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Journal of Experimental Botany, Vol. 47, No. 299, pp. 755-762, June 1996 Journal of Experimental Botany The rea (red embryonic axis) phenotype describes a new mutation affecting the response of maize embryos to abscisic acid and osmotic stress M. Sturaro1, P. Vernieri2, P. Castiglioni1, G. Binelli1 and G. Gavazzi1'3'4 1 2 3 Dipartimento di Genetica e di Bioiogia dei Microrganismi, Universita di Milano, Milano, Italia Dipartimento di Bioiogia delle Piante Coltivate, Universita di Pisa, Pisa, Italia Dipartimento di Fisiologia delle Piante Coltivate e Chimica Agraria, Universita di Milano, Milano, Italia Received 30 May 1995; Accepted 12 February 1996 Abstract During a screen for mutants with defective germination, a new phenotype was observed consisting of red pigmentation of the embryonic axis in the dormant seed. Segregation ratios, as determined in F2 and back-crossed progeny, indicate that the phenotype is due to a recessive single gene mutation that has been symbolized rea to denote red embryonic axis. A closer inspection of the rea phenotype revealed that the mutant is occasionally viviparous, indicating a defect in abscisic acid (ABA) metabolism. The mutation probably affects ABA sensitivity since no difference in ABA content was detected in mutant versus normal tissues. Moreover, when immature mutant and wild-type embryos were incubated on media containing 10 //M ABA, only the mutants germinated. ABA-regulated gene expression in rea embryos differed from that of embryos of the viviparous mutant vp1 which does not respond to the inhibitory action of ABA at the level of immature embryo germination. These results, therefore, indicate that the two genes exert a different role in the control of embryogenesis. Key words: Zea mays L, embryo dormancy, ABA. Introduction Embryogenesis, encompassing the period between the first zygotic division and seed maturity, is an important period of plant development because the principal events involved in the achievement of the basic structure of the plant occur during this stage. In maize, where this process " To whom correspondence should be addressed. Fax: + 39 2 70 630 856. Oxford University Press 1996 lasts about 50 d, two phases can be distinguished: an early phase encompassing the first 16 d after pollination (DAP) where the basic structure of the plant is defined, and the ensuing maturation phase, from 16 d to seed maturity, characterized by an increase in size, synthesis of seed storage products and the acquisition of a dormant state preventing precocious germination (Sheridan and Clark, 1987). Abscisic acid (ABA) plays an important role during the maturation stage of embryogenesis. For example, it prevents precocious germination and is involved in the regulation of several genes (Davies and Jones, 1994). In maize, mutations affecting ABA metabolism are recognized because they fail to induce embryo dormancy, thus resulting in precocious germination, an event referred to as vivipary (Robertson, 1955). These mutants can be grouped into two classes: class I represented by vpl, which have normal levels of endogenous ABA, but are insensitive to the germination suppressive effect of exogenous ABA, and class II which have a reduced level of the growth substance, both in the embryo and in the plant, and are unable to accumulate ABA in response to water deficit (Robichaud and Sussex, 1986; Neill et al, 1986). Similarly, in Arabidopsis, mutants affecting ABA metabolism have been classified as ABA-deficient {aba) or ABAinsensitive (abi) (Koorneef et al, 1984). In both species, the two groups of mutants, respectively, represent defects in ABA synthesis or responsiveness. vpl has been studied extensively and its product has been identified as a transcriptional activator that mediates the ABA response during seed development (McCarty et al, 1991). In Arabidopsis, the ABB gene, whose 756 Sturaro et al. mutation causes germination to be insensitive to abscisic acid, has been isolated and shown to be structurally related to the maize vpl gene (Giraudat et al., 1992). Altered expression of several ABA responsive genes is observed in different vp mutants: in cultured embryos of class II mutants there is usually a reduced basal level of the transcript, but gene expression can be induced by treating embryos with