Differential Gene Expression in Chilling-Acclimated Maize Seedlings and Evidence for the lnvolvement...

Plant Physiol. (1994) 105: 331-339

Differential Gene Expression in Chilling-Acclimated Maize Seedlings and Evidence for the lnvolvement of

Abscisic Acid in Chilling Tolerance'

Marc D. Anderson*, Tottempudi K. Prasad, Barry A. Martin, and Ceci1 R. Stewart

Botany Department and lnterdepartmental Plant Physiology Major, lowa State University, Ames, lowa 5001 1 (M.D.A., T.K.P., C.R.S); and Pioneer Hi-Bred International, Johnston, lowa 50131 (B.A.M.)

An acclimation phenomenon was characterized in seedlings of chilling-sensitive maize (Zea mays L.) inbred C50 (Pioneer). Seed- lings were germinated at 27°C for 3 d and then exposed to chilling treatments of 4, 5, or 6'C for 2, 4, 7, or 10 d in darkness. Damage symptoms in the more severe treatments included a waterlogged appearance and a discoloration of the tissue. l h e symptoms were most obvious in the mesocotyl. After a 10-d grow-out period in the greenhouse, moderately damaged seedlings exhibited chlorotic areas, an occasional disruption in leaf expansion, and a constriction of the mesocotyl. Crowth and survival were improved by first exposing seedlings to a 14°C acclimation treatment for 3 d before applying the chilling treatment. After chilling at 5'C for 7 d, 79% of the acclimated seedlings survived, whereas only 22% of the nonacclimated seedlings survived. Differences in gene expression between acclimated and control seedlings were investigated using subtraction and differential screening techniques. Transcripts corresponding to three genes, car333, car30, and car757 (chilling acclimation responsive), were present in higher levels in seedlings after acclimation. Sequence analysis identified a r333 as cat3, which encodes maize mitochondrial catalase isozyme 3. Charac- terization of these three clones revealed that all corresponding transcripts were elevated in acclimated seedlings in a manner that depended on the organ, i.e. coleoptile, mesocotyl, or root. Although transcripts were elevated in all three organs in response to acclimation, car30 was most abundant in the coleoptile and root, whereas cat3 and car757 were most abundant in the coleoptile and mesocotyl. Catalase activity followed the same general trend as cat3 transcript levels. Exogenous treatment with abscisic acid (ABA) resulted in an improvement in growth and survival of nonacclimated, chilled seedlings. lnhibition of ABA biosynthesis with fluridone abolished acclimation-induced chilling tolerance, and exogenous application of ABA to fluridone-treated seedlings restored chilling tolerance. Exogenous ABA treatment also resulted in increases in cat3, car30, and car757 transcript levels and catalase activity in the same organ-specific manner as in acclimated seedlings. These results indicate that ABA synthesis i s essential for chilling tolerance. However, measurement of ABA levels in mesocotyls during acclimation and chilling revealed only a marginal increase during acclimation and a dramatic increase during chilling, regardless of whether or not seedlings were acclimated. Thus, although ABA-may be required for chilling tolerance, we have no conclusive evidence that the acclimation process is mediated by ABA.

'This research was supported by grants from Pioneer Hi-Bred Intemational (C.R.S.) and U.S. Department of Agriculture National Research Initiative Competitive Grants Program 92-37100-7646 (C.R.S., T.K.P.).

* Corresponding author; fax 1-515-294-1337. 33 1

Chilling damage results from exposure of a chilling-sensitive plant to low, nonfreezing temperatures. Visual symptoms of chilling damage in vegetative tissue often include surface lesions and necrotic areas, a waterlogged appearance, interna1 discoloration, wilting, acceleration of senescence, a reduction in growth, and death (Saltveit and Morris, 1990). On the cellular and molecular levels, chilling stress was generalized by Levitt (1980) as having three direct effects: reduction of metabolic rates, reduction of membrane fluidity, and dena- turation of proteins. Overall, the documented effects of chilling on metabolism are numerous (see reviews by Graham and Patterson, 1982; Wang, 1982; Markhart, 1986) and have been reported as affecting respiration, photosynthesis, phe- nolic metabolism, sugar metabolism, and redox regulation. It is the means by which some plants are able to survive these effects that is of interest, especially in the development of chilling tolerance in agriculturally important plants.

Many plants acclimate to low temperatures. Exposure to noninjurious low temperatures allows plants to acquire the capability to survive damaging temperatures. Changes in gene expression have been observed during acclimation to freezing in Arabidopsis and wheat (Lin et al., 1990), alfalfa (Mohapatra et al., 1987), and spinach (Guy et al., 1985) and to chilling in rice (Hahn and Walbot, 1989). Biochemically, most theories concerning the mechanism of acclimation involve the maintenance of membrane fluidity by higher lipid unsaturation and higher sterol content, the maintenance of enzymes that would otherwise be altered in nonacclimated plants, or increases in protective substances (Levitt, 1980; Guy, 1990). Much remains to be discovered about how these metabolic changes coordinate to produce a leve1 of low- temperature tolerance during acclimation.

