Photoinhibition of Leaves with Different Photosynthetic Carbon Assimilation Characteristics in Maize ( Zea mays )

Strong light decreases the rate of photosynthesis and assimilates production of crop plants. Plants with different carbon reduction cycles respond differently to strong light stress. However, variation in photoinhibition in leaves with different photosynthetic characteristics in maize is not clear. In this experiment, we used the first leaves (with an incomplete C4 cycle) and fifth leaves (with a complete C4 cycle) of maize plants as well as the fifth leaves (C3 cycle) of tobacco plants as a reference to measure the photosynthetic rate (PN) and chlorophyll a parameters under strong light stress. During treatment, PN, the maximal fluorescence (Fm), the maximal quantum yield of PSII photochemistry (Fv/Fm), and the number of active photosystem II (PSII) reaction centers per excited cross-section (RC/CSm) declined dramatically in all three types of leaves but to different degrees. PN, Fm, Fv/Fm, and RC/CSm were less inhibited by strong light in C4 leaves. The results showed that maize C4 leaves with higher rates of photosynthesis are more tolerant to strong light stress than incomplete C4 leaves, and the carbon reduction cycle is more important to photoprotection in C4 leaves, while state transition is critical in incomplete C4 leaves.


Introduction
Strong light is an important factor that reduces photosynthetic activity and lim-its the production of assimilates in crop plants via a process called photoinhibition [1]. The longer the exposure to excess excitation energy, the more damage to the photosynthetic apparatus. To avoid this damage, plants have evolved a series of protective mechanisms [2] [3] [4] [5], including photochemical quenching, fluorescence quenching, and thermal dissipation of excess excitation energy.
Photochemical quenching is related to the activity of photosystem II (PSII) reaction centers (RC), the efficiency of the electron transfer chain, and the capacity of the photosynthetic carbon cycle. As the terminal destination of excitation energy, the photosynthetic cycle affects the amount of surplus excitation energy absorbed by leaves.
Based on the pathway of photosynthetic carbon fixation, higher plants are classified into three types: C 3 , C 4 , and CAM. In C 3 plants, photosynthesis operates in mesophyll cells (MC) via PSII and ribulose bisphosphate carboxylase/ oxygenase (Rubisco). C 4 plants evolved from C 3 plants [6] and have a higher carbon reduction efficiency. In typical C 4 plants, MC and vascular bundle sheath cells (BSC) in the leaves are arranged in specialized Kranz anatomy around vascular tissues. MC chloroplasts have higher PSII activity and lower Rubisco activity. In contrast, BSC chloroplasts have lower PSII activity and higher Rubisco activity [7] [8]. Additionally, C 4 photosynthetic enzymes are distributed in MC and BSC, which cooperate during C 4 photosynthesis.
The responses of plants with different photosynthetic pathways to strong light are different [9] [10] [11]. C 4 plants are less susceptible to strong light stress than C 3 plants [10]. The maximal photochemical efficiency of PSII (F v /F m ) declined more slowly in C 4 maize than that in C 3 plants under strong light [12], while the efficiency of the C 4 photosynthetic cycle varies in maize leaves at different positions. The first to third leaves of maize have not completed the differentiation of MC and BSC and thus have a less efficient C 4 cycle, with lower activity of C 4 photosynthetic enzymes in MC and higher activity of PSII in BSC [13] [14].
However, how these maize leaves differ in photoinhibition is not clear. Knowing this difference and its cause would help to understand the mechanisms of strong light defense in plants. In this paper, we investigated the differences in photoinhibition among the first (incomplete C 4 cycle) and fifth (complete C 4 cycle) leaves of maize and the fifth leaves (C 3 cycle) of the C 3 plant tobacco as a reference and analyzed the basis of the differences.

Experimental Materials
Maize hybrid Zhengdan958 (a widely used Chinese hybrid) was crossed by Zheng58 and Chang7-2 inbred at Experimental Station of Shenyang Agricultrual University in the summer of 2012. Tobacco K326 were from plant immunity institute of Shenyang Agricultrual University. Both maize and tobacco were grown in pots in a growth chamber. The photon flux density (PFD) on the plant canopy was 1000 μmol•m −2 ·s −1 from metal halogen lamps with a 14 h/10h light/dark cycle at 24˚C/22˚C (day/night). The first (M1) and fifth (M5) fully expanded leaves on maize plants and the fifth (T5) fully expanded leaves on tobacco were used for measurements.

