Organ Specificity and Light Regulation of NADH-Dependent. Hydroxypyruvate Reductase Transcript Abundance1. John McC. Greenler2 and Wayne M. Becker*.
Plant Physiol. (1990) 94, 1484-1487 0032-0889/90/94/1 484/04/$01 .00/0
Received for publication July 2, 1990 Accepted July 29, 1990
Communication
Organ Specificity and Light Regulation of NADH-Dependent Hydroxypyruvate Reductase Transcript Abundance1 John McC. Greenler2 and Wayne M. Becker* Department of Botany, University of Wisconsin, Birge Hall, Madison, Wisconsin 53706 ABSTRACT
to increase in parallel with HPR enzyme activity. Data obtained by in vitro translation of poly(A)+ RNA followed by immunoprecipitation with HPR antiserum indicated that the rise in HPR protein depends on an increase in translatable HPR mRNA. HPR transcript levels in cotyledons are regulated both developmentally and by light (9). After 3 to 4 d of seedling development, HPR transcripts become detectable in cotyledons. At this point, they either rise rapidly in the light or remain at a low level in the dark. This pattern is similar to that seen for HPR enzyme activity, HPR protein, and HPR translatable mRNA (1, 1 1). In this report we show that HPR transcript abundance in cucumber seedlings is tissue-specific and strongly regulated by light in leaves.
By probing total RNA blots with an NADH-dependent hydroxypyruvate reductase (HPR) cDNA clone, we have found that HPR transcript abundance is highly regulated in developing cucumber (Cucumis sativus) seedlings. HPR transcript levels in light-grown seedlings are 50-fold more abundant in leaves and cotyledons than in roots. When 12-day light-grown seedlings are shifted to the dark for 4 days, the HPR transcript level in leaves drops 20fold. Upon return of these dark-adapted plants to the light, HPR transcript levels rise to 50% of the previous light-grown level within 2 hours.
MATERIALS AND METHODS
Cucumber seedlings provide a useful model system for the study of the regulation of genes encoding photorespiratory enzymes. The seedling functions heterotrophically for the first several days, utilizing stored triglycerides in the cotyledons as a carbon and energy source. After 3 to 4 d, the cotyledons emerge into the light and enzymes of photosynthesis and the photorespiratory pathway become detectable (1). After 8 to 10 d of growth in the light, the first true leaves begin to develop. HPR3 catalyzes the reduction of hydroxypyruvate to glycerate with the simultaneous oxidation of NADH to NAD+. This reaction is part of the photorespiratory pathway and is localized in leaf peroxisomes (16, 17). The appearance of HPR in the cotyledons of cucumber seedlings is both developmentally and light-regulated (1 1). This pattern is seen for other peroxisomal enzymes, including serine:glyoxylate aminotransferase ( 1) and glycolate oxidase ( 18). The response of peroxisomal enzyme synthesis to light is regulated at least partially by phytochrome (13), although other photoreceptors have also been implicated (6, 14, 15). Previous research in our laboratory has shown that the rise in HPR activity in the cotyledons of greening cucumber seedlings is regulated at a pretranslational level (1 1). Using an immunoprecipitation assay, HPR protein levels were found
Sources
Cucumber seeds (Cucumis sativus cv Improved Long Green) were purchased from L. L. Olds Seed Co., Madison, WI. Unless otherwise noted, all enzymes were from New England Biolabs, all Escherichia coli strains and vectors were from Stratagene Cloning Systems, and all reagents were from Sigma Chemical Company. Growth, Harvesting, and Preparation of Plant Material Cucumber seedlings were germinated and grown either in continuous white light or in darkness at 24 to 26°C on vermiculite saturated with Hoagland solution (10). Harvesting of cotyledons and preparation oftissue homogenates for RNA analysis were as described previously (1 1). RNA Blot Analysis
Total RNA was prepared from cucumber seedlings by homogenization in 4 M guanidine thiocyanate and ultracentrifugation through a discontinuous CsCl gradient (1 1). Total RNA (15 ,ug) was fractionated through 1.2% agarose gels containing 2.2 M formaldehyde, transferred to nitrocellulose or Nytran (Schleicher and Schuell) by blotting, hybridized at 42°C in 50% (v/v) formamide, and washed at high stringency (final wash: 0.1 x SSC and 0.1% SDS at 65°C), all according to the protocol of Schleicher and Schuell. These blots were probed with a[32P]dATP-oligolabeled HPR H18 cDNA (7, 9). The relative amount of transcript level in each RNA sample was determined by cutting out the area on the blot
'This research was supported by National Science Foundation grant DCB-8509852 to W. M. B. 2 Present address: Center for Biology Education, B37 Russell Laboratories, University of Wisconsin-Madison, Madison, WI 53706. 3 Abbreviations: HPR, NADH-dependent hydroxypyruvate reductase; SSC, 0.15 M sodium chloride/0.0 15 M sodium citrate solution (pH 7.0).
