Effects of nitrogen form on growth, CO₂ assimilation, chlorophyll fluorescence, and photosynthetic electron allocation in cucumber and rice plants

Effects of nitrogen form on growth, CO 2 assimilation, chlorophyll fluorescence, and photosynthetic electron allocation in cucumber and rice plants 1 first-author †E-mail:yanhongzhou@zju.edu.cn †E-mail:yanhongzhou@zju.edu.cn yanhongzhou@zju.edu.cn 1 1 1 1 1 1 2 Department of Horticulture, Zhejiang University, Hangzhou 310029, China Department of Horticulture, Zhejiang University, Hangzhou 310029, China Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310029, China Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310029, China Cucumber and rice plants with varying ammonium (NH 4 + ) sensitivities were used to examine the effects of different nitrogen (N) sources on gas exchange, chlorophyll (Chl) fluorescence quenching, and photosynthetic electron allocation. Compared to nitrate (NO 3 − )-grown plants, cucumber plants grown under NH 4 + -nutrition showed decreased plant growth, net photosynthetic rate, stomatal conductance, intercellular carbon dioxide (CO 2 ) level, transpiration rate, maximum photochemical efficiency of photosystem II, and O 2 -independent alternative electron flux, and increased O 2 -dependent alternative electron flux. However, the N source had little effect on gas exchange, Chl a fluorescence parameters, and photosynthetic electron allocation in rice plants, except that NH 4 + -grown plants had a higher O 2 -independent alternative electron flux than NO 3 − -grown plants. NO 3 − reduction activity was rarely detected in leaves of NH 4 + -grown cucumber plants, but was high in NH 4 + -grown rice plants. These results demonstrate that significant amounts of photosynthetic electron transport were coupled to NO 3 − assimilation, an effect more significant in NO 3 − -grown plants than in NH 4 + -grown plants. Meanwhile, NH 4 + -tolerant plants exhibited a higher demand for the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for NO 3 − reduction, regardless of the N form supplied, while NH 4 + -sensitive plants had a high water-water cycle activity when NH 4 + was supplied as the sole N source. Nitrogen form Photosynthetic electron allocation Alternative electron flux Nitrate reductase Introduction 1 Nitrogen (N) is one of the most important nutrients affecting the growth, development, yield, and fruit quality of plants (Fernandes and Rossiello, 1995 1995 ; Gerendás et al., 1997 1997 ). It is required in the greatest quantities at each stage of plant growth during which N level markedly affects the amount of Rubisco content, and therefore photosynthesis (Evans and Terashima, 1988 1988 ; Evans, 1989 1989 ). In addition to the amount supplied, the form of N available has significant effects on the growth and photosynthesis of plants (Cramer and Lewis, 1993 1993 ), and preference for the N sources nitrate (NO 3 − ) and ammonium (NH 4 + ) varies between plant species (Baker and Mills, 1980 1980 ). For example, NH 4 + was reported to be superior to NO 3 − for the growth of rice and conifer (Gerendás et al., 1997 1997 ; Britto and Kronzucker, 2002 2002 ; 2004 2004 ), whereas some plant species such as cucumber, tomato, eggplant, maize, barley, wheat, and bean prefer NO 3 − to NH 4 + nutrition (Bloom et al., 1989 1989 ; Kafkafi, 1990 1990 ; Cramer and Lewis, 1993 1993 ; Claussen and Lenz, 1995 1995 ; Roosta and Schjoerring, 2007 2007 ). Meanwhile, some plant species show enhanced growth when cultivated in both NO 3 − and NH 4 + conditions, compared with growth under sole NO 3 − and NH 4 + conditions (Errebhi and Wilcox, 1990 1990 ). Furthermore, environmental conditions, such as temperature, light intensity, soil water content, pH, and ions in the growth medium, also affect plant sensitivity to NO 3 − and NH 4 + nutritions (Kotsiras et al., 2005 2005 ). For example, the presence of potassium (K + ) may alleviate NH 4 + toxicity (Szczerba et al., 2006 2006 ). The management of the amount and form of N in the nutrient