Effect of low water temperature at reproductive stage on yield and glutamate metabolism of rice (Oryza sativa L.) in China

Field Crops Research 175 (2015) 16–25

Contents lists available at ScienceDirect

Field Crops Research journal homepage: www.elsevier.com/locate/fcr

Effect of low water temperature at reproductive stage on yield and glutamate metabolism of rice (Oryza sativa L.) in China Yan Jia a , Detang Zou a , Jingguo Wang a , Hualong Liu a , Mallano Ali Inayat b , Hanjing Sha a , Hongliang Zheng a , Jian Sun a , Hongwei Zhao a,∗ a b

Rice Research Institute, College of Agriculture, Northeast Agriculture University, Harbin 150030, Heilongjiang, China Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Northeast Agriculture University, Harbin 150030, Heilongjiang, China

a r t i c l e

i n f o

Article history: Received 28 November 2014 Received in revised form 7 January 2015 Accepted 8 January 2015 Keywords: Rice Reproductive stage Low water temperature Glutamate metabolism Yield Spikelet sterility

a b s t r a c t In rice, plants exposure to low water temperature (Tw ) treatment during reproductive stage severely affects the yield-related traits, diminishes physiological and metabolic processes leading to low yield. In higher plants, the glutamate metabolism playes central roles in the amino acid metabolism, plant defense and productivity. In order to understand how low Tw treatment during reproductive stage affects the yield and relative physiological parameters of glutamate metabolism, the rice were subjected to 17 ◦ C low Tw for varying length of time (three, six, nine, twelve, and fifteen days) at reproductive stage (microsporogenesis). The indexes of yield, yield-related traits, amino acid content, enzyme activities of glutamate metabolism, and the relationships among them were investigated, respectively. The results revealed that the changes degree of yield, yield-related traits and glutamate metabolism varies with rice varieties and duration of exposure. Multiple stepwise regression identified glutamate (Glu) content and glutamate dehydrogenase (GDH) activity as important traits that influenced the grain yield and spikelet sterility, respectively. By adjusting the glutamate metabolism level of leaves in rice, we can thus improve rice cold tolerance, and reduce the effect of low Tw (17 ◦ C) treatment on grain yield and spikelet sterility during reproductive stage. © 2015 Published by Elsevier B.V.

1. Introduction Cold temperature causes huge agricultural losses and its effects on crop growth and yield are well known (Beck et al., 2007). Environmental factors, such as climate change, results the augmentation of chilling and involves the inevitable changes in plant metabolism (Oliver et al., 2007). Cold damage has become one of the main factors affecting rice production (Shimono et al., 2002, 2007), causing yield losses of about 10% per year (Wu and Garg, 2003), which is a major concern in areas at high latitudes or high altitudes, especially in northeastern China and in areas with similar climate such as parts of northern Japan (Shimono et al., 2007), Australia (Farrell et al., 2006). On the other hand, over the last 5 decades, scientists have started to search for the most sensitive stage of cold damage in rice (Satake and Hayase, 1970; Shimono et al., 2002, 2007; Wang et al., 2013). Several researches have showed that for

∗ Corresponding author. Tel.: +0086 0451 55190292. E-mail address: hongweizhao [email protected] (H. Zhao). http://dx.doi.org/10.1016/j.fcr.2015.01.004 0378-4290/© 2015 Published by Elsevier B.V.

rice reproductive stage is the most sensitive period for cold damage, for instance, low temperature treatment during reproductive stage caused degeneration of spikelets and an increase in spikelet sterility; affected seed filling, leading to low seed set and ultimately low grain yield (Wang et al., 2013; Jacobs and Pearson, 1999; Sipaseuth et al., 2007). Low water temperature (Tw ) and low air temperature (Ta ) generally limits the nutrient uptake by rice plants (Shimazaki et al., 1963; Zia et al., 1994; Robredo et al., 2011). Shimazaki et al. (1963) observed a reduction in rice growth associated with a decline in uptake of nitrogen (N), phosphorus (P) and potassium (K) under 30-day exposure to low Tw (17 ◦ C) treatment. The nitrogen plays an important role for rice growth and development, the assimilation of inorganic nitrogen can transfer the new uptake N. In higher plants, this process is catalyzed by glutamine synthetase (GS) and controlled by the GS and glutamine–oxoglutarate aminotransferase (GOGAT) cycle (Forde and Lea, 2007). Glutamate metabolism which orchestrates pivotal metabolic functions, played a central role in the amino acid metabolism of plants (Seifi et al., 2013) and plant productivity (Sun et al., 2012; Dubois et al., 2003). Moreover, the

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

prodution from glutamate metabolism, such as Glutamate (Glu), ␥-aminobutyric acid (GABA), arginine, and proline, which play key roles in plant defense (Forde and Lea, 2007; Galili et al., 2001). Recent studies have also revealed that organic N, particularly l-glutamate (l-Glu), plays a regulatory role in modulation of plant growth and development (Walch-Liu et al., 2006; Walch-Liu and Forde, 2008). Glu has also been proved to be associated with signaling cascades of ABA in plants (Khan et al., 2004). On the other hand, ABA is found to increase significantly in response to cold stress (Shinozaki and Yamaguchi-Shinozaki, 2000). It is conceivable that Glu may also be involved in cold stress (Khan et al., 2004). There have been a number of studies regarding the GABA shunt response to abiotic stresses (Kinnersley and Turano, 2000; Bouché et al., 2003; Miyashita and Good, 2008; Song et al., 2010). ␥Aminobutyric acid (GABA) level, as the stress response product in crops, would increase significantly under many abiotic stresses. When glutamine synthetase was inhibited and protein degradation was accelerated under some environmental stresses, including low temperature, salinity, hypoxia and drought, glutamate could be synthesized to GABA in the catalysis of GAD. On the other hand, GABA as a kind of osmotic regulator, could reduce the oxidative damage of crops under many abiotic stresses. (Bouché and Fromm, 2004; Fait et al., 2007). Great efforts have been made to show that alanine (Ala) accumulation increased under hypoxic stress in plants (Reggiani et al., 1988, 2000; Miyashita and Good, 2008; Rocha et al., 2010). Moreover, Wallace et al. (1984) found that the contents of GABA, Glu, aspartate (Asp), and Ala increased under low temperature (6 ◦ C) stress in soybean leaves. However, to our knowledge, there have been no studies to evaluate the role of Glu in physiological processes of leaves in rice under low Tw treatment during reproductive stage. Moreover, few studies have addressed the relationship between transamination of amino acids and GABA shunt under low Tw treatment. Many studies were concentrated at the seedling stage, which could not accurately reveal the effects of cold damage on the physiological metabolism of rice in field and the relationships between yield and glutamate metabolism. Therefore, it seems that an appropriate survey of the relationship between grain yield and defense mechanisms needs particular attention to assess the glutamatemediated central metabolism changes in plants exposed to cold or other stresses. The present study investigated the influence of low Tw (17 ◦ C) treatment during reproductive stage on the content of free amino acid (AA), Glu, Ala, Asp and GABA, glutamate metabolism enzyme activities, yield-related traits and yield. Multiple stepwise regression were applied to determine the most important index for the effects of glutamate metabolism on grain yield and spikelet sterility of rice under low Tw (17 ◦ C) treatment at reproductive stage. The results revealed that the changes degree of yield, yield-related traits and glutamate metabolism varies with rice varieties and duration of exposure. Multiple stepwise regression identified Glu content and glutamate dehydrogenase (GDH) activity as important traits that influenced grain yield and spikelet sterility, respectively. By adjusting the glutamate metabolism level of leaves in rice, we can improve rice cold tolerance and reduce the effect of low Tw (17 ◦ C) treatment on grain yield and spikelet sterility during reproductive stage.

