Effects of nitrogen on photosynthetic characteristics of leaves from two different stay-green corn (Zea mays L.) varieties at the grain-filling stage

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1 Effects of nitrogen on photosynthetic characteristics of leaves from two different stay-green corn (Zea mays L.) varieties at the grain-filling stage G. Li 1, Z.-S. Zhang 2, H.-Y. Gao 2, P. Liu 1,3, S.-T. Dong 1,3, J.-W. Zhang 1, and B. Zhao 1 1 State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai an , China; and 2 College of Life Science, Shandong Agricultural University, Tai an , China. Received 17 February 2012, accepted 5 April Li, G., Zhang, Z.-S., Gao, H.-Y., Liu, P., Dong, S.-T., Zhang, J.-W. and Zhao, B Effects of nitrogen on photosynthetic characteristics of leaves from two different stay-green corn (Zea mays L.) varieties at the grain-filling stage. Can. J. Plant Sci. 92: The effects of nitrogen on the photosynthetic characteristics of two different stay-green corn (Zea mays L.) at the grain-filling stage were studied. Using a stay-green inbred line (Q319) and a non-stay-green inbred line (HZ4) as materials, using fast chlorophyll fluorescence-induction kinetics and 820 nm light-absorption curves, we analyzed the influences of nitrogen supplementation on photosystem I (PS I) and photosystem II (PS II). The results show that nitrogen fertilization has significant effects on promoting the net photosynthetic rate (P n ) in leaves at the grain-filling stage and on single-plant grain yield at the harvest stage (PB0.05) in Q319, whereas there is no significant effect in HZ4. Analyses performed using JIP-Test showed that nitrogen fertilization significantly increased electron donor and acceptor performance in the reaction center of PS II (PB0.05). The increased performance of the electron transport chain at a point after the electron acceptor in the PS II reaction center was greater in Q319 than in HZ4, reducing excessive excitation energy production in PS II and significantly improving the coordination between PS II and PS I. Although the performance of the electron transport chain after the electron acceptor in the PS II reaction center was increased in HZ4, the increase was not substantial enough to improve the coordination between PS II and PS I; therefore, P n and grain yield were still significantly lower than those of Q319. Key words: Nitrogen, corn, stay-green variety, photosynthetic characteristics, photosystem Li, G., Zhang, Z.-S., Gao, H.-Y., Liu, P., Dong, S.-T., Zhang, J.-W. et Zhao, B Les effets de l azote sur des parame` tres photosynthe tiques de feuilles de deux diffe rentes maïs(zea mays L.) au stade remplissage de grain. Can. J. Plant Sci. 92: Les effets de l azote ont e té e tudie s sur des parame` tres de deux diffe rentes maı s (stay-green) au stade du remplissage du grain. Nous avons étudie l influence de la nutrition azote e sur les photosyste` mes I (PS I) et II (PS II) à partir d une lignée stay green (Q319) et d une ligne e dite non-stay green (HZ4) en observant la cine tique d induction de la fluorescence rapide de la chlorophylle et les courbes d absorption de la lumière a` la longueur d onde de 820 nm. Les re sultats montrent que la nutrition azote e sur la lignée Q319 a un effet significatif sur l induction du taux net de photosynthe` se dans les feuilles au stade remplissage de grain and sur la production par unité de plante au stade de la re colte (PB0.05). A l oppose, la ligne e HZ4 ne semble pas être affecte e par le meˆme traitement azote. Les analyses, utilisant le test JIP, ont montre que la nutrition azote e augmentait considérablement les performances de donneur et d accepteur d e lectrons du centre re actionnel PS II (PB0.05). En effet, les performances de la chaine de transport d e lectron dans le PS II était supe rieure dans la ligne e Q319 par rapport a` la HZ4; ceci avait pour conséquence de re duire la production excessive d e nergie d excitation dans le photosyste` me et ame liorait de manie` re significative la coordination entre les deux photosyste` mes PS II et PS I. Bien que la performance de la chaine de transport d e lectron apre` s l e lectron accepteur dans le centre photo re actionnel PS II soit ame liore e dans HZ4, l augmentation n e tait pas assez importante pour induire une meilleure coordination entre les centres PS II and PS I. Par conse quent, le taux photosynthe tique net (P n ) et la production du grain de la ligne e HZ4 étaient plus faibles que ceux de la ligne e Q319. Mots clés: Azote, maïs, varie te stay-green (toujours verte), parame` tres photosynthe tiques, photosyste` me 3 To whom correspondence should be addressed ( address: liup@sdau.edu.cn or stdong@sdau.edu.cn). (P. Liu or S.-T. Dong). The author (Z.-S. Zhang) contributed equally to this work. Abbreviations: JIP-Test, anlysis software of Handy-PEA chlorophyll fluorometer; PS I, photosystem I; PS II, photosystem II; P n, net photosynthesis rate; G s, stomatal conductance; C i, intercellular CO 2 concentration; PAR, photosynthetically active radiation; SG, stay-green; NSG, nonstay-green; F o, fluorescence at 20 ms; F k, fluorescence at 300 ms; F j, fluorescence at 2 ms; F i, fluorescence at 30 ms; F m, maximal fluorescence; V k, relative variable fluorescence at K phase; V j, relative variable fluorescence at J phase; V i, relative variable fluorescence at I phase; OEC, oxygen-evolving complex Can. J. Plant Sci. (2012) 92: doi: /cjps

