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Mitogen-activated protein kinases (MPKs) play roles as critical signal components in the environmental stress responses and developmental processes in plants. Calcium ion (Ca2+) is one of the most essential ubiquitous intracellular second messengers involved in many signal transduction pathways in plants. It was previously known that MPKs are activated by the increasing Ca2+ concentration. However, the mechanism of how Ca2+ activates MPKs is not elucidated yet. In this study, we revealed that Ca2+ could activate MPK signaling pathway via inhibiting the activity of a dual-specificity protein phosphatase1 (DsPTP1) by Ca2+/calmodulin (CaM). We showed that DsPTP1 directly interacts with MPK6 in vitro and in vivo. DsPTP1 was able to inactivate the active MPK6 by dephosphorylation. Interestingly, the DsPTP1-mediated dephosphorylation of MPK6 was strongly inhibited by Ca2+/CaM. Moreover, this inhibition was caused by the binding of CaM to the calmodulin-binding domain II (CaMBDII) of DsPTP1. This study implies that Ca2+/CaM is involved in the activation of MPKs through the inhibition of DsPTP1.

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.

Malathion is an organophosphate chemical (OPC) and a toxic contaminant that adversely impacts food quality, human health, biodiversity, and the environment. Due to its small size and unavailability of sensitive sensors, detection of malathion remains a challenging task. Often chromatographic methods employed to analyze OPCs suffer from several shortcomings, including cost, immobility, laboriousness, and unsuitability for point-of-care settings. Hence, developing a specific and sensitive diagnostic sensor for quick and inexpensive food testing is essential. We discovered four unique malathion-specific ssDNA aptamers; designed two independent sensing strategies using fluorescence labeling and Thioflavin T (ThT) displacement. Selected aptamers formed the G4-quadruplex-like (G4Q) structure, which helped develop a label-free detection approach with a 2.01 ppb limit of detection. Additionally, 3D structures of aptamers were generated and validated using a series of computational modeling programs. Furthermore, we explored structural features using CD spectroscopy and molecular docking, probing ligands' binding mode, and revealed vital intermolecular interactions with aptamers. Subsequently, the novel sensors were optimized to detect malathion from food samples. The novel sensors could be further developed to meet the demands of sensing and quantifying toxic contaminants from real food samples in field conditions.

Journal of laser applications 34(4), 042019 (2022). doi:10.2351/7.0000772, The laser-spot welding process of aluminum alloy 1050A with a limited thickness is observed with the x-ray phase contrast method to investigate the melt dynamic especially when the melt penetrates the material. The laser-spot welding is investigated with two different wavelengths of the laser beam source: 515 and 1030 nm to investigate the influence of the absorptivity. The melt progressively penetrates the material during the spot-welding process until reaching the bottom side of the material and when the melt penetrates the lower side of the material, the so-called “lens-like” melt appears at the lower side due to the surface tension. At a comparable beam intensity value, the oscillation of the “lens-like” melt at the lower side of the material is driven by the expansion of vapor capillary. This expansion occurs inside of the material and directly above the “lens-like” melt. The shape of the expanded vapor determines the volume as well as the geometry of the resulting melt volume. Furthermore, the transition from the heat conduction welding mode to the keyhole welding mode is investigated by defocusing the laser beam for the beam source with a 515 nm wavelength. At a given variation, a clear difference between either mode is observed with the x-ray phase contrast method., Published by Laser Inst. of America, Orlando, Fla.

Fipronil is a broad-spectrum insecticide widely used in agriculture and residential areas; its indiscriminate use leads to environmental pollution and poses health hazards. Early detection of fipronil is critical to prevent the deleterious effects. However, current insecticide analysis methods such as HPLC, LC/MS, and GC/MS are incompetent; they are costly, immobile, time-consuming, laborious, and need skilled technicians. Hence, a sensitive, specific, and cheap biosensor are essential to containing the contamination. Here, we designed two novel biosensors—the first design relied on fluorescent labeling/quenching, while the second sensor focused on label-free detection using Thioflavin T displacement. Altogether, we identified four candidate aptamers, predicted secondary structures, and performed 3D molecular modeling to predict the binding pocket of fipronil in FiPA6B aptamer. Furthermore, the aptameric sensors showed high sensitivity to fipronil of sub-ppb level LOD, attributed to stringent experimental design. The biosensors displayed high specificity against other phenylpyrazole insecticides and demonstrated robust sensitivity for fipronil in real samples like cabbage and cucumber. Notably, to the best of our knowledge, this is the first demonstration of noncanonical G4-quadruplex-like aptamer binding to fipronil, verified using CD spectroscopy. Such aptasensors possess considerable potential for real-time measurements of hazardous insecticides as point-of-care technology.

