Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by -cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones b and d of esterase and zone b of phosphogluconate dehydrogenase coincided with vascular bundle formation.Abbreviations ADH alcohol dehydrogenase - CD -cyclodextrine - CS cell suspension culture - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraaeetie acid - LiBo lithium hydroxide/boric acid - PEG polyethylene glycol - PVP polyvinylpyrrolidone - SEg somatic embryo at the globular stage - SEh heart stage - SEte early torpedo stage - SEtl late torpedo stage - SEce early cotyledonary stage - SEcl late cotyledonary stage - SECD somatic embryo blocked at the torpedo stage with -cyclodextrine - EST esterase - GOT aspartate aminotransferase - IDH isocitrate dehydrogenase - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) - PMS phenazonium methosulfate - PGD phosphogluconate dehydrogenase - PGI glucosephosphate isomerase - PGM phosphoglucomutase - SO dry seed - S1–3 seed after 1–3 days of germination - SP1–2 young and old somatic plantlets - ZE zygotic embryo - ZP4–5 4–5 day-old seedlings     

Characterization of Storage Proteins in Daucus carota L.: Two Novel Proteins Display Zygotic Embryo Specificity

Although maturation-related proteins are well known in the endospermof albuminous seeds, an important question is whether the zygoticembryo possesses its own maturation proteins. We report on theisolation and partial characterization of storage proteins ofcarrot (Daucus carota L. var Nandor) dry achenes and isolatedzygotic embryos, using one- and two-dimensional electrophoresistechniques, HPLC and amino acid sequencing. The presence ofa series of abundant polypeptides showing charge heterogeneity,that are rapidly degraded upon germination, was revealed inthe endosperm. These proteins consisted of glycoproteins, themost abundant of which displayed a molecular mass (M_r) of 58,000,albumins of M_r 42,000 comprising at least one rß-1,3-glucanase,and two globulins of M_r 90,000 and 50,000–55,000 respectively,the second being an oligomer composed of three subunits of M_r13,000, 20,000 and 30,000. None of these storage proteins identifiedin the endosperm were detected in zygotic embryos. In contrast,two novel proteins were isolated from zygotic embryos, namelya globulin family of M_r 50,000 and pI 6.3–6.8, which wasnamed "daucin", and a late embry-ogenesis abundant (LEA) proteinfamily of M_r 25,000 and pI6.3–6.6, named "RAB25". Sincethe latter proteins are apparently absent of the endosperm,these results suggest that the maturation of carrot zygoticembryos requires its own specific set of storage and LEA proteins. (Received July 15, 1997; Accepted October 28, 1997)     

REVIEW ARTICLE: Zygotic embryogenesis versus somatic embryogenesis

This review will summarize molecular and genetic analyses aimedat identifying the mechanisms underlying the sequence of eventsduring plant zygotic embryogenesis. These events are being studiedin parallel with the histotogical and morphological analysesof somatic embryogenesis. The strength and limitations of somaticembryogenesis as a model system will be discussed briefly. Theformation of the zygotic embryo has been described in some detail,but the molecular mechanisms controlling the differentiationof the various cell types are not understood. In recent yearsplant molecular and genetic studies have led to the identificationand characterization of genes controlling the establishmentof polarity, tissue differentiation and elaboration of patternsduring embryo development. An investigation of the developmentalbasis of a number of mutant phenotypes has enabled the identificationof gene activities promoting (1) asymmetric cell division andpolarization leading to heterogeneous partitioning of the cytoplasmicdeterminants necessary for the initiation of embryogenesis (e.g.GNOM),(2) the determination of the apical-basal organization whichis established independently of the differentiation of the tissuesof the radial pattern elements (e.g.KNOLLE, FACKEL, ZWILLE),(3) the differentiation of meristems (e.g.SHOOT-MERISTEMLESS),and (4) the formation of a mature embryo characterized by theaccumulation of LEA and storage proteins. The accumulation ofthese two types of proteins is controlled by ABA-dependent regulatorymechanisms as shown using both ABA-deficient and ABA-insensitivemutants (e.g.ABA, ABI3). Both types of embryogenesis have beenstudied by different techniques and common features have beenidentified between them. In spite of the relative difficultyof identifying the original cells involved in the developmentalprocesses of somatic embryogenesis, common regulatory mechanismsare probably involved in the first stages up to the globularform. Signal molecules, such as growth regulators, have beenshown to play a role during development of both types of embryos.The most promising method for identifying regulatory mechanismsresponsible for the key events of embryogenesis willcome frommolecular and genetic analyses. The mutations already identifiedwill shed light on the nature of the genes that affect developmentalprocesses as well as elucidating the role of the various regulatorygenes that control plant embryogenesis. Key words: Development, marker, mutant, somatic embryogenesis, zygotic embryogenesis