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Ionomic and physiological responses to low nitrogen stress in Tibetan wild and cultivated barley
Plant Physiology and Biochemistry 111 (2017) 257e265
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Ionomic and physiological responses to low nitrogen stress in Tibetan wild and cultivated barley Xiaoyan Quan, Jianbin Zeng, Zhigang Han, Guoping Zhang* Agronomy Department, Institute of Crop Science, Zhejiang University, Hangzhou, 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2016 Received in revised form 26 November 2016 Accepted 4 December 2016 Available online 6 December 2016
In a previous study, we identified the low-nitrogen (LN) tolerant accessions from the Tibetan wild barley (Hordeum vulgare subsp. spontaneum). In this study, two wild barley genotypes (XZ149, LN-tolerant and XZ56, LN-sensitive) and a barley cultivar ZD9 (H. vulgare) were used to determine the LN tolerant mechanism underlying the wild barley in the ionomic and physiological aspects. XZ149 exhibited higher LN tolerance with highest relative dry weight and N accumulation among three barley genotypes under LN stress. When exposed to LN stress, XZ149 had more N transportation from roots to leaves, and remained relatively higher activities of nitrate reductase (NR, EC.1.7.1.1) and glutamine synthetase (GS, EC.6.3.1.2) in leaves than other two genotypes, ensuring its higher capacity of N assimilation and utilization. The ionome analysis showed that LN stress had a significant effect on tissue ionome and the effect was genotypic and tissue-specific difference. On the whole, XZ149 maintained more stable Mn and Cu contents in roots, and less reduction of root P, K and Ca contents than XZ56 and ZD9 when exposed to LN stress. It may be assumed that more N movement into shoots, greater N assimilating capacity and specific rearrangement of nutrient element levels in tissues under LN stress are attributed to LN tolerance in XZ149. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Glutamine synthetase Ionome Nitrogen Nitrate reductase Tibetan wild barley
1. Introduction Nitrogen (N) is an essential mineral nutrient required in quantity and is frequently a key factor limiting crop yield and quality (Marschner, 2012). However, the environmental problems brought by the excessive N fertilizer application in crop production become increasingly severe (Socolow, 1999). Thus, well understanding of the mechanisms of low nitrogen tolerance is imperative for improving N use efficiency. The Tibetan annual wild barley has been proved as one of the ancestors of cultivated barley (Dai et al., 2012), and shows generally better adaption to poor soil fertility, including N deficiency (Quan et al., 2016) and K deficiency (Zeng et al., 2015). Some wild barley genotypes with high LN tolerance have been identified in our previous study (Yang et al., 2014), providing elite materials for understanding the mechanisms of LN stress tolerance. Extensive studies have been performed on plant biomass, nitrate uptake and root architecture to link the traits with LN stress tolerance (Brouwer, 1962; Drew and Saker, 1975; Lea and Azevedo,
* Corresponding author. E-mail address: [email protected] (G. Zhang). http://dx.doi.org/10.1016/j.plaphy.2016.12.008 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved.
2006). Nevertheless, it is less documented about the fine-tuning of plant metabolism under LN stress. Meanwhile, adaptation to steady-state low N in plants is also poorly studied (Forde and Lea, 2007). It would be of significance to obtain an overview of the modifications in plant N metabolism when subjected to N stress. In fact, some N-containing compounds and enzyme activity have been used as diagnostic indicators to reveal the N level response of the different plant genotypes. Meanwhile, plants require at least 13 mineral elements for adequate development, including so-called macronutrients phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) in addition to N, as well as iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), boron (B), chloride (Cl), nickel and molybdenum, which are referred to as micronutrients (Marschner, 2012). All of these elements are associated with plant growth and crop yield, and in many instances, deficiency of one element interconnects with the metabolism of other nutrients under crosstalking regulation (Schachtman and Shin, 2007; Liu et al., 2009). Their interactions have been highlighted in some recent reviews (Amtmann and Armengaud, 2009; Williams and Salt, 2009). For example, N metabolism and allocation could be altered by B and Fe bal deficiency in tobacco and cucumber separately (Camacho-Cristo
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lez-Fontes, 1999; Borlotti et al., 2012). Similarly, improve and Gonza N status enhanced root uptake, remobilization and root-to-shoot translocation of Zn in wheat and ammonium toxicity decreased levels of Ca and Mg in cucumber (Erenoglu et al., 2011; Roosta and Schjoerring, 2007). These results demonstrate that mineral homeostasis in plants is highly monitored, indicating that changes in availability of a single element could exert an effect on the uptake and accumulation of other elements in plants. Thus, LN stress may change mineral balances in plants, directly or indirectly affecting mineral accumulation (Kutman et al., 2011; Xue et al., 2012; Schreiner et al., 2013). However, the genotypic responses of elements and their interactions to LN stress have not been fully elucidated up to date. The ionome has been defined as the mineral nutrient composition of an organism or tissues (Salt et al., 2008). Ionomics is the study of elemental accumulation in living organisms using highthroughput elemental profiling. There are four major techniques in the studies of ionomics, i.e. neutron activation analysis (NAA), inductively coupled plasma-atom/optical emission spectrometry (ICP-AES/OES), inductively coupled plasma-mass spectrometry (ICP-MS) and X-ray fluorescence (XRF) (Wu et al., 2013). Highthroughput elemental profiling has been applied to study the ionome response to the environment or the genetics underlying the changes of ionome over the environments (Baxter, 2009). For instance, multivariable ionomic signatures in Arabidopsis were established to investigate physiological responses using ICP-MS, such as P and Fe homeostasis (Baxter et al., 2008). In addition, ionome has been employed on the studies of element contents under different N supply (Watanabe et al., 2015; Lecourt et al., 2015). However, almost no experiment has been reported on studying the effect of LN stress on plant ionome profiles comprehensively. In the present study, we employed two wild barley genotypes (XZ149, LN-tolerant and XZ56, LN-sensitive) and one barley cultivar (ZD9) to study the effect of LN on N metabolism and ionomic changes using ICP-OES with aims at understanding the mechanisms of LN stress tolerance underlying Tibetan wild barley.
