[Show abstract][Hide abstract] ABSTRACT: Plants use solar energy to produce lipids directly from inorganic elements and are not thought to require molecular systems for lipid uptake from the environment. Here we show that Arabidopsis thaliana Aminophospholipid ATPase10 (ALA10) is a P4-type ATPase flippase that internalizes exogenous phospholipids across the plasma membrane, after which they are rapidly metabolized. ALA10 expression and phospholipid uptake are high in the epidermal cells of the root tip and in guard cells, the latter of which regulate the size of stomatal apertures to modulate gas exchange. ALA10-knockout mutants exhibit reduced phospholipid uptake at the root tips and guard cells and are affected in growth and transpiration. The presence of a phospholipid uptake system in plants is surprising. Our results suggest that one possible physiological role of this system is to internalize lysophosphatidylcholine, a signalling lipid involved in root development and stomatal control.
[Show abstract][Hide abstract] ABSTRACT: Microalgae can serve as useful feedstocks for biofuel production as they can be grown with fresh, brackish, or salt water and their lipid and starch contents can be manipulated to create customized feedstocks for different classes of biofuels. Continuous buoyant density gradient centrifugation (CBDGC) was used to perform reiterative, transgressive selection to isolate wildtype and ethyl methanesulfonate-mutagenized Dunaliella salina cells with enhanced lipid and starch production. Sixty rounds of transgressive selection resulted in the isolation of cell populations with significantly lower or higher buoyant densities. Lipid content in the low-density populations was enhanced by 1.2- to 2.9-fold in wildtype cells and 1.3- to 2.3-fold in mutagenized cells as measured by Nile Red dye staining, but the lipid content differences were not significant when quantified by liquid chromatography–tandem mass spectroscopy possibly due to the composition of the lipid pools measured by these contrasting techniques. In contrast, starch content in the high-density populations was increased by 2-fold in wild type cells and 1.4- to 1.6-fold in mutagenized cells, respectively. The observed alterations in lipid and starch contents appeared to be stable after more than 70 weeks (392 cell generations). CBDGC-based selection provides a useful and accessible technological alternative to genetic engineering approaches for the customization of microbial biofuel feedstocks.
[Show abstract][Hide abstract] ABSTRACT: Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions.
[Show abstract][Hide abstract] ABSTRACT: Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis
thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues—highly conserved in other ACA isoforms—localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.
Full-text · Article · Oct 2013 · Plant Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina and disruption of the multilayered retinal architecture leading to distorted vision and blindness. During disease progression in PVR, retinal pigmented epithelial cells (RPE) lose cell-cell adhesion, undergo epithelial-to-mesenchymal transition (EMT), and deposit ECM leading to tissue fibrosis. The EMT process is mediated via exposure to vitreous cytokines and growth factors such as TGF-β2. Previous studies have shown that Na,K-ATPase is required for maintaining a normal polarized epithelial phenotype and that decreased Na,K-ATPase function and subunit levels are associated with TGF-β1-mediated EMT in kidney cells. In contrast to the basolateral localization of Na,K-ATPase in most epithelia, including kidney, Na,K-ATPase is found on the apical membrane in RPE cells. We now show that EMT is also associated with altered Na,K-ATPase expression in RPE cells. TGF-β2 treatment of ARPE-19 cells resulted in a time-dependent decrease in Na,K-ATPase β1 mRNA and protein levels while Na,K-ATPase α1 levels, Na,K-ATPase activity, and intracellular sodium levels remained largely unchanged. In TGF-β2-treated cells reduced Na,K-ATPase β1 mRNA inversely correlated with HIF-1α levels and analysis of the Na,K-ATPase β1 promoter revealed a putative hypoxia response element (HRE). HIF-1α bound to the Na,K-ATPase β1 promoter and inhibiting the activity of HIF-1α blocked the TGF-β2 mediated Na,K-ATPase β1 decrease suggesting that HIF-1α plays a potential role in Na,K-ATPase β1 regulation during EMT in RPE cells. Furthermore, knockdown of Na,K-ATPase β1 in ARPE-19 cells was associated with a change in cell morphology from epithelial to mesenchymal and induction of EMT markers such as α-smooth muscle actin and fibronectin, suggesting that loss of Na,K-ATPase β1 is a potential contributor to TGF-β2-mediated EMT in RPE cells.
