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Notes: Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pistillate flowers nonetheless develop non-functional reproductive structures of the ompposite sex. Ubiquitin is a small, highly conserved protein found in all eucaryotes: a covalent ATP-dependent attachment of ubiquitin marks proteins for degradation. In the present paper, we used immunoblotting to investigate the presence of free ubiquitin and ubiquitin conjugates during pollen development in male (androfertile) and in female (androsterile) genotypes of kiwifruit. In the male, several high molecular mass protein conjugates were present throughout development. On the contrary, such a pattern characterized only early stages of pollen from the female genotype, where conjugates progressively disamppeared, until they were detectable only in trace amounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were ampproaching the first mitosis. Free ubiquitin increased continuously during development of the male microgametophyte so that mature pollen contained considerable amounts of the ubiquitin monomer at the time of its release from the anther. By contrast, only low levels were detectable in the degenerating microspores in the pistillate flowers. In vitro experiments using labeled ubiquitin indicated that early-uninucleate microspores of the female genotype had a much higher conjugation rate than those of the male genotype at the same stage. However, after feeding α-lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progressively degenerated in the microspores of the pistillate flowers starting from the early-uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity. At anthesis, the microspores in the pistillate flowers either had no nucleus or showed only traces of chromatin. Thus, the ubiquitin system seems to play an important role in protein turnover occurring during the normal developmental pathway of the kiwifruit microgametophyte, while it was mainly involved in regressive events related to microspore degeneration in the female genotype.

Notes: The possibility of a relation between the expression of root inducing (Ri) T-DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast-growing tobacco hairy roots. Transformed root cultures have been established on hormone-fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T′ roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T′ roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T′ roots. Significant differences were found during the progression of growth, particularly in the TCA-insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.

Notes: The changes taking place during fruit development in the concentration of the 3 polyamine fractions, i.e. free, perchloric acid-soluble conjugates and perchloric acid-insoluble bound polyamines, were analyzed in tomato fruits (Lycopersicon esculentum Mill, cv. F121) induced to set by either pollination or auxin application. Before the onset of cell division, total polyamines were 50% higher in auxin-treated fruits than in pollinated ones, most of the polyamines being found as perchloric acid-soluble conjugates in both fruit set treatments. At the onset the level of polyamines in both fruit types was 100 times higher than during cell expansion or ripening. The highest polyamine found during cell division was perchloric acid-soluble conjugated spermidine in both fruit set treatments. After cell division, polyamine concentration was similar in both fruit set treatments. The concentration of polyamines in the jelly was similar in pollinated and auxin-induced fruits during cell expansion but not during ripening. At the onset of ripening there was an increase of one order of magnitude in the concentration of perchloric acid-insoluble bound putrescine in the jelly of pollinated fruits. Polyamines were more than 5-fold higher in unpollinated ovaries than in fruits induced to set by either pollination or auxins. It is suggested that pollinated and parthenocarpic fruits differ in their polyamine metabolism during the initial stages of development, but not after cell division. It is also suggested that polyamines undergo rapid turnover during cell division. Perchloric acid-insoluble bound putrescine might play a role in seed formation in tomatoes.

Notes: The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot (Daucus carota L. cv. Ingrid) was studied. In the presence of 1 mM Ca2+, IAA (10−7 to 10−4M) enchances [14C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca2+. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La3+, known not to penetrate the plasmalemma, exerts the same effect as Ca2+, but gives lower uptake values than Ca2+. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca2+ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca2+ -induced increase of spermidine uptake.

Notes: Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean (Phaseolus vulgaris L. cv. Lingot) and soybean (Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.