2 672 CANADIAN JOURNAL OF PLANT SCIENCE A number of annual cereals exhibit genetic variation in the degree and rate of leaf senescence during grain filling (Thomas and Smart 1993). Thomas and Smart (1993) characterized a stay-green trait (i.e., the phenotypes that exhibit delayed senescence) as having higher water and chlorophyll contents in the leaves at maturity. Expression of the SG trait has been reported in corn (Tollenaar and Daynard 1978; Ma and Dwyer 1998; Rajcan and Tollenaar 1999a, b). Duvick et al. (2004a, b, c) reported that the yield increase was associated with an increase in ears per 100 plants, leaf-angle score, stay-green score, and a decrease in anthesissilking interval and tasselsize scores. Heterosis for dry-matter accumulation during the grain-filling period results from greater light interception as a result of greater maximum LAI and increased stay-green qualities (Tollenaar et al. 2004). Genotypes possessing the stay-green trait have a significant yield advantage compared with genotypes that do not possess this trait, especially under post-anthesis drought conditions (Borrell et al. 2000; He et al. 2001, 2002a, b; Kumudini et al. 2002). As a key component of proteins (both structural and enzymatic) and nucleic acids, nitrogen affects organ formation, root-cap development, photosynthesis, carbonnitrogen ratio, source-sink dynamics, and many other processes (Mi et al. 2007). Lack of nitrogen inhibits crop growth and photosynthesis and is therefore a major cause of yield and quality decline in crops (He et al. 1998; Jin and He 1999). The SG trait is thought to be a consequence of the balance between N demand by the grain and N supply during the grain-filling stage (Borrell et al. 2001). Nitrogen from catabolized ribulose-1, 5-bisphosphate carboxylase/oxygenase and chlorophyll is re-translocated to reproductive organs during the grain-filling stage (He et al. 2003). Nitrogen fertilization decreases nitrogen release from the leaf after blossom, delays leaf senescence, maintains a higher photosynthetic rate, and provides carbohydrates for grain filling. However, nitrogen regulation of corn photosynthesis is dependent on variety, and the regulation is more profound in nitrogen-efficient varieties (Zhang et al. 1997; Mi et al. 2007). The responses of different staygreen corn lines to nitrogen regulation are also different (He and Jin 2000). Previous studies of the relationship between nitrogen and plant photosynthetic characteristics have mainly dealt with the photosystem as a whole with respect to photosynthesis and fluorescence (He et al. 2001, 2002a, b, 2003). However, these studies have not clarified the effects of nitrogen on the two photosystems and have also ignored its effects on individual electron-transfer sites within the electron transport chain. Recently, the fast chlorophyll fluorescence induction kinetics curve has become one of the most useful tools in the study of photosynthesis, especially the initial photochemical reactions (Jiang et al. 2003; Schansker et al. 2005; Ilı k et al. 2006). The abundant and important information contained in this curve can reflect the initial photochemical reactions in PS II and the status changes of the photosynthetic apparatus. In addition, with the application of the PS I 820 nm lightabsorption curve, the coordination between the two photosystems can be further understood (Schansker et al. 2003, 2005). Therefore, the objective of this study was to determine the effects of nitrogen fertilization on PS I and PS II photosynthetic performance and the coordination between these two systems in the leaves of different staygreen corn inbred lines. Elucidating these effects should reveal the physiological mechanisms leading to high yield in stay-green corn. MATERIAL AND METHODS Plant Material and Experimental Design These experiments were performed at the Corn Research Center of Shandong Agricultural University, Shandong Province, China. Experimental materials used included the stay-green inbred line, Qi 319 (Q319), and the nonstay-green inbred line, HuangZao 4 (HZ4); these lines have very similar flowering and reproductive patterns. The single plant pot cultivation method was used, with a pot height and diameter of 50 cm and 37 cm, respectively. The pots were buried 45 cm underground with row spacing of 66 cm and a plant density of ha 1. Field topsoil and fine sand were mixed in a volume ratio of 2:1 and added to the pots, each pot containing kg of soil. The average nutritional composition and content of pot soil in 2 yr is as below: organic material was g kg 1, soil ph was , total N was g kg 1, and available N, P, K was , , mg kg 1, respectively. Urea (N content of 45%) was used