For ionome analysis, only the plants exposed to 2 and 0.2 mM N were used. The plant samples were taken at 18 d after N treatment. Dry roots and shoots were finely ground, and approximately 0.2 g tissue samples were predigested with a mixture of 6 ml HNO3 and 1 ml of H2O2 for about 20 min at 130 C, and then digested in a microwave (Multiwave 3000, Anton Paar GmbH, Australia) after adding 1 ml of H2O2. The digested solution was boiled to eliminate acid for 1.5 h at 160 C. The contents of K, P, Mg, Ca, Zn, Fe, Cu and Mn were determined using an ICP-OES spectrometer (Optima 6000 series, PerkinElmer Inc, USA). 2.3. Data statistics Significant difference for physiological traits and element
2. Materials and methods 2.1. Plant materials and treatments Healthy seeds of the two Tibetan wild barley accessions XZ149 (LN tolerant) and XZ56 (LN sensitive), a cultivar ZD9 were germinated in a plant growth chamber (22/18 C, day/night). Ten-day-old seedlings with uniform size were transplanted into black plastic containers (5 L) with aerated hydroponic solution in a greenhouse with natural light. The hydroponic solution was prepared according to Quan et al. (2016), and renewed every five days. At three-leafstage, seedlings were exposed to 2 mM N (control), 0.2 mM N (LN) and 0 mM (N starvation). 2.2. Sampling and measurement The plant samples were taken and separated into shoots (or leaves and stems) and roots at 14 d after N treatment. Six biological replications were prepared for dry weight, and oven-dried for use in total N content measurement. Meanwhile fresh plant tissues were taken for physiological analysis, with three biological replications. Soluble protein content was determined according to Andrews et al. (1999), and nitrate-N content was determined according to Mosier et al. (1998). NR activity was measured according to Kaiser et al. (1999). GS activity was measured as described by MasclauxDaubresse et al. (2006).
Fig. 1. Effects of low nitrogen stress on dry weight (mg.plant¡1) of three barley genotypes. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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contents among treatments and genotypes was analyzed using a data processing system (DPS) software. Principal component analysis (PCA) of element contents was carried out to separate samples and elements using Metaboanalyst 3.0 (Xia et al., 2015). Relative change was calculated by the value of LN stress/control. N accumulation (mg.plant1) ¼ N concentration dry weight/plant. 3. Results 3.1. Physiological difference among barley genotypes in response to N treatment LN stress (0.2 mM N) and N starvation (0 mM N) inhibited shoot growth and promoted root growth of all genotypes (Fig. 1). There were the distinct differences in dry weight, N accumulation, nitrate N content and soluble protein content among the three genotypes in response to N treatment. In comparison with control (2 mM N), shoot dry weight was reduced by 13%, 32% and 30% under LN and 42%, 51% and 67% under N starvation in XZ149, XZ56 and ZD9, respectively. By contrast, root dry weight was increased by 35%, 28% and 31% under LN stress, and 72%, 38% and 82% under N starvation. In particular, total dry weight of XZ149 was significantly smaller than that of the other two genotypes under normal N condition, but significantly higher under LN and N starvation conditions. In comparison with control, LN and N starvation treatments had significantly lower N concentration and accumulation for all genotypes (Fig. 2). Overall, there was little genotypic difference in N concentration, but the difference was significant in N accumulation (Fig. 2), with XZ149 being significantly higher than other two
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genotypes under LN or N starvation. As shown in Fig. 2, LN and N starvation reduced nitrate N content by 64%e83% in shoots and 33%e88% in roots among the three genotypes, respectively, whereby XZ149 had less reduction than XZ56 under LN and N starvation conditions (Fig. 2). Notably, nitrate N content in ZD9 was significantly higher than that of the two wild genotypes under LN stress. Both LN and N starvation significantly reduced soluble protein contents in leaves and roots of all barley genotypes (Fig. 3). Compared with XZ56, XZ149 showed less reduction of the soluble protein content in leaves and roots under both LN and N starvation. Soluble protein content in leaves of ZD9 remained higher under all N levels, and showed less change under LN and N starvation in comparison with the two wild barley genotypes. LN stress caused a dramatic reduction of nitrate reductase (NR, EC.1.7.1.1) activity (Fig. 3). However the reduced extent differed among three barley genotypes, with XZ149 being slightly lower than the other two genotypes. As expected, no NR activity was detected in the plants exposed to N starvation. There was no significant difference among three barley genotypes in glutamine synthetase (GS, EC.6.3.1.2) activity under control (Fig. 4). However LN and N starvation treatments caused the dramatic increase of GS activity in roots and stems of all genotypes, with X149 and ZD9 having the greatest increase under N starvation in stems and roots, respectively. On the other hand, leaf GS activity showed relatively smaller change in comparison with that in roots and stems. Moreover X149 and XZ56 showed significantly higher leaf GS activity under N starvation than the control, while ZD9 remained little change.
Fig. 2. Total nitrogen accumulation (mg.plant¡1) and nitrate N content (mg.g¡1 FW) of three barley genotypes under different N levels. FW, fresh weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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3.2. Tissue ionomic difference among barley genotypes in responses to LN stress To obtain an overall view of ionomic responses, PCA was conducted on the element contents in shoots and roots of the three genotypes under both control and LN stress. The first PCA components (PC1) may explain 42.1% and 44.8% of the total variance in leaves and roots, respectively, and could separate the samples of control and LN stress, indicating that LN stress had substantial effect on element content (Fig. 5a, c). Ca, Fe and Zn contents in shoots, and P, Mn, Mg, K, Fe, Ca contents in roots were associated with control components (Fig. 5b, d). On the other hand, P, K, Mg and Mn contents in shoots and Zn content in roots were the specific loadings for the LN-related principal components (Fig. 5b, d). Genotypic difference was reflected by the second principal component (PC2), which clearly separated the samples of XZ149 from those of XZ56 and ZD9 in shoots, and the samples of XZ149 and XZ56 from those of ZD9 in roots (Fig. 5a, c). Mn, Zn and Mg contents contributed to the variation of XZ149 in shoots, while Ca, K, Zn and Mg contents were dominant in the root ionome (Fig. 5b, d). Obviously, the response of element contents to LN stress varied with treatments and genotypes. Concerning element contents in shoots, LN stress significantly increased K and P contents and reduced Ca content in all three barley genotypes, and increased Mg content in the two wild genotypes. Whereas Mg content in ZD9 was reduced. In comparison with control, LN-treated plants had much higher K content in XZ149 than that in XZ56 and ZD9 (Fig. 6). The contents of P, K, Ca and Mg in roots were significantly reduced in the three barley
genotypes under LN stress, varying from 6.7% of K to 30.3% of Mg. The P, K and Ca contents in roots was less changed in XZ149 relative to XZ56 and ZD9 under LN stress (Fig. 7). On the whole, microelement contents in shoots decreased under LN stress in comparison with control, with Fe and Cu contents decreasing dramatically in ZD9 and showing less change in XZ149 and XZ56. Mn content decreased in XZ149 and remained unchanged in ZD9 and XZ56; Zn content decreased in XZ149 and ZD9, and remained unchanged in XZ56 (Fig. 8). In roots, LN stress significantly reduced Fe content in ZD9, and Cu and Mn contents in XZ56 and ZD9 (Fig. 9). Zn content in XZ149 and ZD9 increased but unchanged in XZ56 (Fig. 9). In roots, the microelement content was less affected in XZ149 compared with XZ56 and ZD9 (Fig. 9). 4. Discussion In order to understand the mechanisms of LN stress tolerance in Tibetan wild barley, two wild barley genotypes, XZ149 and XZ56, differing in LN tolerance and a barley cultivar, ZD9 were used to determine the genotypic difference in physiological and ionome responses to LN stress. 4.1. More nitrate transportation to shoots and higher GS activity in leaves are the major traits associated with higher LN tolerance in XZ149 XZ149 was identified as a LN-tolerant and XZ56 as a LNsensitive wild barley genotype according to the previous studies (Yang et al., 2014; Quan et al., 2016). In this study, XZ149 again
Fig. 3. Soluble protein content (mg.g¡1 FW) and NR activity (mg.g¡1.h¡1) of three barley genotypes under the different N levels. FW, fresh weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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exhibited higher LN tolerance than the other two genotypes, characterized by its better growth performance and higher capacity of N absorption under LN stress (Figs. 1 and 2). Nitrate taken up via roots would be most reduced and assimilated in leaves (Lemaître et al., 2008). In our previous study, XZ149 transported more nitrate to shoot for assimilation under LN stress in comparison with other two genotypes (Quan et al., 2016). Currently, the leaves of XZ149 remained less change of NRA activity relative to XZ56 under LN stress (Fig. 4), indicating that the former contains high nitrate level under LN stress. Correspondingly, XZ149 remained relatively higher soluble protein content than XZ56 under LN stress (Fig. 3). The ammonium produced from reduced nitrate is assimilated by GS. The current results showed that GS activity was significantly higher in LN-treated plants than those exposed to normal N supply (control) (Fig. 5). The increase of GS activity under LN stress could be attributed to higher GS1 protein content when N is limited (Diaz et al., 2008). GS1 isoforms are regarded as the markers of N remobilization (Martin et al., 2005). In view of reduced NR activity in leaves under LN stress, it may be suggested that leaves start to mobilize nitrogen efficiently during LN stress, which is consistent with the idea that N limitation facilitates N remobilization, acting as an exogenous senescence-triggering factor (Wingler et al., 2004). Therefore, GS is not only the key enzyme in the process of inorganic N assimilation into amino acid glutamine, but also plays an important role in the process of reactivating N (Miflin and Habash, 2002; Bernard and Habash, 2009; Kumada et al., 1993); helping plants to adapt to LN stress (Fuentes et al., 2001; Vincent et al., 1997). In the present study, compared with XZ56, XZ149 had higher leaf GS activity under LN stress, indicating that the genotype has the higher ability of N assimilation or remobilization when N is limited. It is well documented that a high level of GS activity in roots is negatively correlated with above-ground biomass (Limami et al., 1999; Fei et al., 2003). Here, relative root GS activity was higher in XZ56 than XZ149 under LN stress, being consistent with their growth performance. However, root GS activity was increased under LN stress, which seems to be confliction with the previous study that one gene encoding GS1 was down-regulated in root of XZ149 under LN stress. The different GS1 isoforms show the specific locations and function in N assimilation and recycling (MasclauxDaubresse et al., 2008). The function of root GS1 in the primary N assimilation is not well understood, especially for species primarily assimilating N in shoots. Thus, further studies are necessary to confirm contribution of GS1 in total N assimilation, especially under the condition of N limitation.
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et al., 2009; Schlüter et al., 2012). In the present study, P content increased in shoots and decreased in roots under LN stress. Moreover there was a dramatic difference among the three genotypes in the response of P content to LN stress, with XZ149 being least changed by LN stress. LN stress also significantly affected the elements related to photosynthesis. Fe is associated with the carbon assimilation capacity and it plays a key role in the synthesis of chlorophyll and electron transferring in photosynthesis. In addition, Fe is also a coenzyme factor of NR, nitrite reductase and glutamate synthase in the pathway of nitrate reduction (Borlotti et al., 2012), closely related to N metabolism. It was reported that a high concentration of nitrate increased pH in the protoplast, leading to inadequate iron absorption (Smolders et al., 1997; Nikolic et al., 2007). The current
4.2. Specific rearrangement of nutrient contents in XZ149 under LN stress is attributed to its higher LN tolerance N deficiency in plants will disrupt the multiple metabolic and energy pathways, changing energy supply and transporter activity, eventually cause an imbalance in the uptake and translocation of some essential elements in plant tissues. In fact, the results of ionomic analysis indicated that LN stress had a significant effect on the ionome in barley plant tissues. First of all, LN stress causes the significant change in the contents of structure elements. P is one of the important substrates for biomembrane synthesis and energy metabolism, participating in the regulation of electron transport and oxidative phosphorylation in mitochondria and photophosphorylation in chloroplast. It was reported that LN stress increased P content in grape leaves (Schreiner et al., 2013). It was found that N availability affects absorption and accumulation of P in many plant species, whereas the results varied with plant species, treating time and P level in the nutrient solution (Urbanczyk-Wochniak and Fernie, 2005; Tschoep
Fig. 4. GS activity (U.mg¡1 prot) changes of three barley genotypes under different N levels. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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Fig. 5. Analysis of ionome variation using principal component analysis (PCA) and loadings of elements to the PC1 and PC2 in shoots and roots under the two N levels. (a) PCA in shoots; (b) loadings in shoots; (c) PCA in roots; (d) loadings in roots. PC1, the first principal component; PC2, the second principal component.
results showed that LN stress significantly reduced Fe content of barley cultivar ZD9, but had less effect on the two wild barley genotypes in both shoots and roots, suggesting that the wild barley has relatively stable capacity of Fe uptake and translocation under LN stress. In addition, Zn is also essential for chlorophyll synthesis. LN stress caused Zn accumulation in roots of XZ149 and ZD9, limiting its translocation to the shoots, which was consistent with the results found in wheat (Kutman et al., 2011; Erenoglu et al., 2011; Xue et al., 2012). Mn participates in the structure of
photosynthetic enzymes and proteins (Millaleo et al., 2010). In this study, Mn content was reduced in roots of XZ56 and ZD9 under LN stress, but remained less change in XZ149. The current results showed that the specific rearrangement of nutrient element levels in plant tissues of XZ149 under LN stress is attributable to the enhanced LN tolerance. In view of the complicated crosstalk between elements, further researches are required to reveal the ways of the nutrient rearrangement in responses to LN tolerance.
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Fig. 6. Shoot macronutrient content (mg.g¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
Fig. 7. Root macronutrient content (mg.g¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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Fig. 8. Shoot micronutrient content (mg.kg¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
Fig. 9. Root micronutrient content (mg.kg¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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Authors contributions XQ and GZ designed research. XQ, QQ and ZH performed research. XQ analyzed data. XQ, JZ and GZ wrote the paper. All authors have read, edited and approved the current version of the manuscript. Acknowledgments We thank Prof. Dongfa Sun (Huazhong Agricultural University, China) for providing Tibetan wild barley accessions. This work was supported by Natural Science Foundation of China (31330055), China Agriculture Research System (CARS-05) and Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP). References Amtmann, A., Armengaud, P., 2009. Effects of N, P, K and S on metabolism: new knowledge gained from multi-level analysis. Curr. Opin. Plant Biol. 12, 275e283. Andrews, M., Sprent, J.I., Raven, J.A., Eady, P.E., 1999. Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies. Plant Cell Environ. 22, 949e958. Baxter, I., 2009. Ionomics: studying the social network of mineral nutrients. Curr. Opin. Plant Biol. 12, 381e386. Baxter, I.R., Vitek, O., Lahner, B., Muthukumar, B., Borghi, M., Morrissey, J., et al., 2008. The leaf ionome as a multivariable system to detect a plant's physiological status. Proc. Natl. Acad. Sci. U. S. A. 105, 12081e12086. Bernard, S.M., Habash, D.Z., 2009. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol. 182, 608e620. Borlotti, A., Vigani, G., Zocchi, G., 2012. Iron deficiency affects nitrogen metabolism in cucumber (Cucumis sativus L.) plants. BMC Plant Biol. 12, 1. Brouwer, R., 1962. Nutritive influences on the distribution of the dry matter in the plants. Neth J. Agric. Sci. 10, 399e408. bal, J.J., Gonz Camacho-Cristo alez-Fontes, A., 1999. Boron deficiency causes a drastic decrease in nitrate content and nitrate reductase activity, and increases the content of carbohydrates in leaves from tobacco plants. Planta 209, 528e536. Dai, F., Nevo, E., Wu, D., Comadran, J., Zhou, M., Qiu, L., et al., 2012. Tibet is one of the centers of domestication of cultivated barley. Proc. Natl. Acad. Sci. U. S. A. 109, 16969e16973. Diaz, C., Lemaître, T., Christ, A., Azzopardi, M., Kato, Y., Sato, F., et al., 2008. Nitrogen recycling and remobilization are differentially controlled by leaf senescence and development stage in Arabidopsis under low nitrogen nutrition. Plant Physiol. 147, 1437e1449. Drew, M.C., Saker, L.R., 1975. Nutrient supply and the growth of the seminal root system in Barley II. Localized, compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. J. Exp. Bot. 26, 79e90. Erenoglu, E.B., Kutman, U.B., Ceylan, Y., Yildiz, B., Cakmak, I., 2011. Improved nitrogen nutrition enhances root uptake, root-to-shoot translocation and remobilization of zinc (65Zn) in wheat. New Phytol. 189, 438e448. Fei, H., Chaillou, S., Hirel, B., Mahon, J.D., Vessey, K.J., 2003. Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate. Planta 216, 467e474. Forde, B.G., Lea, P.J., 2007. Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339e2358. ndez, G., 2001. Over-expression of Fuentes, S.I., Allen, D.J., Ortiz-Lopez, A., Herna cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations. J. Exp. Bot. 52, 1071e1081. Kaiser, W.M., Weiner, H., Huber, S.C., 1999. Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity. Physiol. Plant. 105, 384e389. Kumada, Y., Benson, D.R., Hillemann, D., Hosted, T.J., Rochefort, D.A., Thompson, C.J., et al., 1993. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc. Natl. Acad. Sci. U. S. A. 90, 3009e3013. Kutman, U.B., Yildiz, B., Cakmak, I., 2011. Effect of nitrogen on uptake, remobilization and partitioning of zinc and iron throughout the development of durum wheat. Plant Soil 342, 149e164. Lea, P.J., Azevedo, R.A., 2006. Nitrogen use efficiency. 1. Uptake of nitrogen from the soil. Ann. Appl. Biol. 149, 243e247. Lecourt, J., Lauvergeat, V., Ollat, N., Vivin, P., Cookson, S.J., 2015. Shoot and root ionome responses to nitrate supply in grafted grapevines are rootstock genotype dependent. Aust. J. Grape Wine R. 21, 311e318. Lemaître, T., Gaufichon, L., Boutet-Mercey, S., Christ, A., Masclaux-Daubresse, C., 2008. Enzymatic and metabolic diagnostic of nitrogen deficiency in Arabidopsis thaliana Wassileskija accession. Plant Cell Physiol. 49, 1056e1065. Limami, A., Phillipson, B., Ameziane, R., Pernollet, N., Jiang, Q., Roy, R., et al., 1999.
