Sucrose assimilation during early developmental stages of chicory (Cichorium intybus L.) plants.

The activities of enzymes of both sucrose and fructan metabolism were measured in chicory (Cichorium intybus L. cv. Turbo) plants during early vegetative growth. From 21 to 42 d after sowing (phase I), carbohydrates were used for structural growth and sucrose was predominantly cleaved by acid invertase whereas neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) activities were low. From 49 to 63 d after sowing (phase II) a cambium formed producing secondary tissues, concomitant with induced sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) and fructan:fructan-1-fructosyl transferase (EC 2.4.1.100) activities, and fructan synthesis in the roots. Accumulation of 1-SST mRNA occurred at the onset of thickening, indicating that 1-SST is controlled at a transcriptional level. Acid invertase activity gradually increased during phase I and remained high during early phase II. It subsequently decreased. The pattern of invertase mRNA accumulation correlated with the enzyme activities, indicating that acid invertase is controlled at the transcriptional level. Both acid invertase and 1-SST probably contributed to the sink strength in the root at the beginning of phase II.

Nitrate assimilation in chicory roots (Cichorium intybus L.) which acquire radial growth.

Nitrate assimilation was analysed in chicory plants (Cichorium intybus L. cv. Turbo) during the early vegetative growth. Nitrate reductase (NR, EC 1.6.6.1) activity (NRA) was measured in roots and leaves at different developmental stages. During phase I, which corresponds to the structural growth (21-42 DAS), nitrate reduction mainly occurred in the roots. At the onset of the tuber formation (phase II), which is characterized by the formation of a cambium inducing a radial growth (42-63 DAS), NRA rapidly decreased in roots and developed in leaves. A tight correlation was found between the nitrate content, the amino acid level and NRA in roots and leaves. Northern blot and ELISA analysis showed that both levels of NR mRNA and NR protein were not modified during the time-course of the experiment suggesting that modification of nitrate assimilation was not controlled at a transcriptional level. In vitro NRA assayed in presence of either Mg2+ ions or EDTA showed that NR was influenced at least in part by a reversible phosphorylation/dephosphorylation reaction. Okadaic acid, a serine-threonine protein phosphatases inhibitor, strongly decreased NRA. Conversely, staurosporine, a serine-threonine protein kinases inhibitor, did not significantly change NRA in roots or leaves. Therefore, NRA was regulated at a post-translational level during the early vegetative growth by modifying the phosphorylation balance of the NR protein in chicory.

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots.

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 nM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.