Result

RGR and Clone Growth

Considered the toxically effect of high concentration ammonium on plant, the effect of ammonium on plant growth was detected in order to set up a specific total nitrogen concentration in this study. In terms of relative growth rate, our results showed that plant responses varied between treatments, depending on supplied ammonium concentration (Fig.

IA). The optimal plant growth (0.072 ± 0.007 gg-1d-1) was found in plants supplied with 5 mM ammonium, and RGR of E. crassipes increased for ammonium concentrations ranging from 0.5 mM to 5mM. RGR (0.069 ± 0.005 gg-1d-1) slightly decreased by 4.2% when ammonium concentrations increased from 5mM to 7mM, but there was no significant difference. According to the above results, specific total nitrogen concentration of 5 mM was selected for further study.

After 28 days of incubation in different nitrogen sources, plants supplied with sole nitrate showed better vegetative appearance than those grown in the other NO3-:NH4+ ratios. The highest RGR was 0.094±0.006 gg-1d-1, and E. crassipes RGR reduced with decrease of partition of nitrate in nutrition solution. Ammonium as the only nitrogen source resulted in relatively low growth rates (0.071±0.005 gg-1d-1) when compared to plants grown with other NO3-:NH4+ ratios (Fig. 1A). Similar nitrogen form effect was observed in clone numbers (Fig.

IB). The clone number slightly increased with increase of partition of nitrate in solution, and the highest clone number was 6.75±0.96 in sole nitrate-supplied plants. Except for nitrate-fed plants, there was no significant difference of clone number among different NO3-:NH4+ treatments.

Figure 1. Effects of mixed nitrogen regimes on growth (A) and generated clone number (B) of E. crassipes. 5 mM of nitrogen was supplied to plants composed with different ratios of NaNO3and NH4Cl. Values are the means of four replicates ± SE. Different letters indicate significant differences

among treatments at P < 0.05 (one-way ANOVA's Tukey test).

Nitrate and Ammonium Concentration

The nitrate concentration was always higher in leaves than that in roots regardless nitrogen form, and it significantly increased in both parts as the nitrate concentration increased in the medium (Fig. 2A). When nitrate was the sole nitrogen source, the highest nitrate concentration in root and leaf were found, which were 6.7 and 7.1 folds higher than that in ammonium-fed plants, respectively. Similarly, higher ammonium concentration was observed in leaves. The free ammonium amounts in both shoot and root tissues were relatively low in nitrate treated plants, and it increased in both parts with increase of ammonium partition in the medium (Fig. 2B). However, changes of ammonium concentration in roots and leaves showed different pattern. Roots ammonium concentration significantly increased with increase of partition of ammonium in solution. The highest ammonium concentration was observed in ammonium-fed plants (1.94±0.14^mol g-1FW), which was four folds than that in nitrate-fed plants. For ammonium content in leaves, it only significantly increased with partition of ammonium when relatively high nitrate form was available in nutrient solution. Under high ammonium condition, there were no significant differences between treatments. For example, the highest free ammonium contents of leaf tissues (2.20±0.15^mol g-1FW) were found in plants grown in the presence of ammonium (NO3-/NH4+ 0:100). The ammonium contents of leaves were not significantly different and increased as ammonium was supplied in greater proportion (NH4+ /NO3- >1).

Nitrate Reductase Activity and Glutamine Synthetase Activity

The youngest fully expanded leaves and new-formed roots were used to detect NRA in E. crassipes. NRA in roots and leaves were given in Fig. 3A. The results showed that root and leaf NRA in E. crassipes were significantly affected by different nitrogen sources, and increased with nitrate partition in media solution. In all NH4+ /NO3- proportions treatments, the activity of NR showed the same pattern of distribution among plant parts, and high proportion of NRA of E. crassipes was observed in leaves. Opposite to NR activity, GS activity in both roots and leaves decreased with increase of nitrate partition in the media (Fig. 3B). The highest rates of GS activity were found in the leaves of E. crassipes plants supplied with sole ammonium. NH4+ /NO3- significantly affected leaf GS activity in plants grown in the media containing NO3- as the major nitrogen form. When partition of ammonium in the media increased, leaf GS activity was only slightly influenced by NH4+ /NO3- ratio. For GS activity in E. crassipes roots, there were significantly differences among various treatments.

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Figure 2. Effects of mixed nitrogen regimes on tissue nitrate concentration (A) and free ammonium content (B) in root (black) and leaf (white). 5 mM of nitrogen was supplied to plants composed with different ratios of NaNO3and NH4Cl. Values are the means of four replicates ± SE. Different letters indicate significant differences among treatments at P < 0.05 (one-way ANOVA's Tukey test).

100:0

75:25

50:50

25:75

NH4 / NO3 in media

100:0

75:25

25:75

0:100

NH4 / NO3 in media

Figure 2. Effects of mixed nitrogen regimes on tissue nitrate concentration (A) and free ammonium content (B) in root (black) and leaf (white). 5 mM of nitrogen was supplied to plants composed with different ratios of NaNO3and NH4Cl. Values are the means of four replicates ± SE. Different letters indicate significant differences among treatments at P < 0.05 (one-way ANOVA's Tukey test).

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