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Calcium Alleviates Temperature Stress by Regulating Nitrogen and Respiratory Metabolism in Malus baccata Roots.

Byline: Hong Su, Lijie Li, Huaiyu Ma, Deguo Lyu and Jing Sun


The effects of calcium on respiratory and nitrogen metabolism of apple roots (Malus baccata Borkh.) exposed to temperature stress (5C ~ 20C ~ 0C) were investigated. Seedlings were treated with distilled water (control), calcium chloride (CaCl2) or calmodulin antagonist trifluoperazine (TFP) before temperature stress. Temperature was increased from 5C to 20C (1C h-1) and then decreased to 0C (1C h-1). Temperature stress decreased root vitality and increased root malondialdehyde (MDA) concentration, the effect of which was exacerbated by TFP treatment. Treatment with CaCl2 improved root vitality and decreased root MDA concentration. At 20C, exogenous CaCl2 alleviated the negative effects of temperature stress on the total respiration rate by enhancing the activity of tricarboxylic acid cycle (TCA). Activities of key enzyme in nitrogen metabolism were strongly inhibited by temperature stress.

Exogenous CaCl2 significantly increased key enzyme activities of nitrogen metabolism compared to the control. However, the TFP treatment markedly reduced the activity of glutamate synthase (GOGAT) at 20C and noticeably inhibited glutamate dehydrogenase (GDH) activity during the entire temperature stress period. The data showed that the Ca2+-calmodulin (Ca2+-CaM) signal system was involved in increase of GOGAT and GDH activity that occurred with an increase in temperature, and played a role in the increase in the total respiration rate and GDH activity which occurred with a decrease in temperature. Cultural practices that improve plant calcium (Ca) status in the early spring may mitigate damage induced by temperature stress.

Keywords: Calmodulin; Glutamate synthase; Glutamate dehydrogenase, Isocitrate dehydrogenase; Respiration rate


Temperature limits the production and geographical distribution of fruit trees. In the cool fruit-growing region of northern China, cold air movement from Baikal Lake and Siberia can cause rapid changes in air temperature in early spring (Meng et al., 2013). Large fluctuations in temperature are detrimental to apple growth and development, damage apple blossoms in northern China. Soil temperature has a significant effect on root function, which in turn affects the growth of the aerial part (Nedlo et al., 2009). However, limited information is available on how rapid changes in temperature alter root function of apple trees.

Root respiration is sensitive to environmental changes. When exposed to a sudden temperature drop, plants invest more carbon (C) in root respiration (Barthel et al., 2014). In addition, low temperature can reduce plant nitrogen (N) uptake capacity and the activity of enzyme in N metabolism, such as glutamate dehydrogenase (GDH) (Lu et al., 2005; Lloyd et al., 2011).

There is close relationship between N metabolism and respiration in plants and these two metabolic systems connect with each other via the tricarboxylic acid cycle (TCA) (Fig. 1). Nitrate (NO -) is converted to amino acids by nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT). In this process, respiration provides energy and C skeleton (2-oxoglutarate) for N metabolism (Foyer et al., 2011). The synthesis of 2- oxoglutarate (2-OG) is catalyzed by isocitrate dehydrogenase (IDH), one of the key enzymes in the TCA cycle. Very little is known on the interaction between N metabolism and respiration in roots in response to rapid changes in temperature.

Stress can trigger a sudden, transient increase in the concentration of calcium ions in the cytosol [Ca2+]cyt initiating a series of physiological and biochemical processes by binding of Ca2+ to Ca2+-binding proteins such as calmodulin (CaM) (White and Broadley, 2003). Previous studies have found that the adaptability of plants to temperature stress could be enhanced by exogenous calcium (Ca) treatment (Ding et al., 2012). Trifluoperazine (TFP) is a CaM antagonist that has been used to explore Ca2+-CaM dependent activation of various enzymes. It is unclear whether Ca2+-CaM dependent enzyme activation occurs during respiratory and/or N metabolism responses in apple roots during the temperature changes.

