Abstract	Results	Fig. 1
Introduction	Discussion	Fig. 2
Material and Methods	References	Fig. 3

Symbols and abbreviations: HL - plants or leaves cultivated under high light, LL - plants or leaves cultivated under low light, NO₃ - nitrate, NH₄ - ammonium, chl - chlorophyll, PS II - photosystem II, LHC - light harvesting complex, NR - nitrate reductase

Introduction Numerous experiments have yet been done to investigate the sensitivity to photoinhibition of variety of plant species under several stresses. Effects of nutrient supply on the sensitivity to photoinhibition has been studied in majority of casesin plants grown at different nutrient availability. This is true especially for nitrogen (N); e.g. Marques da Silva et Arrabaca (1992). Surprisingly, only few studies dealing with photoinhibition as affected by nitrogen supply took into account different form of N: nitrate - NO3 , ammonium - NH4. This can not be omitted because the amount and form of available N varies dynamically in the field in dependence on soil humidity and temperature. In this study, we focused on sycamore (Acer pseudoplatanus L.), a species quite abundant in mixed temperate forests in Europe mainly in highlands and mountainous locations. It is a prospective tree species to be planted instead of Norway spuce at deforested mountainous areas influenced by heavy air pollutants impact. Seedlings of sycamore are adaptable to shade. Typically, they are capable to survive substantial part of their lifetime in forest understory. Thus a question arises how the species will perform at open deforested areas when exposed to high irradiance, low soil pH, and changing N availability. Different form of N supplied combined with low pH of cultivation medium leads to differences in uptake rates of N, and other minerals, basic cations in particular (Gloser and Gloser 1996). Those differences likely lead to altered rate of protein synthesis in all plant parts, and consequently to altered amount of pigment-protein complexes of thylakoid membranes of chloroplasts. Also the amount of the main photosynthetic enzyme (Rubisco) might be affected in this way. We hypothesized that those alternations may lead to changes in functioning of photosystem II and consequently to different sensitivity of A. pseudoplatanus seedlings to photoinhibition. In this study, we addressed two questions: (1) Is the effect of exclusive NO3/NH4 nutrition on PS II diferent in high light- compared to low light-cultivated plants?, (2) Does form of N supplied to seedlings alter the sensitivity of A. pseudoplatanus to photoinhibition? Material and Methods Experimental plants Seeds of sycamore (A. pseudoplatanus L.) were collected in autumn 1996 and stored at 5 o C. In spring 1997, the seeds were germinated in dark between two sheets of wet paper. Germinating plants were cultivated in moist granulated PE for 4 d and then replanted to a modified Hoagland solution where cultivated for following 60 d under pH of 4.8 and different source of nitrogen. The sole source of nitrogen was either nitrate (0.2 mM N - NO3) or ammonium (0.1 mM N - NH4). The plants were grown in a climatic chamber under controlled air temperature (day/night: 18/22 o C), humidity (85 %), and two irradiances (high light = HL, low light = LL). In the cultivation under HL, the plants were exposed to 400 (µmol m-2 s-1 incident on the top of plants. In the cultivation under LL, the irradiance was 35 (µmol m-2 s-1). Chlorophyll, nitrate , and nitrate reductase content determination Fully developed leaves were colected from 60-d-old seedlings and cut in pieces of constant leaf area (LA). Chl extract of was made in 80 % acetone and absorptances in 663 and 640 nm were measured using an UV-B spectrophotometr (UV-B 1601, Shimadzu). Chl content on LA basis was then calculated according to Arnon (1949) using extract absorbances in 663 and 640 nm. Nitrate reductase (NR) activity in root and leaf