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Vincent, R., Fraisier, V., Chaillou, S., Limami, M.A., Deleens, E., Phillipson, B., et al., 1997. Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development. Planta 201, 424e433. Watanabe, T., Urayama, M., Shinano, T., Okada, R., Osaki, M., 2015. Application of ionomics to plant and soil in fields under long-term fertilizer trials. SpringerPlus 4, 1. Williams, L., Salt, D.E., 2009. The plant ionome coming into focus. Curr. Opin. Plant Biol. 12, 247e249. s, M., Pourtau, N., 2004. Spatial patterns and metabolic regulation Wingler, A., Mare of photosynthetic parameters during leaf senescence. New Phytol. 161, 781e789. Wu, D., Shen, Q., Cai, S., Chen, Z., Dai, F., Zhang, G., 2013. Ionomic responses and correlations between elements and metabolites under salt stress in wild and cultivated barley. Plant Cell Physiol. 54, 1976e1988. 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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Ionomic and physiological responses to low nitrogen stress in Tibetan wild and cultivated barley Xiaoyan Quan, Jianbin Zeng, Zhigang Han, Guoping Zhang* Agronomy Department, Institute of Crop Science, Zhejiang University, Hangzhou, 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2016 Received in revised form 26 November 2016 Accepted 4 December 2016 Available online 6 December 2016
In a previous study, we identified the low-nitrogen (LN) tolerant accessions from the Tibetan wild barley (Hordeum vulgare subsp. spontaneum). In this study, two wild barley genotypes (XZ149, LN-tolerant and XZ56, LN-sensitive) and a barley cultivar ZD9 (H. vulgare) were used to determine the LN tolerant mechanism underlying the wild barley in the ionomic and physiological aspects. XZ149 exhibited higher LN tolerance with highest relative dry weight and N accumulation among three barley genotypes under LN stress. When exposed to LN stress, XZ149 had more N transportation from roots to leaves, and remained relatively higher activities of nitrate reductase (NR, EC.1.7.1.1) and glutamine synthetase (GS, EC.6.3.1.2) in leaves than other two genotypes, ensuring its higher capacity of N assimilation and utilization. The ionome analysis showed that LN stress had a significant effect on tissue ionome and the effect was genotypic and tissue-specific difference. On the whole, XZ149 maintained more stable Mn and Cu contents in roots, and less reduction of root P, K and Ca contents than XZ56 and ZD9 when exposed to LN stress. It may be assumed that more N movement into shoots, greater N assimilating capacity and specific rearrangement of nutrient element levels in tissues under LN stress are attributed to LN tolerance in XZ149. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Glutamine synthetase Ionome Nitrogen Nitrate reductase Tibetan wild barley
1. Introduction Nitrogen (N) is an essential mineral nutrient required in quantity and is frequently a key factor limiting crop yield and quality (Marschner, 2012). However, the environmental problems brought by the excessive N fertilizer application in crop production become increasingly severe (Socolow, 1999). Thus, well understanding of the mechanisms of low nitrogen tolerance is imperative for improving N use efficiency. The Tibetan annual wild barley has been proved as one of the ancestors of cultivated barley (Dai et al., 2012), and shows generally better adaption to poor soil fertility, including N deficiency (Quan et al., 2016) and K deficiency (Zeng et al., 2015). Some wild barley genotypes with high LN tolerance have been identified in our previous study (Yang et al., 2014), providing elite materials for understanding the mechanisms of LN stress tolerance. Extensive studies have been performed on plant biomass, nitrate uptake and root architecture to link the traits with LN stress tolerance (Brouwer, 1962; Drew and Saker, 1975; Lea and Azevedo,
* Corresponding author. E-mail address: [email protected] (G. Zhang). http://dx.doi.org/10.1016/j.plaphy.2016.12.008 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved.
2006). Nevertheless, it is less documented about the fine-tuning of plant metabolism under LN stress. Meanwhile, adaptation to steady-state low N in plants is also poorly studied (Forde and Lea, 2007). It would be of significance to obtain an overview of the modifications in plant N metabolism when subjected to N stress. In fact, some N-containing compounds and enzyme activity have been used as diagnostic indicators to reveal the N level response of the different plant genotypes. Meanwhile, plants require at least 13 mineral elements for adequate development, including so-called macronutrients phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) in addition to N, as well as iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), boron (B), chloride (Cl), nickel and molybdenum, which are referred to as micronutrients (Marschner, 2012). All of these elements are associated with plant growth and crop yield, and in many instances, deficiency of one element interconnects with the metabolism of other nutrients under crosstalking regulation (Schachtman and Shin, 2007; Liu et al., 2009). Their interactions have been highlighted in some recent reviews (Amtmann and Armengaud, 2009; Williams and Salt, 2009). For example, N metabolism and allocation could be altered by B and Fe bal deficiency in tobacco and cucumber separately (Camacho-Cristo
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lez-Fontes, 1999; Borlotti et al., 2012). Similarly, improve and Gonza N status enhanced root uptake, remobilization and root-to-shoot translocation of Zn in wheat and ammonium toxicity decreased levels of Ca and Mg in cucumber (Erenoglu et al., 2011; Roosta and Schjoerring, 2007). These results demonstrate that mineral homeostasis in plants is highly monitored, indicating that changes in availability of a single element could exert an effect on the uptake and accumulation of other elements in plants. Thus, LN stress may change mineral balances in plants, directly or indirectly affecting mineral accumulation (Kutman et al., 2011; Xue et al., 2012; Schreiner et al., 2013). However, the genotypic responses of elements and their interactions to LN stress have not been fully elucidated up to date. The ionome has been defined as the mineral nutrient composition of an organism or tissues (Salt et al., 2008). Ionomics is the study of elemental accumulation in living organisms using highthroughput elemental profiling. There are four major techniques in the studies of ionomics, i.e. neutron activation analysis (NAA), inductively coupled plasma-atom/optical emission spectrometry (ICP-AES/OES), inductively coupled plasma-mass spectrometry (ICP-MS) and X-ray fluorescence (XRF) (Wu et al., 2013). Highthroughput elemental profiling has been applied to study the ionome response to the environment or the genetics underlying the changes of ionome over the environments (Baxter, 2009). For instance, multivariable ionomic signatures in Arabidopsis were established to investigate physiological responses using ICP-MS, such as P and Fe homeostasis (Baxter et al., 2008). In addition, ionome has been employed on the studies of element contents under different N supply (Watanabe et al., 2015; Lecourt et al., 2015). However, almost no experiment has been reported on studying the effect of LN stress on plant ionome profiles comprehensively. In the present study, we employed two wild barley genotypes (XZ149, LN-tolerant and XZ56, LN-sensitive) and one barley cultivar (ZD9) to study the effect of LN on N metabolism and ionomic changes using ICP-OES with aims at understanding the mechanisms of LN stress tolerance underlying Tibetan wild barley.
