Gibberellins in Embryo - Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development 1

Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C,9-GAs, GA1, GA4, GA5, GA6, and one C2o GA, GA44, were identified. From the embryo, five C1gGAs GA,, GA4, GAs, GAs, GAo0 and two C20 GAs, GA1,9 and GA4 were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development. Several contributions (1, 16, 22) concerning the endogenous GAs2 in various plant species have highlighted the fact that the GA status ofa plant is rather complicated. Therefore there is the need for a more complete picture of the GA concentration in the specific plant tissue studied, if the physiology of this hormonal group is to become more clear. The developing seed of Phaseolus coccineus is a good demonstration of how complex the GA status can be: at least 12 GAs have been identified in the various seed components. Our work with this species has recently been dedicated to the identification, quantification, and biosynthesis of GAs in specific parts of the seed in an attempt to elucidate their role in the embryo-suspensor system during seed development. With few exceptions, the first division of the zygote in both monocots and dicots originates two cells. The basal cell (micropylar) generates the suspensor, while the other cell develops into the embryo proper. The suspensor is almost ubiquitous in angiosperms. Its structure and morphology vary from rudimentary (few small and/or giant cells; sometimes only one) to massive (many giant cells; in P. coccineus about 200 cells). According to an old interpretation, the suspensor has the function of pushing the embryo into the endosperm to facilitate its nutrition. On the other hand, a massive suspensor develops in some plants which are characterized by minimal endosperm development (Sherardia, Tropaeolum, Medicago, Phaseolus). These massive suspensors show conspicuous cell growth through chromosome endoreduplication or, much less frequently, multinucleation (10). Earlier, a hypothesis (21) was put forward that the suspensor 'This work was supported by M.P.I. 40%, Italy. 2 Abbreviation: GA(s), gibberellin(s); f.w., fresh weight; MVA, mevalonic acid; EtOAc, ethyl acetate; SiO2, silica-gel; TMSi, trimethylsilyl; TMSi-TMSi, trimethylsilyl ester ether; Me-TMSi, methyl ester trimethylsilyl ether. might take over the function of the endosperm. To support this idea, Corsi (8) showed that the Eruca sativa embryo, excised at the early stage, could grow in vitro if attached to the suspensor. Meanwhile, at the late heart stage, the embryo could be easily grown in vitro even though deprived of the suspensor. One postulated function attributed to the suspensor is the synthesis of hormones and their supply to the growing embryo (15). To test this hypothesis, analysis ofGAlike activities in embryo and suspensors were conducted at two different stages of embryogenesis: heart shaped (stage A) and cotyleonary (stage B) embryo. High levels of GA-like substances were found in both tissues (2), but even by extracting 2000 suspensors only GA, was identified (3). The hypothesis that the suspensor could be a site of GA synthesis was confirmed by work done with a cell-free system derived from it. This cell-free system demonstrated the incorporation of MVA into kaurene and ofkaurene into 7 ,B-hydroxykaurenoic acid, and finally 7 ,B-hydroxykaurenoic into GA,, GA5, and GA8 (5, 6). The question was then posed as to whether suspensor and embryo proper would contain other native GAs, besides GA,, at these two stages of embryogenesis. Initially we analyzed suspensors and embryos at stage B of embryogenesis. These results showed the presence of six GAs in the suspensor and five GAs in the embryo (18, 19). In this paper we report the amount and the type of GAs present in suspensor and embryo at stage A (heart-shaped embryo) of P. coccineus, together with a general discussion about GAs in this system. MATERIALS AND METHODS