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Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.

Heat stress is a substantial and imminent threat to plant growth and development. Understanding its adverse effects on plant development at the molecular level is crucial for sustainable agriculture. However, the molecular mechanism underlying how heat stress causes developmental defects in flowers remains poorly understood. Here, we identified Indole-3-Acetic Acid 8 (IAA8), a repressor of auxin signaling, as a substrate of mitogen-activated protein kinases (MPKs) in Arabidopsis thaliana, and found that MPK-mediated phosphorylation of IAA8 inhibits flower development. MPKs phosphorylated three residues of IAA8: S74, T77, and S135. Interestingly, transgenic plants overexpressing a phospho-mimicking mutant of IAA8 (IAA8DDD OX) exhibited defective flower development due to high IAA8 levels. Furthermore, MPK-mediated phosphorylation inhibited IAA8 polyubiquitination, thereby significantly increasing its stability. Additionally, the expression of key transcription factors involved in flower development, such as bZIP and MYB genes, was significantly perturbed in the IAA8DDD OX plants. Collectively, our study demonstrates that heat stress inhibits flower development by perturbing the expression of flower development genes through the MPK-mediated phosphorylation of IAA8, suggesting that Aux/IAA phosphorylation enables plants to fine-tune their development in response to environmental stress., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The unfolded protein response (UPR) is a crucial cellular mechanism for maintaining protein folding homeostasis during endoplasmic reticulum (ER) stress. In this study, the role of IRE1, a key component of the UPR, was investigated in protein translation regulation under ER stress conditions in Arabidopsis. We discovered that the loss of IRE1A and IRE1B leads to diminished protein translation, indicating a significant role for IRE1 in this process. However, this regulation was not solely dependent on the interaction with bZIP60, a key transcription factor in the UPR. Interestingly, while chemical chaperones TUDCA and PBA effectively alleviated the translation inhibition observed in ire1a ire1b mutants, this effect was more pronounced than the mitigation observed from suppressing GCN2 expression or introducing a non-phosphorylatable eIF2α variant. Additionally, the kinase and ribonuclease activities of IRE1B were demonstrated to be crucial for plant adaptation and protein synthesis regulation under ER stress conditions. Overall, this study not only highlights the complex regulatory mechanisms of IRE1 in plant ER stress responses but also provides insights into its multifaceted roles in protein translation regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The plant hormone abscisic acid (ABA) is an important regulator of plant growth and development and plays a crucial role in both biotic and abiotic stress responses. ABA modulates flowering time, but the precise molecular mechanism remains poorly understood. Here we report that ABA INSENSITIVE 2 (ABI2) is the only phosphatase from the ABA-signaling core that positively regulates the transition to flowering in Arabidopsis. Loss-of-function abi2-2 mutant shows significantly delayed flowering both under long day and short day conditions. Expression of floral repressor genes such as FLOWERING LOCUS C (FLC) and CYCLING DOF FACTOR 1 (CDF1) was significantly up-regulated in abi2-2 plants while expression of the flowering promoting genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was down-regulated. Through genetic interactions we further found that ost1-3 and abi5-1 mutations are epistatic to abi2-2, as both of them individually rescued the late flowering phenotype of abi2-2. Interestingly, phosphorylation and protein stability of ABA INSENSITIVE 5 (ABI5) were enhanced in abi2-2 plants suggesting that ABI2 dephosphorylates ABI5, thereby reducing protein stability and the capacity to induce FLC expression. Our findings uncovered the unexpected role of ABI2 in promoting flowering by inhibiting ABI5-mediated FLC expression in Arabidopsis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The ubiquitin/26S proteasome system is a crucial regulatory mechanism that governs various cellular processes in plants, including signal transduction, transcriptional regulation, and responses to biotic and abiotic stressors. Our study shows that the RING-H2-type E3 ubiquitin ligase, Arabidopsis Tóxicos en Levadura 2 ( ATL2 ), is involved in response to fungal pathogen infection. Under normal growth conditions, the expression of the ATL2 gene is low, but it is rapidly and significantly induced by exogenous chitin. Additionally, ATL2 protein stability is markedly increased via chitin treatment, and its degradation is prolonged when 26S proteasomal function is inhibited. We found that an atl2 null mutant exhibited higher susceptibility to Alternaria brassicicola , while plants overexpressing ATL2 displayed increased resistance. We also observed that the hyphae of A. brassicicola were strongly stained with trypan blue staining, and the expression of A. brassicicola Cutinase A ( AbCutA ) was dramatically increased in atl2 . In contrast, the hyphae were weakly stained, and AbCutA expression was significantly reduced in ATL2 -overexpressing plants. Using bioinformatics, live-cell confocal imaging, and cell fractionation analysis, we revealed that ATL2 is localized to the plasma membrane. Further, it is demonstrated that the ATL2 protein possesses E3 ubiquitin ligase activity and found that cysteine 138 residue is critical for its function. Moreover, ATL2 is necessary to successfully defend against the A. brassicicola fungal pathogen. Altogether, our data suggest that ATL2 is a plasma membrane-integrated protein with RING-H2-type E3 ubiquitin ligase activity and is essential for the defense response against fungal pathogens in Arabidopsis .