Malus baccata Borkh. is native to northern China and widely used as apple rootstock in cold regions of the world because of its higher cold tolerance (Wu et al., 2012). In this study, we use M. baccata to characterize how rapid changes in temperature affects respiration and N metabolism in roots and to determine whether Ca plays a role in the regulation of these processes.

Materials and Methods

Experimental Details and Treatments

Experimental material: Seeds of M. baccata were collected from Shenyang, Liaoning, China, stratified at 0-4C. After germination, seeds were planted in 50-hole trays filled with garden soil of pH 6.3; soil organic matter, 22.9 g*kg-1; alkali-hydrolyzable nitrogen, 106 mg*kg-1; available phosphorus, 27 mg*kg-1 and exchangeable potassium of 123 mg*kg-1 and placed inside a greenhouse. After 30 d, seedlings were transplanted into separate black plastic pots (10 x 10 cm) filled with garden soil and kept in the greenhouse.

Treatments: After 60 d, a totoal of 135 seedlings (15 leaves), similar in height and vigor, were randomly divided into three groups and watered with 100 mL of distilled water (control), 100 mL of 2% (w/v) CaCl2, or 100 mL of 0.1 mg*L-1 TFP. Subsquently, these seedlings were kept at 5C in the dark for 16 h and then transferred to growth chamber (MLR-351H, SANYO Elevtric Co., Ltd, Moriguchi, Japan) at 5C for 24 h with a photosynthetic photon flux density of 150 umol*m-2*s-1. After being at 5C for 24 h, roots of 5 plants per replicate per treatment were harvested and pooled samples from 5 plants from each of three replicates (n=3; 15 plants per treatment). Samples from these plants represented the plant condition before the temperature stress event. The remaining plants in the growth chamber were then subjected to temperature stress.

Temperature Stress and Harvesting

Plants were exposed to temperature stress (5C ~ 20C ~ 0C) that mimiced changing air temperature in early spring of northern China. Growth chamber temperature was increased from 5C to 20C (1C h-1) and maintained at 20C for 2 h. After being exposed to 20C for 2 h, roots of 5 plants per replicate per treatment were harvested (n=3; 15 plants per treatment). Subsequently, growth chamber temperature was decreased to 0C (1C h-1) and maintained at 0C for 2 h. After being exposed to 0C for 2 h, roots of 5 plants per replicate per treatment were harvested (n=3; 15 plants per treatment). At each harvest, fine roots, which have a primary structure without lignification, were used for the analyses. All analyses were performed on pooled samples from 5 plants in each of three replicates (n=3) harvested at each targeted temperature (5C, 20C and 0C).

Determination of Root Vitality and Malondialdehyde Concentration

Fresh roots (0.5 g) were homogenized with 5 mL 0.4% triphenyl tetrazolium chloride (TTC) solution and 5 mL 1/15 M Na2HPO4-KH2PO4 (pH 7.0). Root vitality was determined according to the methods of Zou (2000). Root vitality was calculated from: reduced TTC / (h x fresh root weight).

For determination of malondialdehyde (MDA) concentration, roots (0.5 g) were homogenized with 0.25% 2-thiobarbituric acid in 10% trichloroacetic acid and measured using a colorimetric assay (Shah et al., 2001). The MDA concentration was expressed as umol*g-1 FW.

Determination of Total Respiration Rate (Vt)

Total respiration rate (Vt) was measured according to the method of Bouma et al. (2001) using a Clark-type oxygen electrode (Hansatech Oxytherm, Hansatech Instruments Ltd, Norfolk, U.K.). Root samples (0.05 g; diameter, 1.5 mm; length, 2.0-3.0 cm) were cut into 2.0-mm pieces and added to 1.5 mL phosphate buffered saline (20 mM PBS, pH 6.8).

The Vt was defined as the rate of O2 uptake by roots per unit fresh root weight (umol min-1 g-1 FW).

