Proportions of Nitrate and Nitrite in Leaves and Litter

Supplementary Table 1

Proportions of nitrate and nitrite in leaves and litter.

The differing proportions of 3NPA, nitrate and nitrite in fresh leaves compared to litter pose an interesting question. The in vitro reaction catalysed by 3NPA oxidase produces 2 molecules of nitrate for every molecule of nitrite (initial reaction kinetics). However, peroxide is also generated, and additional non-enzymic reactions involving free radicals, catalase and superoxide dismutase are possible in vivo. This is particularly true in senescing leaves when cellular compartments become leaky. We had shown previously (reference 17 in main text) that the addition of catalase and superoxide dismustase to in vitro reactions will change the proportions of nitrate and nitrite produced. Nitrate ratios in litter are actually closer to what is expected, on the basis of the stochiometry of the in vitro reaction. Experimental results indicate that the proportions of nitro-compounds in leaves change as they senesce, i.e. leaves going through the stages from mature (fresh) to moribund (senescing) and finally to the ‘litter’ stage just prior to leaf drop. Data are given below in Supplementary Table 1. This table includes results obtained with water-cultured Hippocrepis comosa, Securigera varia and Coronilla viminalis, and results obtained with the Westcliff population of H. comosa. Note that the results in Supplementary Table 1a indicate large decreases in 3NPA occur as leaves are going through senescence (i.e. pre-leaf drop). In the last stages of senescence, leaves have their water supply cut off and become extremely dry while they remain on the plant. Results from other experiments also suggest that it is during this phase of senescence that the proportions of nitro-compounds change. Thus, when fresh leaves are removed and dried we see similar changes (Supplementary Table 1b).

(a) Effect of senescence on nitrite and nitrate content (μmol g-1 dry weight) of leaves (n=3 ± SE)

Leaf stage NO2 NO3 NO2:NO3

Water Culture

H. comosa

Fresh 12.2 ± 0.8 6.1 ± 1.1 2.0

Moribund 1.1 ± 0.5 1 .0 ± 0.3 1.1

Litter 0.4 ± 0.1 1.7 ± 0.1 0.23

C. viminalis

Fresh 13.6 ± 0.6 6.5 ± 0.7 2.09

Moribund 4.2 ± 0.4 3.8 ± 0.5 1.1

Litter 2.1 ± 0.1 2.5 ± 0.2 0.84

S. varia

Fresh 9.5 ± 1.2 4.4 ± 0.4 2.16

Moribund 0.7 ± 0.2 2.1 ± 0.5 0.33

Litter 0.5 ± 0.1 3.9 ± 0.3 0.13

Westcliff Population (H. comosa)

Leaf stage 3NPA nitrite nitrate

Fresh leaves 473 ± 25.7 49.7 ± 1.5 6.8 ± 1.2

Moribund leaves 185 ± 18.0 17.0 ± 0.8 38.4 ± 2.2

Litter 37 ± 2.6 3.2 ± 0.2 5.6 ± 0.7

(b)Effect of drying on 3NPA, nitrite and nitrate (μmol g-1 dry weight) in Hippocrepis comosa leaves (n=3 ± SE)

Leaf treatment 3NPA NO2 NO3 NO2:NO3

Fresh 138 ± 9.9 22.4 ± 3.4 14.6 ± 0.9 1.53

Air-dried 164 ± 13.8 17.5 ± 1.8 27.0 ± 7.6 0.65

Oven-dried 104 ± 7.4 9.5 ± 0.5 10.4 ± 1.0 0.91