Plant Cell Reports (1984) 3:55-59
Plant Cell Reports © Springer-Verlag 1984
Abscisic acid promotes lectin biosynthesis in developing and germinating rice embryos Hetta M. Stinissen 1, Willy J. Peumans 1, and E. De Langhe 2 1 Laboratorium voor Plantenbiochemie, KULeuven, Kardinaal Merderlaan, 92, B-3030 Leuven, Belgium 2 Laboratorium voor Tropische Plantenteelt, KULeuven, Kardinaal Mercierlaan, 92, B-3030 Leuven, Belgium Received March 4, 1984 - Communicated by D. yon Wettstein
ABSTRACT Immature rice (Oryza sativa, L) embryos isolated about 12 days post anthesis are fully able to develop into young seedlings when cultured in vitro. Concomitantly, they rapidly loose their lectin synthesis activity. Abscisic acid added to the nutrient medium prevents precocious germination of the immature embryos and simultaneously strongly promotes lectin biosynthesis activity. Similarly, abscisic acid keeps mature embryos grown in a nutrient medium in a dormant state and maintains their lectin synthesis activity, whereas control embryos rapidly germinate but also quickly loose their lectin synthesis activity. It appears, therefore, that rice lectin is typically synthesized in embryos which are kept in a dormant state. Abbreviations: ABA = abscisic acid; GA 3 = gibberellic a~id; WG-A ~ Wheat germ agglutinin; DPA = days post anthesis. INTRODUCTION Gramineous lectins are an extented class of closely related proteins, which have been conserved fairly well during the evolution and divergency of this plant family (Stinissen et al. 1983). All grass lectins which hitherto have been purified and characterized into some detail belong to one of the three subgroups that, according to the taxonomic group in which they occur, are designated as cereal, rice or Brachypodium lectins. Although several Gramineae lectins have been studied into detail with respect to their biochemical, physico-chemical and cellular-biological properties, their physiological role in the plant is still a matter of controversy. As early as ]975, it was proposed that wheat germ agglutinin (WGA) (which is the best known and most intensiVely studied gramineous lectin) acts as an antifungal agent against chitin-containing phytopathogens during seed imbibition and early germination (Mirelman et al. 1975). Although in a recent report on the localization of lectin in embryos and adventitious roots of wheat, the association of lectins with those parts of the plant that come in close contact with the soil have been interpreted in favor af a possible defensive role of WGA (Mishkind et al. ]982), evidence is accumulating that gramineous lectins might fulfill a much more specific physiological role. Indeed, the timing and localization of lectin biosynthesis in developing wheat and rice embryos indicate
that these lectins are maturation-specific proteins which are exclusively synthesized in the primary axes (Peumans et al. 1982; Stinissen, Peumans 1983). Moreover, the promoting effect of abscisic acid (ABA) on lectin synthesis in cultured, immature wheat embryos (Triplett, Quatrano 1982), indicates that WGA is not only a maturation-specific protein but could be a dormancy-specific protein. This evidence, taken together with the evolutionary conservatism of gramineous lectins (Stinissen et al. ]983), possibly argues for a specific endogenous physiological role of these proteins. The present paper deals with the effect of ABA on lectin biosynthesis in developing and germinating rice embryos. In both systems this plant growth substance prevents germination and markedly stimulates lectin synthesis. MATERIAL AND METHODS Plant Material Rice (Oryza sativa L. cv. Koshihikari) plants were grown in a greenhouse at 28 °C during the day (]3 h) and 20 °C during the night. Under these conditions the time between anthesis and complete seed maturation took about 40 days. For the experiments with developing embryos, grains were collected between 8 and 12 days post anthesis (DPA) since at this developmental stage lectin synthesis is maximal (Stinissen, Peumans, 1983). Experiments with germinating embryos were carried out with grains harvested around 25 DPA (about 2 weeks before grain maturity) and artificially dried in the laboratory. Such a precocious harvest was recommendable since embryo s from fully mature rice grains synthesize little lectin upon germination (Stinissen, Peumans, ]983). Embryo Culture Grains were collected, surface-sterilized with 0.5 % NaOCI and the embryos isolated manually with a scalpel blade under a laminar air flow. Embryos were plated in sterile Petri dishes on Whatman no.| filter paper soaked with sterile nutrient medium consisting of Linsmaier and Skoog salts (1965) pH 5.6, supplemented with 2 % (w/v) sucrose, 150 mM L-glutamine and 500 Dg/ml ampicillin or 200 Dg/ml earbenicillin. The plant growth substances ABA and gibberellic acid (GA3) were added to the culture medium as indicated in the legends to the figures. Unless stated otherwise, the concentrations of ABA and GA 3 were 25 and 50 ~M respectively. Embryos were incubated at 25 °C during the day and 20 °C during the night under a 12-h photoperiod