ABA (Butler and Cuming, 1993; Paiva and Kriz, 1994; Pla et al., 1991; Thomann et al., 1992; Williamson and Scandalios, 1992). In the vp] mutant, on the other hand, some ABA-inducible genes can not be detected, even following ABA treatment (Butler and Cuming, 1993; Paiva and Kriz, 1994; Thomann et al., 1992) while others show vpl independent expression profiles (Williamson and Scandalios, 1992). Interestingly, some of the genes requiring a functional Vpl product for ABA induction are expressed in vpl mutants in response to an osmotic stress induced by the osmolyte mannitol (Butler and Cuming, 1993; Thomann et al, 1992; White and Rivin, 1995) indicating that different parallel pathways are operating in the perception of ABA and osmotic stress (Bray, 1993). In a chemically mutagenized population, a new maize mutant has been detected, characterized by a strong pigmentation of the embryonic axis in the dormant seed and by an incipient vivipary which occasionally leads to precocious germination and desiccation of the seedling. The mutant, named rea (red embryonic axis), is unaffected in the level of endogenous ABA, but exhibits reduced sensitivity to its presence in the growth medium. These features make rea a vpl-like mutant, although a linkage analysis using RFLP markers appears to exclude allelism between rea and vpl. At the molecular level, the expression of two ABA-responsive genes, as determined in homozygous rea mutant embryos versus their wild-type counterparts, is less affected by ABA and is not induced by osmotic stress. tion of 0.3 M before autoclaving while ABA (Sigma, mixed enantiomers) was added at a concentration of 10 /xM or 100 /uM after autoclaving. Cultures were incubated in a growth chamber at 26 °C in the dark for 8 d. Germination was determined daily on the basis of shoot or root emergence. Standard errors of mean values, when not reported, were less than 5%. RNA gel blot analysis RNA was extracted from 1 -3 g of frozen tissue using the method described by Van Tunen et al. (1988). For Northern blot analysis, 10 //.g of total RNA for each sample was electrophoresed through a 1.5% agarose gel containing 20 mM 3-[iV-morpholino] propane sulphonic acid (MOPS), 5 mM sodium acetate, 0.5 mM EDTA, 18% formaldehyde and blotted on to nylon filters (Pall Europe, Portsmouth, England). Glbl and Rabl 7-specific transcripts were detected using 32P-labelled inserts from the pcGlblS and pMA12 cDNAs, respectively. The cDNAs were kindly provided by Dr M Pages, Consell Superior dTnvestigacions Cientifiques, Barcelona, Spain (Rabl 7) and by Dr AL Kriz, Dekalb Plant Genetics, Discovery Research, Mistic, CT. 06355, USA (Glbl). Hybridization was carried out at 65 °C. Filters were washed twice in 2 x SSC, 1% SDS at 65 °C. ABA determination The ABA content of embryos and leaves was determined by radioimmunoassay using DBPA1 monoclonal antibody as previously described (Bochicchio et al., 1994). Each analysis was performed in triplicate on pools of six embryos. Since there is no difference in mutant versus wild-type embryo size, the results are expressed per unit of fresh weight. ABA accumulation in stressed plants was determined using the detached leaf test (Quarrie et al., 1988). For this analysis, 13 mutant and 26 wildtype kernels from a segregating selfed ear were germinated on filter paper in well-watered conditions. When the developing seedlings reached the third leaf stage, samples of similar size were collected from the second leaf and dissected into two parts, one was immediately frozen to assay the pre-stress ABA level and one left on dryfilterpaper for 2 h before being frozen, to evaluate ABA accumulation under water-stress conditions. To validate the RIA results of this experiment, non-competitive interference was determined by internal standardization as described in Bochicchio et al. (1994). Parallelism between the standard curve and those obtained after adding 10 or 20 ^g of crude extract to each point confirms the lack of competitive interference. Material and methods RFLP analysis Genetic stocks Samples of leaves of each of 93 individuals of a backcrossed (BC) population and from the two parental lines were collected and their DNA extracted as described in Dellaporta et al. (1985). Digestion with restriction endonucleases (EcoRI, BamHI and Hindlll) was carried out on 10 /xg of genomic