Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous...

The Plant Journal (1994) 6(2), 161-175

Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis

Carolyn M. Wstzel 1, Cai-Zhong Jiang 1, LeAnn J. Meehan 1, Daniel F. Voytas = and Steven R. Rodermel 1,* 1 Department of Botany and 2Department of Zoology and Genetics, Iowa State University, Ames, IA 5001 I, USA

Summary

The immutans (ira) variegation mutant of Arabidopsis thaliana contains green- and white-eactored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is Influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are hetero- plasUdic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of irnmutans is 'plastid autonomous'. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally Inher- ited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white t issues of immutans accumulate phytoene, a non-colored C4o carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturaea (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild- type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear--organelle interactions.

Received 29 October 1993; revised 25 March 1994; accepted 26 April 1994. *For correspondence (fax +1 515 294 1337).

Introduction

In plants, as in animals, normal cellular differentiation depends on coordinated interactions between the nuclear and organellar (chloroplast and/or mitochondrial) genomes (reviewed in Bogorad, 1991). A genetic dissection of plant variegation mutants is a powerful means to gain insight into these poorly understood interactions. Variegation mutants have leaves that typically contain green and white (or yellow) sectors. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white (or yellow) sectors have plastids that lack chlorophyll and/or carotenoid pigments. This indicates that these cells are perturbed in some aspect of nuclear-chloroplast communication that manifests itself, either directly or indirectly, in pigment-deficient plastids.

Variegation in plant leaves can arise via several different mechanisms. One common mechanism involves the induction of permanently defective organelles in some cells, but not others, by nuclear gene mutations or by mutations in the organellar DNA (Hagemann, 1986; reviewed in Tilney-Bassett, 1984). The defective organelles subsequently undergo unequal sorting-out, giving rise to sectored leaves containing cells with normal and abnormal plastids. Because mitochondria and chloroplasts are transmitted maternally to offspring in most angiosperms, the probability of transmission of a permanently defective organelle is related to the extent of variegation of the mother plant. Well-known examples of nuclear gene-induced variegation mutants include the Arabidopsis chm (chloroplast mutator), maize NCS (non- chromosomal stripe), and maize iojap mutants (e.g. Coe et al., 1988; Han et al., 1992; Martfnez-Zapater et al., 1992; Newton and Coe, 1986; Rddei, 1973; Roussell et al., 1991; Walbot and Coe, 1979). iojap gives rise to permanently defective plastids, while chm and NCS induce lesions in the mitochondrial DNA that result in permanently defective mitochondria, chin and NCS plants are variegated because the defective mitochondria secondarily affect chloroplast form and function, generating cells with pigment-deficient plastids.

In addition to nuclear gene-induced variegation mutants, a number of variegated 'plastome' mutants have been described, most notably in Oenothera (reviewed in Stubbe and Herrmann, 1982). These mutants have lesions in their plastid DNA that generate permanently defective plastids. Other common leaf variegation mechanisms include transposable element activity and

161

162 Carolyn M. Wetzel et al.

chimerism. Regarding the former, transposon insertion may interrupt a gene required for pigment synthesis (white sectors), while element excision can reconstitute wild-typ~ gene expression (green sectors) (reviewed in Federoff, 1989). In chimeric plants different histological regions of a plant meristem, and consequently the tissues that derive from these regions, have different genotypes. This results in variegation if the genotypic differences affect pigment accumulation (reviewed in Tilney-Bassett, 1986).

The immutans (ira) variegation mutant of Arabidopsis thaliana, first described and partially characterized 30 years ago, has green and white leaf sectors due to the action of a nuclear recessive gene (Chung and R~dei, 1974; R~lei, 1963, 1967a, 1967b, 1975; RSbbelen, 1968) (see Figure 1). R~dei (1967a) and RSbbeten (1968) reported that the green sectors of im contain normal plastids, while the white sectors are homoplastidic for abnormal plastids that lack pigments and internal membrane structures. Progeny from the selfing of im/im plants recapitulate the variegation of the parent, regardless of whether they are derived from all-green or all-white inflorescences, indicating that the mutant seeds have a uniform genetic constitution (R~dei, 1967a). This suggests that sector formation in the mutant is reversible i.e. cells containing chloroplasts can give rise to cells containing white plastids and vice versa--and that transposable element activity is not responsible for the im variegation (or else one would expect that the progeny from green inflorescences would be predominantly wild- type, while the progeny from white branches would be white and/or variegated, depending on the frequency of the excision event). Interestingly, the degree of white- sectoring in the mutant can be enhanced by increased light intensity and temperature during plant development (R~dei, 1963; RSbbelen, 1968; unpublished data).

We originally became interested in immutans as a tool to gain entrance into the complicated circuitry that governs interactions between the nuclear-cytoplasmic and chloroplast genetic systems. The presence of white sectors in the mutant indicated that the IM gene product affects

pigment accumulation in the plastid (either directly or indirectly) and is therefore required for normal chloroplast development. The expression of IM is also influenced by light, an external factor that is required for normal chloroplast differentiation (Bogorad, t991). Consequently, an understanding of the mode of action of this protein might lend insight into the mechanisms by which nuclear genes control plastid function. As a first step toward this goal, we have undertaken an ultrastructural and biochemical re-examination of im plants, and have employed the tools of molecular biology and genetics to gain further insight into the molecular lesion in the mutant. The results of our experiments showed that immutans exhibits an unusual environmentally influenced somatic instability that results in the formation of abnormal white sectors interspersed with phenotypically wild-type green tissue. Significantly, the defect also appears to manifest itself independently among the ptastids in a cell, but the mutation is not maternally inherited, immutans is thus representative of a novel class of Mendelian-inherited, 'plastid autonomous' variegation mutants that should provide great insight into nuclear-organelle interactions.

Results

immutans is not maternally inherited

Redei (1967a) reported that im is a nuclear recessive gene, but it was not clear whether the mutation is also maternally inherited. To examine this question, we reciprocally crossed im/im plants with wild-type plants (Table 1). It was expected that if the im mutation produced permanently defective plastids (e.g. due to chloroplast or mitochondrial gene mutations), then white plastids would be transmitted from variegated females to the F1 progeny plants. We observed that all F1 progeny from these crosses, regardless of parentage of the cytoplasm, had a wild-type phenotype. This indicates that im is not maternally inherited and does not generate permanently defec-

Table 1. Observed phenotype of F1 and F2 progeny resulting from reciprocal crosses, compared with the expected outcome if the variegation was maternally inherited

Observed phenotype Expected phenotype if maternal

inheritance of variegation

Parentals F1 F2 F1 F2

female x male Green Variegated Green Variegated Green Variegated Green Variegated

im X wild-type 16 0 603 181 < 16 > 0 Variable; not 3:1 Wild-type X im 19 0 978 306 19 0 1284 0

Precise predictions about maternal inheritance in im x wild-type progeny cannot be made because of the random nature of sectoring in immutans. Chi-square analyses of F 2 data from both crosses fit a 3:1 ratio (green:variegated) (p > 0.10).

