The major long-term effects of grey poplar exposed to moderate salt concentrations on N metabolism were significantly increased amino acid concentrations in leaves and roots dependent on N nutrition. In leaves, salt treatment resulted in significant higher amino acid concentrations quantitatively independent of N nutrition, but with qualitative differences, whereas in roots, amino acid concentration increased more intensively in ammonium-fed plants. Growth and N metabolism analysed via NR and N transporter transcript accumulation, NR activity, and protein concentration were in general more affected by salt exposure in roots than in leaves. Application of salt reduced root diameter increment as well as stem height more intensively in ammonium-fed plants. This indicates that plants perform better under salt exposure when fed nitrate. These N nutrition-dependent effects on N metabolism of salt-exposed plants were more intensive in roots than in leaves.
N nutrition-dependent long-term response to salt exposure
The different N nutritions had impact on control plants not exposed to salt. Parameters of N metabolism (transcript level of NRT2.1 and NR, NR activity, protein concentration) and root diameter increment analysed in roots were consistently higher in nitrate than in ammonium-fed control plants. However, total N and amino acid concentrations were higher in roots and leaves of ammonium-fed plants. In leaves, protein concentration was also higher upon feeding control plants with ammonium.
Because N nutrition ameliorates tolerance towards salt, the impact of nitrogen versus ammonium on the N metabolism has been analysed in detail with regard to N uptake via transporters, N assimilation and total N accumulation including proteins and amino acids. Poplar plants were exposed to 75 mM NaCl, a concentration that was comparably low in order to study long-term effects and avoid early lethal damages. Na was taken up and distributed within the entire plant, therefore exposing all plant tissues to salt. A more detailed study on ion contents showed that also Cl ions were taken up by the plant, transported and accumulated because of salt treatment in leaves of both nitrate and ammonium-fed plants (Ache, personal communication). Photosynthetic gas exchange of Grey poplar leaves was highly affected by the salt stress and dropped up to 93%, and transpiration rates were reduced to the same extent (data not shown).
Because many studies on the physiological effects of salt stress have been performed in hydroponic solutions, we also preferred to perform our studies in hydroponic solutions. Although differences due to the choice of substrate may occur (Volkov et al. 2004; Kant et al. 2006), similar trends in overall reactions can be expected. Salt stress experiments with other poplar species grown in sand cultures present similar results than we observed with respect to growth rates (Sixto et al. 2005; Chang et al. 2006). For comparison of different data sets, not only the substrate but also the variability between clones of the same species has to be considered (Sixto et al. 2005).
As an indicator for the N uptake, transcript levels of nitrate and ammonium transporters have been analysed. The NO3- and NH4+ transporters analysed in this study as well as NR were mainly induced by NO3-. Mineral N nutrition is strongly dependent on water availability (Gessler et al. 2005), and because salinity reduces osmotic water potential and therefore the water availability, we suggest that nitrogen uptake via NO3- and NH4+ transporters is affected by salt stress. However, mRNA levels of both transporters of NH4+ supplied plants were not affected by salt treatment at all. This was consistent with the NH4+ concentration in roots, which was also not affected by the salt treatment (Supplementary Table S1). Although in nitrate-fed plants NO3- and NH4+ transporter transcripts were lower in the presence of salt than in controls, levels of transporter transcripts were still higher than in ammonium-fed plants (with or without salt). It may be possible that other members of the nitrate or ammonium transporter gene families respond more pronouncedly. However, compared to the two further genes of the NRT2 gene family, only NRT2.1 is root specific and expressed at least at a tenfold higher level than any other members of the NRT2 gene family (Selle, unpublished results), and is thus presumably the most important nitrate importer in fine roots. Furthermore, among three members of the AMT1 gene family that are expressed in fine roots to a comparable extent, AMT1.2 is the only root specifically expressed gene (Selle et al. 2005) and was thus chosen for analysis.
Upon N uptake, nitrate was subjected to reduction. Providing NO3- as the sole nitrogen source resulted in substantially higher NR transcript accumulation and NR activity in roots. Salt had only minor impact on NR transcript accumulation and NR activity. NR is the key enzyme in N assimilation and is therefore highly regulated in its transcriptional and post-transcriptional regulation. From the present experiment, it seems that moderate salt exposure does not negatively affect NR transcript accumulation and NR activity on the long term, and the plants were already adapted after 1 week of salt treatment. Effects of salinity on NR activity in other species are reported contradictory. NR activity was slightly inhibited by salt in tomato roots (Cramer & Lips 1995), in maize leaves (Abd el Baki et al. 2000) and in leaves of Bruguira parviflora (Parida & Das 2004), whereas in soybean, NR activity was stimulated (Bourgeais-Chaillou et al. 1992).
