To shorten the juvenile stage of apple (Malus · domestica Borkh.) the BpMADS4 gene from silver birch (Betula pendula Roth.) was constitutively overexpressed in 25 transgenic apple clones. All clones were characterized by PCR, RT-PCR and Real Time PCR. Solitary flowers were produced on in vitro shoots of eight transgenic clones and most of them appeared to be morphologically normal. Twenty shoots of each clone were rooted and transferred to a glasshouse. Glasshouse plants of clones T1165, T1187 and T1190 developed flowers. Several plants of T1165 and T1187 started floral initiation within 3-4 months following transfer to the glasshouse. Primary flowers were solitary and in a terminal position on the main shoot. Lateral flower clusters, consisting of three to five individual flowers, were also found. Pollen vitality and tube germination of glasshouse-grown flowers were investigated, and there were no significant differences compared to pollen of nontransgenic control plants. Preliminary crosses using flowers of glasshouse plants resulted in small apple fruits. It would seem that this is the first report on in vitro flower induction in transgenic apple.

Despite intensive research on genetic regulation of flower development there are still only a few studies on the early phases of this process in perennial plants like trees. The aim of this study has been to identify genes that regulate early stages of inflorescence development in silver birch (Betula pendula Roth) and to follow the expression of these genes during development of the unisexual birch inflorescences. Here we describe the cloning and characterization of 3 cDNAs representing MADS-box genes designated BpMADS3, BpMADS4 and BpMADS5, all belonging to the AP1/SQUA group of plant MADS-box genes. According to RNA blot analysis, all 3 genes are active during the development of both male and female inflorescences. However, differences in patterns of expression suggest that they play different roles. BpMADS3 is most similar in sequence to AP1 and SQUA, but it seems to have the highest expression at late developmental stages. BpMADS4 is most similar in sequence to the Arabidopsis gene FRUITFULL, but is expressed, in addition to developing inflorescences, in shoots and roots. BpMADS5 is also similar to FRUITFULL; its expression seems to be inflorescence-specific and continues during fruit development. Ectopic expression of either BpMADS3, BpMADS4 or BpMADS5 with the CaMV 35S promoter in tobacco results in extremely early flowering. All of these birch genes seem to act early during the transition to reproductive phase and might be involved in the determination of the identity of the inflorescence or flower meristem. They could apparently be used to accelerate flowering in various plant species.

The phylogenetic relationships within the genus Betula (Betulaceae) were investigated using a part of the nuclear ADH gene and DNA sequences of the chloroplast matK gene with parts of its flanking regions. Two well-supported phylogenetic groups could be identified in the chloroplast DNA sequence: one containing the three American species B. lenta, B. alleghaniensis, and B. papyrifera and the other including all the other species studied. The ADH gene displayed more variation, and three main groups could be identified. In disagreement with the classical division of the genus Betula, B. schmidtii and B. nana grouped with the species in subgenus Betula, and B. ermanii grouped with species in subgenus Chamaebetula, including B. humilis and B. fruticosa. The ADH phylogeny suggests that several independent polyploidizations within the genus Betula could have taken place. The ADH and chloroplast phylogenies were in part incongruent due to the placement of B. papyrifera. The most likely reason for this seems to be cytoplasmic introgression.

In resting grains of Triumph barley (Hordeum vulgare L. cv Triumph) about 40% of the f-amylase could be extracted with a saline solution, the remaining 60% being in a bound form. During seedling growth (200C), the bound form was released mainly between days I and 3. When a preparation containing bound ,-amylase was incubated with an extract made of endosperms separated from germinating grains, release of bound ,#-amylase took place and could be studied in vitro. The release was almost completely prevented by leupeptin and antipain, specific inhibitors of a group of SH-proteinases, but it was not inhibited by pepstatin A or EDTA, which inhibit some other barley proteinases.It is thus very likely that in a whole grain, at least the bulk of the bound f-amylase is released by the proteolytic action of one or several SH-proteinases. When the bound jl-amylase was released by papain, its molecular weight was about 5000 daltons smaller than that of f8-amylase released by dithiothreitol. This indicates that the release is due to removal of a sequence of ,B-amylase itself. A similar decrease in size took place during seedling growth. Bound j0-amylase showed some activity against native starch and it hydrolyzed maltotetraose at a rate that was about 70% of the rate the same amount of bound jl-amylase gave after release. Bound j-amylase is thus not inactive and it is likely that the slower rate of hydrolysis is due to steric hindrances which prevent substrates from reaching the active site.In germinating barley grain, starch present in the starchy endosperm is hydrolyzed into glucose by a concerted action of a-and ,3-amylases, debranching enzyme and a-glucosidase (maltase) (2, 6). In contrast to the three other groups of enzymes, fl-amylase is not synthesized during germination but accumulates during development of the grain (9). At the end of development, when the grain is drying, a portion of the j3-amylase is attached, apparently via S-S-bridges, to insoluble constituents of the starchy endosperm (16,18 Our aim in the present study was to determine whether bound ,B-amylase is released in vivo by the action ofproteolysis and, if so, to identify the proteinase in question.Bound f3-amylase has often been cited to be latent (4, 14, 23). Here we show that it can hydrolyze different substrates with a rate depending on the size of the substrate. MATERIALS AND METHODS Plant MaterialGrains of barley (Hordeum vulgare L. cv Triumph) were obtained from SECOBRA (78580 MAULE, France). They were dehusked with 50% H2SO4, surface-sterilized with 1% NaOCl, and allowed to germinate aseptically on agar gel at 20°C in the dark (20). In these conditions the coleoptile was about 2 cm long after 3 d. Extraction of the Free and Bound ,8-AmylaseTwenty whole resting grains or endosperms from 20 germinating grains were homogenized at 20°C in a mortar with a small amount of quartz sand in the presence of 0.5 to 2 mL of 0.1 M NaCl. After homogenization, more NaCl solution was added, the total volume used being 8 mL. After centrifugation for 1...

a-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after I day of germination at 20C, but they bepn to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaC2, the a-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of a-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of a-amylase activity in the starchy endosperm in a whole grain.Scutella separated after I day of germination released small amounts of a-amylase activity into 10 millimolar CaCl2. This release was linear for at least I hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day I and the total secretion accounted for only 5 to 10% of the increase of a-amylase activity in the starchy endosperm in a whole grain. Since the times from the separation of the parts of the grain to the beginning of the secretion assay (1040 minutes) as well as the duration of the assay itself (2040 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total a-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.At present, there is some controversy concerning the relative roles ofthe aleurone layer and the scutellum in the dissemination of a-amylase into the starchy endosperm in a germinating barley grain. Many studies have shown that aleurone layers separated from embryoless 'half-grains' after imbibition for 3 d are induced by GA3 to synthesize and secrete large amounts of a-amylase activity into the medium (7,10,26 Recently, Gibbons (11-13) has used histochemical staining with fluorescent antibodies specific for all a-amylase to localize this enzyme, which appears only during germination. In grains germinated for 1 to 3 d at 15C, all all a-amylase appeared to be localized in the scutellum and in the zone of starchy endosperm facing the scutellum. Only later was a-amylase detected in the aleurone layer and the adjacent part of the starchy endosperm. On the basis of these results, the author suggested that up to 50% of the a-amylase present in the starchy endosperm after 7 d of germination originated in the scutellum (13). Localization of a-amylase activity by a starch film technique in germinating grains of barley, oat, rice, rye, and wheat also sugges...