Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase

Abstract

Potato tuber pectin is rich in galactan (oligomer of beta-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.

Figure 1

(A) Averaged FTIR spectra of potato tuber cortex from 20 wild-type tubers (solid line) and 20 T_13.1 tubers (dashed line). (B) Exploratory PC analysis discriminates T_13.1 from the wild type, using the third and fifth PC scores. (C) The loadings for PC3 (black line) and PC5 (gray line) show features characteristic of pectins at 1,745, 1,250, 1,150, 1,105, and 1,018 cm⁻¹.

Figure 2

Superose 12 profiles of EPG/PME extracts solubilized from potato tubers. The eluent was monitored by using a refractive index detector. Bars (A, B, C) indicate fractions that were pooled. The asterisk indicates a large peak attributable to the presence of sample buffer salts that is devoid of pectic material. Arrows, elution time of molecular mass markers (kDa). ^a, dextrans; ^b, galacturonic acid.

Figure 3

Sections of wild-type (A and C) and endo-galactanase-expressing (T_13.1) (B and D) potato tubers gold-labeled with monoclonal antibody LM5, silver enhanced and viewed by reflection confocal scanning microscopy (A and B) and transmission electron microscopy (C and D). The walls of wild-type parenchymal cells are strongly labeled (green in A, black particles in C) whereas in T_13.1 tubers, the labeling density is greatly reduced and localized only to some cell corners (arrowheads in B) close to the plasma membrane (arrows in D). Asterisks represent spaces once occupied by starch granules. ML indicates the expanded middle lamella of these filled corners. [Bars = 100 μm (A and B) and 2 μm (C and D).]