There has been considerable interest in the role of ABA in mediating tolerance to low temperature (either chilling or freezing, depending on the plant species under study). Ele- vated ABA levels have been observed in plants exposed to low, nonfreezing temperatures (Riken et al., 1976; Daie and Campbell, 1981; Lalk and Dorffling, 1985), and exogenous application of ABA has been shown to confer freezing toler- ante to alfalfa (Mohapatra et al., 1988) and to Arabidopsis

Abbreviations: ABA, ABA treated; ABACh, ABA treated and chilled; ABA@4, ABA treated at the 4OC chilling temperature; Ac, acclimated; ACABA, ABA treated then acclimated and chilled; AcCh, acclimated and chilled; AT, 3-amino-1,2,4-triazole; cur, chilling acclimation responsive; CAT, catalase; Ch, chilled; Cont, control; SOD, superoxide dismutase.

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332 Anderson et al. Plant Physiol. Vol. 105, 1994

(Lang et al., 1989) and chilling tolerance to maize cell- suspension cultures (Xin and Li, 1992). Heino et al. (1990) demonstrated that ABA-deficient Arabidopsis has low toler- ante to freezing, and Li (1991) showed that artificial induction of endogenous ABA by mefluidide conferred chilling toler- ante to maize, cucumber, and pepper and freezing tolerance to wheat, lemon, and crabapple but not to potato, which is incapable of being acclimated. Lee et al. (1993) attributed the difference between chilling-sensitive and chilling-resistant rice to the ability to increase ABA levels in response to chilling stress. Even though the evidence is strong that ABA is in- volved in low-temperature tolerance, the means by which ABA acts to produce tolerance is poorly understood. Numer- ous studies have shown common induction of genes (Kurkela and Franck, 1990; Guo et al., 1992; Lee and Chen, 1993) and proteins (Lang et al., 1989; Xin and Li, 1993) by both ABA and low temperature; thus it is likely that ABA and low temperature utilize some of the same mechanisms to produce their effect.

In this study, we have described chilling acclimation in seedlings of chilling-sensitive maize inbred G50 (Pioneer). The existence of an acclimation phenomenon provided a means to compare genetically identical plants, differing only in the leve1 of chilling tolerance, for differences in gene expression. We have identified transcripts from three car genes and have characterized their expression. We have also explored the effect of ABA on expression of these three car genes and the role of ABA in chilling tolerance and during chilling acclimation in maize seedlings.

MATERIALS AND METHODS

Seedling Growth and Survival

Chilling-sensitive maize (Zea mays L.) inbred G50 (Pioneer Hi-Bred International Inc., Johnston, IA) was used to test whether acclimation or ABA treatment could improve chilling tolerance. Seeds were planted in Terra-lite (Grace Sierra Horticultural Products Co., Milpitas, CA) and grown at 27OC for 3 d in darkness, and seedlings were then acclimated by exposure to 14OC for 3 d in darkness. Cont seedlings did not receive a 14OC treatment. Little seedling growth occurred during the acclimation treatment, and there was little difference in development between Ac and Cont seedlings despite differences in age. Ac or Cont seedlings were then exposed to chilling treatments of 4, 5, or 6OC (a11 incubator temperatures in this and subsequent experiments fluctuate approximately 50.5OC) for 2, 4, 7, or 10 d. The number of surviving seedlings was counted, and dry weights (five seedlings were sampled when available) were measured both before and after a 10-d grow-out period in the greenhouse (22 f 3OC, 1623 h light/dark, fertilized once with 20/20/20 N/P/K [Peatlite]). Unchilled Conts were also included. Seedlings with extensive necrosis, a severely constricted mesocotyl, or no evident growth were considered dead and were not included in the final counts. Only the survivors were included in the final weight analyses.

Acclimation status and chilling duration were replicated three or four times for each temperature. Percentage survival

and dry weights were analyzed as a factorial design with acclimalion status, chilling temperature, chilling duration, and rep'licate within temperature as the main effects. Because temperalture was not randomized with acclimation status and chilling duration within a replication, replication within temperature was used as the error to test for temperature effects. Unchilled Conts were replicated seven times and were analyzed separately.

TO determine whether exogenous application of ABA affected chilling tolerance, seedlings were exposetl to O, 100, 333, or 1000 PM ABA (mixed isomers, Sigma). Seedlings were germinaited in germination paper at 27OC for 3 d in darkness. The imbibing solution was then replaced with the appropriate ABA solution, and seedlings were allowed to grow for an additiorial 24 h. Seedlings were transplanted into Terra-lite and allowed to become established at 27OC for 12 h, after which they were transferred to 4OC for 7 d. 11s with the acclimation experiment, seedling counts and dry weights were measured both before and after a 10-d grow-out period in the greenhouse. Treatments were replicated three times, and percentage survival and dry weights were analyzed as a randomized complete block design. Means were separated using Tukey's studentized range test.

