Protein Synthesis and Phosphorylation

Plant Physiol. (1994) 105: 1149-1 157

Effect of Aerobic Priming on the Response of Echinochloa crus-pavonis to Anaerobic Stress'

Protein Synthesis and Phosphorylation

Fan Zhang', Jih-Jing Lin3, Theodore C. Fox, Cesar V. Mujer, Mary E. Rumpho*, and Robert A. Kennedy

Departments of Horticultural Sciences (T.C.F., C.V.M., M.E.R.) and Biology (R.A.K.), Texas A&M University, College Station, Texas 77843; and Department of Botany, University of Maryland,

College Park, Maryland 20742 (F.Z., J .4 .L . ) .

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Echinochloa species differ in their ability to germinate and grow in the absence of oxygen. Seeds of Echinochloa crus-pavonis (H.B.K.) Schult do not germinate under anoxia but remain viable for extended periods (at least 30 d) when incubated in an anaerobic environment. E. crus-pavonis can be induced to germinate and grow in an anaerobic environment if the seeds are first subjected to a short (1-18 h) exposure to aerobic conditions (aerobic priming). Changes in polypeptide patterns (constitutive and de novo synthesized) and protein phosphorylation induced by aerobic priming were investigated. In the absence of aerobic priming protein degradation was not evident under anaerobic conditions, although synthesis of a 20-kD polypeptide was induced. During aerobic priming, however, synthesis of 37- and 55-kD polypeptides was induced and persisted upon return of the seeds to anoxia. Further- more, phosphorylation of two 18-kD polypeptides was observed only in those seeds that were labeled with 32P04 during the aerobic priming period. Subsequent chasing in an anaerobic environment resulted in a decrease in phosphorylation of these polypeptides. Likewise, phosphorylation of the 18-kD polypeptides was not observed if the seeds were labeled in an anaerobic atmosphere. These results suggest that the regulated induction of the 20-, 37-, and 55- kD polypeptides may be important for anaerobic germination and growth of E. crus-pavonis and that the specific phosphorylation of the 18-kD polypeptides may be a factor in regulating this induction.

Unlike many Echinochloa (bamyard grass) species, Echin- ochloa crus-pavonis (H.B.K.) Schult does not typically germinate and grow in the absence of oxygen and is not found naturally in flooded rice paddies (Barrett and Seaman, 1980; Kennedy et al., 1990). Rather, E. crus-pavonis is found in

Supported by U.S. Department of Agriculture Competitive Re- search Grant 87-CRCR-1-2595 (R.A.K. and M.E.R.), a Herman Frasch Foundation Grant in Agricultural Chemistry (R.A.K.), the Maryland Agricultural Experiment Station, and the Texas Agricul- tural Experiment Station. This is Texas Agricultural Experiment Sta- tion paper No. TA31543.

Present address: Department of Biochemistry, University of Maryland, College Park, MD 20742.

Present address: Laboratory of Biochemical Physiology, Biolog- ical Response Modifier Program, National Cancer Institute, Frederick Cancer Research Development Center, Frederick, MD 20702.

* Corresponding author; fax 1-409-845-0627.

higher, dryland areas and has generally been considered to be an anaerobic- or flooding-intolerant species (Yabuno, 1983; Grist, 1986). This is in sharp contrast to Echinochloa phyllopogon (Stev.) Koss, Echinochloa o ryzoides (Ard.) Fritsch Clayton, and Echinochloa muricata (Beauv.) Fem, a11 of which readily germinate, produce a shoot, and survive for extended periods of time under anaerobic or flooded condltions (Barrett and Seaman, 1980; Yabuno, 1983; Kennedy et al., 1990). Other than Echinochloa species, the best-studied examples of seeds capable of anaerobic germination include Oryza sativa L. (rice) (Tsuji, 1972; Hook and Crawford, 1978), Erythina caffra (coral tree) (Small et al., 1989), and Zostera marina (seagrass) (Churchill, 1992). Seeds of most other plants that have been examined succumb quickly if incubated in an anaerobic environment (Morinaga, 1926).

