Application of γ‐aminobutyric acid (GABA) and nitrogen regulates aroma biochemistry in fragrant rice

Abstract The 2‐acetyl‐1‐pyrroline (2AP) is a key aroma compound in fragrant rice. The present study assessed the γ‐aminobutyric acid (GABA) and nitrogen (N) application induced regulations in the biochemical basis of rice aroma formation. Four N levels, that is, 0, 0.87, 1.75, and 2.61 g/pot, and two GABA treatments, that is, 0 mg/L (GABA0) and 250 mg/L (GABA250), were applied to three fragrant rice cultivars, that is, Yuxiangyouzhan, Yungengyou 14, and Basmati‐385. Results showed that GABA250 increased 2AP, Na, Mn, Zn, and Fe contents by 8.44%, 10.95%, 25.70%, 11.14%, and 43.30%, respectively, under N treatments across cultivars. The GABA250 further enhanced the activities of proline dehydrogenase (PDH), ornithine aminotransferase (OAT) (both at 15 days after heading (d AH), and diamine oxidase (DAO) (at maturity) by 20.36%, 11.24%, and 17.71%, respectively. Significant interaction between GABA and N for Mn, Zn, and Fe contents in grains, proline content in leaves, GABA content in leaves at 15 d AH and maturity stage (MS), Δ1‐pyrroline‐5‐carboxylic acid (P5C) contents in leaves at 15 d AH, and Δ1‐pyrroline‐5‐carboxylate synthase (P5CS), PDH, and OAT activities in leaves at MS was noted. Moreover, the 2AP contents in grains at MS showed a significant and positive correlation with the proline contents in the leaves at 15d AH. In conclusion, GABA250 enhanced the 2AP, Na, Mn, Zn, and Fe contents, as well as the enzyme activities involved in 2AP biosynthesis. Exogenous GABA and N application improved the 2AP contents and nutrient uptake in fragrant rice.

The biosynthesis and regulations of 2AP are largely affected by not only the genetic factors but also the external environment and crop management factors (Bao et al., 2018;Hasanuzzaman et al., 2019). The genetic factors play an important part in determining the quality of aromatic rice (Bao et al., 2018;Bradbury et al., 2008), which, on the other hand, are much dependent on prevailing environmental conditions during the growth period and also by pre-and postharvest management techniques (Gay et al., 2010). For example, nitrogen fertilization has great impacts on 2AP formation and accumulation. Recently, Ren et al. (2017) showed that 60 kg/ hm 2 nitrogen with water deficit conditions at tillering stage could enhance the 2AP contents in grains significantly, whereas Mo et al. (2018) also demonstrated that different nitrogen application levels affected the 2AP contents in fragrant rice differently. Early studies revealed that BADH induced the biosynthesis of γ-aminobutyric acid (GABA) by γ-aminobutyraldehyde, while the inactivation of BADH in aromatic rice induced the accumulation of △1-pyrroline (Bradbury et al., 2008). Moreover, the 2AP and GABA contents in grain were increased under salt treatment and shading condition (Mo et al., 2015;Poonlaphdecha et al., 2012) and there was a significant positive correlation between 2AP and GABA in the grains of "Yuxiangyouzhan" rice cultivar (Mo et al., 2015). Hence, there might be a possibility for exogenous GABA to regulate the 2AP biosynthesis.
GABA is well known as a plant signaling molecule and is involved in various physio-biochemical processes in plants (Fait, Fromm, Walter, Galili, & Fernie, 2008;Routray & Rayaguru, 2018). GABA has been reported to increase plant resistance ability against several environmental stresses (Yang, Shewfelt, Lee, & Kays, 2008). Regulatory effects of GABA in plant physio-biochemical metabolism have been previously reported in Arabidopsis, tobacco, maize, barley, and rice (Batushansky et al., 2014;Li, Guo, Yang, Meng, & Wei, 2016;Nayyar, Kaur, Kaur, & Singh, 2014;Song, Xu, Wang, Wang, & Tao, 2010;Yu & Sun, 2007), whereas exogenous GABA applications could improve salt stress resistance in wheat seedlings (Li, Guo, et al., 2016) and enhance photosynthesis capacity in maize seedling (Li, Liu, et al., 2016). In sum, GABA plays a crucial role in plant growth to respond to the external environmental conditions (Fait et al., 2008;Li, Guo, et al., 2016); nevertheless, endogenous GABA levels in plants are very low, but it can be accelerated under stress conditions (Kinnersley & Turano, 2000). Hence, GABA may influence the physiological metabolism of fragrant rice by affecting either aroma biosynthesis or plant growth. Reports are available for GABA-induced regulations in stress tolerance in various crops; however, effects of exogenous GABA application coupled with various N levels on aroma biosynthesis are rarely investigated. Therefore, the present study was aimed to evaluate the effects of GABA under various levels of N on physio-biochemical attributes, aroma biosynthesis, and enzyme involved in 2AP formation in fragrant rice.