Quercetin is a flavonol belonging to the flavonoid group of polyphenols. Quercetin is reported to have a variety of biological functions, including antioxidant, pigment, auxin transport inhibitor and root nodulation factor. Additionally, quercetin is known to be involved in bacterial pathogen resistance in Arabidopsis through the transcriptional increase of pathogenesis-related ( PR ) genes. However, the molecular mechanisms underlying how quercetin promotes pathogen resistance remain elusive. In this study, we showed that the transcriptional increases of PR genes were achieved by the monomerization and nuclear translocation of nonexpressor of pathogenesis-related proteins 1 (NPR1). Interestingly, salicylic acid (SA) was approximately 2-fold accumulated by the treatment with quercetin. Furthermore, we showed that the increase of SA biosynthesis by quercetin was induced by the transcriptional increases of typical SA biosynthesis-related genes. In conclusion, this study strongly suggests that quercetin induces bacterial pathogen resistance through the increase of SA biosynthesis in Arabidopsis .

Protein phosphatase 2A (PP2A) is a key regulator of plant growth and development, but its role in the endoplasmic reticulum (ER) stress response remains elusive. In this study, we investigated the function of PP2A under ER stress using loss-of-function mutants of ROOTS CURL of NAPHTHYLPHTHALAMIC ACID1 (RCN1), a regulatory A1 subunit isoform of Arabidopsis PP2A. RCN1 mutants (rcn1-1 and rcn1-2) exhibited reduced sensitivity to tunicamycin (TM), an inhibitor of N-linked glycosylation and inducer of unfolded protein response (UPR) gene expression, resulting in less severe effects compared to wild-type plants (Ws-2 and Col-0). TM negatively impacted PP2A activity in Col-0 plants but did not significantly affect rcn1-2 plants. Additionally, TM treatment did not influence the transcription levels of the PP2AA1(RCN1), 2, and 3 genes in Col-0 plants. Cantharidin, a PP2A inhibitor, exacerbated growth defects in rcn1 plants and alleviated TM-induced growth inhibition in Ws-2 and Col-0 plants. Furthermore, cantharidin treatment mitigated TM hypersensitivity in ire1a&b and bzip28&60 mutants. These findings suggest that PP2A activity is essential for an efficient UPR in Arabidopsis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kyun Oh Lee reports a relationship with National Research Foundation of Korea that includes: funding grants., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Since plants are sessile organisms, developmental plasticity in response to environmental stresses is essential for their survival. Upon exposure to drought, lateral root development is suppressed to induce drought tolerance. However, the molecular mechanism by which the development of lateral roots is inhibited by drought is largely unknown. In this study, the auxin signaling repressor IAA15 was identified as a novel substrate of mitogen-activated protein kinases (MPKs) and was shown to suppress lateral root development in response to drought through stabilization by phosphorylation. Both MPK3 and MPK6 directly phosphorylated IAA15 at the Ser-2 and Thr-28 residues. Transgenic plants overexpressing a phospho-mimicking mutant of IAA15 (IAA15DD OX) showed reduced lateral root development due to a higher accumulation of IAA15. In addition, MPK-mediated phosphorylation strongly increased the stability of IAA15 through the inhibition of polyubiquitination. Furthermore, IAA15DD OX plants showed the transcriptional downregulation of two key transcription factors LBD16 and LBD29, responsible for lateral root development. Overall, this study provides the molecular mechanism that explains the significance of the MPK-Aux/IAA module in suppressing lateral root development in response to drought., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dehydrating stresses trigger the accumulation of abscisic acid (ABA), a key plant stress-signaling hormone that activates Snf1-Related Kinases (SnRK2s) to mount adaptive responses. However, the regulatory circuits that terminate the SnRK2s signal relay after acclimation or post-stress conditions remain to be defined. Here, we show that the desensitization of the ABA signal is achieved by the regulation of OST1 (SnRK2.6) protein stability via the E3-ubiquitin ligase HOS15. Upon ABA signal, HOS15-induced degradation of OST1 is inhibited and stabilized OST1 promotes the stress response. When the ABA signal terminates, protein phosphatases ABI1/2 promote rapid degradation of OST1 via HOS15. Notably, we found that even in the presence of ABA, OST1 levels are also depleted within hours of ABA signal onset. The unexpected dynamics of OST1 abundance are then resolved by systematic mathematical modeling, demonstrating a desensitizing feedback loop by which OST1-induced upregulation of ABI1/2 leads to the degradation of OST1. This model illustrates the complex rheostat dynamics underlying the ABA-induced stress response and desensitization., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from Arabidopsis thaliana , which was designated AtCYS5. RT-qPCR analysis showed that the expression of AtCYS5 in germinating seeds was induced by heat stress (HS) and exogenous abscisic acid (ABA) treatment. Analysis of the expression of the β -glucuronidase reporter gene under the control of the AtCYS5 promoter showed that AtCYS5 expression during seed germination was induced by HS and ABA. Constitutive overexpression of AtCYS5 driven by the cauliflower mosaic virus 35S promoter led to enhanced HS tolerance in transgenic Arabidopsis , which was characterized by higher fresh weight and root length compared to wild-type (WT) and knockout ( cys5 ) plants grown under HS conditions. The HS tolerance of At-CYS5 -overexpressing transgenic plants was associated with increased insensitivity to exogenous ABA during both seed germination and post-germination compared to WT and cys5 . Although no HS elements were identified in the 5'-flanking region of AtCYS5 , canonical ABA-responsive elements (ABREs) were detected. AtCYS5 was upregulated in ABA-treated protoplasts transiently co-expressing this gene and genes encoding bZIP ABRE-binding factors (ABFs and AREB3). In the absence of ABA, ABF1 and ABF3 directly bound to the ABREs in the AtCYS5 promoter, which activated the transcription of this gene in the presence of ABA. These results suggest that an ABA-dependent pathway plays a positive role in the HS-responsive expression of AtCYS5 during seed germination and post-germination growth.

The expression of CBF (C-repeat-binding factor) genes is required for freezing tolerance in Arabidopsis thaliana. CBFs are positively regulated by INDUCER OF CBF EXPRESSION1 (ICE1) and negatively regulated by MYB15. These transcription factors directly interact with specific elements in the CBF promoters. Mitogen-activated protein kinase (MAPK/MPK) cascades function upstream to regulate CBFs. However, the mechanism by which MPKs control CBF expression during cold stress signaling remains unknown. This study showed that the activity of MYB15, a transcriptional repressor of cold signaling, is regulated by MPK6-mediated phosphorylation. MYB15 specifically interacts with MPK6, and MPK6 phosphorylates MYB15 on Ser168. MPK6-induced phosphorylation reduced the affinity of MYB15 binding to the CBF3 promoter and mutation of its phosphorylation site (MYB15S168A) enhanced the transcriptional repression of CBF3 by MYB15. Furthermore, transgenic plants overexpressing MYB15S168A showed significantly reduced CBF transcript levels in response to cold stress, compared with plants overexpressing MYB15. The MYB15S168A-overexpressing plants were also more sensitive to freezing than MYB15-overexpressing plants. These results suggest that MPK6-mediated regulation of MYB15 plays an important role in cold stress signaling in Arabidopsis., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Membrane-delimited ABA signal transduction plays an important role in early ABA signaling, but the molecular mechanisms connecting core signaling components to the plasma membrane remain unclear. Plants have evolved a large number of receptor-like kinases (RLKs) to modulate diverse biological processes by perceiving extracellular stimuli and activating downstream signaling responses. In this study, a putative leucine-rich repeat-RLK gene named RECEPTOR DEAD KINASE1 (AtRDK1) was identified and characterized in Arabidopsis thaliana. RDK1 promoter-GUS analysis revealed that RDK1 is expressed ubiquitously in the various tissues in Arabidopsis, and its expression is mainly induced by ABA. In the presence of ABA, RDK1-deficient rdk1-1 and rdk1-2 lines showed significant resistance in cotyledon greening and root growth, whereas RDK1-overexpressing lines showed enhanced sensitivity. Consistently, the expression of ABA-responsive genes was significantly downregulated in rdk1 mutant seedlings, which were also hypersensitive to drought stress with increased water loss. Interestingly, RDK1 was found to be an atypical kinase localized to the plasma membrane and did not require its kinase activity during ABA-mediated inhibition of seedling development. Accordingly, RDK1 interacted in the plasma membrane with type 2C protein phosphatase ABSCISIC ACID INSENSITIVE1 (ABI1); this interaction was further enhanced by exogenous application of ABA, suggesting that RDK1-mediated recruitment of ABI1 onto the plasma membrane is important for ABA signaling. Taken together, these results reveal an important role for RDK1 in plant responses to abiotic stress conditions in an ABA-dependent manner., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis.

Plant growth-promoting rhizobacteria (PGPR) facilitate the plant growth and enhance their induced systemic resistance (ISR) against a variety of environmental stresses. In this study, we carried out integrative analyses on the proteome, transcriptome, and metabolome to investigate Arabidopsis root and shoot responses to the well-known PGPR strain Paenibacillus polymyxa (P. polymyxa) E681. Shoot fresh and root dry weights were increased, whereas root length was decreased by treatment with P. polymyxa E681. 2DE approach in conjunction with MALDI-TOF/TOF analysis revealed a total of 41 (17 spots in root, 24 spots in shoot) that were differentially expressed in response to P. polymyxa E681. Biological process- and molecular function-based bioinformatics analysis resulted in their classification into seven different protein groups. Of these, 36 proteins including amino acid metabolism, antioxidant, defense and stress response, photosynthesis, and plant hormone-related proteins were up-regulated, whereas five proteins including three carbohydrate metabolism- and one amino acid metabolism-related, and one unknown protein were down-regulated, respectively. A good correlation was observed between protein and transcript abundances for the 12 differentially expressed proteins during interactions as determined by qPCR analysis. Metabolite analysis using LC-MS/MS revealed highly increased levels of tryptophan, indole-3-acetonitrile (IAN), indole-3-acetic acid (IAA), and camalexin in the treated plants. Arabidopsis plant inoculated P. polymyxa E681 also showed resistance to Botrytis cinerea infection. Taken together these results suggest that P. polymyxa E681 may promote plant growth by induced metabolism and activation of defense-related proteins against fungal pathogen., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the interaction between plants and pathogens, carbon (C) resources provide energy and C skeletons to maintain, among many functions, the plant immune system. However, variations in C availability on pathogen associated molecular pattern (PAMP) triggered immunity (PTI) have not been systematically examined. Here, three types of starch mutants with enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000 hrcC were examined for PTI. In a dark period-dependent manner, the mutants showed compromised induction of a PTI marker, and callose accumulation in response to the bacterial PAMP flagellin, flg22. In combination with weakened PTI responses in wild type by inhibition of the TCA cycle, the experiments determined the necessity of C-derived energy in establishing PTI. Global gene expression analyses identified flg22 responsive genes displaying C supply-dependent patterns. Nutrient recycling-related genes were regulated similarly by C-limitation and flg22, indicating re-arrangements of expression programs to redirect resources that establish or strengthen PTI. Ethylene and NAC transcription factors appear to play roles in these processes. Under C-limitation, PTI appears compromised based on suppression of genes required for continued biosynthetic capacity and defenses through flg22. Our results provide a foundation for the intuitive perception of the interplay between plant nutrition status and pathogen defense.

