In Alnus (Wall, Hellsten, Huss-Danell 2000) both nodule number and nodule biomass per plant decreased when N concentration was increased, but only when P was at low or medium level. At high P level nodule number and biomass per plant were stimulated by increased N. At all N levels high P stimulated nodulation. To distinguish between a P effect via general growth stimulation and a P effect specifically on nodulation we analyzed nodule biomass in relation to plant biomass. Still, high P was stimulating at all N levels. The same pattern emerged when nodule biomass was related to root biomass, indicating that the P effect was not only a general growth effect on plants or below-ground plant parts but rather a more specific effect on nodulation.
An interaction between N and P was further seen when nodulation was plotted against N/P ratio in the nutrient solution. In particular the nodule biomass showed a negative correlation with increased N/P ratio. Individual nodule size and nitrogenase activity (ARA per plant) also showed a negative correlation with increased N/P ratio in the solution.
In Trifolium (Hellsten, Huss-Danell 2001) we used only a partial factorial design (low N at low and high P, medium N and P levels, high N at low or high P). Nodule number and nodule biomass per plant were inhibited by high N when P was low. High P was stimulating at low N and at high N, i.e. there was a counteracting effect of high P. Individual nodule size and ARA per nodule biomass was stimulated by low N. High P had no effect at low N but increased individual nodule size at high N.
Growth of the Trifolium plants responded strongly to the different nutrient treatments. Low N and low P gave the smallest plants and high N and high P gave the biggest plants, about a 10-fold difference in plant biomass. Plant given medium levels of N and P were the second biggest plants. In spite of these differences in plant growth, there were marked effects of N and P when nodulation was related to plant biomass. In general, nodulation decreased with increasing N, but there was a stimulation by high P at both low and high N level.
Effects of N and P on nodulation were systemic on both nodule number and biomass. This is concluded from split-root experiments where the two sides of Alnus and Hippophae root systems were given different combinations of N and P in the nutrient solution (F. Gentili, K. Huss-Danell, unpublished). Inhibition by N was strong at the root side receiving high N and almost as strong in the second root side receiving medium N. When high P was given to one side of the root system a stimulation was seen mainly at the second side kept at medium P. There was an interaction between N and P with respect to how large N inhibition or P stimulation was obtained. Again, the effects were specific for nodulation rather than general growth effects.
In Discaria the nodule biomass in relation to plant biomass, but not the number of nodules per plant, was stimulated by P supply. N assimilation was stimulated by P, and the proportion of nodule biomass as part of plant biomass was negatively correlated to the leaf N/P ratio. This suggests that, in Discaria, P interacts with the feedback control of nodule growth that is associated with the plant demand for N, but not with the initiation of nodulation (C. Valverde, L.G. Wall, unpublished).
The importance of N/P ratio in the solution was thus evident in our experiments. We used several species and cultivation systems. On the other hand, we used the same nutrient solution in all our experiments. Moreover, the N/P ratio in solution does not necessarily reflect the internal N/P ratio of plants. Plants can adjust their N/P ratio in tissues by adjusting uptake rates and ^-fixation rates. Further, when young seedlings are studied the seed reserves of N and P, and thus the N/P ratio, varies among species (C. Valverde, L.G. Wall, unpublished). In the literature there are only few nodulation studies where N and P in plant tissues were reported. Nodule biomass as proportion of plant biomass shows a negative correlation with increasing N/P ratio in leaves or in whole plants. This is concluded from data on the actinorhizal plants Casuarina (Yang 1995; Reddell, Yang, Shipton 1997), Discaria (C. Valverde, L.G. Wall, unpublished) and Hippophae rhamnoides (F. Gentili, K. Huss-Danell, unpublished) as well as the legumes Glycine (Israel 1987; Drevon, Hartwig 1997), Medicago (Drevon, Hartwig 1997) and Trifolium (Almeida el al. 2000; Hellsten, Huss-Danell 2001). These studies comprise several species, growing conditions, nutrient solutions, plant ages, etc., and we conclude that N/P ratio in plant tissues has an important role in nodulation. Still, data are not fully conclusive because N and P in leaves or plants were measured at harvest several weeks after inoculation. It is not known how well these data reflect N and P concentrations in seedlings at time of nodule initiation.
Compared to other plant parts actinorhizal nodules are rich in P. A large need for ATP in nitrogen fixation and N assimilation is often proposed as an explanation. Frankia is a gram-positive bacterium with cell walls containing P. Further, Frankia is encapsulated by a membrane continuous with the plasmalemma and thus expected to be rich in P. However, the mechanisms and signals involved in P stimulation of nodulation and its possible interaction with N inhibition (Wall 2000) are not yet known.
Nodulation responds to the interaction between N (inhibiting) and P (stimulating). Effects of P are specific on nodulation, not only acting via enhanced plant growth. N and P act systemically. Nodule biomass as part of plant biomass is negatively correlated with increased N/P ratio in nutrient solution. Nodule biomass as part of plant biomass is negatively correlated with increased foliar or plant N/P ratio in a number of actinorhizal as well as legume species and at a number of experimental conditions. The role of P in nodulation deserves further studies, and this applies to other nutrients as well.
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Financially supported by the Swedish Natural Science Research Council, the Swedish Council for Forestry and Agricultural Research, the Swedish Foundation for International Cooperation in Research and Higher Education, Universidad Nacional de Quilmes, Agencia Nacional de Promoción Científica y Técnológia and CONICET (Argentina). Correspondence to Kerstin.Huss-[email protected]
Section 7: Proteins in Regulation and Development
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