| Plant material and growing conditions
The seeds of three aromatic rice cultivars, that is, Yuxiangyouzhan (YX), were transplanted into the soil-containing pots (31cm in diameter and 29 cm in height) on 4 April. The experimental soil was sandy loam containing soil organic matter 18.65 g/kg, total nitrogen 1.17 g/kg, available phosphorus 32.69 mg/kg, available potassium 185.28 mg/kg, and pH 6.44. The yearly average temperature of the region lies between 21 and 29°C and is characterized by a subtropical monsoonal type of climate.

| Treatments and plant sampling
The treatment without GABA application was denoted as GABA0 (double-distilled water sprinkler application), and the GABA at 250mg/L (GABA250) was applied at panicle initiation stage, whereas four nitrogen rates, that is, 0, 0.87, 1.75, and 2.61 g/pot denoted as ZN, LN, MN, and HN, were applied as basal dose. Flag leaves at the heading stage (HS), 15 days after heading stage (15d AH), and maturity stage (MS), whereas mature panicles at MS were collected and stored at -80°C for physio-biochemical assays.

| Determination of 2AP contents in grains
The 2AP concentration was determined by synchronization distillation and extraction method (SDE) combined with GCMS-QP 2010 Plus (Shimadzu Corporation) according to Huang et al. (2012) and the contents of 2AP were expressed as μg/g.

| Determination of Na, Mn, Zn, and Fe contents in grains
The Na, Mn, Zn, and Fe contents in grains were determined according to the method of Wu et al. (2014) with some modification. In brief, the grain sample was oven-dried at 80°C to constant weight and ground in powder, and then weighed 0.25 g of the sample. The weighted samples were dry-digested in a muffle furnace at 500°C for 6 hr, and then added to 40 ml HNO 3 :H 2 O (1:1). The contents of Na, Mn, Zn, and Fe were determined using a flame atomic absorption spectrometry (SHIMADZU AA-6300C AA spectrometer).

| Measurement of protein contents in grains
After sun drying, about 1.5 kg grains from each treatment were taken to estimate the protein contents by using an Infratec 1241 grain analyzer (FOSS-TECATOR; Mo et al., 2015).

| Determination of proline, GABA, and pyrroline-5-carboxylic acid (P5C) contents in leaves
The proline contents in fresh leave samples were determined by Bates, Waldren, and Teare (1973) by using ninhydrin, and the absorbance was read at 520nm. The final proline contents were expressed as μg/g fresh weight (FW) of leaves. The GABA contents were measured according to the methods described by Zhao et al. (2009), and the GABA contents were expressed as μg/g. The P5C concentration was estimated by following the methods of Wu, Chou, Wu, Chen, and Huang (2009). The reaction mixture contained 0.2 ml supernatant of enzyme extract, 0.5 ml of 10% trichloroacetic acid (TCA), and 0.2 ml of 40 mM 2-aminobenzaldehyde. The absorbance was read at 440 nm, and the contents were expressed as μmol/g. The PDH activity was assayed by following the methods of Tateishi, Nakagawa, and Esaka (2005) and Ncube, Finnie, and Van (2013).
The absorbance after reaction was read, and the reaction mixture contained L-proline (15 mM), cytochrome c (0.01 mM), phosphate buffer (100m M, pH 7.4), 0.5% (v/v) Triton X-100, and the enzyme extract (0.1 ml) in a total volume of 0.5 ml. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by adding 0.5 ml of 10% trichloroacetic acid (TCA). After adding 0.5ml of 0.5% 2-aminobenzaldehyde in 95% ethanol, the mixture was further incubated at 37°C for 10 min and centrifuged at 8,000 rpm for 10 min, and the absorbance of the supernatant was read at 440 nm, the absorbance change of 0.1 in one minute was defined as one unit of enzyme activity, and the activity was expressed as U/g FW.
The activity of P5CS was estimated according to the methods described by Zhang, Lu, and Verma (1995). The reaction mixture comprised of 50 mM Tris-HCL buffer, 20 mM MgCl 2 , 50 mM sodium glutamate, 10 mM ATP, 100 mM hydroxamate-HCL, and 0.5 ml of enzyme extract. The reaction was started by the addition of 0.5ml of enzymatic extracts. After 5 min at 37°C, the reaction was stopped by addition of 0.5 ml of a stop buffer (2.5% of FeCl 3 plus 6% of trichloroacetic acid, dissolved in 100 ml of 2.5 M HCl). The absorbance after the reaction was read at 440 nm, the absorbance change of 0.1 in one minute was defined as one unit of enzyme activity, and the activity was expressed as U/g FW.
The OAT activity was measured according to the methods of Chen, Chen, Lin, and Kao (2001) and Umair, Leung, Bland, and Simpson (2011). The reaction medium contained 100 mM potassium phosphate buffer pH 8.0, 50 mM ornithine, 20 mM α-ketoglutarate, 1 mM pyridoxal 5-phosphate, and the enzyme extract (0.1ml)-the final total volume was 1 ml. The reaction medium was incubated at 37°C for 30 min. The reaction was stopped by adding 0.5 ml trichloroacetic acid (10%), and the color was developed by incubating the reaction mixture with 0.5 ml o-amino benzaldehyde (0.25%) in ethanol (95%) for 1 hr. After centrifugation at 8,000 rpm for 10 min, the clear supernatant fraction was taken to measure the absorbance at 440 nm. The absorbance change of 0.1 in one minute was defined as one unit of enzyme activity; the activity was expressed as U/g FW.
The DAO activity was assayed by using the methods described by Su, An, Zhang, and Liu (2005). The reaction solutions (3.0 ml) contained 2.5 ml 0.1 M sodium phosphate buffer (pH 6.5), 0.1 ml crude enzyme extracts, 0.1 ml peroxidase (250 U/ml), and 0.2 ml 4-aminoantipyrine/ N, N-dimethylaniline reaction solutions. The reaction was initiated by the addition of 0.1 ml 20 mM Put. A 0.01 value of the changes in absorbance at 440 nm was regarded as one activity unit of the enzyme, and the activity was expressed as U/g FW.