tissues were determined in vivo by using the method of Jaworski (1971) as modified by Rothstein et al (1995). Pieces of leaves or roots were placed into a NRA buffer ( 0.1 M NaH2PO4 , 0.04 M KNO3, and propanol). The samples were shortly infiltrated in vacuum and incubated in dark and temperatue of 30 oC for 1 h. Incubation was stoped by a 5 min exposure to boiling water. Thereafter, 0.5 ml 1% sulphanylamide and 0.5 ml 0.1% N-naphtylethylendiamin dihydrochloride were added to the samples. After 15 min, the absorbance at 535 nm was measured using a UV-B spectrophotometer (Shimadzu, Japan). NR activity was then determined using a calibration curve). Content of nitrate (NO3) in leaf tissues was determined by a N selective elecrode (Crytur, CR) in extract. NO3 was extracted from homogenized leaf tissue in 10 % AlNO3 according to Senkyr and Petr (1979). Photoinhibition and chl fluorescence measurements Before photoinhibition, Kautsky kinetics of chlorophyll fluorescence was measured using a PAM 2000 fluorometer (Walz , Germany) and basic parameters were calculated: Fv/Fm, quantum yield of PS II (Yield PS II), photochemical (qP) and nonphotochemical chl fluorescence quenching (qN) using the equations described elswhere: Fv/Fm = ( Fm - Fo ) / Fm qN = ( Fm - F'm ) / ( Fm - Fo ) qP = ( F'm - F) / ( Fm' - Fo ) where, Fo and Fm are the values of backgroud and maximum dark-adapted chl fluorescence, Fs is steady-state chl fluorescence under actinic light, and F'm is maximum chl fluorescence after saturation pulse applied under actinic light. Light intensities and durations are described below. Photoinhibition was induced by a 40 min exposure of plants to strong light provided by a 1000 W halogen lamp (2300 µmol m-2 s-1 incident on upper leaf surface for HL, 1 900 (µmol m-2 s-1 for LL plants). During exposure to light, air temperature was maintained constant (25 oC). Once before and several times after exposition to photoinhibitory light, chl fluorescence parameters were measured using a pulse-amplitude modulated fluorometer with attached fiberoptics probe and leaf clips. Background (Fo) and maximum (Fm) chl fluorescence was measured by an application of saturation pulse on 15 min dark-adapted leaves. Thereafter, leaf was exposed to actinic light of 120 (mol m-2 s-1 for 5 min and another saturation pulse was applied in order to asses quantum yield of PS II (Yield PS II*), photochemical (qP*) and nonphotochemical chl fluorescence quenching (qN*). Then, actinic light was switched on, followed by a single far red pulse after 10 s, and a saturation pulse after following 30 s in order to asses the components of nonphotochemical quenching: energy quenching (qE*) and the quenching related to photoinhibition of photosynthesis and state transition regulated via phosphorylation of LHC II (qI+T*). The following equations were used for calculations: qN* = ( FmB - F'm ) / (FmB - Fo ) qT+I* = ( FmB - F''m ) / (FmB - Fo ) qE* = ( F''m - F'm ) / (FmB - Fo ) where FmB is maximum dark-adapted chl fluorescence of the sample before photoinhibitory treatment, Fo is dark-adapted minimum chl fluorescence on photoinhibited sample, F'm is maximum chl fluorescence on photoinhibited sample after saturation pulse under actinic light, and F''m is maximum chl fluorescence on photoinhibited sample after saturation pulse applied when actinic light is swithced off. Growth conditionsChlorophyll aChlorophyll bChlorophyll tot g m-2 NO3 HL0.2280.0860.314 0.0240.0140.036 NH4 HL0.1570.0610.218 0.0110.0070.017 NO3 LL0.3070.1190.426 0.0250.0090.034 NH4 LL0.2800.1070.386 0.0260.0100.036 Table 1. Chlorophyll content in sycamore seedlings supplied during growth by either NO3 or NH4 ions as sole source of nitrogen. HL indicates plants grown under the irradiance of 400 µmol m-2 s-1, LL indicates plants grouwn under 35 µmol m-2 s-1. The values (bold font)are means of 4 replicates with standard deviations (normal font)