for N fertilization. Two levels of N were applied. represents 640 kg ha 1 of urea, which amounts to approximately 9.48 g per plant (pot), with 50% applied as a base fertilizer before seeding and 50% as a topdressing fertilizer at the V12 stage. represents no fertilizer being applied, with 0 kg ha 1 of urea, or 0 g per plant (pot). The two different fertilization treatments were applied to 30 pots each. At the grain-filling period in 2007 (20 d after flowering), the mid-lower of ear leaves from plants at similar growth stage with no lesions or damage were selected to measure gas exchange parameters, chlorophyll concentration, and leaf nitrogen content. At harvest time, yields and related traits were measured. In 2008, based on repetition of the experiments conducted in 2007, measurements of the photosystem properties were also performed at the grain-filling stage. Chlorophyll Content For determination of chlorophyll content, 0.50 g of fresh leaves were placed in a 100-mL test tube, 15 ml of pure methanol was added, and the mixture was homogenized with a polytron. The homogenate was then filtered and brought to 100 ml with pure methanol. The chlorophyll

3 LI ET AL. * N EFFECTS ON PHOTOSYNTHESIS OF STAY-GREEN CORN 673 concentration in the supernatant was spectrophotometrically determined by measuring the absorbences at and nm for Chl a and Chl b, respectively, and calculated according to Wang (2007). Leaf Gas Exchange Leaf gas exchange measurements, including net photosynthesis (P n ), stomatal conductance (G s ), and intercellular CO 2 concentration (C i ), were measured simultaneously using a portable infrared gas analyzer (CIRAS II, PP System, Hansatech, UK). The infrared gas analysis system was equipped with a clamp-on leaf cuvette that exposed 1.7 cm 2 of leaf area (PLC version, PP System, Hansatech, UK). Gas exchange measurements were performed on fully expanded and sun-exposed leaves throughout the natural photoperiod. Ten replicate measurements per treatment were measured on clear days between 1000 and Measurement conditions were kept consistent: LED light source, PAR1600 mmol m 2 s 1. CO 2 concentration was maintained at a constant level of 360 mmol mol 1 using aco 2 injector with a high-pressure liquid CO 2 cartridge source. Leaf Nitrogen Content Leaf segments were cut and dried at 808C to a consistent weight. Each individual leaf sample was ground to a fine powder, and total leaf N was determined by the Dumas method using a Rapid NIII nitrogen analyzer (Elementar Analysensysteme, Germany). Chlorophyll Fluorescence Assay and Imaging The chlorophyll fluorescence induction kinetics (Kautsky effect) in pre-darkened leaves and peduncles (30 min) were measured at the red chlorophyll fluorescence band (near 690 nm) using a kinetic imaging fluorometer (FluorCam, PSI, Czech Republic) as described by Nedbal et al. (2000). The duration of the F o measurement was 5.04 s. After measuring the minimum fluorescence in the dark-adapted state (F o ), the samples were illuminated with a saturating pulse (1500 mmol m 2 s 1 ) to determine the maximal fluorescence in the dark-adapted state (F m ). For quenching analysis, samples were illuminated after 10 s of darkness with an orange actinic light (130 mmol m 2 s 1 ) using saturating pulses of 60 s. The fluorescence values that were recorded at every saturating pulse were identified as F m?, whereas the fluorescence values recorded immediately prior to each pulse were called F t. The chlorophyll fluorescence emission transients were captured by a CCD camera in a series of images with a resolution of pixels to reveal the heterogeneity of the leaf surface. Numerical analyses of the classical physiological parameters were performed on the maximum PS II quantum yield F v /F m (F m F o )/F m and on the effective PS II quantum yield F PSII (F m?f t )/F m? using more than 10 replicates (Hetherington et al. 1998). Fast Chlorophyll Fluorescence Kinetics Curve and 820 nm Light Absorption Curve Chl a fluorescence transients in dark-adapted attached corn leaves were measured using a Handy-PEA chlorophyll fluorometer (Hansatech, UK). The transients were induced by 1 s illumination with an array of six light-emitting diodes providing maximum light intensity (photon flux density) of 3000 mmol m 2 s 1 and homogeneous irradiation over a leaf area 4 mm in diameter. All measurements were performed on apparently healthy leaves. The fast fluorescence induction kinetics (F o to F m ), known as the OJIP curve, were recorded from 10 ms to 1 s. The fluorescence intensity at 50 ms was considered as F o (Schansker et al. 2003). In this study, 10 plants per treatment were chosen, and five fluorescence measurements per plant were performed. The short duration of experimental measurement (1 s) allowed us to assess many samples in a short time, leading to highly precise statistical data analysis. The 820 nm light-absorption curve measurements that parallel Chl a fluorescence were carried out using a PEA Senior instrument (Hansatech, UK). The excitation light intensity (photons) of 1800 mmol m 2 s 1 