Effects of Copper Deficiency on the Photosynthetic Apparatus of Sugar beet (Beta vulgaris L.)

J. PlantPbysiol. Vol. 135. pp. 453-458(1989)

Effects of Copper Deficiency on the Photosynthetic Apparatus of Sugar beet (Beta vulgaris L.) FERNANDO

S.

HENRIQUES

Sector de Biologia Vegetal, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte da Caparica, Portugal Received April!7, 1989· Accepted July 14, 1989

Summary The effects of copper deficiency on chlorophyll concentration, photochemical activity, polypeptide composition and ultrastructure of chloroplasts from sugar beet (Beta vulgaris L.) leaves were investigated. The chlorophyll content of copper-deficient leaves was reduced to less than half of the control. The chlorophyll a/ b ratio was increased, averaging 3.4 against 3.1 in the controls. Photosystem 1- and II-dependent electron transport activities were differently affected by copper deficiency, the photosystem I-mediated methyl viologen reduction being more drastically decreased than the photosystem II-mediated p-benzoquinone reduction (80 and 65 % inhibition, respectively). The polypeptide pattern of chloroplast internal membranes was qualitatively similar in control and deficient leaves. Quantitative differences appeared in the 70, 55, 25 and 15 kdalton regions. Chloroplasts from copper-deficient leaves exhibited significant changes in their ultrastructure dependent on the strength of the copper deficiency. In such chloroplasts the internal membrane system was greatly reduced. It is our contention that the changes in internal organization account for some of the observed reductions in pigment and photochemical capability shown by chloroplasts from copper-deficient leaves.

Key words: Beta vulgaris L., chloroplast membrane polypeptides, chloroplast ultrastructure, copper deficiency, electron transport, sugar beet. Abbreviations: chI methyl viologen; PS

= =

chlorophyll; DCIP = 2,6-dichlorophenolindophenolj kD photosystemj SDS == sodium dodecyl sulfate.

Introduction Copper has long been known to be an essential micronutrient for higher plants (Sommer, 1931j Lipman and Mackiney, 1931j Arnon and Stout, 1939) but its role in plant metabolism is being more fully understood only in recent years. Several workers reported that copper deficiency affects a diversity of biochemical and physiological processes, ranging from photosynthesis (Katoh and San Pietro, 1966j Botrill et al., 1970j Natr, 1972; Pissarek, 1974j Baszynski et aI., 1978j Droppa et al., 1984 a; Sandman, 1985j Droppa et aI., 1987), lipid and protein synthesis (Rasheed and Seeley, 1966j Droppa et al., 1984 b), dark respiration (Zinkiewicz, 1985) to © 1989 by Gustav Fischer Verlag, Stuttgart

=

kilodaltonj MV

=

lignification (Rahimi and Bussler, 1973) and plant susceptibility to microorganism infection (Wood and Robson, 1984). Photosynthesis is greatly affected by availability of copper, an observation that in the past has been mostly attributed to a decrease in PSI due to lowering of the blue copper-protein plastocyaninj however, recent work indicates that copper may also function at other sites of chloroplast electron transport (Droppa et aI., 1984aj Sandman, 1985j Droppa et al., 1987). Furthermore, it has been shown that copper deficiency decreases pigment and plastoquinone synthesis and changes stoichiometric ratios between functional components of photosynthesis (Droppa et aI., 1984 bj Baszyinski et

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al., 1985; Droppa et al., 1984 a). It seems, thus, that copper deficiency has multiple effects on chloroplast function, some of which are possibly uncovered as yet. There have been some conflicting results on the effects of copper deficiency on chloroplast ultrastructure. Vesk et aI. (1966) reported no changes in the internal structure of eudeficient chloroplasts of tomato and spinach, whereas Baszynski et al. (1978) observed a disintegration of the thylakoid membranes of chloroplasts from eu-deficient plants of oat and spinach. Recently, Droppa et aI. (1984a), working with sugar beet, failed to observe any effects of eu deficiency on chloroplast ultrastructure, contrary to previous results from our laboratory (Henriques, 1984) that showed conspicuous changes in chloroplasts of the same species under copper deficiency. These discrepancies seemed important enough to be pursued further. In this paper data on the photochemistry, polypeptide composition and ultrastructure of control and eu-deficient chloroplasts of sugar beet are presented. Briefly, marked decreases in both PS 1- and PS II-dependent electron transport activities of eu-deficient chloroplasts have been found to exhibit quantitative polypeptide differences compared to the controls and to display gross alterations in their internal membrane system.

anhydrous methanol and dried under vacuum for 3 hours. The dried protein pellet was dissolved in 0,0625M Tris-HCl (pH 6.8), 5 % glycerol, 5 % /3-mercaptoethanol and 2 % SDS at a concentration of 1 mg protein/ml by heating in boiling water for 2 min.

