Plant Physiol. (1994) 106: 1381-1387

The 58-Kilodalton -Binding 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 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 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 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 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 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, 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 (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 (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 .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. 1, A and B). A single EcoRV fragment cording to the manufacturer's guidelines. Membranes were was detected when the whole cDNA was used a:; a probe incubated in a prehybridization solution containing 50% (Fig. 1A). Two hybridizing fragments were found after BamHI formamide (v/v), 4X SSC, 1X Denhardt's solution (Sambrook digestion, as would be the case for a single-copy ,gene that et al., 1989), 10% (w/v) PEG (mol wt 6000), 1%(w/v) SDS, contains a single BamHI site in the coding sequence (Baum et and salmon-sperm DNA (100 pg mL-'; sonicated and boiled) al., 1993). Hind111 and EcoRI digests resulted in a few smaller for 6 h at 42OC. Nick-translated DNA probes were added for hybridizing fragments. These could be interpreted either as hybridizations for 16 h. Washing of membranes was per- an indication of the presence of more than one GAD-like formed as described (Sambrook et al., 1989). gene or as subfragments of a single gene that contains introns Expressio 58-ke th f nDo Glutamate Decarboxylas Petunien i a 1383

However, the 58-kD GAD protein continued to accumulate (per total protein) after levels of its mRNA began declining. The 58-k reacheD DGA d maximal level open si n flowers. GAD activity measure n whole-flowei d r extracts als- in o creased during flower developmen Tabl; B d e tan (FigA , 2 . kb I). However, the overall changes in GAD activity were smaller 23.1 — than change abundance th (2.5 D n si - GA versu f eo s 10-fold, respectively), suggesting that factors othe abunr D than-GA 9.4 — dance also affect GAD activity.

6.5 _

Flower size (cm) 4.4 —

2.3 — in 6 I o 5 3 oi

GAD mRNA Northern blot

GAD protein Western blot Figur . Organizatioe1 f CAD-relateno d genepetunie th - n si ge a nome. Restriction endonuclease digests of total DMA from petunia GABA TLC leaves were hybridized wit followine hth g 32P-labele fragA -DN d ment probess scompleta e th , cDNAA D : 450-ba eCA , A B ; pDN fragmen cDND CA A ' f terminu 5 o tfro e BamHme th th o st I site (cf. Baum et al., 1993). X-Phage DNA digested with H/ndlll was used a sizsa e marker.

(with no EcoRI and Hi'ndlll endonuclease cleavage sites in e codinth g sequence; Bau t mal.e , 1993) resolvo T . e this ambiguity, another genomic Southern blot was probed with a 32P-labeled 450-bp DNA fragment that included only the region from the 5' terminus of the GAD cDNA up to the unique BamHI site (Bau t mal.e , 1993) singlbanA A . deDN detectes wa EcoRVe eacn dth i f ho , EcoRI BflmHd an , I endo- nuclease digests. These bands correspond in size to fragments 0.5 1.0 2.0 2.0-7.0 7.0 (open) detected wit fule hth l cDNA prob Figurn ei . e1A Flower size (cm) We also cloned genomic fragments containing the GAD coding sequence. Their preliminary analysis confirme th s existence of introns (not shown). Therefore, the petunia 58- Figure 2. Expression of the 58-kD CAD in developing petunia kD CaM-binding GAD is most likely encoded by a single flowers. Whole flower t differensa t stage f developmenso t ranging in length from 0.5 to 7.0 cm (fully expanded open flowers) as gene containing introns. However e existencth , f moro e e indicated werproteind an e A collectes RN wer d ean d extracted. distantly related petunia GAD genes that did not hybridize Flowers ranging in length from 2.0 to 7.0 cm (but before opening) to the whole cDNA probe or to the smaller cDNA probe were pooled and tested as a single sample (indicated as 2.0-7.0). cannot be excluded. Protein sample jt0 gs (2 each) were fractionate SDS-PACy db d Ean transferre nitrocelluloso dt e membrane r immunoblosfo t analysis. Developmental Regulation of GAD Expression in Flowers TotasampleeachA g M RN l 0 ) wer(1 s e loade r northerfo d n blot analysis , AutoradiograA . northera f mo n blot D showinCA e gth GAD mRN hardls Awa y detecte youngese th n di t flowers mRNA signal, a western blot showing the 58-kD CAD, and a TLC tested (0.long)m 5c , whereas striking change abune th n si - plate showin e ['4gth C]CAB A produce vitrn i d f owithio h n1 mRND dancGA A f eo wer e detected during flower develop- decarboxylatio f L-[U-'no 4C]Glu (see "Material Methods")d san , B . ment (Fig. 2, A and B) and the highest levels of GAD mRNA Relative abundance of the CAD mRNA, the 58-kD GAD, and CAD were found in 2.0-cm-long flowers, about 10 times more than activity during flower development. The highest level of each parameter in this experiment was set at 100%. Quantitation of levele th 0.5-cn si m flowers mRND levele GA .Th A f so the n mRNA and protein levels was determined by scanning films with a declined rapidly; in fully expanded open flowers the levels Molecular Dynamics (Sunnyvale, CA) 300A Computing Densitom- were simila thoso rt 0.5-cm-lonn ei g flowers. Increasee th n si eter. Quantitatio activitD performes CA f ywa o n describes da n di abundanc 58-ke proteiD th f Deo GA n appeare parallen di l "Materials and Methods." Relative levels of CAD mRNA and protein with the increase in the abundance of the GAD mRNA. wer averagee e th experimentso tw f so . Bars indicate SE values. 1384 Chen et al. Plant Physiol. Vol. 106,1994