was generated by four 650 nm LEDs. Far-red light (718 nm peak wavelength, 200 mmol m 2 s 1 light intensity) and modulated far-red light (820 nm peak wavelength) for measurements were provided by two additional LEDs. Light regimes were performed in the following order: turn on red light for 1 s, far-red light for 10 s, and red light for 2 s. The relative amplitude of the 820 nm light absorption at the 3rd stage was used as the indicator for the maximal oxidation-reduction capability of PS I to represent the activity of PS I. Further technical details and applications are described in several papers by Schansker et al. (2003). JIP Test The concept of the JIP test is based on the Energy Flux Theory of bio-membranes (Strasser et al. 1995). Raw fluorescence OJIP transients were transferred to a spreadsheet using the Handy-PEA program (supplied with the instrument). In this form, the data are available to be treated according to the equations of the JIP test parameters by any tabulation program and were analyzed using the JIP test (Strasser et al. 2000, 2004). These parameters provided structural and functional information (including specific and phenomenological fluxes, quantum yields, and vitality indexes) and permitted us to quantify the PS II behavior of the plants. The following factors were used in the analyses: F o (fluorescence at 20 ms, O phase); F k (fluorescence at 300 ms, K phase); F j (fluorescence at 2 ms, J phase); F i (fluorescence at 30 ms, I phase); F m (maximal fluorescence, P phase); V k, V j, and V i (relative variable fluorescence at K, J, and I); the ratio, W k, of variable fluorescence, F k, to amplitude, F j -F o ; the probability, C o, of transferring electrons to other electron acceptors downstream of Q A in the electron transport chain by captured exciton; and

4 674 CANADIAN JOURNAL OF PLANT SCIENCE the photochemical performance indicator, PI ABS, based on the absorption of light energy. More technical details and applications are described in several papers by Strasser et al. (2000, 2004). Statistical Analyses Analysis of variance was performed with the Data Processing System software (Tang and Feng 2007), and data were presented as means and standard error. Differences among means were evaluated by using one way analysis of variance (ANOVA) with LSD multiple comparison test, and a P level of 0.05 or 0.01 to test statistical significance (PB0.05 or 0.01). RESULTS Biomass and Grain Yield Table 1 shows the changes in biomass and grain yield of two stay-green corn types at two N levels. After N fertilization, the biomass of HZ4 and Q319 had significantly increased above that of the treatment, and the growth rates of HZ4 were 12.63% in 2007 and 16.51% in 2008, which were higher than those for Q319. The harvest index of HZ4 decreased after N addition, but that of Q319 increased. Thus, the grain yield of HZ 4 did not improve in either year. The grain yields of Q319 at the level were and 18.13% higher than the level in 2007 and 2008, respectively. The main reason for this increased grain yield was the improvement in average grain numbers per ear. Compared with the treatment, the increases in grain numbers for Q319 were and 14.31% in 2007 and 2008, respectively. For HZ4, increases in grain numbers were only 5.21 and 6.25% for the same 2 yr, respectively. There was no significant difference in grain weight in either Q319 or HZ4 between the two N levels. Gas Exchange and Chlorophyll Content Table 2 shows the effects of N on gas exchange characteristics and chlorophyll content in the different stay-green corn varieties that were tested. The P n, G s, and content of Chl a, Chl b, and Chl (ab) for Q319 were significantly higher than those for HZ4 in both years. Compared with the level, N improved the P n Table 1. Effects of nitrogen on biomass and grain yield in two different stay-green varieties of corn Harvest year Treatments Biomass per plant (g plant 1 ) Harvest Index of Q319 but had no significant effect on that of HZ4. Nitrogen addition significantly increased the G s of both Q319 and HZ4. Nitrogen addition did not improve the C i in either stay-green type. There was no significant difference in Chl a, Chl b, or total chlorophyll content in leaves at the grain-filling stage under either N level in either variety. This indicates that the increased P n in leaves of stay-green corn at the grain-filling stage after N fertilization was not caused by changes in chlorophyll content. Nitrogen Content in Leaves As shown in Fig. 1, the N content in the ear leaves of Q319 at the grain-filling stage increased by 3.9% and 3.3% in 2007 and 2008, respectively, following N fertilization. There was no significant difference between these two years, and the increase was not remarkable compared with the level. After N fertilization, the N content of ear leaves of HZ4 at the grain-filling stage increased by 23.4% in 2007 and 18.8% in The N content in ear leaves at the grain-filling stage was not consistent with the P n increase in these two types of corn. It suggested that the increase in protein content in