Alcohol dehydrogenase (ADH) is one of a number of enzymes of glycolysis and fermentation known to be synthesized preferen- tially under low O, conditions.
Plant Physiol. (1993) 101: 407-414
Hypoxic and Anoxic lnduction of Alcohol Dehydrogenase in Roots and Shoots of Seedlings of Zea mayd Adh Transcripts and Enzyme Activity David 1. Andrews,
B. Creg Cobb, James R. Johnson, and Malcolm C.
Drew*
Department of Horticultural Science, Texas A & M University, College Station, Texas 77843-21 33 1991b). At least eight of the ANPs have been identified as enzymes of glycolysis and ethanolic fermentation (BaileySerres et al., 1988), among which ADHs and pyruvate decarboxylase are prominent. Transcriptional regulation of protein synthesis is also implicated in the anoxic synthesis of ANPs. O2 deficiency induced an increase in transcript levels in primary roots of maize seedlings for SUCsynthase (Springer et al., 1986; McElfresh and Choury, 1988), pyruvate decarboxylase (Kelley, 1989), Adhl (Ferl et al., 1980; Gerlach et al., 1982; Rowland and Strommer, 1986), andAdk2 (Dennis et al., 1985). For Adhl mRNA, induction over the aerobic background leve1 was as much as 20- to 50-fold after 5 to 10 h of O2 deficiency (Ferl et al., 1980; Gerlach et al., 1982; Rowland and Strommer, 1986). The root tip zone of maize seminal roots, compared with the more mature root tissues, is especially vulnerable to hypoxia or anoxia because of its intense metabolic activity and high respiration rate. The sudden imposition of anoxia with inhibition of oxidative phosphorylation caused a rapid decline in energy metabolism, and death of the apical meristem occurred in 15 to 20 h (Roberts et al., 1984a, 1984b; Saglio et al., 1988; Johnson et al., 1989). During that time, there was no incorporation of I4C-labeledamino acids or 35Slabeled Met into proteins (D.J. Hole, B.G. Cobb, and M.C. Drew, unpublished) or induction of ADH activity (Johnson et al., 1989). By contrast, the anoxia tolerance of the root tip is considerably greater if roots are first exposed to a partia1 deficiency of 02,resulting in tissue hypoxia. Following hypoxic pretreatment, the viability of the apical meristem was extended to at least 96 h, ATP concentrations were greater, ADH activity was induced, and the rate of anaerobic respiration was stimulated (Saglio et al., 1988; Johnson et al., 1989; Hole et al., 1992). These results have led us to question whether anoxia tolerance of cells in the apical meristem is truly different from the remainder of the root, or, alternatively, whether hypoxia had been an inadvertent feature of previously published experiments with primary seedling roots. For maize root tips, hypoxia rather than anoxia induced elevated activity of ADH (Saglio et al., 1988; Johnson et al., 1989). In maize protoplasts (Howard et al., 1987; Walker et al., 1987), there were severalfold increases in Adhl mRNA with hypoxia (O2concentra-
Alcohol dehydrogenase (ADH) is one of a number of enzymes of glycolysis and fermentation known to be synthesized preferentially under low O , conditions. We examined levels of Adhl transcripts and of ADH activity in 5-mm root tips, root axes (the remainder of the seminal root), and shoots of maize (Zea mays 1. cv TX 5855) seedlings. Seedlings with roots averaging about 60mm long were transferred from fully aerobic conditions (solutions sparged with 40% [v/v] O,) to anaerobic (02-free) conditions, or to an intermediate O , concentration. There was no prior acclimation to low O,. In root tips, anoxia induced Adhl transcripts and enzyme activity at 6 h, but this was followed by a rapid decline so that at 12 to 18 h neither were detectable and the root tips were dead. In contrast, higher levels of Adhl transcripts and enzyme activity were maintained for at least 48 h i n root axes and shoots. When induction at 6 h was measured over a wide range of 0, concentrations, a peak i n ADH activity occurred in all tissues at 4% (v/v) O,. Maximum levels of transcripts, however, were i n the range of O to 4% O,, depending on the tissue. The time course of hypoxic induction (at 4% O,) i n root tips showed a peak in transcript levels at 6 h, whereas ADH activity continued to rise throughout the 24-h experiment. These results show that i n root tips, ADH induction by anoxia was small and transient relative to induction by hypoxia.
