Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

Abstract

In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.

Gene expression profiling of systemically wound-induced defenses in hybrid poplar

“…The four TI-like sequences are very distinct, with less than 50% pairwise identity among them, and represent rather diverse members of the Kunitz TI family in the Populus genome. Such multiplicity and sequence diversity of Kunitz TIs may be a general phenomenon; a recent analysis of potato ESTs also found that potato tubers express at least 21 distinct Kunitz TI genes (Heibges et al 2003a). Kunitz TIs are one of several families of serine protease inhibitors, typical of legume seeds but also found in other plant species (Richardson 1991).…”

Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

“…Further differentiation was possible within the group A inhibitors and, to some extent, within inhibitors of groups B and C. GST-A 6 -P2B11 showed weaker trypsin inhibition than the other group A inhibitors tested, and strongly inhibited the growth of F. moniliforme mycelium, in contrast to GST-A 5 -P4E1 which did not inhibit F. moniliforme. The amino acid sequences of A 1 -P2B11 and A 5 -P4E1 differ by a number of non-conservative residue substitutions (see the accompanying paper by Heibges et al 2003). In contrast to the other inhibitors belonging to group A, GST-A 5 -P4E1 clearly inhibited a-chymotrypsin.…”

“…Inhibitors of the same homology group (A, B or C) putatively represent different genes. The deduced amino acid sequences are shown in the accompanying paper by Heibges et al (2003). The sequences corresponding to the mature inhibitor polypeptides were subcloned in the pESP-2 expression vector, transformed into S. pombe and purified from induced cultures by affinity chromatography.…”

Functional Analysis of the Kunitz Trypsin Inhibitor Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against Herbivores

“…These functional differences presumably reflect the sequence diversity of the KTI family. Previous study of a KTI gene family, a biochemical analysis of potato KTIs, also revealed functional diversity both between and within the homology groups (Heibges et al, 2003a(Heibges et al, , 2003b. Thus, potato group A and B KTIs strongly inhibit trypsin, whereas potato group C KTIs do so only weakly.…”

“…Thus, determining a precise function of KTI-like proteins cannot be based only on primary sequence similarities, but requires in vitro assays for confirmation. Whereas the biochemical activity of many individual plant KTIs is known, only in potato (Solanum tuberosum) has extensive analysis of a large KTI family within the same species been reported (Heibges et al, 2003a(Heibges et al, , 2003b. In that study, the KTI family showed surprising sequence variation, including many nonsynonymous substitutions and indels, which appear to translate into functional diversity.…”