In the tolerant species whose anaerobic respiratory metabolism has been characterized, one of two general metabolic mechanisms tends to be induced in the absence of oxygen. The first is exemplified by tolerant Echinochloa species and rice, in which a high energy charge and active metabolism are maintained in the absence of oxygen, and anaerobic germination, growth, and sustained survivability are observed (Mocquot et al., 1981; Rumpho et al., 1984; Kennedy et al., 1992). In E. phyllopogon this metabolism is sustained by the conversion of stored carbohydrates into lipids (Knowles and Kennedy, 1984; Kennedy et al., 1991). In tum, the accumulated lipids may serve as an energy reserve to be metabolized upon retum to oxygenated conditions as demonstrated previously in Euglena gracilis (Inui et al., 1982). The second metabolic mechanism is illustrated by Lacfuca sativa (lettuce) seeds (Raymond and Pradet, 1980) and E. crus- pavonis (Rumpho et al., 1984; Kennedy et al., 1987). Seeds of both of these plants exhibit very low ATP levels under anaerobic conditions and display minimal metabolic activity and no germinability or growth, yet they remain viable for weeks and resume growth and development if retumed to aerobic conditions (Raymond and Pradet, 1980; Cobb and Kennedy, 1987; Kennedy et al., 1987, 1990).

Abbreviations: ASP, anaerobic stress protein; I-D, one dimensional; 2-D, two dimensional; NEpHGE, nonequilibrium pH gradient gel electrophoresis; pI, isoelectric point.

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1150 Zhang et al. Plant Physiol. Vol. 105, 1994

The bamer to anaerobic germination in E. crus-pavonis can be removed by exposing seeds that have imbibed water to an aerobic environment for a short time (1-18 h) (aerobic priming). This aerobic priming can take place at the inception of imbibition or at any time during the anaerobic incubation period. The maximum anaerobic incubation period prior to priming studied thus far is 24 d (F. Zhang, J.-J. Lin, M.E. Rumpho, unpublished observations). Following aerobic priming, a seed placed in an anaerobic environment subsequently genninates and grows both a shoot and a root. Seeds of E. crus-pavonis maintained in an anaerobic environment without aerobic priming do not exhibit any gennination or growth under N2, even after 30 d (Kennedy et al., 1987, 1990).

The regulation of gene expression and protein synthesis in germinating seeds is much Iess well characterized compared to regulation of developing seedlings and mature plants under stressed or unstressed conditions. Most dry seeds, however, do exhibit very low rates of protein synthesis from pre-existing mRNA and ribosomes (reviewed in Botha et al., 1992). This process rapidly increases with imbibition accom- panied by rapid transcriptional activity and translation. The transition in gene expression is linked to hydration of the seeds, but the signaling events remain unknown.

During the past two decades, it has become clear that shifts in cellular metabolism frequently involve the reversible phosphorylation of a transducing protein in response to a developmental or environmental cue (Ranjeva and Boudet, 1987; Budde and Randall, 1990). The basic events of this regulatory network include the perception of an extracellular signal and the coupling of this signal to an effector or secondary mes- senger, altering the phosphorylation state of a particular protein. Ultimately, this serves to modify the activity or function of a target protein or induce transcription of particular genes. At each step of this regulatory cascade, the signal may be amplified severalfold, resulting in the modulation of many downstream processes by protein phosphorylation in response to a single environmental factor. The phosphorylation state of specific proteins has been found to modulate the activity of a wide array of cellular processes; examples include proton pumps, vira1 infection, funga1 elicitors, blue-light- mediated phototropism, phytochrome-mediated events, auxin-mediated responses, light energy distribution in thy- lakoids, and the activation/deactivation of specific chloro- plastic, mitochondrial, and glyoxysomal enzymes (reviewed by Randall and Blevins, 1990). Despite the apparent ubiquity of this regulatory network, relatively little information is available for plant systems with respect to many common environmental stresses.

Here we demonstrate that aerobic pretreatment of E. crus- pavonis seeds results in changes in gene expression and reversible phosphorylation of two 18-kD polypeptides, priming the seeds for subsequent anaerobic gennination and growth. Changes in constitutive protein pattems, polypeptide synthesis, and phosphorylation associated with this priming phenomenon are presented.

MATERIALS AND METHODS

Plant Materials

Echinochloa crus-pavonis seeds were surface sterilized with 50% Clorox for 15 min, then rinsed three times with sterile,

deionized water. Seeds were placed in Petri dishes with two layers of filter paper wetted with 4 mL of sterilc; deionized water. Eloth aerobic and anaerobic treatments were camed out in t:he dark at 28OC. Anaerobic conditions were maintained i n a chamber (Forma Scientific, Inc., Marietta, OH) flushed continuously with a 90% N2-10% H2 gas mixture.

