Golgi-localized exo-β1,3-galactosidases involved in cell expansion and root growth in Arabidopsis

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface– and wall–associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana. GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall–extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.

Golgi-localized exo-β1,3-galactosidases involved in AGP modification and root cell expansion in Arabidopsis

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships between their glycosylation, processing, and functionality are poorly understood. Here we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana. GH43 loss of function mutants exhibit root cell expansion defects in sugar-containing growth media. This root phenotype is associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Recombinant GH43 characterization showed that the exo-β-1,3-galactosidase activity of GH43s is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43s did not release galactose from cell wall extracted glycoproteins or AGP rich gum arabic. These results show that Arabidopsis GH43s are involved in AGP glycan biosynthesis in the Golgi, and suggest their exo-β-1,3-galactosidase activity influences AGP and cell wall matrix interactions, thereby adjusting cell wall extensibility.

Ca2+ pulsation in BY-2 cells and evidence for control of mechanosensory Ca2+-selective channels by the plasmalemmal reticulum

A previously unknown cytoskeletal structure, now named the plasmalemmal reticulum (Gens et al. 2000, Protoplasma 212, 115–134), was found in cultured BY-2 tobacco cells during a search for a force-focusing mechanism that might enhance signal transduction by the cells’ mechanosensory Ca2+-selective cation channels (MCaCs). This polyhedral structure, which links cell wall, plasma membrane, and internal cytoplasm, prominently contains arabinogalactan protein (AGP). To check for reticulum-promoted Ca2+ elevation, the AGP-binding reagent (β-d-glucosyl)3 Yariv phenylglycoside has been applied to BY-2 cells expressing a free cameleon Ca2+ reporter. Ca2+ elevation was substantial and prolonged. Moreover it occurred in the nucleus as well as the cytoplasm. Cells treated with non-binding mannosyl Yariv reagent could not be discriminated from untreated controls or those treated with carrier solution alone. Supply of the MCaC inhibiter Gd3+ just before treatment with Yariv reagent prevented Ca2+ rise. These data strongly support the hypothesis that the plasmalemmal reticulum controls MCaC activity. The massive inward spread of Ca2+ suggested that entry of the ion through the channels initiated a wave of release from the ER, and YCX in the ER showed Ca2+ levels consistent with this premise. Cytosolic and nuclear Ca2+ often pulsed in control cells in near synchrony and at rates ranging from zero to five cycles per ∼20-min recording. (Pulsation was over-ridden by the applied amounts of glucosyl Yariv compound.) Suggestively but very crudely, oscillation rate was assessed as possibly correlating with stage of cell cycle. Because cell Ca2+ was lowered and pulsation was eliminated by Gd3+, MCaCs appear to participate in these endogenous fluctuations. The extent to which pulsing plays regulatory roles in relatively undifferentiated types of cells should be evaluated.