Performance of the panicle photosynthesis P-chamber
The P-chamber is connected to a gas analyzer Li-6400 (Li-Cor Inc., Lincoln, Nebraska, USA) to measure gas exchange rate of a rice panicle (Fig. 1a, b). The panicle is illuminated with LED light source (Fig. 1c, d). The LED light source emits pure white light (light wavelength information is shown in Fig. 1f). The photon flux density (PFD) is in a range of ±10% of set value on 87% of the total 30 × 5 cm light field (Fig. 1e). At a room temperature of 25 oC with a flow rate of 700 μmol s-1, temperature rise in the chamber at PFD of 2000 μmol (photons) m-2 s-1 is 4.3±0.7 oC.
To test the leakage of the P-chamber, we recorded Δ[CO2] (the difference between CO2 concentration in the P-chamber and the set reference CO2 concentration in the reference pipe) at different time after closure of the P-chamber. With an initial Δ[CO2] value of around 500 μmol mol-1, the Δ[CO2] values were stabilized within 8 min under different flow rates and different CO2 concentrations (Fig. 1g). The final Δ[CO2] values were ≤ 1.6 μmol mol-1 and ≤ 1.3 μmol mol-1 at a flow rate of 500 and 700 μmol s-1, respectively (Fig. 1g). During a typical measurement of panicle photosynthetic rate and respiratory rate, the light source was turned on before the rice panicle was enclosed, and the photosynthetic rate was recorded after photosynthetic rate reading was stabilized; then the light source was turned off, and the respiratory rate was recorded after respiratory rate reading was stabilized (Fig. 1h). A comprehensive measurement for photosynthetic and respiratory rates of a rice panicle usually took 10 ~ 20 min after the panicle was enclosed in the P-chamber, depended on size and photosynthetic activity of the panicle.
Quantification of rice panicle photosynthesis
We first used the P-chamber to measure the in situ whole-panicle photosynthetic gas exchange parameters 5 days after heading for different rice cultivars when the panicles were still erect or semi-erect. Specifically, whole-panicle net photosynthetic rate (panicle Anet) and dark respiratory rate (panicle Rd) were measured; and whole-panicle gross photosynthetic rate (panicle Agross) was calculated as a sum of panicle Anet and panicle Rd. In addition, average spikelet Agross, Anet and Rd were calculated by dividing panicle Agross, Anet and Rd by the spikelet number of a panicle.
Notably, substantial inter-cultivar variations in photosynthetic gas exchange parameters among rice cultivars were found, for both values of a whole panicle and values normalized by spikelet number. For example, the spikelet Anet and panicle Anet were 0.012 ~ 0.051 nmol s-1 and 2.7 ~ 12.0 nmol s-1 in 2015, and were -0.014 ~ 0.075 nmol s-1 and -3.3 ~ 15.0 nmol s-1 in 2016, respectively (Table 1). Panicle Agross varied from 17.1 to 53.6 nmol s-1 under PFD of 2000 μmol (photons) m-2 s-1 for the seven rice cultivars (Table 1). Moreover, indica type rice cultivars (YLY900, CY1000, SY63 and 9311) had an overall significantly higher Agross, Anet and Rd than japonica and japonica-indica hybrid type rice cultivars (XS134, YY538 and YY17), both on whole panicle basis and on single spikelet basis (Fig. 2).
In 2016, we further measured area of panicles and area of the corresponding flag leaves, and then quantified Agross, Anet and Rd on an area basis by dividing panicle Agross, Anet and Rd by the area of a panicle (Fig. S1; see detailed procedure used to calculate panicle surface area in Materials and Methods). As a result, we found spikelets contributed the majority of the panicle area, whereas branches only accounted for 24 ~ 33% of the panicle area; moreover, total area of a panicle (single side) was 72 ~ 121 cm2, which was 20 ~ 124% higher than that of a flag leaf (Table 2). On an area basis, Anet of panicles were very low, i.e., only -0.6 ~ 2.2 μmol m-2 s-1, under a PFD of 2000 μmol (photons) m-2 s-1; whereas Agross were 2.1 ~ 7.2 μmol m-2 s-1, much higher than Anet, as a result of high Rd, i.e., 2.1 ~ 6.1 μmol m-2 s-1 (Table 3).
