Exogenous γ-aminobutyric acid (GABA) application at different growth stages regulates 2-acetyl-1-pyrroline, yield, quality and antioxidant attributes in fragrant rice

ABSTRACT

The aim of this study was to investigate the optimum time for γ-aminobutyric acid (GABA) application to improve the yield and quality in fragrant rice. Pot and field experiments were conducted during 2016–17 with two fragrant rice cultivars (for pot experiment), and five fragrant rice cultivars (for field experiment) which were applied with five GABA levels i.e. no GABA application (CK), application of GABA at 250 mg l⁻¹ with 25 ml pot⁻¹ at tillering stage (S1), panicle initiation stage (S2), heading stage (S3), and at tillering, panicle initiation and heading stages (S4) in the pot experiment. Similarly, the same treatments with 100 ml m⁻² were applied to all rice cultivars in the field experiment. The S3 treatment significantly increased the 2-acetyl-1-pyrroline (2AP) contents in Meixiangzhan2 (14.76%) and Yuxiangyouzhan (20.19%) in pot experiment, Meixiangzhan2 (27.27%), Yuxiangyouzhan (40.24%), Basmati (43.07%) and Yungengyou14 (13.66%) in field experiment owing to regulations in the contents of proline, ^Δ1-pyrroline-5-carboxylic acid (P5C), GABA and the activities of enzymes involved in 2AP formation. The GABA treatments improved yield and modulated the antioxidant enzyme activities. This study provides a reference for the GABA application to improve yield and quality in fragrant rice.

KEYWORDS:

• γ-aminobutyric acid (GABA)
• growth stages
• application period
• fragrant rice
• 2-acetyl-1-pyrroline
• yield

1. Introduction

Rice is an important cereal crop that feeds billions of people around the world (Mahajan et al. 2010). Fragrant rice is a unique rice type and world-famous due to its special aroma, appearance, and taste, thus regarded as a superfine grain (Giraud 2013). In recent years, it has become more popular and its demand is increasing in international markets worldwide (Hashemi et al. 2013). However, the grain yield was generally lower in fragrant rice than non-fragrant rice cultivars. Thus, some effective strategies are needed and have also been previously employed to improve grain yield production in fragrant rice (Pan et al. 2013; Mo et al. 2015, 2017).

Rice aroma was regarded as one of the most critical quality characters (Das et al. 2018), aroma intensities are closely related to the mixture of aroma compounds in rice (Sansenya et al. 2018). The 2-acetyl-1-pyrroline (2AP) was reported as a crucial volatile compound involved in the intricate volatile chemistry of aroma in fragrant rice (Ren et al. 2017; Boontakham et al. 2019), which can be determined in all parts of fragrant rice excluding root (Maraval et al. 2010; Mo et al. 2015). In recent times, some studies focusing on the formation of aroma have revealed that the betaine-aldehyde dehydrogenase (BADH)-related gene is responsible for the biosynthesis and up–down regulation of 2AP (Bradbury et al. 2005; Chen et al. 2008). Previous studies reported that catalytic activities of proline dehydrogenase (PDH), ^Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) and ornithine aminotransferase (OAT) convert the proline, glutamate, and ornithine into a common metabolite i.e. pyrroline-5-carboxylic acid (P5C) and thus act as precursors for several related physiological processes involved in the formation of 2AP in fragrant rice (Mo et al. 2016a; Ghosh and Roychoudhury 2018). Moreover, diamine oxidase (DAO) and soluble protein have also been found to be associated with grain 2AP production in fragrant rice (Li et al. 2016b; Deng et al. 2018).

Indeed, the biosynthesis and regulations of 2AP in fragrant rice are genetic-controlled mechanisms, however, agronomic measures and crop management practices can also affect the yield, enzyme activities, and genes involved in 2AP biosynthesis to a great extent (Bao et al. 2018). For example, Mo et al. (2019a, 2019b) showed that the regulations of 2AP were linked to the dynamics in water and nitrogen application in fragrant rice. Additionally, it appears that the production and accumulation of 2AP can also be linked to the external environmental conditions and abiotic stress factors, e.g. an increment of 2AP contents in rice was detected in response to salt stress (Poonlaphdecha et al. 2012). Similarly, Mo et al. (2015) reported that the accumulation of GABA and 2AP were enhanced simultaneously by shading or low light intensity during the grain filling stage in fragrant rice. Besides, Bradbury et al. (2008) indicated that both the biosynthesis of γ-aminobutyric acid (GABA) and the accumulation of Δ1-pyrroline were largely affected by Badh2 gene. Furthermore, it was reported that GABA was closely associated with the plant growth and grain yield (Li et al. 2017; Li et al. 2019b). Consequently, it is quite feasible to regulate 2AP biosynthesis and grain yield by the exogenous application of GABA in fragrant rice.

GABA is recognized as a signaling molecule that is involved in various physio-biochemical processes to regulate plant stress responses (Ramesh et al. 2017; Routray and Rayaguru 2018). In general, the endogenous concentration of GABA in plants is relatively low but its levels may be enhanced in response to the various stress conditions (Li et al. 2017). Previous studies confirmed that GABA is endowed with the ability to regulate physio-biochemical metabolism in different plants (Yu and Sun 2007; Song et al. 2010). Li et al. (2016a) suggested that exogenous GABA improved the salt stress tolerance in growing wheat plants due to the enhancement of photosynthesis and antioxidant enzyme activities. Exogenous GABA application improved the chlorophyll contents and other photosynthetic attributes in rice seedlings (Li et al. 2017). Xie et al. (2019) revealed that GABA application improved the nutrient uptake in rice. Shang et al. (2011) showed that GABA application played an important role in alleviating chilling injury in cold-stored peach fruit. Li et al. (2019b) demonstrated that GABA application could regulate rice yield and yield-related traits under different nitrogen levels. Hence, exogenous GABA applications can regulate either aroma biosynthesis or plant growth and even the grain yield by affecting the physiological metabolism. Previous studies have well documented the GABA-induced regulations in different plant species, however, a little is known about the effects of GABA application during the different growth stages of fragrant rice to find out the suitable application period or growth stage in fragrant rice. Therefore, the present study was conducted to investigate the effects of exogenous GABA application at different growth stages of fragrant rice on the 2AP contents and the physiological and biochemical attributes involved in 2AP biosynthesis, grain yield formation, grain quality as well as antioxidant defense system in fragrant rice.

2. Materials and methods

2.1. Experimental description

The pot and field experiments were performed in the greenhouse and under field conditions at Experimental Research Farm, College of Agriculture, South China Agricultural University (SCAU), Guangzhou, P.R. China from 2016 to 2017. The experimental region has a humid subtropical-monsoon type climate with an average air temperature of 24.4°C and average humidity of 79.3% in the growing season of rice. The seeds were collected from the College of Agriculture, South China Agricultural University, Guangzhou, China. The soil of pot experiment was sandy loam with 21.65 g kg⁻¹ organic matter, 152 g kg⁻¹ total N, 1.02 g kg⁻¹ total P, 19.34 g kg⁻¹ total K, and 6.40 soil pH whereas the soil of the field experiment was sandy loam containing 21.74 g kg⁻¹ organic matter, 1.70 g kg⁻¹ total N, 1.30 g kg⁻¹ total P, 20.10 g kg⁻¹ total K, and 6.34 soil pH.

2.2. Experimental treatments and design

The experiments were conducted with two fragrant rice cultivars (Meixiangzhan2 and Yuxiangyouzhan) for pot experiment, and with five fragrant rice cultivars (Meixiangzhan2, Yuxiangyouzhan, Basmati, Xiangyaxiangzhan and Yungengyou14) for field experiment. Those cultivars were globally /regionally popular due to their special aroma and better cooking qualities. The five GABA levels i.e. no GABA application (CK), application of GABA at 250 mg l⁻¹ with 25 ml pot⁻¹ at tillering stage (S1), panicle initiation stage (S2), heading stage (S3), and at tillering, panicle initiation and heading stages (S4) were applied in the pot experiment. Similarly, the same treatments with 100 ml m⁻² were applied to all rice cultivars in the field experiment. The crop management practices and pest control were carried out according to standard cultural practices.

The pot experiment was arranged in randomized complete block design (RCBD) with seven replications for each treatment. The commercial compound fertilizer (N: P₂O₅: K₂O = 15:15:15) was applied in all the pots with 3.5 g pot⁻¹ as basal fertilizer. Each pot (32 cm in diameter and 24 cm in height) was filled with 12 kg of sun-dried soil and transplanted with 30 days old rice seedlings with 3 seedings per hill and five hills per pot at two days after application of basal fertilizer. An additional dose of fertilizer was applied with 3.5 g per pot at the tillering stage.

