Planta

Planta 149, 454-460(1980)

9 by Springer-Verlag 1980

The Initiation of Legumin Synthesis in Immature Embryos of Pisum sativum L. Grown In Vivo and In Vitro C. Domoney, D.R. Davies, and R. Casey John Innes Institute, Colney Lane, Norwich N R 4 7UH, U.K.

Abstract. A highly sensitive immunoassay has been used for the detection of a major storage protein, legumin, in embryos of Pisum sativum L.; with this technique nanogram quantities could be measured. In the two varieties tested, legumin could be detected in embryos in vivo, when they had attained a fresh weight of 2.10 -a g and 3.10 -3 g, respectively. Contrary to earlier claims, embryos cultured in vitro were shown to be capable of initiating legumin synthesis. This capacity to initiate legumin synthesis was confirmed by two-dimensional isoelectric focusing-electrophoresis and fluorography; embryos harvested before initiation of legumin synthesis and cultured in radioactive medium were shown to have synthesized legumin subunits. The amounts of legumin and total protein synthesized per unit fresh weight were consistently greater in vitro than in equivalent embryos grown in vivo. Key words: Embryo culture - Enzyme-linked immunosorbent assay (ELISA) - Legumin - Pisurn - Protein (storage) - Storage protein.

Introduction Studies of macromolecular synthesis and of the accumulation of storage products in embryos and seeds would be facilitated greatly if they could be undertaken in vitro rather than in vivo. The introduction of labelled precursors, the definition and control of environmental conditions as welt as experimental perturbations are all more easily achieved under the former circumstances. However the value of any axenic culAbbreviations: E L I S A = E n z y m e - l i n k e d i m m u n o s o r b e n t assay; BSA = bovine s e r u m albumin ; IgG = immunoglobulin ; SDS = sodiu m dodecyl sulphate; DSP=Pisum cv. D a r k Skinned Perfection.

0032-0935/80/0149/0454/$01.40

ture system is to a large extent dependent on the extent to which growth and development in vitro simulates that in vivo. Studies by Millerd et al. (1975) of macromolecular synthesis in cultured immature embryos of peas (Pisurn sativum) have shown that as far as one of the storage proteins, legumin, is concerned, at least one aspect of synthesis in vitro is different from that in vivo. They showed that embryos detached from the plant prior to the initiation of legumin synthesis, failed to synthesise this protein even after several days in culture. Those which had initiated synthesis in vivo continued to do so in vitro. In contrast, the synthesis of vicilin, the second major group of storage proteins, could be initiated in vitro. They concluded that an unidentified trigger provided by the pod was necessary for initiation of legumin synthesis, since such initiation occurred in pod culture. We have recently developed an improved method of culturing immature pea embryos (Stafford and Davies 1979) and this has enabled us to re-examine the control of legumin synthesis in vitro. The limit of detection of legumin as with all products, is dependent upon the sensitivity of the technique or assay employed. Hitherto it has been possible to detect legumin only in embryos which have attained a fresh weight of at least approximately 0.1 g, - the exact weight being dependent on the variety used (Guldager 1978; Millerd et al. 1975). The technique most commonly used for legumin detection in developing pea cotyledons has been immunoelectrophoresis (Millerd et al. 1975, 1978; Guldager 1978). This paper reports the use of a novel method of detecting legumin, an enzyme-linked immunosorbent assay (ELISA), which allows the detection of very small amounts of the storage protein; it also presents evidence to show that with the improved culture techniques, initiation of legumin synthesis occurs in embryos grown in vitro.

