CIPK9 targets VDAC3 and modulates oxidative stress responses in Arabidopsis.

Calcium (Ca2+ ) is widely recognized as a key second messenger in mediating various plant adaptive responses. Here we show that calcineurin B-like interacting protein kinase CIPK9 along with its interacting partner VDAC3 identified in the present study are involved in mediating plant responses to methyl viologen (MV). CIPK9 physically interacts with and phosphorylates VDAC3. Co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer experiments proved their physical interaction in planta. Both cipk9 and vdac3 mutants exhibited a tolerant phenotype against MV-induced oxidative stress, which coincided with the lower-level accumulation of reactive oxygen species in their roots. In addition, the analysis of cipk9vdac3 double mutant and VDAC3 overexpressing plants revealed that CIPK9 and VDAC3 were involved in the same pathway for inducing MV-dependent oxidative stress. The response to MV was suppressed by the addition of lanthanum chloride, a non-specific Ca2+ channel blocker indicating the role of Ca2+ in this pathway. Our study suggest that CIPK9-VDAC3 module may act as a key component in mediating oxidative stress responses in Arabidopsis.

FDG PET/CT in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma.

Anaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin's lymphoma, with rearrangements of the anaplastic lymphoma kinase gene in 60%-85% of systemic cases. We report an 11-year-old boy with ALCL in whom serial FDG PET/CT revealed partial response and complete metabolic response at interim and end of treatment, respectively. However, the patient relapsed within 2 weeks, confirmed by cytology. Because of the inherent aggressive nature of ALCL, possibility of an early relapse should always be kept in mind.

A protein phosphatase 2C, AP2C1, interacts with and negatively regulates the function of CIPK9 under potassium-deficient conditions in Arabidopsis.

Potassium (K+) is a major macronutrient required for plant growth. An adaptive mechanism to low-K+ conditions involves activation of the Ca2+ signaling network that consists of calcineurin B-like proteins (CBLs) and CBL-interacting kinases (CIPKs). The CBL-interacting protein kinase 9 (CIPK9) has previously been implicated in low-K+ responses in Arabidopsis thaliana. Here, we report a protein phosphatase 2C (PP2C), AP2C1, that interacts with CIPK9. Fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and co-localization analyses revealed that CIPK9 and AP2C1 interact in the cytoplasm. AP2C1 dephosphorylates the auto-phosphorylated form of CIPK9 in vitro, presenting a regulatory mechanism for CIPK9 function. Furthermore, genetic and molecular analyses revealed that ap2c1 null mutants (ap2c1-1 and ap2c1-2) are tolerant to low-K+ conditions, retain higher K+ content, and show higher expression of K+-deficiency related genes contrary to cipk9 mutants (cipk9-1 and cipk9-2). In contrast, transgenic plants overexpressing AP2C1 were sensitive to low-K+ conditions. Thus, this study shows that AP2C1 and CIPK9 interact to regulate K+-deficiency responses in Arabidopsis. CIPK9 functions as positive regulator whereas AP2C1 acts as a negative regulator of Arabidopsis root growth and seedling development under low-K+ conditions.

Rice phytoglobins regulate responses under low mineral nutrients and abiotic stresses in Arabidopsis thaliana.

Just like animals, plants also contain haemoglobins (known as phytoglobins in plants). Plant phytoglobins (Pgbs) have been categorized into 6 different classes, namely, Phytogb0 (Pgb0), Phytogb1 (Pgb1), Phytogb2 (Pgb2), SymPhytogb (sPgb), Leghaemoglobin (Lb), and Phytogb3 (Pgb3). Among the 6 Phytogbs, sPgb and Lb have been functionally characterized, whereas understanding of the roles of other Pgbs is still evolving. In our present study, we have explored the function of 2 rice Pgbs (OsPgb1.1 and OsPgb1.2). OsPgb1.1, OsPgb1.2, OsPgb1.3, and OsPgb1.4 displayed increased level of transcript upon salt, drought, cold, and ABA treatment. The overexpression (OX) lines of OsPgb1.2 in Arabidopsis showed a tolerant phenotype in terms of better root growth in low potassium (K+ ) conditions. The expression of the known K+ gene markers such as LOX2, HAK5, and CAX3 was much higher in the OsPgb1.2 OX as compared to wild type. Furthermore, the OsPgb1.2 OX lines showed a decrease in reactive oxygen species (ROS) production and conversely an increase in the K+ content, both in root and shoot, as compared to wild type in K+ limiting condition. Our results indicated the potential involvement of OsPgb1.2 in signalling networks triggered by the nutrient deficiency stresses.