When entire primary (seminal) roots of maize seedlings are made anoxic by subjecting them to strictly anaerobic conditions through rigorous exclusion of 02,there is a marked change in the pattern of protein synthesis as determined by incorporation of [3H]Leuor [35S]Metfollowed by two-dimensional SDS-PAGE. In place of severa1 thousand proteins detectable under fully aerobic conditions, total protein synthesis was strongly inhibited and only 20 proteins were resolvable when pulse labeling commenced after 5 h of anoxia (Sachs et al., 1980). This pattern of synthesis of ANPs was maintained for up to 70 h of continuous anoxia, at which point the primary root began to die. An early response to anoxia is suppression of mRNA translation in general, but with preferential translation of transcripts of the genes encoding the ANPs (Sachs et al., 1980; Bailey-Serres and Freeling, 1990; Webster et al., 1991a,
' Texas Agriculture Experiment Station Paper No. 30543. Supported by U.S. Department of Agriculture Competitive Grant No. 90-37264-5523. * Corresponding author; fax 1-409-845-0627.
Abbreviations: ADH, alcohol dehydrogenase; ANP, anaerobic polypeptide.
407
Andrews et al.
408
tion 5-10% [v/v], balance N2)but no increase at 1%0, when cell viability declined. Thus, for some cells or tissues, expression of genes encoding at least some of the ANPs may not be signaled by anoxia per se, but by appreciable, subambient concentrations of 02.Study of this phenomenon is complicated by the fact that hypoxia in an organ as bulky as a maize primary root is imprecisely defined because when respiration is rapid, radial gradients in O2 concentration can result in an anaerobic core of cells surrounded by fully aerobic ones (Armstrong and Beckett, 1987; Thomson and Greenway, 1991). A further complication is variation in the use of existing terminologies by different investigators. In this and earlier publications, we have used the terminology of Pradet and Bomsel (1978), by which tissues or cells are defined as hypoxic when the O, partial pressure limits the production of ATP by mitochondria. Anoxia describes the situation where the O2 partial pressure is so low that ATP production by oxidative phosphorylation is negligible relative to that generated by glycolysis and fermentation. Anaerobic means 0,free. On that basis, "hypoxia" has been used by some (Roberts et al., 1984a, 198413; Bailey-Serres and Freeling, 1990; Webster et al., 1991a, 1991b) for cells that were virtually 02-free and "anaerobic" was applied to situations where there was appreciable 02,more likely associated with hypoxia (Rowland and Strommer, 1986; Howard et al., 1987; Walker et al., 1987). It is for these reasons that we have sought to quantitatively examine the "anaerobic response" using Adkl mRNA levels and ADH enzyme activity in maize (Zea mays L.) seedlings as a model system. The specific aims of this investigation were (a) to examine induction by hypoxia of Adkl mRNA in root tips, more mature root zones, and shoots during exposure of intact seedlings to subambient concentrations of O2 and (b) to determine the relationship between Adkl transcript levels and induction of ADH enzyme activity. MATERIALS A N D METHODS Plant Growth Conditions and Gassing Treatments
Caryopses of maize (Zea mays L. cv TX 5855) were treated for 10 min with a 20% (v/v) Clorox bleach solution, rinsed in tap water for 4 to 6 h, placed in contact with moistened germination paper, and allowed to imbibe in the dark at 25OC in an incubator. After 3 d, seedlings were moved to expanded polystyrene floats in 3.5-L screw-top glass jars with entrance and exit ports for gases in the lids as described (Johnson et al., 1989). The jars contained 2 L of 3 mM CaSO,, which was continuously sparged (200 mL/min) with air for 24 h, followed by a period of sparging with various 0 2 / N 2mixtures to vary the O2 partial pressure, or with prepurified NZ gas (99.97% N2). The toots were in contact with solution while the coleoptile and developing first leaf were in the gas phase. The concentration of dissolved O2 is directly proportional to the O2 partial pressure in the gas mixture used to sparge the solution. For reference, the concentration in water in equilibrium with moist air at 25OC (21.4% [v/v] O,) is 258 p ~ and , the presence of dilute C a s o 4 has negligible effects on 0, solubility. The flow and mixing of gases was controlled by
Plant Physiol. Vol. 101, 1993
electronic gas flow meters, and small, positive pressures were maintained within each jar to avoid contamination with the ambient air (Johnson et al., 1989). Details of the gassing treatments are given in relation to individual experiments described in "Results." FoIlowing the gassing treatment, seedIings were dissected into root tips (the terminal 5-mm portion of the primary root), the root axis (the remaining primary root tissue), and shoots (the coleoptile and emerging first leaf). Tissues were immediately frozen in liquid NZ and stored at -8OOC until they were used for the isolation of total RNA or assay of protein and ADH. Depending on the treatment imposed, the amount (gg mg-' fresh weight) of total RNA obtained from root tips was from 2 to 7, and root axes yielded from 0.2 to 0.6 with a similar amount obtained from shoots. Protein amounts (pg mg-' fresh weight) were 10 to 25 for root tips, 1 to 4 for root axes, and 2 to 10 for shoots. Protein and Enzyme Analysis
