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L-Arabinose (L-Ara) is a plant-specific sugar found in cell wall polysaccharides, proteoglycans, glycoproteins, and small glycoconjugates, which play physiologically important roles in cell proliferation and other essential cellular processes. L-Ara is synthesized as UDP-L-arabinose (UDP-L-Ara) from UDP-xylose (UDP-Xyl) by UDP-Xyl 4-epimerases (UXEs), a type of de novo synthesis of L-Ara unique to plants. In Arabidopsis, the Golgi-localized UXE AtMUR4 is the main contributor to UDP-L-Ara synthesis. However, cytosolic bifunctional UDP-glucose 4-epimerases (UGEs) with UXE activity, AtUGE1, and AtUGE3 also catalyze this reaction. For the present study, we first examined the physiological importance of bifunctional UGEs in Arabidopsis. The uge1 and uge3 mutants enhanced the dwarf phenotype of mur4 and further reduced the L-Ara content in cell walls, suggesting that bifunctional UGEs contribute to UDP-L-Ara synthesis. Through the introduction of point mutations exchanging corresponding amino acid residues between AtUGE1 with high UXE activity and AtUGE2 with low UXE activity, two mutations that increase relative UXE activity of AtUGE2 were identified. The crystal structures of AtUGE2 in complex forms with NAD + and NAD + /UDP revealed that the UDP-binding domain of AtUGE2 has a more closed conformation and smaller sugar-binding site than bacterial and mammalian UGEs, suggesting that plant UGEs have the appropriate size and shape for binding UDP-Xyl and UDP-L-Ara to exhibit UXE activity. The presented results suggest that the capacity for cytosolic synthesis of UDP-L-Ara was acquired by the small sugar-binding site and several mutations of UGEs, enabling diversified utilization of L-Ara in seed plants., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Fasciclin-like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI-anchored, is highly N-glycosylated and carries two O-glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino-proximal fasciclin 1 domain and was unaffected by removal of the GPI-modification signal, a highly conserved N-glycan or the deletion of predicted O-glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)-exit and plasma membrane localization of FLA4, with N-glycosylation acting at the level of ER-exit and O-glycosylation influencing post-secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy-proximal fasciclin 1 domain and that its amino-proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy-proximal Fas1 domain and its normal cellular trafficking depends on N- and O-glycosylation., (© 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)