TH E C ROP J O U R NA L 7 (2 0 1 9) 41 9 –4 3 0

Available online at www.sciencedirect.com

ScienceDirect

Rice-duck co-culture benefits grain 2-acetyl-1-

pyrroline accumulation and quality and yield
enhancement of fragrant rice

Meijuan Lia , Ronghua Lia , Shiwei Liua , Jia'en Zhanga,b,c,⁎, Hao Luoa , Shuqing Qiua
a
College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, Guangdong, China
b
Guangdong Engineering Technology Research Centre of Modern Eco-agriculture and Circular Agriculture, Guangzhou 510642, Guangdong,
China
c
Key Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture, Guangzhou 510642, Guangdong, China

AR TIC LE I N FO ABS TR ACT

Article history: Rice-duck co-culture is an integrated farming technology that benefits rice production, grain
Received 12 November 2018 quality, and ecological sustainability in paddy fields. However, little is known about the effects
Received in revised form 22 January of rice-duck co-culture on enzyme activity involved in the biosynthesis of 2-acetyl-1-pyrroline
2019 (2-AP), the volatile that gives fragrant rice its' distinctive and sought-after aroma. The present
Accepted 14 February 2019 study aimed to examine the influence of rice-duck co-culture on the photosynthesis, yield,
Available online 8 April 2019 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,
Keywords: China. We compared the rice grown in paddy fields with and without ducks. We found that
Rice-duck co-culture rice-duck co-culture not only improved the yield and quality of fragrant rice grain, but also
2-AP promoted the precursors of 2-AP biosynthesis formation and 2-AP accumulation in the grain.
Proline Grain 2-AP content in rice-duck co-culture was noticeably increased with 9.60% and 20.81% in
Yield early and late seasons, respectively. Proline and pyrroline-5-carboxylic acid (P5C) (precursors
Grain quality of 2-AP biosynthesis) and the activity of enzymes such as proline dehydrogenase (ProDH),
Fragrant rice 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.
© 2019 Crop Science Society of China and Institute of Crop Science, CAAS. Production and
hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction large number of rice varieties are grown, with a characteristic

fragrance that is highly regarded [3,4]. These aromatic or
Rice (Oryza sativa L.) is one of the most important cereal fragrant rice varieties are sold at a premium price in local and
grains, feeding more than half the world's population [1,2]. A export markets because of their superior grain qualities and

⁎ Corresponding author: College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, Guangdong, China.
E-mail address: jeanzh@scau.edu.cn. (J. Zhang).
Peer review under responsibility of Crop Science Society of China and Institute of Crop Science, CAAS.

https://doi.org/10.1016/j.cj.2019.02.002
2214-5141 © 2019 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi
Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
420 TH E CR OP J OUR NA L 7 ( 2 0 19 ) 41 9 – 4 3 0
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-2H-
pyrrol-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 rice-
duck co-culture on 2-AP accumulation are still unknown.
The pathway of 2-AP formation has recently been docu-
mented; 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−1 organic matter, 0.80 g kg−1 total nitrogen, 0.58 g kg−1
total phosphorous, 15.75 g kg−1 total potassium, 99.47 mg·kg−1
available nitrogen, 66.50 mg kg−1 available phosphorus,
76.18 mg kg−1 available 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−1 with 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 seed-
lings 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.
 TH E C ROP J O U R NA L 7 (2 0 1 9) 41 9 –4 3 0
450
RF
400
350
300
Rainfall (mm)
250
200
150
100
50
0 0
Jan Feb Mar
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
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.
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 m2 from each plot in both
rice growing seasons. Then the sampled plants were oven-
dried at 70 °C till constant weight and measured for total
above-ground biomass.
2.3.2. Photosynthesis rate
The maximum CO2 assimilation rate per unit area (Photosyn-
thesis rate, μmol m−2 s−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 pre-
liminary trials, the photosynthetic photon flux density was
set at 1000 μmol m−2 s−1 for all plots. The ambient CO2 and air
temperature were maintained at 390 μmol mol−1 and 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 sub-
samples of filled and unfilled grains were used to estimate the
421
35
AT
30
25
Average temperature ( )
20
15
10
5
Apr May Jun Jul Aug Sep Oct Nov Dec
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 num-
ber/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 extrac-
tion 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 pro-
line 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
422 TH E CR OP J OUR NA L 7 ( 2 0 19 ) 41 9 – 4 3 0
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 micro-
gram per gram (μg g−1 fresh weight (FW)).
2.3.6.2. Enzyme extracts preparation. Fresh leaf samples
(0.3 g) were homogenized in 8 mL of 50 mol L−1 Tris-HCl
buffer (pH 7.5), which contained 7.0 mol L−1 MgCl2, 1.0 mol L−1
KCl, 3.0 mol L−1 ethylenediamine tetra-acetic acid (EDTA),
1.0 mol L−1 DLdithiothreitol (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 Δ1 pyrroline 5-carboxylic acid synthetase
(P5CS), proline dehydrogenase (ProDH), and ornithine amino-
transferase (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−1 2-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−1 cm−1.
ProDH (EC 1.5.99.8) activity was assayed following Ncube et
al. [44]. The reaction mixture contained 15 mol L−1 proline,
0.01 mol L−1 cytochrome c, 100 mol L−1 phosphate 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 × 103 min−1 cm−1.
The activity of P5CS (EC 1.5.1.12) was assayed measured
following Zhang et al. [45]. 50 mol L−1 Tris-HCl buffer (pH 7.0),
20.0 mol L−1 MgCl2, 50 mol L−1 sodium glutamate, 10 mol L−1
ATP, 100 mol L−1 hydroxamate-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 FeCl3 plus 6% of trichloracetic acid (TCA))
was added, and dissolved in 100 mL of 2.5 mol L−1 HCl 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−1
potassium phosphate buffer (pH 8.0), 50 mol L−1 ornithine,
20 mol L−1 a-ketoglutarate, 1 mol L−1 pridoxal-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% o-
aminobenzaldehyde 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−1 cm−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 co-
culture 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 rice-
duck 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 co-
culture 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
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
 TH E C ROP J O U R NA L 7 (2 0 1 9) 41 9 –4 3 0 423