SDS·Polyacrylamide gel electrophoresis For electrophoresis, a 1 em long 5 % stacking gel (PH 6.8) and a 8 cm long 9 % separating gel were used as described before in detail (Henriques and Park, 1975). Staining, destaining, densitometric tracings and molecular-weight estimations were performed as before (Henriques and Park, 1975).

Electron Microscopy Pieces of leaf tissue were fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (PH 7.0) containing 1.5 % sucrose for 2 hours, buffer-washed and post-fixed in cacodylate-buffered 1 % osmium tetroxide for 1 hour. After buffer-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, double stained with aqueous uranyl acetate and lead citrate (Reynolds, 1963) and examined in a Philips TEM300 electron microscope at 80KV.

Results and Discussion Material and Methods Plant material and growth conditions 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) and illuminated with 650I'Em-2s-1 over a 16h-day. The seeds were germinated in moist vermiculite and after 2 weeks the seedlings were transferred to plastic vessels which were aerated continuously and contained half-strength Hoagland nutrient solution (Hoagland and Arnon, 1950) with or without copper. The plants were grown for an additional 6 weeks under the conditions described above. Leaf material from copper-deprived conditions used for these studies showed clear symptoms of deficiency.

Chloroplast isolation Chloroplasts were isolated in 0.05 M potassium phosphate buffer (PH 7.4) containing 0.01 M KCl and 0.5 M sucrose, following the method of Sane et al. (1970); after isolation chloroplasts were washed twice in the same buffer without sucrose and resuspended in an appropriate buffer for subsequent assays. For polypeptide analysis the chloroplasts were washed once with 1 mM EDT A (PH 8.0) and centrifuged at 20,000 x g for 20 min.

Electron transport rates PS II-dependent (H20'" pBQ) and PS I-dependent (DCIPH 2'" MV) electron transport rates were measured polarographically using a Clark-type oxygen electrode. The reaction mixture contained 50 mM HEPES (PH 7.6), 250,M p-BQ, 10 mM methylamine, 1 mM sodium ascorbate, 50,M DCIP and 100 I'M MV; chloroplasts were added to a concentration equivalent to about 30 I'g chI.

Data on chlorophyll content, chI a/ b ratio and photochemical activity of chloroplasts from control and eu-deficient sugar beet leaves are shown in Table 1. The chi content, expressed per unit leaf area, of chloroplasts from copper-deficient leaves was less than half of the controls and the chI a/ b ratio was higher, averaging 3.4 against 3.1 in the controls. Droppa et aI. (1984 b) working with sugar beet plants subjected to «severe» copper deficiency (leaf eu levels amounting to 23 % of the control) observed a 63 % reduction in chI content, which compares well with our measured 55 % decrease in chi for the same species and cultivar. Additionally, Droppa et al. (1984 a) found an increase in the chi a/ b ratio in eu-deficient chloroplasts (2.97 to 3.17), similar to what we observed in this work (3.1 to 3.4). The similarity of the findings between these two works, suggests that our eu-deficient leaves have a degree of eu availability comparable to the «severely» deficient leaves of Droppa et al. (1984 a). Table 1 also shows that eu-deficient chloroplasts displayed a great decrease in photochemical capability, their rates of PS 11- and PS I-mediated electron transport reactions being ca. 3- and 5-fold lower, respectively, than the corresponding rates for the controls. The large decreases in PS 1- and PS 11associated photosynthetic activities found in this work agree with previous data from other authors (Baszynski et aI., 1978; Droppa et aI., 1984 a). The observed reduction of PS I activity is somewhat expected in view of the role played by

Table 1: Chlorophyll content and photochemical activity of control and copper-deficient sugar beet chloroplasts.

Protein preparation and solubilization EDTA- washed chloroplasts were lipid-extracted with a chloroform: methanol mixture (1 : 2, v/v), followed by three washes with

Control chlor. Cu-def. chlor.