The field experiment was arranged in spilt-plot design with three replications, with rice cultivars were assigned to the main plot and GABA treatments were kept in the sub-plot. The net plot size was 5 × 3 m. The NPK at 90–150–180 kg hm⁻² was applied as basal fertilizer and no additional fertilizer was applied later on. The rice seedlings were transplanted by using rice transplanter (2Z-8A2-PZ80-HDRT25) at 30 × 14 cm planting distance.

2.3. Sampling and measurement

Plant sampling was done during 9:00–11:00 am at the heading stage (HS), 15 days after heading stage (15 d AH), and maturity stage (MS). At HS, 15 d AH and MS thirty flag leaves of the main stem, and at 15 d AH and MS fifteen panicles were sampled randomly. The panicles were divided into two parts: one was stored at −20°C for determination of 2AP content and the other together with all the flag leaves were frozen by liquid nitrogen and stored at −80°C for physio-biochemical assays.

2.3.1. Determination of grain 2AP contents

The grain 2AP contents were determined by the synchronization distillation and extraction method (SDE) combined with GCMS-QP 2010 Plus (Shimadzu Corporation) as described by Huang et al. (2012). The chromatographic condition was described as following: gas chromatograph equipped with a RESTEK Rxi-5 ms (Shimadzu, Japan) silica capillary column (30 m × 0. 32 mm × 0. 25 μm). The temperature of the GC oven was kept at 45°C (1 min), increased at the rate of 2°C per min to 65°C and kept at 65°C for 1 min, and then increased to 220°C at the rate of 10°C per min, and kept at 220°C for 10 min. The auto-injector was AOC-20i, SPL1. High purity helium gas (99.999%, Guangzhou Gases Co., LTD., China) was the carrier gas at a constant flow rate of 2 ml per min. The temperature of the ion source was 200°C. Under these conditions, the retention time of 2AP was 7.5 min. The contents of 2AP in grains were defined as μg g⁻¹.

2.3.2. Determination of proline, P5C, GABA and soluble protein contents in leaves and grains

The proline contents in leaves and grains were determined according to Bates et al. (1973) and the contents of proline were expressed as μg g⁻¹ FW, whereas the P5C contents were determined by Miller et al. (2009) and were defined as μmol g⁻¹ FW. The GABA contents in leaves and grains were determined according to Zhao et al. (2009) and expressed as mg g⁻¹ FW, whereas the soluble protein contents were detected by using the method as described by Kong et al. (2017) and expressed as μg g⁻¹ FW.

2.3.3. Determination of PDH, P5CS, OAT and DAO activities in leaves and grains

Crude enzymes were extracted according to the method represented previously (Tripathi et al. 2013; Naliwajski and Skłodowska 2014). The PDH activity was assayed by the method devised by Ncube et al. (2013) and was expressed as U g⁻¹ min⁻¹ FW, while the P5CS activity was determined according to Sánchez et al. (2002) and was expressed as U g⁻¹ min⁻¹ FW. The OAT activity was measured by using the method of Umair et al. (2011) and was expressed as U g⁻¹ min⁻¹ FW whereas the DAO activity was estimated according to the method of Su et al. (2005) and was expressed as U g⁻¹ min⁻¹ FW.

2.3.4. Determination of antioxidant enzyme activities and malondialdehyde (MDA) contents

The antioxidant enzyme activities i.e. superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as MDA contents, were assayed according to the procedures of Li et al. (2019a). In brief, fresh leaves (0.3 g) were homogenized with 5 ml of 50 mM sodium phosphate buffer (pH 7.8) and centrifuged at 8000 rpm at 4°C for 15 min and the supernatant was the crude enzyme extract. The SOD activity was defined as U g⁻¹ FW, while the POD and CAT activities were defined as U g⁻¹ min⁻¹ FW, as well as the MDA contents were expressed as μmol g⁻¹ FW.

2.3.5. Determination of grain yield and yield related traits

For the pot experiment, six pots from each treatment at maturity stage were sampled to determine grain yield and yield-related traits. The panicle number per pot was measured and averaged from the six pots. The filled grain number per pot and total grain number per pot were recorded from the panicles threshed manually, the 1000-grain weight was recorded by counting and weighing five random samples from filled grains (Mo et al. 2017).

For the field experiment, at maturity stage, the panicle number from the fifty representative plants was investigated in each plot. Four hills from different locations of each plot were sampled to determine the total grain number, the filled grain number, grain-filling percentage, and the 1000-grain weight (Mo et al. 2015, 2017).

2.3.6. Determination of grain quality

Grains were stored at room temperature for three months to measure grain quality attributes after sun drying. About 1.5 kg grains from each treatment were weighed and brown rice rate was estimated by using FC2 K rice huller (Jiangsu, China), whereas milled and head milled rice rates were assayed by JNMJ6 rice polishing machine (Zhejiang, China). The percentage of chalky grain and chalkiness degree were analyzed by using SDE-A lightbox (Guangzhou, China). The Infratec1241 grain analyzer (FOSS-TECATOR) was used to determine amylose content, protein content, and alkali of grains (Mo et al. 2015).

2.3.7. Statistical analysis

Microsoft office 2013 was used as a tool to log, dispose, and calculate data. The data analysis and the evaluation of relationships among the indexes were conducted using Statistix version 8 (Analytical Software, Tallahassee, Florida, USA). The significance of differences amongst means of different treatments was determined by the least significant difference (LSD) test at the 0.05 probability level.

3. Results

3.1. 2AP contents

For pot experiment, compared with CK, the GABA application improved the grain 2AP contents in both cultivars. The S3 treatment increased the grain 2AP contents of MX2 and YX by 14.76% and 20.19% as compared to CK whereas the highest 2AP contents were recorded under the S3 and S4 treatments for MX2 and YX, respectively.

For field experiment, the application of GABA led to substantial improvements in grain 2AP contents of YX, XY, and YG14. Compared to CK, the grain 2AP contents of MX2, YX, BS, and YG14 were increased by 27.27%, 40.24%, 43.07%, and 13.66% under the S3 treatment. Moreover, the highest grain 2AP contents were recorded under S3 for MX2 and YX, under S4 for BS and XY as well as under S2 for YG14 (Table 1).

Table 1. Effects of different γ-aminobutyric acid application periods on the contents of 2AP in grains.

Cultivar	Treatment
	CK	S1	S2	S3	S4
Pot experiment
MX2	8.13b	8.17b	8.29ab	9.33a	8.70ab
YX	7.23c	7.25c	7.68bc	8.69ab	9.18a
Field experiment
MX2	0.33b	0.31b	0.41a	0.42a	0.42a
YX	3.33c	3.85bc	3.94bc	4.67a	4.33ab
BS	2.67b	2.23b	2.80b	3.82a	4.26a
XY	0.56b	0.58b	0.65ab	0.59ab	0.73a
YG14	12.81c	14.07b	15.29a	14.56ab	14.74ab

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

3.2. The proline, P5C, GABA and soluble protein contents in leaves

For pot experiment, the proline contents were significantly affected by the GABA application, all the GABA treatments substantially increased the proline contents for MX2 at HS and MS. The S1 and S3, S2 and S4 treatments significantly improved the proline contents for YX at HS and MS, respectively. However, the GABA application marginally affected the P5C contents for MX2 and YX, for MX2, the S1, S3 and S4 treatments significantly reduced the P5C contents at HS, while the S1, S2 and S4 treatments significantly improved the P5C contents at 15 d AH. Furthermore, for MX2, the S2, S3 and S4 treatments significantly increased the GABA contents, while for YX, the GABA contents were found higher under the S1 and S4 treatments at MS. Likewise, the S1 and S4 treatments significantly increased the soluble protein contents for YX at HS, however, the S3 and S4 treatments significantly reduced the soluble protein contents for MX2 at HS and 15 d AH, respectively (Table 2).

Table 2. Effects of different γ-aminobutyric acid application periods on the contents of proline, P5C, GABA and soluble protein in leaves.

