Effect of Salt Stress on Nitrogen Assimilation of Functional Leaves and Root System of Rice in Cold Region

Gu Jiao-jiao, Zhao Hong-wei, Jia Yan, Hu Bo-wen, Wang Zhuo-qian, Qu Zhao-jun, and Yu Feng-li

Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region, Ministry of Education Northeast Agricultural University, Northeast Agricultural University, Harbin 150030, China

Abstract: The aims were to investigate the effect of salt stress on key enzyme activity of nitrogen metabolism and the concentration of nitrate nitrogen and ammonium nitrogen response to salt stress. Two rice cultivars, Mudanjiang 30 (sensitive cultivar) and Longdao 5(salt-tolerant cultivar), were treated with different salt concentrations (CK 0%, S1 0.075%, S2 0.15%, S3 0.225% and S4 0.3%).The results showed that the activities of nitrate reductase (NR), glutamine synthase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in the functional leaves and roots of rice in cold region presented a single peak curve change and the peak occurred in the heading stage; compared with those of the CK, the activities of NR, GS and GOGAT of rice in cold region decreased, but the activity of GDH increased in the heading stage under salt stress. The variation for key enzyme activity of nitrogen metabolism was the highest under S4 treatment. The activities of NR, GS and GOGAT in the functional leaves significantly decreased compared with those in roots; the concentrations of nitrate nitrogen and ammonium nitrogen in the functional leaves and roots of rice in cold region presented a single peak curve change and the peak occurred in the heading stage; compared with that of the CK,the concentration of nitrate nitrogen decreased in leaves and roots, the concentration of ammonium nitrogen decreased and the concentration of ammonium nitrogen in roots increased under salt stress. The variations for the activities of NR, GS and GOGAT in the functional leaves and roots of Longdao 5 were less than those of Mudanjiang 30 under the same concentration of salt stress.

Key words: rice in cold region, salt stress, functional leave, root system, nitrogen metabolism

Introduction

Soil salinization is one of the abiotic stress factors limiting crop growth and causing a significant reduction of crop plants yield. The main ions of SA cause nutritional imbalance in plants by affecting the absorption, transport and distribution of nutrients,which significantly affect various processes of plant nitrogen metabolism (Sha, 2018). Nitrogen metabolism is the main metabolic pathway in plants and its process is mainly relate to the process of primary nitrogen assimilation and nitrogen reassimilation, especially for nitrogen absorption,nitrate reduction and ammonia assimilation and so on (Sha, 2018). Sha (2018) found that salt stress significantly inhibites the activity of nitrogen metabolism which is related to enzyme in the functional leaves of rice in the tillering stage and it inhibites nitrogen absorption in rice. In the past study, it has found that salt stress inhibites glutamine synthase and glutamate synthase activity in leaves of rice seedling (Linet al., 2000; Liet al., 1999). Root is the major parts which absorb nitrogen and the process of nitrogen metabolism not only occurs in the leaves, but also in roots. Previous studies have found that nitrogen application promotes the activities of nitrate reductase and glutamine synthase in roots (Wanget al., 2003;Li and Lv, 2017). The activities of glutamate synthase and glutamate dehydrogenase in roots of rice seedlings significantly decrease under salt stress (Linet al.,2000). The previous studies of nitrogen nutrition and ammonium nitrogen have mostly focused on nitrogen nutrition and plant absorption characteristics (Du,2008; Su, 2010; Sun, 2010; Heet al., 1998). There are few studies about the contents of nitrate nitrogen and ammonium nitrogen in rice under salt stress.