The immutans mutant ofArabidopsis thaliana 163

Table 2. Determination of the light-responsive phase of immutans expression in developing cotyledons

Light-shift conditions Observations (hours after plating seeds) Cotyledon pigmentation

0-24 24-48 48-72 72-96 Green Vadegated White

L L L L L L L H H H H H H H H L

L L H H H H L L

L 6 29 0 H 4 34 0 H 0 0 42 H 0 0 43 H 0 0 42 L 1 10 32 L 2 40 2 L 12 27 0

Pigmentation was scored 240 h after plating; the values are averages of two replicates. L, low light treatment (5 I~mol m -2 sec-1); H, high light treatment (100 ~mol m -2 sec-~). The light responsive phase is highlighted by a box.

tive plastids. In addition, the data in Table 1 confirm the finding of Rddei (1967a) that the im phenotype segre- gates in the F2 progeny in a 3:1 ratio (wild-type:im), as expected of a Mendelian recessive mutation.

The light-responsive phase of im action is early in cotyledon development

The light conditional character of immutans vadegation was investigated to determine the developmental stage during which the IM product is active. In a representative light-shift experiment, described in Experimental procedures and in Table 2, a light-responsive phase of immutans expression during cotyledon development was identified between 0 and 24 h after seed coat breakage; seed coat breakage occurs approximately 48 h after seeds are plated out, regardless of light level (data not shown), immutans seeds exposed to low light (5 I~mol m -2 s -1) between 48 and 72 h after plating developed green or variegated cotyledons, while seeds exposed to high light (100 ilmol m -2 s -1) during the same time pedod predominantly developed all-white cotyledons. Some variability in variegation was expected at the end-points of the light-responsive phase due to differences in the germination rates within the seed population, as is seen in the H-H-H-L treatment population (Table 2). Exposure to either low or high light before or after the 48-72 h time period had no measurable effect on ultimate cotyledon pigmentation. Wild-type control seedlings developed green cotyledons under all treatments (data not shown). These data support the suggestion by R6bbelen (1968) that the IM product is active during a discreet light- responsive phase of plant development.

The white tissue of immutans contains several types of plastids

Electron micrographs of chloroplasts from wild-type cells and from white sectors of immutans plants are shown in Figure 2. Figure 2(c) shows a representative abnormal plastid from im white tissue, and Figure 2(b) shows both normal and abnormal chloroplast~plastids from adjacent cells in im white tissue. The abnormal plastids are vacuolated and lack organized thylakoid membranes. Thylakoids from normal chloroplasts of immutans white tissue (Figure 2d) appear the same as those of wild-type (Figure 2a). Chloroplasts from green tissue of immutans are indistinguishable from those of wild-type cells (data not shown).

The presence of green plastids in visually white immutans tissue was unexpected, and is contrary to previous observations (R6dei, 1963, 1967a; RObbelen, 1968). We therefore undertook a microscopic examination of mesophyll cells from immutans leaves to determine the extent of this phenomenon. Figure 3 shows light and chlorophyll fluorescence microscopy photomicrographs of green and white im leaf sectors. Green sectors contain normal chloroplasts with high levels of chlorophyll fluorescence. White sectors, however, contain cells lacking chlorophyll and cells that have a low number of plastids with chlorophyll fluorescence (Figure 3a, b, d and e). The fluorescent plastids of white cells appear to be smaller than those of green cells (e.g. Figure 3d), although occasionally a wild- type-sized chloroplast is present (Figure 3e). The white cells that lack chlorophyll fluorescence do contain some colorless plastids (i.e. Figure 3c and f). These data indicate that cells in the white im sectors can be heteroplastidic for defective and normal plastids.

164 Carolyn M. Wetzel e t al .

Figure 1. Immutans. The plant was grown under continuous illumination (35 pmol m -2 sec-1). Figure2. Electron micrographs of plastids from wild-type and white

immutans tissues. (a) Thylakoids of a wild-type chloroplast. Bar = 500 nm. (b) Two adjacent cells in a white sector of immutans, containing normal chloroplasts and abnormal plastids. Bar = 2 p.m. (c) Abnormal plastid from a white sector of immutans. Bar = 2 p.m. (d) Thylakoids of a chloroplast from a white sector of immutans. Bar = 500 nm.

Rgure 3. Light and corresponding fluorescence microscopy images of variegated immutans tissue. Chloroplasts appear red in the fluorescence photos. (a) Green and white sectors of a leaf. Magnification 80 x. (b) View of a green-white interface, with small fluorescent plastids present in the apparently white tissue. Magnification 410 x. (c) View of a green--white interface, with no fluorescent plastids in the apparently white tissue. Magnification 410 x. (d) White sector that has small fluorescent plastids. Magnification 410 ×. (e) White sector that contains a single wild- type-sized chloroplast. Magnification 410 ×. (f) White sector that contains colorless, non- fluorescent plastids. Magnification 410 x.


Rgure 4. HPLC analyses of leaf pigments from (a) wild-type tissue; (b) green immutans sectors; (c) Norflurazon-treated wild-type tissue; and (d) white immutans sectors. The absorbance spectra for all retention times are shown, with the relative absorbance units displayed as peak height. Note that the green tissu e sample extracts were diluted fivefold relative to the white tissue sample extracts before chromatography; even in higher concentration extracts no phytoene peaks were detectable in the green samples. The peaks were identified (see Experimental procedures) as: 1, neoxanthin; 2, violaxanthin; 3, antheraxanthin; 4, lutein; 5, chlorophyll b; 6, chlorophyll a; 7, pheophytin a; 8, beta-carotene; and 9, 10, 11, phytoene isomers (* indicates position where phytoene peaks would be if it were present in the sample).