C. Domoney et al. : Legumin in Pisum Embryos

Materials and Methods Plant Material. Plants of JI 26 and of JI 1068 (cv. Birte) were grown in John Innes compost or in 1:1 (v/v) chick grit and perlite in a heated greenhouse with supplementary lighting until flowering. Uniform plants were then transferred to a 15~ C growth cabinet with fluorescent lighting (illuminance at bench level of ca. 580 lm m -s, 16 h daily). Single flowers at the second, third and fourth flowering nodes were allowed to develop and these were sampled for developing embryos. Dry, mature seed of cv. Dark Skinned Perfection (DSP) and JI t068, were used for the purification of legumin and vicilin. Embryo Culture Conditions. Pods of a range of developmental stages were sterilized as previously described (Stafford and Davies 1979), and the embryos dissected aseptically by removal of the seed coats. Each embryo (two cotyledons with intact axis) was freed of surface moisture on sterile filterpaper, placed in a previously weighed sterile vial containing a drop of medium (see below) and its fresh weight determined. It was then immediately transferred to a 5 cm plastic petri-dish containing 5 ml of medium. The sucrose (18 % final concentration) was autoclaved but all other components of the medium (Stafford and Davies 1979) were filtersterilized. Cultures were maintained at 20~ C on a gently rotating table for one week with a 24 h photoperiod provided by Gro-Lux fluorescent lights (light intensity of ca. 340 lm m-2). Embryos were then removed from the medium, weighed, immediately frozen in liquid nitrogen and subsequently stored at - 2 0 ~ C. A range of in vivo embryos were similarly weighed and stored. In one culture experiment using embryos of JI 1068, 2 ml of 14C medium (l.85.106 Bq [U-14C]protein hydrolysate (Radiochemical Centre, Amersham) ml-1) were used per petri-dish with 8 embryos per dish. The labelled amino acids, supplied in 2% ethanol were first evaporated to dryness, dissolved in medium and sterilized by ~Millipore' filtration. The labelled embryos were freeze-dried and stored at - 2 0 ~ C. Antibody Preparation and Purification. Monospecific anti-legumin IgG was prepared from high titre rabbit antisera by affinity chromatography as previously described (Casey 1979). The legumin utilized for the affinity matrix was purified from mature seed of cv DSP. The IgG was further purified by passage through a column of vicilin linked to Sepharose, to remove any possible traces of anti-vicilin. Vicilin was prepared by isoelectric precipitation of legurain and minor proteins (Davies 1976) and further purified by zonal isoelectric precipitation (Scholz et al. 1974) prior to covalent linkage to Sepharose-4B as outlined previously (Casey 1979). Before inclusion in the ELISA plates, residual traces of legumin were removed from this vicilin preparation by two successive passages through an anti-legumin IgG affinity column. Purified IgG, at a concentration of 10 -3 g ml 1 50% phosphate buffered saline (PBS, which is 0.8% NaCI, 0.02% KHzPO4, 0.114% Na2HPO4, 0.02% KC1, 0.02% NaN 3 (all w/v), pH 7.4), was stored in 30 btl portions in silicone-treated glass vials at - 2 0 ~ C. The purifies of legumin and vicilin samples were checked throughout their preparation by subunit analyses of SDS/polyacrylamide gels (Laemmli 1970) and by analytical ultracentrifugation (Casey 1979). The specificity of the IgG preparation was checked by immunodiffusion, (Casey 1979) and by the immunoassay (ELISA) described below. The concentrations of pure legumin preparations were determined as previously described (Casey 1979); vicilin concentrations were measured by the dye-binding method of Bradford (1976). Estimation of Legumin and Total Protein in Immature Embryos. Embryos of 1.0. i0 4 g to 1.5-10 2 g fresh weight, were i~dividual-

