Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDPglucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS-and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from wholegenome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-b-glucuronidase/green fluorescent protein reporter lines further uncovered cell-and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.The presence of trehalose in a wide variety of organisms and the existence of different biosynthesis pathways suggest a pivotal and ancient role for trehalose metabolism in nature. The most widely distributed metabolic pathway consists of two consecutive enzymatic reactions, with trehalose-6-phosphate (T6P) synthase (TPS) catalyzing the transfer of a glucosyl moiety from UDP-Glc to Glc-6-phosphate to produce T6P and UDP, and T6P phosphatase (TPP) catalyzing dephosphorylation of T6P to trehalose (Cabib and Leloir, 1958; Avonce et al., 2006). Apart from operating as a (reserve) carbon source and structural component in bacteria, fungi, and invertebrates, trehalose also functions as a major stress protectant of proteins and membranes during adverse conditions such as dehydration, high salinity, hypoxia, and nutrient starvation (Elbein et al., 2003). Trehalose accumulation is also observed in a few lower vascular resurrection plants (e.g. Selaginella lepidophylla). Until about a decade ago, higher vascular plants were believed to have lost the ability to produce trehalose, but with the emergence of more sensitive assays, genome sequencing, and the use of yeast (Saccharo...

Glycoside hydrolases (GH) have been shown to play unique roles in various biological processes like the biosynthesis of glycans, cell wall metabolism, plant defence, signalling, and the mobilization of storage reserves. To date, GH are divided into more than 100 families based upon their overall structure. GH32 and GH68 are combined in clan GH-J, not only harbouring typical hydrolases but also non-Leloir type transferases (fructosyltransferases), involved in fructan biosynthesis. This review summarizes the recent structure-function research progress on plant GH32 enzymes, and highlights the similarities and differences compared with the microbial GH32 and GH68 enzymes. A profound analysis of ligand-bound structures and site-directed mutagenesis experiments identified key residues in substrate (or inhibitor) binding and recognition. In particular, sucrose can bind as inhibitor in Cichorium intybus 1-FEH IIa, whereas it binds as substrate in Bacillus subtilis levansucrase and Arabidopsis thaliana cell wall invertase (AtcwINV1). In plant GH32, a single residue, the equivalent of Asp239 in AtcwINV1, appears to be important for sucrose stabilization in the active site and essential in determining sucrose donor specificity.

Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.

Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.