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.
Treatment Yield (t ha−1) Spikelet per Seed-setting 1000-grain Productive panicle Harvest index
panicle rate (%) weight (g) (104 ha−1)

Early season
CK 5.93 ± 0.14 69.86 ± 0.24 77.13 ± 0.74 21.81 ± 0.12 288.89 ± 4.02 0.40 ± 0.00
DR 6.69 ± 0.03⁎ 72.45 ± 0.47⁎ 82.07 ± 0.24⁎ 22.94 ± 0.57 313.48 ± 3.34⁎ 0.44 ± 0.01⁎

Late season
CK 6.57 ± 0.13 66.14 ± 0.95 68.87 ± 1.39 21.04 ± 0.42 270.14 ± 2.89 0.40 ± 0.00
DR 7.48 ± 0.15⁎ 72.44 ± 0.96⁎ 75.59 ± 1.12⁎ 20.94 ± 0.41 316.83 ± 9.28⁎ 0.47 ± 0.01⁎

Asterisks indicate significance of the Students t-test for each comparison (⁎P < 0.05, ⁎⁎P < 0.01). CK, control; DR, rice-duck co-culture.
424 TH E CR OP J OUR NA L 7 ( 2 0 19 ) 41 9 – 4 3 0

Table 2 – Effect of rice-duck co-culture on grain quality of fragrant rice in the early and late growing seasons in 2016.
Treatments Brown rice rate Milled rice rate Head rice rate Amylose content Length/ Chalky rice rate Chalkiness
(%) (%) (%) (%) width (%) degree (%)

Early season
CK 78.96 ± 0.07 66.08 ± 0.71 51.70 ± 0.61 26.51 ± 0.01 2.80 ± 0.05 10.98 ± 0.33⁎ 5.70 ± 0.26⁎
DR 79.15 ± 0.68 66.95 ± 0.74 54.23 ± 0.54⁎ 26.50 ± 0.00 2.73 ± 0.04 6.32 ± 0.27 2.50 ± 0.17

Late season
CK 78.70 ± 0.14 66.29 ± 0.31 53.18 ± 0.17 25.51 ± 0.24 2.81 ± 0.03 11.50 ± 0.53⁎ 4.52 ± 0.31⁎
DR 79.14 ± 0.13 68.36 ± 0.05⁎ 57.04 ± 0.21⁎ 24.92 ± 0.42 2.82 ± 0.01 8.11 ± 0.53 2.70 ± 0.18

Asterisks indicate significance of the Students t-test for each comparison (⁎P < 0.05, ⁎⁎P < 0.01). CK, control; DR, rice-duck co-culture.

