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We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) 8-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular 8-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short consenred motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular 8-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. lnfection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic 8-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant MOI. Biol. 4,111-1 161. Suspension-cultured carrot cells contain high levels of mRNA for extracellular 0-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

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To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.