Transit peptides play a major role in the preferential import of proteins into leucoplasts and chloroplasts.

The in vitro import characteristics of six different precursors of plastid proteins were assessed to determine differences in the protein import pathways of leucoplasts and chloroplasts. Five of these precursor proteins are destined to different subchloroplast sites, and one is a leucoplast stromal precursor protein. The results indicate that some of these precursors can be imported equally into both plastid types and others preferentially into one type of plastid versus the other. The ability of plastids to import different proteins correlates with the in vivo steady state levels of these proteins. Additional differences were also observed in the intraorganellar portion of the translocation pathway for two thylakoidal proteins. The differences in import characteristics were found to be predominantly governed by information in the transit peptides, since attachment of the various transit peptides to different plastid and foreign proteins demonstrated that the import behavior of the proteins is transferable with the transit sequence. These results indicate that the import mechanisms of leucoplasts and chloroplasts are sufficiently different such that the plastids respond differently to the information present in the transit peptides.

Import characteristics of a leucoplast pyruvate kinase are influenced by a 19-amino-acid domain within the protein.

Two cDNA clones encoding distinct forms of plastid pyruvate kinase (designated Pka and Pkg) have recently been characterized. Pkg is found in both leucoplasts and chloroplasts, whereas Pka is present only in leucoplasts. The precursors of these proteins have different in vitro import characteristics. The Pkg precursor behaves like a typical stromal protein precusor with both types of plastid. In contrast, Pka precursors accumulate on the outer envelope membrane of leucoplasts under the same assay conditions and require a higher level of ATP for import into the organelle. Interestingly, the binding of Pka precursors to chloroplasts cannot be detected at any tested level of ATP even though the precursors are imported into the organelle at higher concentrations of ATP. Various N-terminal deletions and chimeric fusions were used to examine the translocation signaling mechanism of the Pka precursor. The N-terminal 83-amino-acid segment of Pka contains a transit peptide that is capable of directing dihydrofolate reductase and the mature body of Pkg into both types of plastid. Unlike the complete Pka precursor, these fusion proteins behave like typical stromal protein precursors. The behavior of the Pka transit peptide is influenced by a 19-amino-acid domain (-P-S-S-I-E-V-D-A-V-T-E-T-E-L-K-E-N-G-F-) located immediately downstream of the N-terminal 83-residue segment. Deletion of this domain from Pka alters its import properties such that it resembles a typical stromal protein precursor. Re-introduction of the 19-residue domain into the Dhfr fusion protein alters its import characteristics to resemble that of the complete Pka precursor. This 19-amino-acid domain can also influence the function of transit sequences from other precursors when it is placed immediately behind the transit peptide. These results suggest that this 19-amino-acid domain plays an important role in governing the import characteristics of the Pka precursor. We have named this 19-residue segment the "import modifying domain."

Structure of the genes encoding the alpha- and beta-subunits of castor pyrophosphate-dependent phosphofructokinase.

Full-length genomic clones encoding the alpha- and beta-subunits of the pyrophosphate-dependent phosphofructokinase (PFP) from the castor plant have been isolated and sequenced. The gene (PFP alpha) encoding PFP alpha is approx. 5.8 kb in length and contains 19 exons, which collectively encode a protein of 617 amino acids (aa) having a deduced M(r) of 67,360. PFP beta is approx. 4.6-kb long and contains 16 exons. Together, these exons encode a protein (PFP beta), of 552 aa with a deduced M(r) of 60,114. The intron-exon splice junctions in both genes contain the consensus sequences typical for plants. An alignment of intron placement in castor PFP alpha and PFP beta with introns in the 5' portion of the gene encoding the ATP-dependent phosphofructokinase (PFK) from rabbit muscle, indicates that only one intron occupies the same position in all three genes. Furthermore, within castor PFP alpha and PFP beta, only two introns are identically placed. Within the promoter regions of castor PFP alpha and PFP beta, there are short sequences having high homology to each other (up to 65%). The results demonstrate, for the first time, that there is little homology between PFP and PFK, nor are PFP alpha and PFP beta closely related. This lack of homology suggests PFP did not evolve from PFK, but rather, that PFP and PFK have probably evolved from a common ancestral gene.

