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Loss of blade photosynthetic area and of chloroplasts' photochemical capacity account for reduced CO2 assimilation rates in zinc-deficient sugar beet leaves
J. Plant Physiol. 158. 915 – 919 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Loss of blade photosynthetic area and of chloroplasts’ photochemical capacity account for reduced CO2 assimilation rates in zinc-deficient sugar beet leaves Fernando S. Henriques* Plant Biology Unit, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte da Caparica, Portugal Received October 18, 2000 · Accepted January 26, 2001
Summary The effects of zinc deficiency on sugar beet chloroplasts’ ultrastructure and photochemical ability, as well as on whole-leaf CO2 assimilation rates, were evaluated. It is shown that increasing zinc shortage firstly induces progressive disorganisation of the chloroplasts’ internal membrane system, followed by degradation of both lamellae and stromal components. This zinc deficiency-triggered premature senescence process leads to cell death and, ultimately, to chlorotic/necrotic blade lesions, thus reducing the photosynthetically-active leaf area; chloroplasts in the remaining leaf area show slight impairment of their capacity to reduce methyl viologen using water as electron donor. Wholeleaf CO2 assimilation rates expressed on an area basis are observed to decrease with zinc deficiency, but do not do so significantly when expressed per unit weight of chlorophyll. It is proposed that the partial loss of chloroplasts’ photochemical capacity, aggravated by loss of photosynthetically-competent blade area in severely-stressed leaves, accounts for depressed CO2 assimilation rates measured in the zinc-deficient sugar beet leaves. Key words: chloroplast ultrastructure – CO2 assimilation – electron transport – sugar beet – zinc deficiency Abbreviations: CA carbonic anhydrase. – Chl chlorophyll. – MV methyl viologen
Introduction Lately, there has been a renewed interest in zinc deficiency in plants, and its implications for animal and human health are coming to be fully appreciated (Grusak and DellaPenna 1999, Tuormaa 1995). Zinc is a micronutrient essential to plants (Sommer and Lipman 1926), and its presence in concentrations below a critical minimum level hinders plant growth and * E-mail: [email protected]
development (Marschner 1995). Two long-known effects of zinc deficiency are on photosynthesis and on carbonic anhydrase (CA, EC. 4.2.1.1) activity (Botrill et al. 1970, Wood and Silby 1952), but many other cellular processes and components are altered by zinc deprivation (Marschner 1995). We do not yet fully understand how zinc deficiency exerts such multiple and varied effects. The finding of Welch et al. (1982), subsequently corroborated by several workers (Cakmak and Marschner 1988, Pinton et al. 1993), that zinc is required for proper membrane activity could explain reported 0176-1617/01/158/07-915 $ 15.00/0
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disturbances of processes that directly or indirectly depend on membrane integrity, but still leaves many others unexplained. In the past few years, evidence has emerged (Cakmak 2000) indicating that many of the zinc deficiency effects are due to damage resulting from attacks by reactive oxygen species (ROS) on various cell structural and functional components. Indeed, several authors have reported that zinc not only plays a critical role in decreasing or preventing the generation of ROS, but may also be involved in the expression of anti-oxidant enzymes (Cakmak 2000). Chloroplasts are a preferential site for ROS formation, and thylakoid membranes are a prime target for their action. It has been shown (Cakmak and Engels 1999, Marschner and Cakmak 1989) that low zinc levels increase the production of superoxide radicals during photosynthetic electron transport, and these, in turn, could give rise to other highly reactive oxygen species that are even more aggressive to chloroplast constituents (Asada 1999). This enhanced ROS formation could cause severe damage to thylakoid membranes, with eventual impairment of their function, and thus lead to the decreased CO2 assimilation rates that have been reported for zinc-deficient leaves of several species (Graham and Reed 1971, Ohki 1978, Randall and Bouma 1973, Sasaki et al. 1998). We show here that in sugar beet leaves, increasing zinc shortage induces extensive disorganization of chloroplast thylakoids, followed by their degradation as well as that of stromal components. These deteriorative changes resemble those occurring during natural leaf senescence, and ultimately lead to premature cell death and necrosis of blade tissue. We propose that this reduction in the blade’s photosynthetically active area, together with a decline in the photochemical capability of the chloroplasts from the remaining leaf area, are the primary causes for the observed reduction in CO2 assimilation rates. This proposal contrasts with explanations that relate inhibition of photosynthetic CO2 fixation under zinc deficiency to limitations in CO2 availability at the chloroplast carboxylation site, resulting from decreased CA activity, as will be discussed.
Materials and Methods Plant material Sugar beet plants (Beta vulgaris L. cv. F-58-554 HL) were grown in hydroponic culture in a controlled cabinet at 25/20 ˚C (day/night) temperature under a 16-h day and an illumination of 650 µmol m – 2 s –1. The plastic culture vessels were aerated continuously, and contained halfstrength Hoagland’s nutrient solution (Hoagland and Arnon 1950), with 25 µg Zn L –1 added as ZnSO4 ( + Zn treatment) or without zinc ( – Zn treatment).
Chloroplast isolation Chloroplasts were isolated in 25 mmol/L HEPES/NaOH buffer (pH 7.6) containing 10 mmol/L NaCl and 500 mmol/L sucrose; after isolation, chloroplasts were washed once and resuspended in the same buffer.
Chlorophyll was extracted in 80 % acetone and measured according to Arnon (1949).