Cultivar	Treatment	Proline content (μg g^−1 FW)			P5C content (μmol g^−1 FW)			GABA content (mg g^−1 FW)			Soluble protein content (μg g^−1 FW)
		HS	15 d AH	MS	HS	15 d AH	MS	HS	15 d AH	MS	HS	15 d AH	MS
Pot experiment
MX2	CK	12.43d	9.98b	8.79c	0.57a	0.63c	1.19a	0.05c	0.06bc	0.10c	13.42a	12.94a	9.64a
	S1	19.23bc	12.39a	11.61b	0.46b	0.92ab	1.32a	0.05c	0.05c	0.05d	13.88a	13.71a	8.99a
	S2	21.03ab	10.03b	11.62b	0.62a	0.85b	1.45a	0.10b	0.08b	0.25a	13.26ab	13.15a	8.66a
	S3	17.52c	10.30b	12.89b	0.44b	0.76bc	1.47a	0.10b	0.06bc	0.15b	12.23b	13.32a	10.19a
	S4	22.67a	11.08ab	20.42a	0.47b	1.11a	1.19a	0.51a	0.34a	0.05d	14.31a	10.17b	10.46a
YX	CK	14.89b	13.27b	16.26b	0.71b	0.88a	0.93bc	0.11cd	0.11b	0.09d	11.19c	11.92a	11.56a
	S1	17.62a	22.89a	16.19b	0.78b	0.62b	1.13ab	0.17b	0.10bc	0.16ab	12.25a	11.56a	11.22a
	S2	14.85b	13.02b	22.47a	0.75b	0.74ab	0.98abc	0.10d	0.08c	0.10cd	11.29bc	11.01a	9.86b
	S3	17.72a	13.31b	16.09b	0.94a	0.75ab	0.91c	0.20a	0.13ab	0.13bc	12.02abc	11.50a	11.64a
	S4	14.10b	13.66b	19.61a	0.72b	0.72ab	1.16a	0.14c	0.15a	0.18a	12.08ab	11.08a	11.41a
Field experiment
MX2	CK	44.71a	40.63a	4.51a	1.33a	1.57ab	3.19b	3.79a	10.13bc	4.22a	8.07a	7.44a	7.45b
	S1	30.32c	33.09a	2.60c	1.32a	1.61ab	3.20b	2.61c	15.20a	3.09b	8.08a	7.71a	7.45b
	S2	43.54ab	33.97a	4.37a	1.32a	1.84a	3.32ab	3.67ab	12.16b	3.25b	7.91ab	7.58a	7.62a
	S3	38.29b	33.70a	3.30b	1.34a	1.65ab	3.40ab	3.18b	16.54a	3.31b	8.06a	7.60a	7.61a
	S4	41.19ab	20.71b	3.30b	1.33a	1.54b	3.51a	3.27ab	9.06c	3.49ab	7.74b	7.46a	7.53ab
YX	CK	52.57c	3.86b	6.92a	1.49a	1.85c	3.80a	14.21a	2.64cd	2.26c	8.08a	7.42c	7.58b
	S1	41.90d	2.65b	5.78b	1.58a	2.00b	3.95a	2.74e	2.40d	3.07b	7.92a	7.62b	7.64ab
	S2	48.54cd	32.94a	4.87c	1.66a	2.29a	3.96a	4.34d	3.15bc	2.85bc	7.94a	7.38c	7.61b
	S3	77.97a	31.05a	4.83c	1.75a	2.02b	3.95a	9.64b	6.24a	3.35b	7.84a	7.62b	7.72a
	S4	65.46b	31.59a	3.89d	1.54a	1.92bc	3.91a	7.50c	3.70b	4.99a	7.88a	7.73a	7.40c
BS	CK	29.96b	34.89a	19.66a	1.42bc	1.97ab	2.71d	2.17c	3.45cd	2.40b	7.75ab	7.52c	7.57a
	S1	36.79b	28.55b	12.55b	1.46abc	1.95ab	3.13c	4.13b	3.09d	2.51b	7.91ab	7.67b	7.64a
	S2	31.15b	29.54b	10.28c	1.31c	1.93b	3.59a	5.99a	3.98bc	2.82ab	7.84ab	7.84a	7.44a
	S3	32.60b	7.12c	10.52c	1.52ab	1.79c	3.38b	3.26b	4.37b	3.19a	7.97a	7.48c	7.60a
	S4	50.07a	5.71c	8.27d	1.65a	2.03a	3.26bc	3.88b	4.93a	3.10a	7.68b	7.47c	7.57a
XY	CK	35.35b	31.81a	20.32a	1.14ab	1.50a	3.38a	2.29b	4.06bc	5.92ab	7.76ab	7.64b	7.44b
	S1	26.91c	30.98a	12.57b	1.22ab	1.65a	3.49a	7.95a	5.69a	5.04b	8.00a	7.63b	7.55ab
	S2	21.65c	20.47b	18.29a	1.13ab	1.41a	3.24a	2.67b	4.66b	6.95a	7.68b	7.70b	7.44b
	S3	48.38a	19.47b	10.26b	1.33a	1.44a	3.32a	2.47b	3.60c	5.75b	7.70b	7.85a	7.75a
	S4	40.37b	17.03b	4.09c	1.06b	1.70a	3.18a	2.85b	3.31c	5.31b	7.75b	7.69b	7.54ab
YG14	CK	63.55a	31.00b	28.50a	0.34d	0.49c	1.55a	3.81c	4.14c	2.76c	8.01bc	7.57b	7.44b
	S1	51.69b	41.16a	21.38b	0.51bc	0.50c	1.82a	6.59b	2.59d	4.62a	8.14ab	7.61b	7.75a
	S2	35.78c	35.66ab	12.91c	0.46c	0.74a	1.55a	1.68d	9.14a	3.89b	7.94c	7.80a	7.57ab
	S3	39.50c	36.27ab	11.28cd	0.50bc	0.79a	1.62a	2.83c	6.78b	3.46b	8.09abc	7.85a	7.58ab
	S4	35.62c	35.76ab	8.15d	0.57a	0.65b	1.53a	8.47a	2.73d	1.98d	8.26a	7.46c	7.70a

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

For field experiment, all the GABA treatments significantly reduced the proline contents for YX at MS, BS at 15 d AH and MS as well as YG14 at HS and MS, and the S1 and S3 treatments reduced the proline contents for MX2 and XY at HS and MS as well as YX at HS, MX2 at HS, respectively. Additionally, the S3 and S4 treatments significantly increased the proline contents for YX at HS and 15 d AH. Furthermore, all the GABA treatments, S2 and S3 treatments significantly increased the P5C contents for BS at MS and YG14 at HS, YX and YG14 at 15 d AH, respectively. Additionally, the S4 treatment significantly improved the P5C contents for MX2 at MS, BS at HS and YG14 at 15 d AH. Likewise, all the GABA treatments, as well as S2 and S3, S3 and S4 significantly increased the GABA contents for BS at HS, YG14 at 15 d AH and MS, YX and BS at 15 d AH and MS, respectively. Additionally, the S1 treatment significantly improved the GABA contents for YX at MS, XY at HS and 15 d AH as well as YG14 at HS and MS. For the soluble protein contents, the S2 and S3 treatments significantly increased the soluble protein contents for MX2 at MS and YG14 at 15 d AH, while the S1 and S3 treatments significantly increased the soluble protein contents for YX and BS at 15 d AH as well as YG14 at MS, YX and XY at 15 d AH and MS, respectively (Table 2).

3.3. The proline, P5C, GABA and soluble protein contents in grains

Significant effects of GABA application on the proline contents were detected. All the GABA treatments significantly increased the proline contents for YX2 and XY at 15 d AH, YG14 at MS, whereas the S1, S2 and S3 treatments significantly improved the proline contents for BS at 15 d AH. Additionally, the S2 and S4 treatments significantly improved the proline contents for MX2 at MS and YX at 15 d AH, YX at 15 d AH and BS at MS, respectively. However, all the GABA treatments significantly decreased the P5C contents for YX at 15 d AH, while the S1, S2 and S4 treatments significantly increased the P5C contents for MX2 and XY at MS, MX2 at MS, BS at 15 d AH and MS as well as MX2 at MS. Furthermore, significant effects were noted on the GABA contents for MX2, YX, BS and YG14. All the GABA treatments, S3 and S4 treatments significantly increased the GABA contents for MX2 at MS as well as BS and YG14 at 15 d AH, YX at 15 d AH and BS at MS, respectively. Likewise, significant effects were recorded regarding the soluble protein contents for YX, BS, XY and YG14. The S1, S2 and S4 treatments improved the soluble protein contents for BS at 15 d AH and MS as well as YG14 at 15 d AH, BS and XY at 15 d AH, YX and BS at 15 d AH as well as XY and YG14 at 15 d AH, respectively, while the S3 treatment significantly decreased the soluble protein contents for YX at MS and YG14 (Table 3).

Table 3. Effects of different γ-aminobutyric acid application periods on the contents of proline, P5C, GABA and soluble protein in grains in field experiment.

