Field Experiments
Experiments were conducted at the experimental station of the China National Hybrid Rice R&D Center, Changsha, China (28°11′59″N, 113°04′35″E) from 2011 to 2016. We used two cultivars of Indica hybrid rice (Oryza sativa L.), LYP9 (Pai64S/9311), super hybrid rice released in China in 1999 and YLY1 (Y58S/9311), a super hybrid rice cultivar released in China in 2006. Seeds were sown on seedbeds in the field after germination. There were 150 hills per plot and 3 plots were planted for each hybrid rice line. Seedlings were transplanted into the field at the 4th leaf stage, i.e. when the 4th leaf is completely expanded. One plant was sown into each hill with a planting density of 29.4 hills m− 2 (0.20 × 0.17 m) in 2011, 2012, and 25 hills m− 2 (0.20 × 020 m) in 2013, 2014, 2015 and 2016. Fertilizer was supplied according to local standard agronomic practice for growing rice: 250 kg N ha− 1, 150 kg P2O5 ha− 1 and 250 kg K2O ha− 1. All of the potassium and phosphorus fertilizer, and 60% of the total nitrogen were applied before transplantation as basal fertilizer. The remaining 40% N was applied as top dressing at the early panicle differentiation stage. Experiments follow a random block design. YLY1 and LYP9 have similar developmental progressions. So, in this study, we applied the same nitrogen fertilizer quantity and also application scheme. The different nitrogen requirements between YLY1 and LYP9 will be studied separately.
Measurement of Leaf Chlorophyll Contents
The chlorophyll contents of leaves were measured with a SPAD-502(Minolta Camera Co. Ltd., Japan). We used the fully expanded leaves and 10 replicates among the three plots were used for the measurements at TS, PDS, MS and YRS in 2014 and 2015, then the SPAD vs actual chlorophyll content calibration was established for calculating actual chlorophyll content of two rice varieties, respectively.
Measurement of Net Photosynthetic CO2 Uptake Rate and Dark Respiration Rate
Net photosynthetic rate (Pn) was measured with a portable photosynthesis system LI-6400XT (LI-COR, Lincoln, NE, USA). The photosynthetic photon flux density (PPFD) used for measurement of Pn were 1000 μmol m− 2 s− 1 at TS, PDS, HFS, MS and DS. The PPFD for measuring Asat (maximum light saturated rate of leaf photosynthesis) were 1600 μmol m− 2 s− 1 at TS, EPDS, LPDS, MS and DS. The Pn and Asat were recorded after leaves were acclimated in a leaf chamber for about 5 min. The leaf temperature during the measurement was maintained at 25 ~ 30 °C. We alternated the cultivars during the measurements to avoid having leaves from one cultivar experience constant high or low temperature. The ambient CO2 concentration was about 380 ppm from atmosphere (in 2011) and controlled to be around 380 ppm in 2012, 2013, 2014, 2015, and 2016. The measurements were conducted between 9:30 to 12:00 on sunny days for the uppermost fully expanded leaves. Dark respiration of flag leaves in YLY1 and LYP9 were measured by Li-6800 at the MS, with 10 replicates among the three plots between 20:00 to 21:30 at night in 2016.
Net Photosynthesis Rate of Leaves in Different Positions on the Main Panicle Stem
To characterize differences of photosynthetic activity in leaves other than the uppermost leaf, we measured Pn of each leaf on the main panicle stem at the milk (6 green leaves) and dough (4 green leaves) stages in 2013. Measurements were made between 9:30 and 12:00 on sunny days.
Light Response Curves
Net photosynthetic CO2 assimilation rate (Pn) was measured at different light intensities from high light to low light levels, i.e. 2000, 1800, 1500, 1200, 1000, 800, 600, 400, 200, 150, 100, 50, 20 and 0 μmol m− 2 s− 1. The CO2 concentration was maintained at 380 ppm. The light response curve was fitted with a rectangular hyperbola (Long et al. 1994) using SPSS13.0 (SPSS Inc., Chicago, USA).
$$ {P}_n=\frac{aI+{P}_{n\max }-\sqrt{{\left( aI+{P}_{n\max}\right)}^2-4\theta {IP}_{n\max }}}{2\theta }-{R}_d $$
(1)
Where Pn is the net photosynthetic CO2 uptake rate, α is apparent quantum yield (AQY), I is photosynthetic photo flux density (PPFD), θ is curve convexity, Pnmax is the maximal Pn, Rd is the dark respiration rate.
