Multi-year phenotypic identification of yield and taste quality of different panicle types
To investigate the effects of Ep on the grain yield and taste quality under different N fertilizer treatments, we set two N fertilizer treatments as low (L) and high (H) respectively. The state of plants under high nitrogen condition was shown in Fig. 2A. The yield and taste quality traits of tested materials in 4 years were investigated as shown in Fig 1. Under L treatment, LG5 yield had no significant difference with AKI yield in 2020 (Fig. 1C), while LG5 yield was significantly higher than AKI yield in 2018, 2019, and 2021 (Fig. 1A, B and D). However, no significant difference was observed in near-isogenic line materials in L treatment (Fig. 1E-H). LG5 yield in H treatment was significantly higher than AKI in 4 years (Fig. 1A-D), near-isogenic lines showed the same pattern as their parents (Fig. 1E-H).
As for taste quality, in L treatment, there was no significant difference of LG5 and AKI. While in H treatment, the taste quality of LG5 was significantly lower than that of AKI, and AKI did not decrease with the increase of N fertilizer (Fig. 1I-L). The taste quality of near-isogenic lines showed the same pattern as that of their parents under the same treatment. However, compared with L treatment, under H treatment NIL-non Ep also showed a significant decrease in taste quality (Fig. 1M-P).
Yield and yield components
There was no significant difference in yield under L treatment, but there was a significant difference in yield and its components in H treatment (Table 1). Under different treatments, the yield and yield component traits of NILs were consistent with their parents AKI and LG5. The average PNP and GNP in NIL-Ep was 19.9% and 29.5% higher than that of NIL-non Ep (Fig. 2B). In order to clarify the sources of differences in GNP, we divided rice panicles into 24 parts from 1-1 to 12-2 according to the origin positions of branches, the results showed that the significant increase of SGN from 5th spike to 12th spike explained the difference in GNP (Fig. 2D). Subsequently the panicle is divided into 3 parts namely top (top)(from 1-1 to 4-2 panicle locations), middle (mid)(from 5-1 to 8-2 panicle locations) and bottom (bot) (from 9-1 to 12-2 panicle locations) respectively. Results demonstrated that compared with the NIL-non Ep, grain number significant increased mainly in mid to bot locations (Fig. 2E). The panicle weight ratio of each part was changed, and the panicle weight ratio in the mid to bot part increased by 21.5 % and 18.7 % (Fig. 2F). Although Ep showed a low TWG because grain length decreased significantly (Fig. 2C, G), the extremely significant increase of GNP and PNP was the key factor leading to the significant increase of yield, the contribution rates of the three factors were -7.1%, 29.5% and 19.9% respectively.
Table 1 Performance of grain yield related traits for LG5, AKI, NIL-Ep, NIL-non Ep.
|
Treatment
|
Panicle type
|
HD
|
PH(cm)
|
PL(cm)
|
PNP
|
GNP
|
FGN
|
PBN
|
PGN
|
SBN
|
SGN
|
TWG(g)
|
GY(kg/hm2)
|
L
|
LG5
|
115
|
109.82±2.91
|
14.97±0.63
|
448.10±32.71
|
89.50±2.74
|
76.50±2.74
|
10.50±1.05
|
57.67±2.42
|
11.33±1.21
|
31.83±2.64
|
25.21±1.12
|
7446.67±310.05
|
AKI
|
114
|
118.77±1.37
|
17.22±0.86
|
433.29±36.51
|
90.83±6.49
|
78.17±10.76
|
10.67±0.82
|
59.33±4.18
|
11.67±1.75
|
31.50±4.59
|
26.53±0.23
|
7646.67±215.72
|
p
|
n.s.
|
*
|
*
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
*
|
n.s.
|
NIL-Ep
|
110
|
92.58±0.90
|
13.74±0.55
|
474.46±39.23
|
92.94±5.80
|
79.94±5.80
|
10.71±1.21
|
61.53±6.25
|
11.53±0.87
|
31.41±3.55
|
23.61±0.46
|
8106.67±292.97
|
NIL-non Ep
|
111
|
118.29±2.04
|
16.87±1.01
|
464.75±28.82
|
90.50±6.62
|
79.00±11.45
|
10.69±1.08
|
60.25±5.37
|
11.19±1.64
|
30.25±4.01
|
26.32±0.21
|
8116.67±363.64
|
p
|
n.s.
|
***
|
**
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
**
|
n.s.