Assay of ADH (EC 1.1.1.1)was in triplicate, using a standard procedure (Cobb and Kennedy, 1987) in which the conversion of NAD to NADH was determined spectrophotometrically. Protein was assayed by Bradford's method using BSA as standard. A unit of enzyme activity converted 1 pmol of NAD to NADH per min. Northern Analysis
Total RNA for northern analysis was isolated using the lithium chloride method (Stiekema et al., 1988). Yield was determined by spectrophotometric absorbance at a wavelength of 260 nm. After electrophoresis of 25 pg of total RNA per lane on agarose gels, RNA was transferred to GeneScreen Plus membranes according to the manufacturer's specifications. Northern gel blotting was done initially only to verify that the sample was undegraded, and that the probe only hybridized to a single band of proper size. Quantitation was done on samples consisting of 20 pg of total RNA per slot applied directly to GeneScreen Plus, in a slot-blot format, also according to the manufacturer's specifications. An Adkl linear cDNA fragment (approximately 1600 bp) was isolated from bacterial plasmid pZML 793 (Dennis et al., 1984) by band interception onto NA45 DEAE membranes (Schleicher & Schuell) following the manufacturer's suggested protocol. Fragments obtained in this manner were quantitated on agarose gels by comparing ethidium bromide fluorescence with standardized, HindIII-digested lambda DNA fragments (BRL). Radiolabeled probe was prepared from 25 ng of linear DNA fragment by the random primer method (Feinburg and Vogelstein, 1983, 1984). After synthesis, radiolabeled fragment was purified by spin-column chromatography and specific activity and percent incorporation were calculated by counting aliquots of the sample, before and after purification, in standard scintillant. Using this method, probes were routinely obtained with a specific activity of at least 1 x 10' cpm/pg. Because probes of different specific activity were used, quantitation of northern blots obtained in different experiments cannot be compared.
Alcohol Dehydrogenase in Maize Seedlings
409
Table 1. Message levels for Adh 1 and ADH enzyme activity in 5-mm root tips of maize seedlings in 3 mM Caso4 solution sparged with air and with 40% O, Experiment A, After 3 d of germination in an incubator, germinated seedlings were arbitrarily separated into two groups; with roots 2 to 4 cm long and with roots 4 to 6 cm long. Experiment 6, Roots were 4 to 6 cm long when treatment with 40% O2 began. For ADH enzyme activity, values are mean (+SE), n = 3. Adhl mRNA
Treatment
Experiment A: 20.4% oxygen (air) Root length 4 cm Experiment B: 40% oxygen 12 h 24 h 48 h
ADH Enryme Activity
cpm pg-' total RNA
cpm mg-' fresh wt
units mg-' protein
units g-' fresh wt
0.300 0.260
0.94 1.79
0.212 (0.028) 0.115 (0.013)
6.56 (0.77) 3.27 (0.36)
0.303 0.310 0.303
0.808 0.651 0.785
0.107 (0.011) 0.061 (0.006) 0.074 (0.007)
1.63 (0.016) 0.741 (0.008) 1 .O4 (0.009)
Membranes were prehybridized for 2 h in 5 mL of the following solution: 5 x SSC, 50% deionized formamide, 5X Denhardt's solution, 100 pg/mL of calf thymus DNA, 50 mM sodium phosphate (pH 6.8), and 0.5% SDS. After 2 h at 42OC, 2 to 3 mL of solution was removed and probe was added to give an activity of 2 X 107 cpm/mL. Hybridization was at 42OC for 18 h in a temperature-regulated, forced-air incubator. The membrane was then washed in 200 mL of 2X SSC, 0.5% SDS for 10 min at room temperature. A second wash was carried out at 42OC for 30 min in a fresh solution of the same composition. After two intermediate washes in I X SSC, 0.5% SDS for 30 min at 55OC, the membrane was washed twice at 0.2X SSC, 0.1% SDS for 30 min at 68OC. The final washing conditions used have been shown to be specific for Adhl (Strommer et al., 1982). The membrane was visualized and bound radioactive probe was quantitated on a Betascope 603 Blot Analyzer (Sullivan et al., 1987; Schneider-Gadicke et al., 1989) (Betagen Corp., Waltham, MA) and autoradiographed using preflashed, Kodak X-Omat XAR-5 xray film (Eastman Kodak Co., Rochester, NY). Films were developed with a Konica QX-130APLUS x-ray film processor (Konica Corp., Japan). A reagent blank lacking RNA was included in each blot to give the background level for subtraction from 32Passayed by the Betascope Analyzer. To verify quantitation using the Betascope Blot Analyzer, a seria1 dilution was made of two 32P-labeledprobes (3-7000 cpm by liquid scintillation counting) that were slot blotted as described above and assayed using the Betascope Blot Analyzer. Three durations of autoradiographic exposure were quantitated subsequently using a scanning densitometer and by liquid scintillation counting. Relative to the latter method, the correlation against the Betascope estimate gave an ? of 0.999 to 1.000, which applied equally well at low or high levels of 32P, compared with approximately 0.800 against autoradiography/densitometry. The Betascope Blot Analyzer was therefore used for the determination of a11 northern blot data in this paper. We found that the Betascope Analyzer gave highly accurate and reproducible assays of extremely low levels of P-radiation. For example, in a typical run, background was 3.9 f 0.39 cpm, whereas hybridized probe gave 28 f 1 to 114 t- 2 cpm.