leaves at the grain-filling stage is not a major factor affecting changes in P n ; there may be other factors that were not detected. Chlorophyll Fluorescence Assay and Imaging The chlorophyll fluorescence image and parameters of for Q319 and HZ4 varied similarly compared with. After N fertilizer, the F v /F m of Q319 and HZ4 increased 2.83 and 6.92%, respectively (Fig. 2A, B, C and D). The nitrogen improved the values of F PSII of Q319 and HZ4 at the grain-filling stage increased by 7.93 and 18.84%, respectively (Fig. 2E, F, G and H). Compared with F v /F m, the F PSII of Q319 and HZ4 at level increased more than, and this was partly attributable to the nitrogen fertilizer improved the functional activity of PS II. Changes in Fluorescence Transients Figure 3 shows the polyphasic rise of fluorescence transients in the ear leaves of Q319 and HZ4 under Grain yield per plant (g plant 1 ) Number of Grains per ear 1000-grains weight (g) HZ b a a b a HZ a b a a a Q b b b b a Q a a a a a HZ b a a b a HZ a b a a a Q b b b b a Q a a a a a a, b Values within a column are mean9se. Means followed by different letters are significantly different at PB0.05. Data are the averages of 10 plants.

5 LI ET AL. * N EFFECTS ON PHOTOSYNTHESIS OF STAY-GREEN CORN 675 Table 2. Effects of nitrogen on leaf photosynthetic characteristics and chlorophyll contents of two different stay-green corn varieties at grain filling stage Harvest year Treatment P n (mmol m 2 s 1 ) G s (mmol m 2 s 1 ) C i (mmol mol 1 ) Content of Chl a (mg g 1,FW) Content of Chl b (mg g 1,FW) Total content of Chl (mg g 1, FW) HZ a b a b a a HZ a a a a a a Q b b a a a a Q a a a a a a HZ a b a a a a HZ a a a a a a Q b b a a a a Q a a a a a a a, b Values within a column are mean9se. Means followed by different letters are significantly different at PB0.05. Data are the averages of 10 plants. P n : net photosynthesis rate; G s : stomatal conductance; C i : intercellular CO 2 concentration; Chl: chlorophyll; FW: fresh weight. two N levels. Using Q319- as a reference, our results show that fluorescence intensity at the K step and the J step of both Q319 and HZ4 were significantly lowered compared with the level (Fig. 3B). Changes in PS II Donors/Acceptors Analyses of the fast chlorophyll fluorescence induction kinetics curve using the JIP test showed that the ratio (W k ) of variable fluorescence, F k, at the K step to the amplitude, F o F p, decreased by 12.6% and 4.6% for Q319 and HZ4, respectively, at the level compared with the ratio at the level. The ratio (V j ) of variable fluorescence, F j, at the J step to the amplitude, F o F p, decreased by 5.1 and 13.6% for Q319 and HZ4, respectively, significantly lower than the ratio at the level (Fig. 4). This finding indicates that the electron transfer capability of both the donor and the acceptor sides at the PS II reaction center of the electron transport chain were significantly improved after N treatment; the improvement in donor performance at PS II for Q319 was greater than that for HZ4, while the improvement in acceptor performance for HZ4 was greater than that for Q319. Ear leaf N content (%) A * HZ4 Q319 HZ4 Q319 Fig. 1. The effects of nitrogen fertilization on nitrogen content in leaves at the ear position in two different stay-green corn varieties at the grain-filling stage over a 2-yr (2007, A; 2008 B) period. *, the difference between nitrogen treatments was significant at PB0.05 and PB0.01, respectively. * B Changes in PS II Performance The probability (C o ) of transferring electrons to other electron acceptors downstream of Q A in the electron transport chain by captured exciton in the leaves of HZ4 increased significantly at the level, with an increase of 7.9%. For Q319, C o did not show a significant change. There was a significant difference in the performance indicator, PI ABS, which was based on the absorption of light energy, after N treatment in both HZ4 and Q319, with increases of 65.7 and 87.3%, respectively (Fig. 5). The increase of PI ABS caused by N treatment had a significantly higher C o in both HZ4 and Q319. However, the increases in both C o and PI ABS in HZ4 were much lower than those in Q319, indicating that the improvement in PS II function in Q319 is greater than that in HZ4. PS I Performance As shown in Fig. 6, there was no significant change in the 820 nm light-absorption curve of photosystem I (PS I) in the leaves of HZ4 after N treatment. However, Q319 showed a significant difference in PS I 820 nm light absorption between N levels. Analyses showed that the maximal oxidation and reduction activity (DI/I o )of PS I in leaves after N treatment increased by 9.8% and 21.7%, respectively in HZ4 and Q319; both values were significantly higher than those found without N treatment. In addition, the increase in DI/I o in Q319 was significantly higher than that for HZ4 (Fig. 7). This finding indicates that PS I performance in leaves was improved after N treatment in both types of corn, and the increase of PS I function in Q319 was greater than that in HZ4. Coordination of PS II and PS I When PS II and PS I were activated by red light at the same time, there was a significant difference in the relative change value, F (PS I/ PS II),ofDI/I o and C o between different N levels. Compared with the level, F (PS I/PS II) only increased 4.1% in HZ4 at the level, an increase that was not significant. However, in Q319, F (PS I/PS II) at the level increased by 20.2% over the level, a significant increase (Fig. 8). Therefore,