|
H
|
LG5
|
118
|
111.52±1.43
|
15.82±0.48
|
497.73±30.14
|
147.00±6.42
|
126.00±6.51
|
12.60±1.02
|
71.40±3.61
|
25.40±1.62
|
75.60±3.26
|
24.24±0.14
|
11523.33±322.94
|
AKI
|
116
|
127.43±4.27
|
18.25±0.86
|
427.41±44.11
|
120.76±9.32
|
108.71±12.45
|
12.00±0.61
|
68.71±3.85
|
18.41±2.94
|
52.06±10.47
|
25.24±0.37
|
7870.00±315.75
|
p
|
n.s.
|
***
|
**
|
**
|
***
|
***
|
n.s.
|
n.s.
|
***
|
***
|
*
|
***
|
NIL-Ep
|
113
|
94.24±1.83
|
16.28±0.45
|
524.39±25.33
|
167.60±5.50
|
141.80±6.26
|
12.60±0.55
|
60.60±3.58
|
29.00±2.00
|
107.00±4.85
|
23.22±0.13
|
12145.33±947.07
|
NIL-non Ep
|
114
|
115.22±4.80
|
19.16±0.96
|
437.08±24.36
|
129.47±6.22
|
114.53±7.62
|
12.06±0.90
|
69.41±2.76
|
19.76±2.49
|
60.06±5.80
|
24.99±0.20
|
8076.67±285.72
|
p
|
n.s.
|
***
|
**
|
***
|
***
|
***
|
n.s.
|
**
|
***
|
***
|
**
|
***
|
HD, heading date; PH, plant height; PL, panicle length; PNP, panicle number per square meter; GNP, grain number per panicle; FGN, filled grain number per panicle; PBN, primary branches; PGN, primary grain number; SBN, secondary branches number; SGN, secondary grain number; TGW, thousand-grain weight; GY, grain yield . The *, ** and ***denote significance of Student's t test at p < .05, p < .01, and p < .001, respectively.
|
Eating quality and protein content in different panicle positions
The EQ is a complex sensory trait affected by the hardness, viscosity, elasticity and other indicators of rice. In order to accurately and objectively measure the EQ of the tested materials, we adopted two sets of evaluation systems: artificial tasting and machine evaluation.The two evaluation systems showed the same results.Under L treatment, there was no significant difference in the EQ between near isogenic lines, and the eating quality of NIL-Ep was significantly lower than that of NIL-non Ep under H treatment (Fig. 3A). The texture of rice showed that the hardness increased significantly while the viscosity and elasticity decreased significantly (Fig. 3E-G).
Then we identified the EQ of the top, mid and bot parts, and the results showed that the EQ of the mid to the bot part of NIL-Ep was significantly decreased, which was the key factor affecting the overall EQ (Fig. 3B). For RVA characteristics, mid and bot locations exhibited significantly lower breakdown, higher final viscosity and setback values than those of NIL-Ep (Additional file 1: Table S1). Previous studies demonstrated that rice with high palatability has a higher breakdown and a lower final viscosity and setback than low-palatable varieties (Ma et al, 2017). Therefore, it also proves that the eating quality of mid-bot position grains is lower compared with NIL-non Ep.
Starch and protein account for 70–80 and 7–10% of the components in the rice endosperm, respectively, and were considered to be the main factors that affecting eating quality (Chen et al. 2021). Therefore, the amylose content and protein components of different panicle locations in H treatment were detected to analyze the key factors causing the decline of eating quality. The results showed that there was no significant difference in amylose content among different panicle positions, but there was a significant difference in nitrogen content of mid and bot grains, which was significantly higher than that of NIL-non Ep (Fig. 3C, D).
Then we tested the protein content of 24 parts of panicle, and the results were shown that under H treatment, the grain protein content in the mid-bot panicle of NIL-EP was significantly higher than top part, while there was no significant difference among panicle sites of NIL-non Ep (Fig. 3 H, I). Subsequent analysis of the protein components showed that the difference in nitrogen accumulation was due to the significant increase in the prolamint and glutelin contents (Table 2).