solutions of crops is a complex and important part of successful crop production system. In the last two decades, a large number of studies have focused on the effects of NO 3 − and NH 4 + nutritions on plant photosynthetic physiology, which is one of the most important metabolic processes affected by N form (Geiger et al., 1999 1999 ; Guo et al., 2002 2002 ; Lopes et al., 2004 2004 ; Lopes and Araus, 2006 2006 ). The effect of N form on photosynthesis is manifested in differences in the stomatal conductance, and the intercellular carbon dioxide (CO 2 ) partial pressures of NO 3 − and NH 4 + -supplied plants, but the results are not consistent between different plant species. For example, decreases in CO 2 assimilation due to stomatal closure were observed in wheat and barley when NH 4 + was present (Lopes et al., 2004 2004 ; Lopes and Araus, 2006 2006 ), but plants such as French bean and tobacco supplied with NH 4 + had a higher CO 2 assimilation rate and stomatal conductance than those supplied with NO 3 − (Geiger et al., 1999 1999 ; Guo et al., 2002 2002 ). N form also has different effects on the Calvin cycle activity, indicated by the Rubisco content or capacities of Rubisco carboxylation and ribulose-1,5-bisphosphate regeneration. Raab and Terry ( 1994 1994 ; 1995 1995 ) showed that the leaf Rubisco content in NH 4 + -grown sugar beet plants was significantly higher than that in NO 3 − -grown plants, which was similar to those found in tobacco plants by Terce-Laforgue et al. ( 2004 2004 ). Although the effects of N form on stomatal factors and the Calvin cycle in the photosynthesis process have been extensively investigated, relatively few studies have focused on the relationship between photosynthetic electron transport and electron utilization in photosystem II (PSII) in plants with different sensitivities to NO 3 − and NH 4 + nutritions. It is well known that light energy is mainly used by the photosynthetic linear electron transport to produce assimilatory powers [adenosine triphosphate (ATP) and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)] for CO 2 assimilation. When plants absorb energy that exceeds their photosynthetic capacity, they activate some physiologic processes to protect the chloroplast from photodamage, such as xanthophyll cycle-dependent heat dissipation from antennae in PSII, the D1 repair cycle, photorespiration, N assimilation, and the operation of the water-water cycle (Osmond and Grace, 1995 1995 ; Demmig Adams and Adams, 1996 1996 ; Zhou et al., 2004 2004 ; Kanervo et al., 2005 2005 ; Brück and Guo, 2006 2006 ; Wang et al., 2008 2008 ). There are marked differences in the photo-energy consumption and the supply of reducing equivalents between NO 3 − and NH 4 + nutritions. In the processes of NO 3 − uptake, transport, reduction, and assimilation, almost 32 mol photons per mol N were consumed, while the energy cost for NH 4 + is only 9.45 mol photons per mol N (Raven, 1985 1985 ). Accordingly, NO 3 − assimilation may represent a considerable additional reductant sink increasing NADPH consumption (Guo et al., 2007 2007 ). Substantial differences in electron utilization have been found under sole NO 3 − and NH 4 + conditions. However, allocation of photosynthetic electrons to CO 2 , O 2 , and NO 3 − reduction and cyclic electron flow, as influenced by N form, remain poorly understood. In this work, we studied the relationship between the characteristics of NO 3 − /NH 4 + -sensitivity and photosynthetic electron utilization by calculating the electron flux in PSII. Materials and methods 1 Plant materials and treatments 2 Cucumber ( Cucumis sativus L. cv. Jinyou No. 30) seeds were first sown in a substrate containing a mixture of soil and perlite (1:1, v:v) in 72-cell plastic trays. Groups of six seedlings at the two-leaf stage were transplanted into 15-L plastic containers filled with a nutrient solution comprising the following nutrients (in units of mmol/L): 2.0 Ca(NO 3 ) 2 , 2.0 KNO 3 , 2.5 KH 2 PO 4 , 2.0 MgSO 4 , 29.6×10 −3 H 3 BO 3 , 10×10 −3 MnSO 4 , 50×10 −3 Fe-Na 2 EDTA, 1.0×10 −3 ZnSO 4 , 0.05×10 −3 H 2 MoO 4 , and 0.95×10 −3 CuSO 4 . At the same time, rice ( Oryza sativa L. cv. Taiyou No. 1) seeds were germinated in Petri dishes moistened with distill water. Five to seven days later, six seedlings were transplanted into the same containers (15 L) containing a nutrient solution comprising the following nutrients (in units of mmol/L): 3.0 NH 4 NO 3 , 0.6 Na 2 HPO 4 , 0.3 K 2 SO 4 , 0.4 MgCl 2 , 0.2 CaCl 2 , 45×10 −3 Fe-EDTA, 50×10 −3 H 3 BO 3 , 9×10 −3 MnSO 4 , 0.7×10 −3 ZnSO 4 , 0.3×10 −3 CuSO 4 , and 0.1×10 −3 H 2 MoO 4 . The nutrient solution was changed once a week, and its pH was adjusted with 1 mol/L HCl to 6.5 for cucumber plants and 5.5 for rice plants. The environmental conditions in the greenhouse were as follows: a 12-h photoperiod, temperatures of 28/22 °C (day/night), and an average photosynthetic photon flux density (PPFD) of 500 µmol/(m 2 ∙s). The treatments, which differed only in the source of N, were imposed at the three- to four-leaf stages for cucumber and at the four-leaf stage for rice. Two N treatments at constant N concentrations were administered: 6.0 mmol/L N as NO 3 − only or as NH 4 + only for cucumber and rice plants, respectively. The solutions were renewed every two days to minimize variations in pH and nutrient concentrations. Seven days after the treatments were started, gas exchange and chlorophyll (Chl) a fluorescence analyses were carried out. The roots and shoots of cucumber and rice plants were then harvested and oven-dried at 80 °C for three days for the determination of the dry weight of plants. Meanwhile, the leaves were also sampled, snap frozen in liquid nitrogen, and stored at −86 °C until their use for the analysis of NO 3 − reductase activity. Each treatment had 18 plants with four replicates. Gas exchange and Chl a fluorescence measurements 2 Gas exchange and Chl a fluorescence were determined simultaneously on the 2nd leaf with an LI-6400 portable open gas exchange system equipped with an LI-6400 leaf chamber Chl fluorometer attachment (Li-Cor, Lincoln, NE, USA). Leaf net photosynthesis rate ( P n ), stomatal conductance ( g s ), intercellular CO 2 concentration ( c i ), and transpiration rate ( T r ) were measured at 25 °C with PPFD of 500 µmol/(m 2 ∙s) and CO 2 concentration of 360 μmol/mol. Meanwhile, steady state fluorescence yield ( F s ) and light-adapted maximum fluorescence ( F m ′) were recorded for the calculations of the parameters including the quantum efficiency of PSII ( Ф PSII ), photochemical quenching coefficient ( q P ), and the efficiency of excitation capture by open PSII centers ( F v ′/ F m ′) (Zhou et al., 2004 2004 ). The plants were then placed in a dark room for 20 min to determine the maximum photochemical efficiency of PSII ( F v / F m ) (Zhou et al., 2004 2004 ). Electron transport analyses 2 The total electron flux in PSII ( J PSII ) was measured according to the methods of Miyake and Yokota ( 2000 2000 ), and can be calculated as: J PSII = Ф PSII × α ×PPFD, where α is the product of the absorbance coefficient and ratio of allocation of excitation energy to PSII. As Miyake and Yokota ( 2000 2000 ) suggest, J PSII can be divided into four components, which can be determined by measuring P n and chlorophyll parameters under both 21% and 2% O 2 conditions. The four components are electron flux for photosynthetic carbon reduction ( J c ), electron flux for photo-respiratory carbon oxidation ( J o ), O 2 -dependent alternative electron flux [ J a (O 2 -dependent)], and O 2 -independent alternative electron flux [ J a (O 2 -independent)]. Nitrate reductase (NR) activity estimation 2 The activity of NR in the leaves was measured according to Li et al. ( 2006 2006 ) and was expressed as nanomoles of NO 2 − per gram fresh weight (FW) per minute. Statistical analysis 2 All the measurements were repeated at least four times from different individual plants. Data were statistically analyzed using analysis of variance (ANOVA), and tested