Maria Grazia Galli

Sex determination and differentiation in Asparagus officinalis L.

Abstract The paper summarizes the coordinated researches conducted by three Italian groups in the area of sex determination and differentiation in the dioecious species Asparagus officinalis . Morphological evidence indicates that sex differentiation in Asparagus consists essentially of selective abortion of gynoecium or androecium of initially hermaphroditic floral primordia occurring in genotypically determined male and female individuals. Abortion occurs in pollen-mother cells and anthers in females and in megaspore-mother cells but not in the vegetative tissues of the ovary in males. The differential developmental pathway is accompanied by changes in relative abundance of auxin and cytokinins. The genetic ssytem controlling abortion of male or female organs is apparently monogenic (possibly a bipartite gene) with factor(s) associated with the homomorphic chromosome pair L5. Other genes influence the development of reproductive structures as indicated by the presence of genetic factors controlling stylar growth in male plants. The presence of extensive polymorphism in isoenzyme and DNA restriction fragment length patterns (RFLP) allows the search for markers associated with ‘sex genes’: a locus encoding a malic dehydrogenase (MDH) isoenzyme has been found about 20 cM from sex genes implying that chromosomes in which sex factors are located could pair and recombine. Searches for messages specifically expressed in reproductive structures were conducted by 2D-electrophoresis of existing and newly synthesized polypeptides or of in vitro translation products of poly(A) + RNA from male and female flowers and by isolating specific monoclonal antibodies against sex specific floral antigens.

Functional roles of the plant α‐like and γ‐like DNA polymerases

Plant cells are endowed with two distinct DNA polymerases [ 1,2] whose properties closely resemble those of the DNA polymerases cr and y present in animal cells [3,4]. The plant DNA polymerases have consequently been named o-like [ 1 ] and y-like [2]. The B-like DNA polymerase activity is the most abundant in cultured plant cells [l ] and responds to changes in the rate of cell multiplication, whereas experiments with spinach leaves have shown that the y-like DNA polymerase is present in the chloroplast 121. A DNA polymerase has also been isolated from the mitochondria of wheat embryos [S]. Spinach mitochondria may also contain a DNA polymerase whose properties are partially different from those of the y-like DNA polymerase isolated from the chloroplasts of the same cells and are similar to those of the wheat embryo enzyme (unpublished). However, no evidence is available as yet on the existence in plant cells of a DNA repair enzyme similar to the DNA polymerase p of mammalian cells ]4,6]. By analogy with animal cells, the assignment of functions to the DNA polymerases in plant cells is hampered by the lack of conditional mutants defective in DNA synthesis. Thus, we approach this problem by exploiting the properties of aphidicolin and of ethidium bromide. Aphidicolin [7] specifically inhibits the cr-like DNA polymerase purified from plant cells 181, while the

Synchronization of nuclear DNA synthesis in cultured Daucus carota L. cells by aphidicolin

By inhibiting the α‐like DNA polymerase, and therefore nuclear DNA synthesis, aphidicolin induces accumulation of suspension cultured carrot cells at the G1/S boundary of the cell cycle. After a 24‐h treatment with the drug the accumulation is complete, affecting all the cycling cells (95% of the population). Upon removal of the inhibitor, all cycling cells immediately resume nuclear DNA synthesis and move synchronously throughout the S‐phase.

Comparative effects of fusicoccin and gibberellic acid on the promotion of germination and DNA synthesis initiation in Haplopappus gracilis

Abstract We have compared the effects of fusicoccin (FC) and gibberellic acid (GA 3 ) on the promotion of germination, the stimulation of cell elongation and the resumption of nuclear DNA synthesis in dormant seeds of Haplopappus gracilis . Although both FC and GA 3 promote germination and stimulate cell elongation, FC appears to act earlier and to be more effective. On the otherhand, FC has no effect on the resumption of DNA synthesis which, in contrast, is stimulated effectively by GA 3 .