Cultivar	Treatment	Proline content (μg g^−1 FW)		P5C content (μmol g^−1 FW)		GABA content (mg g^−1 FW)		Soluble protein content (μg g^−1 FW)
		15 d AH	MS	15 d AH	MS	15 d AH	MS	15 d AH	MS
MX2	CK	8.76d	6.28bc	3.30a	0.40c	1.08bc	0.52c	7.38ab	7.11a
	S1	19.53b	5.55c	2.97a	0.61ab	1.28ab	0.64ab	7.26b	7.08a
	S2	14.31c	9.77a	2.97a	0.63a	1.21bc	0.67a	7.41a	7.08a
	S3	13.94c	7.13b	2.77a	0.50bc	0.96c	0.60ab	7.38ab	7.13a
	S4	25.66a	5.38c	3.24a	0.63a	1.54a	0.60b	7.33ab	7.13a
YX	CK	18.59b	11.57ab	4.53a	1.05a	0.84cd	0.81b	7.36b	7.14b
	S1	12.11d	10.29b	3.51bc	0.96ab	0.74d	0.82b	7.29b	7.01c
	S2	26.36a	6.53c	3.20c	1.02a	0.98bc	0.93a	7.31b	7.20b
	S3	24.32a	12.23a	3.50bc	1.03a	1.13a	0.88ab	7.26b	6.99c
	S4	15.49c	11.21ab	3.89b	0.88b	1.10ab	0.85ab	7.49a	7.41a
BS	CK	9.32d	8.75a	2.09bc	0.54bc	0.52d	0.54b	7.27b	6.97b
	S1	18.64b	8.47a	2.36b	0.63b	0.87c	0.54b	7.32a	7.14a
	S2	14.78c	7.96a	0.94d	0.59b	1.05ab	0.56b	7.33a	7.00b
	S3	10.51d	9.13a	1.67c	0.46c	0.89bc	0.80a	7.29ab	7.00b
	S4	21.20a	4.68b	4.64a	1.05a	1.13a	0.86a	7.31a	7.13a
XY	CK	13.49c	9.13a	1.95a	0.57b	1.14a	0.56a	7.22b	7.12ab
	S1	17.94b	6.84b	1.36b	2.05a	1.30a	0.49ab	7.30ab	6.98b
	S2	17.09b	6.53b	1.80a	0.53b	1.13a	0.56a	7.34a	7.09ab
	S3	22.08a	9.19a	2.17a	0.40b	1.10a	0.49ab	7.31ab	7.11ab
	S4	16.35b	3.63c	2.01a	0.37b	1.28a	0.43b	7.42a	7.25a
YG14	CK	34.66a	11.38c	3.39ab	0.65ab	1.14c	0.92a	7.48b	7.35a
	S1	28.64bc	18.15a	3.98a	0.72a	2.53a	0.89a	7.62a	7.23ab
	S2	31.02b	16.32b	2.77b	0.61b	2.24ab	0.74b	7.37bc	7.05b
	S3	25.83c	15.20b	3.53ab	0.57b	2.05b	0.87ab	7.33c	7.04b
	S4	27.40c	16.36b	3.79a	0.61b	2.28ab	0.89ab	7.60a	7.15ab

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

3.4. The PDH, P5CS, OAT and DAO activities in leaves

For pot experiment, all the GABA treatments significantly increased the PDH activity for MX2 at HS and MS whereas the S2 and S3 treatments significantly decreased the PDH activity for YX at 15 d AH. For the P5CS activity, the S1 treatment significantly increased the P5CS activity for MX2 at MS as well as YX at HS and MS whereas the S3 and S4 treatments significantly increased the P5CS activity for MX2 at HS, YX at MS and MX2 at HS, respectively. Furthermore, the S3, both the S2 and S4 treatments significantly increased the OAT activity for YX at HS, MX2 at 15 d AH, respectively. Likewise, the GABA treatments showed a significant effect on the DAO activity. All the GABA treatments significantly increased the DAO activity for MX2 at MS, while the S3 and S4 treatments significantly increased the DAO activity for YX at 15 d AH. Additionally, the S1, S2 and S4 treatments substantially improved the DAO activity for YX at HS and MS, MX2 at HS and YX at MS, YX at MS, respectively (Table 4).

Table 4. Effects of different γ-aminobutyric acid application periods on the activities of PDH, P5CS, OAT and DAO in leaves.

Cultivar	Treatment	PDH activity (U g^−1 min^−1 FW)			P5CS activity (U g^−1 min^−1 FW)			OAT activity (U g^−1 min^−1 FW)			DAO activity (U g^−1 min^−1 FW)
		HS	15 d AH	MS	HS	15 d AH	MS	HS	15 d AH	MS	HS	15 d AH	MS
Pot experiment
MX2	CK	4.27c	7.27b	1.99c	11.68cd	14.61a	13.16bc	21.81a	20.17b	25.33a	1.51b	2.26a	2.08c
	S1	6.00b	6.30b	2.79b	10.02d	12.54ab	16.75a	21.38a	22.55ab	23.65ab	1.50b	1.74b	2.87ab
	S2	7.38a	10.73a	2.85b	13.30c	11.33b	14.70bc	20.61a	24.45a	22.15b	2.16a	1.97b	3.11a
	S3	5.32b	6.34b	4.63a	16.98b	12.42ab	12.94bc	20.54a	23.37ab	23.47ab	1.17c	1.84b	2.64b
	S4	5.30b	7.12b	2.72b	29.13a	11.13b	12.80c	21.52a	24.15a	25.29a	1.49b	1.37c	2.71b
YX	CK	6.61bc	7.18a	5.04b	9.50b	11.84a	11.70bc	20.83b	22.19a	25.12a	1.49b	2.10c	3.55c
	S1	7.03b	7.91a	5.70b	11.26a	13.03a	17.06a	19.44b	23.43a	26.38a	1.84a	1.82c	4.31ab
	S2	6.25c	5.81b	5.07b	10.45ab	12.39a	12.41b	20.07b	22.94a	25.05a	1.48b	1.72c	4.26ab
	S3	7.27b	5.70b	7.33a	11.21ab	11.48a	10.00c	23.16a	22.82a	25.01a	1.06c	2.83b	3.75bc
	S4	9.20a	5.44b	7.06a	10.64ab	12.26a	16.76a	20.04b	23.39a	26.55a	0.99c	3.54a	4.71a
Field experiment
MX2	CK	28.80a	36.67a	40.85ab	52.63d	77.89a	70.41a	189.20b	201.98c	165.42b	23.98a	20.96b	29.68a
	S1	29.68a	36.45a	35.08b	62.82b	76.26a	68.75ab	197.39a	211.85bc	161.80b	25.03a	22.92ab	30.05a
	S2	26.87a	38.06a	41.64a	85.93a	64.33b	61.96c	192.89ab	224.44a	162.94b	24.97a	24.99a	30.41a
	S3	27.10a	34.59a	39.51ab	57.30c	52.60c	68.23ab	196.77a	218.67ab	228.72a	25.67a	23.41ab	32.43a
	S4	30.48a	35.44a	40.06ab	63.73b	59.59bc	66.54b	196.82a	202.85c	223.72a	26.18a	24.52a	30.13a
YX	CK	27.68a	72.40b	33.55a	56.96b	42.43b	73.29a	201.67a	214.41ab	171.99a	23.65a	18.32c	30.78a
	S1	27.77a	75.37ab	31.88a	55.68b	42.47b	69.29ab	201.59a	224.80a	155.66ab	24.68a	18.84c	30.30a
	S2	25.56a	73.63b	34.51a	56.21b	38.66bc	62.34c	203.77a	200.02c	146.59b	23.85a	18.41c	26.89b
	S3	25.84a	86.66a	32.28a	60.96a	49.35a	70.33ab	207.61a	210.54bc	155.94ab	24.20a	21.80b	28.21ab
	S4	26.44a	49.91c	34.76a	46.26c	35.50c	67.28b	200.33a	207.95bc	154.50ab	24.02a	23.96a	29.23ab
BS	CK	37.41a	82.61abc	31.02b	89.55a	58.54ab	88.36a	199.73b	236.51a	173.55ab	22.19a	20.44ab	31.17b
	S1	33.05b	74.38bc	34.17ab	86.18ab	51.83b	90.42a	202.27ab	232.35a	172.73ab	23.26a	19.48abc	34.69a
	S2	38.95a	89.23ab	35.67ab	77.14c	63.09a	85.05ab	199.49b	224.30a	188.87a	22.88a	21.30a	33.83ab
	S3	28.65c	96.11a	37.09ab	81.33bc	55.67ab	88.42a	211.14a	221.44a	162.95b	21.39a	18.32c	35.31a
	S4	25.90c	67.24c	40.06a	68.84d	61.24a	76.21b	200.01b	218.89a	176.01ab	22.28a	19.20bc	27.14c
XY	CK	31.23a	38.84a	43.53a	44.26c	68.03b	72.39a	189.75c	208.46a	216.42a	25.15a	24.08ab	30.31ab
	S1	26.53b	34.12a	45.45a	58.31a	73.85a	74.49a	192.38c	207.29a	213.41a	16.00b	23.99ab	28.44ab
	S2	24.68bc	35.43a	41.20a	45.01bc	62.96b	63.28b	187.32c	199.45ab	210.59a	16.61b	22.93b	26.96b
	S3	22.28c	28.68b	38.90ab	49.32b	55.97c	74.29a	256.69a	192.89ab	217.38a	17.35b	25.48ab	31.20ab
	S4	26.18bc	24.92b	33.13b	42.93c	55.35c	61.24b	233.10b	183.49b	210.18a	17.01b	27.67a	32.27a
YG14	CK	23.47c	37.86a	38.35a	41.31b	36.23abc	53.68a	192.18a	196.18a	148.77a	22.69bc	19.74ab	22.56a
	S1	30.51a	37.68a	35.85ab	47.80a	31.50c	52.51a	192.93a	198.04a	155.21a	25.36a	17.23c	23.43a
	S2	24.29c	37.81a	37.27a	39.56b	40.00a	51.49a	188.34a	211.27a	155.63a	21.69c	20.81a	23.79a
	S3	28.02b	32.89a	30.46b	42.99b	33.55bc	52.70a	196.18a	197.37a	157.67a	21.18c	18.50bc	24.52a
	S4	28.48ab	35.98a	34.70ab	42.02b	37.46ab	50.61a	183.73a	204.42a	160.20a	23.83ab	19.97ab	24.36a

