Agricultural and Food Science in Finland, Vol. 10 (2001): 59–64. Vol. 10 (2001): 59–64. 59 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 59–64. © Agricultural and Food Science in Finland Manuscript received August 2000 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 59–64. Research Note Influence of nitrogen deficiency on photosynthesis and chloroplast ultrastructure of pepper plants Snejana Doncheva, V. Vassileva, G. Ignatov, S. Pandev Institute of Plant Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Ramdane Dris and Raina Niskanen Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland, e-mail: ramdane.dris@helsinki.fi Pepper plants (Capsicum annuum L. cv. Zlaten Medal) were grown on nutrient solution without ni- trogen, and photosynthetic response of plants was examined by determination of leaf CO 2 fixation and chlorophyll and carotenoid contents. The absence of nitrogen in the medium resulted in a de- crease of the leaf area and of plant biomass accumulation, and in an increase of the root-shoot dry weight ratio. The photosynthetic activity and chlorophyll and carotenoid contents decreased signifi- cantly under nitrogen deprivation. Examination of nitrogen deficient leaves by transmission electron microscopy showed dramatic changes in chloroplast ultrastructure. The proportion of starch granules and plastoglobules in the stroma matrix was increased and internal membrane system was greatly reduced. It seems that nitrogen plays an important role in the formation of chloroplast structure and hence to the photosynthetic intensity and productivity of pepper plants. Key words: Capsicum annuum, CO 2 assimilation, leaf area, nitrogen deficiency, photosynthetic pigments, plastoglobules, starch Introduction The nitrogen supply is one of the major factors determining the growth of plants. As a constitu- ent of chlorophyll molecule and carboxylating enzymes, nitrogen is essential for the mainte- nance of active photosynthesis and production of carbohydrates (Peoples et al. 1980). When nitrogen availability is low, the leaf cell number and volume (Lawlor et al. 1989), cell wall ex- tensibility (Taylor et al. 1993) and photosynthe- sis (Kutik et al. 1995, Meinzer and Zhu 1998) are markedly reduced. Nitrogen deficiency de- creases CO 2 assimilation capacity (Terashima and Evans 1988) and the quantum yield of pho- tosynthesis (Lawlor et al. 1987). It also affects PSII photochemistry, including the decrease in the quantum yield of PSII electron transport and the sufficiency of excitation energy capture by mailto:ramadane.dris@helsinki.fi 60 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Doncheva, S. et al. Nitrogen deficiency, photosynthesis and chloroplasts of pepper plants open PSII reaction centres (Verhoeven et al. 1997). At the ultrastructural level, the N defi- ciency reduces chloroplast size and the amount of thylakoids and increases the amount of non- nitrogenous substances such as starch and the size of plastoglobuli containing isoprenoid plas- toquinone (Laza et al. 1993, Kutik et al. 1993, 1995). The aim of the present investigation was to study the effect of N deficiency on photosyn- thetic function and ultrastructure of mesophyll cell chloroplasts of pepper plants. Material and methods Five-day-old pepper seedlings (Capsicum annu- um L. cv. Zlaten Medal) were grown hydropon- ically in naturally illuminated greenhouse with photoperiod of 16 h at 25/19°C day/night tem- peratures. The nutrient solution contained per litre of distilled water 0.944 g Ca(NO 3 ) 2 .4H 2 O; 0.738 g MgSO 4 .7H 2 O; 0.816 g KH 2 PO 4 ; 0.170 g NaNO 3 and micronutrients (Hoagland and Arnon 1938). Solution pH was maintained near 6.0 through the addition of 0.1 M KOH. The plant material was divided into two experimental var- iants: (1) plants on full nutrient solution (con- trol); (2) plants with nutrient solution lacking nitrogen. The nutrient solution was continuous- ly aerated and changed twice in a week. Plants were harvested 30 days after planting. Twelve plants were used for each variant and three in- dependent experiments were done. At harvest, leaf area and fresh weight were measured and different plant organs were weighed after dry- ing at 60°C to give a constant weight. The pigments of the photosynthetic appara- tus (chlorophyll a and b and carotenoids) were extracted in 80% acetone (Arnon 1949) and con- centrations were determined spectrophotometri- cally using the extinction coefficients proposed by Mc Kinney (1941). The rate of photosynthetic CO 2 fixation was determined as described by Fedina et al. (1994). The leaf discs of 5 mm in diameter, taken from each variant after 30 day treatment, were kept in a closed chamber for short-term (20 min) expo- sition in the atmosphere containing 