Higher-plant cofactor-independent phosphoglyceromutase: purification, molecular characterization and expression.

Cofactor-independent phosphoglyceromutase (PGM) was purified to homogeneity from developing castor seed endosperm. Immunological characterization using monospecific antisera raised against this protein indicates that the enzyme is located in the cytosol and that there is no immunologically related polypeptide in the leucoplast from this tissue. Isolation and sequence determination of full-length cDNA clones for castor and tobacco PGM demonstrate that the protein is highly conserved in these plants and is closely related to the maize enzyme. A comparison of the amino acid sequence of peptides derived from Neurospora crassa PGM with the cofactor-independent enzyme from higher plants demonstrated that they are related and may have diverged from a common ancestral gene. The previously proposed relationship between higher-plant PGM and alkaline phosphatases is not supported by sequence analysis of the castor and tobacco enzymes. Expression of the single castor cytosolic PGM gene correlates well with other cytosolic glycolytic genes in developing and germinating castor seeds, and with the appearance of enzyme activity and PGM polypeptides in these tissues.

Structure of the gene encoding potato cytosolic pyruvate kinase.

The polymerase chain reaction (PCR) has been used to generate a series of overlapping genomic clones representing 43 bp of 5' untranslated sequence, 63 bp of 3' untranslated sequence and the entire coding sequence of the gene encoding potato cytosolic pyruvate kinase (PKc). This portion of the gene is approximately 4.5 kb in length and is interrupted by three introns, one of which is present in the 5' untranslated region. Southern blot analysis indicates that PKc is encoded by a small gene family, and sequence data from a number of PCR-derived genomic clones indicate that there are as many as six PKc genes. Sequence differences between the PCR-generated genomic clones and a PKc cDNA clone are discussed with respect to the fidelity of Taq polymerase. An alignment of intron placement in the potato PKc gene with intron placement in PK genes from other sources indicates that two of the potato introns correspond to intron positions in other species.

Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase.

The coding sequence of the cytosolic isozyme of potato tuber pyruvate kinase (PK) was attached to the transit peptide of the small subunit of pea ribulose-1,5-bisphosphate carboxylase oxygenase and placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Nicotiana tabacum. Unexpectedly, two primary transformants were recovered in which PK activity in leaves was greatly reduced. The reduction in PK activity appeared to result from the complete absence of the cytosolic form of the enzyme (PK(c)). In addition, no PK(c) could be detected on western blots of leaf extracts. Metabolite analyses indicated that the levels of phosphoenolpyruvate are substantially higher in PK(c)-deficient leaves than in wild-type leaves, consistent with a block in glycolysis at the step catalyzed by PK. PK(c) deficiency in the leaves does not appear to adversely affect plant growth. Analysis of progeny indicates that PK(c) deficiency is a heritable trait. The leaves of PK(c)-deficient transformants have normal rates of photosynthetic O(2) evolution and respiratory O(2) consumption, indicating that these plants are using alternative pathways to bypass PK.

Expression of the Genes for the alpha- and beta-Subunits of Pyrophosphate-Dependent Phosphofructokinase in Germinating and Developing Seeds from Ricinus communis.

Various tissues from both germinating and developing castor seeds (Ricinus communis L.) have been analyzed for the level of expression of the genes for the alpha- and beta-subunits of pyrophosphate-dependent phosphofructokinase (PFP). In tissues in which PFP is expressed, there is a single mRNA species of approximately 2 kilobases for each of the subunits. In germinating endosperm, the gene for the alpha-subunit is expressed at an earlier time after imbibition than that for the beta-subunit, whereas in developing castor seed endosperm, both genes are highly and coordinately expressed. During seedling development, there is tissue-specific expression of the two genes. Tissues in which there is a high level of mRNA correspond with tissues in which both subunits of PFP can be detected. The differential expression of the two subunit genes in germinating endosperm does not result in the presence of the alpha-subunit polypeptide in the absence of the beta-subunit polypeptide. Southern analysis of castor genomic DNA indicates the presence of a single gene for both the alpha- and beta-subunits of PFP in contrast with potato, in which there are at least two genes for each subunit.

Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources.

Two cDNA clones, PK(p)alpha and PK(p)beta, for the leucoplast isozyme of pyruvate kinase have been isolated and characterized. A Southern blot of castor (Ricinus communis) DNA probed with PK(p)alpha indicates the presence of a single gene for PK(p). Most (1610 base pairs) of the sequence of both cDNAs is identical. These 1610 base pairs begin with an ATG translation initiation codon, and have 248 base pairs of 3'-untranslated and 1362 base pairs of coding sequence. The sequences of the two clones 5'- to the identical regions are different but both encode peptides with a high percentage of hydrophobic amino acids. The derived sequence of PK(p)alpha encodes eight amino acid residues which have been identified as the amino-terminus of one subunit of PK(p) from castor seed leucoplasts when the enzyme is purified in the absence of cysteine endopeptidase inhibitors. The sequence upstream of these amino acids is possibly the transit peptide for this protein. When PK(p) is extracted under conditions that eliminate its proteolytic degradation, its alpha-subunit has a relative molecular weight equal to the full-length coding sequence of PK(p)alpha. The data indicate that the transit peptide for the subunit of leucoplast pyruvate kinase encoded by PK(p)alpha is not cleaved until the protein is released from the plastid. The derived amino acid sequences of PK(p)alpha and PK(p)beta are most closely related to Escherichia coli pyruvate kinase. Although the residues involved in substrate binding are conserved in leucoplast pyruvate kinase, there is no phosphorylation site and only 5 of 15 amino acids in the E. coli fructose-1,6-bisphosphate binding site are conserved.

Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase.

Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA. The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical. A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP. A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E. coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar. These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme.

Duplication of the phycocyanin operon in the unicellular cyanobacterium Anacystis nidulans R2.

Two phycocyanin (PC) operons, each containing alpha- and beta-subunit genes, have been isolated from the unicellular cyanobacterium Anacystis nidulans R2. Using oligodeoxyribonucleotide probes for the PC-coding regions, three PstI fragments were obtained and shown to contain the two operons, which are 2.7 kb apart, with a proposed gene order of 5'-(beta I-alpha I)-(beta II-alpha II)-3'. The nucleotide sequences of both alpha-subunit genes are identical, as are the beta-sequences and the 51-bp intergenic regions. However, significant nucleotide sequence differences are found in both the 5' and 3' untranslated regions of the two operons. Two mRNA species of 1.65 and 1.5 kb were detected in A. nidulans R2 RNA when probed with either the alpha-specific or the beta-specific probe. The results demonstrate the existence of two PC operons which are both transcriptionally active.

Characterization of alcohol dehydrogenase in young soybean seedlings.

Molecular properties of alcohol dehydrogenase (ADH) were examined in young soybean seedlings. Soybean radicle tissue is ADH-rich. Enzyme specific activity decreases slowly with the development of roots and becomes almost undetectable when the first true leaves appear. Soybean ADH was not found to be inducible by flooding. 2,4-Dichlorophenoxyacetic acid (2,4-D) treatment increased ADH specific activity as much as 14-fold. Only one ADH isozyme was detected by isoelectric focusing. By DNA-DNA hydridization, soybean ADH genomic sequences were shown to be partly homologous to maize ADH1 cDNA. The presence of more than one Adh gene in soybean is discussed.

Regulation of swelling of etiolated-wheat-leaf protoplasts by phytochrome and gibberellic acid.

The effect of light on the size of intact protoplasts isolated from the primary leaves of etiolated Triticum aestivum was studied. A 2-min red-light irradiation in the presence of 1 mM KCl was sufficient to cause a swelling of protoplasts compared with those maintained in darkness. The effect was photoreversible by far-red light over two light cycles, indicating the involvement of phytochrome. At 4°C, escape from reversibility occurred between 2 and 5 min after the exposure to red light. In exposure-response experiments, 20 s red light at 27 µmol m(-2)s(-1) was sufficient to saturate the response. Exogenous gibberellic acid added in darkness in the presence of KCl also induced protoplast swelling. Gibberellins may act as an intermediate in the phytochrome-induced swelling of protoplasts.