Plant materials and experimental design
Rice plants were grown in a paddy field at Songjiang breeding station of the Institutes of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China (30°56' 44" N, 121°8' 1" E) in 2015 and 2016. Seeds were sown on seedbeds after germination on 1 June 2015 and 5 June 2016, and seedlings were transplanted to the field on 26 June 2015 and 29 June 2016. In both years, one plant was transplanted into each hill with a spacing of 20 cm between hills and 20 cm between rows. The cultivars were planted in plots. Plots were arranged in blocks with five replicates, each containing 56 (7 × 8) plants. Basal fertilizer was applied at a rate of 120 kg N ha−1, 65 kg P ha−1 and 65 kg K ha−1. Additional N fertilizer was top-dressed at a rate of 80 kg N ha−1 3 weeks after transplanting. Weeds, pests and diseases were controlled following common agronomic practice in the region. In 2015, five rice cultivars were grown for experiments: two recently bred indica super high yield hybrid cultivars Y-Liang-You 900 (YLY900; http://www.ricedata.cn/variety/varis/614537.htm) and Chao-You 1000 (CY1000; http://www.ricedata.cn/variety/varis/616642.htm), one widely grown traditional indica hybrid cultivar Shan-You 63 (SY63; http://www.ricedata.cn/variety/varis/601174.htm), one indica inbred cultivar Yang-Dao 6 (9311; http://www.ricedata.cn/variety/varis/600611.htm) and one elite japonica inbred cultivar Xiu-Shui 134 (XS134; http://www.ricedata.cn/variety/varis/603176.htm). In 2016, in spite of the above mentioned 5 cultivars, an additional two japonica-indica hybrid cultivars Yong-You 538 (YY538; http://www.ricedata.cn/variety/varis/613563.htm) and Yong-You 17 (YY17; http://www.ricedata.cn/variety/varis/612347.htm) were grown for experiments. In 2015, the heading date and harvest date of main stems for rice cultivars YLY900, CY1000, SY63, 9311 and XS134 were 2 Sep., 2 Sep., 27 Aug., 27. Aug., 6 Sep. and 25 Oct., 25 Oct., 25 Oct., 25 Oct., 5 Nov., respectively. In 2016, the heading date and harvest date of main stems for rice cultivars YLY900, CY1000, SY63, 9311, XS134, YY538 and YY17 were 4 Sep., 4 Sep., 29 Aug., 29. Aug., 7 Sep., 7 Sep., 15 Sep. and 28 Oct., 28 Oct., 28 Oct., 28 Oct., 8 Nov. , 8 Nov. , 8 Nov., respectively.
Construction of the custom-designed panicle chamber
To enable direct and non-intrusive measurement of panicle photosynthesis, we developed a custom-designed panicle photosynthesis measurement chamber (P-chamber), which can be used together with an infrared gas analyzer to measure photosynthetic and respiratory rates of a whole rice panicle. The chamber body is made of aluminum, with cooling fins and cooling fans on both sides (Fig. 1A). The chamber body has a dimension of 30 × 5 × 5 cm. The air inside the chamber was mixed with two air-mixing fans inside the chamber (F2008ES-05WAV, SHICOH IC FAN, Japan), light source and a light intensity control system (Fig. 1A, B). The inner space is uniformly illuminated with 192 light-emitting diodes (LEDs; CREE XML T6, Guangdong Benbon Electrical Co., Ltd) on an 30 × 5 cm Aluminum substrate (Dongxing electronic technology Co., Ltd) for which the photon flux densities can be precisely controlled to span a photon flux density (PFD) from 0 to 2000 μmol m-2 s-1 (Fig. 1B). The PFDs in the P-chamber under different input voltages were calibrated with an irradiance sensor (LightScout Light Sensor Reader, LightScout, Spectrum Technologies, Inc. USA) to build the relationship between input voltage and PFD. During measurement, the P-chamber is connected to a standard Infra-red gas analyzer, and the rice panicle is held upright and fully enclosed in the chamber (Fig. S1). Flow rate and CO2 concentration of influx air are controlled by LI-6400, and the data are recorded and stored in LI-6400.
Panicle photosynthetic gas exchange parameters measurement
Twenty panicles were randomly selected and labeled at heading for each rice cultivar, which were used to measure photosynthetic gas exchange parameters and yield related agronomic traits. Three and five panicles among these twenty were used to measure whole-panicle net photosynthetic rate and dark respiratory rate in 2015 and 2016, respectively. The photon flux density used for each measurement of net photosynthetic rate was 2000 μmol m-2 s-1; the CO2 concentration was set to 400 μmol mol-1, the flow rate was set to 700 μmol s-1; then dark respiratory rate was measured by setting the PFD to 0. Each time when the readings were stabilized, data were logged after matching the two infrared gas analyzers (IRGAs).
The rice cultivar YY17 was used for photosynthetic light and CO2 response curves measurements in 2016 at early grain filling stage. For photosynthetic light response curve measurement, the CO2 concentration was set to 400 μmol mol-1, the flow rate was set to 700 μmol s-1; and the light levels were manually changed to 2000, 1500, 1000, 700, 500, 300, 200, 100, 50, 25 and 0 μmol m- 2 s-1 during measurement, respectively. For photosynthetic CO2 response curve measurement, the light intensity was set to 2000 μmol m-2 s-1, the flow rate was set to 700 μmol s-1; and the CO2 concentrations were manually changed to 400, 300, 150, 100, 50, 400, 600, 900,1200, 1500, 1800 μmol mol-1 during measurement, respectively.
Agronomic traits measurement
Five of the twenty labeled panicles were sampled and stored in envelops individually for each cultivar at harvest. The panicle length was measured, then the panicles were put in an oven for sample drying. The temperature of the oven was maintained at 110 oC for 1 hour, and then switched to 70 oC and maintained for 3 days until samples were completely dried. Dry weight of each panicle was measured with an electronic balance AL104 (JB / T, METTLER TOLEDO, USA). The panicles were then threshed to determine the total spikelet number and filled grain number. The weight of all filled grains were measured to determine the 1000-grain weight.
Data Analysis
Statistical analyses were carried out using R-project (version 3.6.2). One-way analysis of variance (ANOVA) and a Fisher’s least significant difference (LSD) post-hoc test were used to compare data at a level of 5%. The statistically significant p-values were adjusted using Bonferroni’s method. The Pearson correlation coefficients between panicle photosynthetic gas exchange parameters and grain yield related agronomic traits were calculated using the R package (Corrplot; version 0.84).