Copyright 0 1990 by the Genetics Society of America

Molecular Characterization of a Catalase Null Allele at the Cat3 Locus in Maize

Gregory J. Wadsworth and John G. Scandalios' Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695-7614

Manuscript received January 22, 1990 Accepted for publication May 2, 1990

ABSTRACT Previous analysis has identified line IDS28 of maize (Zea mays L.) as being homozygous for a

Catalase-3 (Cat3) null allele. Catalase-3 (CAT-3) protein-specific antibodies could not detect CAT-3 in extracts of several tissues of IDS28, which in a typical maize line possess CAT-3. The absence of CAT-3 resulted in a significant decrease in total catalase activity in those tissues where CAT-3 is the predominant catalase isozyme. RNA blot analysis indicated that IDS28 does not accumulate Cat3 transcript in any tissues. Genomic DNA blots revealed significant structural alterations in the Cat3 gene in IDS28. The results suggest that the molecular basis for Cat3 null phenotype in IDS28 may Ge a deletion in the 5' end of &I; Cat3 gene.

C ATALASE (H202:H202 oxidoreductase, EC 1.1 1.1.6) is a tetrameric heme-containing en-

zyme that catalyzes the degradation of H202 into 0 2

and H20. In maize, multiple isozymic forms of catalase (CAT-1, CAT-2 and CAT-3) are encoded by three unlinked structural genes, Catl, Cut2 and Cat3 (ROU- PAKIAS, MCMILLIN and SCANDALIOS 1980). Each of the maize catalase genes is differentially expressed in distinct temporal and spatial patterns during the maize life cycle (SCANDALIOS et al. 1984). Cat l is expressed primarily in the tissues of the immature kernel, the postimbibition scutella, and in light- and dark-grown leaf tissue. Cat2 is expressed in the postimbibition scutella, the aleurone of the developing kernel and the bundle sheath cells of green leaves. Cat3 is ex- pressed in coleoptiles, in the light- and dark-grown mesophyll cells and in the pericarp of immature ker- nels. Cell fractionation experiments indicate the cat- alase gene-products are differentially localized within the cell (SCANDALIOS 1974; SCANDALIOS, TONG and ROUPAKIAS 1980). CAT-1 and CAT-2 co-isolate with the glyoxysomes and cytosol of postgerminative scu- tellar cells and with the peroxisomes and cytosol of green leaves. CAT-3 co-isolates only with mitochon- dria.

The physiological role of multiple forms of catalase in maize remains unclear. However, the develop- mental pattern of expression and subcellular localiza- tion may be suggestive of the respective metabolic roles of some of the isozymes. For example, CAT-2 occurs in tissues that have specialized processes known to produce high levels of H202, the postgerminative seedling with the glyoxylate cycle and the bundle sheath cells of green leaves with photorespiration.

' To whom correspondence should be addressed.

Genetics 125: 867-872 (August, 1990)

CAT-3 on the other hand may function to specifically eliminate H202 generated during mitochondrial res- piration (SCANDALIOS, TONG and ROUPAKIAS 1980). However, there is as yet no direct evidence linking the maize catalase isozymes to essential roles in specific metabolic processes.

We have been utilizing genetic approaches to inves- tigate the physiological roles of the maize catalases. Complete null mutations are among the most power- ful genetic tools for studying the functions of gene products. During an investigation of isozyme poly- morphisms in maize a CAT-3 electrophoretic null allele was identified in maize line IDS28 (GOODMAN and STUBER 1986). Herein, we describe the molecular and biochemical basis for this electrophoretic null allele of the Cut3 gene.

MATERIALS AND METHODS

All maize lines used in this study are maintained in our laboratory. Original IDS28 seeds were a gift of M. GOOD- MAN. (The genotype for the catalase genes of W64A is C a t l V , CatZZ and Cat3A and of IDS28, CatlF, Cat2Z and Cat3 null.) Scutella and epicotyls were isolated from seed- lings 7 days postimbibition. Immature kernels were har- vested from greenhouse-grown plants at 9 days after manual pollination. Green leaves for DNA isolation were harvested from 14-day, postimbibition plants. CAT-3 antiserum was raised in rabbits (CHANDLEE, TSAFTARIS and SCANDALIOS 1983). [a-"PIdCTP was obtained from New England Nu- clear. All other chemicals were reagent grade or better.