Determination of Respiratory Pathway Activity (Vr)

Respiratory pathway activity (Vr) was detected according to method of Qin et al. (2014) using Clark-type oxygen electrode. The activities of the glycolytic pathway (EMP), TCA, and pentose phosphate pathways (PPP) were inhibited by addition of NaF (0.5 M), malonic acid (0.5 M), and Na3PO4 (0.5 M), respectively. These inhibitors were dissolved by 20 mM PBS (pH 6.8). Vr was calculated as (Vt - residual respiration rate). Residual respiration rate was defined as the respiration rate after adding the corresponding inhibitor to PBS.

Assay of Nitrate Reductase Activity

Root tissues (0.2 g) were homogenized with 4.0 mL cold 25 mM phosphate buffer (pH 7.5) containing 5 mM cysteine and 5 mM EDTA-Na2 and then centrifuged at 4,000 g for 20 min at 4C. The activitiy of nitrate reductase (NR) was measured according to the method of Zou (2000). The absorbance was measured at 540 nm. The NR activity was expressed as umol*h-1*g-1 FW.

Assay of Glutamine Synthetase Activity

Root (0.5 g) were homogenized with 2.5 mL cold extraction buffer (0.05 M Imidazole-HCl, 0.5 mM EDTA and 1.0 mM dithiothreitol, pH 7.2) and centrifuged at 10,000 g for 20 min at 4C. The glutamine synthetase (GS) activity was measured according to the method of Rhodes et al. (1975).

GS activity was assayed by monitoring the formation of g- glutamyhydroxamate at 540 nm.

Assay of Glutamate Synthase and Glutamate Dehydrogenase Activity

Root tissues (0.5 g) were homogenized in 50 mM KH2PO4 buffer (pH 7.5) containing 2 mM EDTA, 1.5% soluble casein, 2 mM dithiothreitol (DTT), and 1% insoluble polyvinylpolypyrrolidone and centrifuged at 30,000 g for 20 min at 4C. The resulting extract was used to measure enzyme activity of glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) activity. GOGAT and GDH activity was measured according to the method of Groat and Vance (1981). GOGAT and GDH activity was assayed by monitoring the oxidation of NADH at 340 nm for 5 min and 3 min, respectively. One unit of GOGAT and GDH activity is defined as the oxidation of 1 umol NADH per hour.

Assay of Isocitrate Dehydrogenase Activity

Root tissues (0.5 g) were pulverised in 3 mL extraction buffer (100 mM Tris-HC1, pH 7.5). The isocitrate dehydrogenase (IDH) activity was measured according to the method of Collins and Merrett (1975). IDH activity was analyzed by measuring an increase in extinction at 340 nm corresponding to the reduction of NADP+.

Analysis of Calmodulin Concentration

Root tissues (0.2 g) were pulverised in 2 mL extraction buffer (10 mM glyoxaline, 0.25 mM phenylmethylsufonyl fluoride, 1 mM EGTA, 1 mM b-mercaptoethanol, 10 mM MgCl2 and 150 mM NaCl, pH 7.5). The mixture was heated at 100C for 3 min and then cooled in an ice bath. Subsequently, the mixture was centrifuged for 30 min (16,000 g, 4C), and the supernatant was immediately assayed for calmodulin (CaM) concentration according to the method of Sun et al. (1995). The absorbance was measured at 450 nm.

Statistical Analysis

All experiments were performed in triplicate. Data were statistically analyzed by SPSS 17.0 data processing software (SPSS, Inc., Chicago, USA) using Duncan's mutiple range test at a 0.05 level. The data are expressed as the mean standard error (SE). Pearson's correlation analyses were used for analysis of relationships of IDH activity with key enzyme activities of N metabolism.


Effects of Ca and TFP on Root Vitality and Malondialdehyde Concentration

In the control, root vitality significantly decreased with the change in temperature from 5C to 0C. Compared with the control, root vitality was significantly increased by the CaCl2 treatment and reduced by TFP treatment throughout the entire experiment (Fig. 2A). During the temperature stress, the malondialdehyde (MDA) concentration in the control showed an increasing trend. The MDA concentration in the CaCl2 treatment was relatively stable during the temperature stress and lower than of control at 20C and 0C. In contrast, the MDA concentration markedly increased after the addition of TFP, compared to control (Fig. 2B).