2. Material and methods 2.1. Experimental site and design Present research was conducted at the XiangFang experimental site of the Northeast Agricultural University, which is located in Harbin City, Heilongjiang Province, China (longitude: 126◦ 22 –126◦

17

50 E; latitude: 45◦ 34 –45◦ 46 N) from April to early September in 2012 and 2013, separately. The site is located in the cold temperate zone and has a continental climate type with an annual precipitation from 500 to 550 mm, a frost-free period of approximately 140 days. The crop-rotation system was applied with continuous cropping of rice, and the soil was mollisol. The soils were analyzed for selected physical and chemical characteristics before starting the experiment. The average values for selected soil characteristics of composite topsoil samples (0–20 cm) from the main experimental plots during 2012–2013 were that organic matter was 22.34 ± 0.12 g kg−1 ; total N was 1.24 ± 0.06 g kg−1 ; total P was 0.41 ± 0.04 g kg−1 ; slowly available K was 706.5 ± 2.34 mg kg−1 ;available N, 1 mol L−1 NaOH-alkali-hydrolyzed N was 129.8 ± 4.34 mg kg−1 ; available P, 0.5 mol L−1 NaHCO3 -Olsen P was 18.5 ± 0.4 mg kg−1 ; available K, 1 mol L−1 NH4 OAc-exchangeable K was 98.4 ± 0.6 mg kg−1 ; PH was 6.75 ± 0.04. As for as application of fertilizer is concerned, fertilizer were mainly based on the practice of local farmers. N as urea (9 g m−2 ), phosphorus (6 g m−2 as single superphosphate) and potassium (8 g m−2 as potassium sulphate) were applied and incorporated before transplanting. N as urea was also applied at mid-tillering (4.5 g m−2 ) and panicle branch differentiation (1.5 g m−2 ) as top dressing. The total N application was 15 g m−2 . Two rice (Oryza sativa L.) varieties currently used in local production, DN428 (japonica) and SJ10 (japonica) were used. DN 428 has an average grain yield of 843.08 g m−2 , extensive adaptability, medium cold resistance and pest-resistance, growth duration of 137 days (d) and an active accumulated temperature of approximately 2520 ◦ C from emergence to maturity. SJ10 has an average grain yield of 774.2 g m−2 , low cold resistance, growth duration of 137 days (d) and an active accumulated temperature of approximately 2500 ◦ C from emergence to maturity. Seedlings were raised in the seedbed with sowing date on 17 April, and transplanted on 25 May 2012, 20 April and 29 May 2013 with the hill spacing of 29.7 cm × 10.0 cm and three plants per hill. The main growth stages of heading and mature dates were determined, respectively, under cold-water stress (Table 1). Experimental design was a split plot by using the low Tw treatment as the main plot, rice varieties as vice plot. Plants were divided into two subplots, one plot was subjected to well-watered treatment as control (24 ◦ C) and the other plot was subjected to low Tw treatment. Plot dimensions were 4.8 m × 5 m and plots were separated by an alley 0.5 m wide with soil ridges to form a barrier. The well-watered group, serving as the control, was maintained well-watered irrigation during the whole experiment. Low temperature water from the base of large storage dams which is used to maintain a constant temperature of 17 ◦ C was used for irrigation at reproductive stage (microsporogenesis). Microsporogenesis was determined by the distance between the ligule of the flag leaf and that of the penultimate leaf, considering an interval of—1 cm (flag leaf ligule below the penultimate leaf ligule) (Pereira da Cruz et al., 2006), as the indicative of this stage. Rice were subjected to 17 ◦ C low Tw for 3 (D3), 6 (D6), 9 (D9), 12 (D12), 15 (D15) days, respectively. The low Tw was maintained at a 20 cm depth. Normal irrigation was restored immediately after low Tw treatment. A carefully calibrated Pt100 thermometer (R902-31, Chino Co., Tokyo, Japan) was used to measure water temperature. The water temperature was recorded every 20 min in each plot and recorded the average water temperature every one hour. By adjusting the poured volume and flow rate of cold water, the water temperature was maintained at approximately 17 ◦ C in each plot. A canopy analyzer (LAI-2200, Beijing, China) was used to measure canopy temperature, and a climate field meter (YF-Z3-6, Beijing, China) was used to measure field temperature and light radiation. The details of experimental conditions during 2012–2013 are listed in Table 2.

18

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

Table 1 Summary of the treatments and growth stages in both yearsa . Year

2012

Treatment

DN428 SJ10 DN428 SJ10 DN428 SJ10 DN428 SJ10 DN428 SJ10 DN428 SJ10

D0 D3 D6 D9 D12 D15

2013

Treatment dates

Varieties

DN428 SJ10 DN428 SJ10 DN428 SJ10 DN428 SJ10 DN428 SJ10 DN428 SJ10

D0 D3 D6 D9 D12 D15

Start

End

–

–

7/8

7/11

7/8

7/14

7/8

7/17

7/8

7/20

7/8

7/23

–

–

7/12

7/15

7/12

7/18

7/12

7/21

7/12

7/24

7/12

7/27

Heading date

Panicle development Duration (days)

Date Start

End

PIb -HD

8/3 8/3 8/6 8/6 8/8 8/8 8/9 8/10 8/12 8/12 8/13 8/13

7/5 7/5 7/5 7/5 7/5 7/5 7/5 7/5 7/5 7/5 7/5 7/5

9/1 9/1 9/3 9/3 9/5 9/5 9/7 9/7 9/9 9/9 9/10 9/10

30 30 33 33 35 35 36 37 39 39 40 40

8/6 8/6 8/8 8/9 8/10 8/10 8/12 8/13 8/15 8/15 8/16 8/17

7/9 7/9 7/9 7/9 7/9 7/9 7/9 7/9 7/9 7/9 7/9 7/9

9/4 9/4 9/4 9/6 9/8 9/8 9/11 9/12 9/12 9/13 9/13 9/14

29 29 31 33 34 34 36 37 39 39 40 41

PI: panicle initiation, HD: heading date. a Dates represent month/day. b Panicle initiation is defined as panicle length = 1 mm.