In Vivo Protein Labeling and Phosphorylation

For in vivo labeling experiments, E. crus-pavoni:; seeds were incubated for 5 d under anaerobic conditions before transfer to aerobic conditions for 1, 4, 12, or 24 h, and then retumed to N2 for 4 h, 24 h, or 7 d. Labeling took placc during the last 1 to 4 h of aerobic priming or following anaerobic incubation. In each case, 30 seeds were pulse-labeled with 100 pCi of T r a n ~ - ~ ~ S label (ICN Biomedicals, Inc., Costa Mesa, CA; 15% of the label in Cys and 70% in Met) in 800 pL of 50 mM Hepes buffer (pH 7.5). The seeds were rinsed three times with deionized water, blotted dry, frozen in liquid nitrogen, and stored at -8OOC until needed.

For protein phosphorylation, E. crus-pavonis seeds were incubated in N2 for 5 d prior to aerobic priming. Fifty seeds were labeled with 500 pCi of 32P04 or 33P04 (Amersham; E.I. DuPont de Nemours and Co.) in 800 pL of cairier-free 50 mM Hepes buffer (pH 7.5) in air or N2 for 4 11, and then washed three times in 50 mM nonradioactive K-PO, buffer (pH 7.5). The seeds were then blotted dry and frozen in liquid h12 or chased in air or N2 for 2 h in 50 mM nonradioactive K-P04 buffer (PH 7.5), blotted dry, and frozen in liquid N2.

Protein Extraction

Total proteins were extracted according to Hiirkman and Tanaka (1986) with the following modificatiors: 30 to 50 seeds were powdered in liquid N2 and homogenized in 3 mL of extraction buffer (0.7 M SUC, 0.5 M Tris [pH 9.21, 30 m~ HC1, 501 mM EDTA, 0.1 M KC1, 2% [v/v] 2-merc3iptoethanol, 1 mM PMSF, 1 mM N-tosyl-L-Phe chloromethyl ketone, and 0.1 mM leupeptin). The homogenate was transferred to a 15- mL centrifuge tube and an equal volume of wafer-saturated phenol containing O. 1 % hydroxyquinoline was added. The mixture was shaken for 10 min at room temperature and the phases were separated by centrifugation at l0,OOOg for 15 min. Proteins were precipitated from the phenol phase by adding 5 volumes of 0.1 M ammonium acetate in methanol and incubated ovemight at -2OOC. The prelipitate was washed three times with ice-cold 0.1 M ammoriium acetate in methanol and one time with cold acetone. The pellet was vacuum dried and resuspended in 9.5 M urea, 2% Nonidet P-40,5'% 2-mercaptoethanol, and 2% ampholytw (0.4%, pH 3-10; 0.8%, pH 4-6; and 0.8%, pH 5-8; Bio-Lyte, Bio-Rad). The insoluble material was removed by cenbifugation at 15,OOOh: for 5 min. Total protein was measured using the Bio- Rad protein assay reagent with BSA as the stmdard. The labeled protein was measured by liquid scintillatLon counting followiiig TCA precipitation of aliquots.

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Aerobic Priming of Echinochloa crus-pavonis Seeds 1151

Gel Electrophoresis

1-D SDS-PAGE was as described by Laemmli (1970). 35S-or 32P-labeled protein (7000 or 3000-4000 cpm, respectively)was loaded in each lane of a 12.5% (w/v) acrylamide runninggel and 4.5% (w/v) acrylamide stacking gel. 2-D NEpHGE/SDS-PAGE was performed according to O'Farrell et al. (1977)and Mujer et al. (1993). Equal radioactivity (100,000 cpm 35S-and 35,000 cpm 33P-labeled protein) was loaded onto theacidic end for NEpHGE. The anode (upper) buffer was 10mM H3PO4 and the cathode (lower) buffer was 0.02 N NaOH.Gels were run for 0.5 h at 200 V and for 4 h at 500 V for atotal of 2100 V X h. After electrophoresis, the gels wereequilibrated for 30 min in 50 mM Tris-HCl (pH 6.8), 2.3%SDS, 10% glycerol, and 5% 2-mercaptoethanol. The 2-DSDS-PAGE was performed as described by Laemmli (1970)employing a 12.5% (w/v) acrylamide running gel. Gels wereeither stained with Coomassie brilliant blue R-250 or silver-stained using the Bio-Rad silver stain kit, and/or dried forfluorography using the method of Jen and Thach (1982).

