Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies

A plant plasma membrane Ca(2+) pump is required for normal pollen tube growth and fertilization

Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca(2+) transporter as a key regulator of pollen development and fertilization in flowering plants

A Distinct Endosomal Ca2+/Mn2+ Pump Affects Root Growth through the Secretory Process1[C][W][OA]

Ca2+ is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca2+ pumps conferred the ability to grow on Ca2+-depleted medium and tolerance to toxic levels of Mn2+. A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca2+ and Mn2+ loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mm Ca2+, indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 μm Mn2+, indicating that AtECA3 is also important for the detoxification of excess Mn2+. Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca2+-stimulated root growth and the detoxification of high Mn2+, possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall

A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen

Ion signals are critical to regulating polarized growth in many cell types, including pollen in plants and neurons in animals. Genetic evidence presented here indicates that pollen tube growth requires cyclic nucleotide-gated channel (CNGC) 18. CNGCs are nonspecific cation channels found in plants and animals and have well established functions in excitatory signal transduction events in animals. In Arabidopsis, male sterility was observed for two cngc18 null mutations. CNGC18 is expressed primarily in pollen, as indicated from a promoter::GUS (β-glucuronidase) reporter analysis and expression profiling. The underlying cause of sterility was identified as a defect in pollen tube growth, resulting in tubes that were kinky, short, often thin, and unable to grow into the transmitting tract. Expression of a GFP-tagged CNGC18 in mutant pollen provided complementation and evidence for asymmetric localization of CNGC18 to the plasma membrane at the growing tip, starting at the time of pollen grain germination. Heterologous expression of CNGC18 in Escherichia coli resulted in a time- and concentration-dependent accumulation of more Ca(2+). Thus, CNGC18 provides a mechanism to directly transduce a cyclic nucleotide (cNMP) signal into an ion flux that can produce a localized signal capable of regulating the pollen tip-growth machinery. These results identify a CNGC that is essential to an organism's life cycle and raise the possibility that CNGCs have a widespread role in regulating cell-growth dynamics in both plant and animals

Subcellular Targeting of Nine Calcium-Dependent Protein Kinase Isoforms from Arabidopsis

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism