Invertases. Primary Structures, Functions, and Roles in Plant Development and Sucrose Partitioning

Abstract

One of the key features of plants is their ability to reduce carbon dioxide in the presence of sunlight and water to sugars, and the subsequent transport of assimilated carbon to the nonphotosynthetic tissues (sink tissues). In most plants, the transported sugar is Suc, a nonreducing disaccharide, in which Glc and Fru are linked (α1→β2). Depending on the physiological activities and the biochemical needs of the heterotrophic tissues, Suc is channeled into various pathways in different subcellular compartments. It may enter glycolysis and the tricarboxylic acid cycle for the production of ATP and NADH. The carbon of the disaccharide may also be used for the biosynthesis of primary metabolites important for tissue growth and development. Likewise, Suc may become converted into polymers such as starch, triacyl glycerides, or polypeptides for long-term storage, or into secondary compounds, enabling plants to cope with predators and pests or other environmental challenges. Many of these processes may take place in the same cell at the same time and, thus, allocation of Suc to these different processes requires efficient and precise control mechanisms. Utilization of Suc as a source of carbon and energy depends on its cleavage into hexoses, and in plants either Suc synthase (EC 2.4.1.13) or invertase (EC 3.2.1.26) catalyzes this reaction. Suc synthase is a glycosyl transferase, which converts Suc in the presence of UDP into UDP-Glc and Fru. Invertase is a hydrolase, cleaving Suc into the two monosaccharides. Suc synthase is a cytoplasmic enzyme and, in most plants, two closely related isoforms have been identified (for summary, see Sturm et al., 1999). Invertase exists in several isoforms with different biochemical properties and subcellular locations (Sturm, 1996; Tymowska-Lalanne and Kreis, 1998). The specific functions of the different invertase isoforms are not clear, but they appear to regulate the entry of Suc into the different utilization pathways. Because sugars in plants are not only nutrients but also important regulators of gene expression (Koch, 1996), invertases may be indirectly involved in the control of cell differentiation and plant development. Most plant species contain at least two isoforms of vacuolar invertase, which accumulate as soluble proteins (soluble acid invertases) in the lumen of this acidic compartment. Likewise, several isoforms of extracellular invertase (cell wall invertases) that are ionically bound to the cell wall have been detected. Vacuolar and cell wall invertases share some biochemical properties, e.g. they cleave Suc most efficiently between pH 4.5 and 5.0 and attack the disaccharide from the Fru residue. Thus, these so-called acid invertases are β-fructofuranosidases and also hydrolyze other β-Fru-containing oligosaccharides such as raffinose and stachiose. Additionally, plants have at least two isoforms of cytoplasmic invertase with pH optima for Suc cleavage in the neutral or slightly alkaline range. Neutral and alkaline invertases are less well characterized but, in contrast to the acid invertases, these enzymes appear to be Suc specific. Acid invertases have been purified from several plant species (for summary, see Unger et al., 1992). The enzymes have aK m for Suc in the low-millimolar range. Activity is inhibited by heavy metal ions such as Hg2+ and Ag+, suggesting the presence of a sulfhydryl group at the catalytic site. Acid invertases are also inhibited by their reaction products, with Glc acting as a non-competitive inhibitor and Fru as a competitive inhibitor. The mature polypeptides are N-glycosylated and the majority have molecular masses between 55 and 70 kD. Analysis of some of the purified proteins on denaturing SDS gels under reducing conditions revealed the presence of proteolytic fragments. A 70-kD monomeric form of vacuolar invertase of mung bean hypocotyls was found to be split into a 30-kD N-terminal and a 38-kD C-terminal fragment (Arai et al., 1991). Likewise, the 68-kD monomer of isoform I of vacuolar invertase from carrot was fragmented into N- and C-terminal polypeptides of 43 and 25 kD, respectively (Unger et al., 1992, 1994). Under native conditions, these fragments appear to be tightly associated and, in a complex, possess enzyme activity. Fragmentation does not appear to be an artifact of protein purification, but instead seems to be under developmental control. For example, the full-length proteins predominate in very young hypocotyls of bean and seedlings of carrot, whereas with increasing hypocotyl and seedling age the fragments were more abundant. Whether fragmentation has a physiological function is not clear. Like plant invertase, yeast invertase exists in different isoforms with discrete subcellular locations (Carlson and Botstein, 1982). A secreted form of the enzyme is highly N-glycosylated and its expression is regulated by Glc repression. Cytoplasmic yeast invertase is a nonglycosylated polypeptide that is constitutively expre