455 ly homogenized in extract buffer [PBS containing 2% (w/v) polyvinyl pyrrolidone (PVP) (BDH chemicals, M.W. approximately 44,000) and 0.05% (v/v) Tween 20], in 'Reacti-Vials' (Pierce Chemical Co.., USA), in a total volume of 300 ~tl. Embryos of 1.5 to 4.0-10-2 g fresh weight were homogenized in small glass-in-glass homogenizers in a total volume of 500 gl, and those above 4.0.10 -~ g in a total volume of 1 ml. Embryos weighing Iess than 1.0-10 .4 g were pooled and extracted in a total volume of 300 gl. Each embryo sample was first homogenized in a half-volume of extract buffer at 4~ C and the extract centrifuged at 3,000 g for 10 min. The pellet was then re-extracted in the remaining volume of buffer, the extract centrifuged as previously and the combined supernatants stored at - 2 0 ~ C. Legumin was detected and quantified in extracts by ELISA (Voller et al. 1976, Clark and Adams 1977). Polystyrene microfitre plates (Sterilin Ltd.) were coated with purified antibody (freshly diluted to 2 . 1 0 - 6 g m1-1 0.05 M sodium carbonate; 200 gl were added to each well) for 3.5 h at 37 ~ C. Plates were then washed extensively by flooding with PBS containing 0.05% (v/v) Tween 20. Two hundred gl aliquots of extract diluted with extract buffer, or of standards (see below) were added to triplicate wells (single wells in the case of undiluted extracts of small embryos) and the plates placed for 16-18 h at 4~ C. Plates were thoroughly washed as described. Enzyme-labelled antibody (200 I-tl aliquots) was then added to each well. Conjugation of antibody to alkaline phosphatase was essentially as described by Clark and Adams (1977), with the following modifications. Alkaline phosphatase at 5.10 3 g was utilized per 2.10 3 g antibody, the final concentration of glutaraldehyde was 0.06% (v/v) and BSA was added to 0.5% (w/v). Prior to addition to test plates, the enzyme-conjugate was diluted 1:200 with extract buffer containing 0.2% ovalbumin (Sigma). Plates were incubated with enzyme conjugate for 4.5 h at 37~ C. Thereafter all procedures were carried out at 20 ~ C. Plates were washed as before and 300 gl aliquots of the enzyme substrate (p-nitrophenyl phosphate, 6.10-4 g ml i 9.7% (v/v) diethanolamine containing 0.02% (w/v) NaNa, pH 9.8) added to each well. After 15-20 min (the time being the same for each well within a given plate), 50 gl 3 M NaOH was added to each well to terminate the reaction. The quantity of hydrolyzed substrate in each well was measured by spectrophotometric absorbance at 405 rim. The legumin concentration of each extract could therefore be determined from the absorbance values of the standards. Purified legumin was included in every plate at 10 a _ 10 7 g m l - i extract buffer. Controls included buffer alone, pea leaf extracts and vicilin. Initially, vicilin was included at a range of concentrations in test plates to check the specificity of the antibody. The total amount of protein in the extracts was determined using the remaining portions of extracts. The dye-binding method of Bradford (1976) was used and a range of concentrations of BSA in extract buffer used as standards.

Two-Dimensional Isofocusing - Eleetrophoresis and Fluorography. Each sample of freeze-dried cultured embryos (8 embryos per sample) was homogenized in a glass-in-glass homogenizer in 3 successive portions of 1.5 ml 9.5 M urea containing 5% (v/v) 2-mercaptoethanol. Each extract was centrifuged at 3,000 g for 10 rain, the supernatants of each sample combined and concentrated under vacuum. Ampholines (pH range 3.5-10) were added to 2% (v/v), nonidet NP40 to 2% (w/v) and unlabelled legumin, purified from JI 1068, to 5.10-3g ml - t . A 30~tl sample (approximately 3.7. 104Bq 14C) was submitted to two dimensional electrophoresis (O'Farrell 1975). Gels were stained in 0.25% (w/v) Coomassie brilliant blue G250 in 45% (v/v) methanol and 10% (v/v) acetic acid for 1 h, destained in 5% (v/v) methanol and 10% (v/v) acetic acid, and fluorographed (Bonner and Laskey 1974, Laskey and Mills 1975), using Fuji Rx X-Ray film.