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for roots. This high level in leaves is similar to that found in the cotyledons of 5-d light-grown seedlings. In cotyledons, the amount of HPR transcript per unit of total RNA in 5-d lightgrown seedlings is, in turn, 10-fold higher than in 2-d lightgrown seedlings and 4-fold higher than in 5-d dark-grown seedlings.
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RNA blot analysis of total RNA from the leaves of darkadapted plants revealed that HPR transcript abundance is light-regulated in leaves. As shown in Figure 2, the amount of HPR transcript drops significantly when 12-d light-grown plants are transferred to darkness for 4 d. However, the abundance of transcript rapidly returns to, in fact surpasses, previous levels when plants are returned to the light. Quantification revealed that the HPR transcript level drops about 20-fold when plants are moved to darkness (Fig. 3). Upon return of the plants to light, the amount of HPR transcript increases within 2 h to approximately 50% of the original light-grown level. By 24 h after return to the light, the abun-
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Figure 1. Organ specificity of HPR transcript abundance in cucumber seedlings. Cotyledons were harvested from seedlings grown for 2 or 5 d in either the light (L) or the dark (D), whereas leaves and roots were harvested from 12-d light-grown seedlings. Total RNA (15 ,g) was isolated from cotyledon (cot), leaf, or root tissue and subjected to electrophoresis through agarose/formaldehyde and blotted to nitrocellulose. HPR transcript was detected by probing the blot with a[32P]dATP-labeled HPR H18 cDNA. The blot is shown in the inset. Transcript levels were quantified by excising the bands from the blot and determining the amount of bound radioisotope by liquid scintillation spectrometry. All values were normalized to the peak value (cotyledons from 5-d light-grown seedlings = 100 units) and are therefore expressed as relative units of transcript. The bars represent the average values from three separate blots of the same RNA
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corresponding to the visible band on an autoradiogram of the blot and quantifying bound radioisotope by liquid scintillation spectrometry. The relative HPR transcript in each RNA sample was calculated from the average of three replicate blots. RESULTS
Organ Specificity of HPR Transcript Abundance The relative abundance of HPR transcript in different organs of cucumber seedlings was determined by RNA blot analysis (Fig. 1). An equal amount of total RNA (15 ,g) from cotyledon, leaf, or root tissue was loaded in each lane. As shown in the inset, the same size transcript was detected in each organ. The accumulation of HPR transcript in cucumber seedlings is clearly organ specific. Under the growth conditions of this experiment, the amount of HPR transcript in 12d light-grown seedlings is about 50-fold higher for leaves than
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Days of Development Figure 2. HPR transcript in the leaves of dark-adapted seedlings. Cucumber seedlings were grown for 12 d in white light, transferred to the dark for 4 d, then returned to the light for up to 24 h. RNA blot analysis as described in the legend for Figure 1 was used to detect HPR transcript in total RNA extracted from leaves after 12 d in the light (lane 1), after 4 d in the dark (lane 2), and at 1, 2, 4, 8, and 24 h after return to the light (lanes 3-7, respectively).
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