Phosphate fertilizers increase CO2 assimilation and yield of soybean plants in a shaded environment

To study how phosphate fertilizer regulates the photosynthetic of summer soybeans in low light environment, this study used Qihuang 34 as the test variety, two light treatments[ambient sunlight (L1) during the whole growth period and 40% shade (L2) from 40 days after sowing to 24 days after owering] with two phosphate fertilizer treatments including non-phosphate fertilizer application (P0), conventional phosphate fertilizer application (P1) in each light treatment was set up to measure the gas exchange, chlorophyll a uorescence differences of photosynthetic performance as well as the yield and its components in Qihuang 34. The results showed that phosphorus signicantly increased chlorophyll content, photosynthetic rate, and grain yield in shading treatment. The yield of soybean was signicantly reduced after shading treatment, which was 29.9%, but compared with L2P0, L2P1 treatment yield increased by 36.7%, which was signicantly higher than the 27.1% under L1 treatment. The photosynthetic rate under saturated light (Asat) under L2 treatment decreased by 31.5% and 15.6% in R1 and R3 periods, respectively, and average increased by 27.4% and 45% after phosphorus application under two light condition. Furthermore, photosynthetic measurements demonstrated a more signicant increase in the maximum eciency of CO2 xation, maximum rates of RUBP-carboxylation (Vcmax) in phosphorous treatment under shading conditions. Moreover, further analysis of OJIP curves revealed that phosphate fertilizer signicantly improved the electron transfer and light energy absorption performance of the PSII reaction center in shading. The above results demonstrate that phosphate fertilizer contributes to light energy utilization eciency, increase the low light utilization eciency by improving PSII performance, promote RUBP regeneration, ensure the source of carboxylates substrates, and coordinate the balance of the photochemical reaction and the Calvin cycle, then alleviate the reduction of photosynthetic eciency under shading, increase the rate of dry matter accumulation, and greatly increase yield.


Introduction
Soybean, as both a grain and oil crop, is one of the most important crops in the world, which native to China. Many factors are affecting the scale of soybean production, of which the low yield is one of the most prominent factors. Photosynthesis is the basis of crop growth and yield formation. As one of the most important environmental factors in agricultural production [1] , light is not only the driving force of, but also affects the structure and function of photosynthetic mechanisms [2,3] . In the past half-century, the decrease of sunshine hours and solar radiation of crops have shown a persistent decline trend, due to the increase in aerosol concentration caused by human activities and the associated light fog and low clouds, especially in the North China Plain (June-October), severely restricts soybean production [4] .
Photosynthesis is a complex biochemical and biophysical process that comprises photosynthetic pigment synthesis, light energy electron transport, and the Calvin cycle [5,6] , these processes are all affected by the light intensity [7] . Leaf area, chlorophyll content, and antenna pigment-protein under weak light environment improve the light interception and the absorption e ciency [8,9] , but the decrease of light conversion and electron transfer e ciency [10,11] as well as a signi cant decrease in expression level and activity of photosynthetic carbon immobilized enzyme [12][13][14] resulting in the inevitable reduction of photosynthetic capacity under weak light. Increasing nutrients is one of the important ways for crops to improve the use of low light [15] . Phosphorus has the effect of regulating the stability of thylakoid membranes and the activity of photosynthetic proteins penetrating inside and outside the membrane [16,17] , thereby affecting photosynthetic electron transport, photosynthetic phosphorylation, Calvin cycle, assimilation synthesis, and transport e ciency [18] , and then regulate crop photosynthesis. Soybeans are sensitive to phosphorus and require a large amount of phosphorus. Reasonable application of phosphorus fertilizer is an important measure to regulate soybean growth and yield formation [19] .
Previous reports mostly focused on light intensity or phosphorus as a single factor to increase photosynthetic capacity and physiological mechanisms in increased yield [18][19][20] . According to eld test results, there is a limited linear relationship between the amount of phosphorus application and photosynthetic rate. When the amount of phosphorus applied exceeds a certain ratio, the photosynthesis decreases rather than increases [21] , which shows that although the resource attributes of light and phosphorus are inconsistent, there is a certain complementary effect between them. There are few reports on the correlation between whether phosphorus applied or not and the relative inferiority e ciency of utilization of weak light in shading, the complementary mechanism still uncertain. The consequence of phosphate fertilizers compensate for the decrease in light intensity and an increase in photosynthesis needs to be further clari ed. In this regard, we explored the complementary effects between phosphorus and light intensity under different light environments, as well as the connection and mechanism between phosphate fertilizer application and low light utilization e ciency, to innovate the agronomic production model for crops to e ciently use low light.

