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Papers by Wayne Snedden

Plant physiology

Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of pla... more Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohex...

Plant molecular biology, 1998

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, d... more The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflores...

Plant molecular biology, 1997

This report describes the cloning and characterization of a plant cDNA coding for a protein which... more This report describes the cloning and characterization of a plant cDNA coding for a protein which shows high amino acid sequence similarity with prohibitin, whose gene is associated with antiproliferative activity in mammalian cells. Arabidopsis thaliana and Nicotiana tabacum prohibitin complete cDNAs were isolated, and the expression pattern of prohibitin was examined using polyclonal antibodies raised against the Arabidopsis recombinant prohibitin expressed in Escherichia coli. A single immunoreactive protein was detected in various plant species and in all Arabidopsis organs examined. Subcellular fractionation using tobacco leaves revealed prohibitin in a mitochondrial-enriched fraction. Phylogenetic conservation of prohibitin's amino acid sequence and subcellular localization suggests a similar function in plants, yeast and mammals.

Planta, 2010

The de novo biosynthesis of the triphosphopyridine NADP is catalyzed solely by the ubiquitous NAD... more The de novo biosynthesis of the triphosphopyridine NADP is catalyzed solely by the ubiquitous NAD kinase family. The Arabidopsis (Arabidopsis thaliana) genome contains two genes encoding NAD ? kinases (NADKs), annotated as NADK1, NADK2, and one gene encoding a NADH kinase, NADK3, the latter isoform preferring NADH as a substrate. Here, we examined the tissue-specific and developmental expression patterns of the three NADKs using transgenic plants stably transformed with NADK promoter::glucuronidase (GUS) reporter gene constructs. We observed distinct spatial and temporal patterns of GUS activity among the NADK::GUS plants. All three NADK::GUS transgenes were expressed in reproductive tissue, whereas NADK1::GUS activity was found mainly in the roots, NADK2::GUS in leaves, and NADK3::GUS was restricted primarily to leaf vasculature and lateral root primordia. We also examined the subcellular distribution of the three NADK isoforms using NADK-green fluorescent protein (GFP) fusion proteins expressed transiently in Arabidopsis suspension-cultured cells. NADK1 and NADK2 were found to be localized to the cytosol and plastid stroma, respectively, consistent with previous work, whereas NADK3 localized to the peroxisomal matrix via a novel type 1 peroxisomal targeting signal. The specific subcellular and tissue distribution profiles among the three NADK isoforms and their possible non-overlapping roles in NADP(H) biosynthesis in plant cells are discussed.

Trends in Plant Science, 1998

I n any organism, one of the fundamental properties of defense mechanisms is that they must be in... more I n any organism, one of the fundamental properties of defense mechanisms is that they must be inducible in response to the external threat. This ensures that resources are not wasted. Thus, plant cells have evolved to perceive different signals from their surroundings, to integrate them and to respond by modulating various functions via signal transduction pathways. Recently, tremendous progress has been made in understanding the role of Ca 2+ as a second messenger in plants. This has become possible in several ways: by employing innovative types of technology to measure, in real time, changes in the concentration of Ca 2+ in cells 1,2 ; by using novel approaches to introduce signaling components and reporter genes into single plant cells 3 ; and by using methods for the isolation of downstream cellular targets of calmodulin 4 . These studies suggest that Ca 2+ has a vital role in mediating plant responses to external stimuli of both abiotic origin (e.g. light, cold, heat, movement, hypoxia and drought) and biotic origin (e.g. phytohormones, pathogens and interactions with symbionts). Thus, Ca 2+ triggers a myriad of cellular processes that influence growth, development and physiology, which allow plants to adapt to the changing environment.

The Plant Cell, 1994

The identity of a soluble 62-kD Ca2+-dependent calmodulin binding protein (CaM-BP) from fava bean... more The identity of a soluble 62-kD Ca2+-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 1251-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular mames were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and interna1 microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the a-decarboxylation of glutamate to form the stress-related metabolite y-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 pM Ca2+, a 100% stimulation by the addition of 100 pM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca2+-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca*+-mediated signal transduction pathway.

PLANT PHYSIOLOGY, 1992

Addition of L-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus spre... more Addition of L-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake Of L- [U-14C] glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with I millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyndoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H /L-glutamate cotransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H /L-Glu symport may involve both Hz consumption during 4-aminobutyrate production and ATP-driven H+ efflux.