RESULTS

Patterns of Coomassie Brilliant Blue-Stained Polypeptides

The effect of aerobic priming on the pattern of constitutiveand inducible polypeptides in E. crus-pavonis was examinedby separating protein extracts by 2-D NEpHGE/SDS-PAGE(Fig. 1). All seeds were first incubated for 5 d under anoxiaand then either maintained for an additional 6 d under N2(Fig. 1 A) or transferred to air for 4 h (Fig. IB), 12 h (Fig. 1C),or 24 h (Fig. ID) and then back to N2 for another 6 d. Totalproteins were extracted at the conclusion of the experiments.Two observations were noted from these experiments. First,ASP55 and a cluster of proteins around ASP37 increasedwith longer aerobic priming prior to anaerobic incubation.Second, several polypeptides (indicated by the unlabeledarrowheads in Fig. 1) decreased with longer aerobic primingtimes, possibly reflecting increased degradation of storageproteins.

Patterns of de Novo-Synthesized Polypeptides

The patterns of de novo-synthesized polypeptides, as in-fluenced by anaerobiosis and aerobic priming, were examinedby both 1-D SDS-PAGE (Fig. 2) and 2-D NEpHGE/SDS-PAGE (Figs. 3-5), following labeling with [Trans-35S]Met/Cys as detailed for each figure. For the 1-D analysis, seedsof E. crus-pavonis were incubated for 5 d under anoxia,aerobically primed for 1, 4, 12, or 24 h, and either transferredback to anoxia or maintained in air. Incorporation of 35S intoprotein took place during the final 1 h of treatment (signifiedby asterisks in Fig. 2). One of the most notable observationsfrom this analysis was the increased labeling of a 20-kDpolypepride (ASP20) under anaerobic conditions (Fig. 2, com-pare lanes 2 [N2] and 3 [air]). Label in ASP20 disappearedwith increasing periods of aerobic priming and labeling in air

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Figure 1. Effect of aerobic priming on the constitutive expressionof proteins in £. crus-pavonis. E. crus-pavonis seeds were treated asfollows: A, 5 d of N2 + 6 d of N2; B, 5 d of N2 + 4 h of air + 6 d ofN2; C, 5 d of N2 + 12 h of air + 6 d of N2; and D, 5 d of N2 + 24 hof air + 6 d of N2. Total proteins were separated by 2-D NEpHGE/SDS-PACE and stained with Coomassie brilliant blue. The labeledarrowheads highlight ASP55 and a cluster of polypeptides aroundASP37 that increased with longer aerobic priming. Several proteins,indicated by the unlabeled arrowheads, decreased with longeraerobic priming.

(Fig. 2, lanes 4,8,12, and 16). If aerobic priming was followedby labeling under anaerobic conditions, label in ASP20 in-creased with longer anaerobic treatment (Fig. 2, lanes 7, 11,15, and 19).

The 2-D analysis of de novo-synthesized polypepride pat-terns was carried out for several different aerobic primingperiods. Only the results from 0 h (Fig. 3), 4 h (Fig. 4), and24 h (Fig. 5) of aerobic priming are shown here, since theyillustrate the major points observed for all of the analyses. Inthe absence of aerobic priming, polypeptide patterns of seedsincubated for 5 d in anoxia plus another 7 d in either N2 (Fig.3A) or air (Fig. 3B) and labeled for the final 4 h in theirrespective atmospheres differed considerably. ASP20 wasdetected only in the anaerobically labeled seeds, whereasASP37 was detected about equally in both aerobically andanaerobically treated samples. ASP55 was not clearly dis-cernible in these examples.