For ionome analysis, only the plants exposed to 2 and 0.2 mM N were used. The plant samples were taken at 18 d after N treatment. Dry roots and shoots were finely ground, and approximately 0.2 g tissue samples were predigested with a mixture of 6 ml HNO3 and 1 ml of H2O2 for about 20 min at 130 C, and then digested in a microwave (Multiwave 3000, Anton Paar GmbH, Australia) after adding 1 ml of H2O2. The digested solution was boiled to eliminate acid for 1.5 h at 160 C. The contents of K, P, Mg, Ca, Zn, Fe, Cu and Mn were determined using an ICP-OES spectrometer (Optima 6000 series, PerkinElmer Inc, USA). 2.3. Data statistics Significant difference for physiological traits and element
2. Materials and methods 2.1. Plant materials and treatments Healthy seeds of the two Tibetan wild barley accessions XZ149 (LN tolerant) and XZ56 (LN sensitive), a cultivar ZD9 were germinated in a plant growth chamber (22/18 C, day/night). Ten-day-old seedlings with uniform size were transplanted into black plastic containers (5 L) with aerated hydroponic solution in a greenhouse with natural light. The hydroponic solution was prepared according to Quan et al. (2016), and renewed every five days. At three-leafstage, seedlings were exposed to 2 mM N (control), 0.2 mM N (LN) and 0 mM (N starvation). 2.2. Sampling and measurement The plant samples were taken and separated into shoots (or leaves and stems) and roots at 14 d after N treatment. Six biological replications were prepared for dry weight, and oven-dried for use in total N content measurement. Meanwhile fresh plant tissues were taken for physiological analysis, with three biological replications. Soluble protein content was determined according to Andrews et al. (1999), and nitrate-N content was determined according to Mosier et al. (1998). NR activity was measured according to Kaiser et al. (1999). GS activity was measured as described by MasclauxDaubresse et al. (2006).
Fig. 1. Effects of low nitrogen stress on dry weight (mg.plant¡1) of three barley genotypes. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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contents among treatments and genotypes was analyzed using a data processing system (DPS) software. Principal component analysis (PCA) of element contents was carried out to separate samples and elements using Metaboanalyst 3.0 (Xia et al., 2015). Relative change was calculated by the value of LN stress/control. N accumulation (mg.plant1) ¼ N concentration dry weight/plant. 3. Results 3.1. Physiological difference among barley genotypes in response to N treatment LN stress (0.2 mM N) and N starvation (0 mM N) inhibited shoot growth and promoted root growth of all genotypes (Fig. 1). There were the distinct differences in dry weight, N accumulation, nitrate N content and soluble protein content among the three genotypes in response to N treatment. In comparison with control (2 mM N), shoot dry weight was reduced by 13%, 32% and 30% under LN and 42%, 51% and 67% under N starvation in XZ149, XZ56 and ZD9, respectively. By contrast, root dry weight was increased by 35%, 28% and 31% under LN stress, and 72%, 38% and 82% under N starvation. In particular, total dry weight of XZ149 was significantly smaller than that of the other two genotypes under normal N condition, but significantly higher under LN and N starvation conditions. In comparison with control, LN and N starvation treatments had significantly lower N concentration and accumulation for all genotypes (Fig. 2). Overall, there was little genotypic difference in N concentration, but the difference was significant in N accumulation (Fig. 2), with XZ149 being significantly higher than other two
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genotypes under LN or N starvation. As shown in Fig. 2, LN and N starvation reduced nitrate N content by 64%e83% in shoots and 33%e88% in roots among the three genotypes, respectively, whereby XZ149 had less reduction than XZ56 under LN and N starvation conditions (Fig. 2). Notably, nitrate N content in ZD9 was significantly higher than that of the two wild genotypes under LN stress. Both LN and N starvation significantly reduced soluble protein contents in leaves and roots of all barley genotypes (Fig. 3). Compared with XZ56, XZ149 showed less reduction of the soluble protein content in leaves and roots under both LN and N starvation. Soluble protein content in leaves of ZD9 remained higher under all N levels, and showed less change under LN and N starvation in comparison with the two wild barley genotypes. LN stress caused a dramatic reduction of nitrate reductase (NR, EC.1.7.1.1) activity (Fig. 3). However the reduced extent differed among three barley genotypes, with XZ149 being slightly lower than the other two genotypes. As expected, no NR activity was detected in the plants exposed to N starvation. There was no significant difference among three barley genotypes in glutamine synthetase (GS, EC.6.3.1.2) activity under control (Fig. 4). However LN and N starvation treatments caused the dramatic increase of GS activity in roots and stems of all genotypes, with X149 and ZD9 having the greatest increase under N starvation in stems and roots, respectively. On the other hand, leaf GS activity showed relatively smaller change in comparison with that in roots and stems. Moreover X149 and XZ56 showed significantly higher leaf GS activity under N starvation than the control, while ZD9 remained little change.