The total N concentration and the amino acid concentration increased in roots and leaves because of salt. In roots, the increase of total N concentration was independent of the N source, whereas the increase of amino acids occurred only in ammonium-fed roots. In leaves, the increase of total N was favoured in nitrate-supplied plants, while the total concentration of amino compounds increased significantly in leaves of both nitrate- and ammonium-fed plants. This suggests that changes in total N concentrations due to salt are only partially reflected by changes in amino acid concentrations. Accumulation of free amino acids under water stress has been shown in many plant species with different amino acids increasing in different species (summarized by Rai 2002). In addition, poplars grown in aerated aqueous solutions increased the concentration of amino compounds in leaves independent of N nutrition (Dluzniewska et al. 2007). Accumulation of free amino acids in stressed plants could be a consequence of several processes (Mansour 2000), for example, protein degradation (Becker & Fock 1986) and/or growth inhibition (Davies & van Volkenburgh 1983). In the present experiment, the increase of amino compounds was not due to degradation of WSP in leaves or roots (Fig. 6). However, non-soluble proteins could be decomposed and could account for the increase in amino acids (Dluzniewska et al. 2007). There was no growth inhibition of leaves (Table 1 & Fig. 2), but root DW and root diameter increment was smaller upon salt exposure when the plants were fed with ammonium. In parallel, the increase in the concentration of amino compounds in roots only occurred in ammonium-fed plants. It might be possible that the increase of amino compounds correlates with inhibited growth in roots. Other explanations for the accumulation of free amino compounds such as inhibition of protein synthesis (Dhindsa & Cleland 1975) and/or decreases in amino acid export (Tully, Hanson & Nelson 1979) were not investigated in this study. In addition, mobilization of storage proteins in bark and wood (Cooke & Weih 2005) and transport of the constituent amino compounds to the leaves and/or roots cannot be excluded from the present results.
Accumulation of amino compounds in roots and leaves in response to salt stress is thought to be connected to the compensation of salt stress by compatible solutes (Mansour 2000). Proline is 300 times more soluble in water than other amino acids and thus can act as a non-toxic osmoprotectant (Palfi et al. 1974). It is accumulating because of salt stress in many plants (Lee & Liu 1999; Khatkar & Kuhad 2000; Muthukumarasamy, Gupta & Panneerselvam 2000; Singh et al. 2000; Jain et al. 2001; Popova et al. 2003). In this study, proline was not detected in Grey poplar roots, but in leaves, proline increased more than three to four times (experiments I and II, respectively) upon salt stress. However, this increase was not specific, because a general increase of the soluble amino acid content was observed in the same order of magnitude, and other amino compounds, such as serine, increased more than 10-fold. In Populus euphratica, proline also appears to play only a minor role in cell pressure adjustment because its overall concentration is too small to compensate salt stress considerably (Ottow et al. 2005).
When amino compounds are sorted into groups of biosynthetic origin, proline belongs to the glutamate group. The major components of the glutamate biosynthetic group are glutamate and glutamine. Although proline and glutamate accumulated in response to salt stress, the relative abundance of the glutamate biosynthetic group decreased. Accumulation of glutamine upon salt stress was also detected in other plant species (Amonkar & Karmarkar 1995). Both the primary route of NH4+ assimilation as well as reassimilation of photorespiratory NH4+ produce glutamine. Changes in photorespiration may have caused the increase of glutamine in the present study, because other products of photorespiration, that is, serine, also increased substantially as a result of salt treatment independent of the N source. Serine is the precursor for cysteine. Cysteine, glutamate and glycine are important for synthesis of glutathione (GSH), which has been significantly increased because of moderate salt treatment analysed after 1 and 2 weeks (Herschbach, personnal communication). GSH synthesis is linked to photorespiration, probably because photorespiratory glycine is required for GSH synthesis (Noctor et al. 1999). In further experiments, photorespiration needs to be analysed in order to evaluate its actual impact.
In roots, the relative and absolute amounts of amino compounds changed differently upon salt treatment compared to leaves. There were no major effects of salt treatment on total amino compounds in NO3--fed roots. In NH4+-fed roots, amino acids synthesized from aspartate, with asparagine being the main component, were increased because of salt. Asparagine, synthesized via amidation of aspartate, represents an inert amino acid used as storage and/or transport of N from source to sink tissue. In parallel, amino acids of the glutamate group increased as a result of salt stress with glutamine being the major component. Glutamine is also used for long-distance N transport and may indicate more intensive allocation of amino N from the roots to the shoots.
The accumulation of specific amino acids indicates an active process of adaptation and protection in response to salinity, such as the production of osmoprotectants and compounds reactive against oxidative stress, as well as the mobilization and transformation into transport forms. However, there may be more than one function for one particular osmoprotectant, and different osmoprotectants can have different functions (Hasegawa et al. 2000).