Phytoene accumulates in the white tissue of immutans

RL=clei reported that entire immutans plants have less total chlorophyll and carotenoid pigments than wild-type plants (R~dei, 1963). We isolated green versus white sectors to examine the pigment composition of immutans in greater detail. HPLC pigment analyses of these tissues (Figure 4) show that the green tissues of the mutant contain the normal complement of pigments. In contrast, the white tissues contain only very low levels of pigments, but accumulate a compound with the absorption profile of phytoene (Davies, 1976), the first C40 intermediate in the biosynthesis of carotenoids. The identity of this peak was verified by comparison with an extract from Norflurazon- treated tissue, which accumulates phytoene (Bartels and McCullough, 1972; Blume and McClure, 1980; Sandmann and Albrecht, 1990). In both the white immutans and the Norflurazon-treated tissue sample extracts, phytoene apparently elutes after several different retention times, giving rise to three peaks with the same absorption spec- trum. These probably represent different forms of phytoene, e.g. cis-and trans- (Albrecht et al., 1991; Khachik et al., 1989). The presence of pigments in visually white tissue is most likely due to the mixed plastid types that are

Table 3. Pigment content of immutans and wild-type leaves (in Fg g-1 fresh weight of tissue)

immutans

Pigment Wild-type Green sectors White sectors

Chlorophyll 58122 + 42.69 638.93 + 7.49 N.D. B-carotene 11.72 + 0.54 9.68 ± 0.31 N.D. Phytoene N.D. N.D. 22.19 ± 4.17

The values are an average of two determinations. N.D., not detectable.

found in white sectors (as described above). Chlorophylls, beta-carotene, and phytoene were quantified by thin-layer chromatography (Table 3).

immutans does not encode phytoene desaturase

Phytoene desaturase (PDS) catalyzes two successive desaturations of phytoene (to phytofluene and zeta- carotene) in higher plants (Bartley et al., 1991 b; Pecker et aL, 1992). This enzyme, as other carotenogenic enzymes, is encoded in the nuclear DNA and imported into the chloroplast, where all of the steps of carotenoid biosynthe-


sis occur (reviewed in Kleinig, 1989; Sandmann, 1991). The observation that the white sect6rs of im accumulate phytoene suggests that these tissues are blocked in the PDS step of carotenoid biosynthesis. Consistent with this observation, a number bacterial and fungal mutants that accumulate various intermediates in the carotenoid biosynthetic pathway have been shown to have lesions in the corresponding structural genes of the pathway (reviewed in Bartley et aL, 1991a). In contrast, molecular analyses have been performed on relatively few higher plant pigment-deficient mutants that accumulate carotenoid biosynthetic intermediates (Buckner et aL, 1990; Giuliano et aL, 1993). A primary reason for this is that it is difficult, in most plant species, to clone genes for which only a mutant phenotype exists. We have therefore taken advantage of the relative ease of genetic manipulation of Arabidopsis to determine whether immutans is the structural gene for PDS.

When the present studies were initiated, the sequence of a phytoene desaturase (PDS) cDNA clone had been reported from soybean (Bartley et al., 1991b). To obtain an A. tha/iana PDS sequence, we used the polymerase chain reaction (PCR) to amplify an ~300 bp PDS sequence from a soybean cDNA library. This fragment was then used as a probe to isolate a PDS clone from an A. thaliana genomic lambda phage library. A 2.0 kbp subfragment of this clone was isolated and partially

sequenced (data not shown); the exon sequences of this clone bear 100% nucleotide identity to the recently reported A. thaliana PDS cDNA sequence (Scolnik and Bartley, 1993). Use of this subfragment as a probe in Southern hybridization experiments revealed that PDS is a single-copy gene in the A. thaliana genome (Figure 5). Hybridizations carried out under low-stringency conditions (see Experimental procedures) did not reveal any new bands, indicating that there are no other closely related PDS sequences in A. thaliana.

If immutans encodes PDS, then the cloned A. thaliana PDS sequence should map to ira, which has previously been mapped to chromosome 4 (Koornneef, 1987). To test this, 45 plants were identified among the progeny of a cross between im (ecotype Columbia) and ap2, cer2, and bp (ecotype Landsberg) that had undergone recombination between these chromosome 4 markers. The A. tha/iana PDS clone was then used to identify a poly- morphic EcoRI fragment between Landsberg and Columbia DNAs (Figure 5), and the recombinant plants were scored for the PDS polymorphism, as well as for an EcoRI polymorphism that defines the ag locus (Yanofsky et a/., 1990), which resides near im on chromosome 4 (Figure 6). The PDS polymorphism was found to co- segregate with im in only 29 of the 45 plants, indicating that PDS does not map to ira. Chi-square analyses, however, showed that PDS is linked to chromosome 4 and lies close to ag (Figure 6).

The segregation of PDS was only consistent with the gene residing in the interval between ag and bp. To define the map position of PDS more precisely, 43 chromosomes were identified from among the 45 recombinant plants that had undergone recombination between ag and bp. PDS was found to co-segregate with ag in 32 of these 43 chromosomes. This indicates that PDS maps ~7.2 cM from ag, assuming a 28 cM map distance between ag and bp (Figure 6). The upper and lower 95% confidence limits place this gene between 33.4 cM and 26.3 cM on the Koornneef map of chromosome 4 (Koornneef, 1987).

Figure 5. Genomic Southern blot hybridized with an A. thaliana PDS gane. Genomic DNA was isolated from Landsberg (La) and Columbia (Col) ecotypes. The cloned PDS sequence is from Landsberg DNA and does not contain restriction sites for EcoRI, Hindlll, Xhol or ,~s/f.

ap2 cer2 im ag

M 63.5 46.8 43.3 37.6

Ef 2 0.20 1.80 3.75 11.75 0.65 0.16 0.05 0.0005

pds bp

30.4 9.6

1.08 0.30

Figure 6. A map of a portion of A. thaliana chromosome 4. Pooled F 3 progeny from 45 F2 recombinant plants were tested for co-segregation of PDS with chromosome 4 markers. Chi-square (Z 2) analyses tested the hypothesis that PDS is not linked to a given marker. P values < 0.05 indicate deviation from non-linkage (i.e. linkage). The calculated position of PDS is shown.

Figure 7. Steady-state RNA levels of representative nuclear and plastid genes in green (G) and white (W) leaf tissues of immutans and in leaves from wild-type (VVT) plants. Both types of plants were grown under continuous illumination (35 pmol m -2 sec-~), and RNAs were isolated from mature, fully expended leaves just prior to bolting. Each lane of the gel contains 5 mg of RNA.

