Invertases catalyze the irreversible hydrolysis of sucrose to glucose and fructose. Plants contain two unrelated families of these enzymes: acid forms that derive from periplasmic invertases of eubacteria and are found in cell wall and vacuole, and neutral/alkaline forms evolved from the cytosolic invertases of cyanobacteria. Genomes of rice (Oryza sativa) and thale cress (Arabidopsis thaliana) contain multiple genes encoding these two families. Here for rice we identify the member genes of a cell-wall group (designated OsCIN1-9), a vacuolar group (OsVIN1-2), and two ancient neutral/alkaline groups: alpha (OsNIN1-4) and beta (OsNIN5-8). In Arabidopsis these groups contain six, two, four and five members, respectively. It is believed that the vacuolar group evolved from the cell-wall group. We provide evidence that the N-terminal signal peptide that directs cell-wall invertases co-translationally into the endoplasmic reticulum for secretion was replaced in the vacuolar group by a sequence similar to the complex N-terminal motif that targets alkaline phosphatase post-translationally to the vacuolar membrane of yeast. Since the last common ancestor of Arabidopsis and rice, the two invertase families evolved equally rapidly via gene duplication and gene loss, but the acid invertase family underwent approximately 10 events of intron loss compared with a single event of intron gain in the neutral/alkaline invertase family. Transcripts were detected for all rice invertase genes except OsCIN9. The acid invertase genes showed greater spatial and temporal diversity of expression than the neutral/alkaline genes.

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.

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Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and w2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [␤-(2,1)] are much better substrates than levan-type fructans [␤-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a ␤-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.Starch is the most prominent storage carbohydrate in plants, but about 15% of flowering plant species use fructan (a Fru polymer) as a storage compound (Hendry, 1993). Inulin-type fructan consists of linear ␤-(2,1)-linked fructofuranosyl units and occur mainly in dicotyledonous species. Levan consists of linear ␤-(2,6)-linked fructofuranosyl units, but more complex and branched fructan types (graminan, inulin neoseries, and levan neoseries) are common in monocotyledonous species (Vijn and Smeekens, 1999;Pavis et al., 2001b) Next to their obvious role as reserve compounds, fructan might have other functions in plants like stress protectants (drought and cold) or osmoregulators (Vergauwen et al., 2000; Hincha et al., 2002, and refs. therein). Unlike starch, fructans are water soluble and are believed to be stored in the vacuole , although the exclusive vacuolar localization has been questioned .Although the metabolism of inulin has become clear in dicotyledonous species and the respective biosynthetic and breakdown enzymes have been cloned (Edelman and Jefford, 1968;Van den Ende and Van Laere, 1996a; van der Meer et al., 1998;Hellwege et al., 2000;Van den Ende et al., 2000, fructan metabolism in monocots is not yet completely unraveled. So far, four different fructosyltransferases, each with their own specificity, are believed to be involved in monocot fructan biosynthesis. In addition to inulin biosynthesis by Suc:Suc 1-fructosyl tr...

. What shapes amino acid and sugar composition in Mediterranean floral nectars? Á Oikos 115: 155 Á169.We studied the amino acid (AA) composition of the floral nectars of 73 plant species occurring in a phryganic (East Mediterranean garrigue) community and investigated whether AA and sugar composition is shaped by evolutionary (plant phylogeny), ecological (flowering time as a direct effect of summer drought) and coevolutionary (pollinator partnership) constraints. Our study utilised an extensive plant Ápollinator matrix compiled in the same area where the plants had been sampled.Using HPLC we detected 22 AA compounds/groups of compounds, out of which 15 were commonly present in almost all nectars. Among all AAs, phenylalanine was the most abundant, especially in keystone (''cornucopian'') plant species visited by many insect species, such as the majority of the Lamiaceae. Amino acid quantities were transformed into percentages (% of each AA over the total AA content of a flower). Sugar composition was similarly expressed as % of each of the three sugars (glucose, fructose, sucrose) over the total content of these sugars; a number of other sugars, occurring in only a few plant species and in very low quantities were disregarded. The number of insect species of a particular family or guild was taken as a measure of the attraction of a nectar compound for such a family (guild).We found that taxonomical plant group had a weakly significant effect on nectar composition while neither life form nor flowering season had a discernable effect. Pollinators' preference had the most important effect, with phenylalanine being the most consequent discriminatory compound for the response of the nectar consumers in phrygana, predominantly for long tongued bees, especially for Megachilidae. Gamma-aminobutyric acid (GABA) had a similar, even stronger influence on bees (long tongued bees, Anthophoridae, Andrenidae) and flies (Syrphidae and other Diptera), whereas asparagine behaved as a general repellent together with tryptophane (rather as repellent). Considering total sugar and AA contents, as well as the volume of nectar, we found that total AA content was positively related to the number of species of long tongued bees and included families visiting the phryganic species; nectar volume was negatively related to flies (both hover flies and remaining Diptera), whereas total sugar content was not significant for any guild. We argue that due to the highly concentrated nectars in the dry Mediterranean communities that are characterised by outstanding melittophily, sugars play a less important role as phagostimulants compared to AAs in floral nectars. This is why phenylalanine, a phagostimulant tested earlier on honeybees, appears to be of high importance in phrygana, especially with long tongued bees and Megachilids as the main selective agents for phenylalanine-rich nectars. The role of GABA, a strongly NaCl-dependent AA, may be similar, probably because of the associated presence of NaCl.