The aim of this study was to evaluate the putative role of the sucrosyl-galactosides, loliose [␣-d-Gal (1,3) ␣-d-Glc (1,2) ␤-d-Fru] and raffinose 6) ␣-d-Glc (1,2) ␤-d-Fru], in drought tolerance of perennial ryegrass and to compare it with that of fructans. To that end, the loliose biosynthetic pathway was first established and shown to operate by a UDP-Gal: sucrose (Suc) 3-galactosyltransferase, tentatively termed loliose synthase. Drought stress increased neither the concentrations of loliose and raffinose nor the activities of loliose synthase and raffinose synthase (EC 2.4.1.82). Moreover, the concentrations of the raffinose precursors, myoinositol and galactinol, as well as the gene expressions of myoinositol 1-phosphate synthase (EC 5.5.1.4) and galactinol synthase (EC 2.4.1.123) were either decreased or unaffected by drought stress. Taken together, these data are not in favor of an obvious role of sucrosyl-galactosides in drought tolerance of perennial ryegrass at the vegetative stage. By contrast, drought stress caused fructans to accumulate in leaf tissues, mainly in leaf sheaths and elongating leaf bases. This increase was mainly due to the accumulation of long-chain fructans (degree of polymerization Ͼ 8) and was not accompanied by a Suc increase. Interestingly, Suc but not fructan concentrations greatly increased in drought-stressed roots. Putative roles of fructans and sucrosyl-galactosides are discussed in relation to the acquisition of stress tolerance.One of the strategies employed by plants to survive drought stress includes the synthesis of protective compounds, which may act by stabilizing membranes and proteins or mediating osmotic adjustment (Bohnert et al., 1995; Hare et al., 1998; Hoekstra et al., 2001). Included among these protective compounds are the water-soluble carbohydrates (WSCs), Glc, Suc, raffinose, myoinositol, and fructans.Raffinose family oligosaccharides (RFOs) such as raffinose and stachyose accumulate during seed development and are thought to play a role in the desiccation tolerance of seeds (Blackman et al., 1992; Brenac et al., 1997). Raffinose also accumulates in vegetative tissues under drought stress (Taji et al., 2002). RFO biosynthesis requires the presence of galactinol, which is formed by galactinol synthase (GolS; EC 2.4.1.123) from UDP-Gal and myoinositol. Galactinol is the galactosyl donor for the biosynthesis of raffinose from Suc by raffinose synthase (RafS; EC 2.4.1.82). Because galactinol has not been assigned any function in plants other than acting as galactosyl donor for RFOs synthesis, GolS potentially catalyzes a metabolic key step for RFO synthesis. In a recent study, two drought-responsive GolS genes were identified among seven in Arabidopsis (Taji et al., 2002). Overexpression of one of them caused an increase in endogenous galactinol and raffinose as well as an improvement in drought tolerance.In addition to GolS, myoinositol 1-phosphate synthase (INPS; EC 5.5.1.4) is another enzyme that may control the levels of galactinol and raffinose. It repr...

SummaryTwo bursts of H 2 O 2 production have been detected by in situ 3,3 H -diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The ®rst burst, which occurred immediately after wounding was inhibited by Na-diethydithiocarbamate (DIECA), a Cu/Zn±superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G-OXO) activity detected in vitro or visualized in situ by the a-chloronaphtol assay. Four genes encoding G-OXO have been identi®ed from cDNA obtained from wounded L. perenne L. leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G-OXOs of the true cereals and G-OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound-dependent increase of G-OXO activity in¯oated cut leaf blades was the result of differential induction of the four g-oxo genes. The involvement of G-OXOs in wound-induced H 2 O 2 production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G-OXO synthesis. Moreover, expression of g-oxo genes was enhanced by an exogenous supply of H 2 O 2 or methyljasmonate (MeJa). Expression of the four g-oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G-OXOs in H 2 O 2 production in oxalate-producing plant species such as ryegrass. G-OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H 2 O 2 -mediated defences against pathogens and foraging animals.

Changes in the activity of oxalate oxidase (OxO) and of the concentrations of oxalate and H 2 O 2 were investigated during the ageing of leaf sheaths of ryegrass ( Lolium perenne L.) stubble. The accumulation of H 2 O 2 during ageing coincides with the increases of both oxalate level and OxO activity. Western and Northern blot analyses using protein and RNA extracts of the different categories of leaf sheaths suggested that OxO gene expression, as well as Ca-oxalate synthesis, are crucial events of ageing for leaf sheaths. Immunocytochemistry experiments have revealed that OxO, which is an extracellular enzyme, is nearly always present in the parenchymatous cells surrounding the vascular bundles and in the cells of the lower epidermis. Overall, results suggest that in ryegrass that synthesizes both Caoxalate and OxO, the production of H 2 O 2 and Ca 2+ during ageing of stubble might be involved in the constitutive defences against pathogens, thus allowing the phloem mobilization of nutrient reserves from the leaf sheaths towards elongating leaf bases of ryegrass.

Carbohydrate and amino acid composition of phloem sap was studied in the grass Lolium perenne L., before and after defoliation. Leaf exudate was collected in a 5 mmol·L–1 EDTA solution from cut leaf blades or stubble, and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Results indicate that leaf exudates obtained from leaves devoid of petiole might not be relevant predictors of carbohydrate content of pure phloem sap. Sucrose was the dominating carbohydrate, accounting for 93% of the total soluble sugars in the phloem sap. Myo-inositol, glucose, and fructose were present in trace amounts, while fructans, raffinose, and loliose have never been detected. Predominant amino acid in the phloem sap was glutamine followed by glutamate, aspartate, and serine. Phloem sap component concentration declined during the first hours following defoliation. Sucrose was the main sugar transported in the phloem sap of Lolium perenne, despite the fact that the product of fructan degradation was fructose and not sucrose. The results are discussed in relation with the physiological mechanisms that contribute to plant recovery after defoliation.Key words: fructan, sucrose, loliose, defoliation, phloem sap, amino acids.