Fenitrothion is an insecticide belonging to the organophosphate family of pesticides that is widely used around the world in agriculture and living environments. Today, it is one of the most hazardous chemicals that causes severe environmental pollution. However, detection of fenitrothion residues in the environment is considered a significant challenge due to the small molecule nature of the insecticide and lack of molecular recognition elements that can detect it with high specificity. We performed in vitro selection experiments using the SELEX process to isolate the DNA aptamers that can bind to fenitrothion. We found that newly discovered DNA aptamers have a strong ability to distinguish fenitrothion from other organophosphate insecticides (non-specific targets). Furthermore, we identified a fenitrothion-specific aptamer, FenA2, that can interact with Thioflavin T (ThT) to produce a label-free detection mode with a Kd of 33.57 nM (9.30 ppb) and LOD of 14 nM (3.88 ppb). Additionally, the FenA2 aptamer exhibited very low cross-reactivity with non-specific targets. This is the first report showing an aptamer sensor with a G4-quadruplex-like structure to detect fenitrothion. Moreover, these aptamers have the potential to be further developed into analytical tools for real-time detection of fenitrothion from a wide range of samples.

Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised. To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl2 using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15). The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana. These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

Post-translational modification of proteins regulates signaling cascades in eukaryotic system, including plants. Among these modifications, phosphorylation plays an important role in modulating the functional properties of proteins. Plants perceive environmental cues that directly affect the phosphorylation status of many target proteins. To determine the effect of environmentally induced phosphorylation in plants, in vivo methods must be developed. Various in vitro methods are available but, unlike in animals, there is no optimized methodology for detecting protein phosphorylation in planta. Therefore, in this study, a robust, and easy to handle Phos-Tag Mobility Shift Assay (PTMSA) is developed for the in vivo detection of protein phosphorylation in plants by empirical optimization of methods previously developed for animals. Initially, the detection of the phosphorylation status of target proteins using protocols directly adapted from animals failed. Therefore, we optimized the steps in the protocol, from protein migration to the transfer of proteins to PVDF membrane. Supplementing the electrophoresis running buffer with 5 mM NaHSO3 solved most of the problems in protein migration and transfer. The optimization of a fast and robust protocol that efficiently detects the phosphorylation status of plant proteins was successful. This protocol will be a valuable tool for plant scientists interested in the study of protein phosphorylation.

Flavonoids are well known for the coloration of plant organs to protect UV and ROS and to attract pollinators as well. Flavonoids also play roles in many aspects of physiological processes including pathogen resistance. However, the molecular mechanism to explain how flavonoids play roles in pathogen resistance was not extensively studied. In this study, we investigated how naringenin, the first intermediate molecule of the flavonoid biosynthesis, functions as an activator of pathogen resistances. The transcript levels of two pathogenesis-related (PR) genes were increased by the treatment with naringenin in Arabidopsis. Interestingly, we found that naringenin triggers the monomerization and nuclear translocation of non-expressor of pathogenesis-related genes 1 (NPR1) that is a transcriptional coactivator of PR gene expression. Naringenin can induce the accumulation of salicylic acid (SA) that is required for the monomerization of NPR1. Furthermore, naringenin activates MPK6 and MPK3 in ROS-dependent, but SA-independent manners. By using a MEK inhibitor, we showed that the activation of a MAPK cascade by naringenin is also required for the monomerization of NPR1. These results suggest that the pathogen resistance by naringenin is mediated by the MAPK- and SA-dependent activation of NPR1 in Arabidopsis.

The laser beam welding process is not commonly used for joining interconnectors on metallized substrate such as Printed Circuit Board (PCB) due to the presence of the vapor capillary and high energy input which exceed the thermal destruction threshold of the substrate. To perform a welding process between interconnectors and PCBs, a metallization with sufficient thermal mass is required. The cold gas spraying process is used to spray a copper layer on a thin metallization to increase its thickness and the thermal mass. In this paper, a copper interconnector is laser-welded on a metallization of PCB which is cold gas sprayed with copper powders. The characterization and transfer of the welding technology to the joining of spray layer is investigated. The main challenges of the welding process on spray layer on substrate are the uncontrolled surface roughness and the inhomogeneous heat distribution of the spray layer compared to the bulk material. The influence of the surface roughness on the void formation is investigated by considering roughness values of the sprayed layer. A correlation between the void formation and the surface roughness is shown. Also, the void formation at the weld joint increases with the higher laser beam power. The increased laser beam power leads to a deeper vapor capillary and it is assumed that the inhomogeneous heat distribution of the lower joining partner induces varying solidifications speed. By reducing the surface roughness value and lower laser beam power, we could significantly reduce the void formation at the weld root.

Welding in the world 65(3), 463-474 (2021). doi:10.1007/s40194-020-01040-9, Published by Springer, Heidelberg

The laser impulse metal bonding (LIMBO) process is a novel laser spot welding process to form a weld joint between a massive interconnector and thin metallization on a thermally sensitive substrate. As for the LIMBO process, the gap between joining partners in overlap configuration allows energetic separation of the melting and joining processes, whereas the underlying joining partner is only thermally stressed during the welding process. The LIMBO process shows, therefore, potential to substitute the ultrasonic wire bonding and the soldering process for joining interconnectors on metallized substrates such as direct bonded copper or PCB. Due to a separation between joining partners, the LIMBO process is divided into three stages that are achieved with temporal pulse modulation. The initial stage prepares a melt pool on an upper joining partner which penetrates through the material. After achieving a local melt pool through the upper joining partner, the temporal intensity modulation of the laser beam is initialized for the second stage. The enhanced laser beam intensity on the melt pool surface vaporizes the melt surface rapidly and induces a recoil metal plume. Hence, the recoil metal plume pressure accelerates the melt toward the underlying joining partner. After the contact, further energy input is required for the last stage to melt the underlying joining partner and subsequently to form a weld. Therefore, the laser beam intensity is once again temporally modulated to lower the intensity value. In this paper, the LIMBO process is extended with a further stage which combines spatial and temporal pulse modulation. From the outgoing position from the last stage of the LIMBO process, the laser beam is spatially modulated to perform a circular movement around the weld joint. Therefore, the spatial pulse modulation allows us to overcome the limitation of the spot welding process. In combination with temporal pulse modulation during the circular movement, the weld joint is expanded continuously. During the extended stage, the accessed heat is also considered, so the thermal threshold of the sensitive substrate is not exceeded. In addition, the overlap pulsed welding process is applied with the extended LIMBO process to achieve a linear weld joint area.

Diazinon is a widely used organophosphorus neurotoxic insecticide. It is a common environmental contaminant and a hazardous agri-waste. Its detection is critical to control entry into food systems and protect the environment.In this study, three single-stranded DNA aptamers specific for diazinon were discovered using the systematic evolution of ligands by the exponential enrichment (SELEX) process. Since aptamer-based sensors are quick and straightforward to analyze, they could potentially replace the time-consuming and labor-intensive traditional methods used for diazinon detection.Here, we show the engineering of novel sensors for diazinon detection with a high affinity (Kd), specificity, and high sensitivity at the ppb level. Moreover, the aptamers were helpful in the simultaneous detection of two other structurally relevant insecticides, fenthion, and fenitrothion. Furthermore, the real vegetable and fruit samples confirmed the specific detection of diazinon using DIAZ-02.We developed novel biosensors and optimized the assay conditions for the detection of diazinon from food samples, such as vegetables and fruit. The biosensor could be adopted to analyze toxicants and contaminants in food, water, and nature as point-of-care technology.

The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the β-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

Lateral root development is known to be regulated by Aux/IAA-ARF modules in Arabidopsis thaliana. As components, several Aux/IAAs have participated in these Aux/IAA-ARF modules. In this study, to identify the biological function of IAA15 in plant developments, transgenic plant overexpressing the gain-of-function mutant of IAA15 (IAA15P75S OX) under the control of dexamethasone (DEX) inducible promoter, in which IAA15 protein was mutated by changing Pro-75 residue to Ser at the degron motif in conserved domain II, was constructed. As a result, we found that IAA15P75S OX plants show a decreased number of lateral roots. Coincidently, IAA15 promoter-GUS reporter analysis revealed that IAA15 transcripts were highly detected in all stages of developing lateral root tissues. It was also verified that the IAA15P75S protein is strongly stabilized against proteasome-mediated protein degradation by inhibiting its poly-ubiquitination, resulting in the transcriptional repression of auxin-responsive genes. In particular, transcript levels of LBD16 and LBD29, which are positive regulators of lateral root formation, dramatically repressed in IAA15P75S OX plants. Furthermore, it was elucidated that IAA15 interacts with ARF7 and ARF19 and binds to the promoters of LBD16 and LBD29, strongly suggesting that IAA15 represses lateral root formation through the transcriptional suppression of LBD16 and LBD29 by inhibiting ARF7 and ARF19 activity. Taken together, this study suggests that IAA15 also plays a key negative role in lateral root formation as a component of Aux/IAA-ARF modules.