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

For field experiment, all the GABA treatments significantly decreased PDH activity for XY at HS and the S1 and S4 treatments significantly reduced the PDH activity for BS at HS, YX at 15 d AH and XY at MS, respectively, while the S3 and S4 treatments significantly increased the PDH activity for XY at 15 d AH and YG14 at HS. Moreover, GABA treatments significantly affected the P5CS activity for MX2, YX, BS and XY. All the GABA treatments significantly increased the P5CS activity for MX2 at HS, and the S1 and S3 treatments significantly increased the P5CS activity for XY at HS and 15 d AH as well as YG14 at HS, YX at HS and 15 d AH as well as XY at HS, respectively, while the S2, S3 and S4 treatments significantly decreased P5CS activity for BS at HS, and the S2 and S4 treatments significantly reduced the P5CS activity for MX2, YX and XY at MS. Furthermore, the S3 and S4 treatments significantly increased the OAT activity for MX2 at HS and MS as well as XY at HS, while the S2 treatment significantly decreased the OAT activity for YX at 15 d AH and MS. However, all the GABA treatments significantly decreased the DAO activity for XY at HS whereas the S1, S2, S3 and S4 treatments significantly increased the DAO activity for YG14 at HS and BS at MS, MX2 at 15 d AH, YX at 15 d AH and BS at MS, MX2 and YX at 15 d AH, respectively (Table 4).

3.5. The PDH, P5CS, OAT and DAO activities in grains

The GABA application marginally affected the PDH and DAO activities, however, the S1 and S2 treatments substantially improved the PDH activity for MX2 at MS whereas the S2 and S3, S3 and S4 treatments decreased the PDH activity significantly for BS at MS and YG14 at 15 d AH, respectively. All the GABA treatments significantly decreased the DAO activity for BS at 15 d AH. Furthermore, all the GABA treatments significantly increased the P5CS activity for BS and YG14 at 15 d AH and XY at MS whereas the S1, S2 and S4 treatments significantly decreased the P5CS activity for YX at MS. The S2 and S3, S3 and S4 treatments led to decline in the P5CS activity for MX2 at 15 d AH, MX2 at MS and XY at 15 d AH, respectively. Moreover, the S1 treatment reduced the P5CS activity significantly for MX2 at 15 d AH and MS. Likewise, the OAT activities were significantly affected by the GABA treatments. All the GABA treatments significantly decreased the OAT activity for MX2 at 15 d AH and YG14 at MS whereas the S1 and S4, S3 and S4 treatments significantly increased the OAT activity for BS at 15 d AH and MS, XY at 15 d AH, respectively, and the S2 and S3 treatments significantly increased the OAT activity for YX at 15 d AH and BS at MS, MX2 at MS and BS at 15 d AH, respectively (Table 5).

Table 5. Effects of different γ-aminobutyric acid application periods on the activities of PDH, P5CS, OAT and DAO in grains in field experiment.

Cultivar	Treatment	PDH activity (U g^−1 min^−1 FW)		P5CS activity (U g^−1 min^−1 FW)		OAT activity (U g^−1 min^−1 FW)		DAO activity (U g^−1 min^−1 FW)
		15 d AH	MS	15 d AH	MS	15 d AH	MS	15 d AH	MS
MX2	CK	28.15a	38.04b	3.96b	3.66b	63.74a	34.98b	6.79a	6.16a
	S1	27.27a	51.34a	2.34d	2.11c	44.34b	37.39ab	6.57a	6.39a
	S2	28.50	47.34a	3.29c	9.94a	43.37b	37.27ab	6.30a	5.93a
	S3	28.10a	34.31b	2.38d	2.33c	42.97b	41.78a	6.45a	5.91a
	S4	28.29a	34.26b	4.49a	1.61c	41.95b	39.74ab	6.34a	5.81a
YX	CK	30.87a	33.01a	4.88a	2.97a	39.48b	39.48a	5.81a	7.20a
	S1	32.68a	34.54a	3.76b	2.47b	41.86b	36.12a	6.60a	6.81ab
	S2	32.33a	33.60a	3.32bc	2.35b	57.97a	37.53a	6.77a	6.28b
	S3	32.79a	31.53a	3.24bc	3.25a	39.67b	36.31a	6.96a	7.25a
	S4	31.03a	32.93a	2.83c	2.42b	46.87b	37.65a	7.00a	7.31a
BS	CK	28.43b	34.58a	2.35c	1.55cd	34.42c	31.79d	8.41a	5.11b
	S1	28.81b	34.65a	4.89a	1.65c	41.24b	37.54a	6.70b	5.42b
	S2	27.67b	29.43c	3.85b	1.52d	33.62c	34.81bc	7.11b	5.46b
	S3	28.01b	30.75bc	4.85a	2.28a	48.11a	33.29cd	6.01b	5.07b
	S4	30.36a	32.85ab	3.96b	1.93b	45.87ab	36.71ab	6.44b	6.52a
XY	CK	26.34a	32.21ab	3.12a	1.55c	42.56bc	36.40ab	6.50a	5.47a
	S1	26.52a	28.56b	2.37b	2.67a	50.74ab	38.74a	6.38a	5.48a
	S2	28.72a	32.85ab	2.77ab	2.12b	41.22c	33.82b	6.52a	5.56a
	S3	28.39a	35.13a	2.62b	2.33ab	54.50a	36.42ab	6.44a	5.74a
	S4	28.72a	30.77ab	2.60b	2.36ab	52.90a	38.54a	6.59a	5.49a
YG14	CK	28.85a	34.43ab	2.62c	2.27c	49.51a	44.69a	7.21ab	5.55a
	S1	28.86a	33.06ab	3.99ab	2.33c	51.81a	39.53b	7.47a	5.92a
	S2	28.20ab	31.75b	3.45b	2.86b	38.56b	37.79b	7.29ab	5.47a
	S3	27.33b	36.12a	4.24a	3.42a	38.26b	36.77b	6.59b	5.39a
	S4	27.00b	32.77ab	3.55b	1.53d	41.99b	35.72b	7.17ab	6.04a

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

3.6. Grain yield and yield related traits

Significant variations were noted in both yield and yield traits under the different GABA treatments. For MX2 and YX, the S4 treatment enhanced the grains per panicle by 14.40% and 25.06%, respectively, whereas for BS, the 1000-grain weight under the S4 treatment was significantly higher as compared with CK. The grain yield was found higher in YX under the S4 treatment than CK. Moreover, both the S2 and S3 treatments produced 16.70% and 24.77% higher grains per panicle for XY whereas the S3 and S4 treatments improved the filled grain percentage for YG14 by 16.87% and 11.05%, respectively. In addition, the S2 treatment significantly increased the grain yield for MX2 and YX by 16.90% and 22.93% as compared with CK (Table 6).

Table 6. Effects of different γ-aminobutyric acid application periods on yield and yield related traits in field experiment.

Cultivar	Treatment	Panicle number	Grains per panicle	Filled grain percentage (%)	1000-grain weight (g)	Grain yield (g m^−2)
MX2	CK	228.48a	147.51bc	67.28a	21.44b	416.74b
	S1	214.62a	138.09c	66.37a	20.84c	421.61ab
	S2	215.88a	154.55abc	66.03a	19.78d	487.16a
	S3	217.98a	157.93ab	63.58a	21.55b	454.76ab
	S4	212.94a	168.76a	51.49b	22.68a	467.29ab
YX	CK	202.44a	187.43ab	69.47a	21.57ab	561.28cd
	S1	209.16a	176.99b	66.61a	20.83c	540.22d
	S2	204.96a	205.79a	67.92a	21.19bc	689.97a
	S3	197.40a	197.48a	67.35a	21.82a	606.88bc
	S4	200.34a	206.99a	67.18a	20.80c	635.16ab
BS	CK	211.68a	138.69ab	79.90a	24.77b	604.22ab
	S1	205.80a	133.50ab	75.21ab	26.12ab	553.77b
	S2	210.84a	143.30a	72.22bc	25.66b	669.08a
	S3	199.08a	142.35a	77.87ab	25.17b	614.24ab
	S4	206.22a	127.11b	68.14c	27.31a	606.63ab
XY	CK	213.78a	134.88b	63.51ab	21.79a	439.36a
	S1	206.64a	150.43ab	62.59ab	21.52a	422.25a
	S2	211.68a	157.41a	56.46a	21.59a	425.78a
	S3	204.54a	168.29a	67.28b	21.96a	481.21a
	S4	206.22a	168.68a	62.50ab	21.38a	471.56a
YG14	CK	161.70a	129.92a	59.65c	26.83a	452.91a
	S1	153.30ab	126.82a	61.41bc	26.19ab	498.25a
	S2	155.40ab	131.92a	60.22c	25.72b	476.94a
	S3	149.10b	131.27a	69.71a	26.18ab	473.13a
	S4	160.02ab	138.72a	66.24ab	25.61b	447.36a

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

3.7. The SOD, POD, CAT activities and MDA contents

Significant effects of the GABA treatments on the antioxidant enzyme activities and the MDA contents were noted. All the GABA treatments improved the SOD activity for MX2 and XY at HS whereas the S1 and S3 treatments significantly improved the SOD activity for XY and YG14. The S1 and S4, S2 and S3 treatments substantially enhanced the SOD activity for MX2 and YX at 15 d AH, YX at HS, respectively, while the S2 and S4 treatments decreased the SOD activity for MX2 and BS, BS, respectively. Likewise, all the GABA treatments resulted in higher POD activities for YX and BS at MS and MX2 at HS. The S1 and S2, S3 and S4 treatments significantly increased the POD activity for YX at HS and BS at 15 d AH as well as YG14 at MS, XY and YG14, respectively, nevertheless, the S1 and S2 treatments reduced the POD activity for MX2 at 15 d AH and MS as well as XY at MS. Additionally, the S4 treatment significantly increased the POD activity for YX and BS at HS and 15 d AH. Furthermore, the CAT activity was substantially increased under GABA application for YX, BS and YG14 at MS as well as MX2 at 15 d AH, whereas the S3 and S4, S2 and S4 treatments significantly increased the CAT activity for XY at 15 d AH and MS, MX2 at MS and BS at 15 d AH, respectively. The S1 and S3 treatments significantly reduced the CAT activity for MX2 at MS and YX at HS. Moreover, the S2, S3 and S4 treatments significantly reduced the CAT activity for YX and YG14 at 15 d AH as well as XY, YX at 15 d AH and XY at HS, YX at HS and YG14 at MS, respectively. Moreover, the higher contents of MDA were detected for BS and YG14 at HS under the GABA treatments and the S3 and S4 treatments significantly increased the MDA contents for MX2 at MS, YX at HS and 15 d AH as well as XY at 15 d AH, but the S1 and S2, S2 and S3 treatments significantly decreased the MDA contents for YG14 at MS and MX2 at 15 d AH, respectively (Table 7).

Table 7. Effects of different γ-aminobutyric acid application periods on the activities of SOD, POD, CAT and the content of MDA in field experiment.

Cultivar	Treatment	SOD activity (U g^−1 FW)			POD activity (U g^−1 min^−1 FW)			CAT activity (U g^−1 min^−1 FW)			MDA content (μmol g^−1 FW)
		HS	15 d AH	MS	HS	15 d AH	MS	HS	15 d AH	MS(S4)	HS	15 d AH	MS
MX2	CK	12.29c	154.28b	262.76a	63.46d	80.22b	91.00a	887.91b	329.23d	169.61c	3.25b	11.36a	6.48b
	S1	91.44b	223.60a	245.35a	74.92c	60.28d	76.75c	900.66b	374.08c	145.21d	3.62b	7.42c	7.28a
	S2	182.94a	173.78b	184.61b	79.69c	70.09c	79.98bc	800.51c	383.54c	189.86b	3.40b	8.87b	6.89ab
	S3	189.66a	229.22a	262.58a	112.31a	84.22b	84.50b	1085.07a	450.76b	154.59d	4.05b	7.95c	7.32a
	S4	110.20b	252.76a	273.38a	99.31b	97.54a	93.13a	656.50d	477.25a	204.86a	6.14a	11.15a	7.27a
YX	CK	56.21c	126.77b	217.93b	69.65c	50.29c	51.41d	891.77b	247.08c	66.44e	3.72c	6.65c	7.34bc
	S1	43.09d	163.25a	282.69a	73.98b	52.39c	85.70a	770.00d	288.02b	205.56b	4.20c	5.90c	7.90ab
	S2	90.45a	119.21b	173.29c	74.93b	56.27c	71.92b	987.35a	186.17d	134.09c	5.53b	6.65c	6.58c
	S3	79.63b	117.47b	262.70a	71.82bc	74.55b	88.05a	832.12c	170.91d	278.08a	7.48a	8.23b	8.29a
	S4	56.53c	81.35c	132.89d	99.63a	86.72a	66.00c	768.64d	324.13a	104.90d	6.72a	10.04a	8.11ab
BS	CK	248.67a	340.64a	352.23a	80.21d	57.17d	68.08e	959.15a	161.78c	69.83e	5.04c	8.45c	7.32b
	S1	223.51b	295.00a	360.96a	81.87d	65.13bc	78.94b	791.01bc	162.62c	114.37b	6.82b	11.92a	7.28b
	S2	157.25d	337.04a	290.79b	98.64c	68.87ab	72.86d	826.45b	249.62a	94.45c	6.06b	10.23b	8.60a
	S3	199.88c	241.17b	351.07a	111.71b	59.83cd	84.54a	925.26a	166.28c	142.79a	9.12a	7.73d	8.38a
	S4	118.44e	238.36b	234.36c	126.17a	72.94a	76.71c	777.71c	184.08b	83.68d	6.39b	8.54c	7.40b
XY	CK	48.95e	200.50c	250.12d	82.41c	68.97bc	92.66c	831.43c	445.95c	198.63c	3.07b	8.38d	6.45b
	S1	165.84b	264.52b	295.51c	82.85c	71.81b	88.98d	977.52a	430.66c	188.52c	3.17b	9.12cd	6.99b
	S2	75.86d	233.57bc	250.06d	72.79d	65.11c	77.95e	766.76d	404.85d	167.27d	3.10b	9.93bc	6.78b
	S3	113.08c	339.44a	444.88a	109.04a	101.31a	111.19a	748.24d	792.49a	397.58a	4.07a	11.65a	9.38a
	S4	193.19a	357.12a	390.51b	92.99b	100.46a	95.99b	932.47b	656.61b	345.21b	3.35b	10.65b	6.51b
YG14	CK	75.62c	138.78c	159.30c	89.51c	85.06b	58.85d	920.72d	462.31c	208.72d	3.43c	12.37ab	7.50a
	S1	98.16b	205.60b	229.72b	107.59b	87.95b	73.90c	980.39c	529.51b	265.97c	4.47b	12.40ab	6.39b
	S2	67.12c	137.69c	223.19b	89.21c	70.86c	77.80c	951.66cd	384.24e	303.57b	5.70a	11.73bc	5.58b
	S3	120.23a	231.62a	294.29a	110.74b	94.61a	87.12b	1033.13b	749.31a	373.98a	5.35a	11.41c	7.53a
	S4	41.65d	139.27c	267.70a	130.81a	94.44a	95.98a	1097.59a	433.17d	363.29a	5.73a	12.94a	5.95b

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

3.8. Grain quality

Significant effects were noted on the grain quality traits under the GABA treatments. For instance, the S4 treatment significantly increased the grain protein contents in MX2, YX and BS whereas all the GABA treatments significantly reduced the rate of chalky grains and the degree of chalkiness for MX2 and BS as well as the alkali for YG14. The S2, S3 and S4 treatments substantially reduced the rate of chalky grains and the degree of chalkiness for XY. Moreover, higher amylose contents for BS were found under the S2 and S4 treatments, while for YG14, the amylose contents were remained lower under the same treatments. Additionally, the S4 treatment significantly improved the brown and milled rice rate for MX2, BS and XY, whilst both the S2 and S3 treatments caused a significant improvement in brown and milled rice rate for XY (Table 8).

