Nitrate is a negative signal for fructan synthesis, and the fructosyltransferase-inducing trehalose inhibits nitrogen and carbon assimilation in excised barley leaves.

•  Fructan biosynthesis in barley (Hordeum vulgare) has been shown to be upregulated by sugar signalling and downregulated by nitrogen. The relationship between these two regulations is investigated. •  Excised third-leaves of barley were fed nitrate or glutamine under two light intensities. Other leaf blades were supplied in the dark for 24 h with nitrate and trehalose in the presence of validamycin A, a trehalase inhibitor. •  In the light, nitrate, but not glutamine, decreased fructan contents and sucrose:fructan 6-fructosyltransferase protein without affecting the levels of sucrose and other carbohydrates. In darkened leaves, trehalose increased and nitrate decreased the fructan contents and total sucrose:fructosyltransferase activity without altering the concentration of sucrose. The effect on fructan contents of trehalose disappeared, whereas that of nitrate remained in subsequent incubations in water under light. Trehalose decreased and nitrate increased the light- and CO2 -saturated rate of photosynthesis without significantly affecting the initial Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) activity. Trehalose feeding decreased the activation of nitrate reductase and amino acid levels, and blocked the positive effect of nitrate on the maximal activity of this enzyme. •  The results indicate that nitrate, and not a downstream metabolite, is a negative signal for fructan synthesis, independent from the positive sugar signalling and overriding it. Trehalose signalling inhibits nitrogen and carbon assimilation, at the same time, inducing fructosyltransferase activity.

[1]  A. J. Cairns,et al.  Fructan metabolism in grasses and cereals , 1991 .

[2]  Chungui Lu,et al.  Balancing supply and demand: the spatial regulation of carbon metabolism in grass and cereal leaves. , 2003, Journal of experimental botany.

[3]  M. Stitt,et al.  Sucrose-feeding leads to increased rates of nitrate assimilation, increased rates of α-oxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves , 1998, Planta.

[4]  Chang-Sheng Wang,et al.  Effects of nitrogen deficiency on accumulation of fructan and fructan metabolizing enzyme activities in sink and source leaves of barley (Hordeum vulgare) , 1996 .

[5]  Thomas D. Sharkey,et al.  Photometric method for routine determination of kcat and carbamylation of rubisco , 1991, Photosynthesis Research.

[6]  A. Keys,et al.  A comparison between the coupled spectrophotometric and uncoupled radiometric assays for RuBP carboxylase , 1989, Photosynthesis Research.

[7]  Yves Gibon,et al.  Steps towards an integrated view of nitrogen metabolism. , 2002, Journal of experimental botany.

[8]  T. Boller,et al.  Light and sugar regulation of the barley sucrose : fructan 6-fructosyltransferase promoter , 2001 .

[9]  Dm Knoll,et al.  Effect of nitrogen , 1987 .

[10]  W. Kaiser,et al.  Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. , 2001, Journal of experimental botany.

[11]  Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants. , 1989, The Plant cell.

[12]  H. Pontis The Role of Sucrose and Fructosylsucrose in Fructosan Metabolism , 1970 .

[13]  C. Pollock,et al.  Photosynthesis and carbohydrate metabolism in detached leaves of Lolium temulentum L. , 1985 .

[14]  B. W. Wright,et al.  Measurements of starch breakdown as estimates of glycolysis during thermogenesis by the spadix of Arum maculatum L. , 2004, Planta.

[15]  Chi-Lien Cheng,et al.  Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Morcuende,et al.  Effect of sink size on photosynthesis and carbohydrate content of leaves of three spring wheat varieties , 1993 .

[17]  R. Lilley,et al.  An improved spectrophotometric assay for ribulosebisphosphate carboxylase. , 1974, Biochimica et biophysica acta.

[18]  C. J. Nelson,et al.  Fructosyltransferase Activities in the Leaf Growth Zone of Tall Fescue , 1995, Plant physiology.

[19]  O. H. Lowry,et al.  Enzymic Assay of 10−7 to 10−14 Moles of Sucrose in Plant Tissues , 1977 .

[20]  Farrar,et al.  Carbohydrates in individual cells of epidermis, mesophyll, and bundle sheath in barley leaves with changed export or photosynthetic rate , 1998, Plant physiology.