To further investigate the role of ABA in chilling tolerance, fluridone was used to inhibit ABA biosynthesis in an attempt to abolish acclimation-induced chilling toleranctr and to determine whether ABA was required. Fluridone vias administered by allowing seeds to imbibe in 0.1 mM fluiidone/O.l% Tween 20 for 24 h at 27OC in darkness. Fluridone-treated and untreated seeds (soaked in water) were then planted in Terra-lite and watered with the same solution with which they were soaked. The following treatments wwe then applied: Cont, 27OC for 2 d; AcCh, 27OC for 2 d, 14OC for 3 d, 4OC for 7 d; ACABA, irrigation with 300 PM ABA at the time of planting, 27OC for 3 d, 14OC for 3 d, 4OC for ;7 d; ABACh, irrigation with 300 PM ABA at the time of planting, 27OC for 3 d, 4OC: for 7 d; ABA@4, 27OC for 2 d, 4OC for 2 h, irrigation of wet media with cold 1 mM ABA (same amount of ABA applied as ABACh), 4OC for 7 d; and Ch, 27OC for 2 d, 4OC for 7 d.

Fluriclone-treated seeds were not used in the ABA@4 and Ch trea tments. Following the above treatments, seedlings were transferred to the greenhouse for a 7-d grow-out period. Since fluridone-treated seedlings contain no carotenoids, Chl is photooxidized and seedlings survive from their seed re- serves. This precluded extension of the grow-out period to 10 d as in the other survival experiments. Seetlling counts and dry weights were measured as before. Treatments were replicated three times, and percentage survival and dry weights were analyzed as a randomized complete block design. Means were separated with Tukey's studentized range test.

lsolation of Differentially Expressed Transcript!;

Total RNA was extracted from Cont and Ac seedlings (Chomczynski and Sacchi, 1987), and poly(A)' RNA was separated on an oligo(dT) column (Davis et al., 1986). A cDNA isynthesis kit (Amersham) was used to construct a cDNA library in XZAPII (Stratagene) with poly(A)+ RNA




Gene Expression in Chilling-Acclimated Maize 333

from Ac seedlings. Blunt-ended cDNA was ligated to EcoRI adapters before cloning into the EcoRI site of the vector. The library was screened for clones that were differentially expressed in Ac seedlings by hybridizing to a subtraction probe. The subtraction probe was formed by hybridizing 32P-labeled first-strand cDNA from Ac poly(A)+ RNA with biotinylated Cont poly(A)' RNA and precipitating a11 RNA-DNA hybrids with avidin. Putative positives were rescreened by differential hybridization to 32P-labeled first-strand cDNA from either Ac or Cont poly(A)' RNA. Phagemids from three differentially expressed plaques were rescued by in vivo excision according to manufacturer's instructions (Stratagene). Pha- gemid DNA was digested with EcoRI, and the inserts were gel purified. 32P-labeled DNA probes were synthesized using random hexamer primers and were used to hybridize to RNA gel blots containing poly(A)+ RNA from Cont or Ac seedlings.

The three clones were sequenced in both directions by the dideoxy chain termination method using the universal and reverse primers of the pBluescript plasmid. Dideoxyribonu- cleotide triphosphates, tagged with base-specific fluorescent dyes, were detected with a laser in an automated sequencer.

Characterization of Differentially Expressed Transcripts

Seedlings for RNA isolations were grown in darkness and exposed to the following treatments: Cont, 27OC for 3 d; Ac, 27OC for 3 d, 14OC for 3 d; AcCh, 27OC for 3 d, 14OC for 3 d, 4OC for 4 d; Ch, 27OC for 3 d, 4OC for 4 d; ABA, 27OC for 3 d in the presence of 100 PM ABA; ABACh, 27OC for 3 d in the presence of 100 p~ ABA, 4OC for 4 d.

Total RNA was isolated from coleoptile, mesocotyl, and root of seedlings exposed to the above treatments. Total RNA was electrophoresed on 1% formaldehyde agarose gels and transferred to nylon membranes (GeneScreen). 32P-labeled DNA probes were synthesized from the three differentially expressed inserts (as described above) for hybridization to the RNA gel blots.

