Abstract In this study， effects of nitrogen （N） amount applied on photosynthesis behaviors of the summer millet in North China was investigated. Photosynthetic rates （Pn）， chlorophyll contents （Chl）， photosynthetic active duration （PAD）， and chlorophyll relative steady phase （RSP） in flag and the upper third leaves were assessed in cultivars of Baogu 19， Jigu 19， 9050， and 60D under three N treatments [i.e.， 0 （control）， 75， and 150 kg/hm2]. Results indicated that the photosynthesis parameters were drastically regulated by external N levels， all of them showing elevation along with the increased N input in both assayed leaves. Among the cultivars examined， behaviors of the photosynthetic parameters were much better in Baogu 19 and worse in 60D. The plant yields in the cultivars under various N treatments were shown to be in consistent with the behavior of the photosynthesis parameters. Correlation analysis revealed that plant yield is positively correlated with Pn and Chl and significantly positively correlated with PAD and RSP， suggesting that longer effective photosynthetic duration of leaves impacts largely on plant biomass production and the yield formation potential. Our investigation indicates that suitable external N applied can increase the yield of summer millet associating with the improvement of photosynthesis behaviors in upper leaves that contribute to plant biomass at the late growth stage. Baogu 19 exhibited higher plant yield together with improved photosynthetic parameters in upper leaves， suggesting its potential as an elite cultivar in planting in the summer season of North China.
　　Key words Millet （Setaria italica L.）； Summer-sown cultivar； N application level； Photosynthesis parameter； Plant productivity
　　Millet （Setaria italica L.）， an important cereal species， is currently cultivated in various ecological regions worldwide due to its short growing cycle and low water and nutrient requirements[1]. Among the millet types， foxtail type millet is one of the species domesticated earlier and planted in large area in China as well as other East Asia countries， contributing largely to the crop production of these regions[2-4].
　　Photosynthetic function of the leaves determines the carbon transformation efficiency， exerting large impact on the growth， development， and productivity of plants. A line of evidence has revealed the large variation on the photosynthesis parameters among diverse cereal genotypes or cultivars[5-8]. In addition， as one of the indispensible inorganic nutrients， nitrogen （N） input level plays a critical role in regulating the photosynthesis behaviors in cereal crops[9-12]， and modulates drastically the plant biomass and the crop yield potential[13]. To date， although the effects of N input level on photosynthesis behaviors are largely known in various major cereal crops， such as wheat， maize， and rice[6， 14]， and the photosynthesis parameters of leaves under N input treatments are remained to be characterized in millet. In this study， we used four summer millet cultivars released recently in Hebei plain， including Baogu 19， Jigu 19， 9050， and 60D， to investigate the photosynthesis parameters under different N input treatments. Our results indicate that photosynthetic rate （Pn）， chlorophyll content （Chl）， photosynthetic active duration （PAD） and chlorophyll relative steady duration （RSP） in the flag and the upper leaves are elevated by increased N level. PAD and RSP are significantly and positively correlated with plant yield， suggesting their contribution to the productivity of the summer millet cultivars. 　　Materials and Methods
　　Plant materials and treatments
　　The four summer millet cultivars used in this study are Baogu 19， Jigu 19， 9050， and 60D. They were subjected to the field experiments in 2015 and 2016 summer growing seasons at the Experimental Station， Agricultural Sciences of Baoding Academy， Baoding， China （East longitude 115.47 and North latitude 38.47）. The soil type in experiments was clay loam and the arable soil contained follow nutrient amounts： organic matter 1.52%， total N 0.082%， available N 64.30 mg/kg， available phosphorus 13.86 mg/kg， and exchangeable potassium 112.53 mg/kg. The experiments were performed according to a split block design with three replicates， in which cultivars acted as main factor and N input levels [N 0 （0 kg N/hm2）， N 75 （75 kg N/hm2）， and N 150 （150 kg N/hm2）] acted as sub-main factor. For N input treatments of N 75 and N 150， urea was applied as basal before ploughing in June， after harvesting of winter wheat. All plots （each with 6 m long and 4 m width） were supplemented with 45 kg P2O5 （source from superphosphate） and 60 K2O （source from KCl） together with the basal N application. In 2015 and 2016 seasons， sowing dates were June 16 and 18 and harvest dates were September 23 and 25， respectively. For all treatments， row distance was set as 40 cm and plant densities were adjusted to 450 000/hm2 at the four-leaf growth stage. Techniques such as removal of weeds and chemical control of diseases and pests during growth stages were performed as suggested for the regular summer millet cultivation.