The physiological parameters, including the activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), glutamate decarboxylases (GAD), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT), the content of AA, Glu, Ala, Asp and GABA were measured at the end of each low Tw treatment. All measurements were taken on three replicates. 2.2. Measurement items and methods 2.2.1. Yield and yield-related traits Grain yield and days to heading was determined from all plants from a 5.0 m2 site (except border plants) in each plot. A total of 6 traits related to yield, including effective panicles, fertile spikelet number, sterile spikelet number, spikelet number, spikelet sterility, 1000-grain weight were determined from 20 plants (except border plants) sampled randomly from each plot. 2.2.2. Extraction and determination of enzyme activities Full expanded leaves from plants under control and cold water treatment were used to analyze the changes in enzyme activities of glutamate metabolism under low Tw treatment. Leaf tissues for assays of GS, GOGAT and GDH activity were extracted in the

Table 2 Summary of water temperature (Tw ), air temperature (Ta ) and solar radiation (Rd) during low Tw treatment. Year

Treatment

Tw (◦ C)

2012

Control Low Tw treatment Control Low Tw treatment

24.3 17.0 24.5 17.0

2013

± ± ± ±

Ta (◦ C) 2.1 0.8 1.8 0.6

25.1 24.5 23.8 23.6

± ± ± ±

Rd (MJ m−2 day−1 ) 1.8 2.1 1.8 1.5

16.2 16.1 15.6 15.5

± ± ± ±

7.6 7.4 7.5 7.3

same buffer solution containing 0.05 M Tris–HCl buffer solution (pH 7.6), 1 mM ␤-mercaptoethanol, 1 mM MgCl2 , 1 mM EDTA–Na2 was centrifuged at 12,000 × g for 20 min at 4 ◦ C. The resultant supernatant was used for determining GS activity following the method described by O’Neal and Joy (1973). The reaction mixture (1 mL samples each containing 1 mL of 0.125 M Imidazole-hydrochloride buffer (pH 7.4) mixed with 2 mM ␤-mercaptoethanol and 2 mM EDTA–Na2 ; 0.2 mL 10 mM ATP; 0.2 mL Glu-Na2 ; 0.2 mL 0.5 M hydroxylamine; 0.2 mL 0.2 M MgCl2 ·6H2 O and 1 mL of the enzyme extract) was incubated for 30 min at 35 ◦ C, and a blank was adding 1 mL FeCl3 reagent (containing equal volumes of 0.37 M FeCl3 ; 0.67 M trichloroacetic acid; 0.2 M HCl) also run simultaneously. Later on, the reaction was stopped by adding 1 mL FeCl3 reagent, and centrifuged at 5000 × g for 20 min at 4 ◦ C, finally the supernatant was measured at 540 nm (and one unit of GS activity equals to the amount of enzyme catalyzing the formation of 1 mol ␥-glutamyl hydroxamate per min at 37 ◦ C). The resultant supernatant was used for determining GOGAT (EC 1.4.7.1) activity was determined after the method of Singh and Srivastava (1986). The assay mixture contained 0.4 mL 20 mM l-glutamine, 0.05 mL 0.1 M 2-oxoglutarate, 0.1 mL 10 mM KCl, 0.2 mL 3 mM NADH, 1.75 mL 25 mM Tris–HCl buffer (pH 7.6) and 0.5 mL of the enzyme extract in a final volume of 3 mL, The reaction was started by adding L-glutamine and NADH immediately following the enzyme preparation. The decrease in absorbance was recorded for 3 min at 340 nm. One unit of enzyme activity is defined as a decrease of 1 OD340 per min. The resultant supernatant was used for determining GDH (EC 1.4.7.1) activity was determined by using the method of Loulakakis and Roubelakis-Angelakis (1990). The assay mixture contained 0.4 mL 20 mM l-glutamine, 0.3 mL 0.1 M 2oxoglutarate, 0.3 mL1 M NH4 Cl, 0.2 mL 3 mM NADH, 12 mL 25 mM Tris–HCl buffer (pH 8.0) and 1 mL of the enzyme extract in a final volume of 3 mL. The reaction was started by adding l-glutamine and NADH immediately following the enzyme preparation. The

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

decrease in absorbance was recorded for 3 min at 340 nm. One unit of enzyme activity is defined as a decrease of 1 OD340 per min. GAD activity was determined according to Bai et al. (2009). The GABA content in the reaction solution was analyzed using the standard methods. One unit of GAD activity was defined as the release of 1 mol of GABA produced from glutamate per 60 min at 40 ◦ C. The GAD activity of plant tissues is defined as the unit of GAD activity per 1 g protein. 0.2 g of fresh leaves were used for determination of AlaAT and AspAT activities leaves sample were frozen immediately in −80 ◦ C liquid nitrogen, 2.0 mL 0.05 mol L−1 Tris–HCl buffer (pH7.2) was used to grind samples in liquid nitrogen, then centrifuged the mixture at 20,000 rpm for 20 min, the supernatant was used to the measure enzyme activity. We arranged samples in 4 group tubes, 2 group tubes for treatment, with one group containing 0.1 mL enzyme preparation and 0.5 mL AspAT substrate solution (dlAspartate 200 mM, ␣-ketoglutaric acid, 2 mM), and second group containing 0.1 mL enzyme preparation and 0.5 mL AlaAT substrate solution (dl-alanine 200 mM, ␣-ketoglutaric acid, 2 mM). While, the other 2 groups were used as a control (0.1 mL enzyme preparation). All Four groups were incubated for 60 min at 37 ◦ C, and then 0.5 mL 2,4 dinitrophenylhydrazine was added to each group, 0.5 mL AspAT and AlaAT substrate solution was added to the control group. All Four groups were again incubated for 20 min at 37 ◦ C and then 5.0 mL 0.4 M NaOH reagent was added. For enzyme activity analysis, preceding groups were measured at 540 nm after 10 min. One unit of AlaAT and AspAT activity was defined as the amount of enzyme that catalyzes the production of 1 ␮mol pyruvic acid per min. 2.2.3. Amino acids content Amino acid determination was performed by using the Ninhydrin reagent set (Wako Chemical Inc, Japan). An amino acid analyzer (HITACHI L-8900, Japan) was used for analysis of amino acids. Samples were dried to constant weight, and then 0.1 g was added into the hydrolysis tube and hydrolyzed with 6 M HCl at 110 ◦ C for 24 h. Finally, when the hydrolysate was dried, we used 0.02 N HCl to dissolve the sample and centrifuged it at 10,000 rpm for 15 min. We then analyzed the amino acid composition using an amino acid automatic analyzer. All samples were run three times. 2.3. Calculations and statistic analysis Data analyses were performed using the SPSS 18.0 (Chicago, IL) software package. Analysis of variance (ANOVA) was used to evaluate the effects of low Tw treatment on all yield and yield-related traits and physiological parameters. The statistical model used included sources of variation due to year, variety, cold-water stress and recovery, and the interaction of year × variety, year × treatment, variety × treatment and year × variety × treatment. Results are reported as the mean ± standard deviation (SD) values of the three independent experiments, measuring at least three different replicates (plants) in each experiment. SD was calculated directly from crude data. Levels of significance in figures are given by ns, *, ** for not significant, significant at P < 0.05 and P < 0.01, respectively. The graphs were generated with SigmaPlot 2001, and the standard errors of means were calculated and included in the graphs as error bars. Stepwise multiple regression analysis were carried out using SPSS 18.0 (Chicago, IL) software package. The cold-response index (CRI) was calculated using the following formula:

19

Cold-response index (CRI) = S1/C1, where S1 are yield, yield related-traits, physiological parameters under stress and C1 under control.

3. Results 3.1. The effect of low Tw treatment on yield-related traits and yield 3.1.1. The effect of low Tw treatment on yield-related traits The duration of panicle development (DTH) was prolonged. Depending on the low Tw duration, DTH prolonged by 2–11 days under D3, D6, D9, D12 and D15 treatments, respectively, compared with the control (Table 1). There was no difference of yield-related traits in DN428 under D3 treatment, with the exception of fertile spikelet number in 2012 and spikelet number in 2013, compared with the control, which was more affected of SJ10 (Table 3). From 6 days of exposure on, DN428 and SJ10 presented the higher reduction in yield-related traits, especially in fertile spikelet number, spikelet number and 1000-grain weight. DN428 showed a higher fertile spikelet number than SJ10, moreover, the reduction of fertile spikelet number was more severe in SJ10 than in DN428 under low Tw treatment. The reduction rates of fertile spikelet number were ranging from 1.8% to 20.6% and 6.0% to 26.5% in DN428 and SJ10, respectively. The spikelet number and 1000-grain weight showed responses similar to that of fertile spikelet number, as expected. DN428 showed a lower spikelet sterility compare with SJ10 under low Tw treatment, increase from 11.5% to 188.9% (Table 3). In SJ10, the spikelet sterility was extremely increased by low Tw treatments, ranging from 47.8% to 190.0%. The sterile spikelet number showed a similar pattern to that of spikelet sterility in all treatments.