Catalase Activity

Cell-free extracts were prepared from coleoptile, mesocotyl, and root of Cont, Ac, AcCh, and Ch seedlings, grown as previously described for RNA isolations. Extracts were also prepared from coleoptile, mesocotyl, and root of seedlings that had been germinated in germination paper for 3 d at 27OC in darkness and then exposed to O, 300, or 1000 PM ABA for 24 h at 27OC in darkness. An additional set of ABA treatments were transferred to 4OC for 4 d in darkness. Tissue (0.5 g) was ground in 1 mL of 200 mM sodium phosphate buffer (pH 7.8), containing 1 mM EDTA, 1 mM PMSF, and 20 mg of polyvinylpolypyrrolidone. Insoluble material was removed by centrifugation at 14,000 rpm for 15 min in a microcentrifuge. Protein concentration was measured by the Lowry method (Lowry et al., 1951) using BSA as a standard. Catalase activity was measured immediately after extraction according to the method of Beers and Sizer (1952), both with and without 10 mM AT. At this concentration, AT inhibits CAT-1 by 93%, CAT-2 by 98%, and CAT-3 by 32% (Chan- dlee et al., 1983), thus allowing differences in CAT-3 activity

among treatments to be monitored with little interference from other isozymes. A11 acclimation and ABA treatments were replicated three times, and activity was analyzed as a factorial design with acclimation or ABA treatment, chilling, and replication as the factors. Means were separated with Tukey's studentized range test. The acclimation experiment was analyzed separately from the ABA experiment.

ABA Analysis

Cont, Ac, Ch, or AcCh seedlings were grown as previously described for RNA isolations. In addition to the Ac treatment (acclimation at 14OC for 3 d), seedlings were also exposed to acclimation at 14OC for 2, 4, and 8 h and 1 d so that changes in ABA levels during acclimation could be determined. Cont and Ac were replicated three times, and the remaining six treatments were replicated twice. Mesocotyls from a11 treatments were excised directly into liquid nitrogen and stored at -7OOC until ABA analysis. Approximately 2 g of mesocotyls were analyzed for ABA content by the isotope dilution method of Li et al. (1992) using 'H3-ABA as the intemal standard. ABA was quantified by GC-MS using selected ion monitoring. ABA levels were calculated by taking the ratio of ion currents at m/z 190 and m/z 193 times the amount of deuterated standard added. A11 calculations were corrected for the abundance of the m/z 193 fragment in undeuterated standards and of the m/z 190 fragment in the deuterated standard. ABA leve1 was analyzed as a completely randomized design, and means were separated using Tukey's studentized range test.

RESULTS

Seedling Crowth and Survival

Visual symptoms of chilling damage ranged from a slight reduction in growth to a complete browning and decay of the entire seedling, depending on the severity of the chilling treatment. Visual symptoms in moderately damaged seedlings included chlorotic areas, browning and desiccation of leaf margins, and an occasional disruption in leaf expansion. The mesocotyl was frequently constricted and appeared to be the organ most sensitive to chilling. It is likely that these damage symptoms resulted directly from chilling as well as from photooxidation and secondary pathogen infection after seedlings were exposed to greenhouse conditions. Interest- ingly, Ch seedlings that were previously acclimated or treated with a sufficient dose of ABA exhibited a swelling in the mesocotyl. It is possible that this swelling is indicative of some form of protection in the mesocotyl.

Chilling damage in maize seedlings was found to be de- pendent on both temperature and duration of exposure. Survival was unaffected by chilling stress less severe than 5OC for 7 d, and shoot dry weights were reduced with increasing degree of chilling stress (Table I). Acclimation significantly affected survival and shoot dry weights. The effect on survival was most evident in the 5OC for 7-d treatment in which survival in the Ac treatment was 79% and that of the Cont treatment was 22%. In terms of dry weights, Ac seedlings grew better than Cont seedlings in





Table 1. Effect of acclimation, chilling temperature, and chillingduration on survival and shoot dry weight of Ch maize seedlings

PercentageTemp Duration Survival

Dry Wt/Seedling

Ac Cont Ac

d4°Ca 2 97 96

4 97 947 68 22

10 12 3

5°Cb 2 96 994 96 927 79 22

10 14 2

6°Ca 2 97 994 99 977 98 88

10 88 48

Unchilled 98 98Conf

a Three replicates. Percentage survival, SE8. b Four replicates. Percentage survivalSE = 7. c Seven replicates. Percentageweight, SE = 10.

129903827

1831375731

143108

7640

195

Contmg

132642915

146813816

113924633

182

= 4%; dry weight, SE =, SE = 3%; dry weight,survival, SE = 2%; dry

treatments in which the chilling stress was sufficient tosignificantly affect the growth.