Fig. 2. Total nitrogen accumulation (mg.plant¡1) and nitrate N content (mg.g¡1 FW) of three barley genotypes under different N levels. FW, fresh weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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3.2. Tissue ionomic difference among barley genotypes in responses to LN stress To obtain an overall view of ionomic responses, PCA was conducted on the element contents in shoots and roots of the three genotypes under both control and LN stress. The first PCA components (PC1) may explain 42.1% and 44.8% of the total variance in leaves and roots, respectively, and could separate the samples of control and LN stress, indicating that LN stress had substantial effect on element content (Fig. 5a, c). Ca, Fe and Zn contents in shoots, and P, Mn, Mg, K, Fe, Ca contents in roots were associated with control components (Fig. 5b, d). On the other hand, P, K, Mg and Mn contents in shoots and Zn content in roots were the specific loadings for the LN-related principal components (Fig. 5b, d). Genotypic difference was reflected by the second principal component (PC2), which clearly separated the samples of XZ149 from those of XZ56 and ZD9 in shoots, and the samples of XZ149 and XZ56 from those of ZD9 in roots (Fig. 5a, c). Mn, Zn and Mg contents contributed to the variation of XZ149 in shoots, while Ca, K, Zn and Mg contents were dominant in the root ionome (Fig. 5b, d). Obviously, the response of element contents to LN stress varied with treatments and genotypes. Concerning element contents in shoots, LN stress significantly increased K and P contents and reduced Ca content in all three barley genotypes, and increased Mg content in the two wild genotypes. Whereas Mg content in ZD9 was reduced. In comparison with control, LN-treated plants had much higher K content in XZ149 than that in XZ56 and ZD9 (Fig. 6). The contents of P, K, Ca and Mg in roots were significantly reduced in the three barley
genotypes under LN stress, varying from 6.7% of K to 30.3% of Mg. The P, K and Ca contents in roots was less changed in XZ149 relative to XZ56 and ZD9 under LN stress (Fig. 7). On the whole, microelement contents in shoots decreased under LN stress in comparison with control, with Fe and Cu contents decreasing dramatically in ZD9 and showing less change in XZ149 and XZ56. Mn content decreased in XZ149 and remained unchanged in ZD9 and XZ56; Zn content decreased in XZ149 and ZD9, and remained unchanged in XZ56 (Fig. 8). In roots, LN stress significantly reduced Fe content in ZD9, and Cu and Mn contents in XZ56 and ZD9 (Fig. 9). Zn content in XZ149 and ZD9 increased but unchanged in XZ56 (Fig. 9). In roots, the microelement content was less affected in XZ149 compared with XZ56 and ZD9 (Fig. 9). 4. Discussion In order to understand the mechanisms of LN stress tolerance in Tibetan wild barley, two wild barley genotypes, XZ149 and XZ56, differing in LN tolerance and a barley cultivar, ZD9 were used to determine the genotypic difference in physiological and ionome responses to LN stress. 4.1. More nitrate transportation to shoots and higher GS activity in leaves are the major traits associated with higher LN tolerance in XZ149 XZ149 was identified as a LN-tolerant and XZ56 as a LNsensitive wild barley genotype according to the previous studies (Yang et al., 2014; Quan et al., 2016). In this study, XZ149 again
Fig. 3. Soluble protein content (mg.g¡1 FW) and NR activity (mg.g¡1.h¡1) of three barley genotypes under the different N levels. FW, fresh weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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exhibited higher LN tolerance than the other two genotypes, characterized by its better growth performance and higher capacity of N absorption under LN stress (Figs. 1 and 2). Nitrate taken up via roots would be most reduced and assimilated in leaves (Lemaître et al., 2008). In our previous study, XZ149 transported more nitrate to shoot for assimilation under LN stress in comparison with other two genotypes (Quan et al., 2016). Currently, the leaves of XZ149 remained less change of NRA activity relative to XZ56 under LN stress (Fig. 4), indicating that the former contains high nitrate level under LN stress. Correspondingly, XZ149 remained relatively higher soluble protein content than XZ56 under LN stress (Fig. 3). The ammonium produced from reduced nitrate is assimilated by GS. The current results showed that GS activity was significantly higher in LN-treated plants than those exposed to normal N supply (control) (Fig. 5). The increase of GS activity under LN stress could be attributed to higher GS1 protein content when N is limited (Diaz et al., 2008). GS1 isoforms are regarded as the markers of N remobilization (Martin et al., 2005). In view of reduced NR activity in leaves under LN stress, it may be suggested that leaves start to mobilize nitrogen efficiently during LN stress, which is consistent with the idea that N limitation facilitates N remobilization, acting as an exogenous senescence-triggering factor (Wingler et al., 2004). Therefore, GS is not only the key enzyme in the process of inorganic N assimilation into amino acid glutamine, but also plays an important role in the process of reactivating N (Miflin and Habash, 2002; Bernard and Habash, 2009; Kumada et al., 1993); helping plants to adapt to LN stress (Fuentes et al., 2001; Vincent et al., 1997). In the present study, compared with XZ56, XZ149 had higher leaf GS activity under LN stress, indicating that the genotype has the higher ability of N assimilation or remobilization when N is limited. It is well documented that a high level of GS activity in roots is negatively correlated with above-ground biomass (Limami et al., 1999; Fei et al., 2003). Here, relative root GS activity was higher in XZ56 than XZ149 under LN stress, being consistent with their growth performance. However, root GS activity was increased under LN stress, which seems to be confliction with the previous study that one gene encoding GS1 was down-regulated in root of XZ149 under LN stress. The different GS1 isoforms show the specific locations and function in N assimilation and recycling (MasclauxDaubresse et al., 2008). The function of root GS1 in the primary N assimilation is not well understood, especially for species primarily assimilating N in shoots. Thus, further studies are necessary to confirm contribution of GS1 in total N assimilation, especially under the condition of N limitation.
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et al., 2009; Schlüter et al., 2012). In the present study, P content increased in shoots and decreased in roots under LN stress. Moreover there was a dramatic difference among the three genotypes in the response of P content to LN stress, with XZ149 being least changed by LN stress. LN stress also significantly affected the elements related to photosynthesis. Fe is associated with the carbon assimilation capacity and it plays a key role in the synthesis of chlorophyll and electron transferring in photosynthesis. In addition, Fe is also a coenzyme factor of NR, nitrite reductase and glutamate synthase in the pathway of nitrate reduction (Borlotti et al., 2012), closely related to N metabolism. It was reported that a high concentration of nitrate increased pH in the protoplast, leading to inadequate iron absorption (Smolders et al., 1997; Nikolic et al., 2007). The current
4.2. Specific rearrangement of nutrient contents in XZ149 under LN stress is attributed to its higher LN tolerance N deficiency in plants will disrupt the multiple metabolic and energy pathways, changing energy supply and transporter activity, eventually cause an imbalance in the uptake and translocation of some essential elements in plant tissues. In fact, the results of ionomic analysis indicated that LN stress had a significant effect on the ionome in barley plant tissues. First of all, LN stress causes the significant change in the contents of structure elements. P is one of the important substrates for biomembrane synthesis and energy metabolism, participating in the regulation of electron transport and oxidative phosphorylation in mitochondria and photophosphorylation in chloroplast. It was reported that LN stress increased P content in grape leaves (Schreiner et al., 2013). It was found that N availability affects absorption and accumulation of P in many plant species, whereas the results varied with plant species, treating time and P level in the nutrient solution (Urbanczyk-Wochniak and Fernie, 2005; Tschoep
Fig. 4. GS activity (U.mg¡1 prot) changes of three barley genotypes under different N levels. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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Fig. 5. Analysis of ionome variation using principal component analysis (PCA) and loadings of elements to the PC1 and PC2 in shoots and roots under the two N levels. (a) PCA in shoots; (b) loadings in shoots; (c) PCA in roots; (d) loadings in roots. PC1, the first principal component; PC2, the second principal component.