3.1.2. The effect of low Tw treatment on grain yield The effect of low Tw treatment on reduction of rice production was significant, the rate of reduction was caused by duration of exposure and varieties (Table 3). With the increasing days of low Tw treatment, yield of rice was gradually declined. The average reduction of grain yield was 3.6% and 14.8% under D3 and D6 treatment of DN428. Compared with DN428, the average reduction of grain yield under D3 and D6 treatment was 16.7% and 26.8% of SJ10, while the reduction is relatively small. The average reduction of grain yield was 28.3–44.2% of DN 428 under 9–15 d of low Tw treatment, that of SJ 10 was 37.5–49.7%, the biggest reduction of grain yield was under D15 treatment. There was a significantly positive correlation among effective panicles, fertile spikelet number, spikelet number, 1000-grain weight and yield, and all of them were strongly negatively correlated with spikelet sterility and sterile spikelet number (Table 4). In addition, the DTH was significantly negatively related with effective panicles, fertile spikelet number, spikelet number, 1000-grain weight and yield and positively related with spikelet sterility and sterile spikelet number. Correlation analysis between grain yield and yield-related traits indicated that there were highly significantly positive correlations between grain yield and effective panicles, fertile grain number, spikelet number and 1000-grain weight, respectively (Table 4). The correlation coefficients were 0.939** , 0.959** , 0.814** and 0.961** , respectively. There were highly significantly negative correlations between grain yield and sterile spikelet number, spikelet sterility and days to heading, the correlation coefficients were −0.870** , −0.939** , −0.936** , respectively.

20

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

Table 3 Days to heading, yield and yield-related traits of DN428 and SJ10 under low Tw treatment and analysis of variance for yield and yield-related traits in rice between years and treatments and varieties (F values). The experiment was conducted at Northeast Agricultural University farm, Harbin, Heilongjiang Province, northeast China (2012–2013). Year

Varieties

Treatment

Days to heading (d)

Effective panicles (m2 )

Fertile spikelet number (no./panicle)

2012

DN428

D0 D3 D6 D9 D12 D15 D0 D3 D6 D9 D12 D15

30a 33b 35c 36c 38d 39d 30a 33b 35bc 37cd 39de 40e

330.9a 329.9a 326.8b 324.6bc 322.7cd 321.6d 325.4a 321.5b 319.5c 318.1d 317.0e 315.5f

106.4a 103.9b 99.5c 93.8d 88.2e 84.4f 96.3a 90.6b 85.3c 80.7d 76.3e 73.2f

8.7a 10.3a 12.9b 16.4c 20.9d 23.3e 8.9a 13.0b 15.2c 17.7d 20.0e 21.9f

115.1a 114.1a 112.3b 110.1c 109.1c 107.8d 105.2a 103.6b 100.5c 98.4d 96.3e 95.2f

D0 D3 D6 D9 D12 D15 D0 D3 D6 D9 D12 D15

29a 32b 34c 36d 38e 40f 29a 33b 34c 37d 39e 41f

330.2a 329.4a 326.6b 324.6c 322.7d 321.6d 325.4a 321.3b 319.8c 318.5d 317.0e 316.4e

106.7a 104.8a 100.0b 95.2c 91.2d 87.5e 96.2a 89.2b 83.0c 76.0d 72.7e 70.7f

8.4a 9.4a 12.3b 15.5c 18.2d 20.6e 8.7a 13.3b 17.3c 21.0d 21.8de 22.4e

115.1a 114.2b 112.3c 110.7d 109.4e 108.1f 105.0a 102.5b 100.3c 96.9d 94.5e 93.1f

SJ10

2013

DN428

SJ10

F-value

Varieties (V) Treatment (T) Year (Y) V×T V×Y T×Y V×T×Y

64.17** 12.38** 0.00 6.19** 0.09 0.02 0.10

0.45 173.03** 0.08 0.74 0.01 1.77 0.13

53.39** 15.46** 0.03 4.63** 1.03 0.02 3.54*

3.02* 98.27** 0.00 4.78** 1.26 0.64 2.31

Sterile spikelet number (no./panicle)

Spikelet number (no./panicle)

228.39** 3.65** 0.08 14.34** 0.68 0.01 2.69*

9.55** 58.19** 0.01 4.40** 1.30 0.29 2.95*

1000-grain weight (g)

Grain yield (g m−2 )

7.5a 9.0a 11.4b 14.8c 19.2d 21.7e 8.5a 12.6b 15.2c 18.0d 20.7e 23.0f

25.50a 24.95a 23.14b 20.60c 19.30d 18.46e 25.17a 22.77b 21.36c 19.71d 18.32e 17.23f

897.67a 854.89b 752.22c 627.27d 549.35e 501.15f 788.43a 663.04b 581.82c 506.06d 443.17e 397.92f

7.3a 8.2a 10.9b 14.0c 16.6d 19.0e 8.3a 13.0b 17.3c 21.6d 23.1e 24.1e

24.84a 24.76a 23.21b 20.84c 19.58d 18.63d 25.06a 22.57b 21.48c 19.72d 18.27e 17.58e

875.47a 854.52a 758.05b 644.05c 576.12d 524.16e 784.76a 647.07b 570.18c 477.23d 421.29e 393.11e

3.71** 131.36** 0.00 6.87** 0.00 0.22 0.29

19.08** 42.50** 0.01 13.77** 0.11 0.03 1.66

Spikelet sterility (%)

Alphabets indicates differences between low Tw treatments values (P ≤ 0.01). * Significant at P < 0.05. ** Significant at P < 0.01.

3.2. Effects of low Tw treatment on glutamate metabolism in leaves of rice 3.2.1. Effects of low Tw treatment on the ammonia assimilation enzyme activities GS, GOGAT, and GDH are the main enzymes involved in ammonia assimilation of plant. Different low Tw treatments significantly affected ammonia assimilation enzyme activities. In the leaves of DN428, GS activity increased by 60.64–31.50% under D3, D6, and D9 treatments and decreased by 40.62% and 51.00% under D12 and D15 treatment, respectively (Fig. 1). Whereas, GOGAT activity increased by 15% under D3 treatment and decreased by 2.16–19.67% under the other treatments. GDH activity increased by 12.2% under D3

treatment, however, it decreased by 5.38–23.42% under the other low Tw treatments. In the leaves of SJ10, GS activity increased by 42.69% and 14.93% under D3 and D6 treatment, respectively, and decreased by 21.48–50.99% under the other treatments (Fig. 1). No significant changes in GOGAT activity were observed under D3 treatment, while GOGAT activity decreased by 5.41–25.10% under 6–15 days of low Tw treatment. GDH activity decreased by 4.43–32.15% under low Tw treatments. 3.2.2. Effects of low Tw treatment on GAD activity In this assay, GAD activity increased significantly under low Tw treatments in leaves of DN428 and SN10 and exhibiting the average

Table 4 Correlation coefficients among yield and yield-related traits of rice under low Tw treatment at reproductive stage based on the data obtained from on-farm experiments in Harbin, Heilongjiang province, China in 2012 and 2013.

