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.Although biochemical events causing structural changes of cell walls are expected to influence plant growth and development (Pilling et al., 2000;Bouton et al., 2002), the degradation and remodeling of cell wall constituents during development appear to be of considerable complexity and far from being fully understood. For example, the remodeling of pectin, which is thought to impact upon processes such as adhesion between cells and plant growth, requires the action of several different enzyme classes (Ridley et al., 2001). The complex role of pectin in the dynamics of cell walls is further indicated by the presence of large families of these pectic enzymes in plant genomes. For example, more than 50 genes encoding putative polygalacturonases (PGs) have been identified in Arabidopsis (Arabidopsis Genome Initiative, 2000).Indications that the pectic polymers homogalacturonan (HGA), rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II) play an important role in plant development have been obtained by analyzing several cell wall mutants, most of which have been obtained by mutagenesis of Arabidopsis. Among these, Arabidopsis emb30 mutants, having a mutation in a gene putatively involved in the secretory pathway, show an abnormal localization and accumulation of pectin in intercellular/interstitial spaces rather than in the corners. Cells of emb30 mutants are larger than those of the wild type and do not adhere well to each other; the seeds are impaired in the control of cell division, expansion, and polarity and do not develop as wild type (Shevell et al., 2000). The Arabidopsis quartet mutants have microspores that fail to separate during pollen development as a result of the persistence of pectin in the pollen mother cell wall (Rhee and Somerville, 1998). The Arabidopsis mur1 mutation, leading to a deficiency in fucosyl residues, affects RG-II, reducing its capacity to dimerize through the formation of boron diest...

Transgenic Expression of a Fungal endo-Polygalacturonase Increases Plant Resistance to Pathogens and Reduces Auxin Sensitivity

“…Moreover, crossing tobacco PG plants with a line expressing high levels of the bean (Phaseolus vulgaris) PG-inhibiting protein PvPGIP2, an inhibitor of A. niger PGII, completely blocked PG activity and reverted the dwarf phenotype. The observed morphological alterations are associated to a reduced content of HGA in both tobacco and Arabidopsis, suggesting that the phenotype of PG plants is due to an enhanced degradation of HGA (Capodicasa et al, 2004). Interestingly, tobacco PG plants show no alterations of ion-mediated increase in xylem hydraulic conductivity (Nardini et al, 2007) and have a photosynthesis rate and stomatal conductance similar to those of wild-type plants (R. Galletti and D. Pontiggia, unpublished data).…”

“…Tobacco PG plants were inoculated with B. cinerea, and their susceptibility to the fungus was compared to that of wild-type plants and plants expressing the bean PvPGIP2, which inhibits several fungal PGs, including B. cinerea PG (PGIP2 plants; Leckie et al, 1999;Capodicasa et al, 2004). Upon infection, wild-type plants exhibited typical soft rot symptoms with rapidly expanding water-soaked lesions, whereas PG plants displayed lesions with a reduced size (Fig.…”

“…PG plants of both species accumulate the enzyme in their tissues and display morphological alterations, such as dwarfism and slightly curled leaves, but no other severe aberrations (Capodicasa et al, 2004). These phenotypes are dependent on the enzyme activity because expression of an inactive form of A. niger PGII has no obvious impact on Arabidopsis development.…”

“…expressing an attenuated version of PGII of Aspergillus niger (Capodicasa et al, 2004). PG plants of both species accumulate the enzyme in their tissues and display morphological alterations, such as dwarfism and slightly curled leaves, but no other severe aberrations (Capodicasa et al, 2004).…”

The Arabidopsis thaliana Class III Peroxidase AtPRX71 Negatively Regulates Growth under Physiological Conditions and in Response to Cell Wall Damage.

“…We have previously shown that tobacco (Nicotiana tabacum) and Arabidopsis plants expressing an attenuated version of the Aspergillus niger polygalacturonase II (AnPGII; 35S:AnPGII plants) have a reduced content of deesterified HG and a significant reduction of growth that correlates with the levels of expression of the transgene (Capodicasa et al, 2004). Furthermore, 35S:AnPGII plants show constitutive activation of defense responses and accumulation of high levels of ROS in their tissues .…”