| Statistical analyses
The pots were arranged in a completely randomized design (CRD), and the data were analyzed by using Statistix version 8 (Analytical Software). Relationships among the indexes were evaluated using correlation analyses by Statistix version 8 (Analytical Software).
Means among treatments were compared based on the least significant difference test (LSD) at the 0.05 probability level.

| 2AP contents
The 2AP contents in grains were substantially affected by GABA treatment (T), variety (V), nitrogen (N), and V × N. Compared with GABA0, the GABA 250 significantly increased the 2AP contents by 8.44% under nitrogen treatments across cultivars. With the increase in the application dose of nitrogen fertilizer, the 2AP contents in grains of all rice cultivars were increased. Moreover, the highest 2AP contents were recorded in YG, which were in the range of 8.89 to 12.57μg/g FW, while the BS and YX were found statistically similar (p ˃ .05) regarding grain 2AP contents with a range from 3.86 to 5.98 μg/g FW (Figure 1).

| Protein contents
The T, V, N, T × V, and V × N significantly affected the protein contents in grains of aromatic rice cultivars. Compared with GABA0, GABA250 led to a decrease in the protein content by 3.76% under N treatments across cultivars. With the increased application dose of N fertilizer, the mean protein contents in grains were increased. BS showed higher mean protein content than YX and YG ( Figure 2).

| Na, Mn, Zn, and Fe contents in grains
Compared with GABA0, Na content in grains under GABA250 was significantly higher, which was 5.33%. Similar trends were also re-

| P5C concentration
GABA treatment (T) did not affect the P5C contents in leaves, whereas the cultivars were found statistically significant regarding P5C contents in leaves. Significant differences were observed for T × V, N, T × N, V × N, and T × V×N at 15 d AH. Furthermore, YG produced lower P5C content in leaves as compared to YX and BS, while P5C contents substantially differed among different growth stages (Table 3).

| GABA contents
Cultivar significantly differed regarding the accumulation of GABA contents in leaves; however, GABA treatment (T), nitrogen (N), T × V, V × N, and T × V×N did not affect GABA content in leaves significantly. On the other hand, significant differences were observed in GABA content for T × N at 15 d AH and MS, whereas YG produced lower GABA content in leaves as compared to YX and BS, while GABA contents substantially differed (p < .05) among different growth stages (Table 4).

