Journal of Horticultural Science & Biotechnology (2009) 84 (2) 181–186
Excess ammonium in foliar tissue: a possible cause of interveinal chlorosis in strawberry (Fragaria ananassa Duch. cv. Nyoho) By ANAMARIJA PETROVIC*, YUICHI YOSHIDA and TOSHIMASA OHMORI The Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-naka, 700-8530 Okayama, Japan (e-mail:
[email protected]) (Accepted 7 October 2008) SUMMARY Substrate-grown strawberries (Fragaria ananassa Duch. cv. Nyoho) in excessively fertigated peat bags often suffer interveinal chlorosis (leaf yellowing) in their immature leaves shortly after planting. Full recovery was observed in such plants following a 4 – 7 d restraint in the supply of nutrients. Hence, the cause of this phenomenon could reasonably be attributed to excess NH4-N accumulation in plant tissues. As has been previously shown, NH4-N accumulation in plant tissue can be induced by inhibition of glutamine-synthetase (GS). Thus, a GS inhibitor (glufosinate-ammonium) was applied at various dosages to peat bag-grown ‘Nyoho’ plants, foliar NH4-N concentrations were determined and yellowing symptoms were observed. After 7 d of treatment, foliar NH4-N concentrations increased dramatically, 1 – 2 d prior to the onset of severe yellowing. Subsequently, the relationship between nitrogen (N)-source and leaf yellowing was investigated. NH4-fed plants initially had higher NH4-N concentrations in their immature leaves than NO3-fed plants, and later suffered from interveinal chlorosis. Potted plants dipped in the relevant nutrient solutions exhibited seven-fold higher NH4-N concentrations in their immature leaves than plants that were manually supplied with 50 ml of the relevant nutrient solutions twice a day. In this study, we also investigated whether the combined effect of a Nsource under various environmental conditions (e.g., light intensity and air temperature) affected NH4-N accumulation in plant tissues, as has been suggested previously. We observed that, in ‘Nyoho’ plants, elevation of foliar NH4-N concentrations and the appearance of yellowing symptoms began earlier and was more severe under conditions of higher solar radiation and air temperature. The absence of interveinal chlorosis in plants that exhibited low NH4-N concentrations, regardless of treatment, led to the conclusion that high leaf NH4-N concentrations and excess accumulation of NH4-N may play an important role in the leaf yellowing phenomenon.
A
s one of the most popular fruits in Japan, strawberries occupy a large amount of greenhouse space (6,790 ha). Since the introduction of low-cost substrate culture systems in the mid-1990s, substrate-grown strawberry production has shown a constant and significant increase (Ministry of Agriculture, Forestry and Fisheries of Japan, 2007). In forced, June-bearing strawberry production, the nitrogen (N) supply is usually stopped for pot- or traygrown nursery plants in late August, as a way to stimulate early and uniform flower induction. Hence, shortly after planting into highly fertigated substrates in lateSeptember to early-October, N-starved plants undergo drastic changes in their N nutrition.The exact changes that occur in the absorption and metabolism of N in substrategrown strawberries immediately after transplanting are unclear. Strawberry growers often face problems of yellowing (interveinal chlorosis) in young, expanding leaves in such plants, 10 – 20 d after planting. This is reflected in a delay of plant development and consequently in reduced yield. Severe interveinal chlorosis in plants grown in excessively-fertigated peat bags, and their recovery after less severe yellowing caused by a 4 – 7 d restraint in the supply of nutrient solutions, has been observed (Petrovic et al., unpublished data). It was proposed that this phenomenon may be due to abnormal N-metabolism in the plant tissues. *Author for correspondence.