DNA for each sample. The resulting fragments were separated on 1.5% agarose gels and blotted on nylon membranes. Filters were hybridized and washed as described for RNA detection. Clones for RFLP analysis were obtained from Dr D Grand (Pioneer Hi-Bred Int.), Dr B Burr (Brookhaven National Laboratory) and Dr D Hoisington (University of Missoury, Columbia). The fragments recovered after PstI digestion were labelled by random primed extension (Prime-a-Gene kit, Promega). For RFLP analysis, 12 markers located on chromosome 3 (2 on 3S and 10 on 3L arms) were used to detect polymorphism between the two parental lines. The six markers which showed polymorphism (php20558, umc26, bnl5.37, bnl6.16, umc!7, php 10080) were subsequently used to test The rea phenotype first appeared in the progeny of a sample of seeds of the W22 inbred line mutagenized with the alkylating agent ethyl methane sulphonate (Gavazzi et al., 1993). The mutant stock was outcrossed to the inbred line W64A and then selfed for two cycles to obtain a homozygous stock. The viviparous vp] mutant introduced in the W22 background was obtained by Dr DS Robertson, Iowa State University, Ames, IA 50011. Embryo culture Ears were harvested 28 d after pollination (DAP) and seeds were surface-sterilized with 5% sodium hypochlorite for 15 min and then rinsed in sterile distilled water. Embryos were removed aseptically and transferred to Murashige and Skoog (MS) medium, pH 5.6, containing 3% sucrose and solidified with 0.8% agar. When required, mannitol was added at a concentra- rea phenotype of maize embryos association with the rea locus in the BC population. Linkage analysis was performed by multipoint analysis of data using the MAPMAKER/EXP program (Lander et al, 1987). Results Mutant description and genetic analysis The red pigmentation of the embryonic axis conditioned by the rea mutant (Plate 1) is due to accumulation of 757 anthocyanin as deduced from the absorbance spectrum of the pigments extracted from the axis of mature seeds (data not shown). This pigmentation is visible at approximately 25 DAP and by the time the seed is mature it is distributed evenly in the outermost layer of the embryonic axis, but is not detectable in other parts of the embryo, including the scutellum. This pigmentation was a convenient marker for mutant embryos when vivipary was not apparent. In all crosses of the mutant with different inbred lines, the trait disappears, but reappears in the F 2 or in the progeny of back crosses to the parental mutant line. Thus, embryonic pigmentation behaves as a recessive trait. The mutant segregation, while approximating the expected one-half in backcrossed families, is less than the expected one-quarter in F 2 populations (Table 1). The extent is genotype-dependent. However, individual progenies of selfed Fj plants differ in their mutant segregation values (data not shown), an observation that suggests the involvement of an environmental effect on rea expression, even though the contribution of gametophytic selection or the presence of modifiers can not be discounted. The rea phenotype is not associated with the R locus Plate 1. Enlargement of a mature rea embryo showing the red embryonic axis. The distribution of anthocyanin in seed and plant tissues of maize is controlled by a regulatory gene denoted R, which is a transcriptional activator. R consists of a small gene family whose members control pigment distribution in specific tissues. The stocks used in the crosses described below carry all the structural genes necessary for anthocyanin biosynthesis while differing in their R constitution. R-r conditions red pigmentation of the seed (aleurone) and seedling (primary root and coleoptile) tissues while r-r leads to colourless seed but red seedling tissues, r-g, the null allele, accounts for colourless seed and seedling tissues. To test whether the red embryonic axis trait is an R effect, a homozygous rea and r-r line carrying all the structural genes for pigment biosynthesis (colourless seed, red seedling) was crossed with a second line with identical constitution in the structural genes, but homozygous for R-r and Rea (coloured seed and plant). The F 2 progeny was then analysed to test linkage of R with rea. The results indicate that the two genes are independent, since Table 1. Segregation ratios (%) of the rea