ABA treatment had a significant effect on growth andsurvival of seedlings exposed to 4°C for 7 d (Table II). Thelargest effect was observed at 1000 fiM ABA, but 333 LLM alsohad a significant effect. The inhibition of ABA by fluridonedramatically affected the ability of Ac seedlings to toleratechilling stress as shown in Table III. In unchilled seedlings,fluridone treatment had no effect on survival, although seed-ling weights were greatly reduced. AcCh seedlings showed adramatic reduction in survival when treated with fluridone.Exogenous application of ABA to fluridone-treated seedlingsrestored their ability to survive a chilling stress. ABA appliedat 4°C provided some chilling tolerance but less than whenit was applied at the time of planting.

Table II. Effect of exogenous ABA treatment at differentconcentrations on survival and shoot dry weight of Ch maizeseedlings______________________________

ABA Concentration Percentage Survival' Dry wt/Seedling

1000b333b100bOc

6641201

mg80573322

' Three replicates. Percentage survival, SE = 8%. b Three rep-licates. Dry weight, SE = 4. c Survivors present only in onereplicate. Dry weight, SE = 7.

Table III. Effect of fluridone and ABA treatment on survival andshoot dry weight of Ch maize seedlings

Fluridone was administered by allowing seeds to imbibe in 0.1mM fluridone/0.1% Tween 20 for 24 h, followed by planting inTerra-lite using the same fluridone solution to saturate the medium.Fluridone was not used in the ABA@4 and Ch treatments. Becauseof the ephemeral nature of fluridone-treated seedlings, weightswere measured after 7 d in the greenhouse rather than at 10 d asin Table I. All treatments were replicated three times. Percentagesurvival, SE = 8%; dry weight, SE = 3.

Treatment

ContAcChAcABAABAChABA@4Ch

Percentage

No fluridone

987988563914

Survival

Fluridone

989

6651

Dry Wt/Seedling

No fluridone Fluridone

1343741242621

mg

47162018

Differential Gene Expression

Three differentially expressed clones were isolated fromthe cDNA library [prepared with poly(A)"1" RNA from Acseedlings]. These were designated car333 (cat3), car30, andcar757, and transcript levels are illustrated in Figure 1. Asshown, the full-length sizes of cat3, car30, and car757 were1.8, 1.6, and 1.5 kb, respectively. Densitometry showed thattranscript levels in Ac seedlings were 2.6-fold higher than inCont seedlings for cat3, 3.1-fold for Car'30, and 2.4-fold forcar757. car30 and car757 were not homologous to any se-quence in the GenBank data base. car333 had 97.8% identityin a 230-base overlap with the 3' end of maize cat3, whichcodes for maize catalase, isozyme 3 (Redinbaugh et al., 1988).The effect of various low-temperature treatments (Fig. 2) andABA treatments (Fig. 3) on the relative levels of transcriptsfrom these three genes revealed that, although the organ

1.8kb1

1.6kb1.5kb

cat3 car30 car757

Figure 1. RNA gel blots showing car gene transcripts that arepresent in higher abundance in Ac seedlings than in Cont seedlings.





cats

carSO

car757

IT

MfeC M R C M R C M R C M R

Cont Ac AcCh Ch

Figure 2. Transcript levels for cat3, car30, and car757 in coleoptile(C), mesocotyl (M), and root (R) of unchilled seedlings (Cont),acclimated at 14°C for 3 d in darkness (Ac), acclimated and chilledat 4°C for 4 d in darkness (AcCh), or chilled without acclimation(Ch).

response differed among the three, they were induced by thedifferent low-temperature treatments and by ABA. In thecoleoptile, cat3 was induced 3.8-fold by Ac, 5.6-fold by AcCh,and 2.5-fold by Ch. In the mesocotyl, cat3 was induced 4.9-fold by Ac and 7.3-fold by AcCh but only 2.4-fold by Ch.cat3 was undetectable in the root of all treatments. car30 wasinduced 9.6-fold by Ac, 10.4-fold by AcCh, and 4.8-fold byCh in the coleoptile. In the root, car30 was induced 2.4-foldby Ac, 2.3-fold by AcCh, and 1.6-fold by Ch. car30 wasundetectable in Cont mesocotyls but was induced by Ac andmore so by AcCh and remained very low in Ch. car757 wasin lower abundance than cat3 and car30. It was induced 4.7-fold by Ac, 6.8-fold by AcCh, and 5.0-fold by Ch in coleop-tiles; 2.2-fold by Ac, 4.1-fold by AcCh, and 4.2-fold by Ch

cat3

car30

car757

C M R C M R C M R

Cont ABA ABACh

Figure 3. Transcript levels for ca(3, car30, and car757 in coleoptile(C), mesocotyl (M), and root (R) of unchilled seedlings (Cont),seedlings germinated in 100 HM ABA (ABA), and seedlings germi-nated in 100 fiM ABA followed by chilling at 4°C for 4 d in darkness(ABACh).