The green tissue of immutans is phenotypically wild-type

To obtain further information concerning the molecular lesion in immutans, we performed Northern hybridization experiments to assess the steady-state levels of representative mRNAs from plastid and nuclear genes in the white and green sectors of mature leaves of the mutant. These included two nuclear genes for chloroplast proteins, cab (for the light-harvesting chlorophyll a/b-binding proteins of photosystem II) and rbcS (for the small subunit of ribulose bisphosphate carboxylase, or Rubisco); and three chloroplast genes, psbA (for the 32 kD 'DI ' protein of photosystem il), psaA (for the P700 chlorophyll a-binding protein of photosystem I), and rbcL (for the large subunit of Rubisco). The cytoplasmic 18S rRNA and plastid 16S rRNA species served as controls. Spectrophotometric analyses revealed that there was less than 5% contamination of the white sectors by chlorophyll-containing plastids in the im plants used in these experiments (data not shown).


As illustrated in Figure 7, all of the nuclear and plastid RNAs accumulate to a similar extent in the green sectors of the mutant and in wild-type leaf tissue. This supports the data from the pigment (Figure 4) and electron microscopic (Figure 2) analyses showing that cells in the immutans green sectors are phenotypically indistinguishable from cells in wild-type plants. In contrast, the levels of these RNAs (with the exception of the 18S rRNA) are reduced in the white tissues. Phosphorlmage analyses of replicate Northern filters revealed that the three plastid mRNAs are reduced in amount by about 50% in the white tissues (an average of 52% for psbA, 57% for psaA, and 50% for rbcL), whereas rbcS and cab transcript levels are reduced an average of 50% and 80%, respectively. The level of the plastid 16S rRNA is also decreased in the white tissues (~25%), while the fraction of the rRNA pool occupied by the 18S rRNA species is increased by a corresponding amount in these tissues. The increase in the relative abundance of the 18S rRNA species in the white tissues is expected due to the decrease in the relative abundance of 16S rRNA species, since equal amounts of RNA were electrophoresed in each gel lane in these experiments.

Increased acid ribonuclease in immutans white tissue is a secondary effect

In early studies of immutans, R~dei and co-workers found that the activity of a cytoplasmic RNase is enhanced in the mutant plants, and he suggested that the primary lesion in the mutant resides in RNA metabolism (R&dei, 1967a, 1975). However, consistent with the observation that the relative activities of various cytoplasmic enzymes are increased in leaf cells containing white plastids (Oelm011er, 1989), the alterations noted by Redei may be a secondary effect of the mutation. Using a substrate-based gel assay, we examined relative RNase activity in variegated (approximately 50% white) leaves of immutans as compared with wild-type leaves. Figure 8 shows the results of these assays carried out at two pH levels. Table 4 contains the results of densitometric quantitation

Figure8. SDS-PAGE analysis of leaf RNases. Soluble protein extracts were etectro- phoresed and stained for RNase activity as described in Experimental procedures. Lanes were loaded on an equal fresh weight basis for each comparison. Molecular mass markers are shown to the right of the gels. (a) Comparison of wild-type (WT) and variegated immutans (ira) leaves at pH 5.0 and pH 7.5. (b) Comparison of Norflurazon-treated (+NF) and water-treated control (-NF) wild-type plants at pH 5.0 and pH 7.5.


Table 4. Quantitation of RNase levels of leaf soluble protein extracts

Sample pH

Mean total band density + standard error

(O.D. x ram) (a) ~

Wild-type 5.0 4.1 _+ 0.01 immutans 5.0 5.4 + 0.03

WUd-type 7.5 2.4 + 0.20 irnmutans 7.5 2.9 _+ 0.20

Student's t-test (t, df; p-value)

4.35, 6; p< 0.01

1.71,6; 0.10 <,o< 0.30

(b) b Wild-type 5.0 3.6 + 0.00 Wild-type + Norflurazon 5.0 4.4:1:0.10

Wild-type 7.5 2.4 + 0.05 Wild-type + Norflurazon 7.5 2.2 + 0.05

9.02, 2; p< 0.01

2.80, 2; 0.10 <p< 0.30

RNase activity gels, as represented in Figure 8, were quantitated by scanning densitometry and analyzed by Student's t-test. a Wild-type chlorophyll content = 770 ~g chl. per g tissue; immutans chlorophyll content = 275 p.g chl. per g tissue. b Wild-type chlorophyll content = 812 pg chl. per g tissue; wild type + Norflurazon chlorophyll content -- 162 p.g chl. per g tissue.

of the RNA depletion, calculated as the sum total of all bands in a gel lane. Means of replicate lanes were determined and analyzed by the Student's t-test. It is clear from Figure 8 and Table 4 that white immutans leaves have a higher level of acid RNase activity than the wild-type leaves, as detected by degredation of gel-based RNA. No significant difference in the basic pH RNase levels was observed between the tissue types. Similar results were obtained upon repetition of the protein extractions and assays (data not shown). These results are in agreement with those reported by R6dei. However, to carry our analysis one step further and determine if the altered RNase activity is merely due to a secondary effect of putative photooxidation, we also examined the RNase levels of wild-type plants that had been treated with Norflurazon. Norflurazon is a herbicide that blocks carotenoid biosynthesis at the phytoene desaturation step and causes photobleaching of leaves (Bartels and McCuUough, 1972; Blume and McClure, 1980; Sandmann and Albrecht, 1990). As the results in Figure 8 and Table 4 indicate, acid RNase is increased in Norflurazon-treated plants over control levels, while the basic pH RNase activities are not altered by the treatment.

