Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth

Abstract

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

Figure 1.

Immunoblot analyses of AnPGII transgenic tobacco plants. Western-blot analysis of leaf total protein extracts (A) and intercellular fluids (B) of tobacco primary transformants numbers 5, 7, and 16 using an AnPGII-specific polyclonal antibody; AnPGII, 2 ng of purified AnPGII from A. niger. C, Western-blot analysis of leaf total protein extracts (10 μg) of 10-week-old transgenic R2 progeny plants of lines 5, 7, and 16 using the same antibody as in A.

Figure 2.

Purification by affinity chromatography and in-gel activity of AnPGII from transgenic tobacco plants. SDS-PAGE (A) and western-blot analysis using the AnPGII-specific antibody (B) of proteins retained by a bean PGIP2-Sepharose affinity column and eluted with PBS. Affinity chromatography was performed as reported in “Materials and Methods.” Lane 1, AnPGII eluted from the affinity column; lane 2, 30 ng (A) and 1 ng (B) of purified AnPGII from A. niger. C, IEF gel of purified AnPGII stained by silver nitrate. D, IEF gel of AnPGII stained for enzymatic activity.

Figure 3.

Morphological, anatomical, and immunoblot analyses of transgenic tobacco plants. A, Growth characteristics of 10-week-old untransformed (SR1), transgenic homozygous tobacco plants of lines 5, 7, and 16 and a plant obtained by crossing line 16 with transgenic plants expressing P. vulgaris PGIP2 (line 16 × PGIP2). B, Transverse sections of stems of SR1, line 7, and line 16. Arrows in A indicate the approximate region from which the transverse stem sections were taken. c, cortical parenchyma; p, pith parenchyma. Scale bars = 1 mm. C, Western-blot analysis of leaf protein extracts from the same tobacco plants as for A, using the AnPGII-specific antibody, or D, a PGIP-specific antibody. AnPGII, 4 ng of purified AnPGII from A. niger. PGIP2, 2 ng of purified PGIP2 from P. vulgaris.

Figure 4.

Anatomical analysis of transgenic tobacco plants. Stem transverse sections at the level of fourth internode from 8-week-old SR1, line 7, and line 16 plants. A, Cortical region. Line 16 shows smaller and rounder epidermal and subepidermal cells, compared to SR1 plants. B, Central region. Pith parenchyma cells of line 16 are smaller than SR1 and line 7 cells. The number of phloem-associated parenchyma cells in line 16 is greater than in SR1 and line 7 plants. C, Vascular tissue. Along the radial axis in the region of the cambium, line 16 has more cell layers. Cells of line 16 are rounder and smaller than SR1 and line 7 cells. Bars = 100 μm.

Figure 5.

Dehydration of leaves. A, Leaves of 8-week-old SR1 and line 16 plants were collected, transferred in the hood, and their fresh weight was determined at different time intervals. Three independent samples, each formed by three leaves from three different plants, were used. Bars indicate the se for each data point. B, Scanning electron micrographs of abaxial midrib of SR1, line 7, and line 16 leaves of 8-week-old tobacco plants. Cells of line 16 quickly dehydrated when transferred into the vacuum chamber of the electron microscope. A slight dehydration is also evident in line 7 plants. Bars = 100 μm.

Figure 6.

Chemical analysis of cell walls of transgenic tobacco plants expressing AnPGII. Monosaccharide composition of AIS (A) and ChASS (B) fractions prepared from cell wall material of stems of 30-d-old SR1 and transgenic lines 5, 7, and 16. Results are expressed in percentage of total moles. Bars indicate se (n = 3).

Figure 7.

Immunodot analysis of cell wall fractions. ChASS fractions were extracted from cell wall material of untransformed and stems of transgenic tobacco lines 7 and 16 and applied to nitrocellulose in the dilution series indicated. Immunodot analysis with PAM1 indicated that large unesterified blocks of HGA are present in all ChASS fractions, but were 5-fold less abundant in line 16.

Figure 8.

Immunofluorescent labeling of pectic HGA components in transgenic tobacco plants expressing AnPGII. Hand-cut transverse sections of midrib of 10-week-old plants (A–C) and Steedman's wax transverse sections of stems of 9-week-old plants (D–F) were labeled with PAM1 (A–C) and LM7 (D–F). Arrows indicate a corner of an intercellular space (is) in each case. Scale bar = 10 μm.

Figure 9.

Morphological and immunoblotting analyses of transgenic Arabidopsis plants expressing AnPGII. A, Growth characteristics of transgenic Arabidopsis R2 plants of lines 1 and 5 compared to SR1 (control). B, Western-blot analysis of leaf total protein extracts of line 1, line 5, and control using the AnPGII-specific polyclonal antibody.