Rice-duck co-culture benefits grain 2-acetyl-1-pyrroline accumulation and quality and yield enhancement of fragrant rice

Meijun Li, Ronghu Li, Shiwei Liu, Ji'en Zhng,b,c,*, Ho Luo, Shuqing Qiu

aCollege of Natural Resources and Environment,South China Agricultural University,Guangzhou 510642,Guangdong, China

bGuangdong Engineering Technology Research Centre of Modern Eco-agriculture and Circular Agriculture, Guangzhou 510642, Guangdong,China

cKey Laboratory of Agro-Environment in the Tropics,Ministry of Agriculture,Guangzhou 510642,Guangdong, China

Keywords:Rice-duck co-culture 2-AP Proline Yield Grain quality Fragrant rice

A B S T R A C T Rice-duck co-culture is an integrated farming technology that benefits rice production,grain quality,and ecological sustainability in paddy fields.However,little is known about the effects of rice-duck co-culture on enzyme activity involved in the biosynthesis of 2-acetyl-1-pyrroline(2-AP),the volatile that gives fragrant rice its'distinctive and sought-after aroma.The present study aimed to examine the influence of rice-duck co-culture on the photosynthesis, yield,grain quality,rice aroma,and the enzymes involved in 2-acetyl-1-pyrroline biosynthesis in the cultivar Meixiangzhan 2 during the early and late rice growing seasons of 2016 in Guangzhou,China.We compared the rice grown in paddy fields with and without ducks.We found that rice-duck co-culture not only improved the yield and quality of fragrant rice grain, but also promoted the precursors of 2-AP biosynthesis formation and 2-AP accumulation in the grain.Grain 2-AP content in rice-duck co-culture was noticeably increased with 9.60%and 20.81%in early and late seasons,respectively.Proline and pyrroline-5-carboxylic acid(P5C)(precursors of 2-AP biosynthesis) and the activity of enzymes such as proline dehydrogenase (ProDH),ornithine aminotransferase (OAT) and Δ1 pyrroline-5-carboxylic acid synthetase(P5CS)were all improved by 10.15%-12.99%, 32.91%-47.75%, 17.81%-26.71%, 6.25%-21.78%, and 10.58%-38.87% under rice-duck co-culture in both seasons, respectively. Overall, our results suggest that rice-duck co-culture is an environmentally-friendly and sustainable approach to improving rice aroma and grain quality of fragrant rice.

1.Introduction

Rice (Oryza sativa L.) is one of the most important cereal grains, feeding more than half the world's population [1,2]. A large number of rice varieties are grown,with a characteristic fragrance that is highly regarded [3,4]. These aromatic or fragrant rice varieties are sold at a premium price in local and export markets because of their superior grain qualities and pleasant and distinctive aroma [5]. Furthermore, it appears that consumers are increasingly considering aroma when purchasing rice, as global demand for the fragrance in aromatic rice is increasing[6].As such,cultivation techniques for increasing the fragrance,quality,and yield of aromatic rice are becoming more important.

Determining the exact constituents that give fragrant rice its' distinctive aroma is complex, as instrumental analyses have confirmed the presence of ＞200 volatile compounds[7-9]. Likely candidate volatiles included 2-acetyl-l-pyrroline,(E,E)-2,4-decadienal,nonanal,hexanal,(E)-2-nonenal,octanal,decanal, 4-vinyl-guaiacol, 4-vinylphenol, 2-amino acetophenone, and 4,5-epoxy-(E)-2-decenal [8,10]. Further, in some cases similar aroma profiles between cultivars had differences in the levels of various volatile compounds.However, despite this complexity, a single compound, 2-acetyl-1-pyrroline(2-AP)synonymous with 1-(3,4-dihydro-2Hpyrrol-5-yl)ethanone,appears to be the primary source of the fragrance of aromatic rice [8,11]. Various precursors of 2-AP,such as 4-aminobutanal,have also been to accumulate in rice,and cyclisation and dehydration then result in the formation of 1-pyrroline, which is acetylated to form 2-AP [12-14]. 2-AP has been detected in all parts of the rice plant except the roots[15,16].And 2-AP might even be present in other non-fragrant rice varieties,but just at lower concentrations[17].