Table 1. Changes in CAD activity in petunia extracts during organ development CAD Activity' Organ From To pmol GABA p protein h~ •—Te P u Leaf 140 601 Whole flowerb 467 1016 Sepal 541 676 Se Petal-tube 243 601 Petal-limb 423 1142 Stamen 30 36 Ovary 202 322 Style 60 238 B Seed germination 16 39 activitD determines aCA wa y describes da n "Materiali d d an s Methods." The values indicated represent minimal and maximal activities measured in the course of organ development. Wholeb flower Figursn i s rangea . e2 m lengtn c di 0 7. h o frot 5 m0. GAD protein -— Western blot Floral parts were dissected fro stageo mtw f floweso r development: 2.0 cm and 7.0 cm long (open flowers). See Figure 3 for a schematic c presentation of petunia floral organs. Values represent the GABA activity in extracts from whole dry seeds and after 120 h of germi- TLC nation. However activitD CA , wholn i y e seed extracts declined during the first 72 h of germination and increased after that period (cf. Fig. 6). Limb Development We dissected fully expanded, open petunia flowerd an s Flower Length (cm) analyze differene th expressioe n i dth D t floraGA f no l organs c (Fig. 3). Petals were dissected into rubes (the lower cylindrical

part) and limbs (the upper expanding part) (see Fig. 3A, a CO I schematic presentation of petunia floral parts; modified from Van der Krol and Chua, 1993), because differences in the expression of genes between the two parts have been reported GAD protein Western blot (Takatsuj t al.e i , 1992) highese .Th t abundanc 58-ke th f Deo e highes activitth D GA d GA Dtan y were apparene th n i t limbs (Fig. 3B). The abundance of the 58-kD GAD and GAD activity (per total protein) were lowe extractn i r f sepalsso , GABA TLC tubes, ovaries styled an , s (see Tabl . StameneI) s contained tit low levels of the 58-kD GAD (not shown) and GAD activity (Table I). Petals make up about 70% of the biomass of open Figure 3. Expression of the 58-kD CAD in floral organs. A, Sche- flowers of P. hybrida var Mitchell (our analysis). Thus, the matic presentatio longitudinaa f no l sectio matura f o n e petunia stimulatio expressioD GA f o n n observe wholn i d e flowers flower (modifie r KroChuad dde an lfro n , mVa 1993) . Floral parts during development (cf. Fig. 2) is probably a manifestation arfollowss ea , sepal , petalSe : Pe , ;limb Li ;, tube Tu ;, stamen St ; ; expressioD oGA f petalsn ni . Indeed observee w , develda - Sy, style , stigma Sg , ;ovary Ov ,, OrganB . s from fully expanded opmental stimulation of GAD expression in limbs (Fig. 3C). open flowers were dissected and proteins were extracted. Analysis of protein on western blots and off CABA synthesis was as described legene th n i Figurdo t westerA . e2 n blot analysi 58-ke D th f DsCA o ExpressioD GA n during Leaf Development protein and an autoradiogram of a TLC plate showing the [14C]- GAD expressio analyzes nwa leaven di t differensa t stages CABA produce vitrn i d f owithio decarboxylatioh n1 f [o 14nC] - of development (Fig. 4, A and B). The levels of the GAD glutamat presentede ar e , DevelopmentaC . l regulatioD CA f no 58-ke increaseth D mRNf Do GA d parallen Adi an l during expression limbn i f flowero s t differena s t stage f developmenso t leaf development. Maxima mRND lGA A levels were detected (lengths of flowers from which limbs were dissected are indicated proteicm)D n i CA . n level CABd san A synthesis were analyzes da in 6.0-cm-long leaves, and these were about 2.5-fold more described in the legend to Figure 2. than in the youngest leaves (0.5-1.0 cm long). A slight decline in the levels of the GAD mRNA and protein was observed n 9.0-cm-loni g leaves. activitChangeD GA lea n i yn i sf extracts coincided with leveD changeGA l (Fign ; i s 4B . Table I), both showing about 5-fold increases during leaf development. Expression of the 58-kD Glutamate Decarboxylase in Petunia 1385

Leaf development increase GABd dan A accumulated (Inatom Slaughterd an i , 3 1971; Vandewalle and Olsson, 1983). It was proposed that the GABA-shunt pathway provides carbon skeletons for ox- idation in the tricarboxylic acid cycle, a step that is of partic- GAD mRNA Northern blot ular importance during germination (Vandewalle and Olsson, 1983). To examin possibilite eth e b y tha ma e 58-k th D t DGA involve metabolin di c processe seedsn i s analyzee w , s dit GAD protein — < Western blot expression in dry seeds and during the first 5 d of germina- tion. GAD mRNA was undetectable in dry seeds, but the 58- clearls wa yD kapparentDGA explainee .b Thie y th sma y db seedn i depositio D s durinGA f o n g fruit developmente W . GABA tiff TLC indeed detecte 58-ke dth D CaM-bindin developinn i D gGA g petunia fruits (not shown). GAD activity in dry seed extracts was about 10% of that young-lean i f extracts usecontroa s da l (Fig Tabl; .6 Afte. eI) r germinationf o 1 2h sligha , t declin abundance th n ei e th f eo 58-kD GAD protein and in GAD activity was observed. At 48 h of germination, the abundance of the 58-kD GAD was minimal otheo Tw ) .crossr proteikD -6 6 nd bandan 8 (4 s reacted wit anti-GAe hth D seruseedy durind dr an msn i g germinationf o h firs e 0 th 6 t levele .Th thesf s o protein o etw s remained maximal as long as the abundance of the 58-kD protein was minimal. At 60 h of germination, a decline in the abundance of the 48- and 66-kD proteins was apparent, whereas the abundance of the 58-kD GAD increased slightly. At this stage, the major seed proteins (Fig. 6, open arrow- L2 L3 L4 LS L6 heads significantld ha ) y declinerooe alreadd th ha t d dyan Leaf development emerged from the seed coat (not shown). At 72 h of germi- nation, the 48- and 66-kD protein bands were hardly de- Figur . Expressio4 e n developini e 58-k D th f DCA no g leaves. tected, whereas the GAD mRNA and the 58-kD protein were Protei mRNd nan A from leave t differensa t stage f developmenso t more abundant. At this stage the major seed proteins were were extracted and analyzed as described in the legend to Figure o longen f germinatioo r h apparent 0 12 t n A .significan t 2. The lengths of leaves (cm) were: L1, 0.5 to 1.0; L2, 1.0 to 2.0; L3, increases in the abundance of the GAD mRNA, the 58-kD 2.0 to 3.0; L4, 3.0 to 5.5; L5, 6.0; L6, 9.0. A, Autoradiogram of a GAD, and GAD activity were apparent, the cotyledons had northern blo t mRND showinCA Ae gth signal westera , n blot showing the 58-kD CAD protein band, and a TLC plate showing just emerged, and the small and large subunits of Rubisco the [14C]CABA produced in a 1-h reaction with radiolabeled gluta respectively, -LS d an S (FigS , ) 6 wer. e detecte t thada t time. mate. B, Relative changes in the CAD mRNA, 58-kD CAD, and activityD CA , determine describes da legene th n i Figuro dt . e2 DISCUSSION The highest leve f eaco l h paramete thin i r s experiment a t se s wa t 100%. Each point on the activity plot is the average of two experi- In this wor studiee kw expressioe dth 58-ke th f nDo CaM- ments. Bars indicate SE values. binding GAD in different petunia organs and assessed the

GAD Expression in Roots and Stems testes expressioe wa rootn di stemTh d D an sf GA o s f no petunia plants together with samples from leaves (4.5-5.5 cm long) and young flowers (2.0 cm long) from the same GAD protein > — - Western blot plants (Fig. 5). The results show that the 58-kD GAD is expresse l organal n di s tested proteie Th .D n GA leve d an l activity in stem extracts was higher than in leaves, roots, and TLC young flowers (but lower than in open flowers; comparison GABA • tt not shown). Figure 5. Expression of the 58-kD CAD in various organs of petunia GAD Expression during Germination plants. Roots, stems, leaves (4.5-5.5 cm long), and young flowers (2.0 cm long) were dissected and analyzed as described in the Special attentio s beenha n give severay nb l investigators legen Figuro dt representativA . e2 e western blot showin- 58 e gth to the possible roles of GAD and GABA in seeds during proteiD platC CA TL en D k a showinban d d an [e 14gth C]CAB A germination activitD detectes GA . ywa t verda y early stages produce vitrn di withif oo decarboxylatioh n1 f [14o nC]glutamat e of germination, and at the onset of growth GAD activity are presented. 1386 Chen et al. Plant Physiol. Vol. 106, 1994