RESULTS Initial, Uninduced Levels of A D H
At 25OC, the critica1 O2 pressure for the respiration of submerged maize root tips (Saglio et al., 1984) is in excess of the O2 concentration in air-saturated water. Because of this, roots were routinely bubbled with 40% (v/v) O2to avoid any opportunity for induction of enzymes by mild hypoxia (Saglio et al., 1988; Johnson et al., 1989). In the present research, we examined the level of Adhl message and ADH enzyme activity shortly after germination in ambient air and after exposure to solution sparged with 40% 02. When mRNA was hybridized with the Adhl probe (Table I ) , bound radioactivity was essentially equal in a11 samples relative to total RNA, but on a fresh weight basis there was a 2-fold increase in Adhl mRNA in roots >4 cm, perhaps reflecting hypoxic induction during the germination process that was reversed following the 40% O2 pretreatment. Similar amounts of soluble protein (approximately 30 p g g-' fresh weight) were recoverable from root tips at both stages of germination, but this decreased to about half that level after 12 h at 40% 02.The ADH activity per milligram of protein or per gram fresh weight was approximately 2fold greater in roots 4 cm long (Table I). There was clearly a lower level of ADH enzyme activity after the 40% O2 treatment (Table I). The root tips from airbubbled roots >4 cm long had approximately 4-fold more ADH activity (per gram fresh weight) than did those at 40% O2 for 24 h. Pretreatment with 40% O2 avoids any risk of hypoxia and may be a necessary step at 25OC to avoid induction, Adhl Transcript Levels and A D H Activity on Transfer from Aerobic to Anaerobic Conditions
This experiment was designed to observe the effect of the duration of anoxia on Adhl mRNA, ADH enzyme activity, and total RNA and protein content in root tips, root axes, and shoots. Seedlings were pretreated with 40% O2 for 24 h before rapid deoxygenation with 02-free N2 gas. At zero
Andrews et al.
41 O
time, root tips had a significantly higher amount of total RNA (4.4 pg mg-' fresh weight) than did root axes and shoots (0.3 and 0.4 pg mg-', respectively). At zero time, Adhl mRNA level was very low, but at 6 h of anoxia, amounts of Adhl transcripts increased dramatically in the root tips and to a lesser extent in the root axes and shoots (Fig. 1A). The level in root axes stayed fairly constant throughout the experiment but in root tips it declined to barely detectable levels by 18 h. Relative to total RNA (Fig. lB), transcript levels increased in a11 three tissue types at 6 h, followed by a pronounced decline in root tips. At zero time, the root axes had 2.8 times that of the root tips. At 12 h, the ratio had increased to 3.6 times, and at 24 h, to 59 times. Root axes showed a further rise in amounts of Adhl mRNA between 18 and 24 h (Fig. 1B). Thus, there was a loss of the ability to withstand prolonged anaerobic conditions by these root tips and a subsequent acquisition of this ability by the remainder of the primary root tissue. Differences between root tips and root axes were revealed also in terms of ADH enzyme activity. On a fresh weight basis (Fig. 2A), enzyme activity increased during the initial periods of anoxia in a11 three tissue types. The root tips showed appreciable activity at zero time, and activity reached a maximum at 6 h of anaerobic treatment, with a marked decline between 12 and 18 h. When ADH activity was expressed on the basis of protein concentration (Fig. 2B), the root tip showed an initial high activity at zero time, with a
-.-
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Plant Physiol. Vol. 101, 1993
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described for Figure 1. Bars represent exceed symbol size.
