Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.

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I. 1997. Impact of elevated atmospheric CO2 on nitrate reductase transcription and activity in leaves and roots of Plantago major. -Physiol. Plant. 100: 940-948.Vegetative plants of an inbred line, A4, of Plantago major ssp. pleiosperma (L.) Pilger were grown at 350 )al 1"' or at elevated (700 \il T') CO2 in non-limiting nutrient solution with nitrate. Both the relative growth rate (RGR) and the root to total plant weight ratio (RWR) were increased by elevated CO2. However, the stimulation of both RGR and RWR was transient and did not last longer than 8 days. To investigate the physiological mechanisms involved in this stimulation, related changes in C/N metabolism were examined. In the roots soluble sugar concentration increased during the transient period of RGR stimulation (up to 23%), as did the root respiration rate. Changes in nitrogen metabolism were also restricted to this period and consisted of an increase in (1) in vivo and in vitro root nitrate reductase (EC 1.6.6.1) activity. (2) in vitro leaf nitrate reductase activity, (3) leaf and root nitrate reductase mRNA and (4) reduced nitrogen concentration in the roots. The elevated COa-related signal for the increase in nitrate reductase transcript levels in the roots is discussed in terms of the increased availability of soluble sugars. The results suggest that the short-term enhancement of root carbon and nitrogen metabolism may be responsible for the transient effect of elevated CO2 on whole plant RGR.

Doubling the atmospheric CO2 concentration from 350 to 700 μ1 1−1 increased the relative growth rate (RGR) of hydroponically grown Urtica dioica L. and Plantago major ssp. pleiosperma Pilger only for the first 10–14 days. Previous experiments with P. major led to the conclusion that RGR did not respond in proportion to the rate of photosynthesis. The present paper is focussed on the analysis of the impact of changes in leaf morphology, dry matter partitioning, dry matter chemical composition and ontogenetic drift on this discrepancy. Soon after the start of the treatment, carbohydrate concentrations were higher at elevated CO2: a reaction that was largely due to starch accumulation. An increase in the percentage of leaf dry matter and decreases in the specific leaf area (SLA) and the shoot nitrogen concentration were correlated with an increase in the total nonstructural carbohydrate concentration (TNC). A combination of accumulation of soluble sugars and starch and ontogenetic drift explains the decrease in SLA at the elevated CO2 level. A similar ontogenetic effect of elevated CO2 was observed on the specific root length (SRL). Other variables such as shoot nitrogen concentration and percentage leaf dry matter were not affected by correction of data for TNC levels. The net diurnal fluctuation of the carbohydrate pool in P. major was equal for both CO2 concentrations, indicating that the growth response to elevated CO2 may be ruled by variables other than photosynthesis, as for instance sink strength. Elevated CO2 did not greatly influence the partitioning of nitrogen between soluble and insoluble, reduced N and nitrate, nor the allocation of dry matter between leaf. stem and root. The finding that the root to shoot ratio (R/S) was not affected by elevated CO2 implies that, in order to maintain a balanced activity between roots and shoot, no shift in partitioning of dry matter upon doubling of the atmospheric CO2 concentration is required. Our data on R/S are in good agreement with the response of R/S to high CO2 predicted by models based on such a theorem.

The effects of a two weeks soil drying period on the activity of nitrate reductase (NR; EC 1.6.6.6) were studied on Helianthus annuus L. and non-nodulated Lupinus albus L. plants, growing under two nutrient supply regimes. NR activity was assessed in leaf and root extracts, by measuring the activity of the unphosphorylated active form (NR act ), the maximal extractable activity (NR max ) and the activation state. To get insight into potential signalling compounds, nitrate, amino acids and soluble sugars concentrations were also quantified. On both species, foliar NR act and NR max were negatively affected by soil drying and reduced supply of nutrients, the observed changes in NR activity being linearly-correlated with the depletion of nitrate. Similar results were obtained in the roots of sunflower. Conversely, in white lupin roots NR max was found to be independent of tissue nitrate concentration. Regardless of the species and organ, the activation state of the enzyme was unaffected by the nutrient supply regime. In well-watered sunflower roots only about 50% of the existing NR was unphosphorylated, but the activation state increased significantly in response to drought. In contrast, lupin roots always exhibited NR activation state values close to 80% or even higher. At the leaf level, NR activation state was hardly changed in response to soil drying. The contribution of changes in the concentrations of soluble 5 sugars and amino acids to explain the observed variations in NR activity are discussed. AbbreviationsFW, Fresh weight; NR, Nitrate reductase; NR act , activity of the unphosphorylated form of nitrate reductase; NR max , maximal extractable activity of nitrate reductase