The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca(2+)) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca(2+) burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca(2+)-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca(2+) burst, supporting its role as a negative regulator of BIK1

Genome-Wide Association Study for Maize Leaf Cuticular Conductance Identifies Candidate Genes Involved in the Regulation of Cuticle Development.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (g c) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of g c of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with g c were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of g c in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of g c and have the potential to help breeders more effectively develop drought-tolerant maize for target environments

Characterisation of the calcium-dependent protein kinase CPK28 from Arabidopsis thaliana as a regulator of stem elongation and vascular development

Nach einer vegetativen Wachstumsphase vollziehen Pflanzen den reproduktiven Phasenwechsel, durch den bei Rosettenpflanzen wie Arabidopsis thaliana neben dem Blühen auch Elongation der Internodien und damit des Sprosses induziert wird. Diese Arbeit charakterisiert die calcium-anhängige Proteinkinase AtCPK28 als regulatorische Komponente, die die Sprosselongation und vaskuläre Entwicklung kontrolliert und damit spezifisch zur Pflanzenentwicklung nach dem Umschalten in die reproduktive Phase beiträgt. In zwei unabhängigen Mutanten- Allelen der cpk28 konnte, spezifisch nach dem Übergang in die reproduktive Phase, drastische Reduktion der Sprosselongation beobachtet werden, begleitet von der Verkürzung der Petiolen und Anthocyanakkumulation. Anatomische Analyse der basalen Sprossinternodien legte ein verändertes Muster der Sprossanatomie in cpk28 offen, charakterisiert durch Reduktion trachealer Xylemelemente und gleichzeitig verstärktem sekundärem Dickenwachstum mit ektopischer Lignifizierung. Übereinstimmend wurde in cpk28 erhöhte Expression von zellspezifischen Aktivatoren des sekundären Dickenwachstums im Spross beobachtet. Zusätzlich konnte ein Einfluss der CPK28-Funktion auf den Phytohormonstatus der Pflanze gezeigt werden. Der cpk28-Phänotyp konnte durch exogene Applikation von Gibberellinsäure (GA) partiell revertiert werden. Störungen im GA-Metabolismus wurden durch spezifisch in der reproduktiven Entwicklungsphase reduzierte Expression von Schlüsselenzymen der GA- Biosynthese in cpk28 bestätigt. Erste Analysen zum Zusammenhang zwischen CPK28 und dem Jasmonsäure-(JA)-Status der Pflanze lassen verstärkte JA- Signaltransduktion in cpk28 vermuten. Zum einen konnte erhöhte Expression von JA-Markergenen in cpk28 festgestellt werden, zum anderen wurde der cpk28-Sprosselongationsphänotyp in Doppelmutanten durch Aufhebung der JA- Biosynthese bzw. der JA-Signaltransduktion vollständig revertiert. Calcium- abhängige Kinaseaktivität der CPK28 konnte in vitro bestätigt werden. Außerdem führte Expression der aktiven Kinase im cpk28-Hintergrund zur vollständigen Reversion des Entwicklungsphänotyps, während Expression der inaktiven Kinase CPK28-D188A keine Veränderung bewirkte. Dies belegt die essentielle Rolle der CPK28 für die normale Pflanzenentwicklung. CPK28-Expression unter den gewebespezifischen Promotoren pSUC2 und pKNAT1, die Proteinexpression in anderen Geweben vermitteln als in dieser Arbeit für den CPK28-Promoter gezeigt, war ebenfalls ausreichend für die vollständige Komplementation des cpk28-Phänotyps. Dies lässt vermuten, dass mit Hilfe der CPK28-Aktivität möglicherweise ein mobiles Signal zur Sprosselongation generiert oder weitervermittelt wird. Weiterhin konnte in vivo-Autophosphorylierung der CPK28 an drei Phosphorylierungs-stellen gezeigt werden. Austausch der jeweiligen Aminosäuren führte für zwei der drei untersuchten Stellen zu reduzierter, weiterhin calcium-abhängiger Kinaseaktivität in vitro. Allerdings konnte unabhängig von der in vitro-Aktivität für jede der untersuchten Phosphorylierungsvarianten Komplementation der morphologischen Defekte von cpk28 in vivo beobachtet werden. AtCPK28 als Regulator der koordinierten Sprosselongation und des sekundären Dickenwachstums stellt damit eines der wenigen Beispiele dar, das Calcium-Signaltransduktion mittels CDPKs direkt mit einem wichtigen, stadienspezifischen Entwicklungsprozess in Verbindung bringt.After a period of vegetative growth plants undergo a developmental switch to the reproductive phase, inducing flowering and, in plants with a