(Figs. 4, 5a, b). The rice leaf proline content of rice-duck co-
culture 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).
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 rice-
duck 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

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.
 TH E C ROP J O U R NA L 7 (2 0 1 9) 41 9 –4 3 0 425

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.

dry weight), and significant benefits on yield and grain
quality.
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, rice-
duck 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 co-
culture 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 precur-
sors 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. Addition-
ally, 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
426 TH E CR OP J OUR NA L 7 ( 2 0 19 ) 41 9 – 4 3 0

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.

the precursors of 2-AP biosynthesis contributed to the
formation and accumulation of 2-AP in fragrant rice.
However, we also know that the ducks eat weeds, pests,
and apple snails in the paddy fields, and provide supplemen-
tal 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 horticul-
tural settings [58], and some farmers regularly use stress-
treading, trampling, or stamping of wheat and barley seed-
lings 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 stimula-
tion on rice plant by ducks (feeding, trampling, and other
activities) in paddy fields could affect the rice plant physiol-
ogy, including that the permeability of leaf plasma mem-
branes 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 mechan-
ical stimulations which were perceived by the same or
different sensors might be located in cell wall–plasma
membrane–skeleton system, these might trigger the produc-
tion 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,
 TH E C ROP J O U R NA L 7 (2 0 1 9) 41 9 –4 3 0 427

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.

soluble protein, and 2-AP biosynthesis contents were also
increased under rice-duck co-culture cultivation in our
experiment.
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 ap-
peared 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
428 TH E CR OP J OUR NA L 7 ( 2 0 19 ) 41 9 – 4 3 0

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.
Grain 2- Sword leaf Grain
AP
Proline Soluble P5C ProDH OAT P5CS Proline Soluble P5C ProDH OAT P5CS
protein protein

Sword leaf
Proline 0.894 ⁎⁎ 1.000
Soluble 0.771 ⁎⁎ 0.889 ⁎⁎ 1.000
protein
P5C 0.530 0.397 0.171 1.000
ProDH 0.787 ⁎⁎ 0.793 ⁎⁎ 0.651 ⁎ 0.655 ⁎ 1.000
OAT 0.684 ⁎ 0.605 ⁎ 0.366 0.898 ⁎⁎ 0.793 ⁎⁎ 1.000
P5CS 0.463 0.345 0.171 0.850 ⁎⁎ 0.491 0.699 ⁎ 1.000

Grain
Proline 0.849 ⁎⁎ 0.874 ⁎⁎ 0.796 ⁎⁎ 0.641 ⁎ 0.860 ⁎⁎ 0.698 ⁎ 0.580 ⁎ 1.000
Soluble 0.430 0.187 0.015 0.828 ⁎⁎ 0.434 0.728 ⁎⁎ 0.509 0.419 1.000
protein
P5C 0.768 ⁎⁎ 0.660 ⁎ 0.411 0.910 ⁎⁎ 0.838 ⁎⁎ 0.930 ⁎⁎ 0.692 ⁎ 0.807 ⁎⁎ 0.803 ⁎⁎ 1.000
ProDH 0.786 ⁎⁎ 0.690 ⁎ 0.591 ⁎ 0.711 ⁎⁎ 0.864 ⁎⁎ 0.785 ⁎⁎ 0.430 0.776 ⁎⁎ 0.651 ⁎ 0.853 ⁎⁎ 1.000
OAT 0.775 ⁎⁎ 0.450 0.293 0.596 ⁎ 0.560 0.660 ⁎ 0.489 0.565 0.659 ⁎ 0.722 ⁎⁎ 0.608 ⁎ 1.000
P5CS 0.693 ⁎ 0.574 0.324 0.914 ⁎⁎ 0.722 ⁎⁎ 0.949 ⁎⁎ 0.649 ⁎ 0.687 ⁎ 0.846 ⁎⁎ 0.956 ⁎⁎ 0.843 ⁎⁎ 0.677 ⁎ 1.000

2-AP, 2-acetyl-1-pyrroline; P5C, pyrroline-5-carboxylic acid; ProDH, proline dehydrogenase; OAT, ornithine aminotransferase; P5CS, Δ1
pyrroline-5-carboxylic acid synthetase.
⁎ Significant at P < 0.05.
⁎⁎ Significant at P < 0.01.

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.
Conflict of interest
We declare that we have no financial and personal relation-
ships 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 Technol-
ogy 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.
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