Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid 8-fructofuranosidase (BF) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular BF). Using antibodies against isoenzyme I of carrot soluble BF, we isolated several cDNA clones encoding polypeptides with sequences characteristic of BFs, from bacteria, yeast, and plants. l h e cDNA-derived polypeptide of one of the clones contains all partia1 peptide sequences of the purified isoenzyme I and thus codes for soluble acid BF isoenzyme 1. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. lhese two soluble BFs, 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 BF 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 BFs, are preproenzymes with signal peptides and Nterminal 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.

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Soluble 8-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carola). The enzyme hydrolyzed sucrose with a K , of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68000 from a gelfiltration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68,43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble p-fructofuranosidase with the complete sequence of carrot cell-wall P-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.

Abstract. The steady-state levels of transcripts for cellwall 13-fructofuranosidase (cwl3F), for isoenzymes I and II of soluble acid 13-fructofuranosidase (sI, slI), and for sucrose synthase (ss) were determined in the sink and source organs of developing carrot (Daucus carota L.) plants. The expression patterns of the four genes clearly differed. The expression of the gene for cwl3F was development-specific but not organ-specific; high transcript levels were only found in plants with primary roots, with about equal amounts in leaf lamina, petioles and roots. The genes for sI and slI were mainly expressed in roots, sI predominating in primary roots and slI in developing tap roots. Transcripts for ss were found at a low level in all developing plant organs and were markedly up-regulated during the development of young leaves and during the transition of primary roots to tap roots. Developing tap roots contained only transcripts for slI and for ss. Marked alterations in the expression of these two genes after manipulation of the source/sink balance of these plants indicates their importance in sucrose partitioning. We suggest that ss regulates sucrose utilization in developing tap roots, whereas slI located in the vacuole controis sucrose storage and sugar composition.

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