The fragrance in aromatic rice is controlled by a single recessive gene(fgr)on chromosome 8.The dominant fgr allele is associated with loss-of-function mutations in the coding region[18-20].Simultaneously,2-AP biosynthesis in aromatic rice is also affected by external factors, such as geographical locations, crop management, prevailing climatic conditions,and cultivar types [9,21]. For example, significant effects of manganese application [22], illumination intensity [23], and temperature fluctuations [24,25] on 2-AP biosynthesis have been reported. Further, drought [26], salinity [27,28], and crop management practices such as plant populating, harvesting time [29], irrigation regime [30], and storage conditions [3,31]all affect 2-AP accumulation or concentration in aromatic rice grains. Understanding the interacting effects of various cultivation techniques on 2-AP concentration will be key to the future development of this crop.

An important method of integrated cultivation of rice in China is rice-duck co-culture, in which ducks live directly in the rice field [32]. This technique is especially suitable for resource-poor farmers,allowing them to produce organic rice at low cost[33,34].In this system,one-week-old ducklings are released into the rice field at a density of about 225-375 ducklings per hectare 7-10 days after rice transplantation and kept there until the rice heading stage[35].The integration of ducks in rice field creates a mutualistic relationship between the rice and ducks yielding benefits to both entities. For example, ducks eat weeds, weed seeds, insects, pests, and golden apple snail (Pomacea canaliculata) [36-38], thereby reducing the need for manual weeding and the pesticide application.Duck droppings provide nutrients for the growing of rice plants, which could reduce fertilizer application [39].Thus,it is an‘eco-friendly'farming system for increasing rice and duck productivity and providing more economic benefits[33,34,40]. Furthermore, the ducks' activities could induce anatomical changes to the rice stem and enhance the lodging resistance of the rice plant [32]. However, the effects of riceduck co-culture on 2-AP accumulation are still unknown.

The pathway of 2-AP formation has recently been documented; Δ1-pyrroline might be converted into 2-AP when acetyltransferase is catalyzed[22].In this study,we examined 2-AP accumulation in the fragrant rice cultivar Meixiangzhan 2, which is planted widely in south China. We hypothesized that rice-duck co-culture would affect the precursor of 2-AP biosynthesis, increase yield, and improve the grain quality of fragrant rice.

2. Materials and methods

2.1. Experimental site

The field experiment was conducted at Zengcheng Teaching and Research Farm (23°14′N, 113°38′E), South China Agricultural University, Guangzhou, Guangdong province, China, where the subtropical monsoon climate was characterized by warm winters and hot summers, with an annual rainfall of 2 454.7 mm and an average temperature of 31.3 °C (Fig. 1). The soil in the experimental site was sandy loam, containing 17.38 g kg-1organic matter,0.80 g kg-1total nitrogen,0.58 g kg-1total phosphorous, 15.75 g kg-1total potassium, 99.47 mg·kg-1available nitrogen, 66.50 mg kg-1available phosphorus,76.18 mg kg-1available potassium,and had 4.88 pH.

2.2. Agronomic management systems

We used the aromatic rice cultivar Meixiangzhan 2,which was widely grown in south China and was popular due to its special aroma and flavor.Before sowing,seeds were soaked in water for 24 h at room temperature and germinated under moisture conditions. Germinated seeds were sown on March 13, 2016(early growing season)and on July 28,2016(late growing season)for nursery rising. Seedlings were then transplanted at the normal spacing of 20 cm × 20 cm on April 11, 2016 (early growing season) and August 18, 2016 (late growing season). A completely random design was used in this field experiment,which consisted of two treatments,each with three replicates.Experimental plots were arranged in a random design within the field,each with an area of 56 m2(7 m × 8 m).In each plot,organic fertilizer of 500 kg·ha-1with N ≥1.63%, P2O5≥3.53%,K2O ≥1.02%, and organic matter ≥46% were applied before transplanting. No pesticides, herbicides, or other weed control practices were applied for any treatments.