Treatments
Plants were illuminated for 3 h at 28˚C and a PFD of 2000 μmol·m −2 ·s −1 as a strong light treatment. A distance of 0.5m above the top of plant were measured.
The white light source was 400 W SON-T AGRO lamps (Royal Dutch Philips Electronics Ltd., Amsterdam, Netherlands). Each treatment was repeated with six plants.

Photosynthetic Rate
Photosynthetic rate (P N ) was measured each hour during the light treatment using a potable photosynthesis system (CIRAS-1, PP-system, Hitchin, UK) in normal air from 8:00 am to 11:00 am.

Photorespiration Rate and Gross Photosynthetic Rate
The P n was measured at the end of the 3 h light treatment using the CIRAS-1 PP-system in normal air (21% O 2 + 75% N 2 + 380 μmol·mol CO 2 −1 ) and lowoxygen air (2% O 2 + 95% N 2 + 380 μmol·mol CO 2 −1 ). The photorespiration rate (P r ) was calculated as the difference between P N in low-oxygen and normal air, using the equation (Pn2%O 2 -Pn21%O 2 )/Pn2%O 2 [15]. The P N in low-oxygen air was designated the gross photosynthetic rate (GP N ).

Chlorophyll a Fluorescence Parameters
We measured chlorophyll a fluorescence each hour during the light treatment with a Hand-PEA (Hansatech Instruments Limited, UK). After 20 min of dark adaptation, all sample leaves were immediately exposed to a saturating light pulse (3000 μmol·m −2 ·s −1 ) for 2 s. The fluorescence transients in each darkadapted leaf were analyzed according to the JIP-test using the following parameters: 1) the initial fluorescence (F 0 ); 2) the maximal fluorescence (F m ); 3) the difference between F m and F 0 (F v ); 4) the maximal quantum yield of PSII photochemistry (F v /F m ); 5) the quantum yield of fluorescence dissipation (ΦD 0 ); and 6) the number of active PSII RC per excited cross-section (CS m ).

Statistical Analysis
Statistical analyses were performed using SPSS 11.5 (IBM, Chicago, IL, USA). Treatment means were subjected to two-way analysis of variance (ANOVA), and these values and their significant differences (measured by Duncan's significance test) are presented in the figures and table. Design of the experiments was completely randomized with six replications.

Photosynthesis
The three types of leaves had different P N values under control light conditions and varied in their responses to the strong light treatment ( Figure 1). Under control light, M5 showed the highest P N (22 μmol CO 2 ·m −2 ·s −1 ), followed by M1 (18 μmol CO 2 ·m −2 ·s −1 ) and T5 (14 μmol CO 2 ·m −2 ·s −1 ). Under strong light, all three types of leaves showed a decrease in P N , suggesting the occurrence of photoinhibition in all experimental materials. During the treatment period, P N of M5 declined slowly, by 6.8% in the first hour; M1 decreased more rapidly in the first hour (by 44.4%) and then more slowly. A similar pattern was observed in T5, but P N decreased more sharply (by 60.7%) in the first hour. During treatment, M5 maintained a consistently higher P N than did M1 and T5. These results suggested that C 4 leaves (M5) were more tolerant to strong light stress than leaves with an incomplete C 4 (M1) and C 3 leaves (T5).

