Tissue-specific expression of two genes for sucrose synthase in carrot (Daucus carota L.).

Sucrose synthase, which cleaves sucrose in the presence of uridine diphosphate (UDP) into UDP-glucose and fructose, is thought to be a key determinant of sink strength of heterotrophic plant organs. To determine the roles of the enzyme in carrot, we characterized carrot sucrose synthase at the molecular level. Two genes (Susy*Dc1 and Susy*Dc2) were isolated. The deduced amino acid sequences are 87% identical. However, the sequences upstream of the translation initiation codons are markedly different, as are the expression patterns of the two genes. Susy*Dc2 was exclusively expressed in flowers. Transcripts for Susy*Dc1 were found in stems, in roots at different developmental stages, and in flower buds, flowers and maturing seeds, with the highest levels in strong utilization sinks for sucrose such as growing stems and tap root tips. Expression of Susy*Dc1 was regulated by anaerobiosis but not by sugars or acetate. The carrot sucrose synthase protein is partly membrane-associated and this insoluble form may be directly involved in cellulose biosynthesis. Tap roots of the carrot cultivar used accumulated starch in the vicinity of the vascular bundles, which correlated with high sucrose synthase transcript levels. This finding suggests that soluble sucrose synthase in tap roots channels sucrose towards starch biosynthesis. Starch accumulation appears to be transient and may be involved in sucrose partitioning to developing tap roots.

Biochemical, physiological, and molecular characterization of sucrose synthase from Daucus carota.

Sucrose synthase (EC 2.4.1.13) from carrot (Daucus carota) is a tetramer with a molecular mass of 320 kD and subunits of 80 kD. The enzyme has a pH optimum of 7.0 (cleavage direction). Maximal activities were measured at 55 degrees C. The Km for Suc was estimated as 87 mM and for UDP as 0.39 mM. Fructose acts as a noncompetitive inhibitor with an inhibition constant of 17.2 mM. In contrast, glucose inhibits carrot sucrose synthase uncompetitively with an inhibition constant of 4.3 mM. cDNA clones encoding a single class of sucrose synthase polypeptide were isolated and sequenced. DNA gel blot analysis also indicated the occurrence of only one to two genes. The deduced amino acid sequence of the carrot enzyme is highly homologous to the sucrose synthase sequences of tomato, potato, and bean. A comparison of the cDNA-derived amino acid sequence with the SS1- and SS2-type sucrose synthase sequences of the monocot plants maize, rice, and barley showed that the carrot enzyme is neither of the SS1 nor of the SS2 type. High enzyme activity was found in roots and petioles of developing carrot plants, with maximal activity in roots at the transition of primary roots to tap roots. Enzyme activity was highly correlated with both polypeptide and transcript levels, indicating that gene expression is regulated mainly at the mRNA level in the different tissues and organs of developing carrot plants.

Aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Gene cloning, sequence analysis, expression, and purification of the recombinant enzyme.

A cDNA clone encoding a ligninolytic aryl-alcohol dehydrogenase (AAD; EC 1.1.1.91) from the white-rot basidiomycete fungus Phanerochaete chrysosporium was isolated and characterized. The nucleotide sequence obtained reveals an open reading frame encoding a protein of 385 amino acids. Substantial homology (49.3% identity and 67.3% similarity, respectively) was observed between AAD and an open reading frame sequence present on chromosome III of Saccharomyces cerevisiae. A Southern blot analysis showed the presence of multiple AAD gene-related sequences in P. chrysosporium and in other white-rot fungi including Bjerkandera adusta and Fomes lignosus. Northern blot analyses are in line with the view that the levels and appearance of AAD mRNA correlate with the level and appearance of AAD activity and that, under conditions of nitrogen limitation, the AAD mRNA levels are higher than in carbon limited cultures. This is consistent with the regulation of the enzyme by carbon or nitrogen limitation being at the level of transcription. Moreover, the appearance of AAD-specific transcripts correlates with the appearance of lignin peroxidase-specific transcripts in the same cultures. This co-appearance is in line with the proposed synergistic interaction of the two enzymes in lignin biodegradation, which suggests a similar regulation. The AAD encoding cDNA was expressed in Escherichia coli to yield high levels of active enzyme, and the recombinant enzyme was purified by using metal chelate affinity chromatography.

Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa.

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein in media containing n-hexadecane as a carbon source but not in media containing glycerol or glucose. This protein was purified, and the N-terminal amino acid sequence was determined. The amino acid composition of the protein was found to be very similar to that of the so-called protein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7B1. This extracellular protein was previously characterized (K. Hisatsuka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445-450, 1977) and found to stimulate the growth of P. aeruginosa on n-hexadecane and to possess emulsifying activity. To study the role(s) of the PA protein and to make it accessible for possible future applications, we have cloned the PA-encoding (pra) gene and determined its nucleotide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pra gene was inactivated by insertional mutagenesis, and the resulting strain was found to lack extracellular PA protein and to be retarded in its growth in n-hexadecane-containing media. These results are consistent with the growth stimulatory role of the PA protein. The pra gene was expressed in Escherichia coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purified by metal chelate affinity chromatography. The ability to produce secreted PA protein by E. coli provides a simple and safe means to analyze its function(s) in alkane assimilation in the future.

cDNA cloning of carrot (Daucus carota) soluble acid beta-fructofuranosidases and comparison with the cell wall isoenzyme.

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid beta-fructofuranosidase (beta F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). Using antibodies against isoenzyme I of carrot soluble beta F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid beta F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid beta Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.