In Figures 4 and 5, the effect of aerobic priming (4 versus24 h) on subsequent anaerobic protein synthesis is demon-strated. Upon transfer to air after the initial 5 d of anaerobicincubation, aerobic patterns of protein synthesis were evidentwithin 4 h of aerobic priming (Fig. 4A) and synthesis in-creased with longer priming up to 24 h (Fig. 5 A). Upon return www.plantphysiol.orgon April 12, 2019 - Published by Downloaded from




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Figure 2. Effect of aerobic priming on the de novo synthesis ofASP20. E. crus-pavonis seeds were incubated for 5 d under anoxiaand then maintained in N2 an additional 7 d (lane 2), aerobicallyprimed for varying times and labeled in air (lanes 3, 4, 8, 12, and16), or aerobically primed for varying times and then transferredback to N2 before labeling (lanes 5-7, 9-11, 13-15, and 17-19).Seeds or seedlings were labeled with Trans-35S label for the final 1h of treatment (atmospheres for labeling are indicated by asterisks)and the protein extracts were analyzed by SDS-PACE fluorography.Equal counts were loaded per lane. The arrowhead points to a 20-kD polypeptide that disappeared with increasing periods of aerobicpriming (see lanes 4, 8, 12, and 16) and is enhanced with longeranaerobic treatment following the aerobic priming (see lanes 7, 11,15, and 19).

to anoxia following 4 or 24 h of priming, protein synthesiswas greatly repressed during the first 4 h (Figs. 4B and 5B);however, ASP37 and ASP55 were already major anaerobicproteins. Within 24 h after return to anoxia, the synthesis ofmany additional polypeptides resumed (Figs. 4C and 5C) andASP20 was apparent in the 24-h primed samples (Fig. 5C).After 6 or 7 d of anoxia, ASP20 was evident in both the 4-h(Fig. 4D) and 24-h (Fig. 5D) primed samples. Following thelonger (24 h) aerobic priming, the protein patterns in generalresembled more closely those of seedlings labeled in air(compare Figs. 4D and 5D with 3B). It is also noted that onlyafter aerobic priming followed by anaerobic incubation wasan enhancement of expression of ASP37 and ASP55 seen(compare Fig. 4, A with B-D, and Fig. 5, A with B-D).

Protein Phosphorylation

To determine if protein phosphorylation during aerobicpriming may be involved in stimulating the subsequent an-aerobic germination process, seeds were exposed to varyingsequences of atmospheric conditions and incubated with32PO4 for 4 h. Proteins were extracted and separated by 1-DSDS-PAGE (Figs. 6 and 7) and 2-D NEpHGE/SDS-PAGE(Fig. 8). In the first experiment shown in Figure 6, seeds wereincubated anaerobically for 5 d and labeled under anaerobicconditions (lanes 1 and 3) or transferred to and labeled in air(lanes 2 and 4). Equal quantities of protein (200 jig) wereloaded in lanes 1 and 2 and equal counts (4000 cpm) wereloaded in lanes 3 and 4. Several polypeptides of the samemolecular mass were phosphorylated in both N2 and air;however, one particular polypeptide (18 kD) was specificallyphosphorylated only in seeds transferred from N2 to air andlabeled in air (lanes 2 and 4).

Several pulse/chase experiments were also carried out withvariations in the sequence of exposure of the seeds to ananaerobic versus aerobic atmosphere (Fig. 7). In all cases theseeds were first incubated for 5 d under anaerobic conditionsand then either maintained under N2 or transferred to air.The 18-kD polypeptide was specifically phosphorylated

Figure 3. Anaerobic and aerobic proteinexpression in E. crus-pavonis. E. crus-pavonisseeds were incubated in N2 for 5 d and thenmaintained in N2 or transferred to air for 7 dand labeled with Trans-35S label for the final 4h. A, Five days of N2 + 7 d of N2; B, 5 d of N2

+ 7 d of air. Proteins were extracted and ana-lyzed by 2-D NEpHGE/SDS-PACE fluorogra-phy. Equal counts were loaded onto the acidicends for NEpHCE. Equal labeling of ASP37 wasnoted in both N2 and air, whereas ASP20 wasdetected only under anoxia.

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Figure 4. Protein expression after 4 h of aero-bic priming. E. crus-pavonis seeds were incu-bated as follows: A, 5 d of N2 + 4 h of air; B, 5d of N2 + 4 h of air + 4 h of N2; C, 5 d of N2 +4 h of air + 24 h of N2; and D, 5 d of N2 + 4 hof air + 7 d of N2. The proteins were labeledwith Trans-35S label for the final 4 h of the entiretreatment and analyzed by 2-D NEpHGE/SDS-PACE fluorography. Equal counts were loadedonto the acidic ends for NEpHCE.

