ALBERT

Notes: The amount of total ribosome assemblies extractable from the vegetative buds of 2 m high Scots pine (Pinus sylvestrís L.) plants remained more or less constant throughout the sampling period from September to May. The stability of the ribosomes, the shape of the polysome profiles obtained after sucrose density gradient centrifugation and the clustering of material as seen in the scanning electron micrographs suggested the presence of storage formations during the winter.All samples of isolated ribosomes were able to synthesize proteins in vitro. During midwinter the translation capacity, when calculated on a ribosome unit basis, was about one third of that found in September and May. This reflects not only the occurrence of storage formation during the winter, but also the amount of initiated translation processes at any given time. The decrease in the in vitro translation capacity in the autumn ceases around the end of November. Ribosome activity starts to increase as early as the end of January or beginning of February. It seems that the reactions are triggered either by an endogenous clock or by the change in the daylength.

Notes: Scots pine (Pinus sylvetris L.) plants, about 2 m high, were placed in controlled conditions for 2 weeks in January, April and November. During the experiments made in January, the conditions in the climate chambers simulated either a gradual or abrupt advancement of spring. In April they simulated either the advancement of the season or its reversal back to January. In November the plants were transferred to conditions that resembled spring. In January, pieces of buds collected at the end of the experiment were also fixed for electron microscope studies.Isolation of the ribosomes and the determination of their in vitro translation capacity revealed that in January the response to environmental changes was evident. An increase in synthesized proteins was caused by a rise in the translation capacity of ribosome assemblies rather than by an increase in their quantity. The cellular ultras-structure changed in conformity with the changes characteristic of the spring. In April, the plants transferred to the climate chambers maintained their ability to synthesize proteins, but the buds were judged to be under stress. In November die ability of the buds to respond to environmental changes was retarded or inhibited.

Notes: A number of experiments on the effect of wound washing on the development of crown gall were made varying the following factors: Host species; bacterial strain; non-washing versus washing for different periods; sterile versus inoculated wound; time of inoculation. Washing did not have a recognizable effect on the healing of sterile wounds. Neither did it seem to overcome incompatibility between host and bacterial strain. Washing promoted tumor growth significantly under the following conditions: Vicia faba inoculated with T37 20 hours after washing for 1 hour; Helianthus annuus inoculated with B6 immediately after washing for 2 hours.

Notes: DNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with 3H-thymidine in slides in which mitoses had been counted and analyzed before autoradiography.The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two.The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.

Notes: A procedure is described by which the ribosome assemblies isolated from the buds of Scots pine (Pinus sylvestris L.) as well as the ribosome and polysome fractions purified by sucrose density gradient centrifagation can be prepared for study by scanning electron microscopy. The technique has also been used to illustrate ribosomes extracted froai leaves and roots of Nicotiana tabacum L., from roots of Tuiipa gesneriana hybr. and from commercial wheat germ. The sample under study is spread on a piece of coverslip', frozen and dried at room temperature. The coverslip, with attached material, is taken through the critical point drying procedure, glued on a specimen retainer aod coated with gold-palladium. The coverslip serves as a sufficiently stable specimen support to allow the study of ribosomes at magnifications of several hundred thousand. Use of the method makes it possible to study the structure of polysomes, to check the purity of fractioned samples and to investigate differences in the ribosome assembly of the developing plant tissues.

Notes: The ribosome assemblies isolated from buds of Scots pine (Pinus sylvestris L.) containing microsporangiate strobili varied both quantitatively and qualitatively in samples collected from October to April. The seasonal fluctuation in the amount of ribosonnes was more evident in the cytosolic fraction than in the smaller membrane-bound fraction. The profiles obtained after sucrose density gradient centrifugation were of two types. One type was commonly obtained from samples collected late in the autumn and early in the spring, and this type was characterized by a relatively high peak for the large subunits, a low or negligible peak for the dimers, and an even or ascending series of peaks for the polymers. The other type was obtained from samples collected during the winter, and was characterized by small peaks for both subunits, a moderate to large peak for the dimers and a descending series of peaks for the polymers. However, the scanning electron microscope investigations indicated that the winter-time samples did not lack polysomes and clusters of ribosomes. They did not become visible in the polysome profiles because they pelleted too tightly at the bottom of the centrifuge tubes to be removed with gradient fractionation. The au-toradiographic analyses suggested that the cells were capable of synthesizing mRNA throughout the winter, whereas rRNA synthesis was arrested. On the basis of the above results, we postulate that the synthesis of the enzyme proteins needed for the maintenance of winter-time metabolism takes place in the cytosolic ribosome fraction. The possible existence of winter-time polysome stores is also pointed out.

Notes: DNA synthesis was measured during a 30 hour period in stem segments containing a wound — either sterile or inoculated with Agrobacterium tumefaciens— and in control stems of Vicia faba. An identical series of experiments was conducted both on upper, still growing internodes and on lower, mature ones. The incorporation of 3H-thymidine was determined from extracted DNA with a liquid scintillation spectrometer. The results from the two internodes were fairly similar. DNA synthesis was not significantly different in the two types of wounds. It rose above control level at about 10 hours after wounding and reached its peak at about 19 hours. Roughly, this pattern of DNA synthesis seems to correspond to the minimum conditioning and transformation time as found in other plants. However, the decisive factor need not be DNA synthesis, but could be some other process coinciding with it.

Notes: The in vitro translation capacity of total ribosome assemblies isolated from the vegetative buds of small Scots pine (Pinus sylvestris L.) plants depends on the isolation procedure. Good yields and high values for protein synthesis were obtained in experiments in which polyvinyl pyrrolidone (PVP) was added to the grinding buffer. The polysome profiles obtained after sucrose density gradient centrifugation indicated the presence of polysomes in all samples. In addition, large ribosome aggregates were visible in the scanning electron micrographs. The use of an RNase inhibitor (RNasin) together with PVP did not improve the results, and treatment with ribonuclease (RNase, EC 3.1.27.5) destroyed the ability to synthesize protein.D, L-Dithiothreitol (DTT) and mercaptoethanol, if used instead of or together with PVP, gave low yields and also DTT destroyed the in vitro translation capacity of the ribosome assemblies. The polysome profiles had a high peak indicating dimers and often a descending series of peaks indicating polymers. A study of the scanning electron micrographs gave the impression that the largest polymers and aggregates had broken down.Protease K (EC 3.4.21.14) when added to the grinding buffer also destroyed the ability of the ribosomes to maintain protein synthesis in vitro. In this case, the shape of the polysome profiles gave the impression of successful isolation. Clumps of ribosomes, presumably originating from large aggregates, were visible in the scanning electron micrographs. Triton X-100 and 0.25 M NaCl in the grinding buffer extracted chromatin, which affected the results. The material lost during the extraction and purification processes consisted mainly of monosomes and their sub-units.On the basis of the above results it was concluded that the preservation of large polysomes and ribosome aggregates in the isolated ribosome assemblies is necessary if they are to maintain a high translation capacity. The content of the assemblies was best revealed in the scanning electron micrographs. The shape of the polysome profiles did not always correlate with the ability of the isolated ribosomes to synthesize proteins.