Nitrate reductase activity was found allways higher in roots than in leaves. Absolute maximum of nitrate reductase activity was found in roots of NO3-cultivated LL plants (22.2 µg NO2 g-1 FW h-1). Generally, the roots had significantly higher nitrate reductase content (by 20-94 percent) than leaves. Nitrate content (NO3-) in leaves was affected by the form of N nutrition. NO3-supplied plants exhibited much higher NO3- content than NH4-supplied plants: 3.7 fold higher in HL and 3.2 fold in LL plants. Cultivation of A. pseudoplatanus in LL compared to HL led to a 2.7 fold increase in NO3- content: 2.7 fold increase.

NO3 NO3 NH4 NH4 High Light Low Light High Light Low Light NO3-( mg g-1 DW ) 1.71 4.64 0.46 1.44 St. dev. 0.65 1.31 0.06 1.27 Table 2. Content of NO3- in the leaves of A. pseudoplatanus plants cultivated under nitrate (NO3) or ammonium (NH4) nutrition and two levels of irradiance: High Light = 400 µmol m-2 s-1, Low Light = 35 µmol m-2 s-1.

Effects of NO3 and NH4 nutrition on chl fluorescence parameters

30-d-old HL plants exhibited an increase in Fv/Fm in NH4-cultivated compared to NO3-cultivated individuals. LL plants in contrast, showed no difference in Fv/Fm between NH4 and NO3-treatments (Table 3). LL cultivation led to an increase of Fv/Fm both in the presence of NH4 and NO3 in substrate. qP in NH4- compared to NO3-cultivated HL plants exhibited slight decrease while no change was apparent between the two in LL plants. In NH4- and NO3-cultivated plants, qP showed a marked decrease in LL compared to HL plants. In HL plants, NH4 compared to NO3 nutrition increased qN (NH4: 0.800, NO3: 0.321).