Table 8. Effects of different γ-aminobutyric acid application periods on grain quality in field experiment.

Cultivar	Treatment	Brown rice rate (%)	Milled rice rate (%)	Head rice rate (%)	Grains with chalkiness (%)	Chalkiness degree (%)	Length-width ratio	Protein (%)	Amylose content (%)	Alkali
MX2	CK	76.10b	67.92b	61.27a	7.80a	2.10a	2.82d	7.33d	18.97ab	6.50a
	S1	74.57c	65.47c	58.76b	3.73b	0.82c	2.87cd	7.10e	19.80ab	6.43a
	S2	73.39d	63.38d	55.55c	3.45b	0.71c	3.04b	8.00a	21.33a	6.57a
	S3	76.92b	68.04b	61.64a	3.96b	1.06b	2.94bc	7.53c	18.90b	6.60a
	S4	78.66a	69.36a	62.56a	3.50b	0.68c	3.15a	7.67b	20.03ab	6.57a
YX	CK	77.33b	68.19b	62.34b	14.07c	3.42c	2.32bc	7.30c	25.70a	7.33a
	S1	76.26c	67.68b	60.60c	21.02b	5.68a	2.30c	7.53b	26.13a	7.30a
	S2	77.36b	68.48b	64.09a	16.80c	4.44b	2.37b	7.07d	25.67a	7.33a
	S3	79.75a	68.13b	62.39b	27.04a	6.23a	2.24d	6.73e	26.13a	7.23a
	S4	77.35b	69.90a	63.31ab	21.12b	5.31ab	2.53b	7.73a	26.03a	7.20a
BS	CK	73.91d	66.15bc	61.96b	16.00a	2.92a	2.46c	6.37d	20.23a	6.17ab
	S1	75.17c	64.97c	61.89b	7.72c	1.40c	2.60b	6.57c	18.20b	6.00b
	S2	74.33d	66.13bc	61.94b	4.36d	0.65d	2.57b	7.00a	18.13b	6.07b
	S3	76.45b	67.68ab	64.55a	10.70b	2.07b	2.54bc	6.67bc	20.47a	6.30a
	S4	78.01a	68.95a	66.00a	3.12d	0.42e	2.69a	6.83ab	18.00b	6.13ab
XY	CK	75.39c	66.42b	60.02b	8.24b	3.06b	2.99b	7.53a	19.63ab	6.47a
	S1	75.85b	67.33ab	59.58bc	11.50a	3.96a	2.87c	7.07c	19.53ab	6.47a
	S2	76.50a	67.66a	59.84bc	6.76c	1.69c	3.00b	7.37b	19.83a	6.57a
	S3	76.42a	68.01a	62.20a	5.56cd	1.61c	2.93bc	7.50ab	18.30b	6.57a
	S4	76.65a	68.27a	58.24c	5.12d	0.97d	3.19a	7.60a	19.33ab	6.53a
YG14	CK	80.16a	69.78a	68.93a	23.00c	5.11c	1.63b	6.90a	21.67b	6.17a
	S1	79.05b	68.83ab	68.00ab	27.20c	6.77c	1.66a	6.43c	22.70ab	5.97b
	S2	78.92b	67.94b	67.12b	37.40b	10.45b	1.59c	6.43c	23.97a	5.77c
	S3	77.16c	69.13ab	67.97ab	49.04a	13.01a	1.63b	6.30d	24.20a	5.80c
	S4	79.36ab	69.48a	68.63a	46.40a	12.15ab	1.64ab	6.73b	24.23a	5.87bc

The means in the same column followed by different lowercase letters for the same cultivar differ significantly at P = 0.05 according to the LSD test. CK: no spraying of GABA; S1: spraying GABA at tillering stage; S2: spraying GABA at panicle initiation stage; S3: spraying GABA at heading stage; S4: spraying GABA at all the three periods above. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14.

3.9. Correlation analysis

In field experiment, non-significant associations were observed of grain 2AP contents with proline contents in leaves at HS and 15 d AH, GABA contents in leaves, P5CS and OAT activities in grains and OAT activities in leaves at HS and 15 d AH. The grain 2AP contents showed significant and positive correlations with grain P5C contents at 15 d AH, grain DAO activity at 15 d AH, proline contents in leaves at MS, grain proline and GABA contents. However, significant and negative correlations were observed between grain 2AP with DAO activities in leaves at 15 d AH and MS, OAT, P5CS activities and P5C contents in leaves at MS (Table 9). In pot experiment, no significant correlation between 2AP content and other physiological-biochemical attributes were observed (data not shown). Moreover, grain yield was significantly and positively associated with filled grains percentage, and grains per panicle (Figure 1).

Figure 1. Correlation analyses between grain yield and the investigated parameters in field experiment. MX2: Meixiangzhan2; YX: Yuxiangyouzhan; BS: Basmati; XY: Xiangyaxiangzhan; YG14: Yungengyou 14. * Significant at P < 0.05; ** Significant at P < 0.01; ns nonsignificant at P > 0.05 level.

Table 9. Correlation analyses between 2AP contents in grains at maturity and the investigated parameters in field experiment.

Index	Heading stage (HS)	15 days after heading stage (15 d AH)	Maturity stage (MS)
P5C
Grain		0.4072*	−0.0503ns
Leaf	−0.7669**	−0.7675**	−0.8307**
Proline
Grain		0.7112**	0.8447**
Leaf	0.2176ns	0.2952ns	0.3991*
GABA
Grain		0.7385**	0.6693**
Leaf	0.1149ns	−0.2386ns	−0.2709ns
P5CS
Grain		0.1955ns	0.0901ns
Leaf	−0.4417*	−0.7546**	−0.6327**
DAO
Grain		0.4224*	−0.0722ns
Leaf	0.1082ns	−0.5615**	−0.7612**
OAT
Grain		−0.1478ns	0.2150ns
Leaf	−0.3112ns	−0.2202ns	−0.5760**

*Significant at P < 0.05; **Significant at P < 0.01; ns nonsignificant at P > 0.05 level.

4. Discussion

GABA is a non-protein amino acid that acts as a signaling molecule that regulates growth and development, metabolism and stress response in plants (Ramesh et al. 2017). It was found that the GABA concentration tends to be higher in response to environmental stresses such as drought, UV irradiation, mechanical damage, extreme temperature, salinity, and hypoxic conditions (Bouché and Fromm 2004). The positive effects of GABA on different plants under adverse conditions have also been widely studied. For instance, the application of GABA could significantly improve the growth and productivity of black cumin under water deficit conditions (Rezaei-Chiyaneh et al. 2018). Exogenous application of GABA could transiently increase the tolerance to root hypoxia of Prunus rootstock (Salvatierra et al. 2016). In the present study, GABA-induced modulations in 2AP contents and enzymes involved in its biosynthesis as well as grain yield of rice (Tables 1, 4, 5 and 6) were estimated.

GABA application during the different growth stages of fragrant rice could result in regulations in physiological metabolism and growth. For example, the application of GABA at panicle initiation stage could regulate the aroma formation for three aromatic rice cultivars (Xie et al. 2019). Xie et al. (2020) indicated that spraying GABA at the initial heading stage could modulate the grain 2AP contents, grain yield and grain quality in fragrant rice. In the present study, application of GABA at heading stage (S3) or at all the three stages (i.e. tillering stage, panicle initiation stage and heading stage) (S4) promoted the grain 2AP contents whereas application of GABA at panicle initiation stage (S2) improved grain yield of rice (Tables 1 and 6).

The 2AP is considered one of the most important volatiles responsible for the fragrance of aromatic rice (Buttery et al. 1983). Previous studies reported proline as the most important precursor of 2AP (Daygon et al. 2017). The grain 2AP contents are directly related to the enhancement in proline contents in grains (Wang et al. 2013). Besides, proline or 2AP is likely to transport from leaves to grains (Buttery et al. 1983; Maraval et al. 2010). In the present study, GABA application significantly reduced the proline contents in leaves at MS and the contents of proline at MS were substantially decreased as compared with proline contents at 15 d AH (Table 2), the result in field experiment showed the transference of proline from leaves to grains during grain filling thus resulted in more proline contents in grains which involved in 2AP formation. The significant and positive correlation between grain proline and 2AP contents (Table 9) confirms the involvement of proline in 2AP biosynthesis. However, Tang and Wu (2006) revealed the differences in proline contents in leaves at MS and the 2AP contents from 14 days after full heading stage to MS under zinc, iron, and lanthanum application. In the pot experiment, a significant increase or relative stability of proline contents in leaves at HS and MS was noted under the GABA treatments (Table 2), whereas the S3 and S4 treatments also increased grain 2AP contents that were significantly related to the proline contents in leaves at MS.