Cont

Figure 4. Effect of acclimation on catalase activity. Catalase activitywas measured in crude protein extracts of coleoptile (C), mesocotyl(M), and root (R) of various treatments as described in Figure 2.Activity was measured immediately after extraction both with andwithout AT in the assay. Activity after AT treatment is indicative ofrelative CAT-3 activity among treatments. Treatments were repli-cated three times. For uninhibited catalase activity: coleoptile SE =2.8, mesocotyl SE = 1.6, and root SE = 1.6. For AT-inhibited catalaseactivity: coleoptile SE = 1.9, mesocotyl SE = 1.5, and root SE = 0.4.

in mesocotyls; and 2.5-fold by Ac, 6.0-fold by AcCh, and6.1-fold by Ch in roots. Transcripts from all three car geneswere induced by ABA and ABACh in a manner similar to theacclimation treatments. cat3 was induced 3.6-fold by ABAand 3.4-fold by ABACh in coleoptiles and 3.3-fold by ABAand 4.5-fold by ABACh in mesocotyls. Again, cat3 wasundetectable in the roots. carSO was not appreciably inducedby ABA but was induced 2.4-fold by ABACh in the coleoptile.In the roots, it was induced 4.7-fold by ABA and 6.3-fold byABACh. As in the acclimation treatments, carSO was unde-tectable in Cont mesocotyls, but induction was only slight inresponse to ABA alone, whereas ABACh caused moderateinduction. car757 was induced 5.0-fold by ABA and 4.7-foldby ABACh in the coleoptile, 4.2-fold by ABA and 4.1-foldby ABACh in the mesocotyl, and 2.7-fold by ABA and 3.5-fold by ABACh in the root. In terms of differences in organresponse, cat3 was most abundant in the coleoptile andmesocotyl, car30 was most abundant in coleoptile with some-what less in the root, and car757 was induced from a lowlevel in all organs, although to a greater extent in the coleop-tile and mesocotyl than in the root. The different organresponses in the ABA experiment were similar to that of theacclimation experiment.

Catalase activities from the acclimation experiment aregiven in Figure 4 and for the ABA experiment in Figure 5.No differences in total catalase activity were detected amongtreatments in the coleoprile for either experiment. In themesocotyl, total catalase activity was significantly induced byboth acclimation and 1000 /UM ABA treatment. In the root,total catalase activity was significantly induced by both 300and 1000 ^M ABA but not by acclimation. Chilling had nosignificant effect on catalase activity in either experiment.When AT was included in the catalase assay, the inductionwas nearly the same as when uninhibited rates were meas-ured. The root is known to contain primarily CAT-1 (Chan-dlee and Scandalios, 1984), and very little activity was de-





- C a, ._ c

P a . E" ._ E A

I O - v 5 ._ ._ c ü

A Catalase Activity Before Chilling

60

50

6 Catalase Activity After Chilling

ABA conciitration (UM)

Figure 5. Effect of ABA treatment on catalase activity. Catalase activity was measured in crude protein extracts of coleoptile (C), mesocotyl (M), and root (R) of seedlings exposed to O, 300, or 1000 VM ABA for 24 h. Activity was measured both immediately after ABA treatment (A) and after chilling at 4°C for 4 d in darkness (B). Activity was measured both with and without AT. For uninhibited catalase activity: coleoptile SE = 1.9, mesocotyl SE = 4.0, and root SE = 2.0. For AT-inhibited catalase activity: coleoptile SE = 0.6, mesocotyl SE = 1 . I , and root SE = 1.1.

tected in any of the acclimation treatments in the presence of AT. In the ABA experiment, however, there was significantly higher activity in the 1000 PM ABA treatment than the Cont, and the 77% inhibition by AT in the 1000 ~ L M ABA treatment was significantly lower than the 86% inhibition by AT in the Cont. Thus, it is possible that ABA induces CAT-3 in the root to levels detectable by this assay.

ABA Levels

ABA levels in mesocotyls of Cont, Ac, Ch, and AcCh, as well as at various times during acclimation are given in Figure 6. Chilling caused a large increase in free ABA levels, regardless of acclimation status. However, very little ABA accumulation occurred during acclimation. Marginal increases in ABA may have occurred in the first hours of acclimation, followed by a decline to Cont levels, but differences among ABA levels at tlie various times during acclimation were not statistically significant.

DISCUSSION Acclimation and Differential Cene Expression

We have demonstrated the presence of a chilling-acclimation phenomenon in seedlings of chilling-sensitive maize

inbred G50 (Pioneer). Visually, the mesocotyl is the organ that is most sensitive to chilling. Acclimation ancl ABA treatment provided some degree of protection from chilling, which was molst evident in the mesocotyl.