Total chi chI alb (mg/dm 2)

PS II activity "moI02mg-lchlh-l

PS I activity "moI02mg-lchlh-l

4.1 1.8

207± 16 73 ± 9

289±21 58 ± 4

3.1 3.4

Copper Deficiency in Sugar beet Chloroplasts

the copper-containing plastocyanin in this photosystem, but the measured inhibition of PS IT, induced by copper deficiency, is still not well understood. An effect of copper deficiency in PS II-reactions has already been suggested in the past by various authors (Renger, E. et al., 1967; Barr and Crane, 1976) working with copperchelating agents. Spencer and Possingham (1960) found that copper deficiency impaired the Hill reaction in tomato, and tentatively attributed this effect to some change in the acceptor-site ofPSII. More recently, Baszynski et al. (1978) observed a decrease in PS II activity of Cu-deficient spinach chloroplasts, but advanced no explanation for their observation. Thus, a possible role of copper at specific sites in the PS II region has been suggested, but was never unequivocally demonstrated. The recent work of Droppa et al. (1984 a) extends and completes the findings of the above authors. They not only found that Cu deficiency induced-inability of PS II units to accept electrons could not be restored with artificial donors, but from kinetic measurements of fluorescence induction in control and Cu-deficient chloroplasts they proposed that the copper deficiency-induced block of PS II electron transport was located on the acceptor site of this photosystem. This was an interesting proposal which stimu-

455

25 kD

23

20 kD

15

32 9

~ E]

~c ... 0

0\0 41111'1

.D 111III

17

11'1

o· 53 kD

34

25 kD

Fig. 1 B

23

2

20 kD

32

15

53 kD

56

19 16

34

Fig 1 A

2

3

4

Migration

5

6

7

8

3

4

5

6

7

8

9

Migration distance (cm)

9

distance (cm)

Fig. 1 A: SDS-polyacrylamide gel electrophoretograms of chloroplast membrane polypeptides from control sugar beet leaves.

Fig. 1 B: SDS-polyacrylamide gel electrophoretograms of chloroplast membrane polypeptides from Cu-deficient sugar beet leaves.

lated further research and has already been challenged. For example, Sandmann (1985) observed a decrese of PSII activity in Cu-deficient Dunaliella which he explains by a retardation in the development of that alga photosynthetic apparatus, rather than to a direct effect of Cu at a specific site in the PS II unit. This latter interpretation is supported by our data which shows that Cu deficiency clearly inhibits thylakoid membrane synthesis (see discussion below). The polypeptide profiles of EDTA-washed chloroplast membranes from control and Cu-deficient leaves are shown in Fig. 1. The polypeptide pattern of control chloroplasts (Fig. 1 A) exhibit the same diversity of bands already reported for chloroplasts of other higher plants (Henriques and Park, 1975; Henriques and Park, 1976; Henriques and Park, 1978), revealing about 20 discrete components ranging in molecular weight from ca. 15 kD to more than 70 kD. The polypeptide profile of EDTA-washed chloroplast membranes from Cu-deficient leaves (Fig. 1 B) is qualitatively similar to the control, although some quantitative differences are apparent (to allow quantitative comparisons the height of the 15 kD peak was made equal on both densitograms). Compared to the control, the densitogram of Cu-deficient

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chloroplasts shows a large decrease of the 56, 53 and 16 kD peaks and a relative increase in the 25, 23 and 17 kD peaks. Other differences, though less prominent, are the relative decrease of the 32 and 34 kD components and an increase in the 70 kD region components. In a recent study, Droppa et al. (1987), using two-dimensional gel electrophoresis coupled with a silver staining technique, reported that Cu-deficient chloroplasts from sugar beet missed two polypeptides with molecular weights of 23 and 13,5 kD; these workers also found that in spinach a 12,6 kD and two apoproteins of a 29 kD polypeptide were absent, or greatly reduced, in Cu-deficient chloroplasts. It is noteworthy that the 29 kD component was found to be part of a chlorophyll-protein complex (CP 29), which has been suggested to be an internal antenna for PS II (Holdsworth and Arshad, 1977). Droppa et al. (1987) assumed that the 29 kD polypeptide absent in Cu-deficient chloroplasts of spinach was identical to the CP 29 of PS II, but lacked experiI

Fig. 2 b: Cu-deficient chloroplasts showing disintegration of single stroma lamellae and swelling of some thylakoids from grana stacks. x 20,000.