Full-text · Article · Jun 2013 · Experimental Eye Research
[Show abstract][Hide abstract] ABSTRACT: Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.
[Show abstract][Hide abstract] ABSTRACT: The concentrations of mineral nutrients in seeds are critical to both the life cycle of plants as well as human nutrition. These concentrations are strongly influenced by soil conditions, as shown here by quantifying the concentration of 14 elements in seeds from Arabidopsis thaliana plants grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. Each of the modified soils resulted in a unique change to the seed ionome (the mineral nutrient content of the seeds). To help identify the genetic networks regulating the seed ionome, changes in elemental concentrations were evaluated using mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes supports an estimate that as much as 11% of the A. thaliana genome encodes proteins of functional relevance to ion homeostasis in seeds. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280 and CNGC2. In a comparison of nine different accessions to a Col-0 reference, eight accessions were observed to have reproducible differences in elemental concentrations, seven of which were dependent on specific soil conditions. These results indicate that the A. thaliana seed ionome is distinct from the vegetative ionome, and that elemental analysis is a sensitive approach to identify genes controlling ion homeostasis, including those that regulate gene expression, phospho-regulation, and ion transport.
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis thaliana genome contains 20 CNGCs, which are proposed to encode cyclic nucleotide gated, non-selective, Ca(2+)-permeable ion channels. CNGC7 and CNGC8 are the two most similar with 74% protein sequence identity, and both genes are preferentially expressed in pollen. Two independent loss-of-function T-DNA insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., cngc7-1+/-/cngc8-2-/- and cngc7-3-/-/cngc8-1+/-). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both cngc7 and 8 were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring T-DNA disruptions of both cngc7 and 8 displayed a high frequency of bursting when germinated in vitro. The male sterile defect could be rescued through pollen expression of a CNGC7 or 8 transgene including a CNGC7 with an N-terminal GFP-tag. However, rescue efficiencies were reduced ∼10-fold when the CNGC7 or 8 included an F to W substitution (F589W and F624W, respectively) at the junction between the putative cyclic nucleotide binding-site and the calmodulin binding-site, identifying this junction as important for proper functioning of a plant CNGC. Using confocal microscopy, GFP-CNGC7 was found to preferentially localize to the plasma membrane at the flanks of the growing tip. Together these results indicate that CNGC7 and 8 are at least partially redundant and provide an essential function at the initiation of pollen tube tip growth.
[Show abstract][Hide abstract] ABSTRACT: Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3',5'-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca(2+)-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response.
No preview · Article · Feb 2013 · Plant physiology
[Show abstract][Hide abstract] ABSTRACT: Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKβ and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His(6)-GmCDPKβ. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca(2+)/CDPK-mediated processes. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS: GmCDPKβphosphorylatesGmCDPKβ by protein kinase assay (View interaction).
[Show abstract][Hide abstract] ABSTRACT: Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae) were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauri and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a non-vascular moss), Selaginella moellendorffii (a primitive vascular plant), and Arabidopsis thaliana (a model flowering plant). Each organism contained sequences for all five subfamilies of P-type ATPases. Whereas Na(+) and H(+) pumps seem to mutually exclude each other in flowering plants and animals, they co-exist in chlorophytes, which show representatives for two kinds of Na(+) pumps (P2C and P2D ATPases) as well as a primitive H(+)-ATPase. Both Na(+) and H(+) pumps also co-exist in the moss P. patens, which has a P2D Na(+)-ATPase. In contrast to the primitive H(+)-ATPases in chlorophytes and P. patens, the H(+)-ATPases from vascular plants all have a large C-terminal regulatory domain as well as a conserved Arg in transmembrane segment 5 that is predicted to function as part of a backflow protection mechanism. Together these features are predicted to enable H(+) pumps in vascular plants to create large electrochemical gradients that can be modulated in response to diverse physiological cues. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps.
Full-text · Article · Feb 2012 · Frontiers in Plant Science
[Show abstract][Hide abstract] ABSTRACT: Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.
No preview · Article · Feb 2012 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: 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 Ca(2+)-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 K(M) ∼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.