The increasing demand for the substitution of the internal combustion engine vehicles to the battery electric vehicles requires beside battery cells high performance power electronic devices such as power control units (PCU). However, a combined requirement of high junction temperature stability and a large joint area of the interconnection on the PCU is a challenge for the conventional joining method such as soldering and wire bonding process. The Laser Impulse Metal Bonding (LIMBO) process enables a high temperature stable weld joint and large joint area. During the LIMBO process only minimized thermal stress is induced into the underlying substrate by a spatial separation between both joining partners in an overlap configuration with a gap. Hence, an energetic separation between the melting and joining phase is given. In this paper, the LIMBO process is firstly investigated with the disc laser at wavelength λ = 515 nm. Due to the enhanced absorptivity of the laser beam at this wavelength on copper material, the process duration of the LIMBO process is about the half compared to the LIMBO process with wavelength λ = 1064 nm.

Seed germination is a complex biological process controlled by various regulators, including phytohormones. Among these, abscisic acid and gibberellic acid inhibit and promote seed germination, respectively. Many studies have addressed the biological roles of auxin in plant growth and development, but very few have considered its role in seed germination. Here, we identified a novel function of the auxin signaling repressor Aux/IAA8 during seed germination. The IAA8 loss-of-function mutant iaa8-1 exhibited delayed seed germination. The phenotype of iaa8-1 was restored by ectopic expression of IAA8. Interestingly, IAA8 accumulated to high levels during seed germination, which was achieved not only by increased protein synthesis but also by the stabilization of IAA8 protein. We also showed that IAA8 down-regulates the transcription of ABSCISIC ACID INSENSITIVE3 (ABI3), a negative regulator of seed germination. Our study, thus strongly suggest that the auxin signaling repressor IAA8 acts as a positive regulator of seed germination in Arabidopsis thaliana.

11th CIRP Conference on Photonic Technologies, LANE 2020, online, 7 Sep 2020 - 10 Sep 2020; Procedia CIRP 94, 753-757 (2020). doi:10.1016/j.procir.2020.09.142 special issue: "11th CIRP Conference on Photonic Technologies [LANE 2020] / Edited by M. Schmidt, F. Vollertsen, E. Govekar", Published by Elsevier, Amsterdam [u.a.]

Purpose To investigate the efficacy, safety, and tolerability of oro-dispersible film (ODF) formulation of mirodenafil 50 mg and 100 mg for the treatment of patients with erectile dysfunction (ED) in Korea. Materials and methods A multicenter, randomized, double-blind, placebo-controlled, parallel-group study of 129 subjects was performed. Subjects were randomized to either placebo or mirodenafil ODF 50 mg or 100 mg to be taken in an "on demand" manner for 8 weeks. The primary efficacy variable was the International Index of Erectile Dysfunction (IIEF)-5 questionnaire. The secondary efficacy variables comprised Sexual Encounter Profile questions 2 and 3 (SEP2 and SEP3), the Global Assessment Question (GAQ), and the Life Satisfaction Checklist (LSC). Results IIEF-5 was significantly increased in all groups after treatment. However, compared to the placebo group, only the mirodenafil ODF 100 mg group showed a significant difference. SEP2 and SEP3 were increased in both mirodenafil groups; however, the increase was not statistically significant for SEP2. In terms of GAQ and LSC, the mirodenafil ODF groups showed significant increases compared with the baseline. Most treatment-associated adverse events were mild and resolved spontaneously. Conclusions Mirodenafil ODF is an effective and well-tolerated agent for the treatment of patients with ED in Korea.

Sessile plants reprogram their metabolic and developmental processes during adaptation to prolonged environmental stresses. To understand the molecular mechanisms underlying adaptation of plant cells to saline stress, we established callus suspension cell cultures from Arabidopsis roots adapted to high salt for an extended period of time. Adapted cells exhibit enhanced salt tolerance compared with control cells. Moreover, acquired salt tolerance is maintained even after the stress is relieved, indicating the existence of a memory of acquired salt tolerance during mitotic cell divisions, known as mitotic stress memory. Metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy revealed metabolic discrimination between control, salt-adapted and stress-memory cells. Compared with control cells, salt-adapted cells accumulated higher levels of sugars, amino acids and intermediary metabolites in the shikimate pathway, such as coniferin. Moreover, adapted cells acquired thicker cell walls with higher lignin contents, suggesting the importance of adjustments of physical properties during adaptation to elevated saline conditions. When stress-memory cells were reverted to normal growth conditions, the levels of metabolites again readjusted. Whereas most of the metabolic changes reverted to levels intermediate between salt-adapted and control cells, the amounts of sugars, alanine, γ-aminobutyric acid and acetate further increased in stress-memory cells, supporting a view of their roles in mitotic stress memory. Our results provide insights into the metabolic adjustment of plant root cells during adaptation to saline conditions as well as pointing to the function of mitotic memory in acquired salt tolerance.