Nucleic acid probes for DNA and RNA blot analysis were prepared from restriction fragments of the cDNA clones for the three maize catalase genes (BETHARDS, SKADSEN and SCANDALIOS 1986; REDINBAUGH, WADSWORTH and SCAN- DALIOS 1988). Full-length Cat3 cDNA probe was prepared from the EcoRI insert of pCat3.3~. Probes corresponding to the 3' ends of the Catl and Cat3 genes were derived from the 3' HincII/BamHI fragment of pCat. IC and the 3' SacI/

868 G . J. Wadsworth and J. G . Scandalios

FIGURE 1 .-Zymogram analysis of catalase expression in various tissues of \V64A and It)S28. Extracts were prepared fronl the scutellun~, epicotyl ;md inlnlature kernel of iV64.A (14') ; I I ~ IDS28 ( I ) . 'l'hr extracts wc*re ;~n;llyred by starch gel electrophoresis for

I V. and <:AT-!?% Ilolnotetr;~mel-ic isozynles are present ;IS well as the intergenic Ileterotetranler species that form betwen thenl. I n

rpicotyl of \V64)\ o111y CAT-3)\ is evident and 110 catalase isoryme cat1 be secw i n t h t . epicotyl of II)S28. \Z'(i4A immature kernels (9 clays ~ ) o s t ~ ~ o l l i ~ ~ ~ ~ t i o ~ ~ ) l ~ a v e I)oth CAT-I \ ' and CAT-3A activity. The kernels of IDS28 have only CAT-I F. Sote t h a t no CAT-3 w a s detected in ;Illy of the extracts of I I X 2 X .

BcoRl fragment of pCat3.lc, respectively. Nucleic acid hy- bridization probes made from these 3' end restriction frag- ments have been shown to hybridize in a gene-specific manner on DNA or R N A blots (REDINBAUGH, WADSWORTH and SCANDALIOS 1988). Probes for the 5' end of the catalase genes were synthesized from the 5' EcoRI/BamHl fragment of pCat2. l~ and the 5' EcoRI/Aval fragment of pCat3.3c. These 5' end restriction fragments share significant se- quence similarity (REDINBAUGH, WADSWORTH and SCAN- DALIOS 1988) and probes constructed from these fragments cross-hybridize on DNA blots (G. J. WADSWORTH and J . G . SCANDALIOS, unpublished data). Restriction fragments were isolated by preparative gel electrophoresis (BETHARDS, SKAIXEN and SCANDALIOS 1986) and radiolabeled with "'P (FEINRERG and VOGEL~TEIN 1983).

Catalase assays: Tissue extracts were prepared by grind- ing fresh tissue with a mortar and pestle using sand and polyvinylpoly-pyrollidone (PVP) in a buffer of 25 mM gly- cylglycine (pH 7.4). Tissue to buffer ratios (wt/vol) varied w i t h the sample (0.5 g/ml for scutella; 1 g/ml for epicotyl and immature kernel). After grinding, samples were centri- fuged at 15,000 X g for 4 min at 4" and supernatants were recovered.

Catalase enzyme activity was measured spectrophotomet- rically (BEERS and SIZER 1952). Protein concentrations were determined using bovine serum albumin as a standard (LOWRY et al. 1951). Starch gel electrophoresis using the Tris-citrate buffer system was performed as previously de- scribed (CHANDLEE and SCANDALIOS 1984). Catalase activity was detected by incubating gel slices in 0.0 1 % H20n for 20 min then staining in a solution of 1 % FeCls/l % K3Fe(CN),;.