results showed that LN stress significantly reduced Fe content of barley cultivar ZD9, but had less effect on the two wild barley genotypes in both shoots and roots, suggesting that the wild barley has relatively stable capacity of Fe uptake and translocation under LN stress. In addition, Zn is also essential for chlorophyll synthesis. LN stress caused Zn accumulation in roots of XZ149 and ZD9, limiting its translocation to the shoots, which was consistent with the results found in wheat (Kutman et al., 2011; Erenoglu et al., 2011; Xue et al., 2012). Mn participates in the structure of
photosynthetic enzymes and proteins (Millaleo et al., 2010). In this study, Mn content was reduced in roots of XZ56 and ZD9 under LN stress, but remained less change in XZ149. The current results showed that the specific rearrangement of nutrient element levels in plant tissues of XZ149 under LN stress is attributable to the enhanced LN tolerance. In view of the complicated crosstalk between elements, further researches are required to reveal the ways of the nutrient rearrangement in responses to LN tolerance.
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Fig. 6. Shoot macronutrient content (mg.g¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
Fig. 7. Root macronutrient content (mg.g¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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Fig. 8. Shoot micronutrient content (mg.kg¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
Fig. 9. Root micronutrient content (mg.kg¡1 DW) of three barley genotypes under different N levels. DW, dry weight. The different letters mean significant difference among treatments and genotypes according to the Duncan's multiple range, P < 0.05.
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Authors contributions XQ and GZ designed research. XQ, QQ and ZH performed research. XQ analyzed data. XQ, JZ and GZ wrote the paper. All authors have read, edited and approved the current version of the manuscript. Acknowledgments We thank Prof. Dongfa Sun (Huazhong Agricultural University, China) for providing Tibetan wild barley accessions. This work was supported by Natural Science Foundation of China (31330055), China Agriculture Research System (CARS-05) and Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP). References Amtmann, A., Armengaud, P., 2009. Effects of N, P, K and S on metabolism: new knowledge gained from multi-level analysis. Curr. Opin. Plant Biol. 12, 275e283. Andrews, M., Sprent, J.I., Raven, J.A., Eady, P.E., 1999. Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies. Plant Cell Environ. 22, 949e958. Baxter, I., 2009. Ionomics: studying the social network of mineral nutrients. Curr. Opin. Plant Biol. 12, 381e386. Baxter, I.R., Vitek, O., Lahner, B., Muthukumar, B., Borghi, M., Morrissey, J., et al., 2008. The leaf ionome as a multivariable system to detect a plant's physiological status. Proc. Natl. Acad. Sci. U. S. A. 105, 12081e12086. Bernard, S.M., Habash, D.Z., 2009. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol. 182, 608e620. Borlotti, A., Vigani, G., Zocchi, G., 2012. Iron deficiency affects nitrogen metabolism in cucumber (Cucumis sativus L.) plants. BMC Plant Biol. 12, 1. Brouwer, R., 1962. Nutritive influences on the distribution of the dry matter in the plants. Neth J. Agric. Sci. 10, 399e408. bal, J.J., Gonz Camacho-Cristo alez-Fontes, A., 1999. Boron deficiency causes a drastic decrease in nitrate content and nitrate reductase activity, and increases the content of carbohydrates in leaves from tobacco plants. Planta 209, 528e536. Dai, F., Nevo, E., Wu, D., Comadran, J., Zhou, M., Qiu, L., et al., 2012. Tibet is one of the centers of domestication of cultivated barley. Proc. Natl. Acad. Sci. U. S. A. 109, 16969e16973. Diaz, C., Lemaître, T., Christ, A., Azzopardi, M., Kato, Y., Sato, F., et al., 2008. Nitrogen recycling and remobilization are differentially controlled by leaf senescence and development stage in Arabidopsis under low nitrogen nutrition. Plant Physiol. 147, 1437e1449. Drew, M.C., Saker, L.R., 1975. Nutrient supply and the growth of the seminal root system in Barley II. Localized, compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. J. Exp. Bot. 26, 79e90. Erenoglu, E.B., Kutman, U.B., Ceylan, Y., Yildiz, B., Cakmak, I., 2011. Improved nitrogen nutrition enhances root uptake, root-to-shoot translocation and remobilization of zinc (65Zn) in wheat. New Phytol. 189, 438e448. Fei, H., Chaillou, S., Hirel, B., Mahon, J.D., Vessey, K.J., 2003. Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate. Planta 216, 467e474. Forde, B.G., Lea, P.J., 2007. Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339e2358. ndez, G., 2001. Over-expression of Fuentes, S.I., Allen, D.J., Ortiz-Lopez, A., Herna cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations. J. Exp. Bot. 52, 1071e1081. Kaiser, W.M., Weiner, H., Huber, S.C., 1999. Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity. Physiol. Plant. 105, 384e389. Kumada, Y., Benson, D.R., Hillemann, D., Hosted, T.J., Rochefort, D.A., Thompson, C.J., et al., 1993. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc. Natl. Acad. Sci. U. S. A. 90, 3009e3013. Kutman, U.B., Yildiz, B., Cakmak, I., 2011. Effect of nitrogen on uptake, remobilization and partitioning of zinc and iron throughout the development of durum wheat. Plant Soil 342, 149e164. Lea, P.J., Azevedo, R.A., 2006. Nitrogen use efficiency. 1. Uptake of nitrogen from the soil. Ann. Appl. Biol. 149, 243e247. Lecourt, J., Lauvergeat, V., Ollat, N., Vivin, P., Cookson, S.J., 2015. Shoot and root ionome responses to nitrate supply in grafted grapevines are rootstock genotype dependent. Aust. J. Grape Wine R. 21, 311e318. Lemaître, T., Gaufichon, L., Boutet-Mercey, S., Christ, A., Masclaux-Daubresse, C., 2008. Enzymatic and metabolic diagnostic of nitrogen deficiency in Arabidopsis thaliana Wassileskija accession. Plant Cell Physiol. 49, 1056e1065. Limami, A., Phillipson, B., Ameziane, R., Pernollet, N., Jiang, Q., Roy, R., et al., 1999.
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