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Figure 5. Protein expression after 24 h of aero-bic priming. E. crus-pavonis seeds were incu-bated as follows: A, 5 d of N2 + 24 h of air; B,5 d of N2 + 24 h of air + 4 h of N2; C, 5 d ofN2 + 24 h of air + 24 h of N2; and D, 5 d of N2

+ 24 h of air + 6 d of N2. The proteins werelabeled with Trans-35S label for the final 4 h ofthe entire treatment and analyzed by 2-DNEpHGE/SDS-PAGE fluorography. Equalcounts were loaded onto the acidic ends forNEpHGE.




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Figure 6. Phosphorylation of an 18-kD polypeptide. E. crus-pavonisseeds were incubated anaerobically for 5 d and then labeled in N2

with 32PO4 for 4 h (lanes 1 and 3) or transferred to air and labeledin air for 4 h (lanes 2 and 4). Proteins were separated by SDS-PAGEand phosphorylation was observed by fluorography. Equal quan-tities of protein (200 Mg) were loaded in lanes 1 and 2. Equal counts(4000 cpm) were loaded in lanes 3 and 4. Phosphorylation of an18-kD polypeptide was observed only in seeds labeled in air.

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Figure 7. 32PO4 Pulse/chase phosphorylation of an 18-kD polypep-tide. E. crus-pavonis seeds were anaerobically incubated for 5 d andthen treated as follows: transferred to and labeled (indicated withan asterisk) in air with 32PO4 for 4 h (lane 1) followed by a 2-h chase(c) in either air (lane 2) or N2 (lane 3); labeled in N2 for 4 h (lane 4)followed by a 2-h chase in air (lane 5) or N2 (lane 6); or transferredto and incubated in air for 4 h and then labeled in air (lane 7) or N2

(lane 8) for 4 h. The polypeptides were separated by SDS-PAGEand treated for fluorography. Equal counts were loaded in eachlane. Phosphorylation of the 18-kD polypeptide was observed onlyin seeds that had been exposed to air during their treatment.

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Figure 8. 2-D NEpHCE/SDS-PAGE of "PGvlabeled polypeptides.E. crus-pavonis seeds were treated as detailed in Figure 6 exceptlabeling was with 33PO4 during the final 4 h of treatment. Treatmentswere 5 d of N2 + 4 h of N2 (A) and 5 d of N2 + 4 h of air (B). Thefinal extracts were subjected to 2-D NEpHGE/SDS-PAGE fluorog-raphy to identify differences in polypeptide phosphorylation. Equalcounts were loaded onto the acidic ends for NEpHGE. Two 18-kDpolypeptides were phosphorylated only in air.

when £. crus-pavonis seeds were (a) transferred to and labeledin air (lane 1), (b) labeled in air and chased in air (lane 2), (c)labeled in air and chased in N2 (lane 3), or (d) given a 4-haerobic priming prior to labeling in air (lane 7). Phosphoryl-ation of the 18-kD polypeptide was not observed when E.crus-pavonis seeds were (a) labeled in N2 (lane 4), (b) labeledin N2 followed by chasing in N2 (lane 6), or (c) given a 4-haerobic priming prior to labeling in N2 (lane 8). Finally, ifseeds were incubated with label under N2 and then chasedin air, 32P was detected in the 18-kD polypeptide (lane 5).

The phosphorylation of the 18-kD polypeptide was furtherresolved by separating proteins by 2-D NEpHGE/SDS-PAGE. Seeds were incubated for 5 d under anaerobic con-ditions and labeled with 33PO4 for 4 h in N2 (Fig. 8A) or inair (Fig. 8B). The single 18-kD polypeptide observed on 1-DSDS-PAGE separated into two polypeptides of identical mo-lecular mass but slightly different pi values on 2-D gels (Fig.8B). Phosphorylation of the two 18-kD polypeptides waslimited to those seeds labeled in air. The broad 20-kD proteinband phosphorylated in both air and N2 as observed inFigures 6 and 7 was separated by 2-D SDS-PAGE into severalpolypeptides with different pi values (Fig. 8, A and B).

