The potassium channel GhAKT2bD is regulated by CBL-CIPK calcium signalling complexes and facilitates K+ allocation in cotton.

Efficient allocation of the essential nutrient potassium (K+ ) is a central determinant of plant ion homeostasis and involves AKT2 K+ channels. Here, we characterize four AKT2 K+ channels from cotton and report that xylem and phloem expressed GhAKT2bD facilitates K+ allocation and that AKT2-silencing impairs plant growth and development. We uncover kinase activity-dependent activation of GhAKT2bD-mediated K+ uptake by AtCBL4-GhCIPK1 calcium signalling complexes in HEK293T cells. Moreover, AtCBL4-AtCIPK6 complexes known to convey activation of AtAKT2 in Arabidopsis also activate cotton GhAKT2bD in HEK293T cells. Collectively, these findings reveal an essential role for AKT2 in the source-sink allocation of K+ in cotton and identify GhAKT2bD as subject to complex regulation by CBL-CIPK Ca2+ sensor-kinase complexes.

Ca2+ signaling in plant responses to abiotic stresses.

Adverse variations of abiotic environmental cues that deviate from an optimal range impose stresses to plants. Abiotic stresses severely impede plant physiology and development. Consequently, such stresses dramatically reduce crop yield and negatively impact on ecosystem stability and composition. Physical components of abiotic stresses can be, for example, suboptimal temperature and osmotic perturbations, while representative chemical facets of abiotic stresses can be toxic ions or suboptimal nutrient availability. The sheer complexity of abiotic stresses causes a multitude of diverse components and mechanisms for their sensing and signal transduction. Ca2+ , as a versatile second messenger, plays multifaceted roles in almost all abiotic stress responses in that, for a certain abiotic stress, Ca2+ is not only reciprocally connected with its perception, but also multifunctionally ensures subsequent signal transduction. Here, we will focus on salt/osmotic stress and responses to altered nutrient availability as model cases to detail novel insights into the identity of components that link stress perception to Ca2+ signal formation as well as on new insights into mechanisms of Ca2+ signal implementation. Finally, we will deduce emerging conceptual consequences of these novel insights and outline arising avenues of future research on the role of Ca2+ signaling in abiotic stress responses in plants.

Emerging roles of the CBL-CIPK calcium signaling network as key regulatory hub in plant nutrition.

Plant physiology and development essentially depend on sufficient uptake of various essential nutritive ions via their roots and their appropriate transport and distribution within the organism. Many of these essential nutrients are heterogeneously distributed in the soil or are available in fluctuating concentrations. This natural situation requires constant regulatory adjustment and balancing of nutrient uptake and homeostasis. Here, we review recent findings on the role of Ca2+ signals and Ca2+-dependent regulation via the CBL-CIPK Ca2+ sensor-protein kinase network in these processes. We put special emphasis on Ca2+ controlled processes that contribute to establishing the homeostasis of macro-nutrients like potassium (K+), nitrogen (N), and magnesium (Mg2+) and on the micro-nutrient iron (Fe). Increasing experimental evidence indicates the occurrence of nutrient-specific, spatially and temporally defined cytoplasmic Ca2+ elevations as early responses to nutrient fluctuations. Specific CBL-CIPK complexes translate these signals into phosphorylation regulation of important channels and transporters like AKT1, NPF6.3/NRT1.1, AMT1, SLAC1, TPK1 and IRT1. We discuss a crucial and coordinating role for these Ca2+ signaling mechanisms in regulating the sensing, uptake, distribution and storage of various ions. Finally, we reflect on the emerging multifaceted and potentially integrating role of the "nutrient" kinase CIPK23 in regulating multiple nutrient responses. From this inventory, we finally deduce potential mechanisms that can convey the coordinated regulation of distinct steps in the transport of one individual ion and mechanisms that can bring about the integration of adaptive responses to fluctuations of different ions to establish a faithfully balanced plant nutrient homeostasis.

Lewis acid-catalyzed domino generation/[2,3]-sigmatropic rearrangement of ammonium ylides to access chiral azabicycles.