*
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gradual decline to zero activity at 48 h. Presumably, the loss of activity in root tips indicated the death of these tissues. By contrast, root axes had very low activity at zero time, followed by a continuous rise throughout the anoxic period.
4
Adhl Transcript Abundance and Enzyme Activity under Different Percent O 2 Regimens
O
6
12
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24
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Duration of anoxia (h) Figure 1. Changes in Adh J mRNA levels in maize seedlings during anoxia. A, Hybridized Adhl probe (cpm) expressed per milligram fresh weight of tissue; B, hybridized Adhl probe expressed per microgram of total RNA. Seedlings were transferred from a 40% O2 pretreatment (24 h ) to an anaerobic atmosphere at zero time.
The effects of different concentrations of O2 on Adhl mRNA levels and ADH activity were examined in seedlings that had been pretreated with 40% O2 for 24 h. The duration of exposure was 6 h, a time sufficient to give maximal induction of Adhl mRNA in maize roots under anaerobic conditions (Gerlach et al., 1982). At a11 O2concentrations tested, total RNA was found to be more abundant in root tips than in any of the other tissues tested. The total RNA per gram fresh weight obtained from root axes was from 10 to 20% of the amounts isolated from root tips (3.0 pg mg-' fresh weight), and the total RNA in shoots was 20 to 40% that in root tips. The amounts of extractable RNA exhibited no significant change in any of the lower O2 treatments (O, 2, and 4% 02), although among these three treatments, primary root elongation occurred only at 4 % O2 (Saglio et al., 1984). Data resulting from hybridization with the Adhl probe (Fig. 3 ) showed a clear response to hypoxia and anoxia in a11 three tissues. At 0% 02,the amount of mRNA in root tips
Alcohol Dehydrogenase in Maize Seedlings 80
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time in a11 the hypoxic root tips, with maximal induction at 2 to 4% 0 2 . Anaerobically treated root tips showed a decline in ADH activity by 18 h. The difference between 0% O2 (anoxia) and 2% O2 (hypoxia) was particularly striking and suggests that any contamination by molecular O2 in these experiments, which might occur in principle from O2 diffusion through plastic tubing and connectors, was not at a sufficiently high level to cause a biologically significant change in gas composition.
Root tip
t
1
O
10
20 %
41 1
30
40
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Figure 3. Adhl mRNA levels in maize seedlings exposed to different oxygen concentrations. A, Hybridized Adh I probe (cpm)expressed per milligram fresh weight of tissue; B, hybridized Adhl probe expressed per microgram of total RNA. Seedlings were transferred from a 40% pretreatment (24 h) to the stated O2 concentration and sampled after 6 h.
was approximately 20 times that of the 40% O2 treatment on a fresh weight basis (Fig. 3A). Root axis mRNA was also increased at the 0% O2 level, being about 10 times greater than the 40% O2treatments. mRNA levels in shoots increased by 5 - to 10-fold between these two treatments. The highest level of Adhl transcript in root tips was with the O to 2% O2 treatments, the transcript level declining at 16% O2 to a low level, with a gradual further decline up to 40% 02.Relative to total RNA (Fig. 3B), there was a marked induction of Adhl mRNA by O2 deficiency of a11 three tissue types, the peak induction occurring between O and 4% 02.In root tips, activity of ADH on a fresh weight basis was strongly induced by anoxia or hypoxia, with a progressively lower level of induction at 30 or 40% O2 (Fig. 4A). Root axes also showed induction of enzyme activity by anoxia or hypoxia, although the level of response was less dramatic. In the shoot, there was a greater activity of ADH under conditions of hypoxia, with a pronounced rise under anoxia (Fig. 4A). When ADH activity was examined on the basis of extractable protein (Fig. 4B), both root tips and root axes showed a similar response, with maximum induction at 4% O2 and a gradual decline at higher concentrations of 02.Shoots showed a similar level of activity throughout the entire O2 concentration range. In a separate experiment, the response of root tips of intact seedlings to different, low O2 concentrations was examined as a function of time (Fig. 5 ) . Activity of ADH increased with
Changes in Adhl Transcripts and Enzyme Activity in Root Tips during Hypoxia
Because of the peak in Adhl transcripts and ADH enzyme activity at 4% O2 in root tips, we examined their induction as a function of time. Adhl mRNA showed its most rapid increase at 3 h, reaching a peak at 6 h (Fig. 6A). Relative to fresh weight, mRNA declined gradually with time, returning to the zero-time values at 24 h. However, relative to total RNA, mRNA remained at a similar level between 6 and 24 h. Activity of ADH in hypoxic root tips increased perceptibly in the initial 3 h and on a fresh weight basis reached a maximum at 18 h (Fig. 6B). Data expressed on a milligram protein basis were somewhat variable, but an overall increase in activity was apparent over the 24 h.