DNA repair synthesis in plant protoplasts is aphidicolin-resistant

Animal ceils contain 3 distinct DNA polymerases, which, on the basis of studies with isolated •rgane••es and with specific inhibitors [1-9] have been assigned the following main, but possibly not exclusive, functions: ct-polymerase is involved in nuclear DNA replication;/3-polymerase is involved in nuclear DNA repair synthesis; and 7-polymerase is involved in mitochondrial DNA replication. In plant ceils the presence of an a-like DNA polymerase [10,11 ], responsible for nuclear DNA replication [ 12 ], a chloroplast 7-like [ 13 ] and a mitochondrial DNA polymerase [14], possibly involved in organellar DNA replication [ 12], have been reported. However, there is no clear evidence as to the existence of a j3-like DNA polymerase in plants [ 11 ]. Since the repair of radiation-induced DNA lesions also occurs in the ceils of higher plants [ 15 ], we have decided to investigate whether, at variance with animal ceils, this DNA repair synthesis is due to the activity of the nuclear a-like DNA polymerase. To this end, we have used aphidicolin [16], a specific inhibitor of the a-like DNA polymerase of higher plant cells [17], as well as that of the DNA polymerase t~ of animal cells [4,6,9,18]. The results show that the UV light-induced DNA repair synthesis in protoplasts ofNicotiana sylvestris is resistant to this drug and thus is not performed by the a-like DNA polymerase.

Liposome-mediated transfer of DNA to carrot protoplasts: A biochemical and autoradiographic analysis

Abstract The interaction between positively charged multimellar liposomes loaded with [ 3 H]DNA from Bacillus subtilis , and carrot protoplasts has been studied. Molecular sieving analysis of DNA recovered from liposomes after incubation with protoplasts demonstrates that these liposomes can efficiently protect DNA from nucleases present in protoplast suspensions. Autoradiographic analysis of sectioned protoplasts reveals that liposomes can mediate, although at low efficiency, transfer of DNA inside the protoplasts. Transfer efficiency appears to be highly improved by treating with polyethylene glycol protoplasts preincubated with liposomes.

Interaction between abscisic acid and fusicoccin during germination and post-germinative growth in Haplopappus gracilis

Abstract Abscisic acid (ABA) 10 −5 M completely inhibits the germination of seeds of Haplopappus gracilis , and this effect can be effectively reversed by fusicoccin (FC). Nevertheless, when the analysis of reversal promoted by FC is extended to post-germinative growth, it is observed that it results only in a limited elongation of the embryos, perhaps only through cell enlargement, which does not involve any significant resumption of DNA synthesis, cell division or such developmental features as synthesis of chlorophyll and development of root hairs. It is proposed that ABA inhibition of seed germination involves at least two different mechanisms, one mechanism interfering with K + uptake at the level of the cell membrane, which is reversed by FC, promoting cell elongation, the other interfering with the synthesis of nucleic acids and/or protein synthesis and not being affected by FC.

Synthesis of DNA in excised watermelon cotyledons grown in water and benzyladenine

Excised watermelon cotyledons were grown in water and benzyladenine, which greatly promotes growth, breakdown of reserves and development of organelles. In order to investigate the involvement of DNA synthesis in these benzyladenine-induced effects, [3H]thymidine was applied continuously (for 3 d) or administered briefly (5 h) to excised cotyledons at various stages of development. Autoradiographic analysis of squashed and sectioned cotyledons showed that both the cytoplasm (mainly in the region of the plastids) and most of the nuclei were labelled. Both types of labelling were promoted by benzyladenine treatment. The highest percentage of labelled nuclei was found in the early stages of growth (first day after excision of cotyledons), long before the burst of enzymatic activities involved in the germination processes. The possible meaning of the increase of nuclear DNA, apart from the normal replicative synthesis preceding cell division, is discussed.

Synchronization of plant cells in culture and in meristems by aphidicolin

Publisher Summary The study of molecular and structural events occurring during the cell cycle often requires synchronized cell populations. Synchronous populations may also be essential for the isolation of specific cell components, such as metaphase chromosomes. Natural synchrony is an uncommon and incomplete event. Therefore, several methods have been experimented to obtain synchrony in plant tissues and in cultured cells. This chapter describes the methods for the synchronization of plant cells in culture and in the meristems by aphidicolin. Aphidicolin has turned out to be the most effective, least toxic, and most versatile tool for inducing synchronous growth in plant cell populations, both in culture and in differentiated tissues. The drug is effective both in cell-free extracts and in intact cells and tissues, where the addition of aphidicolin causes complete inhibition of nuclear DNA replication while organellar DNA replication, ultraviolet (UV)-induced nuclear DNA repair synthesis, and RNA and protein synthesis are not disturbed. Thus, an appropriate treatment with aphidicolin causes an accumulation of cells at the G 1 /S boundary of the cell cycle, with a small fraction of the cells blocked in the process of DNA synthesis. The blockage is extremely effective, affecting all the cycling cells. It is also reversible, as immediate and synchronous resumption of nuclear DNA synthesis and growth occurs following the removal of the drug. However, inactivation can be controlled by appropriately choosing the plant material and/or the experimental conditions.

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin.Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.