Table 2 Comparison of protein content traits for NIL-Ep and NIL-non Ep in different positions of panicle
|
Locus
|
Panicle type
|
Accumulation amount(mg grain-1)
|
Relative content(%)
|
ALB
|
GLO
|
PRO
|
GLU
|
ALB
|
GLO
|
PRO
|
GLU
|
Total protein
|
TOP
|
NIL-Ep
|
0.196±0.010
|
0.225±0.009
|
0.099±0.003
|
1.440±0.101
|
0.75±0.04
|
0.85±0.04
|
0.38±0.02
|
5.48±0.27
|
7.45±0.37
|
NIL-non Ep
|
0.193±0.015
|
0.227±0.011
|
0.105±0.005
|
1.453±0.073
|
0.71±0.02
|
0.83±0.02
|
0.38±0.01
|
5.30±0.16
|
7.22±0.22
|
p
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
*
|
n.s.
|
n.s.
|
n.s.
|
n.s.
|
MID
|
NIL-Ep
|
0.182±0.013
|
0.214±0.017
|
0.154±0.005
|
1.935±0.116
|
0.70±0.03
|
0.81±0.04
|
0.59±0.03
|
7.64±0.38
|
9.75±0.69
|
NIL-non Ep
|
0.184±0.009
|
0.219±0.011
|
0.092±0.006
|
1.426±0.057
|
0.68±0.02
|
0.80±0.02
|
0.34±0.01
|
5.29±0.16
|
7.11±0.21
|
p
|
n.s.
|
n.s.
|
**
|
**
|
n.s.
|
n.s.
|
***
|
***
|
**
|
BOT
|
NIL-Ep
|
0.184±0.015
|
0.219±0.018
|
0.130±0.010
|
1.677±0.134
|
0.72±0.06
|
0.83±0.07
|
0.51±0.04
|
6.61±0.53
|
8.68±0.49
|
NIL-non Ep
|
0.197±0.010
|
0.228±0.011
|
0.105±0.005
|
1.410±0.070
|
0.72±0.04
|
0.83±0.04
|
0.39±0.02
|
5.18±0.26
|
7.12±0.36
|
p
|
n.s.
|
n.s.
|
**
|
**
|
n.s.
|
n.s.
|
**
|
**
|
**
|
ALB, Albumin; GLO,Globulin; PRO, Prolamint; GLU, Glutelin. The *, ** and ***denote significance of Student's t test at p < .05, p < .01, and p < .001, respectively.
|
|
|
|
Nitrogen use efficiency and grain protein accumulation
Previous studies have shown that dep1 is considered the major gene controlling nitrogen-use efficiency. In the two years of repeated experiments (Supplementary file 2: Table S2), under the high nitrogen condition, the yield of Ep increased by 30.6 and 50.4% than non Ep in 2019 and 2020. In H nitrogen treatment, the nitrogen recovery efficiency and physiological nitrogen use efficiency were significant higher compare with non Ep. There was no significant difference in the yield and nitrogen use efficiency under L nitrogen treatment.
Then we examined the nitrogen transport in different organs of harvest stage under the condition of high nitrogen. The results showed that the nitrogen accumulation of NIL-Ep was significantly higher than that of NIL-non Ep in all organs (Fig. 4A, F, H, J). So that the total nitrogen accumulation increased significantly in the harvest period (Fig. 4O). From booting to full heading stage, nitrogen content in leaves still maintained an upward trend in NIL-Ep, while in NIL-non Ep, this phenomenon only occurred in flag leaves and 2nd leaves. In addition, it is interesting that 80-100 days after transplanting, the leaf nitrogen content of NIL-Ep showed a sharp downward trend (Fig. 4B-E), and the same trend also appeared in stem and sheath organs (Fig. 4G, I). We further analyzed the dynamic changes of the glutelin and prolamint content of the two genotypes during grain filling stage, and the results showed that there were significant differences in the contents of two protein components between different panicle locations in NIL-Ep compared with NIL-non Ep (Fig. 4K-N).
Nitrogen metabolism processes are in fact a series of reactions including inorganic nitrogen and organic nitrogen inter-conversion and protein biosynthesis and are highly regulated by both genetic and environmental factors. Many enzymes involve to catalyze these reactions, including glutamine synthetase (GS), glutamate synthase (GOGAT), asparagine synthetase (AS), glutamate dehydrogenase (GDH), etc., and all of them are well-known to play key roles in the regulation of nitrogen metabolism. We also measured the activities of enzymes encoded by these genes of the two genotypes during grain filling. As shown in Fig. 4P-S, significant differences in GS, NADH-GAGOT activity between two genotypes occurred at the whole grain filling stages, and the activity of AS and GDH increased significantly in 4-28 and 22-44 day after flowing. Origin of nitrogen in grains from various organs are shown that the ability of NIL-Ep to absorb nitrogen was significantly higher than that of NIL-non Ep in filling stage under high nitrogen condition (Fig. 4T).