Germination (hr) mRNA levels and protein levels in leaves, as opposed to

120\.«a2 7 0 6 t8 4 4 2 2 1 'O petals, suggests that posttranslational regulation of GAD may differ in petals and leaves and may play a role in controlling GAD levels. -LS Increased 58-ke durinlevelD th f DGA o sg organ devel- opment and seed germination coincided with increases in the in vitro-measured GAD activity. These results suggest that 31.0 - Protein stain the 58-kD CaM-binding GAD is responsible for a major portion of the overall GAD activity in petunia extracts. How- 21.5 — ever, we cannot exclude the possibility that other GAD protein detectet santibodier no tha e ou tar y db s hav similaea r expression profile and also contribute to the overall GAD activity. This could be one explanation for the fact that during flower development the level of the 58-kD GAD in young 66kDa- flower thaf o abou s open ti % swa n10 t flowers, althouge hth 5- 8 KOD aGA Western blot 48kOa- activit younn yi g flower extract f tha o s abou n i ts% wa 40 t open flowers summarA . e overal activitth D f yo GA l n yi plant extracts during organ development and germination is GABA 'J TLC presente Tabln di . eI We note that the activity in plant extracts does not neces- sarily reflect relative activities in vivo, which may be regu- GAD mRNA Northern blot late severay db l effectors suc cytosolis ha (SneddeH cp t ne al., 1992; Crawford et al., 1994), ABA (Reggiani et al., 1993), calciud an m signalin(BauM Ca mal.t e a ,g vi 1993) . However, Figure 6. Expression of the 58-kD CAD during germination. Seeds recent finding laboratorr ou n si y show that transgenic plants were collected prior to soaking (0 h) and at the times indicated after overproducin 58-ke gth D CaM-bindin undeD trane grth GA - beginnine th soakine th f go g period mRNAD . AnalysiCA e , th f so scriptiona caulifloweS l 35 contro e th f o lr mosaic virus pro- the 58-kD CAD, and CAD activity were performed as described in moter possess higher rate GABf so A synthesi vivon si . Thus, the legend to Figure 2 (0.5- to 1.0-cm leaves from flowering petunia plants were used as a control). Samples containing 20 jig of total the overal activitD GA l regulatee plantn yi b n sca parn di t soluble protein were separate y SDS-PAGb d d staineEan d with by GAD abundance (Baum et al., 1994). Coomassie brilliant blue identica n uses . A electrotransr dwa fo l ge l - GA seedDn havi y ma se unique features when compared fer of proteins to a nitrocellulose membrane and detection of the to other organs addition I .58-ke th o Dt n GAD otheo tw , r 58-k witD hDCA anti-recombinan serumD 58-ke D GA t Th .D CA seed proteins cross-reacted with the anti-GAD serum (48 and band and two other proteins detected with the anti-CAD serum 66 kD; cf. Fig. 6). The identity of these proteins is still obscure, are indicated with their respective molecular massed san (58, 48 , interestins i bu t i t g that they showe expression da n profile 66 kD; full arrows on the left). Arrows on the right show the Rubisco opposite from that of the 58-kD GAD during germination small and large subunits (SS and LS, respectively). Major seed and that they were detected only in developing fruits (not proteins that disappeared during germination are indicated by open shown), in dry seeds, and during the first 60 h of germination. arrowheads. An autoradiogram of a TLC plate showing the [14C]- CABA produced in 1 h of in vitro decarboxylation and a northern Inatom d Slaughtean i r (1975) describe e occurrencth d f o e blot probed with the 32P-labeled CAD cDNA are presented below different form f higo s h molecular complexemasD GA s n si the western blot. embryos and in roots of barley. However, in our study GAD activity during petunia seed germination coincided wite hth abundanc 58-ke th f eDo protein (Fig. .6) With respece possiblth o t tn seed i e functioM s Ca f o n relationships between change e corree levelth th f -n o si s during germination interestins i t i , g that recentl ynumbea r sponding mRNA, protein, and activity during organ of small proteins purified from radish seeds were identified development. as potent CaM antagonists (Polya et al., 1993). Moreover, a course Inth developmentf eo , increase abundance th n i s e significant decreas thesn ei e antagonist simultaneoua d san s 58-kcoincidee oD