56 for the periods indicated in the figures. Radioactive labelling Lots of 25 embryos_were incubated for 12 h at 30 °C with 180 kBq of [3~S] cysteine (SA: 42.9 TBq/ mmol). Labelled cysteine was used in all labelling experiments because of the unusually high cysteine content (about 24 %) of rice leotin (Tsuda 1979). Extraction and determination of total protein synthesis After incubation, embryos were extensively washed with ice-cold distilled water on a Buchner-funnel, blotted dry and weighed. Then, they were homogenized in a mortar and extracted with I ml of 20 ng{ Tris-HCl, pH 7.8. Homogenates were cleared by centrifugation (4 min; 10,000 g) in an Eppendorf microcentrifuge and the supernatant taken off. Total protein s y n t h e s ~ was estimated by measuring the incorporation of [ ~ S ] cysteine into trichloroacetic acid-insoluble material in three aliquots of 10 ~I of the supernatant. Lectin isolation To the 10,000 g supernatant (in Tris), I ml of Naacetate(50mM, pH3.8) was added, heated for 2 min at 60 °C and quenched cold in an ice-bath for I0 min. Any coagulated material was removed by centrifugation (5 min; 3,000 g) and the resulting supernatant applied onto a small column (0.2 ml bed volume) of immobilized N-acetylglucosamine (Selectin I from Pierce Chemical Company, Rockford, Ill. USA) for affinity-purification of the lectin. Unbound protein was eluted with Nascetate buffer (3 ml) and phosphate-buffered saline (2 ml). The bound lectin was desorbed with I ml of a solution of ]00 Ng/ml chitin hydrolysate (prepared according to Rupley 1964). Finally, the radioactive cysteine recovered in this affinity-purified lectin fraction was determined in a liquid scintillator. The purity of the leotin fractions obtained in this way was controlled by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. They were essentially devoid of contaminating proteins (results not shown here). RESULTS Effect of ABA and GA~ on precocious germination and leetin b-~os--~t~esi~ ~ F i n vitro' ~ultu--~ded~ r ~ embryos Immature embryos excised from 8 to 12 DPA rice grains were cultured in vitro in a nutrient medium either or not supplemented with ABA (50 DM), GA3 (25 uM) and a combination of ABA and GA 3 (50 and 25 uM respectively). Under the conditions described in the methods section, control embryos (which did not receive any plant growth substance), were fully able to germinate and eventually developed into seedlings (Fig.la and 2a). GA_-treated embryos behaved almost identically to control embryos except that they germinated somewhat faster. On the contrary, embryos grown in the presence of ABA or ABA + GA 3 did not germinate at all, but nevertheless showed some limited development in vitro as they increased continuously in fresh weight and size. It is worthwhile to mention here that the inhibition of germination by ABA is concentration dependent. At low concentrations, precocious germination was only retarded but not inhibited as is illustrated by Fig. lc, which shows that germination gradually decreases with increasing ABA concentration. At the concentration used in our experiments (25 ~M) no germination was observed. In spite of the obvious differences in germination behaviour between control and GA3-treated embryos on
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Fig.1. a. Effect of plant growth substances on precoclous germination of 12 DPA rice embryos cultured for 3 days in nutrient medium (I) and nutrient medium supplemented with 25 ~M ABA (2), 25 ~M ABA + 50 >M GA3(3) or 50 >M GA 3 (4). b. Effect of plant growth substances on normal germination of dry 25 DPA embryos grown in nutrient medium (I) and nutrient medium supplemented with 25 >M ABA (2), 25 NM ABA + 50 >M GA 3 (3) or 50 DM GA 3 (4). c. Effect of ABA concentration on precocious germination of 12 DPA embryos cultured in vitro for 3 days. ABA concentrations were 0, 0.1, 1 and I0 >g/ml in (I), (2), (3) and (4) respectively. the one hand, and ABA and ABA + GA_-treated embryos on J . . . the other hand, in vivo protein synthesls actlvlty was rather similar over the whole incubation period (up to 48 h). Total protein synthesis, which was measured as amino acid incorporation over a 12 h-period, was high in all embryos during the first 12 h of incubation. Thereafter, it decreased considerably but remained almost at the same level from 12 to 48 h (Fig.2b). The dramatic decrease of protein synthesis activity of the ~n vitro cultured embryos during incubation has to be ascribed most probably to the stress induced by their removal from the intact seeds. It is known, indeed, that, at least in in vitro cultured rye embryos, the polysome content decreases drastically during the first hours of incubation, which of course results in a rapidly declining protein synthesis activity (Peumans et al. ]979). In contrast to the fairly similar total protein synthesis activity in all embryos, the incorporation of labelled cysteine into lectin varied greatly according to the presence or absence of ABA and GA~ in the J nutrient medium (Fig.2c). In control embryos, lectin synthesis activity rapidly deareased as a function of incubation time and eventually fell at the limit of detection after 36 h. Compared to control embryos, GA3-treated samples synthesized much less lectin even
57 a
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Fig.2. Effect of ABA (25 DM), GA 3 ~ ) and a combination of ABA and GA 3 (25 and 50 ~M respectively) on: a. The development of ]2 DPA rice embryos in culture medium, b. Protein synthesis of developing rice embryos. Protein synthesis was determined over the 12 final hours of incubation in nutrient medium (after preincubation periods of 0, ]2, 24 and 36 h respective!x) and is expressed as cpm x i0 ~/embryo. c. Lecti~ synthesis (expressed as cpm x 10-~/embryo)o d. The relative rate of lectin synthesis (%).• • :ABA; O---•: G A _ ; O - - - O ABA + GA3; A A Control.