for significant treatment differences using the Duncan′s multiple range test at P < 0.05 with Origin 7.5 Software (OriginLab, Northampton, MA, USA). Results 1 Effect of N form on plant biomass 2 Cucumber plants supplied with NH 4 + -N showed slower growth compared to those grown with NO 3 − -N. By the final harvest, 7 d after onset of treatment, the dry mass of shoot, root, and total plant for cucumber NO 3 − -grown plants was 79.4%, 144.8%, and 138.7% higher, respectively, than that of NH 4 + -grown plants (Table 1 1 ). In contrast, the N form had little effect on rice plant growth, and there were no significant differences in shoot, root, and total plant biomass accumulations 7 d after the treatment. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments within the same species are indicated by different letters 2 Species 2 N form 3 Dry mass (g/plant) Shoot Root Total plant 2 Cucumis sativus NO 3 − 3.05±0.09a 0.39±0.03a 4.44±0.11a NH 4 + 1.70±0.14b 0.16±0.01b 1.86±0.09b 5 2 Oryza sativa NO 3 − 11.94±0.69a 4.83±0.55a 16.77±0.32a NH 4 + 14.19±2.46a 4.70±0.79a 18.89±1.77a Effect of N form on gas exchange 2 Gas exchange analysis showed that the N form had significant effects on gas exchange in cucumber plants, but not in rice plants. As shown in Fig. 1 1 , NH 4 + nutrition decreased P n by 38.5%, compared with the NO 3 − nutrition in cucumber plants. At the same time, lower g s , c i , and T r were found in NH 4 + -grown plants, which decreased by 64.3%, 14.1%, and 43.1%, respectively, compared with their NO 3 − -grown plants. However, the N form had a minor effect on rice plants, since the changes in these parameters were almost independent of the N form supplied. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments within the same species are indicated by different letters Effect of N form on Chl a fluorescence 2 The changes in F v / F m , Ф PSII , q P , and F v ′/ F m ′ as influenced by N form in cucumber and rice leaves are shown in Fig. 2 2 . For cucumber leaves, the values of F v / F m value declined from 0.82 for the NO 3 − -grown plants to 0.75 for the NH 4 + -grown plants, indicating that a slight photo-inhibition occurred in NH 4 + -grown plants. Significant reductions in Ф PSII and q P were also found in NH 4 + -grown cucumber leaves, which decreased by 22.2% and 20.3%, respectively, compared with their NO 3 − -treated leaves. However, similar values of F v ′/ F m ′ were observed in cucumber plants between NO 3 − and NH 4 + treatments, which suggested that the decline of Ф PSII in cucumber plants was due to declines of q P alone, with no contribution from F v ′/ F m ′. In sharp contrast, there was no significant effect of the N form on the Chl a fluorescence parameters Ф PSII , q P , and F v ′/ F m ′ in rice. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments within the same species are indicated by different letters Effect of N form on allocation of electron flux in PSII 2 For NO 3 − -grown cucumber plants, J c , J o , J a (O 2 -dependent), and J a (O 2 -independent) accounted for 73.0%, 13.4%, 6.1%, and 7.5%, respectively, of the total electron flux in PSII ( J PSII ) (Table 2 2 ). Compared to NO 3 − -grown cucumber plants, J PSII , J c , J o , and J a (O 2 -independent) decreased by 19.8%, 22.8%, 23.5%, and 45.9%, respectively, while J a (O 2 -dependent) increased by 56.1% in NH 4 + -grown cucumber plants. J a (O 2 -independent) accounted for 54.9% of the total J a for NO 3 − -grown cucumber plants compared to 29.6% for the NH 4 + -grown cucumber plants. Interestingly, the N form had negligible effects on the allocation of electron flux in PSII except that J a (O 2 -independent) for NO 3 − -grown rice plants was 92.8% higher than that for NH 4 + -grown rice plants. At last, J a (O 2 -independent) accounted for 78.7% of the total J a for NO 3 − -grown rice plants as compared to 53.3% for the NH 4 + -grown rice plants. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments within the same species are indicated by different letters Species N form J PSII (μmol e/(m 2 ·s)) J c (μmol e/(m 2 ·s)) J o (μmol e/(m 2 ·s)) J a (O 2 -dependent) (μmol e/(m 2 ·s)) J a (O 2 -independent) (μmol e/(m 2 ·s)) 2 Cucumis sativus NO 3 − 106.1±5.8a 77.4±5.0a 14.2±0.3a 6.5±1.0b 7.9±0.3a NH 4 + 85.1±2.9b 59.8±2.6b 10.9±0.9b 10.2±0.9a 4.3±0.7b 7 2 Oryza sativa NO 3 − 111.9±2.9a 80.1±2.1a 18.9±1.3a 2.8±0.6a 10.2±1.3a NH 4 + 107.4±2.4a 80.0±1.9a 17.5±0.5a 4.6±0.8a 5.3±0.8b Effect of N form on NR activity 2 Although cucumber and rice plants received the same N level, the leaves of rice plants exhibited higher NR activity when grown with either N form. Meanwhile, NR activity was consistently higher in leaves of the NO 3 − -grown plants compared to those of NH 4 + -grown plants (Fig. 3 3 ). Interestingly, NR activity was near zero in leaves of NH 4 + -grown cucumber plants, but high NR activity was observed in NH 4 + -grown rice plants. Finally, NR activity in the NO 3 − -grown leaves was 72.6-fold and 1.7-fold higher than that in the NH 4 + -grown leaves for cucumber and rice plants, respectively. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments within the same species are indicated by different letters Discussion 1 Our results provide clear evidence of significant species variation in the susceptibility of plant growth to the N form supplied. In this study, a greater dry mass of shoot, root, and total plant was observed in cucumber under growth conditions using NO 3 − as the sole N source, compared to NH 4 + as the sole N source (Table 1 1 ). In contrast, there was no significant difference in biomass accumulation in rice between NO 3 − and NH 4 + nutritions. Accordingly, rice is an NH 4 + -tolerant plant species, while cucumber is an NH 4 + -sensitive plant species. This is in agreement with earlier studies that cucumber plants are well adapted to NO 3 − as the predominant N form (Roosta and Schjoerring, 2007 2007 ), and that rice plants appear to be adapted to either NH 4 + or NO 3 − as the predominant N form (Britto and Kronzucker, 2002 2002 ). Studying the responses of CO 2 assimilation, Chl a fluorescence, and photosynthetic electron allocation to NO 3 − or NH 4 + nutrient will help in revealing the species differences in NO 3 − /NH 4 + sensitivity. Photosynthesis is one of the primary processes responsible for plant growth, and photosynthetic metabolism will be finely regulated to adapt growth under different N source conditions (Claussen and Lenz, 1995 1995 ; Brück and Guo, 2006 2006 ). In fact, significant effects of the N form on photosynthesis were found in cucumber plants. As shown in Fig. 1 1 , NH 4 + -grown cucumber plants had a significantly lower P n compared with NO 3 − -grown cucumber plants, accompanied by lower g s and c i . This is in agreement with earlier observations that NH 4 + decreased stomatal conductance and transpiration in French beans (Guo et al., 2002 2002 ) and tobacco (Lu et al., 2005 2005 ). However, the N source had no significant effect on the photosynthesis parameters in rice plants in other studies (Guo et al., 2008 2008 ; Li et al., 2009 2009 ). Similar to the changes observed with gas exchange analysis, a higher Ф PSII value was found in cucumber plants grown with the NO 3 − -N source, but no significant differences in Ф PSII were observed between NO 3 − -N and NH 4 + -N sources in rice. Increases in the rate of photosynthetic linear electron transport were also found in barley, pea, and wheat when grown with NO 3 − rather than NH 4 + as the sole N source (Bloom et al., 1989 1989 ; 2002 2002 ; de la Torre et al., 1991 1991 ). It is worthwhile to note that cucumber plants experienced photo-inhibition when they were grown with NH 4 + as the N source, as has been observed in the NH 4 + -grown French bean plants (Zhu et al., 2000 2000 ). Several studies have found that NH 4 + -sensitive plant species exhibit increased photo-respiration to protect the plants from the effects of photodamage (Zhu et al., 2000 2000 ). However, our study showed that photo-respiration (i.e., J o ) was independent of the N