DISCUSSION

E. crus-pavonis is considered to be intolerant to anaerobiosisand flooding as a result of its inability to germinate in a N2atmosphere or under flooded conditions. We have observed,however, that even after 4 weeks of anaerobic incubation,the seeds that had imbibed water remain viable and germi-nate and grow upon return to air. Most recently, we observedthat if £. crus-pavonis seeds are given a short exposure to airin the imbibed state (aerobic priming) they will subsequentlygerminate and grow in an anaerobic environment, similar tothe other naturally flood-tolerant Echinochloa species (Ken-nedy et al., 1980, 1990). However, in the absence of oxygen,the metabolic activity and adenylate levels for E. crus-pavonisare greatly reduced compared with tolerant Echinochloa spe- www.plantphysiol.orgon April 12, 2019 - Published by Downloaded from




cies (Rumpho et al., 1984; Kennedy et al., 1987) and rice (Mocquot et al., 1981). Although E. crus-pavonis does not exhibit the same metabolic activity as naturally tolerant plants, it also does not resemble intolerant plants such as maize, soybean, or peas. Seeds of these plants initially exhibit very elevated respiratory and metabolic fermentative activi- ties in the absence of oxygen, and if the stress is not quickly alleviated metabolic activity decreases and tissue death occurs (Bewley and Black, 1978; Hook and Crawford, 1978; Moc- quot et al., 1981; Cobb and Kennedy, 1987). The tolerance to anaerobic conditions exhibited by lettuce seeds most closely resembles that of E. crus-pavonis. Both seed types are fully capable of aerobic respiration and germination, but when placed under anaerobic conditions in an imbibed state, respiratory and metabolic activity virtually cease and energy charge values drop to C0.3 (Raymond and Pradet, 1980; Kennedy et al., 1987, 1990). Upon reoxygenation both seed types rapidly commence metabolic activity.

Recently, we (Mujer et al., 1993) demonstrated the induction of several classes of ASPs among the tolerant Echinochloa species germinated and grown for 5 d under continuous anaerobic stress. One key aspect that was not addressed in that paper is the array of early metabolic events that accom- panies the onset of anaerobiosis. Such initial events may be important components of the mechanism responsible for imparting anaerobic tolerance and, hence, differential sur- viva1 of anaerobically tolerant and intolerant Echinochloa species. The present study has shown the rapid induction of 37- and 55-kD polypeptides during anaerobic stress, following aerobic priming, in E. crus-pavonis. These polypeptides were also induced under anoxia among tolerant Echinochloa species. ASP55 has subsequently been identified as the gly- colytic enzyme enolase (Fox et al., 1993). In endosperm cells of developing castor oil seeds, a major form of enolase is found in the plastids (Miemyk and Dennis, 1992). This plastidic enolase isozyme is believed to play an indirect role during periods of most rapid accumulation of storage lipids during endosperm development or during periods of elevated membrane lipid synthesis via conversion of pyruvate to ace- tyl-COA, the substrate for de novo fatty acid synthesis. Eno- lase activity was found to be highest during these periods. In E. phyllopogon, lipid biosynthesis and storage in the shoots increases dramatically under anoxia (Knowles and Kennedy, 1984; Kennedy et al., 1991). Similarly, enolase activity is also highest during this period (Fox et al., 1993; T.C. Fox, C.V. Mujer, M.E. Rumpho, unpublished data). Enolase has also been reported as a heat-shock protein in yeast (Iida and Yahara, 1985) and as an important structural protein in the lenses of vertebrate eyes, where it serves a protective role (Wistow and Piatigorsky, 1987; Lebioda and Stec, 1988). The physiological role of enolase in imparting anaerobic tolerance in Echinochloa still needs to be established. We have not yet identified the 37-kD polypeptide but, based on molecular mass, it could possibly be alcohol dehydrogenase.

In contrast to maize (Sachs et al., 1980), no transition polypeptides were detected in E. crus-pavonis seeds immedi- ately following the imposition of anoxia after aerobic priming. Instead, the 37- and 55-kD polypeptides were persistently synthesized for the duration of the anaerobic regime, together with the subsequent induction of the "aerobic" protein com-

plement. It is interesting to note, however, that aerobic priming is essential for protein degradation to commence in E. crus-pavonis. In the absence of this pretreatment, germination is blocked, possibly due to the inability of E. crus-pavonis seeds to mobilize essential energy sources for growth. How- ever, once the gennination barrier is overcome, slow growth of the shoot resumes. In contrast, seeds of the tolerant Echin- ochloa species do not require the aerobic priming, indicating that they possess the metabolic machinery to initiate gennination anaerobically.