14CO 2 with 7.4 MBq 14C under irradiation of 920 µmol m-2 s-1 at 25°C. The radioactivity of the samples was determined by scintillation counting. Each ex- periment was repeated three times. CO 2 assimi- lation was calculated in CO 2 mg dm-2 h-1. Leaf portions (1–2 mm2) from the control and nitrogen deficient plants were fixed with 5% (w/ v) glutaraldehyde in Na-cacodylate buffer (pH 7.2) at 4°C for 3 h, and postfixed with 1.3 % OsO 4 (w/v) in the same buffer. Then, the sam- ples were buffer-washed and dehydrated through a graded ethanol series (25–100%, v/v), ethanol- propylene oxide (1:1, v/v), propylene oxide and propylene oxide-Durcupan ACM (1:1, v/v), and embedded in Durcupan ACM (Fluka AG, Buchs, Switzerland). Ultrathin sections were stained with uranyl acetate and lead citrate (Reynolds 1963) and examined with a transmission elec- tron microscope (JEM 100 B, JEOL, Japan) at 80 kV. A morphometric analysis of the chloroplast structures was performed for the estimation of chloroplast area and length, starch inclusions, plastoglobules and number of granal thylakoids. From 10 randomly chosen ultrathin sections of each treatment, 10–18 fields over the image screen were measured with a semiautomatic im- age analyzer (Morphomat 10, Zeiss, Oberko- chen, Germany). Results and discussion In comparison with the control, the total dry weight and the total and specific leaf area de- creased and the root to shoot dry weight ratio increased significantly in the nitrogen deficient pepper plants (Table 1). Probably, the enhanced root growth and retarded shoot growth in N-de- ficient plants is related to a high cytokinin/ABA ratio in the roots and to a low ratio in the shoots (Mardanov et al. 1998). The acceleration of root 61 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 59–64. growth provides an increasing demand for as- similates and changes the source-sink system in N - d e f i c i e n t p l a n t s i n f a v o u r o f t h e r o o t s (Marschner et al. 1996). Total chlorophyll and carotenoid contents, the chlorophyll a/b ratio and the photosynthetic efficiency, indicated by the rate of CO 2 assimilation, were significantly lower in leaves of nitrogen deficient plants compared with the control (Table 1). These results are in accordance with other studies reporting that N deficiency decreases the chlorophyll content per unit area (Lichtenthaler 1973), the chlorophyll and carotenoid formation (Sundqvist et al. 1980) and the rate of net photosynthesis (Lawlor et al. 1989). At TEM, the chloroplasts of leaves of con- trol plants appeared elongated in shape and with the stroma containing some small starch grains and small dark plastoglobules (Fig. 1a). The sys- tem of thylakoids was organized into grana stacks and single intergranal thylakoids (Fig. 1b). On the contrary, the chloroplasts of nitrogen de- ficient leaves were mostly more rounded and the stroma was filled with massive starch grains (Fig. 1c). Granal thylakoids were distorted and disor- dered with a lower stacking degree, and were pushed towards the periphery of the chloroplasts (Fig. 1d). Some loculi of the granal thylakoids were swollen (Fig. 1d). Morphometric analysis showed that the cross sectional area of the con- trol plant chloroplasts was smaller, but the chlo- roplast length, the number of grana per chloro- plast and the number of thylakoids per granum were higher with respect to N-deficient plant chloroplasts (Table 1).The proportion of stroma matrix of the chloroplasts cross section occupied by starch inclusions and plastoglobules was in- creased in nitrogen deficient plants (Table 1). The formation of large starch inclusions in the nitrogen deficient chloroplasts can be caused by production of photosynthates in excess with respect to the sink capacity. Impaired export from Table 1. Biomass accumulation, leaf area and specific leaf area (total leaf area/fresh weight of leaves), pigment contents, the rate of photosynthetic assimilation of 14CO 2 and the main parameters of chloroplast structure in leaves of pepper plants grown with (control) and without (–N) nitrogen supply (means and standard deviations). Control –N Significance of difference Dry weight (g/plant) 6.62 ± 0.40a 0.79± 0.03 b ** Root dry weight/shoot dry weight 0.23 b 0.59 a ** Leaf area (dm2/plant) 605 ± 31a 65 ± 5 b ** Specific leaf area (dm2/g fresh weight) 32.7 ± 1.1a 30.2± 0.9 b * Chlorophyll a (mg/g fresh weight) 0.97 ± 0.05a 0.21 ± 0.01b ** Chlorophyll b (mg/g fresh weight) 0.51 ± 0.03 a 0.14 ± 0.02 b ** Chlorophyll a/b 1.89 a 1.49 b * Carotenoids (mg/g fresh weight) 0.26 ± 0.01 a 0.18 ± 0.02 b ** Rate of CO 2 assimilation (CO 2 mg /dm2 h) 20.3 ± 1.3a 2.3 ± 0.1 b ** Chloroplast area (µm2) 10.7± 0.8 b 12.9± 0.8 a * Chloroplast length (µm) 6.4 ± 0.5 a 4.3 ± 0.3 b * Number of grana per chloroplast 19.0 ± 1.0 a 3.6 ± 0.2 b ** Number of thylakoids per granum 11.0 ± 0.8 a 4.0 ± 0.3 b ** Number of starch inclusions 1.7± 0.2 b 4.0 ± 0.3a ** Starch inclusions % of chloroplast area 11.0 b 68.0 a ** Plastoglobules % of chloroplast area 5.6 b 6.7a * ** significant at 1% level, according to ANOVA * significant at 5% level, according to ANOVAs 62 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Doncheva, S. et al. Nitrogen deficiency, photosynthesis and chloroplasts of pepper plants the source leaves leads to sucrose accumulation and facilitates the flow of newly fixed carbon into starch by exerting an inhibitory role on en- zymes synthesizing sucrose (Carmak et al. 1994). The abundance of plastoglobuli in the nitro- gen deficient plant chloroplast stroma has often corresponded to a minimum of thylakoid devel- opment (Tevini and Steinmüller 1985). Because plastoglobuli may serve as pools for storage of thylakoid constituents especially of lipids (Stein- müller and Tevini 1985), the increased volume of plastoglobules under nitrogen deficiency could mean a greater accumulation of lipids (Ku- tik et al. 1993). Our results allow to suggest that chloroplast alterations upon nitrogen deficiency were devel- oped as direct or indirect consequences of de- creased chlorophyll and carotenoid contents. Normal thylakoid stacking could be prevented by low content of photosynthetic pigments (Yalovsky et al. 1992, Kuhlbrandt et al. 1994, Wrischer et al. 1998). A strong correlation has been found between N deficiency-induced reduc- tion of photosynthesis and decline in ultrastruc- Fig. 1. Transmission electron micrographs of chloroplasts from leaves of pepper plants grown on nutrient solution: (A, B) with nitrogen (control); (C, D) without nitrogen. (A) Chloroplast from control leaf with well developed grana, intergranal thylakoids, some starch inclusions and plastoglobules. (B) Part of chlo- roplast from control leaf with grana and intergranal thylakoids. (C) Chloroplast from nitrogen deficient leaf with damaged grana, singly thylakoids, highly stained plastoglobules and large starch inclusions forc- ing the thylakoidal membranes to the periphery. (D) Part of chloroplast from nitrogen deficient leaf with large starch inclusions and incompletely stacked and swollen thylakoids. Note: (g) grana; (ig) intergranal thylakoids; (s) starch inclusions; (o) plastoglobules. Bars = 0.1 µm. (Photo: Snejana Doncheva). 63 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 59–64. tural order (Laza et al. 1993). The reduction of CO 2 fixation in nitrogen deficient leaves might also be due to the disruption of chloroplast struc- tures by starch grains formed inside the or- ganelles. Starch grains may decrease photosyn- thesis either by interfering with light transmis- sion to photochemical centres or by increasing the resistance to CO 2 diffusion (Neales and In- coll 1968). In summary, the results demonstrate the im- portance of an adequate nitrogen nutrition for the formation of normal chloroplast structure and hence for the photosynthetic intensity and pro- ductivity of the pepper plants. Arnon, D.I. 1949. Copper enzymes in isolated chloro- plasts. Polyphenol oxidases in Beta vulgaris. Plant Physiology 24: 1–15. 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The role of carotenoids in the structural and functional stabil- ity of thylakoids in plastids of dark-grown spruce seedlings. Journal of Plant Physiology 153: 46–52. Yalovsky, S., Neseman, E., Schuster, G., Paulsen, H., Harel, E. & Nechushtal, R. 1992. Accumulation of a light-harvesting chlorophyll a/b protein in the chloro- plast grana lamellae. Journal of Biology Chemistry 267: 20689–20693. SELOSTUS Typen puutteen vaikutus paprikan fotosynteesiin ja kloroplastien rakenteeseen Snejana Doncheva, V. Vassileva, G. Ignatov, S. Pandev, Ramdane Dris ja Raina Niskanen Bulgarian tiedeakatemia ja Helsingin yliopisto Liuosviljelyssä kasvatetulla paprikalla (Capsicum annuum L. cv. Zlaten Medal) tutkittiin typen puut- teen vaikutusta biomassan tuotantoon, fotosynteesiin ja kloroplastien rakenteeseen. Ilman typpilannoitus- ta lehtipinta-ala pieneni, biomassan kertyminen vä- heni ja juuren kuivapainon suhde verson kuivapai- noon suureni. Typen puute heikensi fotosynteesiä alentamalla hiilidioksidin sitoutumisnopeutta ja leh- tien klorofylli- ja karotenoidipitoisuutta ja aiheutti kloroplasteissa tärkkelysjyvästen ja plastoglobulien osuuden suurenemista sisäisen membraanirakenteen kustannuksella. Tulokset osoittavat, että kloroplastien rakenteen normaali muodostuminen, fotosynteesin tehokkuus ja paprikan sadon tuotto ovat riippuvaisia riittävästä typen saannista. Title Introduction Material and methods Results and discussion References SELOSTUS