D i s c u s s i o n

immutans is a novel type of 'Plastid Autonomous' variegation mutant

We have undertaken an examination of the immutans variegation mutant to gain insight into the mechanisms

that regulate nuclear-organelle interactions. Contrary to prior investigations (Rddei, 1967a; R6bbelen, 1968), we found that the white sectors of im contain cells that are heteroplastidic for defective and normal plastids. This indicates that the mutation is 'plastid autonomous' and has an unequal effect on the plastids within a cell. We also found that the mutation is not maternally inherited, indicating that plastid and mitochondrial DNAs are unaltered in the mutant. Although the phenomenon of heteroplastidic cells appears to be rare in plants (Tilney-Basset, 1984), the occurrence of such cells has been documented in a number of variegated 'plastome' mutants that contain lesions in their plastid DNA, as well as in some variegation mutants that arise due to nuclear gene-induced organelle DNA mutations (Hagemann, 1986; reviewed in Stubbe and Herrmann, 1982). As first suggested by Baur in his classical experiments with Pelargonium in the early 1900s, it is thought that the plastids in the 'mixed' cells of these mutants are in the process of sorting-out to generate pure sectors of white and wild-type tissues (reviewed in Gillham et al., 1991). In all reported cases, however, the abnormal plastids in these mutants are permanently defective due to mutations in the organellar DNA (an 'intrinsic' plastid factor), and the defect is consequently maternally inherited. Our observation that im contains heteroplastidic cells, but that the defective plastids do not arise via mutations in the organellar DNA, indicates that the expression of immutans is plastid autonomous due to an 'extrinsic', nuclear-cytoplasmic factor, rather than to an 'intrinsic' organellar factor (such as plastid DNA). immutans thus represents a novel type of nuclear-gene

induced variegation mutant. Rhoades in 1943 first suggested the possibility that plastids within a cell may respond differently to cytoplasmic factors (reviewed in Granick, 1955). It is now possible to begin to explore the underlying mechanisms of this phenomenon.

The white tissues of immutans are impaired in carotenoid biosynthesis

All of the steps of carotenoid biosynthesis occur in plastids, although the genes for the requisite enzymes are located in the nuclear genome (reviewed in Kleinig, 1989; Sandmann, 1991). Little is understood about the regulation of this pathway because few genes involved in carotenogenesis have been isolated (e.g. Bartley eta/., 1991b; Bird et al., 1991; Buckner eta/., 1990; Pecker et al., 1992; Ray et al., 1992; Scolnik and Bartley, 1993). Genetic approaches have proven successful in isolating structural genes for carotenoid biosynthetic enzymes in several non-plant species (e.g. photosynthetic bacteria) (Sandmann, 1991), but molecular analyses of higher plant carotenoid-deficient mutants have been complicated by the poor survival of albinos and by difficulties associated with cloning genes for which only a mutant phenotype is known. In fact, the yl mutant of maize is the only carotenoid-deficient mutant for which the mutant gene has been isolated and sequenced (Buckner et al., 1990).

We have shown by spectral and chromatographic analyses that the green tissues of immutans accumulate a normal complement of pigments, but that the white tissues accumulate the colorless C40 carotenoid intermediate, phytoene. This suggests that the white tissues lack activity for phytoene desaturase. Therefore, the simplest hypothesis to explain the primary lesion in immutans and the phenotype of white tissues starts with the assumption that the IM gene product directly affects PDS expression, either at the level of the PDS gene or gene product. In this study we have taken advantage of the relative ease of genetic manipulation of Arabidopsis to test whether im is the structural gene for phytoene desaturase. We found that PDS does not map to the im locus, indicating that im does not code for PDS. The validity of this conclusion rests upon the assumption that PDS is a single-copy gene in A. thaliana. Although the duplication of biosynthetic genes is a common phenomenon in plants, we were not able to detect more than one PDS band on genomic Southerns of A. thaliana DNA under low-stringency hybridization conditions, or in multiply redundant screens of an A. thaliana genomic library at low stringency (data not shown). This is in contrast to reported cases of redundant genes in A. thaliana, such as nitrate reductase (Cheng et aL, 1988) and tryptophan synthase [[3 (Last et aL, 1991), where the alternate genes are highly homolo- gous. This suggests that no other related genes with phy-


toe0e desaturase activity exist in the Arabidopsis genome. Consistent with our data, PDS has recently been found to be a single-copy gene in tomato (Hirschberg, personal communication).

While im does not appear to code for PDS, it is still possible that im may affect PDS expression directly. For example, it may be a regulatory factor of the PDS protein; it may be required for the stability of the PDS protein in the chloroplast envelope; or it may be a transcription factor that regulates the expression of the PDS gene. An examination of PDS expression in immutans may provide some insight into which, if any, of these hypotheses is correct. However, these analyses are complicated by our finding that PDS mRNAs from A. thaliana leaves are below the limit of detection in Northern blot analyses (unpublished data), which suggests that PDS expression may be very low in Arabidopsis leaves. This is indeed the case in tomato leaves, where quantitative RT-PCR methods were required to detect PDS mRNA (Giuliano et al., 1993; Pecker eta/., 1992). We are currently designing a quantitative RT-PCR assay for A. tha/iana PDS mRNA.

Unfortunately, due to the conditional light-sensitive nature of the im mutation, it is not possible to test whether im has a direct effect on carotenoid biosynthesis, other than by measuring the levels of biosynthetic intermediates. Carotenoids serve primarily as photo-protective agents in plants, and in the absence of these pigments, non-quenched oxygen radicals are generated by triplet state chlorophyll under normal-to-high light conditions, causing photooxidation of chloroplast components (reviewed in Oelm011er, 1989). Under low light conditions, an absence of carotenoids does not result in photooxidation because excess light is not absorbed by chlorophyll. In fact, the classical test for a carotenoid biosynthetic mutant is that it is capable of forming chlorophyll (in the absence of carotenoids) when germinated under low light conditions (e.g. Robertson et al., 1978); under these conditions the light intensity is not high enough to photo- oxidize plastid contents, including chlorophyll.

In contrast to other carotenoid biosynthetic mutants, immutans produces only green cotyledons with the full complement of carotenoids and chlorophylls under low light conditions; no true leaves form in low light (unpublished data). When germinated in high light, however, immutans produces all-white cotyledons that soon die. Variegated leaves containing white sectors (lacking carotenoids and chlorophyll) and green sectors (containing carotenoids and chlorophyll) only appear when im seedlings are germinated and grown under moderate light conditions, or when low light-germinated seedlings are shifted to growth in moderate-to-high light conditions. In this context, it should be pointed out that plastid-type appears to be irreversible in the cotyledons and fully expanded leaves of im plants, because plastid pheno-


types and pigment compositions do not change in these organs when plants are shifted from high-to-low light (or vice versa)---e.g, white tissues do not produce chlorophyll when shifted from high-to-low light (unpublished data). Therefore, in contrast to various albino mutants with lesions in carotenogenesis, we have not been able to identify a condition where im makes chlorophyll and not carotenoids.