In higher plants, glutamine synthetase (GS) and glutamate synthetase (GOGAT) are the major enzymes responsible for the assimilation of ammonium ions (NH4+) absorbed by roots, generated by nitrate (NO3–) reduction, or endogenously by ammonium-evolving processes such as photorespiration (Frantz et al., 1982). It has been shown that treatment with inhibitors of GS and GOGAT causes an accumulation of ammonium ions by impairing its assimilation into amino acids (Rhodes et al., 1986). Therefore, in order to induce the accumulation of NH4+ in strawberry plant tissue and to test the hypothesis that excess ammonium ion accumulation in strawberry plants may lead to the appearance of foliar yellowing, we treated plants with a non-selective, commercial contact herbicide, BastaTM. Its active ingredient is glufosinate [glufosinate-ammonium; syn. phosphinothricin (PPT)] which acts primarily through inhibiting GS (Leason et al., 1982), inducing ammonium ion accumulation, and disrupting photosynthesis (Wild et al., 1987). The assimilation of N, and its incorporation into amino acids are vital plant processes. The form of N supplied also affects other physiological processes such as root respiration, water relations, photosynthesis, and secondary metabolism (Matsumoto and Tamura, 1981; Ragab, 1980; Shelp and Taylor, 1990; Wang and Below, 1996). Unlike nitrates, the assimilation of ammonium requires lower energy inputs. However, many plant species show reduced growth under high NH4-N
182
Excess ammonium and interveinal chlorosis in strawberry
nutrition and develop an ammonium toxicity syndrome associated with the accumulation of NH4+ ions in tissues, or diminished cation uptake (e.g., Mg2+ or Ca2+; Mehrer and Mohr, 1989). In the present study, strawberry plants were treated with nutrient solutions, having various NH4-N and NO3N ratios, and the effects of N source on foliar NH4-N accumulation and on the incidence of leaf yellowing were examined more closely. Strawberry plants were dipped into the treatment solutions in order to: (i) simulate an excessively-fertigated peat bag environment, limiting the gaseous phase of the rhizosphere; and (ii) provide a continuous supply of nutrients. Foliar ammonium ion concentrations in NH4-N-fed tomato plants increased at high light intensities (Magalhaes and Wilcox, 1984). Unlike NO3-N-fed plants, Zhu et al. (2000) reported a higher sensitivity to light stress in NH4-N-fed plants, as indicated by reduced grown and interveinal chlorosis in French bean. These reports suggested that not only the N source, but also its combined effect with environmental conditions (e.g., light intensity and/or air temperature) may affect ammonium ion accumulation in plant tissues and interveinal chlorosis in strawberries. Therefore, the effects of treatment with nutrient solutions having various NH4:NO3 ratios were examined under two different natural light-temperature environments. This study investigated the relationship between excess NH4-N in foliar tissues and interveinal chlorosis in leaves of ‘Nyoho’ strawberry, a cultivar that often suffers leaf yellowing symptoms. To achieve this, NH4-N concentrations were measured in the leaves of plants treated with: (i) a GS inhibitor that induces NH4-N accumulation in plant tissues or; (ii) nutrient solutions having different ratios of N-forms (NH4-N vs. NO3-N), under different natural light-temperature regimes, and the occurrence of leaf yellowing was observed.
MATERIALS AND METHODS Effect of a GS inhibitor on the incidence of leaf yellowing The experimental strawberry plants (Fragaria ananassa Duch., cv. Nyoho) were transplanted to a greenhouse and planted in peat bags (eight plants per 16 l bag) on 20 September 2004. They were supplied with 50% (v/v) ‘Ohtsuka A’ nutrient solution (Ohtsuka Chemicals, Osaka, Japan) containing: 8.0 mM NO3–, 0.85 mM NH4+, 0.85 mM H2PO4–, 3.9 mM K+, 2.05 mM Ca2+, 0.93 mM Mg2+, 0.93 mM SO42–, plus microelements, fourto-six times a day. The bags were irrigated using a drip irrigation system consisting of two drippers with an output of 2 l h-1 for each 0.80 m-long peat bag. On 24 November 2004, by removing old leaves, leaf numbers were adjusted to two fully-expanded leaves per plant, and plants were divided into five treatments consisting of different concentrations of glufosinateammonium: (i) 18.5 mg l–1; (ii) 37 mg l–1; (iii) 55.5 mg l–1; or (iv) 185 mg l–1; and (v) control (water). The source of glufosinate was BastaTM (Bayer Crop Science, Tokyo, Japan). In each treatment, each plant was sprayed with 10 ml of a solution containing the surfactant polyoxyethylenealkylether (0.1 ml l–1 solution) and one of the above glufosinate-ammonium solutions. After 7 d,
newly-expanded (immature) and fully-developed (mature) leaf samples from all treated plants were removed in order to determine NH4-N concentrations. Each experimental treatment had four replicates with two plants per replicate (n = 8). The date on which visible leaf yellowing symptoms started was recorded, and the progress of interveinal chlorosis was observed until day-14. Effect of N-form on the incidence of leaf yellowing In this experiment, ‘Nyoho’ plants, each with two fullyexpanded leaves, were grown in a greenhouse in tray containers (cell volume = 130 ml), and manually supplied with 50 ml of 25% (v/v) ‘Ohtsuka A’ nutrient solution once every 2 d. Substrate moisture was maintained at field capacity by watering on a regular basis. The potgrown plants were then divided into four treatments each of which contained 5 mM total-N, but with four different ratios of ammonium to nitrate ions: (i) 0:5; (ii) 1:4; (iii) 2:3; and (iv) control (tap water). The sources of NH4-N and NO3-N were NH4NO3 and KNO3, respectively. During preliminary investigations, severe interveinal chlorosis occurred in plants grown in excessivelyfertigated peat bags and in frequently fertigated substrates. Therefore, in order to simulate highlyfertigated substrate conditions, and to avoid leaching and possible nitrification of the medium while continuously supplying nutrients, half of the plants in each treatment were dipped into the relevant nutrient solution (dipped plants). The rest were supplied manually with 50 ml of the relevant nutrient solution twice a day (non-dipped plants). The concentrations of NH4-N in samples of newly-expanded (immature) leaves were determined on day-1 of the experiment and 3 d later (day-4), using the method described below. Each treatment consisted of four continuously-supplied and four control replicates, each containing two plants. The experiment was conducted twice (starting dates: 6 June and 24 June 2005) and the results represent the means of the two experiments (n = 16). The above experiment with dipped and non-dipped pot plants was repeated twice, in mid-Summer 2006, under different natural light-temperature greenhouse conditions, using only “dipped plants”. The experiment was carried out over two cycles of 5 d each: (i) under naturally cloudy conditions, a cloudy weather cycle [average cumulative solar energy (CSR) was 8.7 MJ m–2, average solar energy at the sampling hour (SR 12.00) was 1.1 MJ m–2, average daily mean air temperature (DMT) was 29.63°C, and average air temperature at the sampling hour (T 12.00) was 27.56°C]; and (ii) in a naturally sunny environment, a sunny weather cycle (average CSR was 19.2 MJ m–2, average SR 12.00 was 2.4 MJ m–2, average DMT 38.61°C, and average T 12.00 was 32.18°C), starting on 19 July and 5 August, respectively. Each experimental treatment consisted of four replicates, each containing two plants (n = 8). Immature leaves of all treated plants were sampled to determine their NH4-N content on days -1, 2, -3, -4, and -6 of the experiment. Light intensity and temperature in the greenhouse were recorded using a photodiode sensor (ML-020V; Eiko, Tokyo, Japan) and a thermocouple, respectively. Previous studies on diurnal changes in foliar NH4-N
183
A. PETROVIC, Y. YOSHIDA and T. OHMORI concentrations in ‘Nyoho’ plants (Petrovic et al., 2006), showed that the concentration increased to a maximum at mid-day, then dropped steadily during the second-half of the light period. Therefore, all samples used in these experiments were taken at approx. 12.00 h. The dates on which leaf yellowing symptoms started were recorded.
(ANOVA). Mean separation was based on Duncan’s multiple range test. Differences were considered to be significant at P < 0.05. All statistical analyses were performed using the statistical software package SPSS Version 12.0 (MapInfo Corporation, Troy, NY, USA).
Determination of NH4-N concentrations in plant tissues Preparation of tissue extracts for NH4-N determination: According to Husted et al. (2000), plant material can be stored at –80°C without causing any significant change in its ammonium ion concentration for up to 6 months. Therefore, tissues were weighed and kept at this temperature until being ground in a chilled pestle and mortar with 10 mM formic acid (20 ml g–1 FW) and a small quantity of sea sand (0.50 g) as a grinding medium. The homogenate was centrifuged for 10 min at 14,000 g at 2°C and the supernatant was used to determine the concentration of NH4-N in the leaf tissue.
RESULTS AND DISCUSSION Effect of a GS inhibitor on the incidence of leaf yellowing In ‘Nyoho’ strawberry plants, the extent and pattern of interveinal chlorosis was dependent on the dose of glufosinate-ammonium applied. All plants exhibited faint green discolouration on their newly-expanding leaves (Figure 1) approx. 2 d after application of the herbicide. Nine days after treatment, the symptoms had progressed to severe interveinal chlorosis. Injury was limited to interveinal chlorosis and desiccation of the leaf tips, while the petioles remained green (data not shown). ‘Nyoho’ plants sprayed with glufosinate doses ≥ 18.5 mg l–1, showed an extreme rise in foliar NH4-N concentration (Figure 2). The application of glufosinate to strawberry plants irreversibly inhibited GS, thereby blocking the synthesis of glutamine from glutamate and ammonium (Manderscheid and Wild, 1986). This resulted in the accumulation of potentially toxic levels of NH4+ ions within the plant cells (Kishore and Shah, 1988). Extreme foliar NH4-N concentrations were reached by day-7, 1 – 2 d prior to the onset of severe yellowing symptoms (data not shown). 60 50 NH4 -N (µmol g-1 FW)
Fluorescence spectroscopy at neutral pH: NH4-N concentrations were determined using the fluorimetry method developed by Goyal et al. (1988), and modified by Husted et al. (2000) for compatibility with modern HPLC-systems. The HPLC-system used here (Jasco, Tokyo, Japan) was equipped with a pump (PU-2080; Jasco), an integrated column oven (CO-965; Jasco) and a scanning fluorescence detector (FP-2020 Plus; Jasco). The carrier phase consisted of 3 mM o-phthalaldehyde (OPA), 10 mM 2-mercaptoethanol, and 100 mM phosphate buffer, adjusted to pH 6.8. A sample (20 µl) of the supernatant, generated as described above, was injected into the carrier stream which entered the reaction coil in the column oven at 63°C. At this temperature, ammonium ions react with OPA to form an alkylthioisoindole fluorochrome which was detected at an excitation wavelength of 410 nm and an emission wavelength of 470 nm. For each set of samples, standards containing 50 µM or 200 µM ammonium [as (NH4)2SO4] were used for calibration. Ammonium concentrations were expressed as µmoles NH4-N g–1 fresh weight (µmol g–1 FW).
A
c
40 30
b
20 a
10 a
0
00
a 18.5 18.5
37.0 37.0
55.5 55.5
60
-1
NH4 -N ( µmol g FW)
50
B b
40 30 20 a
10 0
A
B
FIG. 1 The symptomatic pattern of interveinal chlorosis in newly-expanding leaves of ‘Nyoho’ strawberry plants. Panel A, immature leaf without yellowing leaf symptoms. Panel B, immature leaf suffering interveinal chlorosis.
185.0 185.0
Glufosinate (mg l-1)
Data analysis Data were analysed for the differences between treatments using one-way analysis of variance
a
00
a
118.5 8.5
a
3737.0 .0
55.5 55.5
1185.0 85.0
-1
Glufosinate (mg l ) FIG. 2 Foliar NH4-N concentrations in leaves of ‘Nyoho’ strawberry plants 7 d after glufosinate treatment. Panel A, immature leaves. Panel B, mature leaves. The values shown are means ±SD for eight replicate plants in each treatment. Different lower-case letters indicate significant differences at P < 0.05 by Duncan’s multiple range test.
Effect of N-form on the incidence of leaf yellowing Foliar NH4-N concentrations increased in plants dipped for 3 d in a nutrient solution (continuouslysupplied plants) containing the highest rate of ammonium (2 mM NH4+ : 3 mM NO3–) with 0.60 µmol g–1 FW on day-1 and 5.48 µmol g–1 FW 3 d later (Figure 3A). In this treatment, interveinal chlorosis in immature leaves started to develop 2 d after the beginning of the experiment. In contrast, when solutions were supplied twice a day (control plants; Figure 3B), across all treatments, foliar NH4-N concentrations were low (0.78 µmol g–1 FW in the case of the highest NH4+ rate) and there was no yellowing. With the absence of interveinal chlorosis in immature leaves that showed low foliar NH4N concentrations, we concluded that high foliar NH4-N concentrations, and excess ammonium ion accumulation, play an important role in triggering interveinial chlorosis. According to Claussen and Lenz (1999), the stawberry cultivar ‘Senga Sengana’ showed similar leaf yellowing on the lower leaves (leading to wilting, leaf drop, and impaired growth) when plants were supplied with a nutrient solution containing NH4-N instead of NO3-N. In an earlier study, Claussen and Lenz (1995) observed a decrease in net photosynthesis and interveinal chlorosis in leaves of eggplants supplied with nutrient solutions containing 10 mM NH4-N as sole N source during the early stages of vegetative growth. We observed that the symptoms of foliar yellowing increased with exposure to increasing light intensity and air temperature in plants treated with solutions containing NH4-N. During the cloudy weather cycle, under conditions of lower air temperature and solar radiation (Figure 4B), foliar NH4-N concentrations in ‘Nyoho plants started to rise on day-4 of treatment (Figure 4A). Around that time, interveinal yellowing was observed in newly-expanding leaves (data not shown). As a result of increased solar radiation on day-
-1
4
8 7 6 5 4 3 2 1 0
A
b
a a
a 0:0
0:5
1:4
2:3
NH4 -N : NO3 -N ratio
-1
4
Glufosinate doses of 185 mg l–1 were shown to be lethal to ‘Nyoho’, inducing severe interveinal chlorosis that progressed into necrosis and rapid desiccation of the leaf tips. Within 7 d, these plants showed a dramatic increase in NH4-N accumulation, rising to maximum levels of 43.38 µmol g–1 FW and 34.69 µmol g–1 FW in immature and mature leaves, respectively (Figure 2). This resulted in the total deterioration and death of plants 10 d after application. Extremely high foliar NH4N concentrations in these plants suggest that accumulation of toxic levels of this ion in the cells led to this deterioration. According to Puritch and Barker (1967), degeneration of the ultra-structure of chloroplasts caused by ammonium ion toxicity and manifested as ammonium accumulation, decreased photosynthesis and caused foliar chlorosis and necrosis in tomato. Regardless of the dose of glufosinate applied, NH4-N accumulation and interveinal chlorosis were more pronounced in newly-expanding leaves than in mature leaves. This indicates a possible connection between interveinal leaf yellowing and excess NH4-N accumulation in immature leaves of strawberry plants. The following experiments, which investigated a possible parallel between N source (NH4-N vs. NO3-N) and the incidence of foliar chlorosis, support this hypothesis.
NH -N ( µmol g FW)
Excess ammonium and interveinal chlorosis in strawberry
NH -N ( µmol g FW)
184
8 7 6 5 4 3 2 1 0
B
a
b
c
c
0:0
0:5
1:4
2:3
NH4 -N : NO3 -N ratio FIG. 3 NH4-N concentrations in immature leaves of ‘Nyoho’ plants 3 d after being supplied with various nutrient solutions containing different ratios of NH4-N and NO3-N (5 mM total N). Panel A, plants dipped into nutrient solution. Panel B, non-dipped plants. The values are means ±SD for 16 replicate plants in each treatment. Different lower-case letters indicate significant differences at P < 0.05 by Duncan’s multiple range test.
3 (Figure 4B), the air temperature in the greenhouse was significantly higher at 12.00 h (mean = 33.7°C compared to a mean of 30ºC on days -1 and -2), and was more similar to the temperatures recorded during the sunny weather cycle. Photorespiration, and thereby photorespiratory NH4 production, is known to increase with leaf temperature. Husted et al. (2002) reported that photorespiration increased five-fold when the leaf temperature in oilseed-rape plants was raised from 15ºC to 25ºC. This suggests that the higher air temperature recorded in the greenhouse on the previous day, and the subsequent higher leaf temperature, might explain the increase in NH4-N accumulation and the expression of interveinal chlorosis in immature leaves of strawberry plants on day-4. NH4-N concentrations in the immature leaves of plants supplied with 1 mM NH4+ and 4 mM NO3– rose above those in plants of the other treatments, reaching maximum levels on day-6 (4.57 µmol g–1 FW). In plants grown under higher air temperatures and solar radiation (Figure 4D), NH4-N concentrations started to rise on day-3 of the experiment, and reached maximum levels at the highest ammonium rate (Figure 4C). Subsequently, the concentration of NH4-N in immature leaves continued to increase until the last day of treatment (5.65 µmol g–1 FW). Concomitant with the elevation in foliar NH4-N concentration on day-3 of the experiment, symptoms of interveinal leaf chlorosis started to appear. Plants exposed to a higher light intensity have a higher rate of transpiration and have been reported to transport sufficient complexed ammonium to the shoot to trigger ammonium toxicity through uncoupling photophosphorylation (Goyal et al.,
185
A. PETROVIC, Y. YOSHIDA and T. OHMORI
-
0:0
6
0:5
4
2:3
2
b
1:4
a aa a
a a aa
a aa a
b b
c
a a
a
a
B
20
10
20
5
10
0
3 4 Days of treatment
0 3 4 Days of treatment
6
b
Solar energy (MJ m )
C
8 6 4 2
2
6
-2
-1
NH4 -N ( µmol g FW )
2
40 30
1
1
50
CSR DMT
15
0
10
SR 12.00 T 12.00
Temperature (ºC)
+
NH4 :NO3 :
25
c
b a a a a
a a a a
1
2
a a a
a
a
a
a
b a
0 3 4 Days of treatment
6
25 20
50
D
40
15
30
10
20
5
10
0
0 1 SR 12.00
2
3 4 Days of treatment CSR
T 12.00
Temperature (ºC)
8
A
Solar energy (MJ m-2)
-1
NH4 -N ( µmol g FW )
10
6 DMT
FIG. 4 Daily fluctuations in NH4-N concentrations in immature leaves of ‘Nyoho’ strawberry plants dipped into various nutrient solutions containing different ratios of NH4-N and NO3-N (5 mM total N) under cloudy (Panel A) or sunny (Panel C) 5 d weather cycles. Panel B, cloudy weather cycle solar radiation and air temperature. Panel D, sunny weather cycle solar radiation and air temperature. SR 12.00 is solar radiation at approx. 12.00 h (in MJ m–2). CSR is the cumulative solar radiation (in MJ m–2). T 12.00 is the air temperature at approx. 12.00 h (in °C). DMT is daily mean air temperature (in °C). The values are means ±SD for eight replicate plants in each treatment. Different lower-case letters indicate significant difference at P < 0.05 by Duncan’s multiple range test.
1982; Ikeda and Yamada, 1981). If the plant fails to assimilate NH4 taken-up by the roots or generated in the leaves, then toxic levels accumulate (Miflin and Lea, 1980). It can therefore be argued that NH4accumulation led to expression of interveinal chlorosis in immature leaves on the strawberry plants. Another mechanism of ammonium accumulation in shoots grown under high light intensities could be due to photorespiration. Photorespiration, a physiological defence against light-oxygen toxicity, is recognised as quantitatively the most important process generating NH4+ ions in plants during vegetative growth. Since photorespiration takes place in the presence of light (Lodish et al., 2004), and increases with increasing light intensity (Tolbert, 1980), this process can contribute to ammonium ion accumulation and the expression of
interveinal chlorosis in immature leaves of strawberry plants grown in sunny weather. The effects of leaf yellowing symptoms on late growth of substrate-grown strawberry plants was significant. However, the potential mechanisms underlying, and the means of alleviating these symptom are diverse. The combined effects of insufficient oxygen supply, high respiration rates, higher carbohydrate demand at the cellular level in the roots, and initially lower carbohydrate contents in NH4-fed plants (Ganmore-Neumann and Kafkafi, 1983) could all be responsible for foliar yellowing in substrate-grown strawberries. This study highlights the role in excess NH4-N accumulation in triggering interveinal leaf yellowing, and furthers our understanding of the possible mechanisms controlling this phenomenon in strawberry and, potentially, in other crops.
186
Excess ammonium and interveinal chlorosis in strawberry REFERENCES
CLAUSSEN, W. and LENZ, F. (1995). Effect of ammonium and nitrate on net photosynthesis, flower formation, growth and yield of eggplants (Solanum melongena L.). Plant and Soil, 171, 267–274. CLAUSSEN, W. and LENZ, F. (1999). Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant and Soil, 208, 95–102. FRANTZ, T. A., PETERSON, D. M. and DURBIN, R. D. (1982). Sources of ammonium in oat leaves treated with tabtoxin or methionine sulfoximine. Plant Physiology, 69, 345–348. GANMORE-NEUMANN, R. and KAFKAFI, U. (1983). The effect of root temperature and NO3/NH4 ratio on strawberry plants. I. Growth, flowering, and root development. Agronomy Journal, 75, 941-947. GOYAL, S. S., HUFFAKER, R. C. and LORENS, O. A., (1982). Inhibitory effects of ammoniacal nitrogen on growth of radish plants. II. Investigation on the possible causes of ammonium toxicity to radish plants and its reversal by nitrate. Journal of the American Society for Horticultural Science, 107, 130–135. GOYAL, S. S., RAINS, D. W. and HUFFAKER, R. C. (1988). Determination of ammonium ion by fluorometry or spectrophotometry after on-line derivatization with o-phthalaldehyde. Analytical Chemistry, 60, 175–179. HUSTED, S., HEBBERN, C., MATTSSON, M. and SCHJOERRING, J. (2000). A critical experimental evaluation of methods for determination of NH4+ in plant tissue, xylem sap and apoplastic fluid. Physiologia Plantarum, 109, 167–179. HUSTED, S., MATTSSON, M., MOLLERS, C., WALLBROUN, M. and SCHJOERRING, J. K. (2002). Photorespiratory NH4+ production in leaves of wild-type and glutamine synthetase-2 antisense oilseed rape. Plant Physiology, 130, 989–998. IKEDA, M. and YAMADA, Y. (1981). Dark CO2 fixation in leaves of tomato plants grown with ammonium and nitrate as nitrogen sources. Plant and Soil, 60, 213–220. LEASON, M., CUNLIFFE, D., PARKIN, D., LEA, P. J. and MIFLIN, B. J. (1982). Inhibition of pea leaf glutamine synthetase by sulphoximine, phosphinothricin and other glutamate analogues. Phytochemistry, 2, 855–857. LODISH, H., BERK, A., MATSUDAIRA, P., KISER, C. A., KRIEGER, M. and SCOTT, M. P. (2004). Molecular Cell Biology. 5th Edition. W. H. Freeman and Company, New York, USA. 345 pp. MAGALHAES, J. R. and WILCOX, G. E. (1984). Ammonium toxicity development in tomato plants relative to nitrogen form and light intensity. Journal of Plant Nutrition, 7, 1477–1496. MANDERSCHEID, R. and WILD, A. (1986). Studies on the mechanism of inhibition by phosphinothricin of glutamine synthetase isolated from Triticum aestivum L. Journal of Plant Physiology, 123, 135–142.