[21]  T. Boller,et al.  Trehalose and trehalase in Arabidopsis. , 2001, Plant physiology.

[22]  M. Crespi,et al.  Sucrose and fructan metabolism of different wheat cultivars at chilling temperatures , 1990 .

[23]  R. Morcuende,et al.  Influence of nitrogen supply and sink strength on changes in leaf nitrogen compounds during senescence in two wheat cultivars , 1995 .

[24]  H. S. Srivastava Regulation of nitrate reductase activity in higher plants , 1980 .

[25]  A. J. Cairns,et al.  Characterization of the enzymatic polymerization of 2,6‐linked fructan by leaf extracts from timothy grass (Phleum pratense) , 1999 .

[26]  M. Stitt,et al.  Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco. , 1997, The Plant cell.

[27]  H. Pontis,et al.  Protein kinase and phosphatase activities are involved in fructan synthesis initiation mediated by sugars , 2001, Planta.

[28]  T. Boller,et al.  Disaccharide-mediated regulation of sucrose:fructan-6-fructosyltransferase, a key enzyme of fructan synthesis in barley leaves. , 2000, Plant physiology.

[29]  W. Van den Ende,et al.  Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.) , 2000, Planta.

[30]  E. Gallery,et al.  An improved spectrophotometric assay of urinary metanephrines. , 1986, Clinica chimica acta; international journal of clinical chemistry.

[31]  M. Stitt,et al.  Regulation of nitrate reductase expression in leaves by nitrate and nitrogen metabolism is completely overridden when sugars fall below a critical level , 2000 .

[32]  W. V. D. Ende,et al.  Effect of nitrogen concentration on fructan and fructan metabolizing enzymes in young chicory plants (Cichorium intybus) , 1999 .

[33]  Mark Stitt,et al.  Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase , 1997, Planta.

[34]  T. Boller,et al.  Trehalose induces the ADP-glucose pyrophosphorylase gene, ApL3, and starch synthesis in Arabidopsis. , 2000, Plant physiology.

[35]  R. Morcuende,et al.  Contrasting responses of photosynthesis and carbon metabolism to low temperatures in tall fescue and clovers. , 2001, Physiologia plantarum.

[36]  O. H. Lowry,et al.  Enzymic assay of 10 to 10 moles of sucrose in plant tissues. , 1977, Plant Physiology.

[37]  Mark Stitt,et al.  Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in the Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis , 1998, Planta.

[38]  P. E. Hare [1] Subnanomole-range amino acid analysis , 1977 .

[39]  Sjef Smeekens,et al.  Fructans: beneficial for plants and humans. , 2003, Current opinion in plant biology.

[40]  T. Boller,et al.  Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Chang-Sheng Wang,et al.  Fructan accumulation induced by nitrogen deficiency in barley leaves correlates with the level of sucrose:fructan 6-fructosyltransferase mRNA , 2000, Planta.

[42]  A. Wiemken,et al.  Regulation of Fructan Metabolism in Leaves of Barley (Hordeum vulgare L. cv Gerbel). , 1986, Plant physiology.

[43]  R. Simpson,et al.  Fructan formation in transgenic white clover expressing a fructosyltransferase from Streptococcus salivarius. , 2002, Functional plant biology : FPB.

[44]  C. Scrimgeour,et al.  Regulation of growth, water use efficiency and δ13C by the nitrogen source in Casuarina equisetifolia Forst. & Forst. , 1998 .

[45]  A Bach,et al.  Nitrogen metabolism in the rumen. , 2005, Journal of dairy science.

[46]  D. Dickmann,et al.  Carbohydrates in individual poplar fine roots: effects of root age and defoliation. , 2002, Tree physiology.

[47]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[48]  T. Boller,et al.  Trehalose affects sucrose synthase and invertase activities in soybean (Glycine max [L.] merr.) Roots , 1998 .

[49]  D. Arnon COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. , 1949, Plant physiology.

[50]  Chungui Lu,et al.  Rubisco Small Subunit, Chlorophylla/b-Binding Protein and Sucrose:Fructan-6-Fructosyl Transferase Gene Expression and Sugar Status in Single Barley Leaf Cells in Situ. Cell Type Specificity and Induction by Light1 , 2002, Plant Physiology.