Ducklings were released into the treatment plots at a density of 375 duck individuals per hectare (based on the recommended population) seven days after the rice seedlings were transplanted [41]. Each paddy field plot was surrounded by a 50 cm high nylon mesh fence to prevent ducks from escaping. The ducks were fed with cereal once every day, and kept in the field until the rice heading stage,and taken out of the paddy field on June 10 (early growing season) or October 18 (late growing season) 2016. The water layer was kept at 6-8 cm while ducks were in the field, but irrigation was stopped one week before the rice harvest.Control plots were cultivated identically except without ducks.

Fig.1- Rainfall(RF)and average temperature (AT)per month during experimental year 2016 in Zengcheng,Guangzhou,Guangdong,China.

2.3. Samplings and data collection

2.3.1. Total above-ground biomass

Plants were sampled randomly with three replicates at TS,HS,and MS stages from an area of 0.5 m2from each plot in both rice growing seasons. Then the sampled plants were ovendried at 70 °C till constant weight and measured for total above-ground biomass.

2.3.2. Photosynthesis rate

The maximum CO2assimilation rate per unit area(Photosynthesis rate, μmol m-2s-1) was measured between 9:00 and 11:00 AM using a Li-6400/XT portable photosynthetic system(Li-6400/XT, LI-COR, Lincoln, Nebraska, USA). Based on preliminary trials, the photosynthetic photon flux density was set at 1000 μmol m-2s-1for all plots.The ambient CO2and air temperature were maintained at 390 μmol mol-1and 28 °C,respectively. For each plot, we selected three rice plants, and then measured three fully developed leaves or sword leaves(maturity stage) for each rice plant. An average value was calculated from the nine leaves for each treatment.

2.3.3. Yield and yield components

At maturity stage, grain yield and yield components were measured from three plots with an area of 1 m × 1 m for each replicate, threshed manually and sun-dried (adjusted to 14%moisture contents) to obtain the grain yield. Plants with panicles were separated into straw and panicles, then separated into filled grains and unfilled grains. Three subsamples of filled and unfilled grains were used to estimate the total number of spikelets, and all of the half-filled spikelets were taken and averaged. The number of spikelets per panicle, grain-filling percentage (100 × filled spikelets number/total spikelets number), and 1000-grain weight were also calculated from sampled plants and averaged.

2.3.4.Grain quality

About 500 g of grains harvested from each plot were dried in the sun for grain quality analyses.An 80 g sample of rice grain was passed through a de-husker for polishing, and then separated into broken and unbroken grains. The brown rice rate, milled rice rate, and head rice rate were expressed as percentages of the total(80 g)rice grains.Amylose content and soluble protein content were measured using an Infratec-1241 grain analyzer(FOSS-TECATOR, Hilleroed, Denmark). Chalky rice rate,chalkiness degree, and grain length and width were scanned with a Plant Mirror Image Analysis (MICROTEK, Hsinchu,Taiwan,China),and then the resulting images were processed with SC-E software(Hangzhou Wanshen Detection Technology Co.,Ltd.,Hangzhou,Zhejiang,China).

2.3.5.2-AP contents

The 2-AP content of grain samples (10 g) from each plot was estimated using the synchronization distillation and extraction method (SDE) combined with GCMS-QP 2010 Plus(Shimadzu Corporation, Kyoto,Japan)[23].

2.3.6.Biochemical analyses

2.3.6.1. Proline and soluble protein measurements. The proline content was estimated following Bates et al. [42]. Grains(0.3 g) were homogenized in 5 mL of 3% sulfosalicylic acid,and bathed in boiled water for 10 min. Two milliliters of the filtrate were mixed with ninhydrin reagent(3 mL)and glacial acetic acid (2 mL) while the reaction cooled down. The reaction mixture was placed in a boiling water bath again for 30 min. Then extracted by adding 4 mL toluene after the

Plants were sampled randomly with three replicates at physiological tillering (TS), heading (HS), and maturity (MS)stages from each treatment,washed with tap water and then with distilled water. Fresh leaves and grains were separated and immediately put into liquid nitrogen, and then stored at-80 °C for biochemical analyses.reaction mixture cooled down by ice bath. The reaction mixture was centrifuged at 4000 ×g for 5 min.The absorbance of the red chromophore in the toluene extraction was measured at 520 nm,and proline content was determined by comparing with a standard curve and expressed as microgram per gram(μg g-1fresh weight(FW)).