Days to heading (DTH) Effective panicles (EP) Fertile spikelet number (FSN) Sterile spikelet number (SSN) Spikelet number (SN) Spikelet sterility (SS) 1000-grain weight (GW) Grain yield (GY) *

Significant at P < 0.05. ** Significant at P < 0.01.

DTH

EP

FSN

SSN

SN

SS

GW

GY

1

−0.707** 1

−0.757** 0.964** 1

0.915** −0.800** −0.863** 1

−0.518** 0.922** 0.932** −0.621** 1

0.894** −0.868** −0.932** 0.985** −0.739** 1

−0.928** 0.829** 0.854** −0.948** 0.644** −0.945** 1

−0.870** 0.939** 0.959** −0.939** 0.814** −0.969** 0.961** 1

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

GS activity (μmol mg-1 protein h-1)

35

Control low Tw

DN428

30

35

25

25

20

20

15

15

10

10

5

5

3

6

9

12

15

3

6

9

12

3

15

6

9

12

Time under treatment(days)

35

GOGAT activity(μmol mg-1 protein h-1)

2013

2012

0

0

Control Low Tw

DN428 2012

30

15

3

6

9

12

15

Time under treatment(days)

35

25

25

20

20

15

15

10

10

5

5

Control Low Tw

SJ10

30

2013

0

2012

2013

0 3

6

9

12

15

3

6

9

12

15

3

6

9

Time under treatment(days)

80

GDH activity(μmol mg-1 protein h-1)

Control Low Tw

SJ10

30

2013

2012

21

Control Low Tw

DN428

70

2013

2012

12

15

3

6

9

12

15

Time under treatment(days)

80 70

60

60

50

50

40

40

30

30

20

20

10

10

Control Low Tw

SJ10 2013

2012

0

0 3

6

9

12

15

3

6

9

12

15

Time under treatment(days)

3

6

9

12

15

3

6

9

12

15

Time under treatment(days)

Fig. 1. Ammonia assimilation enzyme activities in leaves of rice under low Tw treatments at reproductive stage. Values are means ± SD. The SD was calculated across three replicates for each year. The experiment was conducted at Northeast Agricultural University farm, Harbin, Heilongjiang Province, northeast China (2012–2013).

highest value of 0.21 and 0.15 U ␮mg−1 protein under D6 treatment, which was 2.49-fold and 2.19-fold higher than that of the control (Fig. 2). 3.2.3. Effects of low Tw treatment on amino acids transamination enzyme activities AlaAT activity increased significantly under low Tw treatments in leaves of DN428 and SN10, exhibiting the highest value under

D12 and D9 treatment, which was 3.57-fold and 1.86-fold higher than that of the control, respectively (Fig. 3). While the AlaAT activity was significantly higher in DN428 than that of SJ10 under low Tw treatments. AspAT activity was significantly lower than the control in leaves of DN428 and SN10 under low Tw treatments and D15 treatment, respectively (Fig. 4). While it was significantly higher than the control under D3, D6, D9 treatment in leaves of SN10.

22

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

GAD activity (U/μmg protein)

0.4

Control Low Tw

DN428 2012

0.4

Control Low Tw

SJ10 2013

2012

2013

0.3

0.3

0.2

0.2

0.1

0.1

0.0

0.0 3

6

9

12

15

3

6

9

12

3

15

6

9

Time under treatment(days)

12

15

3

6

9

12

15

Time under treatment(days)

Fig. 2. GAD activity in leaves of rice under low Tw treatments at reproductive stage. Values are means ± SD. The SD was calculated across 3 replicates for each year. The experiment was conducted at Northeast Agricultural University farm, Harbin, Heilongjiang Province, northeast China (2012–2013).

AlaAT activity (μmol g-1 min-1)

20

Control Low Tw

DN428

20

2013

2012 16

16

12

12

8

8

4

4

Control Low Tw

SJ10

2013

2012

0

0 3

6

9

12

15

3

6

9

12

3

15

6

9

12

15

3

6

9

12

15

Time under treatment(days)

Time under treatment(days)

Fig. 3. AlaAT activity in leaves of rice under low Tw treatments at reproductive stage. Values are means ± SD. The SD was calculated across three replicates for each year. The experiment was conducted at Northeast Agricultural University farm, Harbin, Heilongjiang Province, northeast China (2012–2013).

3.2.4. Effects of low Tw treatment on amino acids contents The content of Asp decreased significantly under low Tw treatments in leaves of DN428 and SN10 (Table 5). No significant changes in Asp were observed under different low Tw treatments,

AspAT activity(μmol g-1 min-1)

20

Control Low Tw

DN428

except that of SJ10 in 2012. Whereas, the contents of AA, Glu, Ala, and GABA increased significantly under low Tw treatments in leaves of DN428 and SN10. AA content exhibited the highest value under D9 treatment, which was average increase 89.95%

20

2013

2012 16

16

12

12

8

8

4

4

Control Low Tw

SJ10

2013

2012

0

0 3

6

9

12

15

3

6

Time under treatment(days)

9

12

15

3

6

9

12

15

3

6

9

12

15

Time under treatment(days)

Fig. 4. AspAT activity in leaves of rice under low Tw treatments at reproductive stage. Values are means ± SD. The SD was calculated across three replicates for each year. The experiment was conducted at Northeast Agricultural University farm, Harbin, Heilongjiang Province, northeast China (2012–2013).

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

23

Table 5 Amino acids content in leaves of DN428 and SJ10 under low Tw treatment at reproductive stage in 2012–2013(mg g−1 FW* ). Year

2012

Control

Low Tw treatment

2013

Control

Low Tw treatment

SJ10

DN428

Treatment (days)