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exposed to different oxygen concentrations. A, Activity expressed per gram fresh weight of tissue; B, activity expressed per milligram of soluble protein. Conditions were as described for Figure 3. Bars represent SE (n = 3 ) where these exceed symbol size. Figure 4. ADH enzyme activity in maize seedlings
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Andrews et al.
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enzyme activity in root tips of maize seedlings with duration of anoxia and hypoxia. A, Activity expressed per gram fresh weight of tissue; B, activity expressed per milligram of soluble protein. Conditions were as described for Figure 3 . Bars represent 2 SE ( n = 3 ) where these exceed symbol size. Figure 5. ADH
Plant Physiol. Vol. 101, 1993
with values of the critica1 O2 pressure for submersed maize root tips, which exceeds 20.4% at 25OC (Saglio et al., 1984). When uninduced seedlings were suddenly made anoxic, there was a distinct increase in Adhl mRNA and ADH activity in root tips, root axes, and shoots (Figs. 1 and 2). The ADH assay does not distinguish between Adhl and Adh2, but because the specific activity of Adhl is some 10 times greater than that of Adh2, enzyme activity represents largely the translation product of Adhl. The rise in transcript levels in root tips and root axes (per milligram fresh weight) was about 17-fold at 6 h of anoxia and comparable in magnitude to other estimates (Gerlach et al., 1982; Rowland and Strommer, 1986). However, the response of root tips to continuous anoxia was in marked contrast to the other tissues, for transcript levels greatly declined between 12 and 18 h, by which time root tips were dead (Johnson et al., 1989). The peak in mRNA levels in the root tip cells at 6 h (per milligram fresh weight) coincided with the peak in ADH activity (Fig. 2), but enzyme activity did not decline as rapidly as did the message level, presumably because of its longer half-life, or because of a delay between mRNA release and accumulation of active enzyme following protein synthesis. The more mature cells of the root axis and the shoot maintained or increased their levels of mRNA and ADH activity to 24 or 48 h, in marked contrast with the decline in the root tips. The ability of the root axis to survive extended periods of anoxia, and the proliferation of lateral roots on reoxygenation, has been noted previously (Sachs et al., 1980; Roberts et al., 1984a; Johnson et al., 1989).
DISCUSSION
Although the Adhl gene of maize is one of the most thoroughly characterized genes in higher plants (Gerlach et al., 1982; Walker et al., 1987), the precise O2 partia1 pressures that induce its expression and the simultaneous changes in Adhl transcript levels and ADH enzyme activity that take place in various tissues of maize seedlings have not been previously reported. The present results show that induction in root tips, as well as in the root axis, is maximal with anoxia or extreme hypoxia (O-4% 02). However, under anoxia, root tips, unlike other tissues, showed only a brief induction because of their limited viability under 02-free conditions. Furthermore, transcript levels for Adhl, while increasing with anoxia or hypoxia, were not always closely paralleled by ADH enzyme activity. In the present study, as in earlier ones (Saglio et al., 1988; Johnson et al., 1989; Hole et al., 1992), we were careful to avoid induction of ADH before experimental treatments began. Relatively high levels of ADH activity were assayed in root tips of seedlings shortly after emergence of the primary root (Table I), but following 12 h or more at 40% O*,induction of either a transcriptional or translational nature was minimal. At 25OC, exposure of root tips to ambient air probably induced mild hypoxia because Adhl mRNA and enzyme activity were both lower at 40% (Figs. 3 and 4). A minor induction of ADH in solution sparged with air is consistent
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Duration of hypoxia (h) Figure 6. Message levels for Adh I and ADH enzyme activity in root tips of maize seedlings during hypoxia (4% OJ. A, Adhl mRNA; B, ADH enzyme activity. Bars represent SE ( n = 3 ) .