2.3.6.2. Enzyme extracts preparation. Fresh leaf samples(0.3 g) were homogenized in 8 mL of 50 mol L-1Tris-HCl buffer(pH 7.5),which contained 7.0 mol L-1MgCl2,1.0 mol L-1KCl, 3.0 mol L-1ethylenediamine tetra-acetic acid (EDTA),1.0 mol L-1DLdithiothreitol(DTT),and 5%insoluble polyvinyl polypyrrolidone(PVP).Then the homogenate was centrifuged at 8000 ×g for 20 min at 4 °C. The supernatants were used to determine the content of pyrroline-5-carboxylic acid(P5C)and the activities of Δ1pyrroline 5-carboxylic acid synthetase(P5CS), proline dehydrogenase (ProDH), and ornithine aminotransferase(OAT).

The P5C content in each plant was measured following Wu et al.[43].The reaction mixture contained 0.2 mL supernatant of enzyme solution, 0.5 mL of 10% trichloroacetic acid (TCA)and 0.2 mL of 40 mol L-12-aminobenzaldehyde.The samples were kept at room temperature for 30 min, and then centrifuged at 8000 ×g for 10 min. After centrifugation, the absorbance was measured at 440 nm by spectrophotometer.The concentration of P5C was determined calculated by the extinction coefficient 2.58 mmol L-1cm-1.

ProDH (EC 1.5.99.8)activity was assayed following Ncube et al. [44]. The reaction mixture contained 15 mol L-1proline,0.01 mol L-1cytochrome c, 100 mol L-1phosphate buffer(pH 7.4), 0.5%(v/v) Triton X-100,and 0.2 mL enzyme extract in a total volume of 1 mL was incubated at 37 °C for 30 min and the reaction ceased with the addition of 1 mL of 10%TCA.The P5C formed was measured by the addition of 1 mL of 0.5% 2-aminobenzaldehyde in 95% ethanol. Then the reaction was incubated at 37 °C for 10 min,centrifuged at 8000 ×g for 10 min at 4 °C. The supernatant was measured at 440 nm. ProDH activity was calculated by a molar extinction coefficient of 2.71 × 103min-1cm-1.

The activity of P5CS (EC 1.5.1.12) was assayed measured following Zhang et al. [45]. 50 mol L-1Tris-HCl buffer (pH 7.0),20.0 mol L-1MgCl2, 50 mol L-1sodium glutamate, 10 mol L-1ATP, 100 mol L-1hydroxamate-HCl, and 0.5 mL of enzyme extract were added in turn to the reaction mixture. The tubes were placed in a water bath at 37 °C for 5 min.Then 0.5 mL of a stop buffer (2.5% of FeCl3plus 6% of trichloracetic acid (TCA))was added, and dissolved in 100 mL of 2.5 mol L-1HCl to terminate the reaction, finally the reaction was measured at 535 nm absorbance.

The activity of OAT (EC 4.1.1.17) was measured following Chen et al. [46]. The reaction mixture contained 100 mol L-1potassium phosphate buffer (pH 8.0), 50 mol L-1ornithine,20 mol L-1a-ketoglutarate, 1 mol L-1pridoxal-5-phosphate and the enzyme extract (0.1 mL). After incubation of the reaction mixture at 37 °C for 30 min, the reaction was terminated by adding 0.3 mL 10% TCA and 0.2 mL 0.25% oaminobenzaldehyde and reincubation for 60 min. And then centrifuged at 8000 ×g for 10 min, the supernatant fraction was measured at 440 nm absorbance. Enzyme activity was calculated by the extinction coefficient 2.68 mmol L-1cm-1.

2.4. Data analyses

The Student's t-test was used to evaluate the differences in plant traits, grain quality, and yield between the control and rice-duck co-culture. All statistical analyses were performed by using the software R 3.4.0(R Development Core Team 2017).

3. Results

3.1. Photosynthetic parameter and total above-ground biomass

The benefits of rice-duck co-culture to rice growth were not overwhelming, but certainly not negative. Rice-duck coculture system had a significant and positive but small effect on net photosynthesis of rice plants in both the early and late growing seasons of 2016, but not at all stages of rice development,and in no stage did ducks have a negative effect(Fig. 2a, b). Similarly, the transpiration rate of rice plants was significantly higher in the late growing season under riceduck co-culture at the later stages of development(Fig.2c,d).