Electron transport rates Electron transport rates from water to methyl viologen were measured polarographically as O2 uptake. The reaction mixture contained 25 mmol/L HEPES (pH 7.6), 10 mmol/L NaCl, 3 mmol/L MgCl, 0.5 mmol/L MV, and 2 mmol/L NH4Cl as uncoupler. Chloroplasts were added to a concentration equivalent of about 30 µg chl · mL –1.
Electron microscopy Pieces of leaf tissue were fixed in 2.5 % glutaraldehyde and postfixed in 1 % osmium tetroxide, as described before (Henriques 1989). After washing and dehydration in a graded series of ethanol, the samples were treated with propylene oxide and embedded in Epon (Luft 1961). Ultrathin sections were cut with a LKB ultramicrotome and examined with a Philips 300 electron microscope.
Leaf gas exchange Photosynthetic CO2 uptake rates were measured in intact leaves, as described before (Henriques and Park 1976), under atmospheric CO2 concentration and at an irradiance of 1000 µE m – 2 · s –1.
Metal determinations Leaf samples from Zn-sufficient and Zn-deficient leaves were ovendried for 48 h at 75 ˚C, digested in a nitric/perchloric mixture (5 : 1, v/v), and zinc was determined by atomic absorption spectroscopy, using a Perkin-Elmer model 3030.
Results In sugar beet leaves, zinc deficiency symptoms become progressively more acute at successive nodes of the plant’s shoot, from the outer, larger and only slightly discoloured leaves to the innermost, smaller, extensively necrotic ones, and these changes are accompanied by decreasing blade zinc contents. In this study, we used leaves moderately stressed, with mean chlorophyll and zinc contents of 0.42 g · m – 2 and 9 mg · kg –1 blade dry weight, respectively, and leaves more severely stressed, with mean chlorophyll and zinc contents of 0.34 g · m – 2 and 6 mg · kg –1, respectively (Table 1). The former exhibited mild deficiency symptoms, consisting mainly of a lighter green colouring of their blades, whereas the latter displayed more severe symptoms, with conspicuous development of small chlorotic areas («pits») between the veins. These pits are essentially devoid of chlorophyll and account largely for the reduction in the chlorophyll content of these leaves. Control leaves, from plants grown in full-nutrient medium, were bright green, with a mean chloro-
Photosynthesis in zinc-deficient sugar beet
Table 1. Chl content, PS II + PS I dependent electron flow and photosynthetic CO2 uptake rates of sugar beet leaves with varying zinc concentrations. Numbers in parentheses indicate rates of CO2 assimilation in µmol · mg – 1 chl · h – 1. Results are mean ± SD values of four replicates of three independent experiments. Chl content Zinc content H2O→MV CO2 uptake µg · g – 1 · d.wt. µmol O2 · mg – 1 chl · h – 1 µmol CO2 · m – 2 · s – 1 g · m–2 0.47 ± 0.04 0.42 ± 0.04 0.34 ± 0.07
17 ± 2.0 9 ± 2.1 6 ± 2.6
87 ± 6.1 79 ± 5.0 77 ± 7.0
24 ± 2.6 (184) 21 ± 3.0 (180) 16.5 ± 2.5 (175)
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may extend for variable lengths and involve large numbers of lamellae, giving rise to long, thick bundles (Fig. 4). Fig. 4 also shows the pronounced waviness of the single lamellae and their fuzzy contours; the stromal matrix is completely altered, with a flocculent appearance; plastoglobuli are present and ribosomes cannot be detected. Additionally, chloroplasts are often swollen and their outlines become irregular. Chloroplasts of normal appearance are seen in most of the leaf blade area between the pit zones. Table 1 shows the effects of varying zinc levels on the photochemical activity of isolated chloroplasts and on whole-leaf CO2 assimilation rates. Chloroplasts from control leaves show electron flow rates from water to methyl viologen comparable with values previously reported for sugar beet (Terry 1983), but these rates are reduced by approximately 10 % in zinc-deficient leaves, on a unit weight chlorophyll basis. Whole-leaf net CO2 uptake per unit leaf area is also decreased with increasing zinc deficiency, moderately-stressed leaves having CO2 assimilation rates that are around 90 % of the control, and severely defi-
Figure 1. Chloroplasts from control sugar beet leaves showing typical thylakoid organization; starch grains are visible in the stroma (×13,000)
phyll content of 0.47 g · m – 2 and average zinc concentration of 17mg · kg –1 blade dry weight (Table 1). The ultrastructure of chloroplasts from these three groups of leaves is shown in Figs. 1– 4. Chloroplasts from control leaves (Fig. 1) display a typical thylakoid organization, with grana stacks connected by stroma lamellae, all embedded in a stromal matrix and bounded by a double-membrane envelope. The stroma has a fine granular appearance and starch grains are frequently visible. The majority of chloroplasts from moderately-stressed leaves resemble those of control leaves, but some appear to be undergoing a process of lamellar disintegration, with plastoglobuli accumulating in the stroma; starch grains are still evident (Fig. 2). In severely deficient leaves (Figs. 3, 4), chloroplasts in the immediate vicinity of the pit areas show dramatic changes in the organization of their internal membranes, with complete unfolding of the stacked regions and the appearance of an extensive fretwork of single lamellae, which either run parallel to each other along the long axis of the chloroplast (Fig. 3) or circumvolute randomly throughout the stroma (Fig. 4). Occasionally, the single lamellae overlap to form what appears to be grana stacks, but a closer examination reveals electron transparent gaps between lamellae pairs, different from what occurs in grana stacks of normal chloroplasts. These apposed regions
Figure 2. Chloroplasts from moderately zinc-deficient leaves showing some disintegration of their internal membrane system (× 12,500)
Figure 3. Chloroplasts from severely zinc-deficient leaves showing extensive unstacking of thylakoids, with single lamellae running in parallel rows; denaturation of stromal components is evident (× 12,500)
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Figure 4. Chloroplasts from severely zinc-deficient leaves showing highly circumvoluted single lamellae and some grana-like regions; extensive degradation of both internal membranes and stromal material is evident (× 18,000)
cient leaves showing only 70 % of the control rates. Photosynthetic CO2 uptake rates, expressed per unit weight chlorophyll, are not significantly different for the various treatments.