Field location
A eld experiment was conducted at the Agronomy Station of Shandong Agricultural University in 2018, in Taian City, Shandong Province (117°09′E, 36°09′N). The region receives adequate light and has a suitable temperature, and rain and heat occur within the same season. The precipitation and atmospheric temperature dynamics of 2019 are shown in Figure 1. The major initial properties within the 0-20 cm soil depth were as follows: 12.53 g kg −1 SOC (an Elementar Vario TOC), 1.15 g kg −1 STN (the semi-micro Kjeldahl method), 11.27 mg kg −1 Olsen P (sodium bicarbonate-molybdenum antimony anti-reagent colorimetric method), 80.9 mg kg −1 exchangeable K (NH 4 OAc extraction-ame photometer method) and a pH (calcium chloride solution extraction-potentiometric method) of 6.8.

Experimental design
Two treatments (L1: ambient sunlight; L2: 40% shade) were arranged in the eld. Two light levels (L1: ambient sunlight; L2: 40% shade) and two phosphate levels (P1: 0 kg P 2 O 5 ha -1 ; P2:180 kg P 2 O 5 ha -1 ) were arranged in the eld with a randomized block design. Shade treatment was applied at 40 days after planting and remained until 64 days after planting. Each plot had an area of 35 m 2 (5m×7m), and three replicates were included in the experiment. Qihuang 34 was selected as the plant material in this study with a planting density of 120,000 plant ha -1 , which was seeded on June 17, 2018, and harvested on October 15, 2018. N and K were applied with 300 kg ha −1 urea (N content was 46.7%), 300 kg ha −1 potassium sulfate (K 2 O content was 50.0%), and calcium superphosphate, P 2 O 5 content was 12%, is used as P fertilizer. P and K fertilizer were applied as a base fertilizer. Half of the N fertilizer at sowing and the other half was broadcast at the beginning pod stage. Maintain a water supply throughout the growth period. Disease, weeds, and pests were well controlled in each treatment.

Crop yield
Upon maturity, constantly collected 30 plants in each treatment with three replicates. The samples were air-dried and threshed, and then weighed to calculate the yield at 13% moisture content.

Chlorophyll a and b measurement
Chlorophyll a and b were extracted by 80% acetone from the leaves of the soybean (fully expanded, exposed) at a similar position for each treatment group. The absorbance of the extract s was measured with a UV-2450 spectrophotometer (Shimadzu Suzhou instruments mfg. co., Ltd, China) at 663 nm (A663), 646 nm (A646), respectively. Chlorophyll a and b were calculated using equations established by Lichtenthaler and Wellburn [22] : chl a (mg mL -1 ) = 12.21A663-2.81A646 chl b (mg mL -1 ) = 20.13A646-5.03A663

Gas exchange measurement
Gas exchange measurements were performed using a portable gas exchange system (CIRAS-3, PPSystem Ltd, Ayrshire, UK). Measurements were made on the inverse fourth leaf of beginning owering (R1), beginning pod (R3), and beginning seed (R5) plants. Responses of net CO 2 assimilation rate (A) to intercellular CO 2 concentration (C i ) were measured at a leaf temperature of 25ºC, and a light intensity of 1400 µmol m -2 s -1 . Plants were acclimated to these conditions until steady-state gas exchange was reached (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)  was to allow the plant to re-acclimate to this condition after being exposed to low CO 2 concentrations.
Data points were taken in sequential order with an equilibration time of 180-300 seconds at each CO 2 concentration. A sat was measured on the inverse fourth leaf at R1, R3, R5, at a CO 2 concentration of 400 µmol mol -1 , a leaf temperature of 25ºC, and a light intensity of 1400 µmol m -2 s -1 .

Photosynthetic light response curves
A/Q response curves were performed using a portable gas exchange system (CIRAS-3, PP Systems USA). Measurements were made on the inverse fourth leaf of beginning owering (R1), beginning pod (R3), and beginning seed (R5) plants. The response of the net CO 2 assimilation rate (A) to light intensity (Q) was measured at a leaf temperature of 25ºC, the relative humidity of 50-60%, and a CO 2 concentration of 400 µmol mol-1. Leaves were initially stabilized at saturating irradiance 2000 µmol m -2 s -1 , after which A was measured at the following PPFD levels; 50, 100, 150, 200, 400, 800, 1200, 1600, 2000 µmol m -2 s -1 .
Measurements were recorded after A reached a new steady-state (1-2 min) before changing to the new light levels.