Photorespiration and Gross Photosynthesis
The three types of leaves had different P r values at the end of the 3-h strong light treatment (Table 1). T5 showed the highest P r (2.87 μmol CO 2 ·m −2 ·s −1 ) and P r /GP N ratio (43.50%), followed by M1 (2.60 μmol CO 2 ·m −2 ·s −1 , 17.8%) and M5 (0.47 μmol CO 2 ·m −2 ·s −1 , 2.24%). GP N , the sum of P N and P r , indicates the amount Figure 1. Changes in net photosynthesis rate (P n ) in leaves with different photosynthetic characteristics during strong light treatments. The sample leaves were subjected to strong light (2000 μmol•m −2 •s −1 ) for 3 h. ▲, maize fifth leaves (complete C 4 cycle, M5); △, maize first leaves (incomplete C 4 cycle, M1); •, tobacco fifth leaves (C 3 cycle, T5). Mean ± SD of six replicates. Bars not seen are smaller than the size of the symbols. of energy consumed via carbon reduction and the oxidation cycle in plants. Similar to the pattern seen with P N , at the end of the treatment, M5 had the highest GP N (20.73 μmol CO 2 ·m −2 ·s −1 ), followed by M1 (14.57 μmol CO 2 ·m −2 ·s −1 ) and T5 (6.57 μmol CO 2 ·m −2 ·s −1 ). Despite the higher P r and P r /GP N under strong light stress, GP N in the C 3 leaves (T5) and incomplete C 4 leaves (M1) was still lower than that in the C 4 leaves (M5).

Fv/Fm and ΦD0
F v /F m describes the efficiency of the PSII photochemical reaction. As shown in  Illumimating treatments (hour)
An increase in ΦD 0 can protect PSII against photodamage. As Figure 2

Discussions
The light energy absorbed by leaves is mainly used to drive the photosynthetic carbon reduction cycle. Therefore, surplus energy is generated if carbon reduction is impeded or if light energy absorbed by leaves exceeds that consumed by carbon reduction. The resulting excess energy will lead to photoinhibition, that is, it impairs the photosynthetic apparatus and reduces the photosynthesis rate [1]. The amount of excess energy is related to photosynthetic efficiency. Under the same light intensity, leaves of C 4 plants photosynthesize more efficiently than leaves of C 3 plants, which means that more absorbed light energy flows into the carbon cycle and less excess energy is produced [10]. As a result, C 4 leaves will be less inhibited by strong light than C 3 leaves. In this study, under control light intensity, the C 4 leaves (M5) had the highest rate of photosynthesis, followed by leaves with an incomplete C 4 cycle (M1) and C 3 leaves (T5). Although photoinhibition occurred in all types of leaves under strong light, M5 leaves were more tolerant than M1 and T5 leaves. This result showed that the photosynthetic rate underlies photoinhibition defense in plants.
Photorespiration is a carbon oxidation cycle that consumes light energy like carbon reduction pathways [18]. Increased photorespiration rates have been observed under drought [19], high temperature [20], and strong light stress [9] and are regarded as an important mechanism to prevent photoinhibition. In the present study, a decline in photosynthesis occurred in all types of leaves at the end of the light treatment, but the levels of decline in M1 and T5 were greater than in M5, and their photorespiration rates and the ratio of photorespiration to gross photosynthesis were much higher than those in M5. These results suggested that photorespiration played a larger role in photoinhibition defense in M1 and T5 leaves. Although the photorespiration rates increased in M1 and T5 leaves, the total energy consumption via carbon reduction and oxidation did not increase during photoinhibition. The gross photosynthetic rates at the end of light treatment were significantly lower than at the beginning of treatment. This means that the rise in energy consumption owing to photorespiration only partially compensates for the decline caused by photosynthesis. For C 4 leaves, although the photorespiration rate is very low, the C 4 cycle consumes more energy than the C 3 cycle and reduces the energy surplus.
F v /F m is the photochemical reaction efficiency of PSII and can be used to describe the state of the PSII RC photodamage [17].

Conclusion
In conclusion, C 4 maize leaves, with a higher rate of photosynthesis, are more tolerant to strong light stress than incomplete C 4 leaves, and their PSII RC are less susceptible to intense radiation. In photoprotection, the carbon reduction cycle has an important role in C 4 leaves, while state transition is pivotal in incomplete C 4 leaves. Further investigation will be required to explain the underlying mechanisms of PSII reaction center susceptibility to strong light in maize incomplete C 4 leaves. Interestingly, at present some genus contains both C3, C4 and C3-C4 intermediate species [26] [27] [28] [29], and some genus changes from C3 to C4 in different environments [30] [31]. The studies of these materials under strong light will provide more direct adaptability differences between C3 and C4 pathway.