Discussion We show here that zinc deficiency causes striking alterations in the fine structure of chloroplasts from sugar beet leaves; in leaves displaying severe deficiency symptoms, not only is the chloroplast’s organization grossly disrupted, with complete unfolding of its grana stacks, but the resulting unpaired lamellae also undergo extensive degradational changes. This observation differs from results published by other workers (Vesk et al. 1966), who reported minor or no detectable changes in chloroplasts from Zn-deficient leaves, this probably being due mostly to differences in the plant species used and in the severity of the deficiency they studied. Ultrastructural changes similar to those observed here have been reported for chloroplasts subjected to several experimental treatments (Shaw et al. 1976, Srivastava et al. 1971, Thomsom and Ellis 1972), and have been attributed to various causes such as loss of thylakoid galactolipids or of some protein(s) implicated in thylakoid stacking. Higher plant mutants lacking chlorophyll b or components of the light-harvesting chl a, b-complex (Goodchild et al. 1966, Highkin et al. 1969) have also been found to have none or a reduced number of stacked lamellae, and chloroplasts from bundle sheath cells of some C4 plants undergo loss of their granal regions during development, accompanied by changes of their chemical composition (Bazzaz and Govindje 1973, Leech et al. 1973). It is thus conceivable that chloroplasts from zinc-deficient leaves lack some unidentified constituent that prevents proper thylakoid organization, but our experiments did not aim to shed any light on this matter. We show, rather, that these disar-
ranged chloroplasts undergo an extensive breakdown of their thylakoid lamellae, as well as of their stromal components, paralleled by increases in size and/or number of plastoglobuli. Such deteriorative changes are identical to those occurring during natural leaf senescence (Smart 1994), and strongly suggest that leaf zinc shortage triggers a premature senescence process that precedes and causes cell death, ultimately leading to the formation of the pit and necrotic blade lesions visible in advanced stages of the deficiency. It is well known that senescence is an oxidative phenomenon primarily mediated by activated oxygen species (Thompson et al. 1987), and its early showing in chloroplasts from zinc-deficient leaves can be taken to support previous claims of enhanced formation of such species under zinc deprivation (Cakmak 2000), and is consistent with the view that zinc deficiency effects are mediated by oxidative stress resulting from unchecked ROS formation. Electron transport rates for chloroplasts from both moderately- and severely-stressed leaves are not significantly different, both showing decreases of approximately 10 % relatively to the control. Most of the chlorophyll decrease measured in severely deficient leaves results from the presence of the pigment-devoid pit/necrotic blade spots, rather than from a chl reduction spread over the entire leaf area. It appears, thus, that the photochemical capacity of the photosynthetic units remaining in severely deficient leaves is essentially unchanged relative to those in moderately-stressed leaves, and this observation can explain the decline in the rate of CO2 assimilation measured in these leaves in a direct and straightforward manner: the approximately 30 % reduction in photosynthetic CO2 uptake must result from the ca. 10 % decrease in the photochemical ability of the chloroplasts, aggravated by an approximately 20 % reduction in photosyntheticallycompetent blade area. Such simple interpretation of our results presents an alternative to other explanations put forth that associate the observed inhibition of photosynthesis in zinc-deficient leaves with limitations in CO2 availability within the chloroplasts resulting from decreased CA activity (Ohki 1978, Sasaki et al. 1998). CA is a zinc-containing enzyme that has been suggested as facilitating CO2 transfer from the stomatal cavity to the carboxylation sites, and its activity has been unequivocally shown to be increasingly depressed with progressive lowering of leaf zinc content, down to residual levels in severely deficient leaves (Ohki 1978, Randall and Bouma 1973, Sasaki et al. 1998, Sharma et al. 1995). However, the relationship between CA activity and photosynthetic rate is still uncertain. There are repeated reports (Ohki 1978, Randall and Bouma 1973, Sasaki et al. 1998) of high rates of photosynthesis in leaves with extremely low CA levels, and applications of CA inhibitors to intact chloroplasts were shown not to lower their rates of photosynthesis (Sasaki et al. 1998). Randall and Bouma (1973) failed to find any effect of CA activity on photosynthesis, and from measurements of the process at CO2 levels, both below and above normal atmospheric concentration, concluded the enzyme was probably
Photosynthesis in zinc-deficient sugar beet not involved in the CO2 transfer process to the chloroplast. Recently, Sasaki et al. (1998), measured the components of mesophyll resistance to CO2 assimilation in relation to CA activity and found that low enzyme activities significantly increased the CO2 transfer resistance from the stomatal cavity to the site of carboxylation, but did not observe any corresponding decrease in the rate of photosynthesis. Clearly, a role of CA on photosynthesis has not been conclusively demonstrated, and we have to look for alternative explanations for the effects of zinc deficiency on the photosynthetic process. For sugar beet, we envision these effects as occurring in the following sequence: a) low zinc levels inhibit chlorophyll synthesis and possibly other as yet unknown components of thylakoid membranes; b) this lack of lamellar components impairs proper thylakoid assembly and function; c) these chloroplast disfunctions in turn trigger the organelle’s degradation and lead to premature cell senescence and ultimately to formation of blade pits. These photosynthetically, non-competent blade areas account for most of the measured decrease in the CO2 assimilation rate of zinc-deficient leaves. If additional inhibitory effects were in operation, such as increased resistance to CO2 transfer to the chloroplasts, these would affect the remaining photosynthetically competent blade area and would bring about further declines in net CO2 uptake rates. These results were not observed.
References Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50: 601– 639 Bazzaz MB, Govindjee (1973) Photochemical properties of mesophyll and bundle sheath chloroplasts. Plant Physiol 52: 257– 262 Botrill DE, Possingham JV, Kriedemann PE (1970) The effect of nutrient deficiencies on photosynthesis and respiration in spinach. Plant and Soil 32: 424 – 438 Cakmak I (2000) Possible roles of zinc in protecting plant cells from damage by reactive oxygen species. New Phytol 146: 185 – 205 Cakmak I, Engels C (1999) Role of mineral nutrients in photosynthesis and yield formation. In: Rengel Z (ed) Mineral Nutrition of Crops. Haworth Press, New York, pp 141–168 Cakmak I, Marschner H (1988) Increase in membrane permeability and exudation in roots of zinc-deficient plants. J Plant Physiol 132: 351– 361 Goodchild DJ, Highkin HR, Boardman NK (1966) The fine structure of chloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43: 684 – 688 Graham D, Reed ML (1971) Carbonic anhydrase and the regulation of photosynthesis. Nature 231: 81– 83 Grusak MA, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu Rev Plant Physiol Plant Mol Biol 50: 133–161 Henriques F (1989) Effects of copper deficiency on the photosynthetic apparatus of sugar beet (Beta vulgaris L). J Plant Physiol 135: 453 – 458
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Henriques F, Park R (1976) Development of the photosynthetic unit in lettuce. Proc Natl Acad Sci USA 73: 4560 – 4564 Highkin HR, Boardman NK, Goodchild DJ (1969) Photosynthetic studies on a pea mutant deficient in chlorophyll. Plant Physiol 44: 1310–1320 Hoagland DR, Arnon DI (1950) The water culture method for growing plants without soil. Calif Agric Expt Sta Bull, 347 Leech RM, Rumbsy MG, Thomsom WW (1973) Plastid differentiation, acyl lipid, and fatty acid changes in developing green maize leaves. Plant Physiol 52: 240 – 245 Luft J (1961) Improvement in epoxy resin embedding methods. J Biophys Biochem Cytol 9: 409 – 414 Marschner H (1995) Mineral nutrition of higher plants, 2nd ed. Academic Press, London Marschner H, Cakmak I (1989) High light intensity enhances chlorosis and necrosis in leaves of zinc-, potassium- and magnesium-deficient bean (Phaseolus vulgaris) plants. J Plant Physiol 134: 308 – 315 Ohki K (1978) Zinc concentration in soybean as related to growth, photosynthesis and carbonic anhydrase activity. Crop Sci 18: 79 – 82 Pinton R, Cakmak I, Marschner H (1993) Effect of zinc deficiency on proton fluxes in plasma-membrane enriched vesicles from bean roots. J Exp Bot 44: 623 – 630 Randall PJ, Bouma D (1973) Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach. Plant Physiol 52: 229 – 232 Sasaki H, Hirose T, Watanabe Y, Ohsugi R (1998) Carbonic anhydrase activity and CO2 transfer resistance in zinc-deficient rice leaves. Plant Physiol 118: 929 – 934 Sharma PN, Tripathi A, Bisht SS (1995) Zinc requirement for stomatal opening in cauliflower. Plant Physiol 107: 751–756 Shaw AB, Anderson MM, McCarty RE (1976) Role of galactolipids in spinach chloroplast lamellar membrane. II. Effects of galactolipid depletion on phosphorylation and electron flow. Plant Physiol 57: 724–729 Smart C (1994) Gene expression during leaf senescence. New Phytol 126: 419 – 448 Sommer AL, Lipman CB (1926) Evidence of the indispensable nature of zinc and boron for higher green plants. Plant Physiol 1: 231– 245 Srivastava LM, Vesk M, Singh AP (1971) Effect of chloramphenicol on membrane transformations in plastids. Can J Bot 49: 587– 593 Terry N (1983) Limiting factors in photosynthesis. IV. Iron stressmediated changes in light harvesting and electron transport capacity and its effects on photosynthesis. Plant Physiol 71: 855 – 860 Thompson JE, Ledge RL, Barber RF (1987) The role of free radicals in senescence and wounding. New Phytol 105: 317– 344 Thomsom WW, Ellis RJ (1972) Inhibition of grana formation by lincomycin. Planta 108: 89 – 92 Tuormaa T (1995) The adverse effects of zinc deficiency. J Orthom Med 10: 149–164 Vesk M, Possingham JV, Mercer FV (1966) The effect of mineral deficiencies on the structure of the leaf cells of tomato, spinach and maize. Aust J Bot 14: 1–18 Welch RM, Webb MJ, Loneragan JI (1982) Zinc in membrane function and its role in phosphorus toxicity. In: Scaife A (ed) Proc Ninth Plant Nut Colloq CAB International, Warwick, UK, pp 710–715 Wood JG, Silby PM (1952) Carbonic anhydrase activity in plants in relation to zinc content. Aust J Sci Res Bull 5: 244 – 255
Loss of blade photosynthetic area and of chloroplasts’ photochemical capacity account for reduced CO2 assimilation rates in zinc-deficient sugar beet leaves Fernando S. Henriques* Plant Biology Unit, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte da Caparica, Portugal Received October 18, 2000 · Accepted January 26, 2001