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those who received ABA in combination with GA B (Fig.2c). When in stead of total lectin synthesis the relative rate of lectin synthesis (which is defined as the % of total incorporated [ S]cysteine recovered in the lectin fractions) is considered, similar conclusions can be drawn concerning the effect of ABA and GA 3 but, as is shown in Fig.2d, differences between the treatments are more pronounced. The same results are presented on a logarithmic scale. As is shown in Fig.4a, the relative rate of lectin synthesis decreases exponentially as a function of germination time in GA3-treated embryos. In con-
during the first 12 h of incubation, and, in addition, stopped lectin synthesis about 12 h earlier. ABA and ABA + GA_-treated embryos, on the contrary, exhibited nearly t~e same lectin synthesis activity as control embryos during the first 12 h. Thereafter, however, they synthesized much more lectin than the control embryos and continued synthesizing lectin for at least 48 h. Although both ABA and ABA + GA%-treated samples behaved almost identically with respect to germination behaviour and total protein synthesis activity, embryos grown in the presence of ABA alone exhibited consistently higher levels of lectin synthesis than
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Fig.3. Effect of ABA (25 uM), GA~ ~ M ) and a combination of ABA j and GA 3 (25 and 50 ~M respectively) on: a. Germination of mature rice embryos in culture medium, b. Protein synthesis of germinating rice embryos. Protein synthesis was determined over the 12 final hours of incubation in nutrient medium (after preincubation periods of O, ]2, 24 and 36 h respectively) and is expressed as cpm x 10-~/embryo. c. Lecti~ synthesis (expressed as cpm x 10-~/embryo). d. The relative rate of leetin synthesis (%). A--A: ABA;O---O: GA3~O----O: ABA + G A 3 ; A A: Controi.
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Fig. 4. Effect of ABA, GA 3 and a c~mbination of both on the relative rate of lectin synthesis. Embryos were incubated in nutrient medium and labelled during the 12 final hours• Synthesis of total protein and lectin was determined and the relative rate of lectin synthesis calculated. Results are expressed on a logarithmic scale• a. developing embryos, b. mature embryos• • •: 25 M A B A ; O - - - O : 5 0 M GA3; O---O: 25 M ABA + 50 M GA3; A A: Control•
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Incubation time (h) trol embryos the exponential decrease starts with a certain lag, and in the ABA and ABA + GA~-treatments J a lag of about 24 h was observed. It has been shown previously that such an exponential decrease of the relative rate of lectin synthesis in germinating wheat and rye embryos is due to lectin mRNA depletion as a function of germination time. Dry wheat and rye embryos contain a certain amount of stored lectin mRNAs which aretranslated during early germination and degrade with a half-life of about 3 h (Peumans et al• 1982)• Since no new lectin mRNAs are transcribed during germination, an exponential decrease of the rate of lectin synthesis is observed. Possibly, the exponential decrease of the relative rate of lectin synthesis in in vitro cultured immature rice embryos also can be ascribed to lect~n mRNA depletion during incubation• If so, the maintenance of higher levels of lectin synthesis in response to ABA could be due, at least partly, to an increase of lectin mRNA life-time. Effect of ABA and GA 3 on germination thesis in g-~mi--n~ting rice embryos
and lectin syn-
Although rice lectin can be considered as a maturation-specific protein which is almost exclusively synthesized in developing primary axes (Stinissen, Peumans ]983), some residual lectin synthesis can be observed in germinating embryos (isolated from mature seeds)• To find out whether ABA and GA3.influenced lectin synthesis in germinating embryos in a similar way as in in vitro cultured developing embryos, we looked at the effect of ABA, GA^ and ABA + GA 3 on ger5 . mination, total synthesis and lectln synthesis during early germination of dry rice embryos. These embryos were isolated from 25 DPA grains (which were dried in the laboratory) and grown in a nutrient medium exactly as described for the in vitro culture of developing rice embryos• Under these conditions, control embryos readily germinated and developed into seedlings within a few days. GA3-treated embryos behaved similarly except that they developed somewhat faster than the controls. ABA and ABA + GA3-treated embryos, however, did not germinate• The observed increase of their fresh weight during the initial 12 h of incubation has to be ascribed to aspecific uptake of imbibition water (Fig. Ib and 3a). In contrast to immature embryos, which rapidly loose more than half of their protein synthesis activ i t y d u r i n g i n ~itro culture, germinating embryos, grown under the same conditions, increase their protein synthesis activity as a function of incubation time. A steady increase of protein synthesis activity was not only observed in control and GA -treated em• 3 bryos but also in ABA and ABA + GA3-treated embryos. However, it is evident from Fig.3b that the latter two
treatments resulted in a decreased overall protein synthesis activity. When looking at lectin synthesis, it became evident that in spite of their steadily increasing overall protein synthesis, control and GA3-treated embryos rapidly lost their lectin synthesis activity as a function of germination time (Fig.3c). As already observed for precociously germinating embryos, lectin synthesis activity decreased somewhat faster in GA 3 treated than in control embryos. ABA and ABA + GA 3treated embryos, on the contrary, continued synthesizing lectin during germination at a relatively high rate. In ABA-treated embryos there was even an increase in total lectin synthesis, whereas it remained more or less constant in ABA + GA^-treated embryos. • J • When in stead of total lectln synthesls the relatlve rate of lectin synthesis is plotted against germination time, differences become more pronounced, especially between ABA and ABA + GA3-treated embryos. Indeed, ABA embryos clearly synthesize more lectin than embryos grown in ABA + GA 3 (Fig.3d). The same results pregented on a logarithmic scale (Fig•4b), indicate that in all treatments the relative rate of lectin synthesis decreases exponentially as a function of germination time. As has been discussed above, this exponential decrease can be explained by a lectin mRNA depletion mechanism during germination. The obvious differences between the slope of the graphs corresponding to the four treatments might correspond to differences in lectin mRNA half-life If so, GA~j • • and ABA decrease and increase respectlvely the halflife of the lectin mRNAs.
DISCUSSION The results repo~ted in this paper present evidence that ABA prevents germination of in vitro grown immature and mature rice embryos, and concomitantly strikingly promotes lectin biosynthesis. It is evident therefore that rice lectin, which has been shown previously to be a maturation-specific protein, whose synthesis is almost exclusively confined to a welldefined stage of the development of the embryonic axes (Stinissen, Peumans 1983), is also a dormancy-specific protein. Indeed, rice embryos, irrespective whether they are precociously or normally grown in vitro are fully able to develop into seedlings when grown in a culture medium and thereby rapidly loose their lectin synthesis activity. However, when they are maintained in a dormant state by exogenously added ABA, they continue to synthesize lectin. Similar results have been obtained with precociously germinating wheat embryos by Triplett and Quatrano (]982) who found a marked increase in the
59 biosynthesis of chitin-binding proteins (including WGA) in embryos grown in the presence of ABA. As is demonstrated by Fig.2c and d, and Fig.3c and d, however, the promoting effect of ABA on rice lectin synthesis is not limited to immature embryos hut is also observed for full-grown, normally germinating embryos. ABA is known to inhibit germination of both mature and immature embryos of numerous plant species (Milborrow 1974; Walton 1980). Moreover, it is also well established that ABA promotes specifically the synthesis of typical maturation-specific proteins such as e.g. storage proteins in Brassica, Phaseolus and Gossypi~ ( C r o u c h , S u s s e x 1981; S u s s e x e t a l . 1980; Dure e t a l . 1 9 8 0 ) , and i n some c a s e s e v e n i n d u c e s t h e s y n t h e s i s o f proteins typical for late embryogenesis (e.g. in cotton seeds, Dure et al. 1980). GA 3 could not counteract the effects of ABA, at least not at the concentrations used in our experiments. Indeed, GA 3 + ABA-treated embryos did not germinate and contlnued to synthesize lectin (although less than ABA-treated embryos). Thus GA 3 cannot restore ABA-induced inhibition of germinatlon and abolishes only partially the ABA-induced lectin synthesis. This is clearly in contrast with e.g, the effect of GA on a-amylase-induction in barley aleurone layers, 3 whlch can readlly be reversed by ABA (Mozer, 1980, Higgins et al. 1982). Finally, the finding that both WGA and rice lectin are maturation-specific proteins whose synthesis is somehow related to the maintenance of a naturally or ABA-induced resting state, have to be considered in view of the physiological role of these and other related lectins. Indeed, these observations, taken together with the evolutionary conservatism of Gramineae lectins and the fact that they represent only a minor fraction of the total protein (in contrast to other ABA-promoted proteins such as storage proteins), may argue for a specific and endogenous physiological role in the developing primary axis. Conclusive evidence for such a role awaits experimental proof.
ACKNOWLEDGEMENTS This work is supportedinpart by grants of the National Fund for Scientific Research (Belgium). W.P. is Research Associate of this fund. H.S. acknowledges the receipt of a fellowship of the Belgian 'Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw'.
REFERENCES Crouch ML, Sussex IM (198]) Plants ] 5 3 : 6 4 - 7 4 Dure LSIII, Galau GA, Greenway SC (]980) Isr J Bot 29:293-306 Higgins TJV, Jacobsen JV, Zwar JA (]982) Plant Molec B i o l 1: 191-215 L i n s m a i e r E, Skoog F (1965) P h y s i o l P l a n t 18: 100-127 M i l b o r r o w BV (1974) Annu Ray P l a n t P h y s i o l 2 5 : 2 5 9 - 3 0 7 M i r e l m a n D, G a l u n E, S h a r o n N, L o t a n R (1975) N a t u r e (London) 2 5 6 : 4 1 4 - 4 1 6 M i s h k i n d M, R a i k h e l NV, P a l e v i t z BA, K e e g s t r a K (1982) J Ceil Biol 92:753-764 Mozer TJ (1980) C e l l 2 0 : 4 7 9 - 4 8 5 Penmans WJ, Caers LI, Earlier AR (1979) Plants 144: 485-490 Peumans WJ, Stinissen HM, Carlier AR (1982) Plants 156:41-44 Rupley JA (1964) Biochim Biophys Acta 8 3 : 2 4 5 - 2 5 5 Stinissen HM, Peumans WJ (1983) Plant Cell Reports 2: 277-280 Stinissen HM, Peumans WJ, Carlier AR (1983) Plants 159:105-111 S u s s e x IM, D a l e RMK, C r o u c h ML (1980) I n : L e a v e r CJ (ed) G e n o m e o r g a n i z a t i o n a n d expression in plants, P l e n u m P r e s s , New Y o r k , pp 2 8 3 - 2 8 8 Triplett BA, Q u a t r a n o RS (1982) D e v l B i o l 9 1 : 4 9 1 - 4 9 6 T s u d a M (1979) J Biochem (Tokyo) 86: 1451-1461 Walton De (1980) Annu Rev Plant Physiol 3 1 : 4 5 3 - 4 8 9
Plant Cell Reports © Springer-Verlag 1984
Abscisic acid promotes lectin biosynthesis in developing and germinating rice embryos Hetta M. Stinissen 1, Willy J. Peumans 1, and E. De Langhe 2 1 Laboratorium voor Plantenbiochemie, KULeuven, Kardinaal Merderlaan, 92, B-3030 Leuven, Belgium 2 Laboratorium voor Tropische Plantenteelt, KULeuven, Kardinaal Mercierlaan, 92, B-3030 Leuven, Belgium Received March 4, 1984 - Communicated by D. yon Wettstein