At maturity, rice grown with ducks had about 15 g more total above-ground biomass than control in the early growing season (Fig. 2e, f). In the late season, rice grown with ducks was also larger,but not significantly so.

3.2. Yield and yield components

Rice-duck co-culture significantly increased grain yield and its components in both early and late growing seasons of 2016(Table 1). Rice-duck co-culture produced nearly a ton more grain per ha than the control.This increased yield was due to the rice plants producing more spikelets per panicle, setting more seed,and growing more productive ears.

3.3. Grain quality

Rice-duck co-culture also benefited grain quality in both early and late growing seasons in 2016 (Table 2). Rice grown with ducks had slightly higher milled and head rice rates,but more importantly, had significantly lower chalkiness rice rate and chalkiness degree. No significant effect on brown rice rate,amylose content, soluble protein content and length/width between rice-duck co-culture and control in both early and late growing seasons.

3.4. 2-AP contents

Consistent with our predictions, the 2-AP content in rice grains was significantly higher in rice-duck co-culture in both early and late growing seasons in 2016 (Fig. 3). Rice-duck coculture enhanced grain 2-AP contents noticeably with an increase of 9.60% and 20.81% in the early and late growing seasons,respectively.

3.5. Proline, soluble protein, and P5C content

Fig.2-Influence of rice-duck co-culture on photosynthetic rate(a,b),transpiration rate(c,d),and total above-ground biomass(e,f) of fragrant rice during early and late rice growing seasons at the tillering(TS),heading(HS),and mature(MS)stages.Asterisks indicate the significance of the Students t-test for each comparison(*P ＜0.05, **P ＜0.01).CK,control; DR,rice-duck co-culture.

Table 1-Effect of rice-duck co-culture on yield and its components of fragrant rice in early and late growing seasons in 2016.

In all but three cases, rice-duck co-culture increased rice leaf and grain's proline, soluble protein, and P5C across rice development stage during both growing seasons in 2016(Figs. 4, 5a, b). The rice leaf proline content of rice-duck coculture increased 10%-12% (Fig. 4a, b). The rice leaf soluble protein of rice-duck co-culture also significantly increased by 4.64%-15.38% (Fig. 4c, d). Additionally, the rice leaf P5C content of rice-duck co-culture farmed rice significantly increased at all growing stages during both growing seasons(Fig. 4e, f). Furthermore, rice-duck co-culture resulted in a significant increment on rice grain proline, soluble protein and P5C content of fragrant rice grain during both growing seasons of fragrant rice(Fig.5a,b).

Table 2-Effect of rice-duck co-culture on grain quality of fragrant rice in the early and late growing seasons in 2016.

3.6. Activity of ProDH, OAT, and P5CS involved in 2-AP biosynthesis

Rice-duck co-culture significantly improved the rice leaf and grain's activities of ProDH,OAT,and P5CS(all involved in 2-AP biosynthesis) during both rice growing seasons in 2016 (Figs.5c, d, 6). The activity of ProDH in fragrant rice leaf was increased by 17.81%-26.71%in rice-duck co-culture compared to the control throughout rice development (Fig. 6a, b). The activity of OAT in fragrant rice leaf also increased under riceduck co-culture, by between 6.25% and 21.78% depending on the stage(Fig.6c,d).Similarly,the activity of P5CS in fragrant rice leaf increased under rice-duck co-culture range from 10.58% to 38.87% (Fig. 6e, f). With respect to the activities of rice grain ProDH, OAT, and P5CS, we found a significant difference between the rice-duck co-culture and the control(Fig.5c,d).

3.7. Correlation analysis

The correlation among grain 2-AP, proline, soluble protein,P5C, ProDH, OAT, and P5CS of leaf or grain in fragrant rice at maturity stage showed that there was a positive correlation between grain 2-AP and the biosynthesis of 2-AP in fragrant rice, especially proline and ProDH content of rice leaf and grain. Moreover, positive associations existed among the proline, soluble protein, P5C and the ProDH, OAT, P5CS in the fragrant rice (Table 3).