Summary The effects of zinc deficiency on sugar beet chloroplasts’ ultrastructure and photochemical ability, as well as on whole-leaf CO2 assimilation rates, were evaluated. It is shown that increasing zinc shortage firstly induces progressive disorganisation of the chloroplasts’ internal membrane system, followed by degradation of both lamellae and stromal components. This zinc deficiency-triggered premature senescence process leads to cell death and, ultimately, to chlorotic/necrotic blade lesions, thus reducing the photosynthetically-active leaf area; chloroplasts in the remaining leaf area show slight impairment of their capacity to reduce methyl viologen using water as electron donor. Wholeleaf CO2 assimilation rates expressed on an area basis are observed to decrease with zinc deficiency, but do not do so significantly when expressed per unit weight of chlorophyll. It is proposed that the partial loss of chloroplasts’ photochemical capacity, aggravated by loss of photosynthetically-competent blade area in severely-stressed leaves, accounts for depressed CO2 assimilation rates measured in the zinc-deficient sugar beet leaves. Key words: chloroplast ultrastructure – CO2 assimilation – electron transport – sugar beet – zinc deficiency Abbreviations: CA carbonic anhydrase. – Chl chlorophyll. – MV methyl viologen
Introduction Lately, there has been a renewed interest in zinc deficiency in plants, and its implications for animal and human health are coming to be fully appreciated (Grusak and DellaPenna 1999, Tuormaa 1995). Zinc is a micronutrient essential to plants (Sommer and Lipman 1926), and its presence in concentrations below a critical minimum level hinders plant growth and * E-mail: [email protected]
development (Marschner 1995). Two long-known effects of zinc deficiency are on photosynthesis and on carbonic anhydrase (CA, EC. 4.2.1.1) activity (Botrill et al. 1970, Wood and Silby 1952), but many other cellular processes and components are altered by zinc deprivation (Marschner 1995). We do not yet fully understand how zinc deficiency exerts such multiple and varied effects. The finding of Welch et al. (1982), subsequently corroborated by several workers (Cakmak and Marschner 1988, Pinton et al. 1993), that zinc is required for proper membrane activity could explain reported 0176-1617/01/158/07-915 $ 15.00/0
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disturbances of processes that directly or indirectly depend on membrane integrity, but still leaves many others unexplained. In the past few years, evidence has emerged (Cakmak 2000) indicating that many of the zinc deficiency effects are due to damage resulting from attacks by reactive oxygen species (ROS) on various cell structural and functional components. Indeed, several authors have reported that zinc not only plays a critical role in decreasing or preventing the generation of ROS, but may also be involved in the expression of anti-oxidant enzymes (Cakmak 2000). Chloroplasts are a preferential site for ROS formation, and thylakoid membranes are a prime target for their action. It has been shown (Cakmak and Engels 1999, Marschner and Cakmak 1989) that low zinc levels increase the production of superoxide radicals during photosynthetic electron transport, and these, in turn, could give rise to other highly reactive oxygen species that are even more aggressive to chloroplast constituents (Asada 1999). This enhanced ROS formation could cause severe damage to thylakoid membranes, with eventual impairment of their function, and thus lead to the decreased CO2 assimilation rates that have been reported for zinc-deficient leaves of several species (Graham and Reed 1971, Ohki 1978, Randall and Bouma 1973, Sasaki et al. 1998). We show here that in sugar beet leaves, increasing zinc shortage induces extensive disorganization of chloroplast thylakoids, followed by their degradation as well as that of stromal components. These deteriorative changes resemble those occurring during natural leaf senescence, and ultimately lead to premature cell death and necrosis of blade tissue. We propose that this reduction in the blade’s photosynthetically active area, together with a decline in the photochemical capability of the chloroplasts from the remaining leaf area, are the primary causes for the observed reduction in CO2 assimilation rates. This proposal contrasts with explanations that relate inhibition of photosynthetic CO2 fixation under zinc deficiency to limitations in CO2 availability at the chloroplast carboxylation site, resulting from decreased CA activity, as will be discussed.
Materials and Methods Plant material Sugar beet plants (Beta vulgaris L. cv. F-58-554 HL) were grown in hydroponic culture in a controlled cabinet at 25/20 ˚C (day/night) temperature under a 16-h day and an illumination of 650 µmol m – 2 s –1. The plastic culture vessels were aerated continuously, and contained halfstrength Hoagland’s nutrient solution (Hoagland and Arnon 1950), with 25 µg Zn L –1 added as ZnSO4 ( + Zn treatment) or without zinc ( – Zn treatment).
Chloroplast isolation Chloroplasts were isolated in 25 mmol/L HEPES/NaOH buffer (pH 7.6) containing 10 mmol/L NaCl and 500 mmol/L sucrose; after isolation, chloroplasts were washed once and resuspended in the same buffer.
Chlorophyll was extracted in 80 % acetone and measured according to Arnon (1949).
Electron transport rates Electron transport rates from water to methyl viologen were measured polarographically as O2 uptake. The reaction mixture contained 25 mmol/L HEPES (pH 7.6), 10 mmol/L NaCl, 3 mmol/L MgCl, 0.5 mmol/L MV, and 2 mmol/L NH4Cl as uncoupler. Chloroplasts were added to a concentration equivalent of about 30 µg chl · mL –1.