Moreover, the 2AP contents were also associated with the GABA contents in grains but not with the GABA contents in the leaves of aromatic rice (Poonlaphdecha et al. 2012). Mo et al. (2015) reported that the 2AP contents were significantly and positively correlated with grain GABA contents in fragrant rice. The application of exogenous GABA resulted in more GABA accumulation in grains. In general, the GABA in grains is synthesized via GABald or by the conversion of glutamate (Bouché and Fromm 2004; Fait et al. 2008). Moreover, the biosynthesis and accumulation of 2AP are associated with glutamate and GABald (Xie et al. 2020). In the present study, it was detected that both the S3 and S4 treatments significantly enhanced the GABA contents in grains (Table 3) whereas the leaf GABA contents were significantly increased under the S3 treatment at 15 d AH (Table 2). Therefore, regulation in glutamate and GABald involved in the up/down regulation of aroma formation in rice. Furthermore, a significant and positive correlation between 2AP and GABA contents in grains was noted (Table 9). Therefore, the increase in grain 2AP contents under the S3 and S4 treatments may be attributed to the improvement of GABA contents in grains.

In addition to proline, the P5C and the enzymes i.e. PDH, P5CS, OAT, and DAO were also found to be involved in the biosynthesis of 2AP (Chen et al. 2008; Kaikavoosi et al. 2015). Previously, Li et al. (2016a) and Mo et al. (2016b) reported a significant and positive association between 2AP and P5C contents in rice. Mo et al. (2019a) suggested that P5C content and DAO activity were closely related to grain 2AP contents. In the present study, it was found that the GABA application improved the P5C contents in grains and leaves at MS (Tables 2 and 3), whereas the S2 and S4 treatments substantially increased the P5C contents in leaves at 15 d AH and MS, respectively (Table 2). Moreover, the P5CS and OAT activities in leaves at HS reminded higher under the S3 treatment (Table 4) whilst the P5CS and OAT activities in grains were varied depending upon GABA treatments and rice genotypes (Table 5). Furthermore, the significant and positive correlation between 2AP and P5C contents in grains was also observed (Table 9). Thus, application of exogenous GABA caused higher P5C content and led to more 2AP accumulation in grains. However, further studies at molecular and metabolic levels are still needed to better understand the positive role of GABA application at suitable growth stage in aroma formation in fragrant rice.

Previously, exogenous GABA applications improved the growth, development, and photosynthesis in plants (Luo et al. 2011; Li et al. 2016a). GABA affected grain yield which on the other hand was significantly associated with grain weight (Li et al. 2019b). In the present study, the S2 treatment substantially improved the grain yield owing to an improvement in grains per panicle (Table 6). For example, grains per panicle as well as grain yield were increased under the S2 treatment for YX and BS. Moreover, grain yield was significantly and positively associated with grains per panicle and filled grains percentage (Figure 1). Furthermore, GABA-induced modulations in antioxidant enzyme activities i.e. SOD, POD, and CAT and MDA contents were previously noted (Li et al. 2016a). Malekzadeh et al. (2014) indicated an increase in antioxidant enzyme activities under GABA application which confirms our findings that application of GABA significantly increased the SOD activity at 15 d AH, CAT activity at 15 d AH and MS, as well as POD activity at HS and MS (Table 7). Interestingly, contrary to the reports of Priya et al. (2019), exogenous GABA application enhanced the MDA contents, which indicates that exogenous GABA application might cause stress in fragrant rice. However, AL-Quraan et al. (2015) demonstrated that the significant increase in GABA and MDA accumulation occurs simultaneously under treatment with the three 1,2,3-thiadiazole compounds and discovered GABA molecule may act as a defense mechanism to alleviate oxidative damages. Further, GABA can trap MDA directly and indirectly, as reported by Deng et al. (2010).

Additionally, GABA also plays an important role in ion absorption, nitrate uptake and nitrogen metabolism in plants (Ma et al. 2016). For instance, GABA could regulate the nitrate concentration and metabolism in the leaves of Pakchoi (Brassica campestris ssp. chinensis Makino) (Li et al. 2018). In the present study, different rice cultivars differed in their grain quality attributes, for example, significant influences in grains with chalkiness, chalkiness degree were observed under the GABA treatments, thereinto, down-regulation was noted in MX2, YX, and XY, but in YX and YG14, the two attributes were on the increase, while for protein, a substantial increase was observed in MX2 and BS whereas significant reduction was noted in XY and YG14 (Table 8). The growth and productivity of rice varies among different rice genotype (Li et al. 2019b). These results suggested that the interactive effects of GABA and cultivar influenced grain quality attributes, however, the molecular mechanisms about GABA-cultivar interaction need to be further studied. Overall, exogenous GABA applications during different growth stages of fragrant rice modulated the grain 2AP contents, enzymes involved in its formation and grain yield.

5. Conclusions

In conclusion, GABA application could improve the grain yield and 2AP contents in fragrant rice. Application of GABA at heading stage or at all (tillering, panicle initiation, and heading) stages significantly increased the grain 2AP contents across all cultivars due to the improvement of proline contents in leaves, grain proline and GABA contents. A significant increase in grain yield was noted under the GABA treatments owing to an increment in filled grains percentage, grains per panicle. Besides yield, GABA application also affected the grain quality attributes. Overall, our result suggested that the application of GABA regulate the grain yield formation and 2AP accumulation in fragrant rice.

Authors’ contributions

Z.M. designed the experiments; Z.G., W.X., Y.L., L.M. and R.G. investigated the traits; Z.G., W.X. and U.A. analyzed the data and wrote the manuscript; Z.M., U.A., G.P., H.T., M.D., S.W., and X.T. revised and edited the manuscript. X.T. was the leader of our research team. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

We acknowledge the funding provided by National Natural Science Foundation of China (31601244, 31271646), the Key-Area Research and Development Program of Guangdong Province (2019B020221003) and the financial support of China Scholarship Council (File No. 201908440176).

Notes on contributors

Zifeng Gao

Zifeng Gao is an undergraduate student of the Ding Ying Innovation class of Agronomy, College of Agriculture, South China Agricultural University.

Wenjun Xie

Wenjun Xie is an undergraduate student of the College of Agriculture, South China Agricultural University. Wenjun Xie research directions are rice cultivation and physiology.

Umair Ashraf

Umair Ashraf is an assistant professor at the Department of Botany, Division of Science and Technology, University of Education, Lahore, Punjab, Pakistan. His works include the research on crop production and physiology.

Yuzhan Li

Yuzhan Li is an undergraduate student of the College of Agriculture, South China Agricultural University. Li research directions are rice cultivation and physiology.

Lin Ma

Lin Ma is an undergraduate student of the College of Agriculture, South China Agricultural University. Ma research directions are rice cultivation and physiology.

Runfei Gui

Runfei Gui is an undergraduate student of the College of Agriculture, South China Agricultural University. Gui research directions are rice cultivation and physiology.

Shenggang Pan

Shenggang Pan is faculty of College of Agriculture, South China Agricultural University. Pan main works include the research on crop production and physiology, and the physiology and ecology response of fragrant rice and super rice.

Hua Tian

Hua Tian is faculty of College of Agriculture, South China Agricultural University. Tian main works include the research on crop production and physiology, and the physiology and ecology response of fragrant rice and super rice.

Meiyang Duan

Meiyang Duan is faculty of College of Agriculture, South China Agricultural University. Duan main works include the research on crop production and physiology, and the physiology and ecology response of fragrant rice and super rice.

Shuli Wang

Shuli Wang is faculty of College of Agriculture, South China Agricultural University. Wang main works include the research on crop production and physiology, and the physiology and ecology response of fragrant rice and super rice.

Xiangru Tang

Xiangru Tang is faculty of College of Agriculture, South China Agricultural University. Tang main works include the research on crop production and physiology, and the physiology and ecology response of fragrant rice and super rice.

Zhaowen Mo

Zhaowen Mo is faculty of College of Agriculture, South China Agricultural University. Mo main works include the research on crop production and physiology, and the physiology and ecology response of fragrant rice and super rice.