4. Discussion

The role of rice-duck co-culture in improving rice productivity,quality, and aromatic characters and regulation of enzyme activities involved in 2-AP biosynthesis were studied in an experimental rice paddy system of the cultivar Meixiangzhan 2 in Guangzhou,China,over two growing seasons in 2016.We found variable(but no negative)effects of rice-duck co-culture on rice plant physiology (photosynthesis, transpiration, and dry weight), and significant benefits on yield and grain quality.

Fig.3-Influence of rice-duck co-culture on grain 2-AP content of fragrant rice in early and late rice growing seasons.Asterisks indicate significance of the Students t-test for each comparison(*P ＜0.05,**P ＜0.01).CK,control;DR,rice-duck co-culture.

Fig.4-Effect of rice-duck co-culture on rice leaf proline(a,b),soluble protein(c,d),and P5C(e,f)of fragrant rice in early and late rice growing seasons at the tillering(TS),heading(HS),and mature(MS)stages.Asterisks indicate significance of the Students t-test for each comparison(*P ＜0.05, **P ＜0.01).CK,control; DR,rice-duck co-culture;P5C,proline and pyrroline-5-carboxylic acid.

Our study was consistent with previous studies that had reported significant effects of rice-duck co-culture on the morphology and grain yield of fragrant rice.For example,riceduck co-culture decreased rice stem height but increased root biomass and chlorophyll a & b contents, and improved rice plant lodging resistance and photosynthesis capacity leading to increased rice production [47]. Rice-duck co-culture could also increase yield by 20% by controlling weeds and insects effectively[40].Those increases in yield might also be due to a reduction in the loss of nitrogen and phosphorus[34].Increases in yield,brown rice rate,milled rice rate,head rice rate,and gel consistency,and a reduction in chalkiness due to rice-duck coculture were also reported by Yang et al.[48]and Zhen et al.[49].

We also documented a significant increase in activity of the biosynthesis pathways and concentrations of the precursors of the aromatic volatile 2-AP [50]. Biosynthesis and accumulation of 2-AP in aromatic rice was an important phenomenon which was affected by several factors [51].Proline, glutamic acid, and ornithine were converted into P5C by the enzymes ProDH, P5CS and OAT, respectively, and then converted into 2-AP via enzymatic(acetyl-CoA groups)or non-enzymatic (methylglyoxal) [50]. In this study, we found that the concentrations of soluble protein, proline, and P5C,and the activities of ProDH,OAT,and P5CS in rice plants of the rice-duck co-culture were higher than the control. Additionally, there were positive correlations among the grain 2-AP,soluble protein,proline,P5C,ProDH,OAT,and P5CS in the leaf and grain of fragrant rice. Thus, these results indicated that the precursors of 2-AP biosynthesis contributed to the formation and accumulation of 2-AP in fragrant rice.

Fig.5-Effect of rice-duck co-culture on rice grain proline,soluble protein,and P5C contents(a,b)and grain activities of ProDH,OAT,and P5CS(c,d)of fragrant rice at maturity stage in early and late rice growing seasons.Asterisks indicate significance of the Students t-test for each comparison(*P ＜0.05,**P ＜0.01).CK,control;DR,rice-duck co-culture;P5C,pyrroline-5-carboxylic acid;ProDH,proline dehydrogenase;OAT,ornithine aminotransferase;P5CS,Δ1 pyrroline-5-carboxylic acid synthetase.

However, we also know that the ducks eat weeds, pests,and apple snails in the paddy fields,and provide supplemental nitrogen, phosphorus, and potassium from their faces.Several studies had shown that nitrogen was an important factor increasing the accumulation of 2-AP in fragrant rice[52].Nitrogen was a precursor source of proline to form 2-AP,and the increase of nitrogen in paddy fields would lead to the 2-AP content increasing in fragrant rice[53,54].