456

C. Domoney et al. : Legumin in Pisum Embryos 0.6

Results

Growth of Embryos In Vivo and In Vitro The growth in vivo and in vitro of developing embryos of the two varieties JI 26 and JI 1068 is shown in Fig. 1 and 2, respectively. The term 'embryo' refers throughout to a developing seed with the testa removed. Growth in vivo is expressed as embryo fresh weight in relation to days after flowering (Fig. 1). A more rapid gain in fresh weight was noted with time in embryos of variety JI 1068 than in those of variety JI 26, reflecting the larger seed size of the former. Desiccation and ensuing loss of fresh weight occurred in both varieties at approximately 40 days after flowering. Maximum fresh weight in var. JI 1068 was approximately 0.5 g whereas in var. JI 26 it was approximately 0.33 g. Growth in vitro is expressed as embryo fresh weight after the 7-day culture period as a logarithmic function of initial embryo fresh weight (Fig. 2). The extent of growth in culture is seen to be dependent upon initial embryo fresh weight, as noted previously (Stafford and Davies 1979). Embryos of var. JI 1068 of 10-1 g fresh weight were found to increase approximately 3.5-fold in fresh weight during the culture period, whereas embryos of 2.10-3 g showed approximately a 7-fold increase under the same conditions (Fig. 2). The validity of using embryo fresh weight rather than age as a criterion of developmental stage has been outlined by previous authors (Millerd et al. 1975; Carasco et al. 1978). The latter workers found that seeds of Vigna unguiculata L., which were of

0-4

0"2

a~

E

ua

1; Days

40

50

Fig. 1. The fresh weight attained by embryos of variety JI 26 (~-• and variety JI 1068 ( i n) in vivo in relation to time after flowering. (The plants were maintained at 15~ C and embryos were sampled from single pods at the second, third and fourth nodes)

equal fresh weight, though of different ages, were similar to one another in many respects. Our results (see below) have borne out these observations with respect to quantities of total protein and legumin in embryos of equal fresh weight. Nevertheless, some

J I 1068

JI 26

-1

==

.r

//

lO"!

9 a 9

~0

30 flowering

10O-

1o 0

'-'10

20 after

10 -2

9

i|

lO -2_

dt

9

t |

La.

I0 "3 10-4

I

10"3

i 10"3

I

Initial

i

10 -3

I0 -I 10 -4

10 -2 fresh

weight

i

10-2

i

10 -1

( g )

Fig. 2. The fresh weight attained by embryos of variety JI 26 and variety JI 1068 in vitro. Embryos were harvested from seeds at a range of developmental stages, and cultured for 7 days at 20 ~ C, with one embryo and 5 ml of medium per petri-dish. The lines indicated are calculated from regression analysis (r=0.95 for JI 26; r=0.99 for JI [068)

c. Domoney et al.: Legumin in Pisum Embryos variation in the extent of growth in vitro occurred in embryos of equal initial fresh weight (Fig. 2). Previous workers have failed to correlate such variation with parameters such as age of the plant or pod position (Thompson et al. 1977). Throughout this work, cultured embryos of all sizes maintained a healthy appearance, did not lose chlorophyll, and did not develop a polyphenol layer or show epidermal cell disruption. These symptoms have been noted in material cultured by other workers (Lea et al. 1979; Millerd et al. 1975) but are avoided by the use of 18% sucrose in the medium; this concentration of sucrose also prevents germination of embryos in vitro. It had previously been established that this osmotic pressure was suitable for embryos of any fresh weight, in that even those in the range 1 0 . 4 g to 10- 5 g fresh weight grew as well in medium containing 18% sucrose as they did in medium containing 8%, 10%, 12% or 14% sucrose (data not shown).