6 676 CANADIAN JOURNAL OF PLANT SCIENCE Q A B C D Fv/Fm 1 Q E G F H HZ4 HZ4 1 Φ PSII N treatment significantly increased the coordination between PS II and PS I in Q319, whereas the coordination of these two photosystems did not improve markedly in HZ4. DISCUSSION Dai (2006) studied the photosynthetic performance of wheat after spiking and showed that the decrease in chlorophyll content was not consistent with P n after spiking during the process of senescence. Beginning at the grain-filling stage, the rate of decrease in P n was significantly faster than that of chlorophyll content. Using 17 inbred corn lines, Zhao et al. (1998) reached a similar conclusion by analyzing the relationship between chlorophyll content and photosynthetic rate during 0.5 Fig. 2. The effects of nitrogen on two different stay-green corn variety leaves representative pseudocolour images at the grain-filling stage. A, B, C and D show F v /F m ; E, F, G and H show F PSII. F v /F m : maximum PSII quantum yield; F PSII : PSII effective quantum yield. Chl a relative variable Flu intensity (Vt) ΔVt = Δ[(F t -F o )/(F m -F o )] A O HZ4- HZ4- Q319- Q319- K 0.00 B Time (ms) Fig. 3. The effects of nitrogen on the difference between the variable fluorescence induction kinetics curve (V t, A) and the relative fluorescence induction kinetics curve (DV t, B) of photosystem II (PS II) in leaves of two different stay-green corn varieties at the grain-filling stage. J I P the growing period. They pointed out that chlorophyll content experienced gradual increases and rapid decreases after the V12 stage; the decrease in photosynthetic rate correlated with the decrease in chlorophyll content, but the decrease in photosynthetic rate began earlier and became more dramatic after the grainfilling stage. Therefore, the decline in photosynthetic performance in leaves at the grain-filling stage was not caused simply by changes in pigment in the leaves; there may be other undiscovered reasons. Nitrogen is a key element of chlorophyll and protein in plants, and N fertilization level affects the content and activity of these molecules. Nitrogen fertilization can compensate for N release from the leaves after flowering, delay the decline of leaves, and maintain a higher photosynthetic rate to provide carbohydrates for the filling of grain. The influence of nitrogen on regulation of corn photosynthesis depends on variety (Zhang et al. 1997; Mi et al. 2007), and its influence on photosynthetic performance of different stay-green corn types is also variable (He et al. 2007). Our 2-yr study results show that the accumulation of dry matter per plant increased significantly for both types after N fertilization compared with the untreated control, and the increase in HZ4 was greater than that in Q319; however, Q319, but not HZ4, exhibited a dramatic increase in grain yield per plant (Table 1). There was no significant increase in chlorophyll content in the leaves at the grain-filling stage in either type of corn after N fertilization; however, Q319 showed significantly increased P n at the grain-filling stage after N treatment, while HZ4 did not show a dramatic increase in P n (Table 2). Nitrogen content in the leaves at the ear position indicated that after N fertilization, the increase in N in HZ4 leaves was significantly higher than that in Q319 leaves (Fig. 1). Therefore, the N absorption ability of HZ4 is greater than that of Q319 after N treatment; however, the assimilated N is used mainly for the growth of the plant rather than being 0

7 LI ET AL. * N EFFECTS ON PHOTOSYNTHESIS OF STAY-GREEN CORN * A B Wk 0.28 Vj * HZ4 Q319 HZ4 Q319 Fig. 4. The effect of nitrogen on the ratios of chlorophyll variable fluorescence, F k, to amplitude, F o F j, and variable fluorescence, F j, to amplitude, F o F p, in the leaves of two different stay-green corn varieties at the grain-filling stage. *, the difference between nitrogen treatments was significant at PB0.05 and PB0.01, respectively A PIABS 0 HZ4 Q319 HZ4 Q319 Fig. 5. The effect of nitrogen on the probability, C o (A), and performance index, PI ABS (B), of two different stay-green corn varieties at the grain-filling stage. C o, the probability of transferring electrons to other electron acceptors downstream of Q A in the electron transport chain by captured exciton in the leaves. PI ABS, photochemical performance indicator based on the absorption of light energy. the difference between nitrogen treatments was significant at PB B transported into the grain. Under both N treatment conditions, Q319 demonstrated a higher capacity for N absorption. Compared with the treatment, Q319 used more N