ABSTRACT Immature rice (Oryza sativa, L) embryos isolated about 12 days post anthesis are fully able to develop into young seedlings when cultured in vitro. Concomitantly, they rapidly loose their lectin synthesis activity. Abscisic acid added to the nutrient medium prevents precocious germination of the immature embryos and simultaneously strongly promotes lectin biosynthesis activity. Similarly, abscisic acid keeps mature embryos grown in a nutrient medium in a dormant state and maintains their lectin synthesis activity, whereas control embryos rapidly germinate but also quickly loose their lectin synthesis activity. It appears, therefore, that rice lectin is typically synthesized in embryos which are kept in a dormant state. Abbreviations: ABA = abscisic acid; GA 3 = gibberellic a~id; WG-A ~ Wheat germ agglutinin; DPA = days post anthesis. INTRODUCTION Gramineous lectins are an extented class of closely related proteins, which have been conserved fairly well during the evolution and divergency of this plant family (Stinissen et al. 1983). All grass lectins which hitherto have been purified and characterized into some detail belong to one of the three subgroups that, according to the taxonomic group in which they occur, are designated as cereal, rice or Brachypodium lectins. Although several Gramineae lectins have been studied into detail with respect to their biochemical, physico-chemical and cellular-biological properties, their physiological role in the plant is still a matter of controversy. As early as ]975, it was proposed that wheat germ agglutinin (WGA) (which is the best known and most intensiVely studied gramineous lectin) acts as an antifungal agent against chitin-containing phytopathogens during seed imbibition and early germination (Mirelman et al. 1975). Although in a recent report on the localization of lectin in embryos and adventitious roots of wheat, the association of lectins with those parts of the plant that come in close contact with the soil have been interpreted in favor af a possible defensive role of WGA (Mishkind et al. ]982), evidence is accumulating that gramineous lectins might fulfill a much more specific physiological role. Indeed, the timing and localization of lectin biosynthesis in developing wheat and rice embryos indicate
that these lectins are maturation-specific proteins which are exclusively synthesized in the primary axes (Peumans et al. 1982; Stinissen, Peumans 1983). Moreover, the promoting effect of abscisic acid (ABA) on lectin synthesis in cultured, immature wheat embryos (Triplett, Quatrano 1982), indicates that WGA is not only a maturation-specific protein but could be a dormancy-specific protein. This evidence, taken together with the evolutionary conservatism of gramineous lectins (Stinissen et al. ]983), possibly argues for a specific endogenous physiological role of these proteins. The present paper deals with the effect of ABA on lectin biosynthesis in developing and germinating rice embryos. In both systems this plant growth substance prevents germination and markedly stimulates lectin synthesis. MATERIAL AND METHODS Plant Material Rice (Oryza sativa L. cv. Koshihikari) plants were grown in a greenhouse at 28 °C during the day (]3 h) and 20 °C during the night. Under these conditions the time between anthesis and complete seed maturation took about 40 days. For the experiments with developing embryos, grains were collected between 8 and 12 days post anthesis (DPA) since at this developmental stage lectin synthesis is maximal (Stinissen, Peumans, 1983). Experiments with germinating embryos were carried out with grains harvested around 25 DPA (about 2 weeks before grain maturity) and artificially dried in the laboratory. Such a precocious harvest was recommendable since embryo s from fully mature rice grains synthesize little lectin upon germination (Stinissen, Peumans, ]983). Embryo Culture Grains were collected, surface-sterilized with 0.5 % NaOCI and the embryos isolated manually with a scalpel blade under a laminar air flow. Embryos were plated in sterile Petri dishes on Whatman no.| filter paper soaked with sterile nutrient medium consisting of Linsmaier and Skoog salts (1965) pH 5.6, supplemented with 2 % (w/v) sucrose, 150 mM L-glutamine and 500 Dg/ml ampicillin or 200 Dg/ml earbenicillin. The plant growth substances ABA and gibberellic acid (GA3) were added to the culture medium as indicated in the legends to the figures. Unless stated otherwise, the concentrations of ABA and GA 3 were 25 and 50 ~M respectively. Embryos were incubated at 25 °C during the day and 20 °C during the night under a 12-h photoperiod