Key Message: ZAT11, a Zinc Finger of Arabidopsis Thaliana 11, is a dual-function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni (2+) tolerance. Zinc Finger of Arabidopsis Thaliana 11 (ZAT11) is a C2H2-type zinc finger protein that has been reported to function as an active transcriptional repressor. However, the biological function of ZAT11 remains unknown. Here we show that GFP-tagged ZAT11 is targeted to the nucleus. Analysis of plants expressing ZAT11 promoter-GUS showed that ZAT11 is highly expressed in roots and particularly in root tips. To identify the biological function of ZAT11, we constructed three independent lines of ZAT11 overexpressing transgenic plant (ZAT11 OE). ZAT11 OE enhanced the elongation of primary root but reduced the metal tolerance against nickel ion (Ni(2+)). The reduced Ni(2+) tolerance of ZAT11 OE was correlated with decreased accumulation of Ni(2+) in plants. The decreased accumulation of Ni(2+) in ZAT11 OE was caused by the reduced transcription of a vacuolar Ni(2+) transporter gene. Taken together, our results suggest that ZAT11 is a dual function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni(2+) tolerance.

Phytocystatins are proteinaceous inhibitors of cysteine proteases. They have been implicated in the regulation of plant protein turnover and in defense against pathogens and insects. Here, we have characterized an Arabidopsis phytocystatin family gene, Arabidopsis thaliana phytocystatin 4 (AtCYS4). AtCYS4 was induced by heat stress. The heat shock tolerance of AtCYS4-overexpressing transgenic plants was greater than that of wild-type and cys4 knock-down plants, as measured by fresh weight and root length. Although no heat shock elements were identified in the 5'-flanking region of the AtCYS4 gene, canonical ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs) were found. Transient promoter activity measurements showed that AtCYS4 expression was up-regulated in unstressed protoplasts by co-expression of DRE-binding factor 2s (DREB2s), especially by DREB2C, but not by bZIP transcription factors that bind to ABREs (ABFs, ABI5 and AREBs). DREB2C bound to and activated transcription from the two DREs on the AtCYS4 promoter although some preference was observed for the GCCGAC DRE element over the ACCGAC element. AtCYS4 transcript and protein levels were elevated in transgenic DREB2C overexpression lines with corresponding decline of endogenous cysteine peptidase activity. We propose that AtCYS4 functions in thermotolerance under the control of the DREB2C cascade.

Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50%-70% identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv. Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high-auxin phenotypes such as narrow downward-curled leaves, increased height, erect stature, and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.

C(2)H(2)-type zinc finger proteins (ZFPs) play diverse roles in plant response to abiotic stresses. ZAT6, an Arabidopsis C(2)H(2)-type ZFP, has been reported to regulate root development and nutrient stress responses. However, its roles in regulation of abiotic stress response are incompletely known. Here, we demonstrate that salt or osmotic stress triggers a strong increase in ZAT6 expression in leaves. Transgenic plants overexpressing ZAT6 showed improved seed germination under salt and osmotic stress. Intriguingly, ZAT6 interacts with a stress-responsive mitogen-activated protein kinase MPK6 in vitro and in planta. ZAT6 is phosphorylated by both recombinant and plant endogenous MPK6. Serine 8 and serine 223 in ZAT6 were identified as the sites phosphorylated by MPK6. In contrast to wild-type form of ZAT6, overexpression of phosphorylation mutant form did not display significantly enhanced salt and osmotic stress tolerance. Altogether, our results suggest that phosphorylation by MPK6 is required for the functional role of ZAT6 in seed germination under salt and osmotic stress., (Copyright © 2012. Published by Elsevier Inc.)

Calmodulin (CaM), a key Ca2+ sensor, regulates diverse cellular processes by modulating the activity of a variety of enzymes and proteins. However, little is known about the biological function of CaM in plant development. In this study, an ASYMMETRIC LEAVES1 (AS1) transcription factor was isolated as a CaM-binding protein. AS1 contains two putative CaM-binding domains (CaMBDs) at the N-terminus. Using domain mapping analysis, both predicted domains were identified as authentic Ca2+ -dependent CaMBDs. We identified three hydrophobic amino acid residues for CaM binding, Trp49 in CaMBDI, and Trp81 and Phe103 in CaMBDII. The interactions of AS1 with CaM were verified in yeast and plant cells. Based on electrophoretic mobility shift assays, CaM inhibited the DNA-binding activity of the AS1/AS2 complex to two cis-regulatory motifs in the KNAT1 promoter. Furthermore, CaM relieved the suppression of KNAT1 transcription by AS1 not only in transient expression assays of protoplasts but also by the overexpression of a CaM-binding negative form of AS1 in as1 mutant plant. Our study suggests that CaM, a calcium sensor, can be involved in the transcriptional control of meristem cell-specific genes by the inhibition of AS1 under the condition of higher levels of Ca2+ in plants., (© 2012 Blackwell Publishing Ltd.)

Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the in planta function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in Arabidopsis. A T-DNA insertional mutant (cbnac1) displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae DC3000 (PstDC3000), whereas resistance was reduced in transgenic CBNAC overexpression lines. The observed changes in disease resistance were correlated with alterations in pathogenesis-related protein 1 (PR1) gene expression. CBNAC bound directly to the PR1 promoter. SNI1 (suppressor of nonexpressor of PR genes1, inducible 1) was identified as a CBNAC-binding protein. Basal resistance to PstDC3000 and derepression of PR1 expression was greater in the cbnac1 sni1 double mutant than in either cbnac1 or sni1 mutants. SNI1 enhanced binding of CBNAC to its cognate PR1 promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense.

The phytohormones abscisic acid (ABA) and gibberellic acid (GA) have antagonistic roles in the control of seed germination and seedling development. We report here that the transcriptional regulator MYB44 has a role in the control of seed germination in Arabidopsis thaliana. High levels of the MYB44 transcript are found in dry seeds but the transcript levels decrease during germination. The decrease in transcript level during germination is inhibited by the GA biosynthesis inhibitor paclobutrazol (PAC). MYB44 is phosphorylated by both recombinant and native forms of MPK3 and MPK6 at Ser(53) and Ser(145). Transgenic overexpression of MYB44 results in increased sensitivity of seed germination to ABA or PAC treatment. The PAC-insensitive germination phenotype of the myb44 mutant is complemented by overexpression of wild type MYB44 but not by overexpression of a mutant protein that lacks the MPK-target serines indicating that phosphorylation of MYB44 by MPKs is required for its biological function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mitogen-activated protein kinases (MPKs) are involved in a number of signaling pathways that control plant development and stress tolerance via the phosphorylation of target molecules. However, so far only a limited number of target molecules have been identified. Here, we provide evidence that MYB41 represents a new target of MPK6. MYB41 interacts with MPK6 not only in vitro but also in planta. MYB41 was phosphorylated by recombinant MPK6 as well as by plant MPK6. Ser(251) in MYB41 was identified as the site phosphorylated by MPK6. The phosphorylation of MYB41 by MPK6 enhanced its DNA binding to the promoter of a LTP gene. Interestingly, transgenic plants over-expressing MYB41(WT) showed enhanced salt tolerance, whereas transgenic plants over-expressing MYB41(S251A) showed decreased salt tolerance during seed germination and initial root growth. These results indicate that the phosphorylation of MYB41 by MPK6 is required for the biological function of MYB41 in salt tolerance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mitogen-activated protein kinases (MAPKs or MPKs) are one of the most important and conserved signaling molecules in plants. MPKs can directly modulate gene expression by the phosphorylation of transcription factors. However, only a few target substrates of MPKs have been isolated. Here, we identified a C(2)H(2)-type zinc finger transcription factor from Arabidopsis, ZAT10, as a substrate of MPKs. Using in vitro and in vivo protein-protein interaction analyses, we demonstrated that ZAT10 directly interacted with MPK3 and MPK6. ZAT10 was phosphorylated by recombinant Arabidopsis MPK3 and MPK6 in a kinase assay. Furthermore, ZAT10 was also phosphorylated by native MPK3 and MPK6 prepared from Arabidopsis plants in an in-gel kinase assay. Mass spectrometry analysis of phosphopeptides was used to determine two MPK phosphorylation sites in ZAT10. These sites were verified by site-directed mutagenesis and in vitro kinase assays., (© Springer-Verlag 2011)

Cellular Na(+)/K(+) ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na(+)/K(+) cotransporters in the High-affinity K(+) Transporter (HKT) family from the halophytic Arabidopsis (Arabidopsis thaliana) relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNA interference in T. salsuginea resulted in Na(+) sensitivity and K(+) deficiency. The athkt1 mutant lines overexpressing TsHKT1;2 proved less sensitive to Na(+) and showed less K(+) deficiency than lines overexpressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na(+) or K(+) transporters revealed strong K(+) transporter activity and selectivity for K(+) over Na(+). Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K(+) transporter activity. We consider the maintenance of K(+) uptake through TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

