Galactose induces formation of cell wall stubs and cell death in Arabidopsis roots.

MAIN CONCLUSION: Arabidopsis seedlings growing on low concentration of galactose stop regular root growth. Incomplete cell division with cell wall stubs, binuclear and giant cells and lignified root tips are observed. Galactose is a sugar abundant in root cell walls of Arabidopsis. Nevertheless, we found that the germination of Arabidopsis seedlings on galactose containing media causes a strong modification of the root development, as shown by analysing the root with microscopy methods ranging from the bright field over confocal to transmission electron microscopy. At concentrations of about 1 mM, the growth of the primary root stops after a few days though stem cell markers like WOX5 are still expressed. The root tip swells and forms a slightly opaque, partially lignified structure in parts of the cortex and the central cylinder. The formation of the cell plate after mitosis is impaired, often leading to cell wall stubs and binuclear cells. Some cells in the cortex and the central cylinder degenerate, while some rhizodermal and cortical cells increase massively in size. The galactose toxicity phenotype in Arabidopsis depends on the activity of galactokinase and is completely diminished in galactokinase knock-out lines. From the comparison of the galactose toxicity phenotype with those of cytokinesis mutants and plants treated with appropriate inhibitors we speculate that the toxicity syndrome of galactose is caused by interference with intracellular vesicle transport or cell wall biogenesis.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

Phosphoglucomutase Is Not the Target for Galactose Toxicity in Plants.

Plants synthesize a number of different oligomeric or polymeric sugars containing galactose. During growth and development some of these carbohydrates are metabolized or remodeled releasing galactose as a breakdown product. All plants have established recycling pathways for such sugars, for which they seem to have a limited capacity to cope with. Exceeding these limits results in sugar toxicity, which is observed already at concentrations as low as 1 mmol·l-1 for galactose. The mechanism of galactose toxicity is poorly understood but it seems plausible that the enzymes involved in carbohydrate metabolism also might be the targets responsible for the adverse effects. Data from yeast and bacteria suggests that the enzyme phosphoglucomutase (PGM) is inhibited by galactose-1-phosphate. To test this hypothesis for plants we expressed recombinant cytosolic PGM3 from Arabidopsis in E. coli. Intriguingly, the enzyme was not inhibited by galactose-1-phosphate at physiological concentrations. Furthermore, PGM3 did not convert galactose-1-phosphate to galactose-6-phosphate, which was suggested as the inhibitory mode of action in yeast. In addition, metabolite levels in Arabidopsis roots were analyzed for their galactose-1-phosphate concentration by means of GC-MS. Seedlings grown on MS-media with sucrose contained less than 10 nmol·g FW-1 of galactose-1-phosphate. However, seedlings from plates, in which the sucrose was replaced by galactose, showed a strong increase of Gal-1-P to levels of up to 200 nmol·g FW-1.