　　Assays of plant yield
　　At maturity， thirty representative plants from each plot were collected and air dried in a room. The seeds from spikes were obtained using a mini thresher and the seeds were weighed after removal of the unfertilized ones.
　　Assay of the photosynthetic parameters
　　At the late growth stage， Pn and Chl of the flag and upper third leaves in cultivars across the N input treatments were assessed successively from leaf full expansion to yellowish with a 7-day interval. Pn was assayed using a portable photosynthesis analyser （LiCOR-6200， USA） under follow external conditions： light intensity 1 000 μmol E/（m2·s） and CO2 concentration 360 μ/L that were provided by supplemental facilities. Chl was assessed by recording reads from a chlorophyll analyzer （SPAD-520， Japan）. PAD and CSD， two indices reflecting effective durations of leaf photosynthesis over a leaf growth circle， were calculated based on Pn and Chl as described previously[14-15]. Of them， PAD indicates a length from leaf expansion to a time point at which leaf shows half of leaf expansion Pn， and CSD indicates a length from leaf expansion to a time point at which leaf shows 80% of leaf expansion Chl. 　　Statistical analysis
　　The averages， standard errors， and the regression analysis for data obtained were performed using Excel program of the Window system. Data sets from 2016 growth season were used for analysis in this study given the comparable results in two seasons tested.
　　Results and Analysis
　　Plant yields in cultivars under various N level treatments
　　The plant yields in tested cultivars under different N levels are shown in Fig. 1. With the increase of N input level， plant yields in all of the cultivars were gradually elevated. The average plant yields among cultivars were 6.83， 8.04， and 9.12 g for treatments N 0， N 75， and N 150， respectively， indicating the substantial role of N input level in regulation of the millet productivity. Among the cultivars examined， plant yields in Baogu 19， Jigu 19， 9050， and 60D across N input treatments were 9.53， 8.21， 7.67， and 6.52 g， respectively （Figure 1）， which indicates that Baogu 19 is higher than other cultivars in yield formation.
　　Pn behaviors in the cultivars under various N level treatments
　　The Pn dynamic behaviors in millet cultivars under different N input treatments are shown in Fig. 2. Along with progression of the leaf growth， Pn values in the flag and the upper third leaves gradually decreased. At each assay time， Pn in above leaves was the highest in N 150， followed by N 75， and the lowest in N 0， which was in consistent with the plant yield behavior under N input treatments as described above. For cultivars， Baogu 19 showed the highest Pn at all assay times in two leaves， followed by Jigu 19， 9050， and 60D had the lowest Pn （Fig. 2）.
　　Chl behaviors in the cultivars under various N level treatments
　　The Chl dynamic behaviors in the cultivars under different N input treatments are shown in Fig. 3. Similar to Pn， Chl values were gradually reduced along with leaf growth progression in the tested leaves； however， the reduction rate of Chl over leaf growth circle was much slower than that of Pn. Higher N input increased the Chl values at all assay times， showing to be the highest in N 150， followed by N 75， and the lowest in N 0. Similar to Pn behaviors among cultivars， Chl values were the highest in Baogu 19， followed by Jigu 19 and 9050， and the lowest in 60D over a leaf circle （Fig. 3）. These results suggested the drastic variation on Chl and Pn behavior among the cultivars and N input treatments.