[2,3]-Sigmatropic rearrangement of ammonium ylides represents a fundamental reaction for stereoselective synthesis of nitrogenous compounds. However, its applicability is limited by the scarcity of efficient, catalytic, and mild methods for generating ammonium ylides. Here, we report silver-catalyzed domino generation/[2,3]-sigmatropic rearrangement of ammonium ylides, furnishing chiral azabicycles with bridgehead quaternary stereogenic centers in high enantiomeric purity (up to 99% ee). A combination of density functional theory calculations and experimental studies revealed that residual water in the reaction system is crucial for the mild reaction conditions by functioning as a proton shuttle to assist carbon-silver bond protonation and C2─H deprotonation to generate the ammonium ylide. This reaction has a broad application scope. Besides the diverse substituents, N-fused azabicycles of various ring sizes are also easily accessed. In addition to silver salts, this strategy has also been successfully implemented by using a stoichiometric amount of nonmetallic I2.

The Ca2+ Sensor SCaBP3/CBL7 Modulates Plasma Membrane H+-ATPase Activity and Promotes Alkali Tolerance in Arabidopsis.

Saline-alkali soil is a major environmental constraint impairing plant growth and crop productivity. In this study, we identified a Ca2+ sensor/kinase/plasma membrane (PM) H+-ATPase module as a central component conferring alkali tolerance in Arabidopsis (Arabidopsis thaliana). We report that the SCaBP3 (SOS3-LIKE CALCIUM BINDING PROTEIN3)/CBL7 (CALCINEURIN B-LIKE7) loss-of-function plants exhibit enhanced stress tolerance associated with increased PM H+-ATPase activity and provide fundamental mechanistic insights into the regulation of PM H+-ATPase activity. Consistent with the genetic evidence, interaction analyses, in vivo reconstitution experiments, and determination of H+-ATPase activity indicate that interaction of the Ca2+ sensor SCaBP3 with the C-terminal Region I domain of the PM H+-ATPase AHA2 (Arabidopsis thaliana PLASMA MEMBRANE PROTON ATPASE2) facilitates the intramolecular interaction of the AHA2 C terminus with the Central loop region of the PM H+-ATPase to promote autoinhibition of H+-ATPase activity. Concurrently, direct interaction of SCaPB3 with the kinase PKS5 (PROTEIN KINASE SOS2-LIKE5) stabilizes the kinase-ATPase interaction and thereby fosters the inhibitory phosphorylation of AHA2 by PKS5. Consistently, yeast reconstitution experiments and genetic analysis indicate that SCaBP3 provides a bifurcated pathway for coordinating intramolecular and intermolecular inhibition of PM H+-ATPase. We propose that alkaline stress-triggered Ca2+ signals induce SCaBP3 dissociation from AHA2 to enhance PM H+-ATPase activity. This work illustrates a versatile signaling module that enables the stress-responsive adjustment of plasma membrane proton fluxes.

Enantioselective Synthesis of ABCF Tetracyclic Framework of Daphniphyllum Alkaloid Calyciphylline N.

Efforts toward the enantioselective synthesis of Daphniphyllum alkaloid calyciphylline N which leads to efficient preparation of the ABCF tetracyclic framework containing three bridgehead all-carbon quaternary stereocenters are described. This synthetic work features the utilization of an asymmetric conjugate addition to install the C5 all-carbon quaternary center, an efficient successive inter/intramolecular aldol sequence to build the critical bicyclo[2.2.2]octanone BC core, and a ring closing metathesis reaction followed by stereoselective Nagata conjugate cyanation to deliver the functionalized F ring.

N-myristoylation and S-acylation are common modifications of Ca2+ -regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel.

N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+ -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae.

The WRKY transcription factors are involved in plant resistance against both biotrophic and necrotrophic pathogens. Arabidopsis WRKY46 is specifically induced by salicylic acid (SA) and biotrophic pathogen Pseudomonas syringae infection. To determine its possible roles in plant defense and elucidate potential functional redundancy with structurally related WRKY70 and WRKY53, we examined loss-of-function T-DNA insertion single, double and triple mutants, as well as gain-of-function transgenic WRKY46 over-expressing plants in response to P. syringae. WRKY46 over-expressing plants were more resistant to P. syringae. In contrast, pathogen-infected wrky46wrky70, wrky46wrky53 double mutants and wrky46wrky70wrky53 triple mutants showed increased susceptibility to this pathogen, with increased bacterial growth and more severe disease symptoms. The contrasting responses of gain-of-function plants and loss-of-function mutants were correlated with increased or reduced expression of defense-related PR1 gene. Expression studies of WRKY46, WRKY70, and WRKY53 in various defense-signaling mutants suggested that they are partially involved in SA-signaling pathway. In addition, our findings demonstrated negative cross-regulation among these three genes. These results indicate that WRKY46, WRKY70, and WRKY53 positively regulate basal resistance to P. syringae; and that they play overlapping and synergetic roles in plant basal defense.