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The 58-Kilodalton Calmodulin-Binding Glutamate Decarboxylase Is a Ubiquitous Protein in Petunia Organs and Its Expression Is Developmentally Regulated'

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The 58-Kilodalton Calmodulin-Binding Glutamate Decarboxylase Is a Ubiquitous Protein in Petunia Organs and Its Expression Is Developmentally Regulated' Plant Physiol. (1994) 106: 1381-1387 The 58-Kilodalton Calmodulin-Binding Glutamate Decarboxylase Is a Ubiquitous Protein in Petunia Organs and Its Expression Is Developmentally Regulated' Yali Chen, Gideon Baum, and Hillel Fro"* Department of Plant Genetics, Weizmann Institute of Science, 761O0 Rehovot, Israel et al., 1984); heat shock (Mayer et al., 1990); and water stress A cDNA coding for a 58-kD calcium-dependent calmodulin (Rhodes et al., 1986), which is consistent with this role. (CaM)-binding glutamate decarboxylase (GAD) previously isolated Moreover, GAD activity is enhanced at relatively acidic pH in our laboratory from petunia (Petunia hybrida) (G. Baum, Y. (Snedden et al., 1992; Crawford et al., 1994). It was also Chen, T. Arazi, H. Takatsuji, H. Fromm [1993] J Biol Chem 268: suggested that transamination of a-ketoglutarate by GABA 19610-19617) was used to conduct molecular studies of GAD (producing succinic semialdehyde and glutamate) could reg- expression. GAD expression was studied during petunia organ ulate carbon flow through the tricarboxylic acid cycle by development using the GAD cDNA as a probe to detect the GAD mRNA and by the anti-recombinant GAD serum to monitor the bypassing the direct conversion of a-ketoglutarate to succi- levels of GAD. GAD activity was studied in extracts of organs in nate, a step that may be inhibited under certain physiological the course of development. lhe 58-kD CaM-binding GAD is ex- situations (Dixon and Fowden, 1961). In addition, GABA can pressed in all petunia organs tested (flowers and all floral parts, be transaminated even more effectively with pyruvate to leaves, stems, roots, and seeds). The highest expression levels were succinic semialdehyde and Ala (Dixon and Fowden, 1961; in petals of open flowers. Developmentalchanges in the abundance Streeter and Thompson, 1972b). of GAD mRNA and the 58-kD GAD were observed in flowers and GABA has also been postulated to have a role in nitrogen leaves and during germination. Moreover, developmental changes metabolism and storage in plants. High molecular mass in GAD activity in plant extracts coincided in most cases with GABA conjugates account for more than 6% of the dry weight changes in the abundance of the 58-kD GAD. We conclude that of nitrogen-fixing nodules of legumes (Larher et al., 1983). the 58-kD CaM-binding GAD is a ubiquitous protein in petunia GABA in nutrient solutions can also function as a sole nitro- organs and that its expression is developmentally regulated by transcriptional and/or posttranscriptional processes. Thus, GAD gen source for plant growth (Bames and Naylor, 1959). In gene expression is likely to play a role in controlling the rates addition, GABA is an obligatory intermediate in the assimi- of GABA synthesis during petunia seed germination and organ lation of nitrogen from putrescine in a tobacco cell line that development. can use putrescine as a sole nitrogen source (Baht et al., 1987). The mechanisms regulating GAD activity in vivo are still GAD catalyzes the conversion of glutamate to GABA. The unknown. GAD activity can be stimulated by lowering cy- presence of GAD activity and GABA in plants has been tosolic pH (Crawford et al., 1994). However, reduction of known for at least half a century (refs. in Satyanarayan and cytosolic pH is not necessary for stimulation of GABA syn- Nair, 1990). However, their roles are still obscure. GABA thesis (Crawford et al., 1994). This suggests that mechanisms functions in animals as a major inhibitory neurotransmitter other than cytosolic pH are also involved in the regulation of by modulating the conductance of ion channels (Zhang and GAD activity. A variety of environmental stresses cause Jackson, 1993). The suggestions raised to explain the role of elevations of GAD activity and cytosolic calcium levels. Thus, GABA in plants have not addressed the possibility that GABA Wallace et al. (1984) suggested that GAD may be regulated may function as a regulator of ion channels as in other by calcium signaling pathways. Recently, Reggiani et al. eukaryotes. (1993) showed that wheat root GAD is activated in response Decarboxylation of glutamate to GABA with consumption to treatments with ABA. ABA can itself induce the elevation of a proton has been suggested as a mechanism to stabilize of calcium in plant cells (McAinsh et al., 1990; Gilroy and cytosolic pH in plant cells during stresses that lead to cyto- Jones, 1992; Bush et al., 1993). We recently cloned a cDNA plasmic acidification (Snedden et al., 1992; Crawford et al., encoding a Ca2'-dependent CaM-binding GAD from petunia 1994). The synthesis and accumulation of GABA increase (Petunia hybrida) (Baum et al., 1993). This finding is consistent rapidly in response to anaerobiosis (Streeter and Thompson, with the potential role of calcium signaling in the control of 1972a); mechanical shock, cold shock, and darkness (Wallace GABA synthesis in plants. This calcium-dependent CaM- binding aspect of plant GAD was previously unknown. This research was supported by a grant to H.F. from the Ministry GAD activity and GABA have been detected in virtually of Science and Technology, Israel, and the Gesellschaft fur Biotech- nologische Forschung, GBF Braunschweig, Germany. Abbreviations: CaM, calmodulin; GABA, y-aminobutyric acid; * Corresponding author; fax 972-8-469124. GAD, glutamic acid decarboxylase. 