Given these different representations of gas exchange rates, i.e., on a panicle basis, on a spikelet basis and on an area basis, what’s the relations between them? Positive correlations between values on different bases were found for all the three parameters Agross, Anet and Rd (the Pearson correlation coefficient r=0.59 ~ 0.99; Fig. 3a-i). Interestingly, we found that Anet per panicle was strongly positively correlated with Anet per unit panicle area (the Pearson correlation coefficient r=0.97; Fig. 3e), in contrast to that for Agross and Rd (the Pearson correlation coefficient r=0.77 and 0.59, respectively; Fig. 3b, h). In addition, spikelet based Agross, Anet and Rd were closely related to area based Agross, Anet and Rd (the Pearson correlation coefficient r=0.97-0.99; Fig. 3c, f, i), as a result of a tight relationship between panicle area and spikelet number (the Pearson correlation coefficient r=0.95; Fig. 4).
Characterization of rice panicle photosynthesis
To further characterize rice panicle photosynthesis, we measured dynamic changes of panicle Agross and Rd for seven rice cultivars during grain filling in 2016 (Fig. 5). Firstly, panicle Agross and Rd were highest at early grain filling stage, then slowly decreased over time. Notably, both Agross and Rd could still be detected even 40 days after heading, i.e., panicle photosynthesis and respiration were active throughout the grain-filling season. It is worth mentioning here that the panicle Agross should be interpreted as the maximal photosynthetic potential, as the panicle was held upright during measurement, which was not the natural state after early grain filling stage for some cultivars.
We further measured the photosynthetic light and CO2 response patterns of a panicle using rice cultivar YY17. Although the shapes of both response curves are similar to that of a leaf, there are several differences. Firstly, the apparent CO2 and light compensation points of a panicle were about 180 μmol mol-1 and 390 μmol m-2 s-1 (Fig. 6), much higher than those of a leaf, which are usually <100 μmol mol-1 and <50 μmol m-2 s-1, respectively. Secondly, the panicle photosynthetic rate was still not saturated at very high air CO2 concentration (1800 μmol mol-1; Fig. 6a), which also differed from that of a typical leaf.
Correlation between panicle photosynthetic gas exchange parameters and grain yield related agronomic traits
Finally, we studied the relationship between panicle photosynthetic gas-exchange parameters measured 5 days after heading (Table 1) and six grain yield related agronomic traits collected at harvest across two years, i.e., spikelet number per panicle, panicle length, spikelet density (defined as spikelet number per centimeter of panicle length), 1000-grain weight, grain setting rate and panicle weight (Table 4). Firstly, there were positive correlations between different panicle photosynthetic gas-exchange parameters (Fig. 7). For example, significant positive correlations were found between Agross and Anet of a whole panicle (Pearson correlation coefficient r = 0.87, p-value < 0.001), between Agross and Rd of a whole panicle (r = 0.94, p-value < 0.0001), between Agross and Anet per spikelet (r = 0.93, p-value < 0.0001), and between Agross and Rd per spikelet (r = 0.97, p-value < 0.0001). In contrast, both positive and negative correlations were found between grain yield related agronomic traits, e.g. significant negative correlations were found between 1000-grain weight and spikelet number per panicle, but significant positive correlations were found between 1000-grain weight and grain setting rate (Fig. 7). Notably, strong positive correlations were found between Agross, Anet, Rd per spikelet and grain setting rate, 1000-grain weight. In particular, the Pearson correlation coefficient r is 0.93 (p-value < 0.0001) between Agross per spikelet and grain setting rate (Fig. 7). Concurrently, we found that Agross, Anet and Rd of a whole panicle had a moderate positive correlation with panicle dry weight at harvest (r = 0.39 ~ 0.55); whereas Agross, Anet and Rd per spikelet were not correlated with panicle dry weight at harvest (r = -0.18 ~ 0.12). In addition, all the six panicle photosynthetic gas exchange parameters were found to be positively correlated with panicle length (r = 0.6 ~ 0.85), whereas they were negatively correlated with spikelet density (r = -0.82 ~ -0.28). Finally, no correlations were found between Agross, Anet, Rd of a whole panicle and spikelet number per panicle (r = -0.2 ~ 0.1).