th f DGA d with increase levele th f n so i increase in CaM levels were observed during early phases of mRND . ThesGA A 6) e (cfd th e an result. Figs, 4 , 2 .s suggest germination, implying that CaM-regulated processes are ac- that during petunia organ development transcriptional and/ tivated (Cocucci and Negrini, 1988). It is conceivable that the or posttranscriptional regulatory processes are involved in 58-kD CaM-binding GAD in petunia is one of the enzymes controlling GAD expression. In petals, the 58-kD GAD con- tha activatee ar t Cay db 2+ /Cacourse th M n germinationf i e o . tinued to accumulate after its mRNA began declining, so that The striking increase in the expression of the 58-kD GAD in open flowers the GAD mRNA was hardly detected but during flower development and its accumulation to high GAD abundance was maximal. This observation indicates level petaln si s deserve further attention have obviou.o W en s that in petals, the 58-kD GAD is a relatively stable protein. explanation for these results. A speculative link between Conversely, in large leaves, a decline in GAD mRNA levels GAD activit petad yan l developmen e foune b th y n i d ma t coincided wit declinh a abundanc e th n e i 58-k e th D f eDo GA f petao fac H lt p n importancella thae s i th st t facton i r activitD GA yn i (Fig d Thi. an .4) s apparent coupling between regulating flower pigmentation (Sink, 1984). Furthermore, Expression of the 58-kD Glutamate Decarboxylase in Petunia 1387 genes responsible for controlling petal pH in petunia have Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. al., 1993), but their identity been isolated recently (Chuck et Anal Biochem 132 6-13 has not been revealed. Finally, because GAD has been re- Gilroy S, Jones RL (1992) Gibberellic acid and abscisic acid coordi- ported to be stress activated (Wallace et al., 1984; Rhodes et nately regulate cytoplasmic calcium and secretory activity in barley al., al., 1986; Mayer et a]., 1990), it is noteworthy that other aleurone protoplasts. Proc Natl Acad Sci USA 89 3591-3595 stress-induced proteins are expressed in flowers. These in- Inatomi K, Slaughter JC (1971) The role of glutamate decarboxylase and y-aminobutyric acid in germinating barley. J Exp Bot 22: clude pathogenesis-related proteins (Lotan et al., 1989) and 561-571 heat-shock proteins such as HSP70 (Y. Chen and H. Fromm, Inatomi K, Slaughter JC (1975) Glutamate decarboxylase from unpublished observations). barley embryos and roots. Biochem J 147: 479-484 In summary, we found that the 58-kD CaM-binding GAD Larher F, Goas G, Le Ruddier D, Gerard J, Hamelin J (1983) Bound 4-aminobutync acid in root nodules of Medicago sativa and is a ubiquitous protein in petunia organs and that its expres- other nitrogen fixing plants. Plant Sci Lett 29 315-326 sion is developmentally regulated by transcriptional and/or Logemann J, Schell J, Willmitzer L (1987) Improved method for posttranscriptional processes. These regulatory mechanisms the isolation of RNA from plant tissues. Anal Biochem 163 16-20 are likely to be involved in controlling GABA synthesis during Lotan T, Ori N, Fluhr R (1989) Pathogenesis-related proteins are petunia development. It is not yet known how the regulation developmentally regulated in tobacco flowers. Plant Cell 1: 881-887 of GAD gene expression plays a role in concert with other Mayer RR, Cherry JH, Rhodes D (1990) Effects of heat shock on potential effectors of GAD activity. amino acid metabolism of cowpea cells. Plant Physiol94 796-810 McAinsh MR, Brownlee C, Hetherington AM (1990) Abscisic acid- ACKNOWLEDGMENTS induced elevation of guard cell cytosolic Ca2+precedes stomatal closure. Nature 343 186-188 We thank Ms. Dvora Dolev for excellent technical assistance and Polya GM, Changra S, Condron R (1993) Purification and sequenc- Drs. Esra Galun, Jonathan Gressel, Robert Fluhr, Gad Galili, Adi ing of radish seed calmodulin antagonists phosphorylated by cal- Avni, Julio Salinas, and Hiroshi Takatsuji for critical reading of the cium-dependent protein kinase. Plant Physiol 101: 545-55 l manuscript. Reggiani R, Aurisano N, Mattana M, Bertani A (1993)ABA induces 4-aminobutyrate accumulation in wheat seedlings. Phytochemistry 34 605-609 Received June 14, 1994; accepted August 6, 1994. 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