In support of the idea that im has a defect in carotenoid biosynthesis, our ultrastructural and Northern analyses are consistent with the hypothesis that the white tissues of im are photo-oxidized. As in other species (reviewed in Susek and Chory, 1992), the plastids in the white tissues lack organized lamellar structures and contain reduced amounts of various plastid rRNAs and mRNAs. The levels of rbcS and cab mRNAs are also reduced in these tissues, consistent with the 'plastid signal' hypothesis that the transcription of nuclear genes for plastid proteins is dependent on a signal that requires a functional chloroplast either for its transmission or generation (Susek and Chory, 1992; reviewed in Taylor, 1989). On the other hand, our observation of heteroplastidic cells in the white sectors of im raises the question whether cells in the white tissues represent various stages of plastid photooxidation. Alternatively, these cells could arise due to a complicated secondary effect of #77 on a process such as plastid membrane assembly or quinone biosynthesis (e.g. Schulz et aL, 1993), generating cells with plastids arrested in various early stages of chloroplast development.

Regardless of the precise mechanism, our observation that phytoene accumulates in the white sectors of immutans indicates that the im mutation affects carotenoid biosynthesis, either directly or indirectly. Therefore, an understanding of the molecular lesion in this mutant will provide great insight into the poorly understood mechanisms that regulate carotenoid biosynthesis in higher plants. In this context, it is worth noting that the phenotype of immutans is similar to the well-known ghost variegation mutant of tomato, which also accumulates phytoene in its white sectors (Giuliano and Scolnik, 1988; Giuliano et aL, 1993; Scolnik etaL, 1987). In contrast to immutans, however, ghost is homoplastidic for abnormal plastids in its white tissue.

Increased acid ribonuclease in immutans is a secondary effect of photo-oxidation

Early work on immutans led to the postulation that RNA metabolism may be affected by the mutation. This idea was based on the observation that immutans leaves had greater acid ribonuclease activity than wild-type plants, measured as activity in total soluble protein (Redei, 1967a, 1975). Our results support this observation, and

show that the increase is also apparent when measured on an equal fresh weight basis. The latter is important because several soluble proteins decrease in relative abundance in white tissues (Mayfield et aL, 1986; Oelm~ller, 1989; Tonkyn et aL, 1992), thus potentially leading to a proportional increase in RNase when it is measured in total soluble protein. We found that a similar increase in acid RNase activity occurs in wild-type tissue that has been photobleached by treatment with Norflura- zon. Norflurazon specifically inhibits phytoene desaturase and causes accumulation of phytoene with a concurrent decrease in production of colored carotenoids (Bartels and McCullough, 1972; Blume and McClure, 1980; Sand- mann and Albrecht, 1990). Under normal to high light conditions, treated leaves photobleach because of the lack of carotenoids. We conclude that the increase in RNase in white tissue of immutans is most probably due to secondary effects of photo-oxidation and not to a specific effect of the lesion in the mutant, because the measured increase of RNase in variegated immutans leaves is similar to that in the Norflurazon-treated leaves. This observation is interesting in itself because it indicates that there may be a role for acid ribonuclease in photo-oxidized tissue.

Mechanism of immutans variegation

The primary focus of the present study was to characterize the variegated tissue sectors of immutans. However, it is difficult to separate the primary lesion in the mutant from the conditional nature of its unstable expression. How can a factor that affects carotenoid accumulation and that is required for normal chloroplast differentiation give rise to this variegation?

Any model of the #77 variegation must take into account the conditional nature of the mutation and its dependence on light and temperature, both of which influence the amount of white-sectoring in the mutant. As mentioned above, we have found that plastid-type appears to be irreversible in the cotyledons and fully expanded leaves of im plants. Clonal and positional effects on gene expression can be readily studied in monocots because of their leaf developmental morphology. However, similar analysis is hindered in A. thaliana because the precise lineage of cells is difficult to determine. The variegation in both cotyledons and leaves of immutans has no discernible consistent pattern. Our light-shift experiments show that plastid type in immutans cotyledons is determined by the light environment experienced by the germinating seedling immediately following seed coat breakage. Since the cotyledons of dicots are undergoing very little cell division, but primarily cell expansion at this stage (Lovell and Moore, 1970; Scott and Possingham, 1983), this suggests that the pattern of variegation in immutans

Figure 9. Proposed model of immutans action. See text for details.

cotyledons is not primarily influenced by cell lineage. Unfortunately, similar experiments cannot be performed on maturing leaves of immutans because the level of light perceived relative to the timing of cell differentiation in leaf primordia is highly variable, depending on such factors as anatomy and orientation relative to the light source (Nishio et aL, 1993). Sectoring in the leaves could result from small patches of neighboring primordial cells experiencing similar light levels during their critical phase, and subsequently growing and expanding into larger patches of a common phenotype in the mature leaf.

Taking into account these considerations, as well as the observations in this paper, our current working hypothesis of im variegation is illustrated in Figure 9. There are several assumptions of our model:

(i) The/Mgene product directly or indirectly influences the accumulation of a plastid product that is required for


normal chloroplast differentiation. (For the sake of sim- plicity, we assume that this product is the IM protein.)

(ii) The IM protein is required at a threshold concentration for activity.

(iii) The concentration of IM protein per plastid normally varies as a function of cell-specific IMexpression (e.g. due to endogenous developmental factors, as well as to exogenous factors, such as hormones and light). IM protein concentration per plastid also varies as a function of unequal partitioning during plastid division.

(iv)The mutant IM protein is temperature labile and susceptible to photodama~;c c.g. by direct interaction with electrons, analogous to the 'DI' protein in photosystem II (Krause and Weis, 1991), or by redox interactions coupled to photosynthetic electron flow via a thioredoxin-like component.

(v) The proportion of the mutant IM protein pool that is inactivated depends on the light and/or temperature perceived by a given plastid and/or cell.

Given these assumptions, we would anticipate that only a small proportion of the mutant IM protein pool would be inactivated under low light conditions, and cells with low and high levels of the mutant IM protein would contain the threshold amount required for activity and normal chloroplast differentiation (green sectors in low light). In contrast, a larger fraction of the mutant IM protein pool would be inactivated under high light conditions. In cells with low amounts of the mutant IM protein, we would expect that subthreshold amounts of active protein would not support normal chloroplast development, resulting in colorless plastids (white sectors in high light). However, threshold amounts of active protein would still be obtained in cells with high concentrations of the protein (green sectors in high light). This model explains the sectoring in the mutant, as well as our observation of heteroplastidic cells in the white sectors.