56 for the periods indicated in the figures. Radioactive labelling Lots of 25 embryos_were incubated for 12 h at 30 °C with 180 kBq of [3~S] cysteine (SA: 42.9 TBq/ mmol). Labelled cysteine was used in all labelling experiments because of the unusually high cysteine content (about 24 %) of rice leotin (Tsuda 1979). Extraction and determination of total protein synthesis After incubation, embryos were extensively washed with ice-cold distilled water on a Buchner-funnel, blotted dry and weighed. Then, they were homogenized in a mortar and extracted with I ml of 20 ng{ Tris-HCl, pH 7.8. Homogenates were cleared by centrifugation (4 min; 10,000 g) in an Eppendorf microcentrifuge and the supernatant taken off. Total protein s y n t h e s ~ was estimated by measuring the incorporation of [ ~ S ] cysteine into trichloroacetic acid-insoluble material in three aliquots of 10 ~I of the supernatant. Lectin isolation To the 10,000 g supernatant (in Tris), I ml of Naacetate(50mM, pH3.8) was added, heated for 2 min at 60 °C and quenched cold in an ice-bath for I0 min. Any coagulated material was removed by centrifugation (5 min; 3,000 g) and the resulting supernatant applied onto a small column (0.2 ml bed volume) of immobilized N-acetylglucosamine (Selectin I from Pierce Chemical Company, Rockford, Ill. USA) for affinity-purification of the lectin. Unbound protein was eluted with Nascetate buffer (3 ml) and phosphate-buffered saline (2 ml). The bound lectin was desorbed with I ml of a solution of ]00 Ng/ml chitin hydrolysate (prepared according to Rupley 1964). Finally, the radioactive cysteine recovered in this affinity-purified lectin fraction was determined in a liquid scintillator. The purity of the leotin fractions obtained in this way was controlled by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. They were essentially devoid of contaminating proteins (results not shown here). RESULTS Effect of ABA and GA~ on precocious germination and leetin b-~os--~t~esi~ ~ F i n vitro' ~ultu--~ded~ r ~ embryos Immature embryos excised from 8 to 12 DPA rice grains were cultured in vitro in a nutrient medium either or not supplemented with ABA (50 DM), GA3 (25 uM) and a combination of ABA and GA 3 (50 and 25 uM respectively). Under the conditions described in the methods section, control embryos (which did not receive any plant growth substance), were fully able to germinate and eventually developed into seedlings (Fig.la and 2a). GA_-treated embryos behaved almost identically to control embryos except that they germinated somewhat faster. On the contrary, embryos grown in the presence of ABA or ABA + GA 3 did not germinate at all, but nevertheless showed some limited development in vitro as they increased continuously in fresh weight and size. It is worthwhile to mention here that the inhibition of germination by ABA is concentration dependent. At low concentrations, precocious germination was only retarded but not inhibited as is illustrated by Fig. lc, which shows that germination gradually decreases with increasing ABA concentration. At the concentration used in our experiments (25 ~M) no germination was observed. In spite of the obvious differences in germination behaviour between control and GA3-treated embryos on
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Fig.1. a. Effect of plant growth substances on precoclous germination of 12 DPA rice embryos cultured for 3 days in nutrient medium (I) and nutrient medium supplemented with 25 ~M ABA (2), 25 ~M ABA + 50 >M GA3(3) or 50 >M GA 3 (4). b. Effect of plant growth substances on normal germination of dry 25 DPA embryos grown in nutrient medium (I) and nutrient medium supplemented with 25 >M ABA (2), 25 NM ABA + 50 >M GA 3 (3) or 50 DM GA 3 (4). c. Effect of ABA concentration on precocious germination of 12 DPA embryos cultured in vitro for 3 days. ABA concentrations were 0, 0.1, 1 and I0 >g/ml in (I), (2), (3) and (4) respectively. the one hand, and ABA and ABA + GA_-treated embryos on J . . . the other hand, in vivo protein synthesls actlvlty was rather similar over the whole incubation period (up to 48 h). Total protein synthesis, which was measured as amino acid incorporation over a 12 h-period, was high in all embryos during the first 12 h of incubation. Thereafter, it decreased considerably but remained almost at the same level from 12 to 48 h (Fig.2b). The dramatic decrease of protein synthesis activity of the ~n vitro cultured embryos during incubation has to be ascribed most probably to the stress induced by their removal from the intact seeds. It is known, indeed, that, at least in in vitro cultured rye embryos, the polysome content decreases drastically during the first hours of incubation, which of course results in a rapidly declining protein synthesis activity (Peumans et al. ]979). In contrast to the fairly similar total protein synthesis activity in all embryos, the incorporation of labelled cysteine into lectin varied greatly according to the presence or absence of ABA and GA~ in the J nutrient medium (Fig.2c). In control embryos, lectin synthesis activity rapidly deareased as a function of incubation time and eventually fell at the limit of detection after 36 h. Compared to control embryos, GA3-treated samples synthesized much less lectin even
57 a
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Fig.2. Effect of ABA (25 DM), GA 3 ~ ) and a combination of ABA and GA 3 (25 and 50 ~M respectively) on: a. The development of ]2 DPA rice embryos in culture medium, b. Protein synthesis of developing rice embryos. Protein synthesis was determined over the 12 final hours of incubation in nutrient medium (after preincubation periods of 0, ]2, 24 and 36 h respective!x) and is expressed as cpm x i0 ~/embryo. c. Lecti~ synthesis (expressed as cpm x 10-~/embryo)o d. The relative rate of lectin synthesis (%).• • :ABA; O---•: G A _ ; O - - - O ABA + GA3; A A Control.