mutation as determined in the F2, following crosses of rea to different inbred lines, and in backcrosses (BC) of Rea/rea females to homozygous rea male parents Crosses BC Genetic background No. seeds scored Mutant segregation (%) Mul ±SE W22 W64A B73 A188 K6 W22 B73 ACR line 2554 1226 918 803 1297 287 878 537 19.3 8.9 21.0 15.1 8.1 47.0 65.0 49.3 3.2 2.6 3.9 1.9 2.6 1.0 7.0 3.1 758 Sturaro et al. the pigmented embryonic axis can also be recovered in the R-r background, thus excluding the hypothesis that this pigmentation is an effect of the r allele present in the rea line. The progeny of selfed heterozygous r-g Rea/r-r rea plants (r-g: colourless seed and seedling) was also analysed without recovering any rea non-red seedlings (corresponding genotype: r-g/r-g rea/rea expected, in the F 2 population with a frequency of 1/16th). This indicates that a functional r allele must be present in the plant tissues in order for the rea mutation to be manifested in the dormant embryo. The rea mutant is not affected in ABA synthesis Endogenous ABA levels in developing maize embryos increase from 14 DAP and reach a peak at about 35 DAP (Bochicchio et al., 1994). However, the ABA content of class II vp mutants at 35 DAP is less than 25% of the wild type (Paiva and Kriz, 1994). To determine if the viviparous phenotype of the rea mutant was due to a defect in ABA biosynthesis, the level of this compound was assayed in immature (35 and 40 DAP) embryos segregating from a +/rea selfed ear. The evaluation of the endogenous ABA level in immature embryos does not reveal any significant difference in ABA content of mutant versus normal embryos (Table 2). Similarly, the ABA content of mutant seedlings grown under normal conditions or after 2 h of water stress is not different from wild-type siblings (Fig. 1). Taken together, these results seem to rule out the involvement of the rea gene in ABA synthesis. Table 2. ABA content of immature rea and wild-type F2 embryos Mutant rea embryos are insensitive to exogenous ABA and mannitol Homozygous vp] embryos are much less inhibited in germination than their wild-type counterparts when grown in the presence of ABA (10 /xM) or mannitol (0.3 M) (Robichaud and Sussex, 1986; Neill et al., 1987). To assay whether the rea mutation confers a similar reduction in ABA and osmotic stress sensitivity, immature embryos were exposed to these conditions and their effect was quantified on the basis of germination efficiencies (Fig. 2), growth in culture (Plate 2) and time-course of germination (Fig. 3). Homozygous vpl embryos of identical age were included as a positive control to demonstrate that growth of ABA-insensitive seedlings was not disrupted (Fig. 4), while immature wild-type embryos were used as a negative control to demonstrate inhibition of germination by ABA and mannitol. The results reported | 0Wt • rea # 100 .2 8 » 60 ° I « 20 0 I MS ABA ABA 100(]M Mannitol 0.3M Growth media Fig. 2. Effect of the addition of ABA (10 /xM and 100 /^M) and mannitol (0.3 M) on the germination of immature rea embryos. ABA was determined by radioimmunoassay as described in Quarrie etal. (1988). ABA content (ng g" 1 FW +SE) Embryo age (DAP) 35 40 - 1600 UJ c i", 1200 o i 800 m \ 400 o < a> < rea Wild type 173 + 8 116+15 160 + 1 119 + 7 0 Control [H water stress 1 Ol c 0-> rea rea wt Growth conditions wt Fig. 1. ABA content (ng g ' DW ± S E ) in leaves of F 2 rea and wildtype seedlings grown in well-watered conditions (control) or after 2 h of water stress. Plate 2. Growth of immature rea and normal F 2 embryos on MS basic medium (A) or in the presence of 100 jiM ABA (B), 10 ^M ABA (C), and 0.3 M mannitol (D). rea phenotype of maize embryos to wild-type embryos, even when placed on medium lacking ABA (Plate 2). However, when compared to vpl, the rea embryos were more severely inhibited in their growth when cultured on media supplemented with 10 /nM ABA or 0.3 M mannitol, and their shoot elongation in the presence of 100 ^M ABA was totally prevented. In contrast, in vpl embryos only shoot length was affected (Fig. 4). A MS 100 • 60 20 0 B ABAIOpM rea and vpl are not allelic 100 • —— 60 ma / - 20 t > C | , , , 4wt 1 » Mannitol 0.3 M ^____— rea 100 r 60 •z. 20 00 759 1 __ L— mml 2 3 4 —T 5 wt T 6 7 8 Days of culture Fig. 3. Germination time-course of immature (28 DAP) rea versus normal seedlings on