for grain development following N fertilization. Based on our experiments, in both types of corn, the change in chlorophyll content of leaves at the grain-filling stage following N treatment is not the cause of the increase in P n ; rather, changes in certain characteristics of the photosystems is the major reason for an increase in P n. Although the N content in leaves increased significantly at the grain-filling stage after N fertilization in HZ4, for genetic reasons the N transport system and the synthesis of components of the photosystems may be inhibited by senescence in this variety. Excessive amounts of N still remain in the leaves at the grain-filling stage, thus the increase in P n at the grainfilling stage is not significant after N fertilization. In the harvest stage, this translates to low grain yield per plant and high dry matter per plant. Therefore, HZ4 is a nonstay-green corn with low N efficiency, whereas Q319 is a stay-green type corn with high N efficiency. Studies have shown that changes in chlorophyll fluorescence can, to a certain extent, reflect the effects of environmental factors on the plant (Krause and Weis 1991). The physiological changes in the plant itself, such as senescence (Dai et al. 2006), or stresses, such as iron deficiency or manganese starvation (Jiang et al. 2007), salt stress (Chen et al. 2004), or drought (Eggenberg et al. 1995), can all affect the performance of plant photosystems. Chlorophyll fluorescence has a close correlation with the initial photochemical reactions, representing the oxidation-reduction status of the PS II reaction center, the donor, and the acceptor (Kru ger et al. 1997; Srivastava et al. 1997; Krause and Weis 1991; Relative variable Flu transmission at 820 nm (It) HZ4- HZ4- I 0.4ms Q319- Q319- I 40ms Time (ms) Fig. 6. The effects of nitrogen on the 820 nm relative light absorption curve (I t ) in leaves of two different stay-green corn varieties at the grain-filling stage. A B

8 678 CANADIAN JOURNAL OF PLANT SCIENCE ΔI/Io HZ4 Q319 Fig. 7. The effects of nitrogen on maximal oxidation-reduction activity (DI/I o ) of PS I in the leaves of two different stay-green corn varieties at the grain-filling stage. the difference between nitrogen treatments was significant at PB0.01. Φ(PS I/PS II) HZ4 Maxwell and Johnson 2000). A typical fast chlorophyll fluorescence induction kinetics curve has O, J, I and P phases. J(V j ) and K(W k ) fluorescence increases reflect the excessive accumulation of electrons at the Q A site and inhibition of the activity of the oxygen-evolving complex (OEC), respectively (Strasser et al. 1995; Wu et al. 2003). Using a JIP test data-processing method, comparing the relative fluorescence intensity changes (W k and V j ) at the K and J sites, both the extent of damage to the OEC and the accumulation of Q A can be calculated (Lu and Zhang 1999; Chen et al. 2004). Our experimental results show that the W k and V j of both types of corn decreased dramatically after N fertilization compared with the untreated control. The decrease in W k in HZ4 was significantly greater than the decrease in V j, indicating that the performance Q319 Fig. 8. The effects of nitrogen on the coordination of C o and PS I maximal oxidation-reduction activity of two different stay-green corn varieties at the grain-filling stage. the difference between nitrogen treatments was significant at PB0.01. improvement of electron donors in the PS II reaction center is greater than that of acceptors after N fertilization. In contrast, in Q319, the improvement in performance due to N fertilization is greater on the acceptor side than on the donor side (Fig. 4). The synthesis of 33 kd polypeptide and protein D1 in the leaves might be enhanced after N fertilization, and in turn, OEC activity promoted and the accumulation of Q A \decreased; which may explain the increased performance of acceptors and donors in PS II reaction center. Investigation of PS II performance alone is not sufficient to explain the observed pattern of the characteristics of photosystems. Other studies have shown that when electron transfer from PS II to PS I was blocked, the P n in leaves would decrease (Lu and Zhang 1999). This has been confirmed in a study of the effect of drought stress on photosynthetic performance in corn leaves at the grain-filling stage (Li et al. 2009). C o is one of the most comprehensive indicators of the performance of the PS II reaction center because it relies on the electron supply capability of the electron donor and the receptivity of the acceptor (including PS I). The maximal oxidation-reduction activity of PS I (DI/I o ) reflects the maximal oxidation-reduction activity of P 700 in the PS I reaction center, and it is a comprehensive measure of PS I performance (Schansker et al. 2003). Analyses of the changes of C o and DI/I o at the grain-filling stage in both corn types after N fertilization would clarify the coordination of the two photosystems as well as the reaction site at which N improves photosynthetic performance. Our data show that the C o of Q319 