Legumin Detection and Accumulation in Immature Embryos Grown In Vivo and In Vitro a) Monospecificity of Antibody Preparation. The validity of the results presented here is dependent upon the absolute monospecificity of the anti-legumin preparation used for the detection and measurement of legumin. As outlined in the materials and methods section, anti-legumin was prepared from IgG by passage through Sepharose-legumin and Sepharose-vicilin columns. The purity of the legumin used for column chromatography was established by analytical ultracentrifugation ; this revealed only the 11 S protein with no detectable 7S peak. Ultracentrifugation analysis of the vicilin (7S) preparation, prior to covalent linkage to Sepharose, indicated slight contamination with the 11 S legumin, though this was not apparent on SDS-polyacrylamide gel electrophoresis. Consequently, a proportion of anti-legumin IgG activity was probably lost during this purification step. The legumin contaminant was removed from the vicilin preparation by Sepharose-IgG column chromatography and the eluted protein was identified as vicilin (subunits of approximately 51,000, 47,000 and 33,000 with a minor trace of 70,000 molecular weight). This vicilin sample failed to react with the purified antilegumin IgG preparation, when tested by immunodiffusion or when included at a range of concentrations in ELISA plates (Fig. 3). This fact suggests monospecificity of the antibody preparation in addition to a lack of common antigenic determinants in vicilin and legumin, confirming previous observations (Dudman and Millerd 1975). In addition, extracts of mature leaves of a variety of plants, including pea, failed to react with the IgG preparation, when tested by ELISA.

457

Fig. 3. The reaction between legumin (lanes 2, 3, 4), vicilin (lanes 6, 7), buffer (lanes 5, 8) and the anti-legumin IgG preparation when tested by ELISA. Plates were coated with IgG and then incubated with 200 gl aliquots of test sample. Buffer (PBS containing 2% PVP and 0.05% Tween 20) alone was added as control. Legumin and vicilin were added at 1.10-8 gm1-1 buffer (row B),2.10 8 g ml-l (row C), 3-10 8gml-l(rowD),5.10-Sgml-1 (row E), 0.75-10-7 gml-1 (row F) and 1.10-7 g ml-1 (row G). Sample wells were then incubated with IgG-alkaline phosphatase conjugate. Addition of enzymesubstrate (p-nitropheuylphosphate) resulted in hydrolysisof the substrate and consequent colour development in the case of legumin only. The mean absorbance at 405 nm of the wells used buffer alone was 0.190, while that of the wells used for vicilin samples was 0.185

b) Calibration of ELISA. In this assay, a linear relationship was established between legumin concentrations, in the range 1 - 10- 8 g ml- 1 and 1 910- 7 g ml- 1, and absorbance at 405 nm. All embryo extracts were diluted as necessary, and legumin measured within this range.

c) Legumin Quantities in Embryos Grown In Vivo and InVitro. Figure 4a shows that legumin could be detected in embryos of JI 26 grown in vivo, when they had attained a fresh weight of approximately 2.103 g. Embryos in the range 1- 10 4 g to 2.10 - 3 g fresh weight showed no detectable legumin. In the range 2-10 .3 to 1.10 -~ g fresh weight, the largest embryos measured, an approximately 104-fold increase in amount of legumin was observed (Fig. 4a). This increase was not constant over the weight range tested; a slow increase from 2 . 1 0 - 3 g to 2 . 1 0 - z g fresh weight was followed by a more rapid accumulation of legumin. In this variety legumin was detected in cultured embryos which had attained a minimum of 1 . 6 . 1 0 - 3 g fresh weight. Such embryos would have had an initial fresh weight of approximately 2 - 1 0 - 4 g (Fig. 2) and would not have con-

458

C. Domoney et al.: Legumin in Pisum Embryos

j

10 -3

10 .4

10

-3

b

8

10

10

-4 9

o

-5

10 -5 cD v -0 10

t0

-6 = c: []

'~-

lo ~

10-7

%o

[] ~ ' i

= o-

10

1o "0

-9

-8

-9

10 10.4

I 10.3 Embryo

I

I 10 .2 fresh

10"1 weight

;0 10

(g )