different media1 (A) MS basic medium, (B) basic medium with 10 ^.M ABA and (C) basic medium with 0.3 M mannitol. One possible explanation for the loss of ABA sensitivity in the mutant is allelism between rea and vpl. Vpl besides cotrolling embryo dormancy, regulates anthocyanin synthesis in the aleurone layer of the seed. Therefore, the vpl mutant in the presence of a full set of active genes for anthocyanin production has a non-coloured aleurone (Robertson, 1955). To test the hypothesis of allelism heterozygous +/vpl females were crossed with homozygous rea male parents; the resulting seeds, carrying a full set of genes for anthocyanin production, had dormant non-coloured embryos but a fully coloured aleurone. This result indicates complementation between rea and vpl. The association of the rea phenotype with six RFLP markers of chromosome 3 (see methods) was then checked, all but one of which {phpl0080) were linked to the vpl locus. None of these markers appears to be linked to rea (data not shown). The hypothesis of independence is supported by an LOD score of 2.77 versus the most likely position for association. Gene expression in response to ABA and osmotic stress MS ABAIOuM ABA100uM Mann.0.3M Growth media Fig. 4. Effect of the addition of ABA (10 MM and 100 ^M ) and mannitol (0.3 M) to MS basic medium on rea (A) and vpl (B) shoot elongation. Measurements were taken after 8 d of culture. in Fig. 2 and Plate 2 show that rea mutants were altered in both ABA and stress response in terms of germination and shoot growth. Besides showing a higher germination capacity in the presence of 10 /xM ABA or 0.3 M mannitol, they also exhibited more rapid germination compared The vpl mutation, besides conferring ABA insensitivity, exerts a negative effect on the induction of several ABA responsive genes. A similar, but not identical, effect is observed if the mutant is exposed to high osmotic conditions. Glbl transcription is not activated by ABA or osmotic stress in vpl embryos (Paiva and Kriz, 1994; Rivin and Grudt, 1991), and neither is rabl7 as can be inferred from translational studies (Butler and Cuming, 1993). However, application of mannitol before the onset of vivipary triggers rabl 7 expression in vpl mutants. To assay how the transcription of these two genes is affected in rea mutants, Northern analysis was performed of Glbl and rabl 7 expression in immature embryos cultured for 8 d on media supplemented with 10 [xM and 100 /xM ABA or 0.3 M mannitol. Wild-type embryos from the same segregating ear (F 2 ) or homozygous + / + ears (F 3 ) were placed on the same Petri plates to evaluate the effectiveness of the various treatments in inducing Glbl and rabl 7 gene expression. Homozygous vpl embryos of the same age (28 DAP) were used as a positive control. Both mutant and normal embryos show, at the time of their excision, Glbl and rabl 7 transcripts (data not shown) that are no longer detectable after 8 d 760 Sturaro et al. of culture on the basic MS medium (Plate 3). Addition of ABA to the medium induced Glbl and rabl 7 expression in rea embryos, although to a lesser extent than in wildtype embryos, while mannitol does not have any effect. Thus, while in the vpl mutant the regulation of these genes by ABA and by mannitol is impaired, in rea only the regulation by mannitol is affected. Discussion Genetic dissection by means of single-gene mutants is a powerful tool for the elucidation of different aspects of plant morphogenesis and development. Embryo defective mutants have been isolated in several plant species including Arabidopsis (Meinke, 1991) and maize (Sheridan and Neuffer, 1981). Recent work suggests that a complex network of genetically defined events regulates the onset of embryo dormancy involving on the one hand vegetative growth suppression, and on the other an induction of the synthesis of specific embryo proteins. Abscisic acid has a primary role in the control of embryo dormancy although other ABA-independent pathways might exist. In maize, vivipary is a diagnostic phenotype for the involvement of ABA since all known vp mutants are either ABA-deficient or ABA-insensitive. To establish Mutant MS Mb ABA 1OOuM ABA 1Q M ManrV a 3 M m wt m wt Mm wt I \m wt A Rab17 rea vpl t BGIbi rea vpl Plate 3. Effect of ABA and mannitol on the accumulation of rabl 7 and Glbl transcripts in immature (28 DAP) rea and vpl embryos, determined after 8 d of culture on basic MS medium or with ABA (10 /*M) and mannitol (0.3 M) added. Total RNA was isolated and analysed by RNA blot hybridization with probes complementary to Rabl7 (A) and Glbl (B). RNA from normal sibling embryos was included as a control. The blot was also hybridized with Tub, a 1 kb cDNA clone of maize tubulin (Dolfini el al.. 1993), to verify equal RNA loading (data not shown). whether rea belongs to one of these two classes of mutants, endogenous ABA levels and sensitivity to the hormone was analysed. In normal embryos, the ABA level increases reaching a peak at about 35 DAP (Bochicchio et al, 1994); at the same time, the embryo becomes less sensitive to ABA while progressing towards embryo dormancy. ABA-deficient mutant embryos of class II have ABA endogenous levels that are less than 25% of wild-type levels (Paiva and Kriz, 1994). Seedlings rescued from viviparous kernels contain less ABA than normal siblings and the level does not increase in response to water deficit (Neill et al., 1986). On the contrary, immature rea embryos have a normal amount of endogenous ABA. Similarly, in the seedling, both the basal level and the ABA accumulation in response to water stress are about the same as in non-mutant siblings. These results rule out the involvement of rea in ABA biosynthesis. The mutants responsiveness to ABA during embryogenesis was assayed by placing immature embryos on a culture medium supplemented with ABA. At a concentration of 10 fxM this growth regulator normally prevents germination of wild-type embryos, vpl and some class II vp mutant embryos, however, show a decreased sensitivity to ABA, with vpl being the least sensitive of all (Robichaud et al, 1980). Similarly, it was found that immature rea embryos germinate in the presence of 10 /nM ABA while their non-mutant siblings almost totally fail to germinate. These results, namely reduced ABA sensitivity and precocious germination on basal medium, indicate that the rea mutant does not enter proper developmental arrest during the maturation stage of embryogenesis because of a reduced response to the ABA inhibitory signal. It would be interesting to test whether other rea mutants exhibit reduced ABA sensitivity as a means to establish whether rea mutant represents a leaky or a null mutation. Three new rea alleles have recently been identified that will allow us to address this question. As with exogenous ABA, osmotic stress caused by osmolytes such as mannitol added to the culture medium prevents precocious embryo germination while triggering ABA biosynthesis. The inhibitory effect on germination, however, is not merely a consequence of ABA induction (Neill et al, 1987). Mutant vpl embryos are less affected than wildtype siblings by osmotic stress. Similarly, rea embryos germinate on media supplemented with 0.3 M mannitol, a condition that prevents growth of normal embryos. Both ABA and osmotic stress exert positive regulation on the transcription of a number of genes that seem to be part of a general programme of response to stress and that are normally active during the later stages of embryogenesis (Galau et al, 1987; Morris et al, 1990). The product of one of these, rabl7, accumulates during the maturation phase of embryogenesis and disappears after the onset of germination. ABA treatments and water rea phenotype of maize embryos stress induce its transcription in embryonic and seedling tissues (Goday et al, 1988; Pla et al, 1989). Another ABA inducible gene, GIbl, is only expressed during embryogenesis starting from 20 DAP (Belanger and Kritz, 1989). However, germination in the presence of ABA maintains Glbl expression at high levels (Kriz et al., 1990). In mutant vpl embryos, ABA does not induce the transcription of some of the ABA-inducible genes, including rabll and Glbl (our data and Paiva and Kriz, 1994), although others are induced normally. These results suggest that there is more than one ABA response pathway, and that the Vpl gene product participates in one of these, whilst others are Vpl independent. The response to osmotic stress in terms of ABA-inducible gene expression in homozygous vpl embryos does not always correlate with the ABA response; for some genes, only one of these two effectors is capable of promoting transcription. This suggests