In conclusion, the studies on the nitrogen metabolism key enzymes in the functional leaves and roots of rice under salt stress have mostly focused on the seedling stage and the studies on nitrogen concentration in different forms under salt stress are rarely reported.Therefore, in this study, the salt-tolerant cultivar(Longdao 5) and salt sensitive cultivar (Mudanjiang 30)were used as the materials and the treatments with different salt concentrations were set up to study the variations of key enzyme activities of nitrogen metabolism and the concentrations of nitrate nitrogen and ammonium nitrogen in the functional leaves and roots of rice in cold region under salt stress. The aims were to reveal the mechanism of nitrogen metabolism on the ground and underground response to salt stress and to enrich the physiological basis of salt tolerance in rice.

Materials and Methods

Plant materials

In this experiment, two kinds of rice cold region cultivars with different salt tolerances were selected,Mudanjiang 30 (sensitive cultivar, MDJ30) and Longdao 5 (salt-tolerant cultivar, LD5) (Sunet al., 2012).

Test design

The experiment was carried out at the potting field of Northeast Agricultural University and designed for a completely randomized trial, which was carried out with four salt concentrations. Salt treatments were carried out at one week after seedling transplanting green and each treatment was filled with 25, 50, 75 and 100 mmol · L-1NaCl solution every other day. The concentrations of treatments were enough after 10 days(the amount of NaCl in the soil was 0.075%, 0.15%,0.225% and 0.3%). No salt solution was added since then, and the same ruler was inserted into each basin to control the amount of water and maintained the consistency of concentration between salt treatments.Salt concentration expressed as a percentage of soil salt-containing (NaCl). Therefore, the four salt stress treatments were recorded 0.075% (S1), 0.15% (S2),0.225% (S3) and 0.3% (S4), respectively. No salt application treatment was recorded CK as the control.The experiment was carried out with uniform plastic basin with no damage which was filled with 10 kg soil after sifted and 90 pots were set for each treatment.There were four holes per basin and three seedlings per hole. The other managements were consistent with the field production.

Determination of projects and methods

The pots were sampled during tiller stage, booting stage, heading stage, filling stage and maturity stage.Three pots with uniform plants were selected to sample and the fully expanded functional leaves and roots were selected to determine the physiological indexes which were related to nitrogen metabolism.

The plants were sampled during tiller stage, booting stage, heading stage, filling stage and maturity stage.Three hole plants were selected in each treatment and the plants were divided into different parts, according to different organs. The divided samples were dried in oven and stored to further analyses.

Key enzymes in nitrogen metabolism(1) Nitrate reductase (NR) activity

The measurement of NR activity referred to the living method introduced in Zou (1995).

(2) Extraction and activity determination of gluta-mine synthase (GS), glutamate synthase (GOGAT)and glutamate dehydrogenase (GDH) enzyme liquid.

The extracts of coarse enzyme referred to Zhanget al(1997). Crude enzyme fluids could be used to determine the activities of GS, GOGAT and GDH.The measurement of GS activity referred to method of Rhodeset al(1975). The determination method of GOGAT activity referred to Singh and Srivastava (1986).The activity of GDH was measured by reference of Loulkakais and Ronbelakis-Angelakis (1991).

Nitrogen morphology

Ammonium nitrogen contents and nitrate nitrogen contents were determined by salicylic acid method and Indinavir method, respectively. The specific method referred to theExperimental Guide to Modern Plant Physiology(1999).

Data analysis

All the data were analyzed using Microsoft Excel 2010 and SPSS19.0.

Results

Effect of salt stress on key enzyme activity of nitrogen metabolism in cold japonica rice