did not increase significantly after N treatment at the grainfilling stage, whereas HZ4 showed a dramatic increase. However, the DI/I o of HZ4 did not show a prominent change after N treatment, whereas Q319 exhibited a significant increase. Thus, the two photosystems are regulated differentially by N in these two types of corn. Previous studies have shown that PS I performance relies not only on its own properties, but also on the performance of PS II (Schansker et al. 2003). The electron transfer rate of PS II is controlling, as it takes s for Q A to transfer electrons downstream, while it only takes ps for Q A to obtain an electron from its donor (Krause et al. 1991). Therefore, if electron acceptor performance decreases in the PS II reaction center (a V j increase), feedback inhibition may form, produce excessive excitation energy, and damage the OEC, which would, in turn, lead to a rapid decline in the overall activity of the PS II reaction center. After N fertilization in Q319, the performance of the electron transport chain on the acceptor side of the PS II reaction center increased dramatically, which significantly increased the ratio of DI/I o to C o and the coordination of the two photosystems. Although the performance of the electron transport chain on the acceptor side increased somewhat in HZ4, this increase was not sufficient to improve the coordination of PS II and PS I (Fig. 8). Therefore, after N treatment, the increase of F (PS I/ PS II)

9 LI ET AL. * N EFFECTS ON PHOTOSYNTHESIS OF STAY-GREEN CORN 679 in Q319 at the grain-filling stage was greater than that in HZ4, and the P n increased correspondingly. Previous studies have shown that during photosynthesis, the transport of electrons from the photolysis of H 2 O to NADP relies on the cooperation of PS II and PS I, and generates a proton gradient across the chloroplast membrane to synthesize ATP. However, the synthesis of ATP can also be achieved through PS I cyclic electron transport (Jeanjean et al. 1993). The study by Jiang et al. (2007) on soybean photosynthetic properties under iron deficiency showed that the soybean could enhance PS I cyclic electron transport under iron deficiency for extra ATP synthesis as a response to the metabolic disturbance caused by iron deficiency. The JIP test theory showed that PI ABS can reflect the potential for electron transport between PS II and PS I, and reflect the overall performance of the photosystems (Strasser et al and 2004). Our study used F (PS I/ PS II) to represent the coordination of the two photosystems. Compared with the untreated control, PI ABS and F (PS I/ PS II) in Q319 both showed significant increases after N fertilization, and the increase in PI ABS was significantly greater than that of F (PS I/ PS II). Whereas HZ4 showed a significant increase of PI ABS at the grainfilling stage, F (PS I/ PS II) did not exhibit an obvious change. Our results demonstrate that the PI ABS of plants of both varieties exhibited significant increases after N treatment at the grain-filling stage compared with those without N treatment (Fig. 5B). Only Q319 showed a significant increase in F (PS I/ PS II) after N fertilization, while HZ4 did not show an obvious increase. Compared with PI ABS, the increase in F (PS I/ PS II) of both varieties at the grain-filling stage after N treatment corresponded better with the increase in P n and the yield at the mature stage. We suggest that under the excitation by red light, both photosystems are activated at the same time. The correlation of these two systems determined under this condition is an accurate reflection of the photosystem properties in the leaves. Compared with the untreated control, the increase in F (PS I/ PS II) for both corn varieties at the grain-filing stage was significantly lower than that of PI ABS after N treatment. This could be due to a decline in PS II function under N deficiency at the grain-filling stage (early senescence). PS I cyclic electron transport, to a certain extent, compensates for the loss of photophosphorylation due to the reduced electron supply from PS II caused by N deficiency and early senescence. Compared with HZ4, the increase in cyclic electron transport in Q319 at the grain-filling stage was more significant and therefore improves the coordination of the two photosystems; this is one of the reasons for its increase of P n and yield at the mature stage. In summary, the effect of N on P n at the grain-filling stage and yield at the mature stage of corn is dependent on the stay-green phenotype. The reason that N causes an increase in P n is related to the enhanced coordination of PS II and PS I. After N fertilization, the coordination of these systems in the stay-green variety of corn increased significantly, and P n and grain yield increased dramatically, suggesting that these stay-green varieties possess high N efficiency. Due to genetic limitation in this respect, non-stay-green corn is insensitive to the regulation of N, indicating a low N efficiency phenotype. 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