*
Alcohol Dehydrogenase in Maize Seedlings Both Adhl mRNA and ADH enzyme activity were found to be elevated at low concentrations of 02,but over a wide range of O2 concentrations (Figs. 3 and 4) the amounts of mRNA and enzyme activity did not necessarily correlate. Sometimes an increase in ADH was seen with a much smaller corresponding increase in mRNA. It has been reported that Adhl mRNA is not sequestered in maize and that the increases seen in mRNA levels were due to increased transcription under anaerobiosis (Ferl et al., 1980). Other investigators report an increased half-life of transcripts under "anaerobic" conditions (Rowland and Strommer, 1986). During induction of lactate dehydrogenase activity by hypoxia in barley roots, mRNA levels increased about 20-fold during 24 h (Hondred and Hanson, 1990); a large increase in lactate dehydrogenase activity had been measured in earlier experiments (cited in Hondred and Hanson, 1990). However, in none of these reports was enzyme activity compared with concurrent mRNA levels. . During hypoxia, a sheath of aerobic cells surrounds a core of anaerobic ones (Thomson and Greenway, 1991), so that the tissues used in some of the present experiments must have constituted a heterogeneous population of cells. The decline in ADH expression with increasing O2 (Figs. 3 and 4) probably reflects the smaller number of anoxic cells in the anaerobic core. Because of the faster rate of respiration of root tip cells, a greater requirement for O2 to offset ADH induction would be expected, and that expectation is borne out at both the transcriptional (Fig. 3) and translational (Fig. 4) levels. Hypoxia brought about by the 4% O2 treatment seems to be a key point in the induction of both Adkl mRNA and ADH enzyme activity. At that O2 concentration, induction was close to maximal for a11 tissues, viability of the root tips was not impaired, and root extension continued slowly. It would be interesting to know whether there is a transfer of ATP from aerobic to anoxic cells under such conditions. The time-course study of the 4% O2 treatment (Fig. 6) suggests a delay between the peaks of mRNA synthesis and enzyme activity for ADH similar to that reported for the induction of phenylalanine ammonia lyase in cultured parsley cells (Hahlbrock et al., 1982). In that system, approximately 16 h elapsed between mRNA synthesis and maximal enzyme levels, with reaction products approaching a maximum after an additional40 h. In our experiments with ADH, maximum activity was found to follow maximum mRNA expression by about 15 h. This increase in transcription and subsequent enzyme activity of ADH and presumably other ANPs may provide a mechanism that permits the survival of the anoxic cells of the primary root during periods of O2 deficiency . During anoxia, there was a gradual loss of the ability of the root tips to acclimate to such conditions, but the root axes became more acclimated. Perhaps loss of viability of the apical meristem influences the induction and anaerobic metabolism of the more anoxia-resistant cells of the root axis. Future experiments will include a detailed examination of the mechanism by which a low O2 pretreatment, before the imposition of anoxia, extends the duration of viability under anaerobic conditions for root tip cells. Such information, together with the present results, should throw light on the
41 3
relationship between the root tip and the remainder of the plant in metabolic and developmental strategies for dealing with O2 deficiency. Received April 28, 1992; accepted October 2, 1992. Copyright Clearance Centtr: 0032-0889/93/101/0407/08. LITERATURE ClTED
Armstrong W, Beckett PM (1987) Interna1 aeration and the development of stelar anoxia in submerged roots: a multishelled mathematical model combining axial diffusion of oxygen in the cortex with radial losses to the stele, the wall layers and the rhizosphere. New PhytollO5: 221-245 Bailey-Serres J, Freeling M (1990) Hypoxic stress-induced changes in ribosomes of maize seedling roots. Plant Physiol94 1237-1243 Bailey-Serres J, Kloeckener-Gruissem B, Freeling M (1988) Genetic and molecular approaches to the study of the anaerobic response and tissue specific gene expression in maize. Plant Cell Environ 11: 351-357 Cobb BG, Kennedy RA (1987) Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings. Plant Cell Environ 1 0 633-638 Dennis ES, Gerlach WL, Pryor AJ, Bennetzen JL, Inglis A, Llewellyn D, Sachs MM, Ferl RJ, Peacock WJ (1984) Molecular analysis of the alcohol dehydrogenase (Adhl) gene