Plants have developed various regulatory pathways to adapt to environmental stresses. In this study, we identified Arabidopsis MKKK20 as a regulator in the response to osmotic stress. mkkk20 mutants were found to be sensitive to high concentration of salt and showed higher water loss rates than wild-type (WT) plants under dehydration conditions. In addition, mkkk20 mutants showed higher accumulation of superoxide, a reactive oxygen species (ROS), compared to WT plants under high salt condition. In contrast, transgenic plants overexpressing MKKK20 displayed tolerance to salt stress. MKKK20 transcripts were increased by the treatments with NaCl, mannitol, MV, sorbitol, and cold, suggesting that MKKK20 is involved in the response to osmotic, ROS, and cold stresses. In-gel kinase assay showed that MKKK20 regulates the activity of MPK6 under NaCl, cold, and H(2)O(2) treatments. Taken together, our results suggest that MKKK20 might be involved in the response to various abiotic stresses, especially osmotic stress, through its regulation of MPK6 activity.

Our recent work demonstrated that chitin treatment modulated the expression of 118 transcription factor (TF) genes in Arabidopsis. To investigate the potential roles of these TF in chitin signaling and plant defense, we initiated an interaction study among these TF proteins, as well as two chitin-activated mitogen-activated protein kinases (MPK3 and MPK6), using a yeast two-hybrid system. This study revealed interactions among the following proteins: three ethylene-responsive element-binding factors (ERF), five WRKY transcription factors, one scarecrow-like (SCL), and the two MPK, in addition to many other interactions, reflecting a complex TF interaction network. Most of these interactions were subsequently validated by other methods, such as pull-down and in planta bimolecular fluorescence complementation assays. The key node ERF5 was shown to interact with multiple proteins in the network, such as ERF6, ERF8, and SCL13, as well as MPK3 and MPK6. Interestingly, ERF5 appeared to negatively regulate chitin signaling and plant defense against the fungal pathogen Alternaria brassicicola and positively regulate salicylic acid signaling and plant defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Therefore, ERF5 may play an important role in plant innate immunity, likely through coordinating chitin and other defense pathways in plants in response to different pathogens.

Plants have developed disparate regulatory pathways to adapt to environmental stresses. In this study, we identified MKK4 as an important mediator of plant response to osmotic stress. mkk4 mutants were more sensitive to high salt concentration than WT plants, exhibiting higher water-loss rates under dehydration conditions and additionally accumulating high levels of ROS. In contrast, MKK4-overexpressing transgenic plants showed tolerance to high salt as well as lower water-loss rates under dehydration conditions. In-gel kinase assays revealed that MKK4 regulates the activity of MPK3 upon NaCl exposure. Semi-quantitative RT-PCR analysis showed that expression of NCED3 and RD29A was lower and higher in mkk4 mutants and MKK4-overexpressing transgenic plants, respectively. Taken together, our results suggest that MKK4 is involved in the osmotic-stress response via its regulation of MPK3 activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Arabidopsis MAP kinase phosphatase 1 (AtMKP1) is a member of the mitogen-activated protein kinase (MPK) phosphatase family, which negatively regulates AtMPKs. We have previously shown that AtMKP1 is regulated by calmodulin (CaM). Here, we examined the phosphorylation of AtMKP1 by its substrate AtMPK6. Intriguingly, AtMKP1 was phosphorylated by AtMPK6, one of AtMKP1 substrates. Four phosphorylation sites were identified by phosphoamino acid analysis, TiO(2) chromatography and mass spectrometric analysis. Site-directed mutation of these residues in AtMKP1 abolished the phosphorylation by AtMPK6. In addition, AtMKP1 interacted with AtMPK6 as demonstrated by the yeast two-hybrid system. Finally, the phosphatase activity of AtMKP1 increased approximately twofold following phosphorylation by AtMPK6. By in-gel kinase assays, we showed that AtMKP1 could be rapidly phosphorylated by AtMPK6 in plants. Our results suggest that the catalytic activity of AtMKP1 in plants can be regulated not only by Ca(2+)/CaM, but also by its physiological substrate, AtMPK6.

