Plasma membrane calcineurin B-like calcium-ion sensor proteins function in regulating primary root growth and nitrate uptake by affecting global phosphorylation patterns and microdomain protein distribution.

The collective function of calcineurin B-like (CBL) calcium ion (Ca2+ ) sensors and CBL-interacting protein kinases (CIPKs) in decoding plasma-membrane-initiated Ca2+ signals to convey developmental and adaptive responses to fluctuating nitrate availability remained to be determined. Here, we generated a cbl-quintuple mutant in Arabidopsis thaliana devoid of these Ca2+ sensors at the plasma membrane and performed comparative phenotyping, nitrate flux determination, phosphoproteome analyses, and studies of membrane domain protein distribution in response to low and high nitrate availability. We observed that CBL proteins exert multifaceted regulation of primary and lateral root growth and nitrate fluxes. Accordingly, we found that loss of plasma membrane Ca2+ sensor function simultaneously affected protein phosphorylation of numerous membrane proteins, including several nitrate transporters, proton pumps, and aquaporins, as well as their distribution within plasma membrane microdomains, and identified a specific phosphorylation and domain distribution pattern during distinct phases of low and high nitrate responses. Collectively, these analyses reveal a central and coordinative function of CBL-CIPK-mediated signaling in conveying plant adaptation to fluctuating nitrate availability and identify a crucial role of Ca2+ signaling in regulating the composition and dynamics of plasma membrane microdomains.

A role for calcium-dependent protein kinases in differential CO2 - and ABA-controlled stomatal closing and low CO2 -induced stomatal opening in Arabidopsis.

Low concentrations of CO2 cause stomatal opening, whereas [CO2 ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2 -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+ -binding proteins that function in the stomatal CO2 response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2 , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2 -induced stomatal closure and, unexpectedly, also in low CO2 -induced stomatal opening. Furthermore, K+ -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2 -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.

Golgi α1,4-fucosyltransferase of Arabidopsis thaliana partially localizes at the nuclear envelope.

We analyzed plant-derived α1,4-fucosyltransferase (FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc-green fluorescent protein (GFP) or tomato LeFucTc-GFP restored Lewis-a formation in a fuctc mutant, confirming functionality in the trans-Golgi. AtFucTc-GFP partly accumulated at the nuclear envelope (NE) not observed for other homologs or truncated AtFucTc lacking the N-terminus or catalytic domain. Analysis of At/LeFucTc-GFP swap constructs with exchanged cytosolic, transmembrane and stalk (CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino-acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N-terminal GFP copies did, indicating localization at the inner nuclear membrane (INM). Tunicamycin treatment of AtFucTc-GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N-glycosylation. Yet neither expression in protoplasts of Arabidopsis N-glycosylation mutants nor elimination of the N-glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum (ER)-to-Golgi transport by co-expression of Sar1(H74L) trapped tunicamycin-released AtFucTc-GFP in the ER, however, without NE localization. Since recovery after tunicamycin-washout required de novo-protein synthesis, our analyses suggest that AtFucTc localizes to the NE/INM due to interaction with an unknown (glyco)protein.

Two spatially and temporally distinct Ca2+ signals convey Arabidopsis thaliana responses to K+ deficiency.

In plants, potassium (K+ ) homeostasis is tightly regulated and established against a concentration gradient to the environment. Despite the identification of Ca2+ -regulated kinases as modulators of K+ channels, the immediate signaling and adaptation mechanisms of plants to low-K+ conditions are only partially understood. To assess the occurrence and role of Ca2+ signals in Arabidopsis thaliana roots, we employed ratiometric analyses of Ca2+ dynamics in plants expressing the Ca2+ reporter YC3.6 in combination with patch-clamp analyses of root cells and two-electrode voltage clamp (TEVC) analyses in Xenopus laevis oocytes. K+ deficiency triggers two successive and distinct Ca2+ signals in roots exhibiting spatial and temporal specificity. A transient primary Ca2+ signature arose within 1 min in the postmeristematic stelar tissue of the elongation zone, while a secondary Ca2+ response occurred after several hours as sustained Ca2+ elevation in defined tissues of the elongation and root hair differentiation zones. Patch-clamp and TEVC analyses revealed Ca2+ dependence of the activation of the K+ channel AKT1 by the CBL1-CIPK23 Ca2+ sensor-kinase complex. Together, these findings identify a critical role of cell group-specific Ca2+ signaling in low K+ responses and indicate an essential and direct role of Ca2+ signals for AKT1 K+ channel activation in roots.

Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast.

Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.

Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid.

