Abstract
BackgroundArabinogalactan-proteins (AGPs) are ubiquitous components of cell walls throughout the plant kingdom and are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan polysaccharides (AG) to Hyp residues. AGPs are implicated to function in various aspects of plant growth and development, but the functional contributions of AGP glycans remain to be elucidated. Hyp glycosylation is initiated by the action of a set of Hyp-O-galactosyltransferase (Hyp-O-GALT) enzymes that remain to be fully characterized.ResultsThree members of the GT31 family (GALT3-At3g06440, GALT4-At1g27120, and GALT6-At5g62620) were identified as Hyp-O-GALT genes by heterologous expression in tobacco leaf epidermal cells and examined along with two previously characterized Hyp-O-GALT genes, GALT2 and GALT5. Transcript profiling by real-time PCR of these five Hyp-O-GALTs revealed overlapping but distinct expression patterns. Transiently expressed GALT3, GALT4 and GALT6 fluorescent protein fusions were localized within Golgi vesicles. Biochemical analysis of knock-out mutants for the five Hyp-O-GALT genes revealed significant reductions in both AGP-specific Hyp-O-GALT activity and β-Gal-Yariv precipitable AGPs. Further phenotypic analysis of these mutants demonstrated reduced root hair growth, reduced seed coat mucilage, reduced seed set, and accelerated leaf senescence. The mutants also displayed several conditional phenotypes, including impaired root growth, and defective anisotropic growth of root tips under salt stress, as well as less sensitivity to the growth inhibitory effects of β-Gal-Yariv reagent in roots and pollen tubes.ConclusionsThis study provides evidence that all five Hyp-O-GALT genes encode enzymes that catalyze the initial steps of AGP galactosylation and that AGP glycans play essential roles in both vegetative and reproductive plant growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0670-7) contains supplementary material, which is available to authorized users.
Highlights
Arabinogalactan-proteins (AGPs) are ubiquitous components of cell walls throughout the plant kingdom and are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan polysaccharides (AG) to Hyp residues
In silico analysis of GALT1, GALT3, GALT4, and GALT6 This study focused on the six-member gene/protein family in Arabidopsis, which is found within the carbohydrate active enzyme database (CAZy) GT31 family and distinguished by the presence of both a GALT and a GALECTIN domain
Hydrophobic cluster analysis (HCA) was performed by submitting the protein sequences to the drawhca server and used to identify the hydrophobic pockets containing the “DXD” motifs of the six GALTs; this analysis included two previously characterized AGPrelated GT31 members, GALT31A and At1g77810, which are involved with the elongation of β-1,6-galactan side chains and the β-1,3 backbone of AG polysaccharides, respectively (Additional file 1: Figure S2) [18, 19, 27, 28]
Summary
Arabinogalactan-proteins (AGPs) are ubiquitous components of cell walls throughout the plant kingdom and are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan polysaccharides (AG) to Hyp residues. Based on bioinformatics studies, Arabidopsis contains 85 AGP genes, while rice contains 69 AGP genes [2, 3] These genes are spatially and temporally expressed in a variety of patterns, which likely relates to their multiple functions. There remains a lack of understanding of the biophysical and biochemical modes of action of any individual AGP. This lack of understanding regarding function extends to the carbohydrate moieties or AG polysaccharides, which extensively decorate AGP core proteins and largely define their interactive surfaces
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