Wayne M. Becker

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds.

SummarySunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.

Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber

A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a λgt11 cDNA library made by reverse transcription of poly(A)+ RNA from cucumber cotyledons. In vitro transcription and translation of this clone yielded a major polypeptide which was identical in size, 43 kDA, to the product of in vitro translation of cotyledonary poly(A)+ RNA and subsequent immunoprecipitation with HPR antiserum. Escherichia coli cultures transformed with a plasmid construct containing the cDNA insert were induced to express HPR enzyme activity. RNA blot analysis showed that HPR transcript levels rise significantly in the first eight days of light-grown seedling development. This closely resembles the pattern seen for HPR-specific translatable mRNA. DNA blot analysis indicated that a single HPR gene is likely present per haploid genome. Nucleotide sequence analysis revealed an open reading frame of 1146 bases which encodes a polypeptide with a calculated molecular weight of 41.7 kDa. The derived amino acid sequence from this open reading frame is 26% identical and 50% similar to the amino acid sequence of the E. coli enzyme phosphoglycerate dehydrogenase, which catalyzes a similar reaction and functions in a related pathway. Statistical analyses show that this similarity is significant (z>10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.

Light-stimulated accumulation of the peroxisomal enzymes hydroxypyruvate reductase and serine:glyoxylate aminotransferase and their translatable mRNAs in cotyledons of cucumber seedlings

The development of peroxisomal enzymes in cotyledons of cucumber seedlings is strongly dependent on light. In light-grown seedlings, activities of two peroxisomal enzymes, hydroxypyruvate reductase (HPR) and serine: glyoxylate aminotransferase (SGAT), were barely detectable until three days postimbibition, after which time both activities increased rapidly and linearly for at least three days. In the dark, the activities of these enzymes increased slightly over the same time period, but only to about 5% to 10% of 7-day light-induced levels. When 51/2-day dark-grown seedlings were transferred into white light, activities of HPR and SGAT began to increase after approximately 8 h. HPR protein was shown by an immunoprecipitation assay to increase concurrently with enzymatic activity in both light- and dark-grown cotyledons. Immunoblotting results suggested that the amounts of SGAT-A and SGAT-B, the two subunits of SGAT, also developed along with SGAT activity. The relative levels of translatable mRNAs encoding HPR, SGAT-A, and SGAT-B were also light-dependent, and increased with a developmental pattern similar to enzyme activity and protein levels in light- and dark-grown cotyledons. In 51/2-day dark-grown cotyledons that were transferred to the light, translatable mRNAs for SGAT-A and SGAT-B began to increase within 1 h of illumination and continued of increase rapidly and linearly for the next 24 h in the light to a new steady-state level that was 45 times that of dark controls. Translatable HPR mRNA exhibited a biphasic pattern of accumulation, with a three-fold increase during the first 6 h of illumination, followed by an additional six-fold increase between 8 and 24 h. The accumulation of translationally active mRNA for both enzymes preceded the accumulation of the corresponding protein and enzyme activity by about 8 h. Our data suggest that the rise in enzyme activity depends on an increase in translatable mRNA for these enzymes and is regulated at a pretranslational level, most likely involving transcription of new mRNA.

A polyethylene glycoldextran procedure for the isolation of chromatin proteins (histones and nonhistones) from wheat germ

A new procedure is described for the isolation of both histone and non-histone chromatin proteins, based on a polyethylene glycol (PEG)/dextran two-phase partition system. Chromatin is solubilized in high salt (5 M NaC1) and mixed with PEG and dextran to separate proteins (partitioned into the upper, PEG-rich phase) from nucleic acid (DNA recovered almost exclusively in the lower, dextran-rich phase). The proteins are then absorbed onto Bio-Rex 70 by dialysis to low salt (0.05 M NaC1), followed by salt elution to recover first non-histone proteins (less than 0.55 M NaC1), then histones (2 M NaC1). Cross-contamination is not detectable in either group of proteins. The procedure is rapid, gentle, and lends itself well to scale-up. The proteins are kept in pH range 7.0--8.1, and are not exposed to the denaturing reagents characteristic of most preparative procedures for chromatin proteins. Though developed specifically for the isolation of proteins from chromatin of wheat germ, the procedure should be readily applicable to other sources as well. Wheat germ (isolated wheat embryos) appears to be an excellent source of chromatin proteins; 100 g yields 225--300 mg histones and 30--45 mg nonhistone proteins, depending on technique.

Characterization of genes encoding hydroxypyruvate reductase in cucumber.

