Accumulation of multiple-repeat starch-binding domains (SBD2–SBD5) does not reduce amylose content of potato starch granules

Abstract

This study investigates whether it is possible to produce an amylose-free potato starch by displacing the amylose enzyme, granule-bound starch synthase I (GBSSI), from the starch granule by engineered, high-affinity, multiple-repeat family 20 starch-binding domains (SBD2, SBD3, SBD4, and SBD5). The constructs were introduced in the amylose-containing potato cultivar (cv. Kardal), and the starches of the resulting transformants were compared with those of SBD2-expressing amylose-free (amf) potato clones. It is shown that a correctly sized protein accumulated in the starch granules of the various transformants. The amount of SBD accumulated in starch increased progressively from SBD to SBD3; however, it seemed as if less SBD4 and SBD5 was accumulated. A reduction in amylose content was not achieved in any of the transformants. However, it is shown that SBDn expression can affect physical processes underlying granule assembly, in both genetic potato backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic Kardal plants were similar to those from untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is severely affected. In both the Kardal and amf background, apparently normal oval-shaped starch granules were composed of multiple smaller ones, as evidenced from the many "Maltese crosses" within these granules. The results are discussed in terms of different binding modes of SBD.

Fusion proteins comprising the catalytic domain of mutansucrase and a starch-binding domain can alter the morphology of amylose-free potato starch granules during biosynthesis

“…GtfICAT with an appended SBD alters granular morphology in amf background Consistent with previous results, it was found that more GtfICAT with appended SBD accumulated in the amf than in the Kardal background (Ji et al 2004;Nazarian et al 2006a); a protein of the expected size was evidenced in the starch granules of transgenic amf plants (Fig. 3).…”

“…Transformation and regeneration of potato plants pPFSBDIC and pPFICSBD were transformed into Agrobacterium tumefaciens strain LBA 4404 using electroporation (Takken et al 2000). Internodal stem segments from the tetraploid (cv.…”

“…Finally, the blot was stained for 15 min in dark with a 0.1 M NaHCO 3 solution of pH 9.8, containing 1% NBT/BCIP (Roche Molecular Biochemicals, Germany), and 0.01 M MgCl 2 . The amount of SBD-containing protein in potato tuber juice was determined as described by Nazarian et al (2006a).…”

“…The supernatant was subjected to a microconcentrator column (Millipore, USA). Twenty-five ll of the concentrated samples were analyzed using a 12% SDSpolyacrylamide gel (BioRad, UK), and detection of the fusion proteins was carried out as described by Nazarian et al (2006a). The Precision Plus Protein All Blue standards (BioRad, UK) was used for calibration.…”

“…For this, SBD was fused to the N-or C-terminus of GtfICAT, since our previous studies have pointed out that the position of the SBD in the fusion protein can influence the activity of the appended effectors. The amf background was used for transformation, because we have demonstrated before that accumulation of SBD was most efficient in the amylose-free (amf) potato background (Ji et al , 2004Nazarian et al 2006a). Moreover, the constructs were introduced into the wild type (Kardal) background, in order to facilitate a more direct comparison with the GtfICAT potato transformants that had already been made (Kok-Jacon et al 2005a).…”