Effects of Ca and TFP on Total Respiration Rate and Respiratory Pathway Activity

Compared with the control, total respiration rate (Vt) was significanlty increased by the CaCl2 treatment while it was significanlty inhibited by TFP treatment at 0C (Fig. 3A).

Compared with the control, the CaCl2 treatment significantly enhanced the respiratiory pathway acitvity (Vr) of EMP, TCA and PPP, except for EMP Vr at 0C (Fig. 3B). Similarly, the TFP treatment significantly supressed EMP Vr at 0C compared to the control, but inhibited the Vr of TCA throughout the entire experiment.

Effects of Ca and TFP on Nitrate Reductase, Glutamine Synthetase, Glutamate Synthase, Glutamate Dehydrogenase and Isocitrate Dehydrogenase Activity

During the temperature stress, after the exogenous application of CaCl2, the nitrate reductase (NR) activity increased significantly by 34% at 20C and 8.1% at 0C, compared to control, while the TFP treatment had no effect on NR activity at 20C or 0C (Fig. 4A).

In the CaCl2 treatment, the glutamine synthetase (GS) activity was significantly higher than of control at each temperature and no effect of TFP treatment during the entire experiment was observed (Fig. 4B).

The glutamate synthase (GOGAT) activity in the control and CaCl2 treatment was lower at 20C and 0C than at 5C (Fig. 4C), but it was significantly higher in CaCl2 treatment than control at each temperature. Compared with the control, TFP treatment significantly suppressed GOGAT activity at 20C.

Glutamate dehydrogenase (GDH) activity in the control and TFP treatment decreased from 5C to 0C and was significantly lower in TFP treatment than the control at each temperature (Fig. 4D). While, with CaCl2 treatment GDH activity was greater than control throughout study period.

Compared with control, after the exogenous application of CaCl2, isocitrate dehydrogenase (IDH) activity was increased significantly by 2.2-fold at 0C and it was inhibited by TFP treatment at 5C and 20C (Fig. 4E). During the temperature stress, IDH activity of the control was positively correlated with GOGAT (r = 0.987; P less than 0.01) and GDH (r = 0.795; P less than 0.05) activity.

Effects of Ca and TFP on Calmodulin Concentration

Compared to the control, the CaCl2 treatment significantly improved the calmodulin (CaM) concentration at 5C and 0C, and reduced the CaM concentration at 20C. In addition, CaM concentration decreased significantly by 46% at 20C and 53.3% at 0C after the addition of TFP, compared to the control.


Temperature stress can decrease root vitality and aggravate lipid peroxidation (Zhang and Ervin, 2008).

We found that rapid changes in temperature depressed root vitality and increased MDA concentration (Fig. 2), that created a "temperature stress" condition in apple roots. Exogenous calcium treatment could alleviate the negative effect of rapid changes in temperature on root vitality and MDA concentration, however, the effect was aggravated by TFP treatment. The effects of exogenous calcium and TFP treatment on MDA concentration were similar to those described in tomato leaves [Solanum lycopersicum L.] under heat stress, where Ding et al. (2012) found that Ca2+- CaM signaling pathways were involved in resistance to heat-induced oxidative stress through the induction of antioxidant enzymes and a reduction in the accumulation of oxygen-free radicals and a further reduction of MDA concentration in leaves.

Root respiration provides the energy and C skeletons necessary for biosynthesis and is easily influenced by soil temperature (Atkin et al., 2000; Foyer et al., 2011).

In present study, total respiration rate significantly decreased when temperature rapidly increased from 5 C to 20C. Interestingly, the calcium treatment markedly increased the total respiration rate and the TCA activity compared with the control (Fig. 3). Previous studies have shown that TCA provide C skeleton for biosynthesis and electron donors for the mitochondrial electron transport pathway (Fernie et al., 2004). Therefore, the exogenous calcium treatment strengthened root respiratory metabolism could be attributed to the increase in the activity of TCA. Moreover, the increase in PPP activity with CaCl2 treatment is beneficial for alleviating low-temperature stress (Lin et al., 2005).