1381 1382 Chen et al. Plant Physiol. Vol. .06,1994 all tissues of numerous plants (Satyanarayan and Nair, 1990). Protein Extractions from Plant Tissues However, characterization of the GAD enzyme(s)responsible Plant material was frozen in liquid nitrogen and ground for this ubiquitous GABA synthesis in plant tissues remains under liquid nitrogen to a fine powder in a mortar. Sodium incomplete. To leam about the possible involvement of the 58-kD CaM-binding GAD in catalyzing GABA synthesis in ascorbate and polyvinylpolypyrrolidone (100 mg g-' plant material) were added, and proteins were extracted by further different plant organs, we analyzed its expression profile in grinding in extraction buffer mL g-' plant material) con- different petunia organs during development. We detected (4 taining 100 m Tris-HC1, pH 7.5, 10% (v/v) glycerol, 1 the 58-kD CaM-binding GAD in all petunia organs. Further- m Na2-EDTA, 1 PMSF, 2.5 pg mL-' leupeptin and antipain more, we found that transcriptional and/or posttranscrip- rm (Sigma). The homogenate was centrifuged at 4OC, 30,OOOg tional processes are involved in regulating GAD expression for 30 min and the supematant was collected. Protein and thus may play a role in controlling GABA synthesis in concentrations were determined with a Bradford reagent petunia. (Bio-Rad). MATERIALS AND METHODS lmmunodetection of GAD on Western Blots Plant Material and Growth Protein samples were separated on SDS-PAGE and trans- ferred to nitrocellulose for immunodetection as described Petunia hybrida (var Mitchell) plants were grown in a (Baum et al., 1993). The anti-GAD serum raised against a greenhouse at 22 to 25OC, 16 h light/8 h dark. Plant material recombinant GAD was the same as that described by Baum was collected into liquid nitrogen and stored at -8OOC. For et al. (1993). germination studies, petunia seeds were soaked for 6 h in water after which they were plated on wet filter paper and Determination of GAD Activity kept under a 16-h light/8-h dark cycle under cool-white fluorescent light (approximately 50 pE m-' s-'). Plant extracts were passed through Sephadex 1;-50 col- umns and reactions were performed with L-[LJ-'~C]G~U(250 mCi "01-'; Amersham) incubated with protein extracts as DNA Isolation and Southern Blot Hybridization described (Baum et al., 1993). Amino acids were extracted and fractionated by TLC as described (Baum et d., 1993). Genomic DNA was extracted from petunia leaves as de- [I4C]GABAlevels were determined by exposing TLC plates scribed (Dellaporta et al., 1983). Samples of DNA (about 60 to a BAS-111s Imaging Plate (Fuji Photo Film Co.) and analyz- pg) were digested with the indicated restriction enzymes (100 ing radioactive [14C]GABA in a Fujix BAS1000 Bio-Imaging units each) at 37OC overnight and separated on a 0.8% Analyzer. Quantitation of [14C]GABA levels was done by (w/v) agarose-Tris acetate-EDTA gel (Sambrook et al., 1989), including samples containing different amounts of a ["C]- stained with ethidium bromide, and blotted onto Genescreen GABA standard (Amersham) on each TLC plate. After quan- Plus (NEN) nylon membranes. The blot was prehybridized titative analysis, TLC plates were exposed to Kodak XAR in a solution containing 10% (w/v) PEG (mol wt 6000), 50% films. The position of [14C]GABA on TLC plates was deter- (v/v) formamide, 5X SSPE (Sambrook et al., 1989), 2% mined according to the mobility of a [l4CJGABAstandard (w/v) SDS for 6 h at 42OC, then hybridized under the same (Amersham), which was treated in the same buffers and conditions overnight with a random-primed 32P-labeled extraction solutions as the other samples. probe (Feinberg and Vogelstein, 1983). RESULTS RNA Extractions and Analysis on Northern Blots Genomic Organization of Petunia GAD Genes .RNA was extracted from plant tissues according to The petunia GAD cDNA clone was isolated front a cDNA Logemann et al. (1987) except that RNA pellets were dis- library of petals (Baum et al., 1993). Prior to analyzing the solved in 7 M urea, 2% sarkosyl and extracted once with expression of the 58-kD GAD, we assessed the number of phenol and then with chloroform:isoamylalcohol(24:1, v/v). genes coding for GAD. Total petunia leaf DNA was digested The aqueous phase was collected and RNA was precipitated with restriction endonucleases, size-fractionated, and trans- and kept at -7OOC. RNA samples were fractionated on ferred to Genescreen Plus membranes (NEN). 32P-labeled formaldehyde-agarose gels as described (Sambrook et al., GAD cDNA fragments were used as probes to derect GAD 1989) and transferred to Genescreen Plus membranes ac- genomic fragments (Fig.
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