~,~

10 10

-4

! 10 "3 Embryo

[-2 10 fresh

! -1 10 weight

I 10 0

(g)

Fig. 4a and b. Legumin amounts in embryos developing in vivo ( g - i) and in vitro ([]- []), and total amounts of protein in embryos developing in vivo ( * - * ) and in vitro (0-~>) in logarithmic relation to embryo fresh weight. Legumin was measured in embryo extracts by ELISA and total protein by the dye-bindingmethod of Bradford (1976). a JI 26, b JI 1068

tained legumin prior to culture (Fig. 4 a). Therefore, these results, in contrast to those of others, (Millerd et al. 1975) show that immature embryos can initiate legumin synthesis in culture. In addition, although the patterns of legumin accumulation were similar for embryos grown in vivo and in vitro, cultured embryos were found to accumulate quantities of this protein in excess of their in vivo fresh weight counterparts (Fig. 4a). A similar pattern of legumin accumulation in embryos of variety JI 1068 grown in vivo is shown in Fig. 4b. Legumin was not detected in these embryos before they had attained a fresh weight of approximately 3-10-3 g. However, when embryos which had a smaller initial fresh weight than this were cultured, they were found to contain legumin at the end of the culture period. Cultured embryos of approximately 2.5.10-3 g fresh weight i.e. those which had an initial fresh weight of approximately 2 . 5 - 1 0 - 4 g (Fig. 2) were found to contain legumin. Again, cultured embryos had higher quantities of legumin than embryos of similar fresh weight grown in vivo.

Embryos of variety J1 26 and of variety JI 1068 had similar quantities of legumin at a given embryo fresh weight in vivo and also when grown in vitro. In both varieties, a 10 4 tO 10S-fold increase in legumin content was observed over the range of embryos tested (Fig. 4). Total Protein in Immature Embryos In Vivo and In Vitro

The dye-binding method of Bradford (1976) for protein quantitation was adopted, since the extraction buffer was found to interfere with the method of Lowry et al. (1951). The dye-binding ability of BSA was only slightly affected by the extraction buffer, resulting in a steeper calibration graph than BSA in 0.15 M phosphate buffer containing 5% (w/v) NaC1. In addition, the assay in the presence of the extraction buffer was linear only up to 2 . 1 0 - s g BSA. Embryos of equal fresh weight extracted in extraction buffer or in 0.15 M phosphate buffer containing 5% NaC1 yielded similar amounts of total protein when mea-

C. Domoneyet al. : Leguminin Pisum Embryos

459

10 0

Jr ,.,~ 10 1]

c w.

10.2

10-3 10 .4

I

I

10-3 Initial

fresh

10-2 w e i g h t (g)

I

10-1

Fig. 5. The growth of embryos of variety JI 1068 in vitro (z~) with 8 embryos and 2 ml of medium containing 1.85-10 6 Bq 14C ml-1 per petri dish. Each point represents the mean final fresh weight of 8 embryos as a function of mean initial fresh weight. The growth of control embryos in vitro (A-A) with 1 embryo and 5 ml of mediumper petri-dish. The line indicated is calculated from regression analysis of the values for the control embryos (t = 0.99) sured by the dye-binding method. Efficient protein extraction was therefore achieved in the first buffer. In addition, no significant quantities of protein could be detected in embryo tissue after extraction and centrifugation. The total protein present in extracts of JI 26 and JI 1068 embryos is shown in Figs. 4a and 4b, respectively. More protein is accumulated by embryos of both varieties grown in vitro than those in vivo, reflecting the similar relationship found for legumin. The amounts of protein in vivo and in vitro in JI 26 are similar to those of equivalent embryos in JI 1068.