Responses of Pn to CO2 Concentration
The responses of Pn under different CO2 concentrations (A/Ci curve) were measured under a saturated PPFD of 1600 μmol m− 2 s− 1. The CO2 concentrations used first decreased from 425 to 50 ppm, i.e. 425, 350, 250, 150, 100, 50, and then set to 425 ppm for at least 15 min, and then increased to 1800 ppm, i.e. 500, 700, 900, 1100, 1300, 1500 and 1800 ppm. To fit the maximal rate of carboxylation at RuBP and CO2 saturation (Vcmax) and light saturated rate of electron transfer (Jmax) in the Farquhar model (Farquhar et al. 1980) (Eq. 2–4), least squared method in Gnumeric software was applied by setting Kc to 404 mbar, Ko to 278 mbar, O to 210 mbar and * to 45 ppm. In the Farquhar model, Wc is RuBISCO limited photosynthesis rate and Wj is RuBP regeneration limited photosynthesis rate. Rd is the dark respiration rate of leaf. Ci is intercellular CO2 concentration and Kc is RuBISCO Michaelis menten constant for CO2 and Ko is RuBISCO Michaelis menton constant for O2. * is the CO2 compensation point in the absence of dark respiration.
$$ {P}_n=\min \left({W}_c,{W}_j\right)-{R}_d $$
(2)
$$ {W}_c={V}_{c\max}\frac{Ci}{Ci+{k}_c\left(1+O/{k}_o\right)} $$
(3)
$$ {W}_j=\frac{J_{\mathrm{max}}\cdot Ci}{4\cdot Ci+8\cdot {\Gamma}^{\ast }} $$
(4)
Leaf Area
Leaf areas of the flag leaf, and the basipetal 2rd, 3rd, and 4th leaves of main stems were measured at the milk stage using a handheld laser leaf area meter (Ci-203, CID, Inc., Vancouver, WA, USA). We also measured lag leaf, and the basipetal 2rd, 3rd leaves from 10 stems among the three plots at TS, PDS, MS and YRS in 2013, 2014 and 2015.
Canopy Photosynthesis
Canopy photosynthesis was measured using a specialized system named as canopy photosynthesis and transpiration system (CAPTS) (MilletHill Biotech, Shanghai, China) contain 10 chambers with a controller. CAPTS is an automatic closed-chamber system and a detailed description of the performance and protocol to use CAPTS was provided in (Song et al. 2016; Song and Zhu 2018). CAPTS was used in previous study (T.-G. Chang et al. 2019) for measuring rice canopy photosynthesis. The size of each chamber is (1*1*1.5) m3 (L*W*H), cover a rice canopy with a ground area of 1 m2. The top of each chamber can be automatically open and close by the controller. The top covers of the chambers were first closed and then open in a consecutive cyclic way. The time duration to open or close the top of the chamber can also be set on the controller. Here, we select 45 s duration to close a chamber for one measurement and the chamber was kept open for 495 s when doing measurement on the other chambers. Once the chamber is in closed form, the gas in the chamber was pumped out by the controller for measuring CO2 concentration [CO2] (logging [CO2] for each second), and then the gas returned back to the chamber. The slope of [CO2] change against time is calculated with a data analyzing software CAPTS Suite that follow linear regression method (Song et al. 2016; Song and Zhu 2018). The CAPTS Suite is a specialized software for analyzing raw data of CAPTS and convert into canopy gas exchange rate.
The Weight of Superior, Middle and Inferior Grains and Grain Filling Rate
A total of 200 panicles that headed on the same day were chosen and tagged. The flowering date and the position of each spikelet on the tagged panicles were recorded in 2015 and 2016. Ten tagged panicles from each plot were sampled at a 5-day interval from anthesis to maturity. The middle grains determined as those locating at the middle of a panicle, the superior and inferior grains were identified following (Zhu et al. 1988). The progression of grain filling was fitted using a modified Richards growth equation (Richards 1959).
Agronomic Parameters
The diameter of stem was measured with a vernier caliper (instrument precision, 0.01 mm) at YRS in 2014 and 2015. The methods for measuring stem diameter follow (Chang et al. 2016) with five replicates for each leaf and stem. The height of rice and length of stem were measured by a ruler with 1 mm scale.
Rice plants from ten hills (for year 2011, 2012, 2014, 2015) were harvested at TS, PDS, MS and YRS for the measurements of agronomic parameters. The above ground biomass was measured after plants were heated at 70 °C from ten hills for the year 2011 and 2012, the leaf, sheath, stem and panicle were weighted after departing from the plants, the ratio of organs dry matter = the organ (leaf, sheath, stem, panicle) weight / the above ground biomass × 100%; the productive tiller percentage = the productive panicle number per hill at yellow ripe stage / the max tiller number at tilling stage × 100%. The grain yield was calculated based on measurements from 50 hills each plot for the year 2013, 2014, 2015 and 2016, with three plots as replicates. The harvest index was measured with a ratio of weight of grain to above ground biomass with ten hill replicates. The grain length and width were evaluated by SC-Grice grain appearance quality image analysis system developed by Hangzhou WSeen Detection Technology Co., Ltd., China (Yin et al. 2015).
Statistical Analysis
Analysis of variance (ANOVA) of Pn data was conducted and significance of mean differences was determined using the LSD method in DPS7.05 (Zhejiang University, China).