Rocket immunoelectrophoresis was carried out as de- scribed (CHANDLEE and SCANDALIOS 1984). Antiserum was used at a concentration of 1:2000 (v/v). After electropho- resis catalase activity was detected by first incubating the gel 20 min i n 50 mM KPO, (pH 7.0); 0.1 mM EDTA; 0.4 mg/ ml N B T (nitroblue tetrazolium), then rinsing the gel in HsO and incubating it in 50 n1M KPO.I (pH 7.0); 0.1 mM EDTA; 15 mM HsOs; 10 mM Na-ascorbate, under illumination, until

~. .ILI . 1. . I W . ' woryme conqmsition. I n the s c u t e l l u ~ ~ ~ of iV64A. the C A T -

the Sctltellutll of IDS28. CAT-I I- ; I I I ~ ~ C.A.f-2% > I W prescl1t. 111 the

FIGURE !?."Rocket imn~unoelectrophoresis analysis of CAT-3 in WB4A xnd II)S'.'X. Immunologic;ll determination of CAT-3 protein levels in the tissues of M'B4A ; ~ n d II)S!?X was m;& using rocket imtlltlt~oclectrol,llolesis. Solul,le CS~I'BCIS of the scutellunl (SCUT), the epicotyl (EPIC), ant1 inlmature (9 days postpollination) kernel (KERN) were ;Iss;lyetl. CAI. -3 WIS detected i n the tissues of W64A I X I I 1101 II)S'LX.

the desired level of staining contrast was achieved (about 15 min).

RNA blot analysis: Total RNA was isolated from epico- tyls (9 days postimbibition) and immature kernels (9 days postpollination) of W64A and IDS28 as described (WADS- WORTH, REDINRAUCH and SCANDALIOS 1988). R N A was separated by denaturing agarose electrophoresis (PAVE, CRKVENJAKOV and BOEDTKER 1979), transferred to nitro- cellulose (THOMAS 1980) and hybridized to radiolabeled nucleic acid probes (REDINBAUGH, WADSWORTH and SCAN- DALIOS 1988).

DNA blot analysis: Total DNA was isolated from 14- day, postimbibition green leaves (REDINRAUGH, WADS- WORTH and SCANDALIOS 1988). digested with restriction endonucleases, and separated by agarose gel electrophoresis (MANIATIS, FRITSCH and SAMBROOK 1982). DNA was trans- ferred to nitrocellulose paper and hybridized to radiolabeled nucleic acid probes as described (MANIATIS, FRITSCH and SAMBROOK 1982). Final wash conditions were 0.1 X SSC: 0.1 % SDS at 60" for 1 hr unless otherwise specified.

RESULTS

Zymogram analysis: T h e catalase isozyme content of the tissues of IDS28 was determined by zymogram analysis. Soluble extracts were prepared from various tissues of IDS28 (the CAT-3 null line) and W64A (a maize line with typical catalase expression) and sub- jected to zymogram analysis (Figure 1). T h e postger- minative scutellum (8 days postimbibition) of W64A has the CAT1 V and CAT2Z homotetrameric iso- zymes and the characteristic intergenic heterotetra- mers that form between them (SCANDALIOS et al. 1984). In IDS28 the CAT-IF and CAT-2Z isozymes are seen; however, because they comigrate in this buffer no homotetrameric and heterotetrameric forms can be distinguished. As expected, no catalase activity could be attributed to CAT-3 in the scutellar extracts of either maize line. In extracts of the epicotyl of W64A only CAT-SA is detected on the zymogram. No catalase activity can be detected on the zymograms in the epicotyl extracts of IDS28. T h e immature ker- nel of W64A (9 days postpollination) has both CAT- 1V and CAT-SA activity. In the IDS28 immature

Maize Cat3 Locus 869

TABLE 1

A comparison of catalasespecific activity in the tissues of W64A and IDS28

Tissur \\‘ti414 I DSYU

Scutellunl 1 .ox f 0.16 1 .00 f 0.05 Epicotyl 0.20 f 0.0 1 0.15 f 0.01 lierne1 0 . 1 3 f 0.0 1 0.023 f 0.003

l ’ o t ; ~ l catatme ;Ictivity \GIS tneasured in soluble extracts of the scutell;~, epicotyls (X clays ~~ostimbibition) and immature kernels (9 days I,ostl’ollin;ltion) of inbred nlaize lines M’64A and I IX28 (CAT- 3 n u l l ) . Specificactivity values reported I-epresent the nlean of three indcpentlent estr;~ctions ; I n d catalase assays. Catalase-specific activ- ity is reported ;IS units/nlg protein f the standard error of the n1e;In.

kernel, a single band of catalase activity was detected that comigrates with the CAT-IF isozyme. N o band of activity was detected in the IDS28 immature kernel extracts that comigrate with the two previously iden- tified CAT-3 isozyme allelic variants (A and B alleles) (SCANDALIOS, TONG and ROUPAKIAS 1980).