Effect of salt stress on nitrate reductase activity in rice in cold region

Figs. 1 and 2 showed that the NR activity in the functional leaves and roots of rice in cold region under each treatment condition presented a single peak curve which occurred in the heading stage. The NR activity in the functional leaves and roots of rice in cold region decreased due to salt stress. Compared with that of the CK, the NR activity in the functional leaves of MDJ30 significantly decreased from the tillering stage to the filling stage under S1 treatment and that of LD5 significantly decreased from the booting stage to the filling stage. The NR activity in the functional leaves of rice in cold region significantly decreased from the tillering stage to the maturity stage under the treatments of S2, S3 and S4. However, the NR activity in the functional leaves of LD5 was significantly higher than that of MDJ30 in the heading stage under the treatments of S2, S3 and S4 (Fig. 1). Compared with that of the CK, the NR activity of the roots of rice in cold region decreased from the tillering stage to the maturity stage under S1 treatment. The NR activity of the roots of rice in cold region significantly decreased from the heading stage to the filling stage under S2 treatment. There was a significant decrease from the tillering stage to the maturity stage under the treatments of S3 and S4 (Fig. 2). The decline of NR activity of LD5 was less than that of MDJ30 under the same salt concentration stress.

Effect of salt stress on activity of glutamine synthase of rice in cold region

Figs. 3 and 4 showed that the GS activity of the functional leaves and root system of rice in cold region presented a single peak curve which occurred in the heading stage. Compared with that of the CK, the GS activity in the functional leaves of rice in cold region significantly decreased from the booting stage to the filling stage under S1 treatment. The GS activity in the functional leaves of MDJ30 significantly decreased from the booting stage to the filling stage and that of LD5 significantly decreased from the tillering stage to the filling stage under under S2 treatment. The GS activity in the functional leaves of rice in cold region decreased from the tillering stage to the filling stage under the treatments of S3 and S4. The GS activity in the functional leaves of LD5 was significantly higher than that of MDJ30 in the heading stage under the treatments of S2, S3 and S4 (Fig 3).

Compared with that of the CK, the GS activity in roots of MDJ30 significantly decreased from the tillering stage to the filling stage under S1 treatment,that of LD5 significantly decreased from the tillering stage to the heading stage. The rice in cold region significantly decreased from the tillering stage to the maturity stage under S2, S3 and S4 treatments. The GS activity in roots of LD5 was significantly higher than that of MDJ30 in the heading stage under S4 treatment(Fig. 4). The decline of LD5 GS activity was less than that of MDJ30. The decline of LD5 GS activity was less than that of MDJ30.

Effect of salt stress on glutamate synthase activity of rice in cold region

Figs. 5 and 6 showed that the GOGAT activity in the functional leaves and roots of rice in cold region presented a single peak curve which occurred in the heading stage. Salt stress inhibited the activity of GOGAT in the functional leaves and roots. Compared with that of the CK, the GOGAT activity in the functional leaves in MDJ30 significantly decreased from the booting stage to the maturity stage under S1 treatment. The GOGAT activity in the functional leaves in LD5 significantly decreased from the booting stage to the filling stage. And the activity of GOGAT in the functional leaves of rice in cold region decreased significantly from the tillering stage to the maturity stage under the treatments of S2, S3 and S4. The GOGAT activity in the functional leaves of LD5 was significantly higher than that of MDJ30 in the heading stage under the treatments of S2, S3 and S4 (Fig. 5).

Fig. 1 Change trend of NR activity in functional leaves of rice in cold region in main fertility process under salt stress

Fig. 2 Change trend of NR activity in roots of rice in cold region in main fertility process under salt stress

Fig. 3 Change trend of GS activity in functional leaves of rice in cold region in main fertility process under salt stress

Fig. 4 Change trend of GS activity in roots of cold region rice in main fertility process under salt stress

Fig. 5 Change trend of GOGAT activity in functional leaves of rice in cold region in main fertility process under salt stress

Fig. 6 Change trend of GOGAT activity in roots of rice in cold region in main fertility process under salt stress

Compared with that of the CK, the GOGAT activity in roots of rice in cold region significantly decreased from the heading stage to the maturity stage under S1 treatment. The activity of GOGAT in roots of rice in cold region significantly decreased from the tillering stage to the maturity stage under the treatments of S2,S3 and S4. The activity of GOGAT of roots of LD5 was significantly higher than that of MDJ30 in the heading stage under the treatments of S2, S3 and S4(Fig. 6).

The decline of the activity of LD5 was greater than that of MDJ30.