Studies on the light regulation of anthocyanin variegation in maize vegetative organs (Cone et aL, 1993a, 1993b; Cocciolone and Cone, 1993) and in white mustard cotyledons (Nick et aL, 1993) provide biological precedent for the model proposed for immutans pigmentation variegation. In maize, light induces a regulatory gene that in turn activates transcription of anthocyanin biosynthetic genes if its product is present above a threshold level (Cone et aL, 1993a, 1993b; Cocciolone and Cone, 1993). There is some indication that methylation of the regulatory locus is involved with its transcription level, and that the degree of DNA methylation in a given cell may be determined stochastically, resulting in cell-to-cell variation and ultimately in variegated leaves. Nick et aL (1993) invoke the heterogeneity of cells within an organ to explain the patchiness of anthocynanin production in white mustard cotyledons as a result of various light treatments. The light


perceived by an individual cell in this heterogeneous population of cells is the overriding determinant in pigmentation pattern, according to their model.

Our ability to test the immutans variegation model will require cloning the gene and characterizing its expression in mutant and wild-type plants. Nevertheless, our finding that immutans is plastid autonomous and impaired in carotenoid biosynthesis makes this mutant an attractive system to investigate nuclear-organel le interactions. The information we can gain from a study of immutans should therefore help elucidate general patterns of gene expression and intracellular communication during tissue and organ development.

Experimental procedures

Plant material

The allele of immutans used in the present experiments (irn- 'spotty') was isolated following ethyl methane sulphonate (EMS) mutagenesis of wild-type seeds from the Columbia ecotype; this allele has been simply designated 'ira' in the present discussion. The mutant plants were beck-crossed several times, and comple- mentation tests were performed to verify that this mutation is allelic to other immutans alleles. Specifically, im- 'spotty' did not complement the EMS-induced allele im-52 (provided by J.M. Martinez-Zapater), nor the X-ray induced allele im gi 2 (provided by G.P. Redei). Reciprocal crosses between #77 and Columbia wild-type were performed to test for inheritance of the variegation. Wild-type and im plants were grown at 25°C under a mixture of fluorescent and incandescent lights, and illuminated continuously at a constant intensity in the range of 35 to 100 p.mol m -2 sec -1, These conditions promote the formation of large white leaf sectors, in contrast to lower light intensities, which promote the formation of green sectors.

Determination of the light responsive phase of im expression

Immutans seeds were spread in petri plates on Murashige and Skoog basal salt mixture pH 5.7, with 50 seeds per plate and two replicates per light treatment. Plates containing wild-type Colum- bia seeds were used as controls. Each plate was placed in a 21 °C growth chamber with continuous light provided by a mixture of fluorescent and incandescent bulbs, either at 5 ~mol m -s sec -~ or at 100 ~mol m -2 sec -~. At 24 h intervals a given plate was either left at its current light level or moved to the alternate light level, according to the pattern described in Table 2. By monitoring the germination of individual seeds, it was observed that almost all seed coats were broken open by 48 h after plating, regardless of the light treatment; this represents the time when the emerging cotyledons are first exposed to white light. Therefore, seeds with no seed coat breakage at 48 h were marked and eliminated from the experiment. The seedlings were left to grow at their day 4 light level (72-96 h after plating), and final observations were made 10 days after plating.

Microscopy

Samples for electron microscopy were obtained from expanding leaves of wild-type and immutans plants. Both sets of plants were

maintained under identical growth conditions (35 I~mol m -2 sec -~) and harvested 3 weeks after germination (6-7 leaves per rosette). Small leaf punches were obtained from visually white or green sectors of the mutant and from wild-type leaves. The samples were fixed, stained, and examined as in Homer and Wagner (1980). Light and chlorophyll fluorescence microscopy was carried out on an Olympus Vanox-T research microscope. Fluores- cence emission was passed through a 515 nm cutoff filter, with excitation light of 380-490 nm (475 nm peak). Fresh variegated leaves were placed (adaxial side up) on slides with water and a cover slip for viewing. The photographed focal plane was in the mesophyll layer of the leaf; plastids in this tissue area were the only ones in focus, thus visual 'contamination' from other tissue or cell layers was minimal. All photos were taken within 30 min of slide preparation to minimize artifacts due to cell damage.

HPLC analysis of leaf pigments

Pigments were extracted (modified from the procedure of Almela et al., 1992) from leaves of wild-type plants, from green sectors and visually white sectors of immutans plants, and from photobleached leaves of wild-type plants that had been treated with Norflurazon (see Ribonuclease measurements). Samples of fresh leaf tissue (0.1 g) were frozen in liquid nitrogen, ground to a powder, and homogenized in 2 ml of 100% acetone in the presence of sodium ascorbate. Sodium ascorbate prevents artifactual pheophytin formation during the extraction procedure (Valet aL, 1986). The extracts were centrifuged (1500 x g, 5 min) and the supernatants were dded under nitrogen gas. The residues were dissolved in 100% methanol. During initial analyses, all tissue types were dissolved in 200 I~1 of methanol, but this resulted in too high a concentration of pigments in the green samples for effective separation and identification. For subsequent analyses, the white tissue extracts (immutans and Norflurazon-treated wild- type) were dissolved in 200 Id, while the green tissue extracts (wild-type and immutans) were dissolved in 1 ml of methanol. Each sample was passed through a C18 Sep-Pak filter (Waters Assoc., Watertown, MA) before HPLC analysis. All extraction operations were performed in dim light using precooled (0°C) equipment.

Chromatographic analysis was performed according to the procedure of Wright and Shearer (1984), using a Hewlett- Packard HP-1090 liquid chromatograph equipped with a photodiode array detector and a 25 x 0.46 cm Alltech C18 reverse- phase column. Each extract was injected (25 pl) into the column and then eluted using a linear gradient from 100% solvent A (90% acetonitrile) to 100% solvent B (100% ethyl acetate) over 25 min, with a flow rate of 1 ml min -~. The column was washed between samples by running a blank (methanol injection) gradient. Each elution was simultaneously monitored at 286 nm and 430 nm, while the photodiode array detector measured the full absorbance spectra for later computer-aided analysis. The peaks were identified by comparison of the absorbance spectra and the relative retention times with published values (Davies, 1976; Wright and Shearer, 1984). The identity of the phytoene peak was verified by comparison of the spectra with that of the Norflurazon- treated sample. Standards for phytoene are not commercially available, so Norflurazon-treated tissue is commonly used for this purpose (i.e. Mayer etaL, 1990).