rosette habit like Arabidopsis thaliana, the transition to bolting and elongation of the inflorescence stem. This work identified the calcium-dependent protein kinase AtCPK28 as a regulatory component controlling stem elongation and vascular development, specifically contributing to plant development after this switch to the generative phase. In two independent mutant alleles of cpk28, a severe reduction of stem elongation, accompanied by shorter leaf petioles and enhanced anthocyanin levels, was observed upon the transition to the reproductive phase. Anatomical analysis of the basal internode of the stem revealed an altered vascular pattern in cpk28, characterised by fewer xylem tracheary elements and increased secondary growth and ectopic lignification. Coincident, cpk28 mutants showed enhanced expression of cell type-specific key activators of secondary growth in the stem. Additionally, an impact of CPK28 function on the phytohormone status of the plant was demonstrated. The cpk28 phenotype was partially reverted by exogenous application of gibberellic acid (GA). Disturbances in GA metabolism were confirmed by transcriptional repression of key regulators of GA homeostasis in cpk28, specifically at later stages of plant development. First analyses of the influence of CPK28 on the jasmonic acid (JA) status indicate increased JA signalling in cpk28, since expression of JA marker genes was enhanced in cpk28 and its shoot elongation phenotype could be fully reverted in double mutants blocking JA synthesis or JA signalling. In vitro protein kinase activity of CPK28 was demonstrated to be strictly calcium-dependent. Expression of active kinase in the cpk28 background led to a complete restoration of the phenotype while inactive protein CPK28-D188A did not, proving the essential role of CPK28 for plant development. CPK28 expression under the tissue specific promoters pSUC2 and pKNAT1, driving protein expression in defined tissues different from the expression pattern conferred by the CPK28 promoter, was also sufficient for complementation of the phenotype. This might be due to a mobile signal generated or propagated with the help of CPK28 activity. Furthermore, CPK28 was phosphorylated in vivo at several sites. Site-specific amino acid substitutions at two of the three phosphorylation sites resulted in reduced in vitro activity. However, when introduced into a cpk28 mutant background, all phosphorylation site variants complemented the morphological and developmental defects. AtCPK28 as a regulator for coordinated stem elongation and secondary growth represents one of the few examples directly linking calcium signalling via CDPKs to an important stage-specific developmental process

Experimental Data: Endocytosis is a means of uranium(VI) uptake in tobacco (Nicotiana tabacum) BY-2 cells

The interaction of tobacco (Nicotiana tabacum) BY-2 cells with uranyl(VI) nitrate in phosphate-deficient medium was investigated. The hypothesis was that endocytosis is a means of uranium uptake in these cells. Analysis was in the form of physiological studies (growth and viability), electron microscopy, proteomics and biochemical studies

Integrating GWAS and TWAS to elucidate the genetic architecture of maize leaf cuticular conductance

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments

Genome-Wide Association Study for Maize Leaf Cuticular Conductance Identifies Candidate Genes Involved in the Regulation of Cuticle Development.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (g c) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of g c of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with g c were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of g c in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of g c and have the potential to help breeders more effectively develop drought-tolerant maize for target environments

Genome-Wide Association Study for Maize Leaf Cuticular Conductance Identifies Candidate Genes Involved in the Regulation of Cuticle Development

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of gc of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with gc were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of gc in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of gc and have the potential to help breeders more effectively develop drought-tolerant maize for target environments

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation

The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover

Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase