Marcel Lüscher

Antisense Repression of Vacuolar and Cell Wall Invertase in Transgenic Carrot Alters Early Plant Development and Sucrose Partitioning

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.

Inulin synthesis by a combination of purified fructosyltransferases from tubers of Helianthus tuberosus

Sucrose‐sucrose 1‐fructosyltransferase (1‐SST) was purified 100‐fold from tubers of Helianthus tuberosus L. The purified enzyme was essentially devoid of invertase activity and could be separated by isoelectric focusing into five isoforms which all were composed of two subunits (59 and 26 kDa). Fructan‐fructan 1‐fructosyltransferase (1‐FFT) was purified from the same source [M. Lüscher et al. (1993) New Phytologist 123, 437–442). When incubated individually with sucrose, 1‐FFT was inactive while 1‐SST formed isokestose (trimer) and, upon prolonged incubation, some nystose (tetramer). When a combination of the two enzymes was incubated with sucrose, a series of oligofructosides with a degree of polymerization of up to 20 was formed. Amino acid sequences of tryptic peptide fragments from both 1‐SST and 1‐FFT indicate that these enzymes are highly homologous with plant invertases.

Expression of a functional barley sucrose‐fructan 6‐fructosyltransferase in the methylotrophic yeast Pichia pastoris

The cDNA encoding sucrose‐fructan 6‐fructosyltransferase (6‐SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZαC, containing the N‐terminal signal sequence of Saccharomyces cerevisiae α‐factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6‐SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1‐SST activity when incubated with sucrose.