The 20-kD polypeptide is another protein that preferen- tially accumulates under anoxia in E. crus-pavonis. This could be due to either de novo synthesis or the accumulation of a stable degradation product of a higher molecular mass protein that is rapidly tumed over under anoxia. The low molecular mass of the polypeptide suggests that it could be a monomeric enzyme or a subunit of a multimeric enzyme or structural protein. The decrease in synthesis of the 20-kD polypeptide upon exposure of seedlings to an aerobic environment suggests that it may tum over very rapidly in air and/or that it is specifically involved in metabolic processes only under anoxia. As noted in Figures 6 to 8, several polypeptides with varying pI values but a molecular mass of about 20 kD were phosphorylated in both air and N1. However, the relationship (if any) between the 20-kD polypeptide induced by anaerobiosis and those phosphorylated in air or N2 is not known.

Protein phosphorylation as a ubiquitous regulatory mechanism is recognized among both prokaryotes and eukaryotes. The reversible phosphorylation of proteins is implicated in regulating gene expression, metabolism, and responses of organisms to biotic and abiotic stresses. Among plants, this modification of proteins has been shown to occur in nuclei, plastids, mitochondria, and microsomal membranes, and its influence has been observed at the transcriptional, posttranscriptional, translational, and posttranslational levels (for re- view, see Ranjeva and Boudet, 1987; Randall and Blevins, 1990; Gallie, 1993). Studies leading to the discovery that reversible phosphorylation of proteins is a rapid and flexible response to environmental insult were prompted by observations of a lack of correlation between levels of mRNA and transcription and between mRNA levels and protein synthesis upon shifting of plants to a stressful environment. For example, the imposition of low-oxygen stress on the intolerant plant maize results in the rapid inhibition of normal protein synthesis (Sachs et al., 1980). This is apparently due to the repression of translational initiation and/or elongation of stable aerobic messages, since these mRNAs are not initially degraded (Bailey-Serres and Freeling, 1990; Webster et al., 1991a, 1991b). After a short period, however, the preferential translation of stress-induced messages results in the synthesis of a characteristic set of ASPs (Sachs et al., 1980). Associated with these hypoxia-induced changes in translational activity in maize is a decrease in ribosomal protein synthesis and a decrease in phosphorylation of a 31-kD ribosomal protein (Bailey-Serres and Freeling, 1990). Fur- thermore, Webster et al. (1991a) reported an increased phosphorylation of one isoform of eIF-4A, a eukaryotic initiation factor, upon exposure of maize seedlings to hypoxia. The exact mechanisms by which these modified proteins influence selective translation of mRNAs during hypoxic stress remain

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to be determined. Different hypotheses are being pursued, however, based on parallel changes in hypoxia-induced phosphorylation and changes in metabolism and pH flux (Bailey-Serres and Freeling, 1990; Webster e t al., 1991a, 1991b).

One of the best examples of transcriptional control of low- oxygen-induced gene expression is seen in the aerobic respiratory control (Arc) system of the prokaryote Escherichia coli (Iuchi and Lin, 1988; Spiro and Guest, 1991). In this case, under anoxic conditions, altered respiratory electron flux directly induces the phosphorylation of the membrane-bound protein ArcB. ArcB in tum phosphorylates ArcA, which binds DNA to induce the transcription of Cyt d oxidase (an anaerobic respiration enzyme) and inhibits transcription of aerobic respiratory enzymes as well as metabolic enzymes of the citric acid and glyoxylate cycle. In this system, differential protein phosphorylation is involved in a global cascade system to efficiently regulate and coordinate metabolic path- ways.

The preferential phosphorylation of two 18-kD polypeptides during aerobic priming of E. crus-pavonis seeds suggests that modification by phosphorylation may be an important regulatory control signal for initiating anaerobic germination, perhaps by promoting early events in the germination process. These findings provide a n excellent system to explore the molecular regulation of anaerobic gene expression during germination. Furthermore, differences between E. CTUS-

pavonis and E. phyllopogon (e.g. the ability to germinate without oxygen, adenylate levels, metabolic activity) provide a comparative basis for elucidating the mechanisms responsible for switching between aerobic and anaerobic metabolism in germinating seeds.

Received December 17, 1993; accepted March 20, 1994. Copyright Clearance Center: 0032-0889/94/105/1149/09.

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