source, suggesting that other photo-protection mechanisms are involved in NH 4 + -sensitive plants. It has been well established that excess NADPH in the light reaction, which is not completely used during CO 2 assimilation, is either exported from the chloroplasts or consumed by the reductant sinks (e.g., leaf NO 3 − assimilation or water-water cycle) (Brück and Guo, 2006 2006 ). Previous studies have indicated that a substantial portion of photosynthesis or respiration electron transport generates reducing equivalents for NO 3 − reduction rather than for carbon fixation (Bloom et al., 1989 1989 ; Noctor and Foyer, 1998 1998 ; Lewis et al., 2000 2000 ), and thus, NO 3 − reduction may represent a considerable reductant sink (Brück and Guo, 2006 2006 ). However, this sink for NADPH would not be expected when NO 3 − is absent from the leaves, and it would decrease with a decrease in the NR reaction. Although J a (O 2 -independent) driven by NO 3 − reduction has been suggested as an important sink for electrons in chloroplasts (Miyake and Yokota, 2000 2000 ; Zhou et al., 2004 2004 ), few studies have attempted to quantitate this contribution. With the methods developed by Miyake and Yokota ( 2000 2000 ), we divided the total J PSII into J c , J o , J a (O 2 -dependent), and J a (O 2 -independent). In this model, J a (O 2 -dependent) and J a (O 2 -independent) were most driven by the water-water cycle and NO 3 − reduction, respectively (Miyake and Yokota, 2000 2000 ). J a (O 2 -independent) accounted 5.0% to 7.5% of the total J PSII for plants fed with NO 3 − -N and NH 4 + -N in cucumber, and 4.9% to 9.1% for plants fed with NO 3 − -N and NH 4 + -N in rice (Table 2 2 ). It is interesting to note that J a (O 2 -independent) was detected in both species plants fed with NH 4 + -N and there is a close relation between NR activity and J a (O 2 -independent) (Table 2 2 ; Fig. 3 3 ). It is likely that NH 4 + absorbed was converted to NO 3 − in roots or shoots during growth since this reaction is frequent in plants. It is also possible that other O 2 -independent metabolism pathway was also involved in the utilization of NADPH and ATP. Taken together, these results suggest that NR-driven J a (O 2 -independent) works as an important NADPH sink in electron transport during photosynthesis. When the combined operation of the electron-consuming processes described above fails to dissipate safely, all of the electrons generated by PSII and the water-water cycle activity [ J a (O 2 -dependent)] will increase. In this study, a higher J a (O 2 -dependent) for NH 4 + -grown cucumber plants was observed compared to NO 3 − -grown plants, which indicates a greater rate of electron transport to O 2 . Compared with cucumber plants, rice exhibited a lower proportion of J a (O 2 -dependent), irrespective of the N form, and almost the same values in J a (O 2 -dependent) were found in NO 3 − and NH 4 + nutrition treatments. Normally, enhancement of reactive oxygen species (ROS) generation was correlated with the increase of J a (O 2 -dependent), as has been found in chilled cucumbers (Zhou et al., 2004 2004 ). In fact, impaired plant growth together with photoinhibition occurred in cucumber leaves when grown with NH 4 + as the sole N source. It is likely that NH 4 + -induced ROS generation leads to photo-inhibition and impairs plant growth. Further work on NH 4 + -N source and the associated N metabolism in roots and leaves will help us to elucidate how photo-inhibition and the water-water cycle activity are altered by the N form. 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Different tolerance to light stress in NO3−- and NH4+-grown Phaseolus vulgaris L 10.1631/jzus.B1000059 Project supported by the National Basic Research Program (973) of China (No. 2009CB119000), the National High-Tech R&D Program (863) of China (No. 2008BADA6B02), and the National Natural Science Foundation of China (Nos. 30771471 and 30972033) 2010-02-23 2011-01-10 2010-08-08 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 12