These authors contributed equally to this work. SUMMARYWe have characterized a b-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to b-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to b-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the b-1,6-and b-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-b-1,6-Gal and b-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6-and 3,6-linked galactose (Gal) and reduced levels of 3-, 2-and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched b-1,3-and b-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.

Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/) involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

Under heat stress, polyunsaturated acyl groups, such as α-linolenate (18:3) and hexadecatrienoate (16:3), are removed from chloroplastic glycerolipids in various plant species. Here, we showed that a lipase designated HEAT INDUCIBLE LIPASE1 (HIL1) induces the catabolism of monogalactosyldiacylglycerol (MGDG) under heat stress in leaves. Using thermotolerance tests, a T-DNA insertion mutant with disrupted was shown to have a heat stress-sensitive phenotype. Lipidomic analysis indicated that the decrease of 34:6-MGDG under heat stress was partially impaired in the mutant. Concomitantly, the heat-induced increment of 54:9-triacylglycerol in the mutant was 18% lower than that in the wild-type plants. Recombinant HIL1 protein digested MGDG to produce 18:3-free fatty acid (18:3-FFA), but not 18:0- and 16:0-FFAs. A transient assay using fluorescent fusion proteins confirmed chloroplastic localization of HIL1. Transcriptome coexpression network analysis using public databases demonstrated that the homolog expression levels in various terrestrial plants are tightly associated with chloroplastic heat stress responses. Thus, encodes a chloroplastic MGDG lipase that releases 18:3-FFA in the first committed step of 34:6 (18:3/16:3)-containing galactolipid turnover, suggesting that has an important role in the lipid remodeling process induced by heat stress in plants.

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non-cyanogenic HNGs are the β-HNG epidermin and the γ-HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP-glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co-expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi-enzyme complex.

SignificanceWithanolides form a major class of plant steroids with unique side-chain modifications. Withanolides are one of the main active components in an Indian Ayurvedic medicinal plant, ashwagandha, which has been used for over 3,000 y. Because of their highly diversified structures, withanolides are promising pharmacological compounds with proven antiinflammatory and anticancer properties. We identified a sterol Δ24-isomerase (24ISO) catalyzing the first committed step in the biosynthesis of withanolides and related compounds. Identification of 24ISO paves the way for targeted manipulations to increase withanolide yields and as a starting point to elucidate the downstream pathway of yet-unknown withanolide biosynthesis. This study also demonstrates how the evolution of enzymes catalyzing double-bond modifications of triterpene side chains lead to diversity in structures and functions.