Electron microscopy Pieces of leaf tissue were fixed in 2.5 % glutaraldehyde and postfixed in 1 % osmium tetroxide, as described before (Henriques 1989). After washing and dehydration in a graded series of ethanol, the samples were treated with propylene oxide and embedded in Epon (Luft 1961). Ultrathin sections were cut with a LKB ultramicrotome and examined with a Philips 300 electron microscope.
Leaf gas exchange Photosynthetic CO2 uptake rates were measured in intact leaves, as described before (Henriques and Park 1976), under atmospheric CO2 concentration and at an irradiance of 1000 µE m – 2 · s –1.
Metal determinations Leaf samples from Zn-sufficient and Zn-deficient leaves were ovendried for 48 h at 75 ˚C, digested in a nitric/perchloric mixture (5 : 1, v/v), and zinc was determined by atomic absorption spectroscopy, using a Perkin-Elmer model 3030.
Results In sugar beet leaves, zinc deficiency symptoms become progressively more acute at successive nodes of the plant’s shoot, from the outer, larger and only slightly discoloured leaves to the innermost, smaller, extensively necrotic ones, and these changes are accompanied by decreasing blade zinc contents. In this study, we used leaves moderately stressed, with mean chlorophyll and zinc contents of 0.42 g · m – 2 and 9 mg · kg –1 blade dry weight, respectively, and leaves more severely stressed, with mean chlorophyll and zinc contents of 0.34 g · m – 2 and 6 mg · kg –1, respectively (Table 1). The former exhibited mild deficiency symptoms, consisting mainly of a lighter green colouring of their blades, whereas the latter displayed more severe symptoms, with conspicuous development of small chlorotic areas («pits») between the veins. These pits are essentially devoid of chlorophyll and account largely for the reduction in the chlorophyll content of these leaves. Control leaves, from plants grown in full-nutrient medium, were bright green, with a mean chloro-
Photosynthesis in zinc-deficient sugar beet
Table 1. Chl content, PS II + PS I dependent electron flow and photosynthetic CO2 uptake rates of sugar beet leaves with varying zinc concentrations. Numbers in parentheses indicate rates of CO2 assimilation in µmol · mg – 1 chl · h – 1. Results are mean ± SD values of four replicates of three independent experiments. Chl content Zinc content H2O→MV CO2 uptake µg · g – 1 · d.wt. µmol O2 · mg – 1 chl · h – 1 µmol CO2 · m – 2 · s – 1 g · m–2 0.47 ± 0.04 0.42 ± 0.04 0.34 ± 0.07
17 ± 2.0 9 ± 2.1 6 ± 2.6
87 ± 6.1 79 ± 5.0 77 ± 7.0
24 ± 2.6 (184) 21 ± 3.0 (180) 16.5 ± 2.5 (175)
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may extend for variable lengths and involve large numbers of lamellae, giving rise to long, thick bundles (Fig. 4). Fig. 4 also shows the pronounced waviness of the single lamellae and their fuzzy contours; the stromal matrix is completely altered, with a flocculent appearance; plastoglobuli are present and ribosomes cannot be detected. Additionally, chloroplasts are often swollen and their outlines become irregular. Chloroplasts of normal appearance are seen in most of the leaf blade area between the pit zones. Table 1 shows the effects of varying zinc levels on the photochemical activity of isolated chloroplasts and on whole-leaf CO2 assimilation rates. Chloroplasts from control leaves show electron flow rates from water to methyl viologen comparable with values previously reported for sugar beet (Terry 1983), but these rates are reduced by approximately 10 % in zinc-deficient leaves, on a unit weight chlorophyll basis. Whole-leaf net CO2 uptake per unit leaf area is also decreased with increasing zinc deficiency, moderately-stressed leaves having CO2 assimilation rates that are around 90 % of the control, and severely defi-
Figure 1. Chloroplasts from control sugar beet leaves showing typical thylakoid organization; starch grains are visible in the stroma (×13,000)
phyll content of 0.47 g · m – 2 and average zinc concentration of 17mg · kg –1 blade dry weight (Table 1). The ultrastructure of chloroplasts from these three groups of leaves is shown in Figs. 1– 4. Chloroplasts from control leaves (Fig. 1) display a typical thylakoid organization, with grana stacks connected by stroma lamellae, all embedded in a stromal matrix and bounded by a double-membrane envelope. The stroma has a fine granular appearance and starch grains are frequently visible. The majority of chloroplasts from moderately-stressed leaves resemble those of control leaves, but some appear to be undergoing a process of lamellar disintegration, with plastoglobuli accumulating in the stroma; starch grains are still evident (Fig. 2). In severely deficient leaves (Figs. 3, 4), chloroplasts in the immediate vicinity of the pit areas show dramatic changes in the organization of their internal membranes, with complete unfolding of the stacked regions and the appearance of an extensive fretwork of single lamellae, which either run parallel to each other along the long axis of the chloroplast (Fig. 3) or circumvolute randomly throughout the stroma (Fig. 4). Occasionally, the single lamellae overlap to form what appears to be grana stacks, but a closer examination reveals electron transparent gaps between lamellae pairs, different from what occurs in grana stacks of normal chloroplasts. These apposed regions
Figure 2. Chloroplasts from moderately zinc-deficient leaves showing some disintegration of their internal membrane system (× 12,500)
Figure 3. Chloroplasts from severely zinc-deficient leaves showing extensive unstacking of thylakoids, with single lamellae running in parallel rows; denaturation of stromal components is evident (× 12,500)
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Figure 4. Chloroplasts from severely zinc-deficient leaves showing highly circumvoluted single lamellae and some grana-like regions; extensive degradation of both internal membranes and stromal material is evident (× 18,000)
cient leaves showing only 70 % of the control rates. Photosynthetic CO2 uptake rates, expressed per unit weight chlorophyll, are not significantly different for the various treatments.