Chlorophyll uorescence
Chlorophyll uorescence measurements were performed using a Handy PEA uorometer (Hansatech Instruments Ltd., King's Lynn, Norfolk, Great Britain). The leaves were dark-adapted using leaf clips for 20 min. The dark-adapted leaf samples of 4 mm diameter within each clip were illuminated with 660 nm light of 3500 μmol m −2 s −1 for 1 s. Descriptions and equations for calculating JIP-test parameters are explained in Table 1.
The approximated initial slope of the fluorescence transient The probability that a trapped exciton moves an electron into the electron transport chain beyond Q A increased signi cantly with the application of phosphate fertilizer, and its increase was signi cantly higher than that under full light conditions. Phosphate fertilizer can promote the owering and pod formation of soybean, which increases soybean yield via promoting the number of effective pods per plant and the number of seeds per plant.

Phosphorus fertilizer increases chlorophyll content of plant leaves under low light
As shown in Figure 2, the content of chlorophyll a and b at each growth stage after anthesis increased signi cantly with the decrease of light intensity and the application of phosphate fertilizer. To further explore the reasons for the signi cant increase in the A sat of the L2P1 treatment during the R3 period, CO 2 assimilation rates were determined as a function of light intensity. From these light response curves the CO 2 assimilation rates were shown to be signi cantly lower in L2 treatment when compared to L1 treatment, showing a trend of L1P1> L1P0> L2P1> L2P0, but as the shading treatment time increased, the CO 2 assimilation rate of the L2P1 treatment gradually increased, and showed to be L1P1> L2P1> L1P0 > L2P0 (Figure 4), and the CO 2 assimilation rate of L2P1 remains at a high level after re-lighting.
We focused on the response of the CO 2 assimilation rate to low light intensity (≤200 mmol m -2 s -1 ) to make a thorough probe of change in apparent quantum yield (PQY) during low light. The initial slope of the A / Q curve re ects the plant's weak light utilize the ability. The PQY of plants under weak light shows a signi cant upward trend, especially with L2P1 treatment. Figure 5 compares the photosynthetic capacity unit light intensity, the weak light (100 mmol m -2 s -1 ) utilization capacity of plants in a shading environment was signi cantly higher than that in the full light environment. As the adaptability to weak light improves, its high light intensity (800 mmol m -2 s -1 ) utilization capacity also increased synchronously. The low light utilization capacity of L2P1 plants after relighting maintaining high, indicating that phosphorus application under shading makes the plants quickly adapt to low light environments while ensuring the restoration of photosynthetic capacity of plants after re-lighting, ensure the accumulation of photosynthetic products, and signi cantly increase yield.

Phosphate fertilizer increases the rate of CO 2 assimilation in shading
To explore the changes in the carboxylation system under different treatments, we also determined A as a function of internal CO 2 concentration (C i ) in the same plants. The CO 2 assimilation rate of L2P0 and L2P1 decreased signi cantly after shade treatment. As the plant's adaptability increased, the CO 2 assimilation rate of L2P1 increased signi cantly during the R3 period, which maintaining a high level compared to L2P0 treatment when re-lighting at R5 (Figure 6).

Further analysis of the A/C i curves using the equations published by von Caemmerer and Farquhar
(1981) [23] illustrated that the light environment signi cantly affects the maximum rate of carboxylation by Rubisco (V cmax ) and maximum electron transport rates (J max ). As shown in gure 7, V cmax and J max in the natural light environment were signi cantly higher than in the shading environment, showing to be L1P1> L1P0> L2P1> L2P1, except for the R5 period. V cmax and J max respond differently to phosphate fertilizer under two light conditions. periods under L1 treatment, and 38.8% and 32.3% in L2 treatment, respectively. It can be seen that the application of phosphate fertilizer signi cantly improves the regeneration rate of RUBP in shading.