Demonstration of Legumin Synthesis in Cultured Embryos by Two-Dimensional Isoelectric Focusing Electrophoresis and Fluorography In order to confirm the results obtained with ELISA, in particular the ability of cultured embryos to initiate legumin synthesis, embryos were cultured in the presence of radio-isotoPe. Embryos of JI 1068 with a fresh weight of less than 3- 10- 3 g were cultured in medium containing 14C amino acids. The growth of these embryos in culture under the altered conditions (presence of isotope, a reduced volume of medium and many embryos per petri-dish) was comparable to that of embryos under previous conditions (Fig. 5). Two-dimensional gel electrophoresis of the concentrated extracts of the combined embryos from each petri-dish

revealed on staining a complex pattern of embryo polypeptides. Most of these were not identifiable with the major storage protein subunits as seen in extracts of mature seed, reflecting the preponderance of other proteins in embryos at early stages of development. The position of the 40,000 molecular weight subunits of legumin was recognized on gels of the labelled extracts by addition of unlabelled carrier JI 1068 legurain, and by comparison with similar gels of purified JI 1068 legumin. Three major legumin subunits of approximately 40,000 molecular weight were identified, and their positions noted after impregnation and drying of the radioactive gels. Fluorographs developed after a 17-h exposure of the gels to X-ray film revealed radioactivity in the position of the three major 40,000 molecular weight subunits of the unlabelled carrier legumin. In addition, a complex array of highly labelled polypeptides were seen which were not obvious on the stained gels. These results show that legumin synthesis is initiated in embryos cultured in vitro and confirm the results previously obtained by ELISA.

Discussion

Accumulation of total protein and of legumin was seen to proceed at comparable rates in vivo and in vitro. An increase in legumin content of approximately 4 orders of magnitude was observed between embryos of2-10 -3 g and those of 1-10 1 g fresh weight, while total protein increased by approximately 2 orders of magnitude in equivalent embryos. This reflects an increasing proportion of iegumin per unit protein during development, although the largest embryos used in this study had not yet attained the stage of maximum storage protein synthesis, and the total protein measured in these embryos probably represents predominantly enzymatic and structural proteins. It has been assumed previously that initiation of legume storage protein synthesis coincides with the cessation of cell division or follows the onset of cell expansion and D N A endoreduplication in the developing embryo (Millerd and Spencer 1974 ; Dure 1975 ; Lea et al 1979). The detection of legumin in embryos o f 2 . 1 0 - 3 g (J126)and 3- 10-3 g (JI 1068) fresh weight, in this work suggests that storage protein synthesis occurs at low levels even during the very early stages of embryogenesis, whereas a substantial level of endoreduplication cannot be detected until the embryos are approximately 5- 10- 2 g fresh weight (Cullis 1978). Our ability to pinpoint the onset of synthesis of a particular protein is directly related to the sensitivity of the technique employed in its detection. Had an even more sensitive method than ELISA been avail-

460

able for detection of legumin in developing embryos it is possible that even earlier synthesis of legumin would have been detected ; storage protein gene activity and expression could be initiated conceivably at the onset of embryo formation. On the other hand, it is still possible that storage protein synthesis occurs only in cells that have ceased to divide, and that the early detection of legumin in embryos in this study reflects its synthesis in a small proportion of endoreduplicated cells. If the synthesis of legumin begins when embryos in vivo attain a fresh weight of 2.10 .3 g to 3.10 - 3 g then the results presented here show that contrary to previous observations (Millerd et al 1975). legumin synthesis is initiated in embryos grown in vitro. The failure by Millerd et al. (1975) to detect legumin in cultured embryos isolated from seeds weighing 0.15 g may have been due to the low sucrose level utilized in their medium. These workers routinely used 4% sucrose, with 14% the highest concentration tested. In our experience, embryos grow very poorly at 4%, and elongation of the radicle occurs even in medium containing 14% sucrose. It would be expected that, under conditions in which radicle development is occurring, other features of germination, such as degradation of storage proteins might also take place. It is suggested, therefore, that both synthesis and degradation of legumin may have been taking place concurrently in the embryos used by Millerd et al. (1975). Furthermore the variety they used, (cv. Greenfeast), has a fairly low level of legumin, exacerbating the problems of detection. When Millerd et al. (1975) used older embryos (0.16 to 0.22 g seed weight), then legumin could be detected in cultured embryos; presumably the accumulation of larger quantities of legumin in such embryos would ensure its detection in spite of some loss through hydrolysis in culture. Pod culture allowed the production of legumin in the in vitro experiments of Millerd et al. (1975); this may have been due to the prevention of germination in those circumstances. This exploitation of the ELISA technique, together with the improved method of embryo culture, will greatly facilitate studies of many aspects of storage protein synthesis in peas. Miss C. Domoney thanks the John Innes Charity for the award of a studentship.

References Bonner, W.M., Laskey, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88

C. Domoney et al.: Legumin in Pisum Embryos Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Carasco, J.F., Croy, R., Derbyshire, E., Boulter, D. (1978) The isolation and characterization of the major polypeptides of the seed globulin of cowpea (Vigna unguiculata L. Walp) and their sequential synthesis in developing seeds. J. Exp. Bot. 29, 309323 Casey, R (1979) Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds. Biochem. J. 177, 509-520 Clark, M.F., Adams, A.N. (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34, 475483 Cullis, C.A. (1978) Chromatin-bound DNA-dependent RNA polymerase in developing pea cotyledons. Planta 144, 57-62 Davies, D.R. (1976) Variation in the storage proteins of peas in relation to sulphur amino-acid content. Euphytica 25, 717-724 Dudman, W.F., Millerd, A. (1975) Immunochemical behaviour of legumin and vicilin from Vicia faba: a survey of related proteins in the Leguminosae subfamily Faboideae. Biochem. Syst. Ecol. 3, 25-33 Dure, L.S. (1975) Seed formation. Annu. Rev. Plant Physiol. 26, 25%278 Guldager, P. (1978) Immunoelectrophoretic analysis of seed proteins from Pisurn sativum L. Theor. Appl. Genet. 53, 241-250 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of 3H and t4C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335 341 Lea, PJ., Hughes, J.S., Miflin, B.J. (t979) Glutamine- and asparagine-dependent protein synthesis in maturing legume cotyledons cultured in vitro. J. Exp. Bot. 30, 529-537 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275 Millerd, A., Spencer, D. (1974) Changes in RNA-synthesizing activity and template activity in nuclei from cotyledons of developing pea seeds. Aust. J. Plant Physiol. 1, 331-341 Millerd, A., Spencer, D., Dudman, W.F., Stiller, M. (1975) Growth of immature pea cotyiedons in culture, Aust. J. Plant Physiol. 2, 51-59 Milierd, A., Thomson, J.A., Schroeder, H_E. (1978) Cotyledonary storage proteins in Pisurn sativum. III. Patterns of accumulation during development. Aust. J. Plant Physiol. 5, 519-534 O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 400%4021 Scholz, G., Richter, J., Manteuffel, R. (1974) Studies on seed globulins from legumes. 1. Separation and purification of legumin and vicilin from Vicia faba L. by zone precipitation. Biochem. Physiol. Pflanzen 166, 163-172 Stafford, A., Davies, D.R. (1979) The culture of immature pea embryos. Ann. Bot. 44, 315-321 Thompson, J.F., Madison, J.T., Muenster, A.E. (1977) In vitro culture of immature cotyledons of soya bean (Glycine max L. Merr.). Ann. Bot. 41, 29-39 Voller, A., Bartlett, A., Bedwell, D.E., Clark, M.F., Adams, A.N. (1976) The detection of viruses by enzyme-linked immunosorbent assay (ELISA). J. Gen. Virol. 33, 16~167

Received 30 January; accepted 21 March 1980