PLANT PHYSIOLOGY, 2010

Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of or... more Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of orthophosphate (Pi)-starved (-Pi) plants. APases catalyze Pi hydrolysis from a broad range of phosphomonoesters at an acidic pH. The largest class of nonspecific plant APases is comprised of the purple APases (PAPs). Although the biochemical properties, subcellular location, and expression of several plant PAPs have been described, their physiological functions have not been fully resolved. Recent biochemical studies indicated that AtPAP26, one of 29 PAPs encoded by the Arabidopsis (Arabidopsis thaliana) genome, is the predominant intracellular APase, as well as a major secreted APase isozyme up-regulated by -Pi Arabidopsis. An atpap26 T-DNA insertion mutant lacking AtPAP26 transcripts and 55-kD immunoreactive AtPAP26 polypeptides exhibited: (1) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (2) a 40% decrease in secreted APase activity during Pi deprivation, (3) 35% and 50% reductions in free and total Pi concentration, respectively, as well as 5-fold higher anthocyanin levels in shoots of soil-grown -Pi plants, and (4) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function occurred under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. Transient expression of AtPAP26-mCherry in Arabidopsis suspension cells verified that AtPAP26 is targeted to the cell vacuole. Our results confirm that AtPAP26 is a principal contributor to Pi stress-inducible APase activity, and that it plays an important role in the Pi metabolism of -Pi Arabidopsis.

PLANT PHYSIOLOGY, 2004

NAD kinase (NADK; ATP:NAD 2#-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryote... more NAD kinase (NADK; ATP:NAD 2#-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca 21 -dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaMdependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (K m NAD 5 0.20 mM, K m Mg 21 2ATP 5 0.17 mM). The enzyme was fully activated by conserved CaM (S 0.5 5 2.2 nM) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins.

Plant Molecular Biology, 2007

Various aspects of plant development and stress physiology are mediated by Ca(2+) signaling. Ca(2... more Various aspects of plant development and stress physiology are mediated by Ca(2+) signaling. Ca(2+) sensors, such as calmodulin, detect these signals and direct downstream signaling pathways by binding and activating diverse targets. Plants possess many unique, putative Ca(2+) sensors, including a large family (50 in Arabidopsis) of calmodulin-like proteins termed CMLs. Some of these CMLs have been implicated in Ca(2+)-based stress response but most remain unstudied. We generated transgenic plants expressing CML::GUS reporter genes for members of a subfamily of CMLs (CML37, CML38 and CML39) which allowed us to investigate their expression patterns in detail. We found that CML::GUS genes displayed unique tissue, cell-type, and temporal patterns of expression throughout normal development, particularly in the flower, and in response to a variety of stimuli, including biotic and abiotic stress, hormone and chemical treatments. Our findings are supported by semiquantitative reverse-transcription PCR as well as analyses of microarray databases. Analysis of purified, recombinant CMLs demonstrated their ability to bind Ca(2+) in vitro. Collectively, our data suggest that these CMLs likely play important roles as sensors in Ca(2+)-mediated developmental and stress response pathways and provide a framework of spatial and temporal expression to direct future studies aimed at elucidating their physiological roles.

Plant Molecular Biology, 2005

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. ... more Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca 2+ is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca 2+ -binding proteins such as calmodulin (CaM) detect Ca 2+ signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virusinduced gene silencing (VIGS), compromises the plant's immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca 2+ in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca 2+ -dependent signals during the plant immune response to bacterial pathogens.

Physiologia Plantarum, 1995

1995. Gaba shunt in developing soybean seeds is associated with hypoxia. -Physiol. Plant. 94: 219... more 1995. Gaba shunt in developing soybean seeds is associated with hypoxia. -Physiol. Plant. 94: 219-228. In the present study we investigated the proposal that Ihe )'-aminobutyrate (Gaba) shunt in developing soybean (Glycine max [L.] Merr.) seeds is associated with hypoxia. The ontogeny and pH profile of enzymes associated with glutamate metabolism (glutamate decarboxylase [EC 4.1.1.15]. Gaba transaminase [EC 2.6.1.19], succinic semialdehyde dehydrogenase [EC 1.2.1.16], glutamate dehydrogenase {EC 1.4.U], glutamate:oxaloacetate transaminase [EC 2.6.1.1], glutamate:pyruvate transaminase [EC 2.6.1.2] and 2-cxoglutarate dehydrogenase complex [EC 1.2.4.2]) and hypoxia (aicohol dehydrogenase [ADH, EC 1.1.1.1] and pyruvate decarboxylase [PDC, EC 4.1.1.1])