AA

Glu

Ala

Asp

GABA

AA

Glu

Ala

Asp

GABA

3 6 9 12 15 3 6 9 12 15

1.92a 1.95a 1.99a 2.03a 2.05a 3.09b 3.61cd 3.77d 3.22bc 3.02b

0.21a 0.22ab 0.25ab 0.26ab 0.27b 0.34c 0.43d 0.40de 0.37ce 0.34c

0.13a 0.15a 0.16a 0.17a 0.18a 0.48b 0.60c 0.63c 0.65c 0.49b

0.18b 0.20bc 0.21cd 0.23de 0.24e 0.06a 0.06a 0.06a 0.06a 0.05a

0.28a 0.30ab 0.31ab 0.35b 0.36b 0.67c 0.82d 0.78d 0.70c 0.68c

1.96a 1.94a 1.96a 1.98a 1.98a 2.98b 3.49bc 3.57bc 3.64bc 3.81c

0.24a 0.23ab 0.24ab 0.25ab 0.24ab 0.30b 0.38c 0.37c 0.37c 0.37c

0.16a 0.15a 0.16a 0.17a 0.16a 0.39b 0.49c 0.58d 0.57d 0.57d

0.20a 0.20a 0.20a 0.22b 0.21c 0.06d 0.07e 0.06d 0.05f 0.07e

0.31a 0.30a 0.30a 0.32a 0.30a 0.63b 0.75c 0.71cd 0.67bd 0.67bd

3 6 9 12 15 3 6 9 12 15

1.94a 1.96a 1.99a 2.01a 2.04a 3.10b 3.45bc 3.79c 3.26b 3.15b

0.20a 0.21a 0.23a 0.25a 0.26a 0.35bd 0.45c 0.40cd 0.37bd 0.33b

0.12a 0.14a 0.16a 0.18a 0.20a 0.49b 0.61b 0.63b 0.65b 0.50b

0.17b 0.19bc 0.21cd 0.23d 0.23d 0.06a 0.07a 0.07a 0.06a 0.05a

0.30a 0.29a 0.30a 0.31a 0.32a 0.68b 0.83b 0.79b 0.71b 0.68b

2.10a 2.11a 2.13a 2.15a 2.15a 3.13b 3.46b 3.57b 3.88b 3.92b

0.23a 0.24ab 0.25ab 0.25ab 0.26ab 0.30bc 0.38d 0.37d 0.35cd 0.33cd

0.14a 0.15a 0.16a 0.16a 0.18a 0.39b 0.48c 0.57d 0.54cd 0.53cd

0.21a 0.20a 0.21a 0.22a 0.23a 0.06b 0.06b 0.07b 0.06b 0.06b

0.29a 0.28a 0.29a 0.30a 0.30a 0.61b 0.75b 0.70b 0.66b 0.65b

Alphabets indicates differences between low Tw treatments values (P ≤ 0.01). * FW—fresh weight.

and 87.37% in DN428 and SJ10, respectively. The content of Glu and GABA touched their peak under D6 treatment. The average increment of Glu content was 104.87% and 61.77% in DN428 and SJ10, respectively, that of GABA content was 179.99% and 158.93% in DN428 and SN10, respectively. Ala content exhibited the highest value under D12 and D9 treatments, which was average increase 2.71-fold and 2.59-fold in DN428 and SN10, respectively. 3.3. Comprehensive analysis The cold response index of grain yield and spikelet sterility were included as the dependent variables (Y), 11 indicators as variables (x), with each variable used in the cold response index transformation. The multiple linear stepwise regression was used to analyze the effects of glutamate metabolism on of rice under low Tw treatment at reproductive stage (Table 6). Likewise, x1 , x2 , and x3 in the regression equation of the grain yield (YGY ) were used to represent the content of Glu, AA, and Asp, respectively, whereas, R2 was 0.976, and F-value was 219.477 > F0.01 (5.29). All of these results indicates a close relationship between Glu, AA, Asp content and grain yield in rice. While, coefficient of YGY and the prediction grain yield was 0.965, which indicated that the accuracy of the grain yield regression model were adequate. Thus glutamate, AA and Asp content can be used as the comprehensive evaluation index of the glutamate metabolism effect on grain yield of rice, with a reliability of 97.6%. In addition, x4 , x5 , and x6 in the regression equation of the spikelet sterility (YSS ) were used to represent the activity of GOGAT, AlaAT, GDH, respectively, the coefficient of the equation R2 was 0.900, and F-value was 47.911 > F0.01 (5.29). These results showed a close relationship between these three indexes and spikelet sterility in rice. The coefficient of YSS and the prediction spikelet sterility was 0.945, which suggest that the accuracy of the spikelet sterility regression model were adequate. All in all, the activity of GOGAT, AlaAT, GDH could be used for the comprehensive evaluation of glutamate metabolism effects on the spikelet sterility under cold-water stress, with a reliability of 90.0%.

4. Discussion 4.1. Changes in yield and yield-related traits of rice under low Tw treatment at reproductive stage Water temperature (Tw ) is a major determinant for growth and yield of rice (O. sativa L.) grown under cool climates (Shimono et al., 2002). Tw has effects on rice growth and yield, amongst which yield and spikelet sterility can play pivotal roles (Pereira da Cruz et al., 2006; Thakur et al., 2010). The results in this study showed that the average reduction of grain yield was 3.6–44.2% of DN 428 under 3–15 d of low Tw treatment, that of SJ 10 was 16.7–49.7%, the biggest reduction of grain yield was under D15 treatment (Table 3), compared with the control, indicating that the grain yield of rice was declined with the increasing duration of exposure. Gunawardena et al. (2003) demonstrated that when rice plants encountered low temperature during the reproductive stage, both panicle and root temperature affected spikelet sterility, implying that both low Ta and low Tw affected rice growth. Pereira da Cruz et al. (2006) reported that spikelet sterility of rice significant increased due to 17 ◦ C low air temperature (Ta ) applied for seven days at anthesis. Our date revealed that spikelet sterility was extremely increased under 6–15 d of low Tw treatment at reproductive stage in DN428 and SJ10. Therefore, the effect of low Ta and Tw on spikelet sterility in rice at reproductive stage is similar. In rice, after 5days of low Ta treatment there was a huge reduction in spikelet fertility (Pereira da Cruz et al., 2006). As previously reported, effective panicles, sterile spikelet number, spikelet sterility and 1000-grain weight showed no difference in DN428 under 3 days of exposure, but significant reduction under 6–15 d of low Tw treatment in DN428, whereas, that of SJ10 showed significant reduction under all low Tw treatments, which was more severe in SJ10 than that of DN428 (Table 3), showing that shorter periods of cold stress allow for the detection of genotypes that are more sensitive and longer periods favors tolerant genotypes. Our date also showed that the duration of panicle development (DTH) was prolonged, varying with rice varieties and the duration of exposure. Meanwhile, DTH was significantly negatively related with grain yield and positively related with spikelet sterility and

24

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

Table 6 Identification model of cold-region rice under low Tw treatment at reproductive stage. Dependent variables (Y)

Stepwise regression

Correlation coefficients (r)

R2

Grain yield (YGY ) Spikelet sterility (YSS )

YGY = −0.159 + 1.123x1 − 0.123x2 + 0.216x3 YSS = 8.150 − 3.6024 + 0.033x5 − 5.936x6

0.988 0.949

0.976 0.900

x1 : Glu content, x2 : AA content, x3 : Asp content, x4 : GOGAT activity, x5 : AlaAT activity, x6 : GDH activity.

sterile spikelet number (Table 4), implying that prolonged DTH could be another important reason for agricultural yield loss. However, Shimono et al. (2007) reported that the duration of panicle development varied less under low Tw (19.5 ◦ C) treatment during panicle development, compared with the control, implying that the stage and the temperature of cold stress treatment is also an important factor that caused different results.