Alternative Splicing of CIPK3 Results in Distinct Target Selection to Propagate ABA Signaling in Arabidopsis.

Calcium (Ca2+) signaling is pivotal in transmission of information in the cell. Various Ca2+ sensing molecules work to sense and relay the encrypted messages to the intended targets in the cell to maintain this signal transduction. CBL-interacting protein kinases (CIPKs) are crucial components of Ca2+ signal transduction during various abiotic stresses. Although there are intron rich CIPKs in the plant genome but very little has been reported about their alternative splicing. Moreover the physiological significance of this event in the Ca2+ signaling is still elusive. Therefore in this study, we have selected CIPK3, which has highest number of splice variants amongst Arabidopsis CIPKs. Expression profiling of five splice variants of CIPK3 by qRT-PCR in four Arabidopsis thaliana ecotypes revealed preferential transcript accumulation but similar subcellular localization of the variants and interaction with similar CBLs. ABA and drought treatment resulted in the higher accumulation of the alternately spliced transcripts of CIPK3 in Arabidopsis ecotype Wassilewkija. The transcripts of CIPK3.1 and CIPK3.4 are relatively more induced compared to other alternative splice variants. Out of four splice variants studied, we found CIPK3.1 and CIPK3.2 showing preference for ABR1, a previously reported interactor of CIPK3. We conclude that the differential expression and choice of downstream partner by CIPK3-splice variants might be one of the mechanisms of Ca2+ mediated preferential regulation of ABA and other stress signals.

G-protein Signaling Components GCR1 and GPA1 Mediate Responses to Multiple Abiotic Stresses in Arabidopsis.

G-protein signaling components have been implicated in some individual stress responses in Arabidopsis, but have not been comprehensively evaluated at the genetic and biochemical level. Stress emerged as the largest functional category in our whole transcriptome analyses of knock-out mutants of GCR1 and/or GPA1 in Arabidopsis (Chakraborty et al., 2015a,b). This led us to ask whether G-protein signaling components offer converging points in the plant's response to multiple abiotic stresses. In order to test this hypothesis, we carried out detailed analysis of the abiotic stress category in the present study, which revealed 144 differentially expressed genes (DEGs), spanning a wide range of abiotic stresses, including heat, cold, salt, light stress etc. Only 10 of these DEGs are shared by all the three mutants, while the single mutants (GCR1/GPA1) shared more DEGs between themselves than with the double mutant (GCR1-GPA1). RT-qPCR validation of 28 of these genes spanning different stresses revealed identical regulation of the DEGs shared between the mutants. We also validated the effects of cold, heat and salt stresses in all the 3 mutants and WT on % germination, root and shoot length, relative water content, proline content, lipid peroxidation and activities of catalase, ascorbate peroxidase and superoxide dismutase. All the 3 mutants showed evidence of stress tolerance, especially to cold, followed by heat and salt, in terms of all the above parameters. This clearly shows the role of GCR1 and GPA1 in mediating the plant's response to multiple abiotic stresses for the first time, especially cold, heat and salt stresses. This also implies a role for classical G-protein signaling pathways in stress sensitivity in the normal plants of Arabidopsis. This is also the first genetic and biochemical evidence of abiotic stress tolerance rendered by knock-out mutation of GCR1 and/or GPA1. This suggests that G-protein signaling pathway could offer novel common targets for the development of tolerance/resistance to multiple abiotic stresses.