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those who received ABA in combination with GA B (Fig.2c). When in stead of total lectin synthesis the relative rate of lectin synthesis (which is defined as the % of total incorporated [ S]cysteine recovered in the lectin fractions) is considered, similar conclusions can be drawn concerning the effect of ABA and GA 3 but, as is shown in Fig.2d, differences between the treatments are more pronounced. The same results are presented on a logarithmic scale. As is shown in Fig.4a, the relative rate of lectin synthesis decreases exponentially as a function of germination time in GA3-treated embryos. In con-
during the first 12 h of incubation, and, in addition, stopped lectin synthesis about 12 h earlier. ABA and ABA + GA_-treated embryos, on the contrary, exhibited nearly t~e same lectin synthesis activity as control embryos during the first 12 h. Thereafter, however, they synthesized much more lectin than the control embryos and continued synthesizing lectin for at least 48 h. Although both ABA and ABA + GA%-treated samples behaved almost identically with respect to germination behaviour and total protein synthesis activity, embryos grown in the presence of ABA alone exhibited consistently higher levels of lectin synthesis than
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Fig.3. Effect of ABA (25 uM), GA~ ~ M ) and a combination of ABA j and GA 3 (25 and 50 ~M respectively) on: a. Germination of mature rice embryos in culture medium, b. Protein synthesis of germinating rice embryos. Protein synthesis was determined over the 12 final hours of incubation in nutrient medium (after preincubation periods of O, ]2, 24 and 36 h respectively) and is expressed as cpm x 10-~/embryo. c. Lecti~ synthesis (expressed as cpm x 10-~/embryo). d. The relative rate of leetin synthesis (%). A--A: ABA;O---O: GA3~O----O: ABA + G A 3 ; A A: Controi.
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Fig. 4. Effect of ABA, GA 3 and a c~mbination of both on the relative rate of lectin synthesis. Embryos were incubated in nutrient medium and labelled during the 12 final hours• Synthesis of total protein and lectin was determined and the relative rate of lectin synthesis calculated. Results are expressed on a logarithmic scale• a. developing embryos, b. mature embryos• • •: 25 M A B A ; O - - - O : 5 0 M GA3; O---O: 25 M ABA + 50 M GA3; A A: Control•
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Incubation time (h) trol embryos the exponential decrease starts with a certain lag, and in the ABA and ABA + GA~-treatments J a lag of about 24 h was observed. It has been shown previously that such an exponential decrease of the relative rate of lectin synthesis in germinating wheat and rye embryos is due to lectin mRNA depletion as a function of germination time. Dry wheat and rye embryos contain a certain amount of stored lectin mRNAs which aretranslated during early germination and degrade with a half-life of about 3 h (Peumans et al• 1982)• Since no new lectin mRNAs are transcribed during germination, an exponential decrease of the rate of lectin synthesis is observed. Possibly, the exponential decrease of the relative rate of lectin synthesis in in vitro cultured immature rice embryos also can be ascribed to lect~n mRNA depletion during incubation• If so, the maintenance of higher levels of lectin synthesis in response to ABA could be due, at least partly, to an increase of lectin mRNA life-time. Effect of ABA and GA 3 on germination thesis in g-~mi--n~ting rice embryos
and lectin syn-
Although rice lectin can be considered as a maturation-specific protein which is almost exclusively synthesized in developing primary axes (Stinissen, Peumans ]983), some residual lectin synthesis can be observed in germinating embryos (isolated from mature seeds)• To find out whether ABA and GA3.influenced lectin synthesis in germinating embryos in a similar way as in in vitro cultured developing embryos, we looked at the effect of ABA, GA^ and ABA + GA 3 on ger5 . mination, total synthesis and lectln synthesis during early germination of dry rice embryos. These embryos were isolated from 25 DPA grains (which were dried in the laboratory) and grown in a nutrient medium exactly as described for the in vitro culture of developing rice embryos• Under these conditions, control embryos readily germinated and developed into seedlings within a few days. GA3-treated embryos behaved similarly except that they developed somewhat faster than the controls. ABA and ABA + GA3-treated embryos, however, did not germinate• The observed increase of their fresh weight during the initial 12 h of incubation has to be ascribed to aspecific uptake of imbibition water (Fig. Ib and 3a). In contrast to immature embryos, which rapidly loose more than half of their protein synthesis activ i t y d u r i n g i n ~itro culture, germinating embryos, grown under the same conditions, increase their protein synthesis activity as a function of incubation time. A steady increase of protein synthesis activity was not only observed in control and GA -treated em• 3 bryos but also in ABA and ABA + GA3-treated embryos. However, it is evident from Fig.3b that the latter two
treatments resulted in a decreased overall protein synthesis activity. When looking at lectin synthesis, it became evident that in spite of their steadily increasing overall protein synthesis, control and GA3-treated embryos rapidly lost their lectin synthesis activity as a function of germination time (Fig.3c). As already observed for precociously germinating embryos, lectin synthesis activity decreased somewhat faster in GA 3 treated than in control embryos. ABA and ABA + GA 3treated embryos, on the contrary, continued synthesizing lectin during germination at a relatively high rate. In ABA-treated embryos there was even an increase in total lectin synthesis, whereas it remained more or less constant in ABA + GA^-treated embryos. • J • When in stead of total lectln synthesls the relatlve rate of lectin synthesis is plotted against germination time, differences become more pronounced, especially between ABA and ABA + GA3-treated embryos. Indeed, ABA embryos clearly synthesize more lectin than embryos grown in ABA + GA 3 (Fig.3d). The same results pregented on a logarithmic scale (Fig•4b), indicate that in all treatments the relative rate of lectin synthesis decreases exponentially as a function of germination time. As has been discussed above, this exponential decrease can be explained by a lectin mRNA depletion mechanism during germination. The obvious differences between the slope of the graphs corresponding to the four treatments might correspond to differences in lectin mRNA half-life If so, GA~j • • and ABA decrease and increase respectlvely the halflife of the lectin mRNAs.
