Discussion

Nitrate and ammonium are the most important inorganic N sources for plants, but they induce different growth effects in most plants studied (Britto and Kronzucker, 2002). Results of this study clearly demonstrated invasive plant E. crassipes was able to utilize both nitrate and ammonium as nitrogen sources effectively. However, plants showed different responses in plant performance including relative growth rate and clone number, after four weeks of growth in different NH4+:NO3- sources. This differences indicates that distinct N sources modulate the growth rates of E. crassipes, with NO3- being the most efficient source and NH4 + the least efficient. NO3- gave even better growth results than NH4+:NO3- and NH4+ alone. It is well established that various plant preferences of N sources are one of the nutritional aspects affecting plant community establishment at sites where the NH4+/NO3 substrate ratio changes during succession (Kronzucker et al., 1997). While some studies reported plant prefers for NO3- over NH4+ in wetland plant, and others reported the opposite effect in submerge macrophytes (Tylova-Munzarova et al, 2005; Tylova et al, 2008). For invasive plant E. crassipes, it floats on the surface of disturbed water body but showed different preference from submerge macrophytes. Previous study demonstrated again that different plant species require different forms of N, and plant preference for different forms of N is influenced by environmental factors such as root- or air-temperature and solution pH. Both pH and temperature have been shown to affect N uptake rates and differentially affect ammonium and nitrate uptake rates in some cases (Garnett and Smethurst, 1999). For example, low root temperature generally reduce uptake rate, but may reduce nitrate uptake to a greater extent than ammonium uptake (Macduff and Jackson, 1991). As a tropic origin plant species, it may be an adaptation of E. crassipes to grow in open water columns where nitrate is the predominant N source. On the other hand, we chose pH 5.8 as being representative of acidic water columns. In general, high value of pH depressed ammonium uptake than nitrate uptake, and the preference of nitrate in E. crassipes may also result from the relatively high value of pH in nutrient media in present study.

Significant amounts of NO3- usually occur in the bulk water, top sediment layers in wetland, and nitrate as nitrogen resource is available for floating macrophytes. Our previous study showed that growth and tissue NR activity of E. crassipes was significantly stimulated by increase of nitrate availability (Li and Wang, 2007). In accordance with this, significant increases of the RGR and clone number with nitrate availability in nutrient solution were found in present study, and E. crassipes proved as a leaf nitrate reducer. On the other hand, capability of NH4+ utilization is documented in wetland plants, and NH4+ preference is often suggested as the feature of plants adapted to habitats in which NH4+ is prevalent (Cedergreen and Madsen, 2003). However, high ammonium, especially sole ammonium as nitrogen resource, results in potential toxicity to vascular plants, and this NH4 + toxicity are often correlated with increased concentrations of NH4 + in plant tissues. Although the free ammonium amounts in both shoot and root tissues of E. crassipes increased with partition of ammonium in our study, the significant increase was observed only in root especially in high NH4+/NO3- conditions. Schoerring and his coworkers (2002) reported that one of the main factors responsible for the difference observed in the sensitivity and tolerance of plants to ammonium is related to the ammonium concentration present in the root. The retention of ammonium in the roots of E. crassipes might prevent transport of ammonium to the shoot, which is more sensitive to ammonium accumulation. Results of NH4+ accumulation in roots under high NH4+/NO3- condition reported here suggested that E. crassipes is tolerant to high ammonium stress.

Figure 3. Effects of mixed nitrogen regimes on nitrate reductase activity (A) and glutamine synthetase activity (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).

Figure 3. Effects of mixed nitrogen regimes on nitrate reductase activity (A) and glutamine synthetase activity (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).

NR and GS activity was detected in leaves and roots of E. crassipes under different NH4+/NO3- conditions. Since NR is a kind of substrate-induced enzyme, the end products of nitrate assimilation such as ammonium usually inhibit NR activity (Sivasankar and Oaks, 1996). With the increase of ammonium in media, decreases of NRA in roots and leaves of E. crassipes were likely attributed to this inhibited effect of ammonium in our study. Conversely, increase of GS activity in tissues with increase of NH4+/NO3- ratio. Many studies reported that the influence of nitrogen form on GS activity may be specie-specific in plant. For instance, Kant et al. (2007) reported that GS activity increased in barley when NH4+ instead of NO3- was used as a nitrogen source. In ryegrass, however, nitrogen ionic form had no effect on GS activity (Sagi et al., 1998). Our results indicated that the mixed nitrogen regime led to increased GS activity compared to NO3- alone in E. crassipes. It has been suggested that the GS could work as an ammonium stress-adaptive mechanism and play important role in alleviation of ammonium toxicity by incorporation of ammonium to amino acids (Given, 1978). However, relatively high NH4+ concentration in leaves was observed in present study in spite of nitrogen forms. Although it has been thought that, mainly under NH4+ nutrition conditions, NH4+ had to be preferably assimilated in roots because of its possible toxic effects (Tobin and Yamaya, 2001). Significant quantities of NH4+ in xylem and apoplastic sap in NO3--fed as well as in NH4+ -fed plants was reported in previous study (Schjoerring et al. 2002). A possible explanation for higher free NH4+ content in leaves of E. crassipes is that this situation probably originated from metabolic processes in the leaves and xylem translocation. If root and leaf GS activity of E. crassipes keeps at a high level under high ammonium condition, accumulation of ammonium in tissue, which might lead to toxicity, was likely prevented. In general, the works on ammonium assimilation by distinct plant species show that plants with high GS activities are more tolerant to ammonium (Glevarec et al. 2004). Recently, Cruz et al (2006) reported that GS activity was higher in ammonium-tolerant than that in ammonium-sensitive plant species, resulting in that the amount of ammonium translocated from the roots to the shoots and the production of high ammonium concentrations in the shoots would thus decrease. Therefore, our results suggested that the main location of nitrogen metabolism (shoots or roots) and the high levels of GS activity are an important strategy for ammonium tolerance of E. crassipes.

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