.0kGy. To further investigate the mechanisms by which gamma irradiation inhibits fungal growth, the membrane integrity and cellular leakage of conidia were tested, indicating that gamma irradiation results in the loss of plasma membrane integrity, causing the release of intracellular contents such as soluble proteins. In vivo assays demonstrated that established doses can completely inhibit the growth of fungal pathogens, but such high doses cause severe fruit damage. Thus, to eliminate the negative impact on fruit quality, gamma irradiation at lower doses was evaluated for inhibition of P. digitatum, in combination with a chlorine donor, sodium dichloro-s-triazinetrione (NaDCC). Interestingly, only a combined treatment with 0.4kGy of gamma irradiation and 10ppm of NaDCC exhibited significant synergistic antifungal activity against green mold decay. The mechanisms by which the combined treatment decreased the green mold decay of mandarin fruits can be directly associated with the disruption of cell membrane of the fungal pathogen, which resulted in a loss of cytoplasmic material from the hyphae. These findings suggest that a synergistic effect of combining treatment with gamma irradiation with NaDCC has potential as an antifungal approach to reduce the severity of green mold in mandarin fruits.

Lateral Organ Boundaries Domain16 and 18 Act Downstream of the AUXIN1 and LIKE-AUXIN3 Auxin Influx Carriers to Control Lateral Root Development in Arabidopsis.

Several members of the Lateral Organ Boundaries Domain (LBD)/Asymmetric Leaves2-Like (ASL) gene family have been identified to play important roles in Arabidopsis (Arabidopsis thaliana) lateral root (LR) development during auxin response, but their functional relationship with auxin transporters has not been established yet. Here, we show that the AUXIN1 (AUX1) and LIKE-AUXIN3 (LAX3) auxin influx carriers are required for auxin signaling that activates LBD16/ASL18 and LBD18/ASL20 to control LR development. The lax3 mutant phenotype was not significantly enhanced when combined with lbd16 or lbd18. However, LBD18 overexpression could rescue the defects in LR emergence in lax3 with concomitant expression of the LBD18 target genes. Genetic and gene expression analyses indicated that LBD16 and LBD18 act with AUX1 to regulate LR initiation and LR primordium development, and that AUX1 and LAX3 are needed for auxin-responsive expression of LBD16 and LBD18. LBD18:SUPERMAN REPRESSIVE DOMAIN X in the lbd18 mutant inhibited LR initiation and LR primordium development in response to a gravitropic stimulus and suppressed promoter activities of the cell cycle genes Cyclin-Dependent Kinase A1;1 and CYCLINB1;1. Taken together, these results suggest that LBD16 and LBD18 are important regulators of LR initiation and development downstream of AUX1 and LAX3.

Inducible expression of Arabidopsis response regulator 22 (ARR22), a type-C ARR, in transgenic Arabidopsis enhances drought and freezing tolerance.

The Arabidopsis two-component signaling system, which is comprised of sensor histidine kinases, histidine phosphotransfer proteins, and response regulators, mediates cytokinin response as well as various other plant responses including abiotic stress responses. Arabidopsis response regulators (ARRs) are classified into type-A, -B, and -C. Although the roles of type-A and -B ARRs are well established in Arabidopsis plant signaling, roles of type-C ARRs, ARR22 and ARR24, remain elusive. ARR22, a preferentially cytosolic protein, interacts with certain Arabidopsis histidine phosphotransfer proteins (AHPs) and displays phosphatase activity on AHP5. ARR22 is induced by cold and dehydration. Here, we show that inducible overexpression of ARR22 in Arabidopsis enhanced dehydration, drought, and cold tolerance in a dexamethasone-dependent manner, whereas mutation of the putative phospho-accepting Asp to Asn in ARR22 (ARR22(D74N)) abolished these tolerance phenotypes. Overexpression of ARR22 decreased electrolyte leakage in dehydration-, drought-, or cold-stressed transgenic Arabidopsis plants compared with that of ARR22(D74N) or compared with wild-type plants. Transpiration rates and stomatal apertures were not affected by ARR22 overexpression. No significant difference in both dehydration and freezing tolerance was observed between wild-type and arr22 mutants with or without cytokinin preincubation, consistent with the lack of phenotypes of arr22 mutants in their vegetative development. Meta-profile analyses of the microarray data on ARR22-responsive genes indicate that ARR22 modulates expression of a variety of abiotic stress-responsive genes, which might contribute to increasing drought and freezing tolerance. Taken together, these results suggest that ARR22 plays a positive role in the stress tolerance response in part via enhancing cell membrane integrity and that phospho-histidine phosphatase activity of ARR22 may be required for this function.

Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana.

Cytokinin signaling in Arabidopsis thaliana utilizes a multi-step two-component signaling (TCS) system comprised of sensor histidine kinases (AHKs), histidine phosphotransfer proteins (AHPs), and response regulators (ARRs). Recent studies have suggested that the cytokinin TCS system is involved in a variety of other signaling and metabolic pathways. To further explore a potential function of the cytokinin TCS in the Arabidopsis dehydration stress response, we investigated the expression of all type-A ARR genes and a type-C ARR, ARR22, in both wild type and ahk single, double, and triple mutants in response to dehydration compared to cytokinin as well as dehydration tolerance of ahk mutants. We found that drought significantly induced the expression of a subset of ARR genes, ARR5, ARR7, ARR15, and ARR22. The results of expression analyses in ahk single, double, and triple mutants demonstrated that the cytokinin receptors AHK2 and AHK3 are redundantly involved in dehydration-inducible expression of ARR7, but not that of ARR5, ARR15, or ARR22. Dehydration tolerance assays showed that ahk2 and ahk3 single mutants exhibited enhanced dehydration tolerance compared with that of wild-type plants and ahk4 mutants, and that ahk2 ahk3 double mutants exhibited stronger drought tolerance than that of ahk3 ahk4, which exhibited more enhanced drought tolerance than that of wild-type plants and ahk single mutants. Taken together, these results demonstrate that while the cytokinin receptors AHK2 and AHK3 are critically involved in the dehydration tolerance response, both cytokinin receptor-dependent pathway and receptor-independent pathway occur in the dehydration response regulating ARR gene expression. In addition, preincubating ahk2, ahk3, ahk4, and the wild-type plants with cytokinin induced enhanced dehydration stress tolerance in these plants, demonstrating that cytokinins are involved in regulating plant response to dehydration stress.

A subset of cytokinin two-component signaling system plays a role in cold temperature stress response in Arabidopsis.

A multistep two-component signaling system is established as a key element of cytokinin signaling in Arabidopsis. Here, we provide evidence for a function of the two-component signaling system in cold stress response in Arabidopsis. Cold significantly induced the expression of a subset of A-type ARR genes and of GUS in Pro(ARR7):GUS transgenic Arabidopsis. AHK2 and AHK3 were found to be primarily involved in mediating cold to express A-type ARRs despite cytokinin deficiency. Cold neither significantly induced AHK2 and AHK3 expression nor altered the cytokinin contents of wild type within the 4 h during which the A-type ARR genes exhibited peak expression in response to cold, indicating that cold might induce ARR expression via the AHK2 and AHK3 proteins without alterations in cytokinin levels. The ahk2 ahk3 and ahk3 ahk4 mutants exhibited enhanced freezing tolerance compared with wild type. These ahk double mutants acclimated as efficiently to cold as did wild type. The overexpression of the cold-inducible ARR7 in Arabidopsis resulted in a hypersensitivity response to freezing temperatures under cold-acclimated conditions. The expression of C-repeat/dehydration-responsive element target genes was not affected by ARR7 overexpression as well as in ahk double mutants. By contrast, the arr7 mutants showed increased freezing tolerance. The ahk2 ahk3 and arr7 mutants showed hypersensitive response to abscisic acid (ABA) for germination, whereas ARR7 overexpression lines exhibited insensitive response to ABA. These results suggest that AHK2 and AHK3 and the cold-inducible A-type ARRs play a negative regulatory role in cold stress signaling via inhibition of ABA response, occurring independently of the cold acclimation pathway.