DISCUSSION The results repo~ted in this paper present evidence that ABA prevents germination of in vitro grown immature and mature rice embryos, and concomitantly strikingly promotes lectin biosynthesis. It is evident therefore that rice lectin, which has been shown previously to be a maturation-specific protein, whose synthesis is almost exclusively confined to a welldefined stage of the development of the embryonic axes (Stinissen, Peumans 1983), is also a dormancy-specific protein. Indeed, rice embryos, irrespective whether they are precociously or normally grown in vitro are fully able to develop into seedlings when grown in a culture medium and thereby rapidly loose their lectin synthesis activity. However, when they are maintained in a dormant state by exogenously added ABA, they continue to synthesize lectin. Similar results have been obtained with precociously germinating wheat embryos by Triplett and Quatrano (]982) who found a marked increase in the
59 biosynthesis of chitin-binding proteins (including WGA) in embryos grown in the presence of ABA. As is demonstrated by Fig.2c and d, and Fig.3c and d, however, the promoting effect of ABA on rice lectin synthesis is not limited to immature embryos hut is also observed for full-grown, normally germinating embryos. ABA is known to inhibit germination of both mature and immature embryos of numerous plant species (Milborrow 1974; Walton 1980). Moreover, it is also well established that ABA promotes specifically the synthesis of typical maturation-specific proteins such as e.g. storage proteins in Brassica, Phaseolus and Gossypi~ ( C r o u c h , S u s s e x 1981; S u s s e x e t a l . 1980; Dure e t a l . 1 9 8 0 ) , and i n some c a s e s e v e n i n d u c e s t h e s y n t h e s i s o f proteins typical for late embryogenesis (e.g. in cotton seeds, Dure et al. 1980). GA 3 could not counteract the effects of ABA, at least not at the concentrations used in our experiments. Indeed, GA 3 + ABA-treated embryos did not germinate and contlnued to synthesize lectin (although less than ABA-treated embryos). Thus GA 3 cannot restore ABA-induced inhibition of germinatlon and abolishes only partially the ABA-induced lectin synthesis. This is clearly in contrast with e.g, the effect of GA on a-amylase-induction in barley aleurone layers, 3 whlch can readlly be reversed by ABA (Mozer, 1980, Higgins et al. 1982). Finally, the finding that both WGA and rice lectin are maturation-specific proteins whose synthesis is somehow related to the maintenance of a naturally or ABA-induced resting state, have to be considered in view of the physiological role of these and other related lectins. Indeed, these observations, taken together with the evolutionary conservatism of Gramineae lectins and the fact that they represent only a minor fraction of the total protein (in contrast to other ABA-promoted proteins such as storage proteins), may argue for a specific and endogenous physiological role in the developing primary axis. Conclusive evidence for such a role awaits experimental proof.
ACKNOWLEDGEMENTS This work is supportedinpart by grants of the National Fund for Scientific Research (Belgium). W.P. is Research Associate of this fund. H.S. acknowledges the receipt of a fellowship of the Belgian 'Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw'.
REFERENCES Crouch ML, Sussex IM (198]) Plants ] 5 3 : 6 4 - 7 4 Dure LSIII, Galau GA, Greenway SC (]980) Isr J Bot 29:293-306 Higgins TJV, Jacobsen JV, Zwar JA (]982) Plant Molec B i o l 1: 191-215 L i n s m a i e r E, Skoog F (1965) P h y s i o l P l a n t 18: 100-127 M i l b o r r o w BV (1974) Annu Ray P l a n t P h y s i o l 2 5 : 2 5 9 - 3 0 7 M i r e l m a n D, G a l u n E, S h a r o n N, L o t a n R (1975) N a t u r e (London) 2 5 6 : 4 1 4 - 4 1 6 M i s h k i n d M, R a i k h e l NV, P a l e v i t z BA, K e e g s t r a K (1982) J Ceil Biol 92:753-764 Mozer TJ (1980) C e l l 2 0 : 4 7 9 - 4 8 5 Penmans WJ, Caers LI, Earlier AR (1979) Plants 144: 485-490 Peumans WJ, Stinissen HM, Carlier AR (1982) Plants 156:41-44 Rupley JA (1964) Biochim Biophys Acta 8 3 : 2 4 5 - 2 5 5 Stinissen HM, Peumans WJ (1983) Plant Cell Reports 2: 277-280 Stinissen HM, Peumans WJ, Carlier AR (1983) Plants 159:105-111 S u s s e x IM, D a l e RMK, C r o u c h ML (1980) I n : L e a v e r CJ (ed) G e n o m e o r g a n i z a t i o n a n d expression in plants, P l e n u m P r e s s , New Y o r k , pp 2 8 3 - 2 8 8 Triplett BA, Q u a t r a n o RS (1982) D e v l B i o l 9 1 : 4 9 1 - 4 9 6 T s u d a M (1979) J Biochem (Tokyo) 86: 1451-1461 Walton De (1980) Annu Rev Plant Physiol 3 1 : 4 5 3 - 4 8 9