Exposure of seedlings to the 14OC acclimation temperature likely induces numerous changes that act in ccincert to increase chilling tolerance. The metabolic systrins that are affected by acclimation in maize are unknown. We isolated cDNAs representing three differentially expressed genes and found that they differed in organ specificity. Thus, even though the mesocotyl was the most sensitive to chilling in nonacclimated seedlings, it is apparent that acclimation causes changes throughout the seedling. Since the functions of car30 and car757 are unknown, we cannot assign a role to their gene products during acclimation or chilling. The fact that car30 transcript abundance (Fig. 2) in the ccdeoptile and root of Ac and AcCh seedlings is higher than that of Ch suggests that its corresponding gene product functions during acclimation. The appearance of low levels of car30 in the mesocotyl of Ac seedlings, then higher levels in AcCh but not Ch, is of interest. It is as if acclimation initiates the mechardsm of induction, but high levels of transcript are not observed until chilling stress is imposed. The initiation during acclimation suggests that car30 may function in protecting the mesocotyl during chilling. car757 abundance (Fig. 2) is higher iin coleoptiles and mesocotyls of AcCh and Ch seedlings than in Ac as if it is responding to the degree of temperature stress to which the seedling is expojed.

Although we do not know the function of car30 or car757, the function of cat3 is clear. cat3 codes for catalase isozyme 3, whiclh catalyzes the conversion of H202 to H20 and 02. In maize, four catalase isozymes are present, which differ both spatially and temporally during development (C handlee and Scandalios, 1984; Scandalios et al., 1984; Acevedo and Scan- dalios, 1991). Coleoptiles contain CAT-1 and CAT-3, and roots cclntain CAT-I. CAT-4 is expressed only transiently in the pericarp of germinating seeds. Etiolated leaves contain CAT-1 and CAT-3, and in the light, CAT-1 disappears and CAT-2 appears as the seedling matures. In the developing

I Cont' 2 4 ' 8 ' 24 72 'AcCh' Ch '

Acclimation Duration (h)

Figure 6. ABA levels in mesocotyls of seedlings expos,ed to various acclimatiion and chilling treatments. Treatments were as described in Figure 2 except for the Ac treatment in which the duration of acclimatiion was varied. ABA was measured according to the method of Li et al. (1992). Cont and Ac/72 h were replicated three times; SE = 2.8. All other treatments were replicated twice; SE = 3.4. Fr. LYt., Fresh weight.





stem, only CAT-3 activity is detectable, although both catl and cat3 transcripts are present. Catalase activity in these stems is located primarily in the sclerenchyma beneath the epidermis and around the vascular bundles (Acevedo and Scandalios, 1991). Tsaftaris et al. (1983) demonstrated that in light-grown maize leaves CAT-2 was present in the peroxisomes of bundle sheath cells, whereas CAT-3 was associated with the mitochondria of the mesophyll cells. We observed the abundance of cat3 (Fig. 2) to be elevated in coleoptiles and especially mesocotyls in response to acclimation and chilling. Catalase activity in the presence of AT (Fig. 4), representing relative CAT-3 activity, generally followed the same pattern as cat3 transcripts with two exceptions. These are the low activity in Ch mesocotyls in which transcripts showed significant induction and the high activity in Cont coleoptiles, which have low transcript levels. These discrepancies may be the result of posttranscriptional events.

lnvolvement of Catalase during Acclimation

We have previously shown that H202 levels are elevated in mesocotyls of Ch seedlings (Prasad et al., 1994a). Elevation of H202 is indicative of a state of oxidative stress under which lipid peroxidation and other deleterious effects on membranes (McKersie, 1991) and inactivation of various enzymes in the photosynthetic apparatus (Asada, 1992) occur. Ele- vated H202 can also result in an increased occurrence of OH' (from H202 and 02- in the Haber-Weiss reaction), which is highly reactive and can contribute significantly to cellular damage (Elstner, 1987). The primary means of defense against active oxygen is via production of active oxygen scavengers such as catalase, peroxidase, SOD, GSH, ascorbate, carotenoids, and various phenolics and alkaloids (Lar- son, 1988). In chilling-sensitive plants, the ability to defend against oxidative damage has been shown to be affected by the chilling-induced reduction of antioxidants such as ascorbate, GSH, and a-tocopherol (Wise and Naylor, 1987), catalase (Omran, 1980), and SOD (Michalski and Kaniuga, 1982). Chilling tolerance has been enhanced in chilling-sensitive plants by artificially elevating levels of GSH, peroxidase, and catalase (Upadhyaya et al., 1989) and SOD (Sen Gupta et al., 1993). In our system, we show that chilling-sensitive maize can be made more tolerant by acclimation, and this may involve up-regulating catalase. Furthermore, we have evi- dente that the up-regulation of catalase is the primary means of maintaining low H202 levels in Ac, dark-grown, maize seedlings when subjected to a chilling stress (Prasad et al., 1994a).