Full-text · Article · Aug 2011 · Frontiers in Plant Science
[Show abstract][Hide abstract] ABSTRACT: The cytoplasmic Ca(2+) signals that participate in nearly all aspects of plant growth and development encode information as binary switches or information-rich signatures. They are the result of influx (thermodynamically passive) and efflux (thermodynamically active) activities mediated by membrane transport proteins. On the influx side, confirming the molecular identities of Ca(2+)-permeable channels is still a major research topic. Cyclic nucleotide-gated channels and glutamate receptor-like channels are candidates well supported by evidence. On the efflux side, CAX antiporters and P-type ATPase pumps are the principal molecular entities. Both of these active transporters load Ca(2+) into specific compartments and have the potential to reduce the magnitude and duration of a Ca(2+) transient. Recent studies indicate calmodulin-activated Ca(2+) pumps in endomembrane systems can dampen the magnitude and duration of a Ca(2+) transient that could otherwise grow into a Ca(2+) cell death signature. An important challenge following molecular characterization of the influx and efflux pathways is to understand how they are coordinately regulated to produce a Ca(2+) switch or encode specific information into a Ca(2+) signature.
Full-text · Article · Aug 2011 · Current opinion in plant biology
[Show abstract][Hide abstract] ABSTRACT: Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.
[Show abstract][Hide abstract] ABSTRACT: Controlling elemental composition is critical for plant growth and development as well as the nutrition of humans who utilize plants for food. Uncovering the genetic architecture underlying mineral ion homeostasis in plants is a critical first step towards understanding the biochemical networks that regulate a plant's elemental composition (ionome). Natural accessions of Arabidopsis thaliana provide a rich source of genetic diversity that leads to phenotypic differences. We analyzed the concentrations of 17 different elements in 12 A. thaliana accessions and three recombinant inbred line (RIL) populations grown in several different environments using high-throughput inductively coupled plasma- mass spectroscopy (ICP-MS). Significant differences were detected between the accessions for most elements and we identified over a hundred QTLs for elemental accumulation in the RIL populations. Altering the environment the plants were grown in had a strong effect on the correlations between different elements and the QTLs controlling elemental accumulation. All ionomic data presented is publicly available at www.ionomicshub.org.
[Show abstract][Hide abstract] ABSTRACT: Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-beta plays a central role in the induction of EMT, the targets of TGF-beta signaling are poorly defined. We have shown earlier that Na,K-ATPase beta(1)-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-beta(1)-mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-beta(1), the surface expression of the beta(1)-subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase beta(1)-subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase beta(1)-subunit attenuated TGF-beta(1)-mediated EMT. We further show that both Na,K-ATPase alpha- and beta-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-beta(1)-mediated EMT and is associated with the progression of EMT in cancer and fibrosis.
Full-text · Article · Jun 2010 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: The reproductive (gametophytic) phase in flowering plants is often highly sensitive to hot or cold temperature stresses, with
even a single hot day or cold night sometimes being fatal to reproductive success. This review describes studies of temperature
stress on several crop plants, which suggest that pollen development and fertilization may often be the most sensitive reproductive
stage. Transcriptome and proteomic studies on several plant species are beginning to identify stress response pathways that
function during pollen development. An example is provided here of genotypic differences in the reproductive stress tolerance
between two ecotypes of Arabidopsis thaliana Columbia (Col) and Hilversum (Hi-0), when reproducing under conditions of hot days and cold nights. Hi-0 exhibited a more
severe reduction in seed set, correlated with a reduction in pollen tube growth potential and tropism defects. Hi-0 thus provides
an Arabidopsis model to investigate strategies for improved stress tolerance in pollen. Understanding how different plants cope with stress
during reproductive development offers the potential to identify genetic traits that could be manipulated to improve temperature
tolerance in selected crop species being cultivated in marginal climates.
Full-text · Article · Mar 2010 · Journal of Experimental Botany
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.
[Show abstract][Hide abstract] ABSTRACT: Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca(2+) signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca(2+)-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17-yellow fluorescent protein (YFP) and CPK34-YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34-YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca(2+)-activation mechanism of CPK34 (CPK34-YFP-E465A,E500A), providing in vivo evidence linking Ca(2+) activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca(2+) signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.
Full-text · Article · May 2009 · The Plant Journal