Induction of Callose Formation by Manganese in Cell Suspension Culture and Leaves of Soybean (Glycine max L.)

J. Plant Physiol. Vol.

142. pp. 67-73 {1993}

Induction of Callose Formation by Manganese in Cell Suspension Culture and Leaves of Soybean (Glycine max L.) A. I

2

H. WISSEMEIERl,

A.

1 HERGENRODER ,

G. MIX-WAGNER2, and W. J.

HORST

1

Institute of Plant Nutrition, University of Hannover, Herrenhauser Str. 2, DW-3000 Hannover 21, Germany Institute of Crop Science and Plant Breeding, FAL, Bundesallee 50, DW-3200 Braunschweig, Germany

Received November 20, 1992 . Accepted January 30, 1993

Summary

The effect of 0.02 11M - 20,000 11M manganese (Mn) supply to suspension-cultured soybean (Glycine max L.) cells in liquid B 5 medium was evaluated with respect to growth, Mn concentration, Mn oxidation and callose formation. Mn concentrations of cells as low as 11lg Mn (gdry weightt l were not associated with a growth reduction over a 6-day culture period. However, higher callose concentrations were found compared with Mn concentrations of cells between 5 and 300 Ilg Mn (g dry weightt I. At cell Mn concentrations higher than 300llg Mn (g dry weight)-I again callose synthesis was induced well before any growth reduction. In a time study with 5000 11M Mn supply, Mn oxidation was completed within the first 4 h. Later on only total cell Mn concentrations further increased up to 32,0001lg Mn (g dry weightt I after a 4-day culture period. No correlation existed between the degree of browning of the cells and their concentrations of oxidized Mn. The callose concentrations increased after an initial lag phase, reached a maximum after 3 days and then declined. In intact leaves of soybean plants (cv. Sito) grown in nutrient solution at Mn supplies ranging from deficiency (0.01 11M Mn) up to toxicity (100 11M), again at low and high Mn tissue concentrations, callose formation was induced. At high Mn tissue concentrations callose formation was more sensitive than visible Mn toxicity symptoms or growth effects. The comparison of cultured cells with leaves of intact plants indicated a much lower critical deficiency but higher critical toxic Mn concentrations with regard to growth and callose formation.

Key words: Glycine max, callose, cell culture, critical manganese concentration, manganese deficiency, manganese oxidation, manganese toxicity. Abbreviations: EDDHA = Ethylendiaminedihydroxophenylacetate; EDTA acetate; DMSO = Dimethyl sulfoxide; LE = Laminarin equivalents.

Introduction

The callose synthesizing plasma membrane bound /3-1,3glucan synthase is considered to be present in all living plant cells and can be activated by a variety of abiotic and biotic factors (Fincher and Stone, 1980). Recent biochemical and physiological interest in the enzyme has mainly concentrated on the structure of the enzyme complex and its regulation. The significance of inducible callose formation in higher plants is primarily investigated and discussed in relation to fungal and viral attack, where callose formation © 1993 by Gustav Fischer Verlag, Stuttgart

=

Ethylenediaminetetra-

seems to be part of the defence mechanisms of the host (Kauss, 1990). Cultured cells of soybean are a widely used tool in physiological and ecotoxicological studies (e.g. Langebartels and Harms, 1986), and are also used to study callose formation (e.g. Kohle et al., 1985). As growth media of cultured cells, liquid B5 (Gamborg et al., 1968) or MS medium (Murashige and Skoog, 1962) with 60 and 100 11M Mn, respectively, are often used. However, Mn requirements of cultured plant cells are reported to be low (Ohira et al., 1975) and vary between plant species (Ojima et al., 1977). For intact leaves of

68

A. H. WISSEMEIER, A. HERGENRODER, G. MIX-WAGNER, and W. J. HORST

cowpea we could demonstrate that induction of callose formation is a very sensitive response to high Mn concentrations, which occurs well before any other toxicity symptom or growth reduction (Wissemeier and Horst, 1987, 1992). In the present study the effect of a wide range of Mn concentrations on growth and callose formation is evaluated in liquid B 5 medium. We report that not only a high, but also a low Mn supply can stimulate callose formation in cultured cells and leaves of intact soybean plants.

and Israelstam (1979). The remaining part of the leaf was used for Mn determination (see above). The weight of the plants was determined after drying to constancy.

Callose quantification Callose was extracted in hot NaOH by the method of Kohle et al. (1985) and Kauss (1989) as described by Wissemeier and Horst (1992). For cultured cells about 300 mg fresh weight was extracted in 10 mL NaOH. It was confirmed that even extremely high Mn tissue concentrations did not interfere with the assay.

Material and Methods

Cell suspension culture The soybean cell suspension culture was initiated from root meristems of cv. Mandarin in 1964 and was kindly provided by Dr. Harms (FAL, Braunschweig, Germany), who cultured the cells since 1970 in liquid B 5 medium (Langebartels and Harms, 1986). To reduce the Mn supply in the B 5 medium originally containing 60 J.1M Mn (Gamborg et al., 1968) suspended cells were transferred into fresh B 5 medium without Mn. For experiments with higher Mn supplies MnS04 was added to B 5 medium. Culture conditions for the heterotrophic grown cells in 100 mL liquid medium at pH 5.5 were 25°C with a 16 h photoperiod of 2000 lux in 250 mL Erlenmeyer flasks on a rotary shaker with 150 cycles per minute. At harvest, three aliquots of 10 mL were taken from each flask for determinations of (i) weight, (ii) oxidized and total Mn, and (iii) callose. Samples for callose determination were chemically fixed in a solution with (v/v) 70 % ethanol,S % formaldehyde and 5 % propionic acid. For weight and Mn determinations cells were squashed on ashless filter paper and rinsed 3 times with 10 mL demineralized water. To determine oxidized Mn three volumes of 5 mL of 0.75 % (w/v) hydroxylamine hydrochloride in 20 mM acetate buffer, pH 4.5, were poured onto the cells, allowed to react for 30 sec at a time and than sucked off. This treatment completely reduces commercial Mn02. In the eluate Mn was determined by atomic absorption spectroscopy (AAS). The cells including the filter paper were weighed, dried, weighed again and analyzed for Mn after dry ashing according to White (1969) using flame or flameless AAS.

Dry weight [g flask-I]

0.12

A

-

a

..... a

.......

....a

0.08

0.04

o~~~~~~~~~~~~~~

Mn concentration (Jtg (g dry weight)"!] 1~.-------------------------~

B

9

100 10

Intact plants Soybean seeds (cv. Sito) were germinated in a growth chamber for 4 days between filter papers soaked with 1 mM CaS04. Seven seedlings per 22 L pot were then transferred to constantly aerated nutrient solution of the following composition [J.1M]: KN0 3 1500, Mg(N0 3)2 650, CaS04 500, CaCh 500, KH 2P04 200, H 3B03 10, CUS04 0.4, ZnS04 0.4, (NH4)6M07024 0.1. Since at Mn supplies of 0.01 and 0.1 J.1M (MnS04) FeEDDHA (<
Callose concentration [mg LE (g fresh weigh!)-!]

0.6

r---------------,

0.4 ab

0.2

Mn supply [/LM] Fig. 1: Effect of Mn supply (low concentration range) on final dry weight (A), Mn concentration (B) and callose concentration (C) of cultured soybean cells after a 6-day culture period. Means followed by different letters are significantly different at p < 0.05.

69

Induction of Callose Formation by Manganese

Statistics

Dry weight [g flask-I]

The cell suspension culture experiments were performed with 5 replicates, while for the experiments with intact plants 5 plants were randomly chosen from each of the two pots per Mn supply and treated as 10 independent replicates. Following analysis of variance means were compared with the Tukey-test (Sachs, 1984).

0.10

A

0.08 0.06 0.04

Results

0.02

Cell suspension culture The original Mn concentration of the liquid growth medium B 5 (60 JlM) was stepwise reduced by transferring aliquots of suspended cells to Mn-free B 5 medium, so that media with Mn supplies from 60 down to 0.02 JlM Mn were established. Decreasing Mn supply in the medium reduced Mn concentrations of the cells from 230 to 0.6 Jlg Mn (gDwtl (Fig. 1 B) without any effect on growth (Fig. 1 A) after a 6day period since the last transfer to a new medium. Callose concentrations (Fig. 1 C) substantially increased with decreasing Mn supply. Decreasing the Mn supply from 60 JlM to 0.06 or 0.02 JlM Mn, corresponding to Mn concentrations of 0.6 and 1.2 Jlg Mn (gDwtl, almost doubled callose concentrations. The results indicate that in soybean cell culture Mn concentrations as low as 1 Jlg Mn (gDwtl are sufficient to sustain optimum growth, and callose formation is enhanced at low Mn concentrations well before growth is retarded. To study the effects of high Mn supply in the culture medium on growth, Mn concentrations, and callose formation, Mn was supplied at concentrations of 1 JlM up to 20,000 JlM. After 96 h treatment, distinct growth depressions occurred at 5000 JlM Mn supply and higher (Fig. 2 A), corresponding to final Mn concentrations of the cells of 15,000, 20,000 and 32,000 Jlg Mn (gDwtl (Fig. 2 B). These Mn concentrations were extremely high compared with Mn concentrations found in intact plants. Therefore, the question arises whether a large part of this Mn is precipitated on the cell surfaces. A brown colour of the cells at high Mn supply (Table 1) suggested oxidation of Mn2+ to Mn02. However, at high Mn concentrations only about a quarter of the total Mn could be removed with the reducing agent hydroxylamine hydrochloride, and thus classified as oxidized Mn (Fig. 2 B). The high percentage of thus duated Mn at low Mn supplies

Table 1: Colour classification of soybean cell suspension cultures growing at different Mn supplies up to 96 h. Colour categories: 0 = white, 1 = yellowish, 2 = yellowish-brown, 3 = light brown, 4 = brown,S = deep brown. Treatment duration [h] Mn supply [11M] 1

5000 20000

o

4

9

o o

o o o

000

o

24

48

[Colour category]

012 233

72

o

2 3

96

o 3 5

Mn concentration [J-Ig (g dry weight)-I] I~r-------------------------.

B

d

10000

(24)

(29)(26) (51)

1000 (101)

100

( ) * % oxidized Mn

10

of total Mn

(29)*

Callose concentration [rug LE (g fresh weight)-I]

0.8

C

0.6

0.4 b

0.2

10

100

1000

10000

I~

Mn supply [J-IM] Fig. 2: Effect of Mn supply (high concentration range) on dry weight (A), Mn concentration and percentage of oxidized Mn (B) and callose concentration (C) of cultured soybean cells after a 4-day culture period. Means followed by different letters are significantly different at p < 0.05.

indicated that even some leaching of Mn 2+ may have occurred during the reduction procedure. Callose concentrations (Fig. 2 C) started to increase at 100 JlM Mn supply and, therefore, appeared to be a more sensitive indicator of Mn toxicity than growth. At 5000 JlM Mn supply, which did not negatively affect growth but did induce callose formation (compare Fig. 2), kinetics of growth, Mn uptake and callose formation were

70

A. H.

A.

WISSEMElER,

HERGENRODER,

G.

MIx-WAGNER,

and

Dry weight [g flask-!]

A

0.08 0.06 0.04

0.02

o~--~----~----~--~~--~

Mn concentration (JLg (g dry weight)-!] 1~r-------------------------~

B

8000 4000 ·

OL-__

~

____

~

____

~

____

~

__

~

Oxidized Mn concentration (JLg (g dry weight)"!] ,----------------------------,

C

12000 8000 a a

4000

J. HORST

studied in more detail (Fig. 3). In spite of the accumulation of large amounts of Mn (Fig. 3 B), the cells grew well and doubled their dry weight within 4 days (Fig. 3 A). The steep increase in Mn concentration of the cells within the first 4 hours of Mn treatment was due to oxidation of Mn (Fig. 3 B, C). Net oxidation of Mn, however, was completed thereafter and there was even a tendency towards declining concentrations of oxidized Mn, although total Mn concentrations continued to increase linearly with time. Callose formation showed a lag phase of at least 8 h and then increased linearly with time until 72 h. A longer Mn treatment led to a significant drop in callose concentration. At 20,000 IlM Mn supply a similar pattern of responses in growth, Mn concentration, Mn oxidation and callose formation was found (not shown). Intact plants

12000

l~

W.

a

a

•

!L,ij

a

•

•

•

0 Callose concentration [mg LE (g fresh weight)"!]

D

0.6

In order to test the hypothesis that cells in suspension culture respond to Mn supply in a similar manner to intact plants soybean plants were treated with a range of Mn concentrations in solution culture (Fig. 4). Shoot growth (Fig. 4 A) was depressed at the lowest and the highest Mn supply, indicating that the range of Mn supplies covered both Mn deficiency and Mn tQxicity. Chlorophyll concentrations (Fig. 4 B) reflect the appearance of chlorosis as a symptom of Mn deficiency and toxicity. Compared with cell suspension cultures, in leaf tissue growth depressions due to Mn deficiency occurred at much higher Mn supplies and Mn concentrations, but growth depression due to Mn toxicity occurred at much lower Mn supplies and Mn concentrations (Fig. 4 C compared with Figs. 1 and 2). In addition to chlorosis dark brown speckles occurred on the leaves as typical symptoms of Mn toxicity (Fig. 4 B). As with cell suspension cultures, callose formation was enhanced under conditions of Mn deficiency (significant negative correlation between Mn and callose concentrations at 0.Q1 and 0.11lM Mn supply) and Mn toxicity, but again at much higher or lower Mn concentrations, respectively (Fig. 4 D). Callose formation was a much more sensitive indicator of Mn toxicity than the appearance of brown speckles or dry matter production. Discussion

0.4

b

a

OL---~----~----~----~--~

o

20

40

60

80

Joo

Treatment duration [h] Fig. 3: Time course of dry weight accumulation (A), Mn concentration (B), oxidized Mn concentration (C) and callose concentration (D) of cultured soybean cells after transfer from 1 ~M to 5000 ~M Mn in the medium. Means followed by different letters are significantly different at p < 0.05.

The presented results indicate that in soybean callose formation is induced both at very low as well and at high Mn concentrations. While previous reports have also shown that high Mn concentrations induce callose formation (Wissemeier and Horst, 1987, 1992) this is the first report of low Mn concentrations inducing callose. Similar results were obtained with suspension-cultured cells and intact plants; however, some notable differences were observed. In suspended cells provided with very low Mn supplies, leading to cell Mn concentrations of about 11lg (g DWt t, growth effects were absent (Fig. 1), while in young expanded leaves of intact plants with about 71lg Mn (gDW)-1 growth was largely reduced (Fig. 4). This is in agreement with classical work by Pirson and Bergmann (1955) and Eyster et al.

Induction of Callose Formation by Manganese

Shoot dry weight [g plant-I] 0.8

A

b

0.6 0.4

0.2 o~~~~--~~~~-W~~~

Chlorophyll concentration [mg (g fresh weight)-!] 3

B b

2

b

~ Density of brown speckles [number cm-2]

o

o

o

o

20.3

Mn concentration (Jtg (g dry weight)"!] 1~.-------------------------.

c

1000 d

100 b

10

Callose concentration [mg LE (g fresh weigh!)-!] 7

6

D

d

5 4

c

3

2

0 0.001

0.01

0.1

10

100

1000

Mn supply [I'M] Fig.4: Effect of Mn supply for 13 days on shoot dry weight (A), chlorophyll concentration and density of brown speckles (Mn toxicity symptom) (B), Mn concentration (C) and callose concentration (D) in the second oldest trifoliate leaf of intact soybean plants. Means followed by different letters are significantly different at p <0.05.

71

(1958) with Chiarella, showing that the Mn requirement for optimum growth depends on whether the algae grew autotrophically or heterotrophically in the dark. The latter situation corresponds to the soybean cell suspension culture used here. The essential role of Mn in photosystem II (Renger and Wydrzynski, 1991) can explain the higher Mn demand for autotrophic growth. An additional Mn demand of photosynthetically active cells for the Mn containing superoxide dismutase (SOD) is not suggested by itself since MnSOD is not located within chloroplasts but preferentially in mitochondria Qackson et al., 1978; Corpas et al., 1991). In suspended soybean cells, lower (Figs. 1 and 4) or higher (Figs. 2 and 4) Mn concentrations in the deficiency or toxicity range, respectively, are required for callose formation compared with the leaves of intact plants. Whereas the former case will reflect the different Mn requirements of the systems, the situation at high Mn concentrations will decisively be influenced by the different physiological ages of the cells. The predominant young, meristematic cells in suspension culture will have a much higher Mn tolerance than the mature cells of leaves (Horst, 1988; Wissemeier and Horst, 1990). In tobacco a distinctly higher Mn tolerance of calli compared with seedlings has also been demonstrated (Petolino and Collins, 1985). A clear picture of how very low or high Mn concentrations induce callose synthesis cannot be drawn, although in recent years a lot of work has been published on the regulation of callose synthesis. Using microsomal preparations or suspended cells it appears that besides direct ionic interactions with the callose synthesizing enzyme a variety of compounds like polyamines and polyunsaturated fatty acids, f3linked disaccharides, phosphorylation/dephosphorylation steps, protease and phospholipase action, transmembrane electrical potentials, as well as mechanical/physical burdens on membranes, can be factors in stimulating the always present but mostly inactive enzyme (Kauss, 1985; Mullins, 1990; Kauss and Jeblick, 1991). A generally recognized key factor for callose synthesis is the perturbation of the plasma membrane. With regard to stimulated callose formation at very low Mn concentrations work from Poovaiah and Leopold (1976), indicating a function of Mn in stabilizing the plasma membrane, may be of relevance. Also an altered composition of the plasma membrane might be causally related to callose induction. Constantopoulus (1970) reported reduced glycolipid and polysaturated fatty acid concentrations in membranes of isolated chloroplasts in algae cells suffering from Mn deficiency. For the induction of callose synthesis at high Mn concentrations several modes of actions might be proposed. Manganese can directly stimulate (1,3)-f3-glucan synthase, although Ca is more effective (Morrow and Lucas, 1986). Since at toxic Mn concentrations K efflux can be increased (Waldren et al., 1987), indicating perturbation of membrane properties, the usually low cytoplasmic Ca activities may also no longer be able to be maintained. This too would be an important factor triggering callose synthesis (Kauss, 1985). A further possibility is that in cells and tissues with elevated Mn concentrations the metabolism is altered so that organic elicitors of callose synthesis are formed. A model for this as-

72

A. H. WISSEMEIER, A. HERGENRODER, G. MIX-WAGNER, and W. J. HORST

sumption might be that millimolar concentrations of Mn are known to stimulate digitoxigenin accumulation in Digitalis tissue culture (Ohlsson and Berglund, 1989), which is chemically related to the effective callose-elicitor digitonin (Kauss and] eblick, 1991). Only recently an endogenous activator of callose synthase has been identified in different plant species, which is thought to be inactive under non-stress conditions owing to compartmentalization in the vacuole (Ohana et al., 1992). At high Mn concentrations not only a stimulation of callose synthesis but also a decomposition of callose with time have been demonstrated (Fig. 3). The enzyme {1,3}-,6-glucanase, which can be active in every part of intact plants (Moore and Stone, 1972) and cultured cells Oouanneau et al., 1991), is responsible for callose decomposition. Ethylene production is stimulated at a late stage of Mn toxicity in intact leaves (Horst, 1988) and has been shown to stimulate {1,3)-,6-glucanase activity in leaves of tobacco in a cell-type specific way (Keefe et al., 1990; Mauch et al., 1992). Not only a stimulated decomposition can reduce callose concentrations after long exposure times to high Mn concentrations, but also a reduced ability to synthesize callose, which would dilute the callose concentration in growing cells. Oxidized phenols have been shown to be inhibitors of glucan synthase (Mason and Wasserman, 1987). The time study on extraction of oxidized Mn and discolouration of the cells (Fig. 3 and Table 1) indicates that the brown colour of the cells at high Mn supply is not due to oxidized Mn but most likely due to oxidized phenols. This has also been indicated by histochemical work on Mn-intoxicated cowpea leaves (Wissemeier and Horst, 1992). The present study shows that for suspension-cultured soybean cells in B 5 medium 60 J.1M Mn is optimal for growth; however, there is a risk of elevated levels of background callose (Fig. 1 C). Mn supplies lower than 60 J.1M also sustained optimal growth and are recommended for studies where callose formation is followed as a sensitive stress response. Further evaluations of the influence of nutrient supply in tissueculture media on callose formation should concentrate on boron, since like Mn, non-constitutive callose can also be induced by boron deficiency (Van de Venter and Currier, 1977) and boron toxicity (McNairn and Currier, 1965) in intact plants. Acknowledgements

We thank Mrs. Maria Ahlgrimm for technical help and Dr. Stephen Waters for critical reading and correcting the English text. The spectrofluormetric callose measurements were performed at the Institute of Botany, University of Hannover; the support of Prof. Dr. Frank Herzfeld is greatly appreciated.

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RENGER, G. and T. WYDRZYNSKI: The role of manganese in photosynthetic water oxidation. BioI. Metals 4, 73-80 (1991). SACHS, L.: Angewandte Statistik, 6. Auflage, Springer-Verlag, Berlin (1984). VAN DE VENTER, H. A. and H. B. CURRIER: The effect of boron deficiency on callose formation and HC translocation in bean (Pha· seolus vulgaris L.) and cotton (Gossypium hirsutum L.). Amer. J. Bot. 64, 861-865 (1977). WALDREN, S., M. S. DAVIES, and J. R. ETHERINGTON: The effect of manganese on root extension of Geum rivale L., G. urbanum L. and their hybrids. New Phytol. 106, 679-688 (1987). WHITE, R. P.: Hydroxylamine hydrochloride as a reducing agent for atomic absorption determinations of manganese in dry-ashed plant tissue. Soil Sci. Soc. Amer, Proc. 33, 478 - 479 (1969). WISSEMEIER, A. H. and W. J. HORST: Callose deposition in leaves of cowpea (Vigna unguiculata (L.) Walp.) as a sensitive response to high Mn supply. Plant Soil 102, 283-286 (1987). - - Manganese oxidation capacity of homogenates of cowpea (Vigna unguiculata (L.) Walp.) leaves differing in manganese tolerance. J. Plant Physiol. 136, 103-109 (1990). - - Effect of light intensity on manganese toxicity symptoms and callose formation in cowpea (Vigna unguiculata (L.) Walp.). Plant Soil 143, 299-309 (1992).