Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Abstract

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance.Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-D-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1-2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development.

Effect of Arabinogalactan Proteins from the Root Caps of Pea and Brassica napus on Aphanomyces euteiches Zoospore Chemotaxis and Germination

“…An indication for the production of arabinogalactan proteins by pea roots has been made previously by several authors, and Ara and Gal have been reported as major sugar residues in root-secreted slime (Bacic et al, 1986;Moody et al, 1988;Knee et al, 2001;Xie et al, 2012). Furthermore, a pea border cell-specific b-galactosidase has been reported to be required for pea root growth, and although the endogenous substrate of this enzyme has not been determined, arabinogalactan proteins were proposed as candidates (Wen et al, 2008). Also, arabinogalactan protein epitopes were found to be highly expressed in Arabidopsis border-like cells (van Hengel and Roberts 2002;Vicré et al, 2005).…”

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

“…As such, the in situ analysis of GH activities responsible for the degradation of plant cell wall polysaccharides has received comparatively little attention, primarily due to a paucity of convenient assay methods (Vicente et al, 2007). Some notable exceptions include the use of commercially available X (5-bromo-4-chloro-3-indolyl glycoside) substrates for the detection of exoglycosidase activity (Monroe et al, 1999;Chantarangsee et al, 2007;Macquet et al, 2007;Wen et al, 2008). Likewise, transglycosylase activity has been visualized in higher plant and yeast cell walls using sulforhodamine-oligosaccharide acceptor substrates (Vissenberg et al, 2000;Bourquin et al, 2002;Nishikubo et al, 2007;Cabib et al, 2008).…”

Root Border Cells and Their Role in Plant Defense

“…Plants with altered border cell numbers, border cell delivery, or border cell gene expression would be predicted to exhibit altered susceptibility to infection. Experiments therefore were designed to develop tools to test the impact of border cells on root infection using plants with altered expression of genes controlling border cell development (103)(104)(105)(109)(110)(111)(112)(113)(114). Altered border cell development was achieved in transgenic plants, but pleiotropic effects precluded their use in field studies.…”

“…Curiously, transgenic roots with altered expression of a flavin-binding protein gene that is expressed only in border cells markedly altered root morphology as well as rate and direction of growth (103). A second border cell-specific gene was found to encode a galactosidase that functioned outside the cell at only the low pH characteristic of the root tip extracellular environment (105). This seemed to be a likely prospect for altered gene expression without pleiotropic effects on root development.…”