Discussion We show here that zinc deficiency causes striking alterations in the fine structure of chloroplasts from sugar beet leaves; in leaves displaying severe deficiency symptoms, not only is the chloroplast’s organization grossly disrupted, with complete unfolding of its grana stacks, but the resulting unpaired lamellae also undergo extensive degradational changes. This observation differs from results published by other workers (Vesk et al. 1966), who reported minor or no detectable changes in chloroplasts from Zn-deficient leaves, this probably being due mostly to differences in the plant species used and in the severity of the deficiency they studied. Ultrastructural changes similar to those observed here have been reported for chloroplasts subjected to several experimental treatments (Shaw et al. 1976, Srivastava et al. 1971, Thomsom and Ellis 1972), and have been attributed to various causes such as loss of thylakoid galactolipids or of some protein(s) implicated in thylakoid stacking. Higher plant mutants lacking chlorophyll b or components of the light-harvesting chl a, b-complex (Goodchild et al. 1966, Highkin et al. 1969) have also been found to have none or a reduced number of stacked lamellae, and chloroplasts from bundle sheath cells of some C4 plants undergo loss of their granal regions during development, accompanied by changes of their chemical composition (Bazzaz and Govindje 1973, Leech et al. 1973). It is thus conceivable that chloroplasts from zinc-deficient leaves lack some unidentified constituent that prevents proper thylakoid organization, but our experiments did not aim to shed any light on this matter. We show, rather, that these disar-
ranged chloroplasts undergo an extensive breakdown of their thylakoid lamellae, as well as of their stromal components, paralleled by increases in size and/or number of plastoglobuli. Such deteriorative changes are identical to those occurring during natural leaf senescence (Smart 1994), and strongly suggest that leaf zinc shortage triggers a premature senescence process that precedes and causes cell death, ultimately leading to the formation of the pit and necrotic blade lesions visible in advanced stages of the deficiency. It is well known that senescence is an oxidative phenomenon primarily mediated by activated oxygen species (Thompson et al. 1987), and its early showing in chloroplasts from zinc-deficient leaves can be taken to support previous claims of enhanced formation of such species under zinc deprivation (Cakmak 2000), and is consistent with the view that zinc deficiency effects are mediated by oxidative stress resulting from unchecked ROS formation. Electron transport rates for chloroplasts from both moderately- and severely-stressed leaves are not significantly different, both showing decreases of approximately 10 % relatively to the control. Most of the chlorophyll decrease measured in severely deficient leaves results from the presence of the pigment-devoid pit/necrotic blade spots, rather than from a chl reduction spread over the entire leaf area. It appears, thus, that the photochemical capacity of the photosynthetic units remaining in severely deficient leaves is essentially unchanged relative to those in moderately-stressed leaves, and this observation can explain the decline in the rate of CO2 assimilation measured in these leaves in a direct and straightforward manner: the approximately 30 % reduction in photosynthetic CO2 uptake must result from the ca. 10 % decrease in the photochemical ability of the chloroplasts, aggravated by an approximately 20 % reduction in photosyntheticallycompetent blade area. Such simple interpretation of our results presents an alternative to other explanations put forth that associate the observed inhibition of photosynthesis in zinc-deficient leaves with limitations in CO2 availability within the chloroplasts resulting from decreased CA activity (Ohki 1978, Sasaki et al. 1998). CA is a zinc-containing enzyme that has been suggested as facilitating CO2 transfer from the stomatal cavity to the carboxylation sites, and its activity has been unequivocally shown to be increasingly depressed with progressive lowering of leaf zinc content, down to residual levels in severely deficient leaves (Ohki 1978, Randall and Bouma 1973, Sasaki et al. 1998, Sharma et al. 1995). However, the relationship between CA activity and photosynthetic rate is still uncertain. There are repeated reports (Ohki 1978, Randall and Bouma 1973, Sasaki et al. 1998) of high rates of photosynthesis in leaves with extremely low CA levels, and applications of CA inhibitors to intact chloroplasts were shown not to lower their rates of photosynthesis (Sasaki et al. 1998). Randall and Bouma (1973) failed to find any effect of CA activity on photosynthesis, and from measurements of the process at CO2 levels, both below and above normal atmospheric concentration, concluded the enzyme was probably
Photosynthesis in zinc-deficient sugar beet not involved in the CO2 transfer process to the chloroplast. Recently, Sasaki et al. (1998), measured the components of mesophyll resistance to CO2 assimilation in relation to CA activity and found that low enzyme activities significantly increased the CO2 transfer resistance from the stomatal cavity to the site of carboxylation, but did not observe any corresponding decrease in the rate of photosynthesis. Clearly, a role of CA on photosynthesis has not been conclusively demonstrated, and we have to look for alternative explanations for the effects of zinc deficiency on the photosynthetic process. For sugar beet, we envision these effects as occurring in the following sequence: a) low zinc levels inhibit chlorophyll synthesis and possibly other as yet unknown components of thylakoid membranes; b) this lack of lamellar components impairs proper thylakoid assembly and function; c) these chloroplast disfunctions in turn trigger the organelle’s degradation and lead to premature cell senescence and ultimately to formation of blade pits. These photosynthetically, non-competent blade areas account for most of the measured decrease in the CO2 assimilation rate of zinc-deficient leaves. If additional inhibitory effects were in operation, such as increased resistance to CO2 transfer to the chloroplasts, these would affect the remaining photosynthetically competent blade area and would bring about further declines in net CO2 uptake rates. These results were not observed.
References Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50: 601– 639 Bazzaz MB, Govindjee (1973) Photochemical properties of mesophyll and bundle sheath chloroplasts. Plant Physiol 52: 257– 262 Botrill DE, Possingham JV, Kriedemann PE (1970) The effect of nutrient deficiencies on photosynthesis and respiration in spinach. Plant and Soil 32: 424 – 438 Cakmak I (2000) Possible roles of zinc in protecting plant cells from damage by reactive oxygen species. New Phytol 146: 185 – 205 Cakmak I, Engels C (1999) Role of mineral nutrients in photosynthesis and yield formation. In: Rengel Z (ed) Mineral Nutrition of Crops. Haworth Press, New York, pp 141–168 Cakmak I, Marschner H (1988) Increase in membrane permeability and exudation in roots of zinc-deficient plants. J Plant Physiol 132: 351– 361 Goodchild DJ, Highkin HR, Boardman NK (1966) The fine structure of chloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43: 684 – 688 Graham D, Reed ML (1971) Carbonic anhydrase and the regulation of photosynthesis. Nature 231: 81– 83 Grusak MA, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu Rev Plant Physiol Plant Mol Biol 50: 133–161 Henriques F (1989) Effects of copper deficiency on the photosynthetic apparatus of sugar beet (Beta vulgaris L). J Plant Physiol 135: 453 – 458
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Henriques F, Park R (1976) Development of the photosynthetic unit in lettuce. Proc Natl Acad Sci USA 73: 4560 – 4564 Highkin HR, Boardman NK, Goodchild DJ (1969) Photosynthetic studies on a pea mutant deficient in chlorophyll. Plant Physiol 44: 1310–1320 Hoagland DR, Arnon DI (1950) The water culture method for growing plants without soil. Calif Agric Expt Sta Bull, 347 Leech RM, Rumbsy MG, Thomsom WW (1973) Plastid differentiation, acyl lipid, and fatty acid changes in developing green maize leaves. Plant Physiol 52: 240 – 245 Luft J (1961) Improvement in epoxy resin embedding methods. J Biophys Biochem Cytol 9: 409 – 414 Marschner H (1995) Mineral nutrition of higher plants, 2nd ed. Academic Press, London Marschner H, Cakmak I (1989) High light intensity enhances chlorosis and necrosis in leaves of zinc-, potassium- and magnesium-deficient bean (Phaseolus vulgaris) plants. J Plant Physiol 134: 308 – 315 Ohki K (1978) Zinc concentration in soybean as related to growth, photosynthesis and carbonic anhydrase activity. Crop Sci 18: 79 – 82 Pinton R, Cakmak I, Marschner H (1993) Effect of zinc deficiency on proton fluxes in plasma-membrane enriched vesicles from bean roots. J Exp Bot 44: 623 – 630 Randall PJ, Bouma D (1973) Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach. Plant Physiol 52: 229 – 232 Sasaki H, Hirose T, Watanabe Y, Ohsugi R (1998) Carbonic anhydrase activity and CO2 transfer resistance in zinc-deficient rice leaves. Plant Physiol 118: 929 – 934 Sharma PN, Tripathi A, Bisht SS (1995) Zinc requirement for stomatal opening in cauliflower. Plant Physiol 107: 751–756 Shaw AB, Anderson MM, McCarty RE (1976) Role of galactolipids in spinach chloroplast lamellar membrane. II. Effects of galactolipid depletion on phosphorylation and electron flow. Plant Physiol 57: 724–729 Smart C (1994) Gene expression during leaf senescence. New Phytol 126: 419 – 448 Sommer AL, Lipman CB (1926) Evidence of the indispensable nature of zinc and boron for higher green plants. Plant Physiol 1: 231– 245 Srivastava LM, Vesk M, Singh AP (1971) Effect of chloramphenicol on membrane transformations in plastids. Can J Bot 49: 587– 593 Terry N (1983) Limiting factors in photosynthesis. IV. Iron stressmediated changes in light harvesting and electron transport capacity and its effects on photosynthesis. Plant Physiol 71: 855 – 860 Thompson JE, Ledge RL, Barber RF (1987) The role of free radicals in senescence and wounding. New Phytol 105: 317– 344 Thomsom WW, Ellis RJ (1972) Inhibition of grana formation by lincomycin. Planta 108: 89 – 92 Tuormaa T (1995) The adverse effects of zinc deficiency. J Orthom Med 10: 149–164 Vesk M, Possingham JV, Mercer FV (1966) The effect of mineral deficiencies on the structure of the leaf cells of tomato, spinach and maize. Aust J Bot 14: 1–18 Welch RM, Webb MJ, Loneragan JI (1982) Zinc in membrane function and its role in phosphorus toxicity. In: Scaife A (ed) Proc Ninth Plant Nut Colloq CAB International, Warwick, UK, pp 710–715 Wood JG, Silby PM (1952) Carbonic anhydrase activity in plants in relation to zinc content. Aust J Sci Res Bull 5: 244 – 255