Rocket immunoelectrophoresis: Rocket immunoe- lectrophoresis provides a mare sensitive and specific assay for the presence of a specific isozyme in different tissue extracts. Extracts of IDS28 and W64A tissues were analyzed by electrophoresis through an agarose gel containing anti-CAT-3 Ab under conditions where CAT-3 will precipitate during electrophoresis (Figure 2). The “rocket” of immunoprecipitated CAT-3 that forms has a height that is proportional to the amount of CAT-3 cross-reacting materials (CRM) in the crude extracts (LAURELL 1966). Because the immunoprecip- itated complex retains enzymatic activity, it was pos- sible to stain directly for catalase activity that greatly increased the sensitivity and specificity of this assay. CAT-3 CRM was detected in all the W64A samples (Figure 2). There were approximately equal levels of CAT-3 in the epicotyl and immature kernel and low levels in the postgerminative scutella as evidenced by rocket height. No active CAT-3 protein could be detected in any of the IDS28 tissues. This result is consistent with the zymogram analysis.

Catalase enzyme activity: Catalase activity was measured in crude extracts of various tissues to quan- tify the impact of the CAT-3 null allele on total catalase activity (Table 1). The total catalase-specific activity in the scutella of IDS28 and W64A was ap- proximately the same (1 .OS f 0.16 units/mg protein and 1 .OO f 0.05 unit/mg protein, respectively). The specific activity of catalase in the epicotyls was lower in IDS28 (0.15 f 0.0 1 unit/mg protein) than in W64A (0.20 -I- 0.01 unit/mg protein). The residual catalase activity in the extracts of IDS28 epicotyls may be due to low levels of CAT-1 not detected on the zymo- grams. Less catalase activity was also found in the immature kernels (9 days postpollination) of IDS28 (0.023 f 0.003 unit/mg protein) than in W64A im-

A: Cat3gsp B: Cat3 flp c : Cat7 gsp v-7

1 2 3 4 I 2 3 4 1 2 3 4 FIGUR~.: 3.-RNA blot analysis of Cal l and Caf3 transcripts in

W64A and IDS28. Total R N A ( 1 0 pa) from the immattlre kernel (9 d a y s 1)os‘l’ollin;rtiotl) of l I X 2 X (lane 1 ) and W64A (lane 2). and from d;lrk-grown epicotyls (X clays postimbibition) of Ills28 (lane 3) and from W64A (Ianc 4 ) were examined by R N A blot analysis. R N A wils hybridized t o the Caf3 gene-specific probe (gsp) (pnel A), the full-length Caf3 cDNA probe (111’) (p;rncl R). and the C a f l gene-specific probe (asp) (panel C) .

mature kernels (0.13 f 0.0 1 unit/mg protein). RNA blot analysis: RNA blot analysis was per-

formed to compare the level of Cut3 transcripts in various tissues of IDS28 to the standard line W64A. RNA blots of total RNA from dark-grown epicotyls and immature kernels of W64A and IDS28 were hybridized with a full-length Cut3 cDNA probe and a Cut3 gene-specific probe derived from the 3’ end of the cDNA (Figure 3, A and B). These probes strongly hybridized with an RNA species from the W64A epicotyl and weakly hybridized with an RNA species from the W64A immature kernel. The Cut3 probes did not hybridize at detectable levels with the IDS28 RNA samples from either the epicotyls or the imma- ture kernels, indicating the Cut3 mRNA does not accumulate in the IDS28 tissues. When an identical RNA blot was hybridized to the Cutl gene-specific probe as a control, a hybridization signal was detected for all the RNA samples and the signal was similar for both lines in both tissues (Figure 3C). This indicates that the IDS28 RNA was intact and that the level of Cutl gene expression in IDS28 is equivalent to W64A.