Effect of salt stress on glutamate dehydrogenase activity of rice in cold region

The activity of GDH in the functional leaves and roots of rice in cold region presented a single peak curve which occurred in the heading stage. The variation of activity of GDH increased which was different with that of NR, GS and GOGAT under salt stress.Compared with that of the CK, the activity of GDH in the functional leaves and roots of rice in cold region was significantly improved from the tillering stage to the maturity stage under the treatments of S1, S2, S3 and S4.

Effect of salt stress on nitrogen morphology of rice in cold region

The concentration of nitrate nitrogen in leaves and roots of rice in cold region presented a single peak curve which occurred in the heading stage. The concentration of nitrate nitrogen in the leaves and roots of rice in cold region decreased under salt stress.Compared with that of the CK, the concentration of nitrate nitrogen in leaves and roots of rice in cold region significantly decreased from the tillering stage to the maturity stage under the treatments of S1, S2,S3 and S4. The concentration of nitrate nitrogen in the functional leaves of LD5 was significantly higher than that of MDJ30 in the heading stage under the treatments of S2, S3 and S4.

The decline of nitrate nitrogen content in leaves and roots of LD5 was less than that of MDJ30. The ammonia nitrogen content in leaves and roots of rice in cold region presented a single peak curve which occurred in the heading stage. Salt stress caused different effects on the functional leaves and roots of rice in cold region.Compared with that of the CK, the concentration of ammonium nitrogen in the leaves of rice in cold region significantly decreased from the tillering stage to the maturity stage under the treatments of S1, S2, S3 and S4. The average decline of ammonium nitrogen content in leaves of MDJ30 was greater than that of LD5 under the same salt concentration stress. The concentration of ammonium nitrogen in leaves of LD5 was significantly higher than that of MDJ30 in the heading stage under the treatments of S2, S3 and S4. Compared with that of the CK, the concentration of ammonium nitrogen in roots significantly increased (except for LD5 in the tillering stage under S1 treatment).

Discussion

In general, the nitrate nitrogen which is absorbed by the crop plants needs to be restored before it can be utilized by crop plants. NR is the first key enzyme in nitrate nitrogen reduction and assimilation reaction and its activity is influenced by many factors. This study found that compared with that of the CK, the activity of NR in the functional leaves of rice in cold region decreased during each fertility process under salt stress.With the increase of salt concentration (0.075%-0.3%),the decline of activity of NR in the functional leaves increased. However, the speci fic decline was different due to the salt tolerance of cultivars and the period of fertilization. NR was an inductive enzyme whose activity was affected by the level of NO3-supplied and the concentration of nitrogen assimilation products(Wu, 2013). In this study, the concentration of nitrate nitrogen of rice in cold region significantly decreased in each growth period under salt stress. Therefore,after the supply of NO3-was inadequate, the activity of NR was reduced by the concentration of the feature. At present, the activity of NR in roots of rice response to salt stress was relatively few. This study found that the variation of activity of NR was basically consistent with the variation of nitrate nitrogen content. The activity of NR and nitrate nitrogen in the functional leaves and roots of MDJ30 decreased mostly in the heading stage under the highest salt concentration (S4 treatment, 0.3%). NR activity and nitrate nitrogen in the functional leaves and roots of LD5 decreased mostly in the filling stage under S4 treatment. There was a certain synergy between NR activity and nitrate nitrogen content. It was observed that there were competitive absorptions among the salt ions, NO3-and the salt ions which inhibited the absorption and transshipment of nitrate nitrogen under salt stress (Abouelsaadet al., 2016).So the concentration of nitrate nitrogen decreased in the leaves and roots of rice in cold region led to the decline of NR activity.