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.

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.

Phosphorylation of substrate proteins by mitogen-activated protein kinases (MPKs) determines the specific cellular responses elicited by a particular extracellular stimulus. However, downstream targets of plant MPKs remain poorly characterized. In this study, 29 putative substrates of AtMPK3, AtMPK4 and AtMPK6 were identified by solid-phase phosphorylation screening of a λ phage expression library constructed from combined mRNAs from salt-treated, pathogen-treated and mechanically wounded Arabidopsis seedlings. To test the efficiency of this screening, we performed in vitro kinase assay with 10 recombinant fusion proteins. All proteins were phosphorylated by AtMPK3, AtMPK4 and AtMPK6, indicating the efficiency of this screening procedure. To confirm phosphorylation of isolated substrates by plant MPKs, we performed in-gel kinase assays. All test substrates were strongly phosphorylated by wounding or H2O2-activated AtMPK3 and AtMPK6. Three substrates, encoded by genes At2g41430, At2g41900, and At3g16770, were strongly phosphorylated, suggesting a function as AtMPK substrates. The type of screening provides a powerful way for identifying potential substrates of MAP kinases responsive to biotic and abiotic stresses.

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.

Osmotic stress is induced by several environmental stresses such as drought, cold and salinity. Osmotic stress may finally leads to oxidative damage, ionic imbalance and growth inhibition in plants. Gene expression analyses indicated that ZAT10 transcription factor, a novel substrate of Arabidopsis MPKs, could be induced by environmental stresses that result in osmotic stress. As previously reported, ZAT10 overexpressing transgenic plants showed enhanced tolerance to osmotic stress. However, in contrast to previous report, a zat10 knockout mutant showed an osmotic stress sensitive phenotype. To determine the biological function of EAR domain and phosphorylation sites of ZAT10, we constructed two transgenic plants expressing two ZAT10 mutant proteins having EAR domain mutations (ZAT10EAR) and phosphorylation site mutations (ZAT10AA). The phenotype of zat10 was complemented by the expression of ZAT10EAR mutant, however not by the expression of ZAT10AA mutant, indicating that the phosphorylation sites in ZAT10 by MPKs are involved in stress tolerance but the EAR domain is not. In this report, we suggest that ZAT10 function as a positive regulator in osmotic stress tolerance and the phosphorylation of ZAT10 is required for its function 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.

To survive salt stress, plants must maintain a balance between sodium and potassium ions. High-affinity potassium transporters (HKTs) play a key role in reducing Na+ toxicity through K+ uptake. Eutrema parvula (formerly known as Thellungiella parvula), a halophyte closely related to Arabidopsis, has two HKT1 genes that encode EpHKT1;1 and EpHKT1;2. In response to high salinity, the EpHKT1;2 transcript level increased rapidly; by contrast, the EpHKT1;1 transcript increased more slowly in response to salt treatment. Yeast cells expressing EpHKT1;2 were able to tolerate high concentrations of NaCl, whereas EpHKT1;1-expressing yeast cells remained sensitive to NaCl. Amino acid sequence alignment with other plant HKTs showed that EpHKT1;1 contains an asparagine residue (Asn-213) in the second pore-loop domain, but EpHKT1;2 contains an aspartic acid residue (Asp-205) at the same position. Yeast cells expressing EpHKT1;1, in which Asn-213 was substituted with Asp, were able to tolerate high concentrations of NaCl. In contrast, substitution of Asp-205 by Asn in EpHKT1;2 did not enhance salt tolerance and rather resulted in a similar function to that of AtHKT1 (Na+ influx but no K+ influx), indicating that the presence of Asn or Asp determines the mode of cation selectivity of the HKT1-type transporters. Moreover, Arabidopsis plants (Col-gl) overexpressing EpHKT1;2 showed significantly higher tolerance to salt stress and accumulated less Na+ and more K+ compared to those overexpressing EpHKT1;1 or AtHKT1. Taken together, these results suggest that EpHKT1;2 mediates tolerance to Na+ ion toxicity in E. parvula and is a major contributor to its halophytic nature.