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca(2+) signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca(2+)-dependent electrophoretic mobility shift and Ca(2+) binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.

Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.

Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.

Cadmium (Cd) is a non-essential toxic heavy metal that influences normal growth and development of plants. However, the molecular mechanisms by which plants recognize and respond to Cd remain poorly understood. We show that, in Arabidopsis, Cd activates the mitogen-activated protein kinases, MPK3 and MPK6, in a dose-dependent manner. Following treatment with Cd, these two MAPKs exhibited much higher activity in the roots than in the leaves, and pre-treatment with the reactive oxygen species (ROS) scavenger, glutathione, effectively inhibited their activation. These results suggest that the Cd sensing signaling pathway uses a build-up of ROS to trigger activation of Arabidopsis MPK3 and MPK6., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The dehydration responsive element binding protein 2C (DREB2C) is a dehydration responsive element/C-repeat (DRE/CRT)-motif binding transcription factor that induced by mild heat stress. Previous experiments established that overexpression of DREB2C cDNA driven by the cauliflower mosaic virus 35S promoter (35S:DREB2C) resulted in increased heat tolerance in Arabidopsis. We first analyzed the proteomic profiles in wild-type and 35S:DREB2C plants at a normal temperature (22 degrees C), but could not detect any differences between the proteomes of wild-type and 35S:DREB2C plants. The transcript level of DREB2C in 35S:DREB2C plants after treatment with mild heat stress was increased more than two times compared with expression in 35S:DREB2C plants under unstressed condition. A proteomic approach was used to decipher the molecular mechanisms underlying thermotolerance in 35S:DREB2C Arabidopsis plants. Eleven protein spots were identified as being differentially regulated in 35S:DREB2C plants. Moreover, in silico motif analysis showed that peptidyl-prolyl isomerase ROC4, glutathione transferase 8, pyridoxal biosynthesis protein PDX1, and elongation factor Tu contained one or more DRE/CRT motifs. To our knowledge, this study is the first to identify possible targets of DREB2C transcription factors at the protein level. The proteomic results were in agreement with transcriptional data.

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.

Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca(2+)-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.

In plant cells, the vacuole functions as a major calcium store. Although a calmodulin-regulated Ca2+-ATPase (ACA4) is known to be present in prevacuolar compartments, the presence of an ACA-type Ca2+-ATPase in the mature vacuole of a plant cell has not been verified. Here we provide evidence that ACA11 localizes to the vacuole membrane. ACA11 tagged with GFP was expressed in stable transgenic plants, and visualized in root cells and protoplasts by confocal microscopy. A Ca2+-ATPase function for ACA11 was confirmed by complementation of yeast mutants. A calmodulin binding domain was identified within the first 37 residues of the N-terminal autoinhibitory region.

2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.

Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca(2+)-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Delta1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.

P58IPK has been implicated in eukaryotic ER stress responses and viral pathogenesis, however, its biological functions and molecular mechanism in plants are unclear. Prolonged ER stress produced by tunicamycin (TM) increased P58IPK mRNA and protein levels in Arabidopsis. Although the growth of 2 × 35S:P58IPK-myc plants was less severely inhibited than that of Col-0 plants, TM inhibited the growth of p58ipk-2 mutants more severely than that of Col-0 plants. Under prolonged ER stress conditions, the unfolded protein response (UPR)-related genes were expressed at a higher level in the p58ipk-2 mutants than in Col-0 plants. Protein synthesis inhibition by TM in 2 × 35S:P58IPK-myc plants was lower than in Col-0 plants under prolonged ER stress conditions, however, not significantly different in p58ipk-2 mutants. The GST-P58IPK protein exhibited both chaperone and RNA-binding activities in a dose-dependent manner. P58IPK has been shown to interact with ribosomes, allowing for enhanced protein production on the ER membrane. Following ER stress, 2 × 35S:P58IPK-myc plants recovered better than Col-0, but p58ipk-2 mutants recovered less than Col-0. These findings reveal that P58IPK can promote protein translation in association with ribosomes and contribute to stress recovery in Arabidopsis when induced during the last phase of ER stress. [ABSTRACT FROM AUTHOR]