DNA blot analysis: Genomic Southern blotting was used to examine the structure of the Cut3 gene in IDS28. DNA was isolated from IDS28 and W64A, digested with restriction endonucleases, separated on agarose gels and transferred to a nitrocellulose filter. Identical Southern blot filters were then hybridized with the Cat1 gene-specific probe, the Cat3 gene- specific probe, the Cut3 5’ end probe or the Cut2 5‘ end probe (Figure 4).

The Cutl gene-specific probe hybridized to a single- size restriction fragment in the DNA of both W64A and IDS28 digested with various restriction enzymes (Figure 4; panel A). The level of hybridization to the DNA of the two lines was approximately the same, indicating nuclear DNA was almost equally repre- sented in the total DNA sample from each maize line.

G . J. Wadsworth and J. G . Scandalios 870

Panel A

Lanes 1 2 3 4 5 6 2 1 - 9.1 - "

66- 0

W

J " 22-

Enzyme H E B H E B

Maize Line W64A IDS28

"

Probe Carigsp

H E B H E B

W64A IDS28

"

Catasp

H E B H E B

W64A IDS28

"

Caw 5end

The size of the restriction fragments homologous to the Catl gene-specific probe were different between the two lines for EcoRI and HindIII as might be expected for allelic variants.

The Cat3 gene-specific probe that corresponds to the 3' end of the Cat3 gene, also hybridized to a single-size restriction fragment in the DNA of the two lines (Figure 4; panel B). A restriction fragment length polymorphism was observed between the two lines for BamHI. The Cat3 gene-specific probe, unlike the Catl gene-specific probe, showed a different hybridization intensity between to the DNA of W64A and IDS28 (compare the BamHI digest from each line). This weaker hybridization signal in IDS28 indicates that the region of the Cat3 gene corresponding to the 3' gene-specific probe has diverged significantly either in sequence or in the presence or absence of certain regions in the 3' end of the gene.

The Cat2 and Cat3 5' probes hybridized with ge- nomic DNA to generate a slightly more complex pattern. We believe the added complexity of the 5' end probes over the 3' end probes arises from the sequence similarity of these two probes and their ability to cross-hybridize among the three catalase genes (REDINBAUCH, WADSWORTH and SCANDALIOS 1988). With W64A DNA, the Cat3 5' end probe hybridized to two size restriction fragments with BamHI and HindIII and a single-size restriction frag- ment when digested with EcoRI (Figure 4; panel C). In lanes where two bands of hybridization could be distinguished (Figure 4; panel C, lanes 1 and 3), one of the bands always has a stronger hybridization signal than the other. Additionally, the less intense band was always the same molecular weight as the restriction fragment that hybridized most strongly to the 5' end of the Cat2 gene (Figure 4; panel D). Therefore, we believe that in the two-banded pattern of hybridiza- tion observed when the 5' end of Cat3 is hybridized to W64A genomic DNA, the more intense band is due to homologous hybridization of the Cat3 probe to the Cat3 gene, whereas the less intense band is due to cross-hybridization of the Cat3 probe with the Cat2 gene.

D

1 2 3 4 5 6

*. . .

H E B n t u "

W64A IDS28

Car2 Send

Lanes

FIGURE 4.-Genomic DNA blot analysis of IDS28 and W64A. Total cellular DNA isolated from W64A (lanes 1-3) and IDS28 (lanes 4-6) was digested with Hind111 (H), EccoRI (E), EatnHI (B) and analyzed by Southern blot hybridimtion. Each lane contains 20 ag of digested DNA. Filter-bound DNA was hybrid- ized to the Cat1 gene-specific probe (gsp) (panel A), the Cat3 gene-specific probe (gsp) (panel B), the Cat3 5' end probe (panel C), and the Cat2 5' end probe (panel D).