　　PAD and RSP in the cultivars under various N level treatments 　　PAD and CSD in the flag and the third upper leaves in cultivars under different N input treatments are shown in Table 1. Results indicated that increased N level drastically extended the PAD and RSP of these leaves， indicating that enhanced N level can extend the effective duration of leaf carbon assimilation and slow the degradation of photosynthetic pigments. Among the cultivars， Baogu 19 had the highest PAD and CSD values in tested leaves， followed by Jigu 19 and 9050， and 60D showed the lowest PAD and CSD values （Table 1）， suggesting that the difference on leaf effective photosynthetic function among the summer millet cultivars.
　　Correlation analysis results between plant yield and the photosynthetic parameters
　　Liner regression analysis between plant yield and the photosynthetic parameters， including Pn at leaf expansion （Pnmax）， Chl at leaf expansion （Chlmax）， PAD， and RSP， was performed to understand the contribution of these parameters to plant productivity. The regression coefficients showed that all of the photostnthesis parameters were positively correlated with plant yield， with coefficients （R2） between plant yield and Pnmax， PAD， Chlmax， and RSP to be 0.190， 0.591*， 0.036， and 0.535* in flag leaves and 0.103， 0.497*， 0.083， and 0.393* in the upper third leaves， respectively （Fig. 4）. Of them， PAD and RSP in tested leaves were significantly positively correlated with plant yield， suggesting much more contribution of them to the plant productivity.
　　Discussion and Conclusions
　　Photosynthetic capacity impacts largely on the potential of biomass accumulation and production of the crop plants[16-17]. In cereals， over 90 percentage of the plant biomass derives from the photosynthetic assimilates， which control the plant yield behavior[18]. Moreover， at late growth stage of cereals， grain filling is the central biological process. Improving the photosynthetic functions of upper leaves can increase grain weight via promoting the kernel filling rate. In this study， using four of the summer millet cultivars released in Hebei plain as materials， we assessed the behaviors of Pn and Chl in flag and the upper third leaves in millet plants under various N input treatments. We found that Pn and Chl in the millet cultivars exhibited similar patterns to those in other cereals， such as wheat[14-15]. Across a leaf growth circle， Pn and Chl values in flag and the upper leaves reached the highest values at leaf expansion， then gradually decreased along with the progression of leaf growth， with a large reduction rate on Pn relative to that on Chl. This suggests the unsynchronized characterization on attenuation rate between carbon assimilation and chlorophyll degradation. Based on regression analysis， we observed the consistent patterns and positive correlation between plant yield and Pnmax， Chlmax， PAD， and RSP， which suggest that the photosynthetic parameters in plant upper leaves contribute greatly to the yield potential capacity. In addition， significant positive correlation were found between PAD and RSP values and plant yield， indicating that extended effective photosynthetic durations are crucial in regulating the plant biomass production and yield potential in summer millet. 　　Similar to other external factors， such as light intensity， plant density， temperature and CO2 conditions that affects drastically the physiological processes of plants due to their roles in regulating the carboxylation efficiency and electron transport capacity in photosynthesis[19-20]， nitrogen supply level acts as a critical factor in the regulation of leaf area expansion[21]， photosynthesis efficiency， and leaf senescence[20，22-25]. In this study， the Pn， Chl， PAD and CSD in the upper leaves of millet cultivars indicate to be upregulated by increased N input level. Along with the increase of the N input amount， Pn and Chl were gradually elevated and PAD and RSP extended in the tested leaves， which contributed to the improved plant yield under the high N input treatment （i.e.， N 150 kg/ha）. Thus， suitable N application is an effective technique in improving the production of summer millet in Hebei plain.
　　Drastic variation is present in photosynthetic function across cereal crop cultivars[5]. In this study， we found significant variations on the Pn， Chl， PAD， and RSP behaviors among the summer millet cultivars examined. Among them， Baogu 19 had the highest Pn and Chl in upper leaves and the longest PAD and RSP under various N input levels， whose plant yield was the highest among the cultivars under the N input treatments. These results indicate its potential in largely cultivation in North China and other similar ecological regions.
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