species as Lithospermum erythrorhizon (Mizukami H. et al. 1978) and Daucus earota (Dougall D.K. et al. 1980). In spite of the numerous examples relating to.
Plant Cell Reports
Plant Cell Reports (1988) 7:78-81
© Springer-Verlag 1988
Anthocyanin and proteins as biochemical markers in maize endosperm cultures M . L. Racchi 1 and L. A. M a n z o c c h i 2 1 Dipartimento di Genetica e di Biologia dei Microrganismi, Universit~ degli Studi di Milano, via Celoria 26, 1-20133 Milan, Italy 2 Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, 1-20133 Milan, Italy Received August 8, 1987 / Revised version received November 25, 1987 - Communicated by P. J. King
ABSTRACT Endosperm maize cultures derived from a strain homozygous for all genes required for anthocyanin synthesis develop an intense pigmentation. Pigmenting ability is generally maintained in successive subcultures, altough colourless areas are frequently observed in pigmented cultures. The isolated colourless cell clusters show a growth rate higher than the eoloured ones. These calli nevertheless do not lose the ability to synthesize anthocyanins, and in successive subcultures turn red again. The different growth rates associated with the ability of cells to accumulate pigments suggest the existence of different physiological states of the culture. To investigate this possibility we analyzed the polypeptide patterns of coloured and colourless cultures. SDS gel electrophoresis has demonstrated differences in soluble protein fractions, among which a 26 kD peptide, characteristic of pigmented tissues, has been evidenced. Zein, the major storage protein of malze endosperm is present, although at very low levels, both in pigmented and in unpigmented cultures, confirming that its synthesis occurs continuously in vitro. Abbreviations: 2-4D, 2,4-dichlorophenoxyacetic acid; SDS, sodium dodecyl sulphate, PMSF, Phenylmethyl sulphonyl fluoride; DAP, days after pollination.
INTRODUCTION
Endosperm cultures are a useful tool for studing biosynthetic pathways specific for this tissue and their regulation. Anthocyanin and starch production have been particularly studied using this technique (Strauss J. 1959; Gavazzi G. and Racchi M.L. 1981; Chu L.J.C. and Shannon J.C. 1975; C.H. Saravitz and C.D.Boyer 1987), and more recently long term endosperm cultures capable of producing zeins have been described (Shimamoto et.al. 1983). As far as anthocyanin production is concerned, it has been observed to be unstable in endosperm culture. In fact, spontaneous changes in the ability of cultures to produce pigment have been described (Strauss J. 1958, 1959; Racchi M.L. 1985), so that it is possible to isolate from pigmented cultures unpigmented sectors and to restore their pigmenting ability in subsequent subcultures . Both in Black Mexican Sweet
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and in other strains studied, the loss of pigmentation is associated with an increase in growth rate (Strauss J. 1958, Racchi M.L. 1985). This observation might imply a different level of dedifferentiation of the growing tissue. In this paper we analyze polypeptide patterns of pigmented and unpigmented cultures, in an attempt to characterize them in terms of different levels of differentiation.
MATERIALS and METHODS
Plant Material A strain of maize in a K55/W23 hybrid background, homozygous for all genes required for anthocyanin synthesis, originally obtained from Dr. E.H. Coe, was used in this study. Plants were self pollinated and ears were harvested at 10 and 20 DAP (days after pollination) and at maturity; ears at 10 DAP were used for initiating cultures. Endosperm callus culture. Callus was initiated and maintained according to the procedure previously described (Racchi M.L. 1985) except for that 2-4D was added t o the induction medium at Img/l. The cultures were kept in continuous light at 25oC (light source:OSRAM L40/20 Sa with a fluence rate of 43W m -R) and subcultured every 3-4 weeks. Immature endosperms started to develop pigment at six, seven days of culture. After three weeks, a homogeneous deep purple pigmentation appeared all over the callus. In successive subcultures, white areas consisting of clusters of colourless cells appeared in the mass of the red tissue; these white sectors were then isolated from the red calli and cultured separately in the same experimental conditions. Callus growth, determined as fresh weight increase per month, is expressed as relative growth calculated from (WtWi)/Wi where Wi and Wt stand for initial and total weight, respectively. Anthocyanins were extracted using cold I% Hcl in 95% ethanol by grinding the tissue in a pre-cooled mortar. The homogenate was centrifuged for ten minutes at 13000 g and the supernatant used for quantitative determination. Pigment content was expressed as absorbance at 530 nm per mg fresh weight.
