Bot. Bull. Acad. Sin. (1995) 36: 175-179

Lu et al. — Fe- and Mg-deficiency and thylakoid membranes

Influence of Fe- and Mg-deficiency on the thylakoid membranes of a chlorophyll-deficient ch5 mutant of Arabidopsis thaliana

Yih-Kuang Lu¹, Yung-Reui Chen¹, and Chi-Ming Yang^2,3

¹ Department of Botany, National Taiwan University, Taipei, Taiwan 106, Republic of China

² Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China

(Received January 7, 1995; Accepted May 13, 1995)

Abstract: We characterized the thylakoid membranes of a chlorophyll-deficient ch5 mutant and a wild type of Arabidopsis thaliana grown in Murashige and Skoog medium with and without Fe or Mg by their composition and surface tension, and by the influence of surfactant on the thylakoid membranes. Deficiency in Fe and Mg decreases the amount of chlorophyll and protein in the ch5 mutant and wild type, but dramatically increases the amount of carotenoid. The pattern of the first-derivative absorption spectra of chloroplast revealed that the composition and organization of thylakoid membranes were very different in the two biotypes. In both biotypes, the surface tension of Fe- or Mg-deficient thylakoid membranes was greater than that in plants grown in normal media, indicating that the thylakoid membranes of Fe- or Mg-deficient plants are more easily accessed by surfactants than those of normally-grown plants. In both biotypes, chlorophyll was more easily solubilized from the thylakoid membranes of Mg-deficient plants than that of Fe-deficient plants, which was more easily solubilized than normally grown plants. We concluded that Fe- or Mg-deficiency results in a looser organization of thylakoid membranes than in the controls, and that Mg-deficiency results in a more disorganized thylakoid membrane than does Fe-deficiency. Results suggest that Fe- and Mg-deficient thylakoids contain smaller amounts of the light-harvesting complexes associated with photosystem II (LHCII) and an abnormal ratio of monogalactosyl diglyceride to digalactosyl diglyceride (MGDG/DGDG).

Keywords: Chlorophyll-deficiency; Fe-deficiency; Light-harvesting complex II; Mg-deficiency; Surface tension; Surfactant; Thylakoid membranes.

Introduction

Iron (Fe) is intimately involved in oxidation-reduction reactions in plants. Magnesium (Mg) is an integral component of the chlorophyll molecule and is a divalent cation cofactor for many enzymatic reactions (Evans and Sorger, 1966). Fe-deficiency causes changes in the structure and composition of thylakoid membranes (Terry and Abadia, 1986; Abadia, 1992), such as a drastic reduction in the amount of thylakoid membrane that has few grana stacks, a general decrease of chlorophyll and carotenoid contents (Stocking, 1975; Pushnik and Miller, 1982; Pushnik et al., 1984; Abadia et al., 1988; Abadia et al., 1989; Guller and Krucka, 1993), a decrease of the ratio of monogalactosyl diglyceride to digalactosyl diglyceride (MGDG/DGDG) (Nishio et al., 1985b), and a depletion of thylakoid membrane proteins—especially pigment-protein complexes (Nishio et al., 1985a). Plants suffering from Mg-deficiency suffer pronouned changes in the chloroplasts. Photosynthesis, net assimilation, and transpiration rates are decreased under Mg-deficiency (Terry and Ulrich, 1974; Cao and Tibbits, 1992).

The chlorophyll-deficient ch5 mutant of Arabidopsis thaliana was originally induced by ethylmethane-

sulphonate (Koornneef et al., 1983). In a previous study, we examined the composition and biophysical characteristics of thylakoid membranes and concluded that the thylakoid membranes of ch5 mutant were less compact than those of the wild type (Lu et al., 1995a). In a further study, we presented evidence to show that the drastic reduction of light harvesting complex I and II and of MGDG/DGDG ratio affected the grana stacking and swelling (Lu et al., unpublished). This chlorophyll-deficient mutant is able to operate the xanthophyll cycle to respond to the alteration of light intensity, but within a narrower range than does the wild type (Lu et al., 1995b). The aim of our research is to investigate the influence of Fe- and Mg-deficiency on the structure and organization of thylakoid membranes of the ch5 mutant and wild type.

Materials and Methods

Seeds were germinated and grown in Murashige and Skoog (1962) medium for 4 weeks in a growth chamber providing a photoperiod of 12 h L/12 h D, a photoflux of 250_300 mmol m^-2 S^-1, a relative humidity of 60%, and a temperature of 25°C. Leaves were harvested and the chloroplast and thylakoid membranes isolated as previously described (Markwell, 1986). The concentrations of chlorophyll and carotenoid were determined according to the methods of Arnon (1949) and Jaspers (1965), respectively,

³ Corresponding author.

Botanical Bulletin of Academia Sinica, Vol. 36, 1995

following extraction of liquid-nitrogen-frozen leaf with 80% acetone. Protein concentration was determined using the Bio-Rad protein assay of Bradford (1976). Absorbance and the first derivative spectra were obtained with a Hitachi U3200 UV-visible spectrophotometer.

To study the interaction of surfactants with the thylakoid membranes, 1 ml of fresh digitonin solution in 50 mM Tris-HCl (pH 8.0) was added to an equal volume of fresh thylakoid membrane containing 15 mg ml^-1 of chlorophyll, both being twice the desired final concentration (Bartzatt et al., 1983). The mixture was incubated at 25°C for 10 min prior to the determination of surface tension with a Kyowa face automatic surface tensiometer model CBVP-A3. After measuring the surface tension, the mixture was centrifuged at 5,000 g for 10 min at room temperature. The chlorophyll concentration released in the supernatant fraction was determined as described above.

Results and Discussion

Chloroplasts

The first-derivative of the absorption spectrum can reveal details that are not seen in a straightforward absorption spectrum (Figure 1). In both biotypes, the intensity of all peaks and troughs in the first-derivative absorption spectrum of Fe- and Mg-deficient chloroplasts was much lower than that associated with normally-grown plants. That of Mg-deficient chloroplast was weaker than that of Fe-deficient chloroplast. The peak at 652 nm and the trough at 676 nm found in normally-grown plants were shifted to 656 and 673 nm, respectively, in Fe- and Mg-deficient wild type. In the ch5 mutant, the peak at 656 nm and the trough at 676 nm were shifted to 652 and 673 nm, respectively. The peak ratio of 390/430 nm and trough ratio of 440/483 nm in the wild type were different than those in the ch5 mutant, but the blue-region peak and trough patterns of Fe- and Mg-deficient plants were similar to those of normally-grown plants. These data strongly suggest that in both biotypes the thylakoid membrane components of Fe- and Mg-deficient plants are different than those in the control plants.

Influence of Fe- or Mg-Deficiency on Thylakoid Components

We determined the concentrations of chlorophyll, carotenoid, and protein in thylakoid membranes of Fe- and Mg-deficient ch5 mutant and wild type (Table 1). Fe- and Mg-deficiency resulted in a decrease in chlorophyll and protein and an increase in carotenoid in both biotypes. Our results conflict with reports that chlorophyll and carotenoid are simultaneously decreased by Fe- and Mg-deficiency, and that the decrease of carotenoid is less than that of chlorophyll (Terry and Abadia, 1986; Abadia, 1992). They also conflict with a report that a deficiency in carotenoids may cause a decrease in chlorophyll content (Mayfield and Taylor, 1984). The chlorophyll content of the ch5 mutant is 85% of that of wild type, whereas the carotenoid content of the ch5 mutant is approximately 4.3-fold greater than that of wild type. It is obvious that Fe- and Mg-deficiency cause a higher ratio of carotenoid to chlorophyll. In both biotypes, Mg-deficiency seems more

Figure 1. The first-derivative absorption spectra of chloroplasts isolated from wild type and ch5 mutant of Arabidopsis thaliana grown in Murashige and Skoog solid medium in the absence and presence of Fe or Mg.

Table 1. Effect of Fe- or Mg-deficiency in Murashige and Skoog medium on the components of thylakoid membrane isolated from wild type and ch5 mutant of Arabidopsis thaliana. The results are the mean of three determinations. Numbers in parenthesis present percent.

Strain Treatment Total Chl Carotenoids Protein Carotenoid/

(mg g^-1 leaf) (mg g^-1 leaf) (mg g^-1 leaf) Chl ratio (%)

Wt Control 454±21 (100) 15.5±1.1 (100) 16.3±1.2 (100) 100

-Fe 441±31 (97) 70.9±4.9 (457) 14.9±1.8 (91) 474

-Mg 317±26 (70) 63.6±6.2 (410) 8.2±1.9 (50) 591

ch5 Control 387±18 (100) 67.3±3.1 (100) 18.3±1.4 (100) 100

-Fe 317±24 (82) 80.6±5.3 (120) 11.1±1.3 (61) 154

-Mg 172±13 (44) 95.3±7.6 (142) 10.0±1.4 (55) 336

Lu et al. — Fe- and Mg-deficiency and thylakoid membranes

effective than Fe-deficiency in decreasing chlorophyll and protein levels, but the ratio of carotenoid to chlorophyll in Mg-deficient plant is higher than that in Fe-deficient plant. The above data suggest that in both biotypes, Mg-deficiency causes more changes in the composition and organization of thylakoid membranes than does Fe-deficiency, which indicates that the deficiency in chlorophyll is not directly accompanied by a loss of carotenoids.

Influence of Fe- or Mg-Deficiency on Surface Tension

Surface tension was used to monitor the composition and orientation of the molecules and the structure of biological membranes (Tinoco et al., 1978). The surface tension of thylakoid membranes at various concentrations was measured (Figure 2). Deficiencies of Fe and Mg caused higher surface tension than in the control at all concentrations of thylakoid membranes in wild type and at concentrations less than 50 mg ml^-1 in the ch5 mutant. This indicates that the quality and quantity of constituents of thylakoid membranes, especially the large molecules such as pigment-protein complexes, and the orientation of constituents differ greatly between Fe- and Mg-deficient and control plants in both biotypes. This further indicates that the thylakoid membranes of Fe- and Mg-deficient ch5 mutant are more accessible to surfactants than are those of the wild type.

Influence of Fe- or Mg-Deficiency on Chlorophyll Solubilization

Digitonin was used to study the constituents and organization of thylakoid membranes in the ch5 mutant and wild type (Lu et al., 1995a). The influence of various digitonin concentrations on the solubilization of Fe- and Mg-deficient thylakoid membranes is shown in Figure 3. In the wild type, Mg-deficient thylakoids were solubilized when digitonin concentration was 10^-3%, whereas Fe-deficient and control thylakoids were solubilized at a digi

Figure 3. Influence of digitonin concentration on the solubilization of the thylakoid membrane isolated from wild type and ch5 mutant of Arabidopsis thaliana. Amount of total chlorophyll remaining in the supernatant fraction following centrifugation is shown versus digitonin concentration. Thylakoid membrane concentration was 15 mg ml^-1.

tonin concentration of 10^-2%. Fe-deficient thylakoid is more easily solubilized than the control at digitonin concentrations greater than 10^-1%. In the ch5 mutant, Fe- and Mg-deficient thylakoids were slightly solubilized at a concentration of 10^-4%, whereas the control thylakoids were solubilized at a concentration of 10^-3%. All kinds of thylakoids, however, were greatly solubilized when digitonin concentration was greater than 10^-2%. In both biotypes, Mg-deficient thylakoids are more easily solubilized than Fe-deficient thylakoids, which are more easily solubilized than the controls. The solubilization data suggest that Fe- and Mg-deficient thylakoids are more loosely arranged than the controls in both biotypes.

In the present investigation, we examined certain biochemical and biophysical characteristics of Fe- and Mg-deficient thylakoid membranes isolated from a chlorophyll-deficient mutant. Our results show that: a) the absorbance in the first derivative spectra of Fe- and Mg-deficient chloroplasts is lower than that of the control; b) a deficiency in chlorophyll is not directly accompanied by the loss of carotenoids; and c) Fe- and Mg-deficient thylakoids are more accessible to surfactants.

In conclusion, we found that the Fe- and Mg-deficient thylakoids are more disorganized or swollen than the controls, suggesting that mineral deficiency may result in the presence of only very small amounts of LHCII, which is assumed to regulate the grana stacking (Bennett, 1991; Allen, 1992), and that MGDG and DGDG and the ratio of MGDG to DGDG, which was proposed to affect the grana stacking and lumen swelling (Lu et al., unpublished), may greatly change.

Acknowledgements. This research was supported by National Science Council of the Republic of China grants NSC81-0211-B-002-04 (to Y.R.C.) and NSC82-0211-B-001-051 (to C.M.Y.).

Figure 2. Surface tension of various concentrations of the thylakoid membrane isolated from wild type and ch5 mutant of Arabidopsis thaliana grown in Murashige and Skoog solid medium in the absence and presence of Fe or Mg.

Botanical Bulletin of Academia Sinica, Vol. 36, 1995

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Lu et al. — Fe- and Mg-deficiency and thylakoid membranes