Chlorophyll uorescence analysis reveals increased photosynthetic e ciency in plants in shading
Variable uorescence curves and DV t [For ΔV t analysis (ΔV t =D(F t -F o )/ (F m − F o )) uorescence in L1P0 treatment on each day of the experiment (was a used as a reference and) equaled 0.] were constructed to compare differences between treatments on each measuring day during the experiment ( Figure 8). Compared with plants under ambient light, where the K and J-bands showed signi cantly higher values for both shading treatments, indicating that the PSII donor side and acceptor side were reduced to different degrees in shading and it has alleviated during the R3 period. J-bands were relatively low with phosphorus treatment, especially in the shade environment, which means phosphate fertilizer can alleviate the effect of low light on the performance of the leaf receptor. Figure 9 shows the change of the relative variable uorescence intensity at points K and J of chlorophyll a uorescence transient (OJIP) curves. After shade treatment, W k and V j both increased, especially V j , indicating that the performance of the donor side and the receptor side was reduced to varying degrees, and the receptor side was signi cantly affected by the low light. W k and V j of phosphorus-applied plants showed a downward trend in two light treatments. The effect of phosphate fertilizer on W k was not signi cant, but V j was signi cantly reduced. After re-lighting, the V j of L2P1 was signi cantly lower than that of L2P0 treatment, but not signi cantly different from that of L1P1 treatment. The above results indicate that phosphorus application improves the electron transport performance on the receptor side in shading stress, and is bene cial to the retention of the receptor side performance after the plant was relighted.
The analysis of the relative variable uorescence at points K and J of the OJIP curve found that both light and phosphate fertilizer had a signi cant effect on V j . The change in the relative uorescence at point J is closely related to the electron transport performance of the PSII acceptor side, so we studied the maximum quantum yield of PSII primary photochemistry (F v /F m ), the probability that a trapped exciton moves an electron into the electron transport chain beyond QA -(y o ), the quantum yield of electron transport (j Eo ) and performance index on absorption basis (PI ABS ). Figure 10 shows that F v /F m , y o , j Eo , and PI ABS all decreased signi cantly after shading. With the improvement of the plant's low light adaptability, the photosynthetic mechanism changed adaptively.
During the R3 period, P1 F v /F m , y o , j Eo and PI ABS increased by 0.9% 3.4% 9.9%, and 20.5% in L1 treatment compared to P0, and which were1.5%, 6.4%, 48.5%, and 69.7% under L2, respectively. Phosphate fertilizer can strengthen PSII performance, especially under low light. Compared with other treatments, L2P1 has the least decreased in the R5 period after re-lighting, which was not signi cantly different from the L1P1 treatment, indicating that phosphorus application bene ts maintain the performance of the photosynthetic system after relighting, ensuring that the absorbed light energy is fully used to promote electron transfer, effectively improve the soybean photosynthetic capacity in the later period.