4.2. Changes in glutamate metabolism in leaves of rice under low Tw treatment at reproductive stage Our date revealed that glutamate metabolism demonstrated varying degrees of changes in leaves under low Tw treatment at reproductive stage in rice. Considering the changes of glutamate metabolism is suppose to typically dependent on the length of exposure to low Tw . The response of GAD and the concomitant accumulation of GABA have been described for many plant species under abiotic stresses (Bouché and Fromm, 2004; Fait et al., 2007; Song et al., 2010). However, the increasing proportion of GABA and GAD increased firstly and then decreased, exhibited the highest value under D6 treatment (Fig. 2 and Table 5). Probably, the products of glutamate metabolism implement the hardening effects induced by chilling and as a consequence the stress tolerance increases. GABA could reduce the oxidative damage of crops under abiotic stress (Song et al., 2010). In this study, GABA level increased strongly under 3–6 d low Tw treatment, however, GABA failed to persistently increase under long-time of cold stress (9–15 d). This can be explained that GABA shunt was inhibited under long-time of low Tw treatment. The induction of AlaAT and the concomitant accumulation of Ala have been described previously for many plant species under hypoxic stress (Reggiani et al., 1988, 2000; Miyashita and Good, 2008; Rocha et al., 2010). Besides, the content of Ala may be controlled by the changes in the levels of GABA shunt under hypoxia. (Miyashita and Good, 2008; Rocha et al., 2010). Moreover, Wallace et al. (1984) found that the contents of GABA, Glu, aspartate (Asp), and Ala increased under low temperature (6 ◦ C) stress in soybean leaves. However, a better understanding of the relationship between GABA shunt and Ala metabolism under low Tw treatment of rice is essential. In this study, AlaAT activity had the same trends as GAD activity. However, the levels of Glu, GABA, Ala increased strongly, the increasing proportion of Glu, GABA and Ala increased firstly and then decreased (Table 5). It is most intriguing that the content of Ala appears to occur at the cost of GABA and Glu. Glu and GABA content exhibited the highest value under D6 treatment for DN428 and SN10, whereas, Ala content exhibited the highest value under D12 and D9 treatments for DN428 and SN10, respectively, the change of Ala can be explained that Alanine could maintain osmotic potential in rice under low Tw treatment. These changes of glutamate metabolism were best explained as part of the adaptive response of plants to chilling: the levels of GS/GOGAT cycle directly affected GABA shunt and transamination of amino acids under low Tw treatment, highlighting the important role of glutamate metabolism under low Tw treatment. The activities of GAD, AlaAT and the levels of Glu, GABA, Ala increased strongly under low Tw treatment. Metabolic equilibriums are expected to drive the metabolic flux from glycolysis, via Ala

synthesis and GABA shunt. Probably, more amount of ATP could be gained via these pathways. 4.3. Relationship between yield or spikelet sterility and glutamate metabolism in leaves under low Tw treatment at reproductive stage Low temperature stress caused considerable yield loss in rice, grain yield and spikelet sterility could play pivotal roles to characterize the effects of cold damage at reproductive stage (Thakur et al., 2010). Our date showed that cold stress also caused different degree of dramatic changes to metabolism in rice. Glutamate metabolism which orchestrates pivotal metabolic functions, played a central role in the amino acid metabolism of plants (Seifi et al., 2013) and key role in plant defense (Forde and Lea, 2007; Galili et al., 2001). In addition, the ammonium assimilation enzyme activities were significantly related to grain yield (Sun et al., 2012). Multiple stepwise regression could be appropriate ways to found out the relationship between grain yield or spikelet sterility and glutamate metabolism in leaves under low Tw treatment at reproductive stage. Our date identified glutamate (Glu) content and glutamate synthase (GDH) activity as important traits that influenced grain yield and spikelet sterility, respectively, indicating that the higher content of Glu and GDH activity in leaves, the lower reduction of grain yield or lower spikelet sterility of rice. Recent studies have also revealed Glu plays a regulatory role in modulation of plant growth and development (Walch-Liu et al., 2006; Walch-Liu and Forde, 2008). Glu has also been proved to associate with signaling cascades of ABA in plants (Khan et al., 2004). ABA is found to increase significantly in response to cold stress (Shinozaki and Yamaguchi-Shinozaki, 2000). It is conceivable that Glu could be involved in low Tw treatment at reproductive stage in rice. Masclaux-Daubresse et al. (2006) have confirmed that in both young and old tobacco leaves, Glu is synthesized via the combined action of GS and GDH, whilst GDH is responsible for the deamination of Glu. GDH levels increase under various stress conditions (Dubois et al., 2003; Robredo et al., 2011). The increase of GDH has been proposed that ammonia could be assimilated by the aminating activity of GDH when the GS/GOGAT cycle pathway is inhibited under stress conditions (Oaks, 1995), such as salinity (Skopelitis et al., 2006) or drought (Mena-Petite et al., 2006). The possible role of GDH as a stress related enzyme was also suggested by Robredo et al. (2011) found that NAD-GDH activity of leaves in barley plants rose rapidly in response to drought, such an increase is thought to be required to provide energy for stress tolerance mechanisms. Under cold stress in which photosynthesis is reduced, there is a strong demand for energy, obtainable from carbon released by amino acids. The increased level of GDH activity could deaminate these amino acids. However, compared with the control, GDH activity only increased by 12.2% under D3 treatment in DN428, it decreased in DN428 and SJ10 under the other low Tw treatments (Fig. 1), it showed that the increased GDH activity could provide carbon skeletons for use in respiration under shorter periods of low Tw treatment. However, more amount of energy could be gained via other pathways under longer duration of exposure. GDH also plays a role in controlling plant productivity (Ameziane et al., 2000). In this study, GDH activity showed negative correlation with spikelet sterility under low Tw treatments

Y. Jia et al. / Field Crops Research 175 (2015) 16–25

(Table 6), showing that GDH activity could play a major role in controlling rice productivity under low Tw treatment at reproductive stage. 5. Conclusions The results revealed that the changes degree of yield, yieldrelated traits and glutamate metabolism varies with rice varieties and duration of exposure. Multiple stepwise regression identified Glu content and GDH activity as important traits that influenced the grain yield and spikelet sterility, respectively. By adjusting the glutamate metabolism level of leaves in rice, we can thus improve rice cold tolerance, and reduce the effect of low Tw (17 ◦ C) treatment on grain yield and spikelet sterility during reproductive stage. Acknowledgements We are grateful to the National Science and Technology Project (2011BAD16B11; 2013BAD20B04); the project of Heilongjiang province “science and technology project” (GA10B102-5); the Science and Technology Innovation Project of Northeast Agricultural University (201410224013). References Ameziane, R.K., Bernhard, R.B., Lightfoot, D., 2000. Expression of the bacterial gdhA gene encoding NADPH glutamate dehydrogenase in tobacco affects plant growth and development. Plant Soil 221, 47–57. Bai, Q.Y., Chai, M.Q., Gu, Z.X., Cao, X.H., Li, Y., Liu, K.L., 2009. Effects of components in culture medium on glutamate decarboxylase activity and gamma-aminobutyric acid accumulation in foxtail millet (Setaria italica L.) during germination. Food Chem. 116, 152–157. Beck, E.H., Fettig, S., Knake, C., Hartig, K., Bhattarai, T., 2007. Specific and unspecific responses of plants to cold and drought stress. J. Biosci. 32, 501–510. Bouché, N., Lacombe, B., Fromm, H., 2003. Gaba signaling: a conserved and ubiquitous mechanism. Trends Cell Biol. 13, 607–610. Bouché, N., Fromm, H., 2004. GABA in plants: just a metabolite? Trends Plant Sci. 3, 110–115. Dubois, F.T., Gonzalez-Moro, M.B., Estavillo, J.M., Sangwan, R., Gallais, A., Hirel, B., 2003. Glutamate dehydrogenase in plants: is there a new story for an old enzyme? Plant Physiol. Biochem. 41, 565–576. Fait, A., Fromm, H., Walter, D., Galili, G., Fernie, A.R., 2007. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci. 13, 14–19. Farrell, T.C., Fox, K.M., Williams, R.L., Fukai, S., 2006. Genotypic variation for cold tolerance during reproductive development in rice: screening with cold air and cold water. Field Crops Res. 98, 178–194. Forde, B.G., Lea, P.J., 2007. Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339–2358. Galili, G., Tang, G., Zhu, X., Gakiere, B., 2001. Lysine catabolism: a stress and development super-regulated metabolic pathway. Curr. Opin. Plant Biol. 4, 261–266. Gunawardena, T.A., Fukai, S., Blamey, F.P.C., 2003. Low temperature induced spikelet sterility in rice. I: Nitrogen fertilization and sensitive reproductive period. Aust. J. Agric. Res. 54, 937–946. Jacobs, B.C., Pearson, C.J., 1999. Growth, development and yield of rice in response to cold temperature. J. Agron. Crop Sci. 182, 79–88. Khan, M.A., Gul, B., Weber, D.J., 2004. Action of plant growth regulators and salinity on the seed germination of Ceratoides lanata. Can. J. Bot. 82, 37–42. Kinnersley, A.M., Turano, F.J., 2000. Gamma aminobutyric acid (GABA) and plant responses to stress. Crit. Rev. Plant Sci. 19, 479–509. Loulakakis, K.A., Roubelakis-Angelakis, K.A., 1990. Intracellular localization and properties of NADH–glutamate dehydrogenase from Vitis vinifera L.: purification and characterization of the major leaf isoenzyme. J. Exp. Bot. 41, 1223–1230. Masclaux-Daubresse, C., Reisdorf-Cren, M., Pageau, K., Lelandais, M., Grandjean, O., Kronenberger, J., Valadier, M.H., Feraud, M., Jouglet, T., Suzuki, A., 2006. Glutamine synthetase–glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink source nitrogen cycle in tobacco. Plant Physiol. 140, 444–456. Mena-Petite, A., Lacuesta, M., Mu˜noz-Rueda, A., 2006. Ammonium assimilation in Pinus radiata seedlings: effects of storage treatments, transplanting stress and water regimes after planting under simulated field conditions. Environ. Exp. Bot. 55, 1–14.