Fig. 2 a: Control chloroplast of sugar beet showing the internal membrane system differentiated into single stroma lamellae and stacks of grana lamellae. x 20,000.

mental evidence for such identification. More recently, Sibbald and Green (1987) showed that ~ of the Cu found in PS II units was associated with this antenna and all these results appear to indicate that at least part of the role played by Cu in PS II is CP 29-mediated, although additional evidence is required to confirm such a supposition. Thin-sections of control and Cu-depleted sugar beet leaves are shown in Fig. 2. The chloroplasts from control leaves (Fig. 2 a) exhibit an arrangement of their internal membranes typical of higher plants, with differentiation of grana stacks and single lamellae. Chloroplasts from control leaves harvested during the light period always contained large starch grains, which were sometimes quite abundant and almost completely filled the organelle. Under these circumstances, the internal membranes were grossly displaced and pressed together, not displaying their typical organization. The chloroplasts of Cu-deficient leaves (Fig. 2 b, c and d) showed conspicuous alterations of their internal membranes,

Copper Deficiency in Sugar beet Chloroplasts

Fig. 2 c: Cu-deficient chloroplasts showing reduction of internal membranes and numerous electron-clear areas with fibrillar material. x25,000.

the extent of which depended on the severity of Cu deficiency. Although these changes may not occur in any ordered sequence, it appears that they can be meaningfully interpreted assuming some major stages of events occurring under increasing Cu-deficiency, which we tentatively depict in figures 2 b, c and d. In the initial stage (Fig. 2 b) the inter-grana lamellae disintegrate and completely disappear, leaving freed grana stacks dispersed throughout the matrix. The number of thylakoids per granum is reduced and most stacks show some swelling, particularly of the end thylakoids. Plastoglobulii are always visible and starch grains are no longer apparent in the sections. In the second stage (Fig. 2 c) the chloroplast internal membrane system is greatly decreased, the few grana are reduced in size and the thylakoids are often swollen; numerous electron-clear areas with fibrillar-like material appear in the stromal matrix and plastoglobulii increase in number and size. Chloroplasts display a general appearance of advanced senescence. In the third stage (Fig. 2 d) the chloroplasts can be recognized only by their size and

457

Fig. 2 d: Cu-deficient chloroplasts showing the internal membrane system reduced to a few, single vesicles, scattered throughout the stromal matrix. x 40,000.

shape, as either they have practically no internal membrane system or this is limited to a few, single vesicles scattered throughout the matrix. The chloroplasts show no osmiophylic globuli and numerous ribosome-like structures are clearly visible. Droppa et al., (1984 a, b) claimed that no significant ultrastructural changes occurred in Cu-deficient chloroplasts of sugar beet except under extreme deficiency conditions; they used this claim to support their proposal that the observed reductions in the photochemical activities of Cu-deficient chloroplasts were not caused by changes in the organelle's structure. On the basis of our data we have to differ with Droppa et a1. (1984 a). Indeed, it is easily anticipated that chloroplasts with such pronounced changes in their fine structure as we show in our micrographs must have dramatic reductions in their photosynthetic capabilities, if they exhibit any at all. Since the chloroplasts used in photochemical studies are always a mixture of organelles with different de-

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grees of functional competence, inevitably the values found in those measurements apply to a heterogeneous population. Only if it were possible to separate organelles with distinct degrees of membrane development, which we show here to co-exist in the same leaf, could a correspondence between chloroplast structural integrity and functional capability be unequivocally established. It appears that Droppa et al. (1984 a) were too cautious in denying the occurrence of significant structural changes in Cu-deficient chloroplasts, with the consequent possibility that such changes may be responsible, at least partially, for the observed decreases in their photochemical activities, as Sandmann (1987) recently proposed. Acknowledgement The author thanks the Director of the EM Laboratory of the EAN for the use of the facilities.

References ARNON, D. and P. STOUT: The essentiality for certain elements in minute quantity for plants with special reference to copper. Plant Physiol. 9, 371-375 (1939). BARR, R. and F. CRANE: Organization of electron transport in PSII of spinach chloroplasts according to chelators inhibition sites. Plant Physiol. 57, 450-453 (1976). BASZYNSIU, T., M. RUSZKOWSRA, M. KR6L, A. TUKENDORF, and D. W OUNSKA: The effect of copper deficiency on the photosynthetic apparatus of higher plants. Z. Pflanzenphysiol. 89, 207-216 (1978). BOTRILL, D., J. POSSINGHAM, and P. KRIEDEMANN: The effect of nutrient deficiencies on photosynthesis and respiration in spinach. Plant Soil 32, 424-438 (1970). DROPPA, M., N. TERRY, and E. HORVATH: Effects of Cu deficiency on photosynthetic electron transport. Proc. Natl. Acad. Sci. USA 81, 2369-2373 (1984 a). - - - Variation in photosynthetic pigments and plastoquinone in sugar beet chloroplasts with changes in leaf copper content. Plant Physiol. 74,717 -720 (1984 b). DROPPA, M., J. MASI]IDEK, Z. R6sZA, A. WOLAK, L. HORVATH, I. FARKAS, and E. HORVATH: Characteristics of Cu deficiency-induced inhibition of photosynthetic electron transport in spinach chloroplasts. Biochim. Biophys. Acta 891, 75-84 (1987). GREEN, B. and E. CAMIN: The nature of the light-harvesting complexe as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Biochim. Biophys. Acta 681,256-282 (1982). HENRIQUES, F.: Ultrastructura dos cloroplastos de folhas de beterraba sacarina deficientes em micronutrientes. 1. Deficiencias de ferro e de cobre. Aetas del I Congresso Iberico de Microscopia Electronica, 75 (1984). HENRIQUES, F. and R. PARK: Further chemical and morphological characterization of a chlorophyll b-Iess mutant of Hordeum vulgare. Plant Physiol. 55, 763 -767 (1975).

