A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a lambda gt11 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 greater than 10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.

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The suspensor is the first differentiated structure produced during plant embryogenesis. In most angiosperms, the suspensor functions early in development to provide nutrients and growth regulators to the embryo proper. In Arabidopsis, the suspensor undergoes programmed cell death at the torpedo stage and is not present in mature seeds. We have identified at least 16 embryo-defective mutants of Arabidopsis that exhibit an enlarged suspensor phenotype at maturity. In this report, we focus on seven abnormal suspensor mutants, which define three genetic loci (sus1, sus2 and sus3). Recessive mutations at each of these loci disrupt morphogenesis in the embryo proper and consistently result in the formation of a large suspensor. Defects in the embryo proper appear by the globular stage of development; abnormalities in the suspensor are detected soon after at the heart stage. Storage protein and lipid bodies, which normally accumulate only in the embryo proper late in embryogenesis, are present in both the arrested embryo proper and enlarged suspensor. Therefore, cell differentiation in the embryo proper can proceed in the absence of normal morphogenesis, and the suspensor can be transformed into a structure with features normally restricted to the embryo proper. These observations are consistent with a model in which normal development of the embryo proper limits growth and differentiation of the suspensor. Altered development of the embryo proper in mutant seeds leads indirectly to proliferation of suspensor cells and expression of properties characteristic of the embryo proper. Ultimately, growth of the transformed suspensor is limited by the same genetic defect that disrupts development of the embryo proper. The availability of multiple alleles of sus1 and sus2, including T-DNA tagged alleles of each, will facilitate the cloning of these essential genes and molecular analysis of interactions between the embryo proper and suspensor early in development.

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.

SummaryHydroxypyruvate reductase (HPR) is a peroxisomal enzyme that catalyzes the NADH‐dependent reduction of hydroxypyruvate to glycerate in the photorespiratory glycolate pathway. HPR gene expression in cucumber seedlings is organ‐specific and light‐regulated. A 5.7 kb fragment of cucumber genomic DNA containing the entire HPR‐encoding gene, hpr‐A, plus 1069 bp OF 5′ flanking sequence was introduced into tobacco, as was a construct consisting of the upstream region (positions ‐ 1069 to +43) of the hpr‐A gene fused to the ß‐glucoronidase (GUS) reporter gene. Both constructs were expressed in the transgenic plants in an organspecific and light‐dependent manner. A deletion analysis of the 5’flanking sequence of the hpr‐A gene was conducted in transgenic tobacco. A construct in which the 5′‐flanking region had been deleted to within 299 bases of the transcription start site was sufficient to confer a high level of light‐regulated expression on the hpr‐A gene in the transgenic plants. However, deletion to position ‐218 resulted in a substantial reduction in expression. The region between positions ‐299 and ‐218 contains sequences resembling the 1 box and G box elements that are associated with other light‐regulated genes.