MATSUMOTO, H. and TAMURA, K. (1981). Respiratory stress in cucumber roots treated with ammonium or nitrate nitrogen. Plant and Soil, 60, 195–204. MEHRER, I. and MOHR, H. (1989). Ammonium toxicity: description of the syndrome in Synapis alba and the search for its causation. Physiologia Plantarum, 77, 545–554. MIFLIN, B. J. and LEA, P. J. (1980). Ammonia assimilation. In: Nitrogen in the Environment. (Nielsen, D. R. and MacDonald, J. G., Eds.). Academic Press, New York, USA. Volume 2. 157–189. MINISTRY OF AGRICULTURE, FORESTRY AND FISHERIES OF JAPAN (2007). http://www.maff.go.jp/toukei/sokuhou/data/shiteisakutsukesyukaku2006/shitei-sakutsukesyukaku2006.pdf. (In Japanese). PETROVIC, A., YOSHIDA, Y. and OHMORI, T. (2006). Changes in ammonium concentrations in leaf tissue of strawberry plants. Abstracts of the 27th International Horticultural Congress and Exhibition, Seoul, Korea. 202. PURITCH, G. S. and BARKER, A. V. (1967). Structure and function of tomato leaf chloroplasts during ammonium toxicity. Plant Physiology, 42, 1229–1238. RAGAB, S. M. (1980). Water uptake and trans-potential in sunflower roots as influenced by ammonium ions. Journal of Agricultural Science, 94, 145–150. RHODES, D., DEAL, L., HAWORTH, P., JAMIESON, G. C., REUTER, C. C. and ERICSON, M. C. (1986). Amino acid metabolism of Lemna minor L. I. Responses to methionine sulphoximine. Plant Physiology, 82, 1057–1062. SHELP, B. J. and TAYLOR, D. C. (1990). Carbon and nitrogen partitioning in young nodulated pea wild type and nitrate reductase deficient mutant plants exposed to NO3– or NH4+. Canadian Journal of Botany, 69, 1780–1786. TOLBERT, N. E. (1980). Photorespiration. In: The Biochemistry of Plants. (Stumpf, P. K. and Conn, E. E., Eds.). Academic Press, New York, USA. Volume 2. 487–523. WANG, X. and BELOW, F. E. (1996). Cytokinins in enhanced growth and tillering of wheat induced by mixed nitrogen source. Crop Science, 36, 121–126. WILD, A., SAUER, H. and RUHLE, W. (1987). The effect of phosphinothricin (glufosinate) on photosynthesis. I. Inhibition of photosynthesis and accumulation of ammonia. Zeitschrift fur Naturforschung C: Journal of Biosciences, 42, 263–269. ZHU, Z., GERENDAS, J., BENDIXEN, R., SCHINNER, K., TABRIZI, H., SATTELMACHER, B. and HANSEN, U. P. (2000). Different tolerance to light stress in NO3– and NH4+-grown Phaseolus vulgaris L. Plant Biology, 2, 558–570.