Quantitation of chlorophylls, beta-carotene, and phytoene

Analyses of chlorophyll and carotenoid content of leaf samples excised from white im sectors, green im sectors, and wild-type

plants were carded out by procedures described in Holden (1976) and Davies (1976). In brief, leaf samples from totally white im sectors, totally green im sectors, and wild-type plants were excised and extracted two times in the dark at 4°C with a mixture of 50% (v/v) ethyl ether and methanol. Following each extraction, the samples were centrifuged (1500 x g, 10 min), and the supernatants were dried under N2gas and resuspended in 1.0 ml petroleum ether (bp 37.5- 57°C). To quantitate pigment contents, the petroleum ether extracts were chromatographed on thin-layer silica gel-plates (60 A pore size, 250 mm layer thickness, Fisher Scientific) with 0.5% (v/v) acetone in petroleum ether (bp 37.5-57°C) as the solvent (Giuliano et al., 1986). Bands containing chlorophylls a and b, beta-carotene, and phytoene were scraped from the plates and extracted in 80% acetone (for the colored pigments) or hexane (for phytoene); after centrifugation, absorbances of the supernatants were determined. Pigment contents (l~g pigment per g fresh weight of tissue) were calculated using equations and extinction coefficients given in Davies (1976) and Holden (1976), with the exception that a specific extinction coefficient of 2293 at 480nm was used for beta- carotene in 80% acetone (Ben-Amotz and Avron, 1983).

Molecular analyses

A phytoene desaturase (PDS) gene sequence was obtained by PCR amplification (Ausubel et al., 1987) of a soybean cDN~ library (provided by Dr R. Shoemaker, Iowa State University) using two oligonucleotides generated to nucleotides 1335-1354 and nucleotides 1644-1663 of the soybean PDS cDNA sequence (Bartley et al., 1991b). The amplified fragment was cloned and sequenced (Ausubel et al., 1987), then used as a probe to identify PDS-oontaining clones from an Arabidopsis thaliana lambda phage library constructed from Landsberg ecotype DNA (Voytas et aL, 1990). Several clones were isolated and mapped (Ausubel et aL, 1987), and a 2.0 kbp subfragment that hybridized to the soybean PDS probe in Southern blotting experiments (Ausubel et al., 1987; Church and Gilbert, 1984) was cloned and partially sequenced. The A. thaliana PDS clone was used to probe a blot of genomic DNA from Landsberg and Columbia ecotypes that had been cut with different restriction enzymes (Figure 5). South- ern hybridizations were carried out under high-stringency conditions (65°C; 7% SDS, 0.5 M NaPO4 pH 7.2, 1 mM EDTA, 1% BSA) and low-stringency conditions (55°C, same buffer).

RNA isolation and Northern (RNA) hybridization procedures have been described (Rodermel et aL, 1988). The probes used in the present hybridizations included an A. thatiana cab gene, AB140 (Leutwiler et aL, 1986); an A. thaliana rbcS gene, pATS22 (Krebbers et al., 1988); a soybean 18S rRNA gene, pSRI.2B3 (Shirley and Meagher, 1990); a tobacco rbcL gene, pTB5 (Roder- mel et aL, 1988); and the maize psbA, psaA and 16S rRNA genes in plasmids pZmc427, pZmc556, and pZmc518, respectively (Rodermel and Begorad, 1985). Following hybridization, the relative amounts of transcription products on the Northern hybridization filters were quantified using a Molecular Dynamics Phosphorlmager 400E (Sunnyvale, CA).

Genetic analyses

To determine whether PDS maps to the im locus on chromosome 4, a homozygous immutans plant (ecotype Columbia) was crossed to a chromosome 4 marker line carrying the recessive mutations ap2, cet'2, and bp (ecotype Landsberg) (Koornneef, 1987). The resulting F~ plants were selfed and the F2 population


was screened for recombinants. The genotype of recombinant plants was verified in the F3, and DNA was prepared from pooled F3 plant families (Doyle and Doyle, 1990).

Ribonuclease measurements

Variegated immutans leaves (approximately 50% white) and wild- type leaves were harvested at maturity and ground in liquid nitrogen. Buffer (10 p.1100 p.g-~ tissue) containing 250 mM NaPO4 pH 7.2, 5 mM EDTA, 4 mM PMSF, 25 pg m1-1 leupeptin, and 25 lig m1-1 antipain (Green, personal communication) was added to each sample. Chlorophyll was measured in the homogenate (Arnon, 1949). Soluble protein was extracted according to Yen and Green (1991). The protein samples were run on SDS-PAGE gels (loaded with extracts of equal tissue fresh weight per lane) containing purified torula yeast RNA, and were assayed for RNase activity at two different pH levels, 5.0 and 7.5 (Yen and Green, 1991 ), with an assay incubation time of 30 rain at 51 °C. Separate assays were run to ascertain that this incubation time was in the linear range of substrate depletion in the gel. The gels were stained in toluidine blue, destained, dried in cellulose, and used for scanning densitometric measurement (Quantity One version 2.2, pdi, Inc.) of RNase depletion of gel-based RNA. The desitometer curve integration for each band in a lane was summed over the lane, providing a total RNase value for a given sample and pH. Replicate samples were run on the same gel.

Photobleached leaves were obtained by treating wild- type plants with 5 pM Norflurazon (Sandoz 9789; 4-chloro-5- (methyl-amino)-2-(c~,(x,a-trifluoro-m-tolyl)-3(2H)-pyridazinone). Control plants were treated with water. Leaves were harvested and assayed for RNase activity as described above.

Acknowledgments

The authors wish to express their appreciation to Drs Joanne Chory (Salk Institute), Sabeeha Merchant (UCLA) and Martin Spalding (Iowa State) for careful review of the manuscript; to Drs G.P. Redei and Fred Ausubel for providing A. thaliana seeds; to Dr Pamela Green (Michigan State) for helpful discussion about plant ribonucleases; to Charles Rose (Iowa State) for assistance with HPLC analysis of pigments; and to Drs Jack Homer and Bruce Wagner of the Bessey Microscopy Facility (Iowa State) for electron microscopy. This work was supported by grants from the Iowa State Biotechnology Council and DOE (DE-FG02-94ER 20147) to S.R.R. and by a NIH Postdoctoral Fellowship (GM-16487) to C.M.W. Journal paper no. J-15869 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 2987.

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Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous...

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