Ammonium nitrogen is one of the main sources of nitrogen absorption and utilization by rice. GS/GOGAT cycle and GDH are important ways to assimilatein plants, but GS/GOGAT cycle is the main way to assimilateduring normal conditions. This study found that compared with that of the CK, the activities of GS and GOGAT in the functional leaves and roots of rice in cold region decreased in each fertility process under salt stress, but that of GDH increased. Although the activities of GS and GOGAT in the functional leaves decreased more than those in roots, the activity levels of GS and GOGAT in the functional leaves were higher than those in roots. The GDH activity levels of roots were higher suggestted that the concentration of ammonium nitrogen in roots and the functional leaves might be different under salt stress. In this study, it was found that the concentration of ammonium nitrogen in the functional leaves of rice in cold region decreased under salt stress. The reason for this result might be that salt stress accumulated a large amount of salt ions on the ground of rice which injuried the chloroplast structure in leaves of rice and affected the light respiration of leaves and led to the decline ofin the functional leaves (Chenet al.,2007; Wang, 2013). In the functional leaves, although the activities of GS and GOGAT decreased under salt stress treatment, GS/GOGAT pathway was still the main way of ammonia assimilation. This study found that the activities of GS and GOGAT in roots of rice in cold region decreased under salt stress and that of GDH increased. The former decreased more than the latter causing the nitrogen assimilation ability in roots become weak and promoting the accumulation ofin roots which resulted in an increase of the concentration of ammonium nitrogen in roots. And this result was consistent with Lin and Kao (1996).GS/GOGAT circulation was the main pathway of ammonia assimilation in roots under normal treatment, but it was obviously weakened in ammonia assimilation under salt stress.

This study found that the response to salt stress of different tissues were diverse. The declines of the activities of NR and GS in the functional leaves of rice in cold region were greater than those in roots in the heading stage under salt stress. The activity of NR in the functional leaves was regulated by the concentration of nitrate nitrogen in the leaves. This study found that the decline of the activity of GS in the functional leaves was greater than that in roots,because the environment was different between functional leaves and roots. The GSrb in roots was synthesized by exogenous nitrogen (Zhanget al.,1998). However, the GS2 in the functional leaves was synthesized by light induced (Hirel and Gadal, 1980).The decline degree of GS isozyme in the functional leaves and corresponding protein level were greater than those in roots (Liet al., 1999). In this study, it was found that the decline of the activity of GOGAT in the functional leaves of rice in cold region was greater than that in roots, which was consistent with the results of Linet al(2000).

In this study, it could be found that the decline of the activities of NR, GS, GOGAT and nitrate nitrogen content in the functional leaves and roots of LD5 were greater than those of MDJ30 under salt stress. The activities of NR, GS and GOGAT in the functional leaves of LD5 were significantly higher than those of MDJ30 in the heading stage under the treatments of S2, S3 and S4. The decline of ammonium nitrogen in the functional leaves of MDJ30 was greater than that of LD5, and the concentration of nitrate nitrogen in roots of MDJ30 increased more than that of LD5 under salt stress. The above showed that the variations of key enzyme activity of nitrogen metabolism, the concentrations of nitrate nitrogen and ammonium nitrogen under salt stress were related to salt tolerance.The LD5 with strong salt tolerance could promote the occurrence of nitrate reduction reaction more efficiently and its ammonia assimilation ability was relatively stronger under salt stress.

Conclusions

Compared with that of the CK, the activities of NR,GS, GOGAT and the concentration of nitrate nitrogen in the functional leaves and roots of rice in cold region decreased and the activity of GDH increased in the heading stage under the salt stress. The variations of the activities of NR, GS, GOGAT, GDH and the concentration of nitrate nitrogen in the functional leaves and roots of rice in cold region increased with the adding of salt concentration. The concentration of ammonium nitrogen decreased in the functional leaves of rice in cold region, but the concentration of ammonium nitrogen in the roots of rice in cold region increased under salt stress. Compared with that of the roots, the decline of the activities of NR, GS and GOGAT in the functional leaves increased. Nitrogen assimilation ability of LD5 was stronger than that of MDJ30.