Growth conditions Fv/Fm ratio qP photoch. q. qN non-photoch. q. NO3 HL 0.755 0.916 0.321 0.007 0.008 0.039 NH4 HL 0.766 0.878 0.800 0.006 0.088 0.052 NO3 LL 0.784 0.259 0.497 0.006 0.055 0.122 NH4 LL 0.784 0.262 0.488 0.007 0.070 0.097 Fig.1). While full (100 %) recovery was observed in NH4 plants 2 h after photoinhibitory treatment, full recovery was not reached in NO3 HL plants even after 5 h (about 90 %). In LL plants, Fv/Fm decreased to 40-50 % after photoinhibitory treatment (slightly lower values in NH4 plants) and the recovery was similar in NO3- and NH4-cultivated plants. After 5 h, a 65 % recovery was reached in NO3 and NH4-cultivated plants, respectively. Photoinhibitory treatment increased qN* both in NO3- and NH4-supplied plants. The increase was much more pronounced in LL compared to HL plants (Fig.3. After photoinhibitory treatment, qN* increased mainly due to increase in qI*, proportion of which represented 82-85 % of qN* in HL, and 93-95 % in LL plants. Recovery to the initial proportion between qE* and qT+I* was reached after 2 h in NH4 HL plants while much slower recovery was apparent in NO3 HL plants. In LL plants, no recovery of qE* to qT+I* proportion was seen even after 5 h. Generally, in spite of the fact that qN* showed slight recovery after 5 h (see Fig. 2), the proportion of qT+I* remained high throughout whole recovery period (with an exception of NH4 HL plants). Discussion Increase of chl a, chl b, and total chl contents in LL compared to HL plants is a general phenomenon related to promoted synthesis of LHC chlorophylls in low light-cultivated plants, well documented e.g. for forest understory compared to gap plants (e.g. García-Nunez et al. 1995). Decrease in chl contents induced by NH4 nutrition suggests that N available in leaves for protein synthesis might be limited in NH4-cultivated plants. In this concept, a synthesis of pigment-protein complexes, LHCs in particular, is reduced which results in a decrease in total chlorophyll content. This is, however, not very likely in A. pseudoplatanus since the earlier observations (Barták, unpublished data) showed rather increase than decrease in total N content in the leaves of NH4-cultivated compared to NO3-cultivated plants. Nitrate reductase (NR) content in plants tissues is strongly dependent on nitrate concentration in growth medium and, consequently, in tissues. Thus, high NR activity can not been expected in plants cultivated without nitrate. In A. pseudoplatanus, high activity of NR found in roots indicates that most of N reduction happens there. Nitrogen is then transferred to aboveground parts as ureides. In NO3-cultivated A. pseudoplatanus, however, a N reduction may partially exist in leaves which might be supported by a markedly higher concentrations of NO3 found in the leaves of NO3 HL, NO3 LL plants contrastingly to NH4 plants (see Table 3). Since NR is a typical substrate-induced enzyme we may expect NR activity in tissues with high availability of NO3 (Knaff 1996). Increase in Fv/Fm with decreasing PPFD availability is comparable to the results found by Groninger et al. (1996) for Acer rubrum. This phenomenon suggests that more efficient transfer of absorbed light energy from LHCs to PS II is reached in LL compared to HL plants. The response of Fv/Fm to shade is, however, species specific and may show both increase and decrease in different tree species (Castro et al. 1995). Effect of NO3 / NH4 nutrition on Fv/Fm might be attributed to N content in leaves. It is believed that higher leaf N brings higher Fv/Fm (e.g. Nunes et al. 1993). Low leaf N, on contrary, decreases Fv/Fm. Schafer et al (1993) reported a decrease in Fv/Fm in N-defficient compared to N-supplied cells of Chenopodium rubrum cultivated in HL. This would suggest increased sensitivity to photoinhibition of N-deficient plants. The alternation in Fv/Fm was, however, not related to the loss of D1 protein (Schafer et al 1993) and thus some other mechanism than D1 degradation and synthesis must be considered. Our results suggest that higher Fv/Fm in NH4-cultivated plants may be attributed to N content in leaves. Fv/Fm drop after photoinhibitory treatment and consequent recovery was similar to the experimental data presented for numerous species (for review see e.g. Nishio et al. 1994). The recovery in HL plants of A. pseudoplatanus had a typical fast phase found within 1-2 h after photoinhibitory treatment followed by a much longer slow phase. Fast phase of recovery does not require protein synthesis and is associated with only changes in transthylakoidal pH (Horton and Ruban 1994) while the slow phase needs activation of reparatory mechanisms of proteins, D1 in particular. In our study, much faster recovery was found in NH4- compared to NO3-cultivated plants. We may attribute it to likely higher content of NH3/NH4+ in NH4-cultivated HL plants. Since Tischenko et al. (1995) reported markedly higher NH4+ content in chloroplasts of NH4-cultivated Zea mays, Amarantus paniculatus, Triticum aestivum we may expect high NH4 content in NH4 HL plants of A. pseudoplatanus. Earlier proposed mechanism (e.g. Rottenberg 1979) suggests that NH3 and NH4+ ion in chloroplast may interact with of H+ transport from thylakoid lumen to stroma of chloroplasts. The proposed mechanism may accelerate a decrease in transthylakoidal pH (induced by high light) and likely also the recovery from photoinhibition. Hypothesis of high NH3/NH4+ amounts in chloroplast might be supported indirectly by the evidence that NH4+ content in chloroplast is increasead under high light and low pH inside chloroplast (Rottenberg 1979). Absolute amount of NH3 and NH4+ in chloroplast, however, might be under 1 mM since higher concentration leads to inactivation of ATP synthesis due to H+ depletion and, consequently, in uncoupling of electron transport chain in thylakoid membrane (Lea and Miflin 1979). In this study, we discuss only likely direct mechanism (NH3/NH4+ in thylakoids) leading to changes in chl fluorescence parameters but some indirect regulatory mechanism (e.g. effect of phytohormones, secondary messangers) induced by NH3/NH4 nutrition leading to changes in chl fluorescence parameters can not be excluded. In LL-cultivated plants, the absence of full recovery of Fv/Fm indicates that photoinhibitory treatment led to destruction of pigment-protein complexes of PS II, D1 protein in particular. Since no difference both in the extent of Fv/Fm decrease and Fv-Fm recovery was found between NO3 and NH4 plants, we may conclude that NH4+ form of N supplied has no effect on the sensitivity of LL-cultivated plants of A. pseudoplatanus to photoinhibition. Under high PPFDs, proportion of qT to qI or qE is very small (Krause and Weis 1991). In our results, therefore, an increase in qT+I* after photoinhibitory treatment and its recovery might be attributed almost exclusively to the increase in qI component. The changes in qT+I* are related to both structural and functional changes in PS II and LHC of PS II. Proposed mechanism (Krause and Weis 1991) considers (1)inactivation of LHCs II in terms of inability to transfer excitation energy to PS II centers resulting in heat dissipation, (2) dysfunction and degradation of D1 protein, and (3) formation of free oxygen and other highly reactive species. 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