Living organisms sense and respond to changes in nutrient availability to cope with diverse environmental conditions. Nitrate (NO3-) is the main source of nitrogen for plants and is a major component in fertilizer. Unraveling the molecular basis of nitrate sensing and regulation of nitrate uptake should enable the development of strategies to increase the efficiency of nitrogen use and maximize nitrate uptake by plants, which would aid in reducing nitrate pollution. NPF6.3 (also known as NRT1.1), which functions as a nitrate sensor and transporter; the kinase CIPK23; and the calcium sensor CBL9 form a complex that is crucial for nitrate sensing in Arabidopsis thaliana. We identified two additional components that regulate nitrate transport, sensing, and signaling: the calcium sensor CBL1 and protein phosphatase 2C family member ABI2, which is inhibited by the stress-response hormone abscisic acid. Bimolecular fluorescence complementation assays and in vitro kinase assays revealed that ABI2 interacted with and dephosphorylated CIPK23 and CBL1. Coexpression studies in Xenopus oocytes and analysis of plants deficient in ABI2 indicated that ABI2 enhanced NPF6.3-dependent nitrate transport, nitrate sensing, and nitrate signaling. These findings suggest that ABI2 may functionally link stress-regulated control of growth and nitrate uptake and utilization, which are energy-expensive processes.

Visualization and translocation of ternary Calcineurin-A/Calcineurin-B/Calmodulin-2 protein complexes by dual-color trimolecular fluorescence complementation.

Fluorescence complementation (FC) techniques are expedient for analyzing bimolecular protein-protein interactions. Here we aimed to develop a method for visualization of ternary protein complexes using dual-color trimolecular fluorescence complementation (TriFC). Dual-color TriFC combines protein fragments of mCherry and mVenus, in which a scaffold protein is bilaterally fused to C-terminal fragments of both fluorescent proteins and combined with potential interacting proteins fused to an N-terminal fluorescent protein fragment. For efficient visual verification of ternary complex formation, TriFC was combined with a cytoplasm to plasma membrane translocation assay. Modular vector sets were designed which are fully compatible with previously reported bimolecular fluorescence complementation (BiFC) vectors. As a proof-of-principle, the ternary complex formation of the PP2B protein phosphatase Calcineurin-A/Calcineurin-B with Calmodulin-2 was investigated in transiently transformed Nicotiana benthamiana leaf epidermal cells. The results indicate a Calcineurin-B-induced interaction of Calmodulin-2 with Calcineurin-A. TriFC and the translocation of TriFC complexes provide a novel tool to investigate ternary complex formations with the simplicity of a BiFC approach. The robustness of FC applications and the opportunity to quantify fluorescence complementation render this assay suitable for a broad range of interaction analyses.

Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid.

Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B-like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA.

The vacuolar calcium sensors CBL2 and CBL3 affect seed size and embryonic development in Arabidopsis thaliana.

Stimulus-specific calcium (Ca(2+) ) signals have crucial functions in developmental processes in many organisms, and are deciphered by various Ca(2+) -binding proteins. In Arabidopsis thaliana, a signaling network consisting of calcineurin B-like (CBL) protein calcium sensors and CBL-interacting protein kinases (CIPKs) has been shown to fulfil pivotal functions at the plasma membrane in regulating ion fluxes and abiotic stress responses. However, the role of tonoplast-localized CBL proteins and especially their function in regulating developmental programs remains largely unknown. In this study, we analyzed single and double mutants of the closely related tonoplast-localized calcium sensors CBL2 and CBL3, which show either reduction of function (rf) or complete loss of function (lf). While single cbl2 or cbl3 mutants did not display discernable phenotypes, cbl2/cbl3 mutants exhibited defects in vegetative growth and were severely impaired in seed development and morphology. Seeds of the cbl2/3rf mutant were smaller in size and exhibited reduced weight and fatty acid content compared to wild-type, but accumulation of sucrose was not altered. Moreover, accumulation of inositol hexakisphosphate (InsP6 ), the major storage form of phosphorus in seeds, was significantly reduced in mutant seeds. In addition, complete loss of CBL2 and CBL3 function in cbl2/3lf resulted in a high frequency of severe defects in embryonic development. Together, our findings reveal a crucial function of Ca(2+) -controlled processes at the vacuolar membrane as determinants of seed yield and size, and demonstrate the importance of vacuolar CBL calcium sensors for plant embryogenesis.

Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex.

Potassium (K(+)) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K(+) channels remain poorly understood. Here, we show that the calcium (Ca(2+)) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K(+) channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca(2+)-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca(2+) sensor modulates K(+) channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.