Discussion
Photosynthesis is the basis of crop growth and yield formation, which is susceptible to light intensity [25] and decreases signi cantly under low light [3] . By increasing the chlorophyll content, the plants can improve the light absorption ability of chloroplasts [26] , make full use of weak light, and adapt to it. In this experiment, the chlorophyll a (chl a) and chlorophyll b (chl b) in the leaves increased signi cantly after shading (Figure 2), but the A sat decreased signi cantly, indicating that the increase in chlorophyll was insu cient to compensate for the photosynthetic rate caused by the decrease in light intensity. However, the leaves chl a, chl b, and A sat all increased signi cantly in P1 treatment, especially in the shading environment (L2), indicating that the plants increased the photosynthetic rate of the leaves by increasing the light energy capture capacity (Figure 3). After a long period of shading treatment, the plant has an adaptive response. The initial slope of the A/Q curve (PQY) is considered to be a symbol of the plant's low light usability. Under shading treatment, the PQY of the plant was signi cantly higher than that of the ambient light environment, and the phosphorus application was signi cant to improve its weak light utilization ability, the PQY of L2P1 treatment was signi cantly higher than that of other treatments in the R3 period (Figure 4), and the plant's light energy utilization e ciency (P n /PAR) increased synchronously (including PAR=100 mmol m -2 s -1 and PAR=800 mmol m -2 s -1 ) ( Figure 5), which shows that the reasonable application of phosphorus in the shading environment can slow down the reduction of the CO 2 assimilation rate, and quickly improve the plant's adaptability to shading environment simultaneously and make full use of light energy to produce photosynthetic products, thereby signi cantly increasing production. Compared with P0, the output of P1 treatment under shading increased by 36.7% (Table 2), but due to the restricted of light, the phosphate fertilizer can only partially alleviate but not make up for the decrease of CO 2 assimilation rate and product caused by insu cient light energy.
One question we aimed to address was whether the high CO 2 assimilation rates observed in soybean under shading condition is a consequence of the signi cant improvement of the photosynthetic system or the activity of the related reaction process of the Calvin-Benson cycle. Related studies have shown that C3 cycle-related enzymes are notably affected by light intensity [12,14] . As the most abundant enzyme in plants, the proportion of activated Rubisco in C3 plants is about 25% [27,28] , the content may be excessive, so the reduction of Rubisco carboxylation rate can be compensated by rapid activation. FBPA and SBPase are the key enzymes in the C3 cycle for RUBP regeneration, small changes in its content will have a signi cant impact on CO 2 assimilation, and the activity is affected by the light intensity [29][30] . The activity of FBPA and SBPase signi cantly decreased in the low light, affecting RUBP regeneration, reducing the source of the Rubisco carboxylation substrate [31] . It can be seen that the carboxylation e ciency was reduced after shade treatment from the A/C i curve, and it is effectively relieved in phosphorus application treatment ( Figure 6). Further analysis showed that Rubisco's maximum carboxylation e ciency (V cmax ) and RUBP maximum regeneration rate (J max ) decreased signi cantly with the decrease of light. The V cmax difference between P0 and P1 treatment was not signi cant under the same light environment, while J max increased signi cantly and the increase in shading environment is signi cantly higher than that in the ambient light environment (Figure 7), indicating that the application of phosphate fertilizer in shading mainly increases CO 2 assimilation by increasing the regeneration rate of RUBP rather than the e ciency of Rubisco carboxylation, which also shows that phosphorus application in shading the increase of CO 2 assimilation rate may be closely related to the improvement of the performance of the photosystem.
Based on the above results, we have analyzed PSII. As the primary site of photosynthesis, photosystem II has the function of absorbing and converting light energy [32][33] , and its performance is signi cantly affected by the light intensity [34] . The chlorophyll a uorescence transient (OJIP) curves can re ect the process and state of the original photochemical reaction of PSII. The maximum photochemical e ciency (F v /F m ) is an important parameter to measure the activity of PSII under various environmental stresses [35] . This experiment found that the changes of F v /F m <5% before and after phosphorus application in L1 and L2 treatment ( Figure 10A). Therefore, the effect of applying phosphate fertilizer on the photosynthetic rate of soybean under shading conditions seems to be very small according to the maximum photochemical e ciency of photosystem II. However, after weak light treatment, compared with P1 treatment, the relatively variable uorescence at point J (2 ms) in the OJIP curve treated with P0 was signi cantly increased ( Figure 8-9), indicating that the electron transfer from Q A to Q B is obstructed, that is, the PSII receptor Side electron transfer is blocked. Besides, we also observed that the relative uorescence yield of P0 treatment at point K also showed a signi cant increasing trend, but not as obvious as point J, which proved that the application of phosphate fertilizer can improve the activity of the oxygen-evolving complex, but the in uence on the smoothness of electron transfer is more signi cant.
To further explore the effect of phosphate fertilizer on photosystem activity, we compared j Eo , y o , and PI ABS . Studies on the PSII reaction center showed that electron transfer e ciency and light energy absorption performance were signi cantly reduced under shading (Figure 10 B-D). The performance of the receptor side of the plant, electron transfer e ciency, and light energy conversion e ciency of the PSII reaction center were all signi cantly improved after applying phosphorus fertilizer in shading, which is consistent with the changing trend of A sat . Indicating that the increase in the rate of CO 2 assimilation after the application of phosphate fertilizer in shading is closely related to the improvement of PSII electron transport performance. The higher performance of the photosystem provides more assimilation force (NADPH, ATP) for RUBP regeneration, use the available photochemical energy to provide Rubisco with a su cient source of the substrate.
After resuming light in the R5 period, the V cmax and J max of the L2P1 treatment showed an upward trend, and the performance of the PSII reaction center has a minimum reduction. There was no signi cant difference in A sat compare to L1P1, and the light energy utilization e ciency was signi cantly higher than other treatments. Such results indicate that after restoring the light, L2P1 treatment can use more absorbed light energy for photochemistry due to restored balance between the light-dependent and lightindependent reactions, maintain the electron transfer e ciency of the photosystem, and improve the regeneration rate of RUBP to provide su cient substrate for Rubisco and ensures that soybeans can make full use of light energy after re-lighting, and increase the rate of photosynthesis products accumulation to increase yield.