Maize line I W I W I W

Temperature 60 c 68 C 75 c FIGURE 5.-High stringency genomic DNA blot analysis IDS28

and W64A DNA. DNA was digested with BanHI and hybridized to the 5' end probe of Cat3 under standard conditions. After hybridilation the filters were washed as before but the final wash was performed at varying temperatures (SO0, 68", 75").

In IDS28 DNA the 5' end Cat3 probe hybridized with a single-size restriction fragment with all three restriction enzymes (Figure 4; panel C; lanes 4-6). Those restriction fragments that hybridized with the Cat3 5' probe in IDS28 have the same molecular weight as the restriction fragments that hybridized to the Cat2 5' end probe (Figure 4; panel D; lanes 4-6). This suggests that the hybridization detected in IDS28 DNA using the 5' end Cat3 probe was not due to homologous hybridization to the Cat3 gene but was due to cross-hybridization to the Cat2 gene.

To further investigate the restriction fragment in IDS28 that is hybridized to the 5' end of the Cat3 cDNA, DNA blots hybridized to the 5' Cat3 probe were washed at higher stringencies (Figure 5). DNA blots with BamHI digested IDS28 and W64A DNA were hybridized with the 5' Cat3 probe under stand- ard conditions. Identical blots were then washed at increasing stringencies. At 68", the pattern was the

Maize Cat3 Locus 87 1

same but the relative intensity of the bands is differ- ent. The more intense W64A hybridization band, which we believe to be due to homologous hybridiza- tion, has a stronger signal relative to the second W64A hybridization band and the single IDS28 hybridization band. At 75”, the only hybridization detected was to the homologous band in W64A. The less intense band in W64A and the only band in IDS28 that were seen at the lower wash stringencies did not hybridize to the Cut3 5‘ probe at this higher stringency. This is con- sistent with a model that the single hybridization signal in IDS28 and the lesser hybridization signal in W64A was due to cross-hybridization with the Cat2 gene. If this is correct, the 5’ end of the Cat3 gene is absent from the IDS28 genome.

DISCUSSION

In this communication we have described the bio- chemical and molecular characterization of the CAT- 3 null phenotype of the inbred maize line IDS28. Phenotypically, the Cut3 null allele for IDS28 was identified by the lack of CAT-3 isozyme activity on zymograms of maize tissues in which CAT-3 is ex- pected (e.g., the dark-grown epicotyl). Rocket immu- noelectrophoresis confirmed the lack of active CAT- 3 protein in the tissues of IDS28. CAT-3 was found at high levels in the dark-grown epicotyls and early immature kernels and at very low levels in postger- minative scutella of our standard maize lines. How- ever, CAT-3 was completely absent in these same tissues in the CAT-3 null line, IDS28.

To investigate the molecular basis for the absence of CAT-3 protein in IDS28, we compared the levels of Cut3 mRNA in IDS28 and W64A. Moderate levels of Cut3 mRNA were detected in the W64A epicotyls and low levels in the immature kernel as previously reported (REDINBAUGH, WADSWORTH and SCANDA- LIOS 1988; WADSWORTH and SCANDALIOS 1989). However, no Cut3 mRNA was detected in total RNA isolated from IDS28 dark-grown epicotyls and imma- ture kernels using RNA blot analysis. Therefore, CAT-3 protein cannot accumulate in IDS28 because it lacks the mRNA template on which to synthesize it. Additionally, the RNA blot analysis showed that the CAT-3 null mutation did not affect the expression of the Cutl gene at the mRNA level. The level of Cutl mRNA in IDS28 RNA samples was about the same as in our standard line W64A.

Analysis of gene structure using DNA blot experi- ments indicates that the Cut3 gene in IDS28 may have a partial deletion. When probed with a restriction fragment from the 3’ end of a Cat3 cDNA clone, a single hybridization signal is detected with the DNA of IDS28 and of W64A. Nevertheless, the intensity of the signal in IDS28 is much lower than in W64A. This suggests that the region of the Cat3 gene corre-

sponding to this probe in IDS28 has diverged in sequence from W64A. Alternatively, the lower hy- bridization signal may result because part of the re- gion corresponding to the probe may be deleted from the IDS28 genome. If either of these possibilities was correct, it could contribute to the nonexpression of the Cat3 gene in IDS28.

