Fructan biosynthesis in excised leaves of Lolium temulentum L. III. A comparison of the in vitro properties of fructosyl transferase activities with the characteristics of in vivo fructan accumulation*

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Abstract
SUMMARY: The induction of fructan biosynthesis in excised, illuminated leaves of Lolium temulentum L. is accompanied by a concomitant increase in extractable sucrose: sucrose fructosyl transferase (SST) activity. In leaves treated with the L‐isomer of 2‐(4,methyl‐2,6,‐dinitroanilino)‐N‐methyl propionamide (L‐MDMP) fructan biosynthesis in vivo occurs in the absence of such an increase in extractable SST. suggesting that the activity measured in vitro is not an obligatory component of fructan biosynthesis in vivo.Sucrose:sucrose fructosyl transferase preparations from L. temulentum catalyse the formation of isokestose and kestose from sucrose. The rates of synthesis of these trisaccharides were determined in the range of sucrose concentration 10‐700 mol m−3. Increase in total trisaccharide synthesis was linear with respect to substrate concentration. Isokestose was Formed over the entire range whilst kestose synthesis was inactive below 8O mol m−3. Neither isokestose nor kestose synthesis was saturated at 700 mol m −3. The Km for isokestose synthesis was high at 380 mol m−4. Conventional kinetic analysis of kestose data failed, indicating that this activity does not obey classical Michaelis‐Menten kinetics.The intracellular sucrose concentration in leaf tissue was estimated to be 100 mol m−3. At this substrate concentration, isokestose synthesis would be Functioning well below the measured Km and kestose synthesis would be operating at the extreme lower limit of its activity range.Higher oligosaccharide products of fructosyl transferase incubations were not representative of the oligofructans which accumulate in the leaf. This data casts further doubt on the physiological significance of the measured fructosyl transferase activities.Isokestose synthesis was localized in vacuoles derived from leaf mesophyll protoplasts, confirming current theories of fructan biosynthesis. However, in view of the anomalous characteristics of fructosyl transferases in vitro, we propose that unambiguous assignment of the subcellular compartmentation of fructan metabolism in leaves of Lolium temulentum should await a fuller understanding of the enzymology of fructan biosynthesis in this species.