79 Extraction of proteins
The amount of anthocyanin is significantly different in the two types of ealli. Colourless calli grow four times faster than coloured ones and show a higher content of total protein. As preliminary analysis of total proteins on SDS PAGE had shown differences in the pattern of colourless and coloured calli, mainly in low molecular weight peptides (20000-30000 m.w.), we decided to fraotionate proteins of endosperm cultures by methods used to separate different classes of endosperm proteins and to analyze salt-soluble (albumins and globulins) and ethanol-soluble proteins separately. The presence of zein in ethanolic extracts from calli (and endosperms) was revealed by a dot -blot experiment (Fig.l).
Ten to thirty mg of lyophilized calli and endosperms were utilized for protein extraction. Total proteins were extracted by incubation for one hour at room temperature in 0.5 ml of 200 mM Tris HCI pH 7.6; 5% SDS; 2% Dithiothreitol; 2 mM Phenylmethyl-sulphonyl fluoride (PMSF). The experimental conditions for protein fractionation are those described by Landry and Moureaux (1981) for albumins, globulins and zein; 2 mM PMSF was added to all solvents. Protein concentrations were determined according to the Lowry method, as modified by Peterson (I777). SDS-polyacrylamide gel electrophoresis was performed on 15% acrylamide gel slabs according to Laemmli (I770) and the gels were stained with Coomassie blue R-250. Identification of zein by immunoblotting Zein was identified in total protein extracts and ethanol soluble fractions by cross-reactivity with anti-zein antiserum (prepared by giovanna Viale of the Dipartimento di Biologia e Genetioa per le Scienze Mediche, Universit& degli Studi di Milano, as described by Coraggio et al. I786). The antiserum did not demostrate cross reactivity with other maize endosperm proteins, as shown in western blot experiment (~ig.2 lane C). Dot-blot experiments were performed according to Esen et al. I783. For westernblot analysis (Towbin et al 1979), the proteins separated by SDS-gel electrophoresis were electrophoretically transferred to a nitrocellulose sheet using a Biorad Tran5 Blot cell according to the instructions of the manufacturer. The immobilized proteins were then reacted with purified 1:1000 antizein antiserum. Secondary incubation was with 1:1000 horseradish peroxidase-conjugated goat antirabbit IgG (Miles-Yeda Ltd). Peroxidase localization was revealed by reaction with 4-chloronaphtol and hydrogen peroxide according to Hawkes et.al.(1982). Quantitative determination of zein on dot or western blots was made by comparing known amounts of zein extracted with ethanol from endosperms with extracts of coloured and colourless calli.
Fig.1 Dot blot analysis of ethanol extracts from endosperms and endosperm cultures, reacted with antizein antiserum. A: extracts from 25 DAP endosperms (containing zeins) were applied as control markers in increasing amounts ( corresponding to 0.25, 0.5, I, 2, 4, 6 ~g zein/spot) Row B: extracts from three different red callus cultures Row C: extracts from three different white callus cultures. Ethanolic extracts from calii were applied twice, the first spot deriving from 140 ~g of callus dry weight (positions I-2-3) the second from 280 ~g of callus d r y weight (positions 1 " - 2 " - 3 " ) . Row
RESULTS
In normal developing seed, zein synthesis start around 15 DAP and inceases linearly until 40 DAP (Soave and Salamini,1984). Our primary explants (endosperm 10 DAP) were completely devoid of the protein, as confirmed by our preliminary data (not reported); nevertheless, long term endosperm cultures show presence of small amounts of zein.
Data referring to growth rate, protein and anthocyanin content in coloured and colourless calli and in endosperms at different stages of development are presented in Table I.
Tab. I Protein and anthocyanin content of intact endosperms and endosperm cultures with different pigmentation and growth rate
GROWTH
RATE
PROTEINS
mg/1OOmg d.w.
ANTHOCYANIN
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(Wt-W~/W±)
Salt soluble
Ethanol soluble
A 530/100 mg f.w.
colorless
3.42
?.57
0.06
0.115
colored
0.86
2.67
0.02
3.450
-
11.36
traces
0.009
-
4.50
7.70
2.109
CALLUS
10
DAP
ENDOSPERM 20 DAP
80 Fast growing unpigmented callus appears t o accumulate more zein than callus in which there is. pigment accumulation. The identity of zein in ethanolic extracts was confirmed also by western blot. Ethanolie extracts from pigmented and colourless calli and from endosperm 25 DAP as well as a SDS total protein extract from the same endosperms (added to check the specificity of the antibody), were separated by SDS PAGE, blotted on nitrocellulose sheets and reacted with anti zein antiserum. In all cases bands typical of zein polypeptides were shown (Fig.2).
Kd 94 67 40 30 20.1 14.4
EIO E20 R W S Pig.2 Western blot analysis of ethanolic extracts from a 25 DAP endosperm (E), pigmented (R) and unpigmented (W) endosperm cultures and of a SDS total protein extracts from a 25 DAP endosperm (C). The ethanolic extracts of calli were derived from 20 dry weight; the extracts from 25 DAP endosperms were obtained from 0.25 mg dry weight, containing 13 gg zein.
Pig.3 SDS PAgE polyaerylamide gel electrophoresi5 of salt-soluble proteins (60 pg/lane) from 10 and 20 maize endosperm, pigmented and unpigmented endosperm cultures and from embryogenie callus cultures. E 10, E 2 0 : 1 0 and 20 DAP maize endosperms R: pigmented, W: umpigmented, endosperm callus cultures, S: embryogenic callus culture from the same strain.
SDS-PAGE patterns of salt-soluble extracts from 10 and 20 DAP endosperms, coloured and colourless calli and from an embryogenie scutellar callus from the same strain are presented in Fig.3. Polypeptide patterns of endosperm calli are different from those of the tissue from which they derive; moreover, differences are evident between eolourless and eoloured ealli. In particular, the peptide pattern of coloured calli is characterized by the presence of a 26 kD peptide that represents more than 50% of the protein; this peptide is absent in developing endosperms and is present only as traces in unpigmented callus. Polypeptide patterns and zein contents of red and white calli were essentially the same in different experiments, with minor differences in different callus lines.
secondary metabolism repression in cultured plant cells may be the close relationship between the expression of secondary metabolism and morphological differentiation. Actively dividing cultured cells are under conditions of dedifferentiation and therefore synthesis of secondary metabolites is repressed. In the developing endosperm the differentiation of cells begins 12-15 days after pollination, when mitotic activity is almost terminated and an increasing nuclear size without cell division is observed (Phillips R.L. et al. 1985). At this time, storage protein and anthocyanin biosynthesis begin. These biosynthesis are therefore under a strict developmental control. In our in vitro system the umpigmented sectors appear to have a more active metabolism, suggested by a faster growth rate and a higher salt-soluble protein content than the coloured tissues from which they derive, where protein synthesis seems to be mainly involved in the production of a 26K polypeptide. The presence of zein both in pigmented and in umpigmented calli, even if in small amounts (confirming the observations of Shimamoto) indicates that endosperm cells in culture are able to synthesize a storage protein while continuing active proliferation, in contrast with the in vivo situation where both zein and anthocyanin synthesis start after the end of cell division. Zein synthesis, at a very reduced level, seems to proceed in endosperm cultures in a "constitutive" way, unaffected by variation in the physiological conditions of the tissue. As far as the accumulation of a 26 Kd peptide in pigmented cultures is concerned, it must be recalled that proteins with
DISCUSSION Spontaneous changes in the ability to produce pigment observed in maize endosperm cultures are a common feature in cell cultures producing secondary metabolites. In fact such differences have been reported in callus and cell cultures of different species as Lithospermum erythrorhizon (Mizukami H. et al. 1978) and Daucus earota (Dougall D.K. et al. 1980). In spite of the numerous examples relating to high and low metabolite-yielding cell cultures, there is still little information on changes in secondary metabolism in plant cell culture. According to Ozeki and Komamine (1981) the reason for
8] similar molecular weight have been associated, in tobacco cell culture, with late stages of culture (Singh et al., 1985; King et al., 1986); two immunologically related 26kd peptides have been described, one synthetized after the mid log phase of unadapted cells, the other accumulating during adaptation to NaCI and characteristic of salt-adapted cultures (Singh et al., 1985). The physiological role of both remains unknown. In our maize endosperm callus cultures the apparent absence of a 26 Kd peptide in actively proliferating "white" tissues and its accumulation in pigmented, slowly dividing pigmented tissues; might suggest the presence of proteins with a role similar to that of tobacco 26 kd peptides. The 26 Kd band appears, therefore, to mark a precise "physiological stage" of our culture, in which anthocyanin synthesis is derepressed.
ACKNOWLEDGEMENTS
Gavazzi G and Racchi ML (I780)
Hawkes R, Niday E, Cordon J Biochem. 119:142-147 King GJ, Hussey CE Jr, Molec. Biol. 7:441-449 Lemmli UK
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Landry J and Moureax T (1981) Chem., 29:1205-1212
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Peterson GL (1977)
The authors are grateful to Dr A. Viotti for kindly providing anti-zein antiserum and to Prof. G. Gavazzi for critical reading of the manuscript.
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