Influences of the GABA and N application regulations in the 2AP
and the activities of the enzymes involved in its biosynthesis were assessed in this study. The 2AP is recognized as the key compound for the fragrance of aromatic rice in many previous reports (Buttery, Ling, Juliano, & Turnbaugh, 1983;Magnus, Juliano, & Peter, 2002;Poonlaphdecha et al., 2012). In this study, the 2AP content in grain increased with the improvement of N level and the 2AP content was significantly increased under GABA250 treatment ( Figure 1).
Previous studies have indicated that salt and shading treatment increased the 2AP and GABA contents in grain (Mo et al., 2015;Poonlaphdecha et al., 2012) and a significant positive correlation between 2AP and GABA in the grains was also observed for the "Yuxiangyouzhan" (Mo et al., 2015). Additionally, nitrogen fertilization has great impacts on 2AP formation and accumulation (Mo et al., 2018;Ren et al., 2017). Therefore, the main reasons that GABA and N regulate the aroma formation are as follows: (a) GABA is directly related to 2AP formation; and (b) the GABA and N regulate the physio-chemical parameters of 2AP formation.
Moreover, compared with GABA0, Na, Mn, Zn, and Fe contents in grains under GABA250 were substantially increased. This was supported by the study of Kinnersley and Lin (2000), TA B L E 6 Effect of γ-aminobutyric acid (GABA) and nitrogen on PDH activity in leaves in different fragrant rice genotypes (U/g FW) accumulation in grain. So, correlation analysis revealed that the 2AP content in fragrant rice was significantly and positively associated with proline content in leaves at 15 d AH (Table 9). Our results confirm the previous reports in which proline is reported as the precursor of 2AP in fragrant rice, and its higher level often results in more 2AP contents Poonlaphdecha et al., 2012). Generally, genetic factors largely determine the aroma formation in fragrant rice (Bradbury et al., 2008;Fitzgerald et al., 2008); nevertheless, many environmental factors and the cultivation practices may have a significant influence on aroma volatiles in rice Mo et al., 2015;Yang et al., 2012). Nitrogen fertilizer is also one of the important factors that could lead to the improvement of 2AP accumulation significantly (Ren et al., 2017;Sikdar, Rahman, Islam, Yeasmin, & Akhter, 2008), even there are still different arguments on whether or not N could improve the 2AP content in fragrant rice (Itani, Tamaki, Hayata, Fushimi, & Hashizume, 2004;Li et al., 2014;Yoshihashi, 2005). Moreover, GABA and nitrogen application did not TA B L E 8 Effect of γ-aminobutyric acid (GABA) and nitrogen on DAO activity in leaves in different fragrant rice genotypes (U/g FW) affect the GABA contents in leaves significantly (Table 4), whereas significant but negative correlations between 2AP content in grain and GABA in leaves were detected (Table 9). Differences in opines exist among scientists regarding relationships of GABA with 2AP.
For example, Poonlaphdecha et al. (2012) revealed that 2AP was correlated with proline content but not with the GABA content in leaves, whereas the positive correlation between 2AP and GABA contents in fragrant rice grains was detected by Mo et al. (2015).
Furthermore, GABA treatment did not affect the P5C contents, whereas the cultivars were found statistically significant regarding P5C contents in leaves of all rice cultivars. Significant differences in P5C contents were observed for T × V, N, T × N, V × N, and T × V×N at 15 d AH (  (Tables 5-8).
Moreover, the 2AP contents in grains at maturity showed significant negative associations with some of the investigated enzyme activity in leaves at some growth stage (Table 9). The difference between this study and other previous study is mainly due to the difference in cultivars, application treatments, the correlation analysis that was between 2AP, and the biochemistry parameters in different plant parts. Previous studies reported that grain 2AP contents in fragrant rice are directly associated with the activities of PDH, P5CS, OAT, and DAO , whereas Ghosh and Roychoudhury (2018) reported that aromatic rice types have higher activities of PDH, P5CS, and OAT and P5C contents than nonaromatic rice types.
Furthermore, Mo et al. (2017) also demonstrated that PDH activity and proline content were connected to the 2AP formation and accumulation. Differences in the concentration of 2AP in different plant parts showed their differential abilities to accumulate the 2AP contents (Buttery et al., 1983;Maraval et al., 2010). Micronutrients such as Mn, Zn, and Fe also play important roles in modulation of 2AP contents (Hu, Xu, & Huang, 2001;Huang et al., 2008;Huang, Xiao, & Tang, 2010;Tang & Wu, 2006). Present study indicated that exogenous GABA application improved the uptake of Na, Mn, Zn, and Fe in all rice cultivars. Overall, GABA application in interaction with N substantially modulated the 2AP contents in grains by affecting the enzyme activities involved in the 2AP formation; however, further studies are needed to better understand the involvement of and/or mechanism of GABA and N to regulate the 2AP biosynthesis and the enzymes involved in the whole process.

| CON CLUS ION
GABA250 can increase 2AP, Na, Mn, Zn, and Fe contents, but decrease in protein content in grains as compared to GABA0. The GABA250 treatment further enhanced the activities of PDH and OAT at 15 d AH and DAO activity at maturity but reduced the proline contents at maturity. Significant interactive effect of GABA and nitrogen was observed for Mn, Zn, and Fe content in grains, proline content in leaves, P5C content in leaves at 15 d AH, GABA content in leaves at 15 d AH and MS, and P5CS, PDH, and OAT activities in leaves at MS. Overall, GABA treatment improved the 2AP content and nutrient uptake in all rice cultivars, whereas GABA and nitrogen revealed significant interaction effect on nutrient content in grains and some physiological parameters in leaves that involved in 2AP formation.

ACK N OWLED G M ENTS
We acknowledge the funding provided by the National Natural Science Foundation for Young Scientists (31601244) and the Special Fund for National Natural Science Foundation of China (31271646).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
The study did not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.