Furthermore, it seemed likely that the activities of the ducks had some stimulating effects on the rice plants in the paddy fields. Mechanical stimulation, such as touching,bending and shaking, was an environmental stress factor that affected the growth and development of other plants[55-57]. Mechanical stimulation had also been suggested as a way to controll the plant growth in agricultural and horticultural settings [58], and some farmers regularly use stresstreading, trampling, or stamping of wheat and barley seedlings as a way to prevent spindly growth,strengthen the roots,shorten plant height, and ultimately to improve yield [59].Mechanical stimulation could increase growth rate and soluble protein content but decrease the fluidity of cell membranes and the activity of IAA in Gerbera jamesonii Bolus[60], and the finite IAA would promote the accumulation of proline in wheat[61,62].Specifically,the mechanical stimulation on rice plant by ducks (feeding, trampling, and other activities) in paddy fields could affect the rice plant physiology, including that the permeability of leaf plasma membranes increased, the leaf chlorophyll-a content, and POD,SOD,and PPO activities improved,as well as the ABA content,and the IAA concentrations decreased[63]. Li and Gong [64]reported that when plants were subjected to various mechanical stimulations which were perceived by the same or different sensors might be located in cell wall-plasma membrane-skeleton system, these might trigger the production of second messenger existing cross-talk among each other, which might in turn alter gene expression, then induced the synthesis of osmolytes (such as proline, soluble sugar), as well as other stress proteins. Furthermore, the research of Li and Gong [65] indicated that mechanical stimulation could increase the activity of P5CS and induce accumulation of endogenous proline in tobacco cells. Thus,due to the mechanical stimulation of ducks, the proline,soluble protein, and 2-AP biosynthesis contents were also increased under rice-duck co-culture cultivation in our experiment.

Fig.6-Effect of rice-duck co-culture on the activity of ProDH(a,b),OAT(c,d),and P5CS(e,f)for fragrant rice in early and late rice growing seasons at the tillering(TS),heading(HS),and mature(MS)stages.Asterisks indicate significance of the Students t-test for each comparison(*P ＜0.05,**P ＜0.01).CK,control;DR,rice-duck co-culture;ProDH,proline dehydrogenase;OAT,ornithine aminotransferase;P5CS,Δ1 pyrroline-5-carboxylic acid synthetase.

Overall, rice-duck co-culture might have great potential not only to improve the grain yield and grain quality of fragrant rice, but also to promote 2-AP accumulation and thereby increase rice fragrance. Rice-duck co-culture appeared to have these effects via several processes. First,ducks increased the supply of nutrients for plant growth by their consumption and excretion of animal material which greatly enhanced rice plant photosynthesis and promoted total above-ground biomass accumulation, and improved microclimate conditions [66,67]. Second, as ducks moved around the field, they would shake and peck the rice plants and trample the litter and roots which would also improve the microclimate for rice plant growing [32], as well as stimulate plant physiological mechanisms. And then those changes would occur in soluble protein content, proline and other osmotic regulating substances,and enzyme activity(included ProDH),as well as P5C content,and P5CS and OAT activity[68].

5. Conclusions

We found that rice-duck co-culture not only improved the yield and grain quality of fragrant rice, but also promoted both the precursors of 2-AP biosynthesis and 2-AP accumulation itself.These benefits might be due to several factors. First, the ducks increased nutrient availability in the fields. Second, the ducks stimulated rice plant physiology as they moved around the paddy field.Both these activities appeared to promote the precursors of 2-AP biosynthesis formation and to increase 2-AP content of the rice grain itself.Thus,the rice-duck co-culture might be used to increase the yield and aroma of fragrant rice.

Table 3-Pearson correlations among grain 2-AP, proline, soluble protein, P5C, ProDH, OAT, and P5CS of sword leaf and grain in fragrant rice at maturity stage in 2016.

Conflict of interest

We declare that we have no financial and personal relationships with other people to this work. We declare that we do not have any associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgments

This work was supported by the Science and Technology Project of Guangdong Province (2015B090903077, 2016A020210094,2017A090905030), China; the Science and Technology Project of Guangzhou (201604020062), China; the Innovation Team Construction Project of Modern Agricultural Industry Technology System of Guangdong Province (2016LM1100), China; and the Overseas Joint Doctoral Training Program of South China Agricultural University (2018LHPY010), China. In addition, we would also like to thank Prof. Simon Queenborough at - Yale University for helping us edit the language of this manuscript.