the existence of discrete parallel pathways mediating ABA and stress responses (Thomann et al, 1992; Butler and Cuming, 1993). When assayed in the rea mutant, the expression of both rabl7 and Glbl is promoted by ABA, though to a lesser extent than in normal embryos, while mannitol has no such effect. These results, and those related to germination in the presence of applied ABA and mannitol, can be explained by assuming that rea is mutated in a gene involved in the response to both ABA and osmotic stress. What is still unclear is the relationship between the reduced ABA and stress sensitivity of the rea mutant and the induction of anthocyanin biosynthesis in the embryonic axis. Pigmentation could merely be the result of a general precocious activation of some post-germination specific biosynthesis caused by a limited or improper onset of embryo dormancy in the rea mutant or, alternatively, there could be a direct effect of ABA perception on the regulation of anthocyanin biosynthesis. It is noteworthy to observe that Vpl, besides modulating ABA responses in the embryo, exerts a positive regulation upon the Cl gene, which itself controls genes involved in anthocyanin biosynthesis (McCarty et al, 1991). In this context the Rea gene would prevent the pigmentation of the embryonic axis. A specific interrelationship between anthocyanin synthesis and seed maturation processes has also been demonstrated in Arabidopsis. In this species mutants have been identified, like led (Meinke et al, 1994) and/wsJ (Baumlein et al, 1994) that appear to be desiccation-intolerant, viviparous, accumulate anthocyanin, and contain reduced amounts of storage proteins. These mutants together with the pleiotropic viviparous mutants of maize may identify intrinsic factors that couple the maturation pathway to embryo morphogenesis. Summarizing, Vpl can be envisaged as a member of a network of genes involved, through separate pathways, in the control of critical events associated with embryogenesis. According to this model, Vpl mediates the expres- 761 sion of some but not all ABA regulated genes while rea exerts its effect on ABA perception, normally leading to the suppression of precocious germination. Other ABAassociated effects could be mediated by genes still uncharacterized. Another possibility is to assume that Vpl and rea exert independent control on embryogenesis while sharing some functions such as ABA sensitivity or osmotic stress induction of specific genes. Indeed, the recent discovery of Lee genes in Arabidopsis (Meinke et al, 1994) demonstrates that normal perception of ABA by the Abi3 gene is necessary, but not sufficient, for many important events associated with embryogenesis, the two genes operating through different pathways to ensure that the developing seed is prepared for desiccation and dormancy. Acknowledgements This work was supported by a grant to G Gavazzi from Ministero delle Risorse Agricole Alimentari e Forestali. We are grateful to M Pages and AL Kritz for their generous provision of probes and to C Bowler for critical reading of the manuscript. References Baumlein H, Miser a S, Luerben H, Kolle K, Horstmann C, Wobus U, Muller AJ. 1994. The FUS3 gene of Arabidopsis thaliana is a regulator of gene expression during late embryogenesis. The Plant Journal 6, 379-87. Belanger FC, Kritz AL. 1989. Molecular characterization of the major maize embryo globulin encoded by Glbl gene. Plant Physiology 91, 636-43. Bochicchio A, Vernieri P, Puliga S, Balducci F, Vazzana C. 1994. Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid. Physiologia Plantarum 91, 615-22. Bray EA. 1993. Molecular responses to water deficit. Plant Physiology 103, 1035-40. Butler WM, Cuming AC. 1993. Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos. Planta 189, 47-54. Davies WJ, Jones HG. (eds) 1994. Abscisic acid: physiology and biochemistry. Oxford: BIOS Scientific Publishers. Dellaporta SL, Wood J, Hicks JB. 1985. A plant DNA minipreparation: version 2. Plant Molecular Biology Reporter 1, 19-22. Dolfini S, Consonni G, Mereghetti M, Tonelli C. 1993. Antiparallel expression of the sense and antisense transcripts of maize tubulin genes. Molecular and General Genetics 241, 161-9. Galau GA, Bijaisoradat N, Hughes DW. 1987. Accumulation kinetics of cotton Late-Embryogenesis-Abundant mRNA and storage protein mRNA: co-ordinate regulation during embryogenesis and the role of abscisic acid. Development Biology 123, 198-212. Gavazzi G, Dolfini S, Galbiati M. Helentjaris T, Landoni M, Pelucchi N, Todesco G. 1993. Mutants affecting germination and early seedling development in maize. Maydica 38, 265-74. Giraudat J, Hauge BM, Valon C, Smalle J, Parcy F, Goodman 762 Sturaro et al. HM. 1992. Isolation of the Arctbidopsis ABI3 gene by positional cloning. The Plant Cell A, 1251-61. Goday A, Sanchez-Martinez D, Gomez J, Puigdomenech P, Pages M. 1988. Gene expression in developing Zea mays embryos: regulation by abscisic acid of a highly phosphorylated 23 to 25 kDa group of proteins. Plant Physiology 88, 564-9. Koorneef M, Reuling G, Karssen CM. 1984. The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana. Physiologia Plantarum 61, 377-83. Kriz AR, Wallace MS, Paiva R. 1990. Globulin gene expression in embryos of maize viviparous mutants. Plant Physiology 92, 538-42. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newberg L. 1987. MAPMAKER: an interactive computer package for constucting primary linkage maps of experimental and natural populations. Genomics 1, 174-81. McCarty DR, Hartori T, Carson CB, Vasil V, Lazar M, Vasil IK. 1991. The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell 66, 895-905. Meinke DW. 1991. Perspectives on genetic analysis of plant embryogenesis. The Plant Cell 3, 857-66. Meinke DW, Franzmann LH, Nickle TC, Yeung EC. 1994. Leafy cotyledon mutants of Arabidopsis. The Plant Cell 6, 1049-64. Morris PC, Kumar A, Bowles DJ, Cuming AC. 1990. Osmotic stress and ABA regulate expression of the wheat Em genes. European Journal of Biochemistry 190, 625-30. Neill SJ, Horgan R, Parry AD. 1986. The carotenoid and abscisic acid content of viviparous kernels and seedlings of Zea mays L. Planta 169, 87-96. Neill SJ, Horgan R, Rees AF. 1987. Seed development and vivipary in Zea mays L. Planta 171, 358-64. Paiva R, Kriz A. 1994. Effect of abscisic acid on embryo-specific gene expression during normal and precocious germination in normal and viviparous maize {Zea mays) embryos. Planta 192, 332-9. Pla M, Goday A, Vilardell J, Gomez J, Pages M. 1989. Differential regulation of ABA-induced 23-25 kDa proteins in embryo and vegetative tissue of the viviparous mutants of maize. Plant Molecular Biology 13, 385-94. Pla M, Gomez J, Goday A, Pages M. 1991. Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants. Molecular and General Genetics 230, 394-400. Quarrie SA, Whitford PN, Wang TL, Cook SH, Henson IE, Loveys BR. 1988. A monoclonal antibody to (S)-abscisic acid: its characterization and use in a radioimmunoassay for measuring abscisic acid in crude extracts of cereal and lupin leaves. Planta 173, 330-9. Rivin CJ, Grudt T. 1991. Abscisic acid and the developmental regulation of embryo storage proteins in maize. Plant Physiology 95, 358-65. Robertson DS. 1955. The genetics of vivipary in maize. Genetics 40, 745-60. Robichaud CS, Sussex IM. 1986. The response of viviparous-1 and wild type embryos of Zea mays to culture in the presence of abscisic acid. Journal of Plant Physiology 126, 235-42. Robichaud CS, Wong J, Sussex IM. 1980. Control of in vitro growth of viviparous embryo mutants of maize by abscisic acid. Developmental Genetics 1, 325-30. Sheridan WF, Neuffer MG. 1981. Maize mutants altered in embryo development. In: Subtelney S, Abbot U, eds. Levels of genetic control in development. New York: Alan R. Liss, 137-56. Sheridan WF, Clark JK. 1987. Maize embryogeny: a promising experimental system. Trends in Genetics 3, 3-6. Thomann EB, SoUinger J, White C, Rivin CJ. 1992. Accumulation of group 3 late embryogenesis abundant proteins in Zea mays embryos. Plant Physiology 99, 607-14. Van Tunen AJ, Koes RE, Spelt CE, van der Krol AR, Stuitje AR, Mol NM. 1988. Cloning of two chalcone flavanone isomerase genes from Petunia hybrida: co-ordinate, lightregulated and differential expression of flavonoid genes. EMBO Journall, 1257-63. White CN, Rivin CJ. 1995. Sequence and regulation of a late embryogenesis abundant group 3 protein of maize. Plant Physiology 108, 1337-8. Williamson JD, Scandalios J. 1992. Differential response of maize catalases to abscisic acid: Vpl transcriptional activator is not required for abscisic acid-regulated Cat! expression. Proceedings of the Natural Academy of Science USA 89, 8842-6.