- Development of the photosynthetic unit in lettuce. Proc. Natl. Acad. Sci. USA 73, 4560-4564 (1976). - - Polypeptide cross-linking in chloroplast membranes. Arch. Biochem. Biophys. 189, 44-50 (1978). HOAGLAND, D. and D. ARNON: The water culture method for growing plants without soil. Calif. Agric. Expt. Sta. Bul. 347 (1950). HOLDSWORTH, E. and J. ARSHAD: A manganese-copper-pigment-protein complex isolated from the photosystem II of P. tricornutum. Arch. Biochem. Biophys. 183, 361-373 (1977). KATOH, S. and A. SAN PIETRO: The role of plastocyanin in NADP photoreduction by chloroplasts. In: PEISACH, J. et al. (eds.): The Biochemistry of Copper Academic Press, N.Y. (1966). LIPMAN, C. and E. MACKINEY: Proof of the essential nature of copper for higher green plants. Plant Physiol. 6, 593-599 (1931). LUFT, J.: Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414 (1961). MAKOVEC, P. and A. VOLFOVA: Influence of senescence and nitrogen fertilization in the ultrastructural characteristics of barley chloroplasts. Photosynthetica 15, 145-147 (1981). NATR, L.: Influence of mineral nutrients on photosynthesis of higher plants. Photosynthetica 6,88-89 (1972). PISSAREK, H.: Investigation of the anatomical changes in oats and sunflower caused by copper deficiency. Z. Pflanzenerachr. 137, 224-234 (1974). RAHIMI, A. and W. BUSSLER: The effect of copper deficiency on the tissue structure of higher plants. Z. Pflanzenerachr. 135, 183-195 (1973). RASHEED, M. and R. SEELEY: Relationship between the protein and copper content of some plants. Nature 212,644-645 (1966). RENGER, G., J. VATER, and A. WITT: Effect of salicyl-aldoxime on the complete electron transport system of photosynthesis and on isolated reaction cycle II. Biochem. Biophys. Res. Commun. 26, 477 - 480 (1967). REYNOLDS, E.: The use of lead citrate at high pH as an electronopaque stain in electron microscope. J. Cell BioI. 17, 208-212 (1963). SANDMANN, G.: Photosynthetic and respiratory electron transport on Cu-deficient Dunaliella. Physiol. Plant. 65, 481-486 (1985). SANE, P., J. GOODCHILD, and R. PARK: Characterization of chloroplasts photosystems 1 and 2 separated by a non-detergent method. Biochim. Biophys. Acta 216, 162-168 (1970). SIBBALD and GREEN: Copper in photosystem two. In: BIGGINS, J. (ed): Progress in Photosynthesis Research, Maninus Nijhoff, Dordrecht, Vol. II, pp 573-576 (1987). SOMMER, A.: Copper as an essential for plant growth. Plant Physiol. 6,339-345 (1931). SPENCER, D. and J. POSSINGHAM: The effect of nutrient deficiencies on the Hill reaction of isolated chloroplasts from tomato. Aust. J. BioI. Sci. 13, 441-455 (1960). VESK, M., J. POSSINGHAM, and F. MERCER: The effect of mineral nutrient deficiencies on the structure of leaf cells of tomato, spinach and maize. Aust.J. Bot. 14, 1-18 (1966). WOOD, M. and D. ROBINSON: Effect of copper deficiency in wheat on the infection of roots by Gaeumannomyces graminis var. tritici. Aust. J. Agric. Res. 35,735-742 (1984). ZINKIEWlCS, E., M. RuszKowA, and H. SAMIEC: Rate of dark respiration of oats at various levels of copper supply. Acta Agrobot. 38, 23 -32 (1985). -