New Phytologist, 2001

CaM, clamodulin; Ca 2 + /CaM, Ca 2 + -bound CaM; CaMBD, CaM-binding domain; CCaMK, chimeric Ca 2 ... more CaM, clamodulin; Ca 2 + /CaM, Ca 2 + -bound CaM; CaMBD, CaM-binding domain; CCaMK, chimeric Ca 2 + /CaM-dependent protein kinase; CDPK, calcium-depentent (calmodulin-independent) protein kinase; CNGC, cyclic nucleotide gated channel; cNMP, cyclic nucleotide monophosphate; KCBP, kinesin-like CaM-binding protein.

Journal of Biological Chemistry, 2002

Screening of cDNA expression libraries derived from plants exposed to stress, with 35 S-labeled r... more Screening of cDNA expression libraries derived from plants exposed to stress, with 35 S-labeled recombinant calmodulin as a probe, revealed a new family of proteins containing a transcription activation domain and two types of DNA-binding domains designated the CG-1 domain and the transcription factor immunoglobulin domain, ankyrin repeats, and a varying number of IQ calmodulin-binding motifs. Based on domain organization and amino acid sequence comparisons, similar proteins, with the same domain organization, were identified in the genomes of other multicellular organisms including human, Drosophila, and Caenorhabditis, whereas none were found in the complete genomes of single cell eukaryotes and prokaryotes. This family of proteins was designated calmodulin-binding transcription activators (CAMTAs). Arabidopsis thaliana contains six CAMTA genes (AtCAMTA1-AtCAMTA6). The transcription activation domain of AtCAMTA1 was mapped by testing a series of protein fusions with the DNA-binding domain of the bacterial LexA transcription factor and two reporter genes fused to LexA recognition sequences in yeast cells. Two human proteins designated HsCAMTA1 and HsCAMTA2 were also shown to activate transcription in yeast using the same reporter system. Subcellular fractionation of Arabidopsis tissues revealed the presence of CAMTAs predominantly in the nucleus. Calmodulin binding assays identified a region of 25 amino acids capable of binding calmodulin with high affinity (K d ‫؍‬ 1.2 nM) in the presence of calcium. We suggest that CAMTAs comprise a conserved family of transcription factors in a wide range of multicellular eukaryotes, which possibly respond to calcium signaling by direct binding of calmodulin.

Journal of Biological Chemistry, 2009

Calcium (Ca(2+)) is a key second messenger in eukaryotes where it regulates a diverse array of ce... more Calcium (Ca(2+)) is a key second messenger in eukaryotes where it regulates a diverse array of cellular processes in response to external stimuli. An important Ca(2+) sensor in both animals and plants is calmodulin (CaM). In addition to evolutionarily conserved CaM, plants possess a unique family of CaM-like (CML) proteins. The majority of these CMLs have not yet been studied, and investigation into their physical properties and cellular functions will provide insight into Ca(2+) signal transduction in plants. Here we describe the characterization of CML42, a 191-amino acid Ca(2+)-binding protein from Arabidopsis. Ca(2+) binding to recombinant CML42 was assessed by fluorescence spectroscopy, NMR spectroscopy, microcalorimetry, and CD spectroscopy. CML42 displays significant alpha-helical secondary structure, binds three molecules of Ca(2+) with affinities ranging from 30 to 430 nm, and undergoes a Ca(2+)-induced conformational change that results in the exposure of one or more hydrophobic regions. Gene expression analysis revealed CML42 transcripts at various stages of development and in many cell types, including the support cells, which surround trichomes (leaf hairs) on the leaf surface. Using yeast two-hybrid screening we identified a putative CML42 interactor; kinesin-interacting Ca(2+)-binding protein (KIC). Because KIC is a protein known to function in trichome development, we examined transgenic CML42 knockout plants and found that they possess aberrant trichomes with increased branching. Collectively, our data support a role for CML42 as a Ca(2+) sensor that functions during cell branching in trichomes.