Genomic DNA blot analysis using the 5’ end of the Cut3 cDNA suggested that this highly conserved re- gion of the Cat3 gene may be absent from the IDS28 genome. The 5’ end restriction fragment of the Cut3 cDNA clone hybridized to a single-size fragment on genomic DNA blots of IDS28 DNA. Evidence was presented that this hybridization was likely due to cross-hybridization with the Cut2 gene. First, it was previously shown that these regions of the Cut2 and Cut3 cDNA shared significant sequence similarity and G/C content (REDINBAUGH, WADSWORTH and SCAN- DALIOS 1988). Second, in Southern blot analysis of typical maize lines the 5’ Cat3 cDNA probe generally hybridized to two restriction fragments: one of which could be accounted for by homologous hybridization to the Cat3 gene and the second could be accounted for by cross-hybridization to the Cat2 gene. Third, the genomic restriction fragment to which the 5’ end Cat3 probe hybridized in IDS28 DNA migrates iden- tically as the major restriction fragment to which the 5’ end of the Cat2 gene hybridized. This is consistent with the 5‘ end Cat3 probe cross-hybridizing with the Cut2 gene in IDS28. Finally, under stringencies where the Cat3 5’ end probe hybridizes in a gene-specific manner to W64A DNA, no hybridization was de- tected with the IDS28 DNA. These results indicate that a 5’ end fragment of the Cat3 gene is probably deleted from the IDS28 genome.

A deletion of the 5’ end of the Cut3 gene could have two effects that would contribute to the CAT-3 null phenotype. First, the 5’ portions of protein-en- coding plant genes, like animal genes, are associated with the structural information necessary for tran- scription of the genes (KUHLEMEIER, GREEN and CHUA 1987). If this region were altered or deleted, it could affect the ability of the gene to be transcribed. This lack of transcription would contribute to the lack of Cat3 transcript accumulation seen with the RNA blots of IDS28 RNA. Additionally, the 5’ Cut3 probe that was used to detect the deletion corresponds to a catalytically important region of the CAT-3 protein- encoding portion of the Cut3 transcript, including the heme-binding domain (REDINBAUGH, WADSWORTH and SCANDALIOS 1988). Thus, even if the remaining 3‘ portion of the Cut3 gene that may be present in the IDS28 genome were transcribed and translated at a very low level, it would not produce a functional CAT-3 protein because it lacks this essential heme- binding domain.

872 G . J. Wadsworth and J. G . Scandalios

There are no discernible physiological effects asso- ciated with the CAT-3 null allele in maize line IDS28. IDS28 has a growth rate and reproductive capacity typical of highly inbred maize lines (personal obser- vation). It is possible that other enzymes may be compensating for the lack of CAT-3 in IDS28. These compensating enzymes might include the other cata- lase isozymes or maize peroxidases. Indeed, a residual level of catalase activity was found in all the tissues lacking CAT-3, presumably associated with low levels of CAT-1 and CAT-2. Alternatively, it is possible that CAT-3 is not essential under the conditions in which we have tested IDS28 but may play an important physiological role under environmental conditions that increase oxygen stress.

It is presently impossible to measure subtle and quantitative effects of the CAT-3 null allele because of the broad genetic differences between IDS28 and our standard inbred lines. T o make these analyses, we have begun the genetic manipulation (recurrent back- crosses) to put the Cat3 null allele into the genetic background of our standard maize line W64A. When the CAT-3 null allele is in a standard genetic back- ground, then we can specifically analyze the pheno- typic consequences of the null allele under normal and stressful environmental conditions.

We thank STEPHANIE RUZSA for expert technical assistance and SUZANNE QUICK for skillful typing of the manuscript. This research was supported, in part, by Research Grants GM22733 from the National Institutes of Health and R812404 from the U.S. Environ- mental Protection Agency to J.G.S. This is paper No. 12541 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina.

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