Several clones corresponding to the gene encoding NADH-dependent hydroxypyruvate reductase have been isolated from a cucumber genomic library. Restriction mapping indicates the presence of two HPR genes, hpr-A and hpr-B, in the cucumber genome. Examination of the DNAs of individual plants suggests that hpr-A and hpr-B are most likely alleles at a single locus. The sequence of a 6.7 kb genomic fragment that includes the entire transcribed region, 2.2 kb of 5′ flanking sequence, and about 0.8 kb of 3′ flanking sequence reveals the presence of 12 introns in hpr-A. These introns are AT-rich relative to the exons. The donor sequence at the 5′ end of the sixth intron contains an unusual dinucleotide, GC, rather than the nearly invariant GT. Primer extension analysis maps the transcription initiation site to 61 nucleotides upstream of the translation initiation codon. An AT-rich stretch is centered at position −31 with respect to the transcription initiation site, and a potential CCAAT box is centered at position −138. Several elements that are homologous to regulatory elements of other plant genes have been identified in the flanking regions of hpr-A.

Isolation, purification and characterization of RNA polymerases from wheat germ

Abstract Two DNA-dependent RNA polymerases (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) have been isolated from wheat germ. The enzymes were solubilized by high salt extraction with sonication and were resolved by DEAE-cellulose chromatography. Enzyme I eluted at 0.11 M (NH 4 ) 2 SO 4 , was insensitive to α-amanitin, and was relatively labile. Enzyme II eluted at 0.22 M (NH 4 ) 2 SO 4 , was inhibited by α-amanitin, and was stable after DEAE-cellulose chromatography. Both enzymes were more active with denatured than native DNA as template, but differed in their relative activities with Mg 2+ and Mn 2+ and in their ionic strength requirements. The quiescent wheat embryo appears to be a rich source of RNA polymerases and their properties are similar to nuclear RNA polymerases from other eukaryotes.

INTERACTION OF DNA-BINDING PROTEINS WITH THE 5'-FLANKING REGION OF A CYTOKININ-RESPONSIVE CUCUMBER HYDROXYPYRUVATE REDUCTASE GENE

Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (−382 to −67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (−382 to −285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5′-AAATGACGAAAATGC-3′, that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5′-AAGATTGATTGAG-3′, of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.

The occurrence of microbodies and peroxisomal enzymes in achlorophyllous leaves.

SummarySeveral types of leaves of leaf parts lacking chlorophyll were fixed and embedded according to conventional procedures and examined electron-microscopically for microbodies. Comparisons of relative abundance of microbodies, plastids and mitochondria were made by computing the average numbers of organelle profiles per cell section. Similar leaves were homogenized and assayed for three enzymes characteristic of leaf peroxisomes. The localization of these enzymes in microbodies was indicated for the achlorophyllous tissues by the positive result obtained when 3,3′-diaminobenzidine was used as an electron cytochemical stain for catalase activity.Microbodies were present in all non-photosynthetic leaves or leaf parts examined, including yellowish-white segments of variegated leaves, albino leaves, and etiolated leaves of two species. In several cases, the numbers of microbody profiles per cell section were as great in the achlorophyllous leaves as in the chlorophyllous. The levels of peroxisomal enzyme activity in the yellowish-white leaves were substantial, although often not as high as in the green leaves. It was concluded that enzymatically these microbodies are probably similar to the peroxisomes characterized from chlorophyllous leaves. In the absence of the photosynthetic product, glycolate, however, it seems unlikely that the organelle is performing the same functions as in green leaves. It is also apparent that the initial formation of peroxisomes in leaves can occur when neither light nor a photosynthate such as glycolate is present as an inducer.

Particulate nature of glycolate dehydrogenase in euglena: possible localization in microbodies.

Abstract By using a gentle grinding procedure prior to differential and equilibrium centrifugation, glycolate dehydrogenase was found to be particulate in a streptomycin-bleached strain of Euglena gracilis . Its inhibition by cyanide, ability to oxidize D-lactate, and apparent insensitivity to oxygen indicate it to be the same enzyme previously characterized as a soluble enzyme in several species of green algae, including Euglena . It is speculated that this algal glycolate dehydrogenase may be localized in microbodies as is the glycolate oxidase of higher plants.

Effects of Light Fluence and Wavelength on Expression of the Gene Encoding Cucumber Hydroxypyruvate Reductase

We have investigated the regulation of cucumber (Cucumis sativus) hydroxypyruvate reductase mRNA abundance in response to white-, red-, and far-red-light treatments. Following irradiation of dark-adapted cucumber seedlings with 15 min to 4 h of either white or red light and return to darkness, the mRNA level for the gene encoding hydroxypyruvate reductase (Hpr) in cotyledons peaks in the darkness 16 to 20 h later. The response of the Hpr mRNA level to total fluence of white light depends more directly on irradiation time than on fluence rate. In addition to this time-dependent component, a phytochrome-dependent component is involved in Hpr regulation in dark-adapted green cotyledons as shown by red-light induction and partial far-red-light reversibility. Parallel measurements of mRNA levels for the ribulose bisphosphate carboxylase/oxygenase small subunit and for the chlorophyll a/b-binding protein show that Hpr is the most responsive to short (about 60 min) white- and red-light treatments and that each mRNA has a characteristic pattern of accumulation in dark-adapted cotyledons in response to light.