The TCA cycle is a key metabolic hub for the interacting pathways of N assimilation and respiration metabolism (Foyer et al., 2011). In this experiment, the activities of NR, GS, GOGAT and GDH were inhibited to some extent by rapid changes in temperature (Fig. 4). Similar changes have been reported for fescue (Festuca arundinacea) under high temperature stress (Cui et al., 2006). The decrease in GS activity under temperature stress might inhibit NO - reduction and NH + assimilation (Chien and Kao, 2000) and consequently, force a large number of electrons to enter into the TCA via NADP+, i.e., feedback inhibited the normal process of this pathway. Such feedback inhibition on the TCA would lead to excessive accumulation of electrons and produce a series of reactive oxygen species, resulting in aggravated membrane peroxidation.

Hence, the decrease in GS and GOGAT activity when temperatures increased from 5C to 20C might be one of the reasons that the MDA concentration increased and the activities of respiratory pathways decreased with increased temperature from 5C to 20C. The GS/GOGAT cycle requires 2-OG as a C skeleton to produce glutamate. Thus, IDH plays an important role in the production of 2-OG for N metabolism. In present study, IDH and GOGAT activities were positively related to each other. For this reason, it is highly likely that the decrease in GOGAT activity can be attributed to the decrease in 2-OG synthesis catalyzed by IDH.

According to recent studies, NR and GS activities were activated by Ca2+ (Zhang et al., 2011). In this experiment, the CaCl2 treatment significantly increased the activities of NR, GS, GOGAT and GDH under temperature stress compared to the control (Fig. 4). The effects of applied exogenous calcium in our study are similar to those described in muskmelon (Cucumis melo L. var. reticulates Naud) under hypoxia-stressed conditions (Gao et al., 2011). Moreover, exogenous calcium treatment significantly enhanced the IDH activity at 0C. These results suggest that exogenous calcium may promote the consumption of NADP+ and 2-OG, weaken the feedback inhibition of N metabolism on respiration induced by rapid temperature decrease, enhance the activity of TCA, increase the total respiration rate and thus, effectively alleviate the damage caused by temperature stress.

Ca2+-CaM complexes play an improtant role in the process of plant response to temperature stress (White and Broadley, 2003). The increase in CaM concentration was found when temperatures rose from 5C to 20C in the control, but decreased with CaCl2 or TFP treatments (Fig. 5). Exogenous calcium treatment markedly enhanced the activities of GOGAT and GDH compared to controls, but these activities were significantly blocked by the TFP treatment. These results indicate that the Ca2+-CaM signal system was involved in the increased activities of GOGAT and GDH when temperature increased from 5C to 20C. When temperatures dropped from 20C to 0C, the CaM concentration increased in control and CaCl2 treatment, and the exogenous calcium treatment had greater CaM concentration, total respiration rate and GDH activity than controls, but the result was reversed in case of TFP treatment. Ca2+ and CaM in the presence of "protein factor" could also directly regulate the activity of GDH (Das et al., 1989).

Hence, it is speculated that the Ca2+-CaM signaling system is involved in improving the total respiration rate and GDH activity in M. baccata roots when temperatures rapidly drop.


Exogenous calcium counteracted the negative effects of temperature stress which was partially mediated by Ca2+- CaM signaling. However, it is possible that there may have been additional Ca2+ sensor proteins that participated in this process which warrants further studies.


This work was supported by grants from the Nation Natural Science Foundation of China (No. 31000887), China Agriculture Research System (No. CARS-28), and Research and Demonstration of the Main Natural Disasters Prevention of Fruits Trees (No. 2014BAD16B0703).


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Author:Su, Hong; Li, Lijie; Ma, Huaiyu; Lyu, Deguo; Sun, Jing
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Apr 30, 2016
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