of maize. Nucleic Acids Res 10: 3983-3999 Dennis ES, Sachs MM, Gerlach WL, Finnegan EJ, Peacock WJ (1985) Molecular analysis of the alcohol dehydrogenase 2 (Adh2) gene of maize. Nucleic Acids Res 13: 727-743 Feinburg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1 3 2 6-11 Feinburg AP, Vogelstein B (1984) Addendum: a technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137: 266-271 Ferl RJ, Brennan M, Schwartz D (1980) Zn vitro translation of maize ADH: evidence for the anaerobic induction of mRNA. Biochem Genet 18: 681-691 Gerlach WL, Pryor AJ, Dennis ES, Ferl RJ, Sachs MM, Peacock WJ (1982) cDNA cloning and induction of the alcohol dehydrogenase (Adhl) gene of maize. Proc Natl Acad Sci USA 7 9 2981-2985 Hahlbrock K, Kreuzaler F, Ragg H, Faultz E, Kuhn DN (1982) Regulation of flavonoid and phytoalexin accumulation through mRNA and enzyme induction in cultured plant cells. In Biochemistry of Differentiation and Morphogenesis. Springer-Verlag, Berlin, pp 34-43 Hole DJ, Cobb BG, Hole PS, Drew MC (1992) Enhancement of anaerobic respiration in root tips of Zea mays following low-oxygen (hypoxic) acclimation. Plant Physiol99: 213-218 Hondred D, Hanson AD (1990) Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis. Proc Natl Acad Sci USA 8 7 7300-7304 Howard EA, Walker JC, Dennis ES, Peacock WJ (1987) Regulated expression of an alcohol dehydrogenase chimeric gene introduced into maize protoplasts. Planta 1 7 0 535-540 Johnson J, Cobb BG, Drew MC (1989) Hypoxic induction of anoxia tolerance in roots of Zea mays. Plant Physiol 91: 837-841 Kelley PM (1989) Maize pyruvate decarboxylase mRNA is induced anaerobically. Plant Mo1 Biol 1 3 213-222 McElfresh KC, Chourey PS (1988) Anaerobiosis induces transcription but not translation of sucrose synthase in maize. Plant Physiol 87: 542-546 Pradet A, Bomsel JL (1978) Energy metabolism in plants under hypoxia and anoxia. In DD Hook, RMM Crawford, eds, Plant Life in Anaerobic Environments. Ann Arbor Science, Ann Arbor, Ml, pp 89-118 Roberts JKM, Callis J, Jardetsky O, Walbot V, Freeling M (1984a) Mechanism of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia. Proc Natl Acad Sci USA 81: 3379-3383
414
Andrews et al.
Roberts JKM, Callis J, Jardetsky O, Walbot V, Freeling M (1984b) Cytoplasmic acidosis as a determinant of flooding intolerance in plants. Proc Natl Acad Sci USA 81: 6029-6033 Rowland LJ, Strommer JN (1986) Anaerobic treatment of maize roots affects transcription of Adhl and transcript stability. Mo1 Cell Biol 6: 3368-3372 Sachs MM, Freeling M, Okimoto R (1980) The anaerobic proteins of maize. Cell20 761-767 Saglio PH, Drew MC, Pradet A (1988) Metabolic acclimation to anoxia induced by Iow (2-4 kPa partia1 pressure) oxygen pretreatment (hypoxia) in root tips of Zea mays. Plant Physiol 86: 61-66 Saglio PH, Rancillac M, Bruzau F, Pradet A (1984) Critica1 oxygen pressure for growth and respiration of excised and intact roots. Plant Physiol76 151-154 Schneider-Gadicke A, Beer-Romero P, Brown LG, Nussbaum R, Page DC (1989) ZFX has a gene structure similar to ZFY, the putative human sex determinant, and escapes X inactivation. Cell 57: 1247-1258 Springer BW, Werr P, Starlinger DC, Bennett M, Zokolica M, Freeling M (1986) The Shrunken gene on chromosome 9 of Zea mays L. is expressed in various plant tissues and encodes an anaerobic protein. Mo1 Gen Genet 205: 461-468
Plant Physiol. Vol. 101, 1993
Stiekema WJ, Heidekamp F, Dirkse WG, van Beckum J, deHaan J, ten Bosch C, Louwerse JD (1988) Molecular cloning and analysis of four potato tuber mRNAs. Plant Mo1 Biol 11: 255-269 Strommer JN, Hake S, Bennetzen J, Taylor WC, Freeling M (1982) Regulatory mutations of the maize Adhl gene caused by DNA insertion. Nature 300 542-544 Sullivan DE, Auron PE, Quigley GJ, Watkins P, Stanchfield JE, Bolon C (1987) The nucleic acid blot analyzer. I. High speed imaging and quantitation of 32P-labeled blots. BioTechniques 5: 672-678 Thomson CJ, Greenway H (1991) Metabolic evidence for stelar anoxia in maize roots exposed to low O 2concentrations. Plant Physiol96: 1294-1301 Walker JC, Howard EA, Dennis ES, Peacock WJ (1987) DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase gene. Proc Natl Acad Sci USA 8 4 6624-6628 Webster C, Gaut RL, Browning KS, Ravel JM, Roberts JKM (1991a) Hypoxia enhances phosphorylation of eukaryotic initiation factor 4A in maize root tips. J Biol Chem 266: 23341-23346 Webster C, Gaut RL, Browning KS, Ravel JM, Roberts JKM (1991b) Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system. Plant Physiol96 418-425