The fact that we are dealing with an induction of cat3 suggests that the source of oxidative stress in dark-grown seedlings is the mitochondria. In the light, a major source of oxidative stress is in the chloroplast, where the ascorbate/ GSH mechanism, as well as SOD, is most important in dealing with excess H202 and 02- (Asada and Takahashi, 1987). Catalase undergoes photoinactivation in the light when plants are exposed to low-temperature stress, which was correlated with the low-temperature sensitivity of the plant species (Feierabend et al., 1992). Sources of oxidative stress in the dark are not as well studied, but it appears likely that, if the membrane-associated Cyt pathway is impaired by

chilling, excess electrons may lead to production of Hz02 and 02-. Indeed, we have evidence that respiratory activity, the Cyt pathway, and F,-ATPase activity are irreversibly impaired by chilling but are protected from irreversible damage by acclimation, H 2 0 2 treatment, and ABA treatment (Prasad et al., 1994b). The ability of dark-grown seedlings to tolerate chilling-induced oxidative stress may well depend on the ability to protect the mitochondria. Induction of CAT-3 during acclimation prevents the accumulation of H 2 0 2 to damaging levels in the mitochondria.

lnvolvement of ABA during Acclimation and in Chilling Tolerance

To further investigate the mechanism of acclimation, we determined the effects of ABA on chilling tolerance and on the induction of catalase. We observed that (a) treatment with exogenous ABA improved the growth and survival of Ch seedlings and (b) fluridone treatment abolished and fluridone treatment in the presence of ABA restored acclimation- induced chilling tolerance. These results indicated that ABA is essential for survival in the face of a chilling stress. Fur- thermore, the fact that ABA induced the three car genes indicated that ABA and acclimation have common effects. However, this is not conclusive evidence that the acclimation process is mediated by ABA. Even though ABA improved the survival of Ch seedlings, it was not as effective as acclimation in inducing chilling tolerance (Table 111). It is likely that ABA-independent events occur during acclimation that contribute to the observed level of chilling tolerance. In addition, the fact that fluridone abolished acclimation-induced chilling tolerance does not exclude the possibility that ABA and acclimation operate through separate mechanisms, both of which are required for survival of Ch seedlings. Finally, the fact that ABA levels are, at best, only marginally elevated during acclimation is also evidence against ABA accumulation as an essential component of the acclimation mechanism.

Other works have suggested similar conclusions. Nordin et al. (1991) found that an Arabidopsis low-temperature- induced gene, lti240, was also induced by ABA, but low- temperature induction of Iti240 was unaffected in ABA-deficient and ABA-insensitive mutants and after treatment with fluridone. Gilmour and Thomashow (1991) found that ABA- induced expression of three cor genes was greatly inhibited in the abil (ABA insensitive) mutant of Arabidopsis, but cold- induced expression was unaffected. These facts point to the existence of separate mechanisms for cold- and ABA-induced gene expression. Waldman et al. (1975) found no significant increase of ABA levels in acclimated alfalfa and suggested that a decline in GA3 conferred tolerance by increasing the ABA/GA3 ratio. Mohapatra et al. (1988) concluded that, although ABA may play a role in acclimation to freezing tolerance in alfalfa, the acclimation mechanism was not mediated by ABA given differences in protein profiles, transcripts, and the level of freezing tolerance between ABA- treated and Ac plants.

We observed a dramatic elevation in free ABA levels in response to chilling, both in Ch and AcCh treatments. These





results also constitute evidence against the direct involvement of ABA during acclimation, since it appears that ABA is responding to something other than acclimation. It is as if ABA levels correspond to the leve1 of stress (possibly chilling- induced water stress) experienced by the plant. Capell and Dorffling (1989) concluded that chilling-induced water stress was responsible for the observed elevation of.ABA in chilled cucumber. Other works have shown that exogenous ABA treatment alleviated chilling-induced water stress (Markhart, 1984; Pardossi et al., 1992). Thus, in our experiments, it is possible that ABA is responding to the water status of the seedlings and that the ABA-induced chilling tolerance observed in our survival experiments is due, in part, to alleviation of chilling-induced water stress.

On the other hand, maintenance of low ABA levels during acclimation does not necessarily mean that it has no role in the acclimation process. Perhaps there is a redistribution of ABA toward its site of action, or perhaps the sensitivity to ABA is enhanced during acclimation. Thus, although our data point to separate pathways of induction for ABA- induced and acclimation-induced chilling tolerance, we cannot rule out the possibility that ABA plays a role during acclimation.

ACKNOWLEDCMENTS

We wish to thank Drs. C.E. La Motte and X. Li, Plant Hormone Analysis Facility, Iowa State University, for assistance in the quan- tification of ABA and Dr. J.T. Colbert for helpful suggestions during the construction and subsequent screening of the cDNA library.

Received November 22, 1993; accepted January 28, 1994. Copyright Clearance Center: 0032-0889/94/l05/0331/09.

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