Atmospheric NH₃ deposition, S and N metabolism in curly kale

A well-regulated nutrient uptake is of crucial importance for every organism. Plants, as sessile organisms have developed different strategies to cope with environmental changes. Therefore, the uptake of nitrogen and sulfur are regulated in a coordinate manner and adjusted to meet the nutrient requirement for growth. Nitrate and ammonium are the major N forms that plants roots can use as nitrogen source for growth, while sulfate is the major sulfur source. Both nitrate and sulfate uptake are mediated by groups of nitrate and sulfate transporter proteins. The nitrate assimilatory pathway shares various similarities with the sulfate assimilatory pathway. Both nitrate and sulfate have to be reduced prior to assimilation into amino acids, via the GS-GOGAT cycle, and into cysteine, respectively. The synthesis of cysteine is one of the primary coupling reactions between nitrogen and sulfur metabolism. Atmospheric NH3 and H2S both can be used as reduced nitrogen and sulfur sources. The present thesis was aimed to get more insight into the possible interaction between atmospheric NH3 and pedospheric nitrate nutrition in plant growth and functioning and the interaction between nitrogen and sulfur assimilation on a whole plant basis (Chapter 1). In Chapter 2, a description of the material and methods used in this thesis is presented. In Chapters 3 and 4, comparative studies with different cultivars of Brassica oleracea L. were carried out, in order to search for plants differing in nitrogen and sulfur requirement for growth and to establish to what extent glucosinolates can contribute to the total sulfur pool, respectively. In Chapter 3, only minor differences were found in terms of growth and biochemical composition between the cultivars, while in all cultivars, the sulfur was mainly present as sulfate, representing 70-88 % of the total sulfur content. In that context, it was suggested that organic sulfur rather than total sulfur is the better parameter to calculate sulfur requirement for growth. The data in Chapter 4 showed that in all cultivars total glucosinolate content was highest in the roots. In addition, the glucosinolate fraction expressed on a total sulfur basis was very low (1-2 %) whereas the glucosinolate fraction ranged from 10-23 % when expressed on an organic sulfur basis. Therefore, expression on an organic sulfur basis, rather than on a total sulfur basis, appears to be a better parameter to assess the fraction of glucosinolates. Due to the minor differences in growth and biochemical parameters between the cultivars investigated in Chapters 3 and 4, no more comparative experiments were carried out. All further experiments were carried out with one cultivar only, Brassica oleracea L. (curly kale), the cultivar with a relatively low nitrogen content and a relatively high sulfur content. In Chapters 5 and 6, atmospheric NH3 was introduced as a tool to investigate the interaction between foliar N uptake and root nitrate uptake. In Chapter 5, B. oleracea was exposed to 0, 2, 4, 6 and 8 µl l-1 NH3 during one week and the impact on growth and N compounds was determined. Exposure to NH3 increased shoot biomass production at 2 and 4 µl l-1, but resulted in an inhibition of shoot and root growth at 6 and 8 µl l-1. Shoot total N content was increased at all levels, mainly due to the increase in free amino acids. Even at atmospheric NH3 levels, at which the foliarly absorbed NH3 would cover a proportion of the N requirement there was already an increase in the nitrogen content of the shoots and roots. Apparently, there was no direct regulatory control of and/or interaction between atmospheric and pedospheric nitrogen utilization in B. oleracea. Chapter 6 was aimed to establish to what extent NH3 can contribute to the N budget of the plant or can be regarded as a toxin. B. oleracea was exposed to 0, 4 and 8 µl l-1 NH3, with and without nitrate in the nutrient solution. Growth, N compounds, nitrate uptake rate, soluble sugars and cations were measured. In nitrate-sufficient plant biomass production was not affected at 4 µl l-1 NH3, but reduced at 8 µl l-1 NH3. The measured nitrate uptake rates agreed well with the plant’s N requirement for growth. In nitrate-deprived plants, shoot biomass was increased at both concentrations, but root biomass decreased at 8 µl l-1 NH3. The data presented do not support the hypothesis that NH3 toxicity is caused by a shortage of sugars or a lack of capacity to detoxify NH3. In Chapter 7, seedlings of B. oleracea were exposed to 4 µl l-1 NH3, in the absence and presence of nitrate and sulfate in the nutrient solution. Growth, morphological parameters of the root system and nitrate uptake were measured. In the absence of an atmospheric N source, nitrate as well as sulfate deprivation led to an increase in total root length but did not affect root surface area, volume or average diameter. NH3 exposure in combination with sulfate deprivation resulted in a less branched and thinner root system. NH3 exposure, which affects the internal N status of the plant, counterbalanced the effect of nitrate deprivation, which led to the conclusion that the N status of the plant might be important in controlling root length of B. oleracea. Nitrate uptake was expressed on a weight basis and a root length basis. The results show that nitrate uptake is rather independent of the unit of expression. In Chapter 8, B. oleracea was exposed to 4 μl l-1 NH3 and 0.15 μl l-1 H2S simultaneously, in the presence and absence of nitrate and sulfate in the nutrient solution, for one week, and growth and biochemical parameters were assessed. Unexposed plants, which were deprived of nitrate or sulfate for a week, became N deficient, but not yet S deficient. In the absence of nitrate and sulfate, combined NH3+H2S exposure was beneficial for B. oleracea, since the atmospheric N and S sources were used for growth. However, exposure to both atmospheric nutrient sources did not change biomass allocation in favor of the root in the nitrate- and sulfate-deprived plants. Biochemical analysis revealed that nitrogen nutrition, either by nitrate supply to the root or NH3 exposure to the shoot, mainly affected total N and free amino acid content, whereas sulfate supply to the roots and H2S exposure mainly affected total S and thiol content. The lack of correlation between changes in thiol and OAS pools, under different atmospheric conditions, suggests that their role in the coordination between the nitrate and sulfate metabolic pathways is limited. In Chapter 9, nutrient requirement for growth and the effects of sulfate deprivation in B oleracea were aspects selected for discussion. In addition, the dual role of atmospheric NH3, as nutrient and toxin, is discussed