25

Miyashita, Y., Good, A.G., 2008. Contribution of the GABA shunt to hypoxia-induced alanine accumulation in roots of Arabidopsis thaliana. Plant Cell Physiol. 49, 92–102. O’Neal, D., Joy, K.W., 1973. Glutamine synthetase of pea leaves. 1. Purification stabilization and pH optima. Arch. Biochem. Biophys. 113, 113–122. Oaks, A., 1995. Evidence for deamination by glutamate dehydrogenase in higher plants: reply. Can. J. Bot. 73, 1116–1117. Oliver, S.N., Dennis, E.S., Dolferus, R., 2007. ABA Regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility in rice. Plant Cell Physiol. 48, 1319–1330. Pereira da Cruz, R., Milach, S.C.K., Federizzi, L.C., 2006. Rice cold tolerance at the reproductive stage in a controlled environment. Sci. Agric. 63, 255–261. Reggiani, R., Cantú, C.A., Brambilla, I., Bertani, A., 1988. Accumulation and interconversion of amino acids under anoxia. Plant Cell Physiol. 29, 981–987. Reggiani, R., Nebuloni, M., Mattana, M., Brambilla, I., 2000. Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle. Amino Acids 18, 207–217. Robredo, A., Usue, P.L., Jon, M.A., Lacuesta, M., Amaia, M.P., Alberto, M.R., 2011. Elevated CO2 reduces the drought effect on nitrogen metabolism in barley plants during drought and subsequent recovery. Environ. Exp. Bot. 71, 399–408. Rocha, M., Licausi, F., Araújo, W.L., Nunes-Nesi, A., Sodek, L., Fernie, A.R., van Dongen, J.T., 2010. Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicas. Plant Physiol. 152, 1501–1513. Satake, T., Hayase, H., 1970. Male sterility caused by cooling treatment at the young microspore stage in rice plants. V. Estimation of pollen developmental stage and the most sensitive stage to coolness. Proc. Crop Sci. Soc. Jpn. 39, 468–473. Seifi, H.S., Van Bockhaven, J., Angenon, G., Höfte, M., 2013. Glutamate metabolism in plant disease and defense: friend or foe? Mol. Plant Microbe Interact. 26, 475–485. Shimazaki, Y., Satake, T., Watanabe, K., 1963. Studies on the growth and nutrient absorption of the rice plant reared with cool water irrigation during its early growth stage. Res. Bull. Hokkaido Natl. Agric. Exp. Stn. 80, 1–12 (in Japanese, with English summary). Shimono, H., Hasegawa, T., Iwama, K., 2002. Response of growth and grain yield in paddy rice to cool water at different growth stage. Field Crop Res. 73, 67–79. Shimono, H., Okada, M., Kanda, E., Arakawa, I., 2007. Low temperature-induced sterility in rice: evidence for the effects of temperature before panicle initiation. Field Crop Res. 101, 221–231. Shinozaki, K., Yamaguchi-Shinozaki, K., 2000. Molecular responses to dehydration and low temperature: differences and crosstalk between two stress signaling pathways. Curr. Opin. Plant Biol. 3, 217–223. Singh, R.D., Srivastava, H.S., 1986. Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen. Physiol. Plant. 66, 413–416. Sipaseuth, Basnayake, J., Fukai, S., Farrell, T.C., Senthonghae, M., Sengkeo, Phamixay, S., Linquist, B., Chanphengsay, M., 2007. Opportunities to increasing dry season rice productivity in low temperature affected areas. Field Crops Res. 102, 87–97. Skopelitis, D.S., Paranychianakis, N.V., Paschalidis, K.A., Pliakonis, E.D., Delis, I.D., Yakoumakis, D.I., Kouvarakis, A., Papadakis, A.K., Stephanou, E.G., Roubelakis Angelakis, K.A., 2006. Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 18, 2767–2781. Song, H., Xu, X., Wang, H., Wang, H., Tao, Y., 2010. Exogenous ␥-aminobutyric acid alleviates oxidative damage caused by aluminium and proton stresses on barley seedlings. J. Sci. Food Agric. 90, 1410–1416. Sun, Y.J., Ma, J., Sun, Y.Y., Xu, H., Yang, Z.Y., Liu, S.J., Jia, X.W., Zheng, H.Z., 2012. The effects of different water and nitrogen managements on yield and nitrogen use efficiency in hybrid rice of China. Field Crop. Res. 127, 85–98. Thakur, P., Kumar, S., Malik, J.A., Berger, J.D., Nayyar, H., 2010. Cold stress effects on reproductive development in grain crops: an overview. Environ. Exp. Bot. 67, 429–443. Walch-Liu, P., Forde, B.G., 2008. Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises l-glutamate-induced changes in root architecture. Plant J. 54, 820–828. Walch-Liu, P., Liu, L.H., Remans, T., Tester, M., Forde, B.G., 2006. Evidence that glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana. Plant Cell Physiol. 47, 1045–1057. Wallace, W., Secor, J., Schrader, L.E., 1984. Rapid accumulation of ␥-aminobutyric acid and alanine in soybean leaves in response to an abrupt transfer to lower temperature, darkness, or mechanical manipulation. Plant Physiol. 75, 170–175. Wang, J., Lin, X., Sun, Q., Jena, K.K., 2013. Evaluation of cold tolerance for japonica rice varieties from different country. Adv. J. Food Sci. Technol. 5, 54–56. Wu, R., Garg, A., 2003. Engineering rice plant with trehalose-producing genes. improves tolerance to drought, salt and low temperature. ISB news report, March 2003. Covering agricultural and environmental biotechnology developments. In: ISB News Report (February). Zia, M.S., Salim, M., Aslam, M., Gill, M.A., 1994. Effect of low temperature of irrigation water on rice growth and nutrient uptake. J. Agron. Crop Sci. 173, 22–31.