Notes: Biondi, S., Torrigiani, P., Sansovini, A. and Bagni, N. 1988. Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata. - Physiol. Plant. 72: 471–476.The effect of 1 mAf dicyclohexylamine (DCHA) on the synthesis of spermidine and spermine was examined in excised cotyledons of radiata pine (Pinus radiata D. Don) cultured under shoot-forming (with cytokinin) and non-shoot-forming (minus cytokinin) conditions by incubation with [14C]-putrescine. In control cotyledons incorporation into spermidine showed a peak at day 2 in the presence and at day 5 in the absence of N6-benzyldenine (BA). DCHA-treated cotyledons gave the same labeling pattern, both in the presence and absence of benzyladenine, with a much smaller peak at day 2. The incorporation into spermidine and spermine was insignificant at day 5 and later. The total radioactivity in the trichloroacetic acid supernatant indicated that precursor uptake was strongly reduced by the drug. In addition, the percentage label found in the benzene phase and combined in the 3 polyamines was lower in DCHA-treated cotyledons. Thus, treatment with DCHA not only inhibited the conversion from putrescine to spermidine and spermine, but also reduced its conversion to other benzene-extractable compounds. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity, which furnishes the propylamine group to spermidine and spermine synthases (EC 2.5.1.16 and EC 2.5.1.-), was not significantly affected by DCHA and appeared to be independent of the spermidine and spermine synthase reactions, suggesting that spermine synthesis decreased as a result of substrate depletion. The correlation between morphological development and polyamine biosynthesis is discussed.

Notes: Ornithine decarboxylase (ODC, EC 4.1.1.17) was studied in crude extracts of parenchyma slices of dormant tubers activated for 12 h, tuber shoots and shoot apices. It was highest in shoot apices. The enzyme activity was measured by the production of 14CO2 from labelled ornithine; Vmax was 450 nmol (mg protein)-1h-1, Km for ornithine and pyridoxal phosphate were, respectively, 30 mM and 5μM. Only when partially purified, the 14CO2 production was inhibited by α-difluoromethylornithine, while in crude extracts dithiothreitol was inhibitory. Ornithine and arginine decarboxylase (ADC, EC 4.1.1.19) activities from parenchyma tubers were not greatly altered by exogenously supplemented ornithine, even though its endogenous pool increased. Exogenously supplemented arginine enhanced ornithine decarboxylase activity, whereas putrescine decreased it slightly. The possibility of artifactual activities in the crude extract is also discussed.

Notes: The effects of exogenous ornithine, arginine and polyamines added to media leading to root, callus or bud initiation of Datura innoxia Mill. leaf explants growing in vitro were examined. Ornithine and arginine decarboxylase activities (ODC, EC 4.1.1.17; ADC, EC 4.1.1.19) as well as endogenous polyamine levels were also determined during the course of in vivo differentiation of the leaves and their subsequent in vitro dedifferentiation under rooting, callusing, or budding conditions. Decarboxylase activities were determined by measuring the 14CO2 released and the polyamines were quantified after dansylation by thin-layer chromatography. In vivo, ODC and ADC activities decreased from shoots to young to old leaves. In vitro, synergistic effects between ornithine and indole-3-acetic acid on rhizogenesis were detected, while arginine was not effective. Exogenous putrescine also acted synergistically with auxin by promoting root growth. A close relationship was found between rhizogenesis, ODC activity and increase in endogenous putrescine and spermidine levels. ODC increased during the induction and time course of cell dedifferentiation and seemed to support these events, while ADC seemed to support only the later events involving redifferentiation.

Notes: The occurrence of polyamines in different RNAs was investigated in view of the importance of these organic cations in RNA function. RNA was extracted at low ionic strength and separated on Sephadex columns.tRNA and rRNA of plants (Spinacia oleracea leaves, etiolated epicotyls of Pisum sativum, and Neurospora crassa mycelia) contain polyamines although in a different ratio than they are found in a complete cell. Contrary to spermine and putrescine, spermidine is always present.The binding of polyamines to tRNA and rRNA is discussed applying the Liquori model to the double stranded regions, where it was possible.Polyamines were also detected in 5S RNA.

Notes: Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto ‘a grappolo lungo’ (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.