Silvana Dolfini

Light-Dependent Spatial and Temporal Expression of Pigment Regulatory Genes in Developing Maize Seeds.

Both light and developmental stimuli are directly involved in the regulation of plant gene expression. In maize, activation of the anthocyanin pathway represents an excellent model system for studying the interactions between an external factor, such as light, and internal factors that regulate plant and seed development. By analyzing in detail the aleurone and pericarp seed layers, different developmental windows for light have been found in the two tissues[mdash]the former in the advanced stages of development and the latter in the early stages of seed development. Transcriptional control of the structural genes involved in anthocyanin deposition within the pericarp is known to be exerted by the Sn and pl genes, whereas the aleurone is controlled by the R and C1 regulatory genes. By using in situ hybridization analysis, we detected tissue-specific expression of Sn and R in the seed layers, revealing a correlation between structural gene activation and anthocyanin accumulation. In addition, RNA gel blot analysis revealed that Sn expression is enhanced by light, whereas the R gene expression is not. However, the light-induced expression of the myb-type genes C1 and pl, detected by reverse transcriptase-polymerase chain reaction, was found to be the limiting factor for conferring the developmental competence of the pericarp and the aleurone layers to light responsiveness.

Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize.

SummaryThe Sn locus of maize is functionally similar to the R and B loci, in that Sn differentially controls the tissue-specific deposition of anthocyanin pigments in certain seedling and plant cells. We show that Sn shows molecular similarity to the R gene and have used R DNA probes to characterize several Sn alleles. Northern analysis demonstrates that all Sn alleles encode a 2.5 kb transcript, which is expressed in a tissue-specific fashion consistent with the distribution of anthocyanins. Expression of the Sn gene is light-regulated. However, the Sn: bol3 allele allows Sn mRNA transcription to occur in the dark, leading to pigmentation in dark-grown seedlings and cob integuments. We report the isolation of genomic and cDNA clones of the light-independent Sn: bol3 allele. Using Sn cDNA as a probe, the spatial and temporal expression of Sn has been examined. The cell-specific localization of Sn mRNA has been confirmed by in situ hybridization using labelled antisense RNA probes. According to its proposed regulatory role, expression of Sn precedes and, in turn, causes a coordinate and tissue-specific accumulation of mRNA of structural genes for pigment synthesis and deposition, such as A1 and C2. The functional and structural relationship between R, B, Lc and Sn is discussed in terms of an evolutionary derivation from a single ancestral gene which gave rise this diverse gene family by successive duplication events.

Antiparallel expression of the sense and antisense transcripts of maize α-tubulin genes

In all eukaryotes α- and β-tubulins are encoded by small families of closely related genes and are highly conserved. In Zea mays, at least six different α-tubulin coding sequences are known. We describe the isolation from scutellar nodes of the maize inbred line W22 of a clone (CTM5) coding for an α-tubulin. On the basis of the 3′ end nucleotide sequence, this clone can be assigned to the already reported tua4 gene. Northern analysis demonstrates that CTM5 encodes a 1.5 kb transcript, which is expressed in different tissues of the seed and of the seedling. In order to define the spatial and temporal expression of α-tubulin genes, in situ hybridization experiments were performed on these tissues. Unexpectedly, a specific signal was detected with both antisense and sense RNA strands. Temporal and spatial distribution of the two RNAs, however, shows that high levels of the two transcripts are always discordant. In tissues where sense transcripts are highly abundant (embryos at various developmental stages, root tips, pollen grains), the antisense transcripts are expressed in relatively small amounts, while in pericarp, coleoptile, leaves, and scutellar node, where antisense transcripts accumulate, the sense transcript only reaches a very low level. Northern analysis using single-stranded DNA probes confirmed the presence of an antisense transcript of 1.5 kb, prompting speculation about the role of this transcript in the regulation of the expression of α-tubulin genes.

Morphological and chromosomal characterization of three new continuous cell lines of Drosophila melanogaster

Three continuous cell lines (GM1, GM2 and GM3) were obtained from embryos of Drosophila melanogaster. Karyotypic analysis revealed characteristics distinguishing each line. Except for some minor variations GM1 cells had an X and a centric heterochromatic fragment (which is a portion of the Y). GM2 line was characterized by XO cells showing two “new” telocentric chromosomes while an autosome of the II pair was missing. GM3 cells were XY; the Y chromosome, however, was shorter than the normal, having a deletion of the terminal section of the short arm. Several problems concerning the origin of these different genomes are discussed.

The cell cycle of an established line ofDrosophila melanogaster cells in vitro

La durata media delle fasi del ciclo cellulare (G1, S e G2) e del tempo di generazione totale (G T ) in una linea stabilizzata di cellule diDrosophila melanogaster risulta essere rispettivamente di 1.8, 10.0, 7.2 e 18.8 ore.

Karyotype polymorphism in a cell population of Drosophila melanogaster cultured in vitro

The karyotypic variation of a cell line (KC) established from primary cultures of embryonic cells of Drosophila melanogaster was studied. Aneuploidy and structural rearrangements were found consisting mainly in: 1) loss of one of the chromosomes of the IV pair, 2) presence of a heterochromatic centric fragment (Y ?), 3) enlargement of the heterochromatic portion of one X, 4) shortening of an arm of one element of the two large autosome pairs. Moreover, the presence of tetraploid cells (3–12%) was noticed. The polymorphism in this cell population can be explained as a result of different mechanisms, including hybridization between different cell types. Replication of DNA was studied by autoradiography with special reference to the timing of replication of heterochromatin.

Dap (D efective a leurone p igmentation) mutations affect maize aleurone development

Dap (Defective aleurone pigmentation) is the designation for mutations in maize that give rise to a characteristic dappled endosperm phenotype, consisting of patches of purple tissue, of variable size and shape, on a yellow background. Features shared by all Dap mutants are: dominant expression when they are maternally derived, lack of expression or transmission when they originate from pollen, failure to recover homozygous Dap genotypes, reduced frequency of Dap seeds in the progeny of outcrosses of Dap/+ females, association of the dappled phenotype with reduction in seed size. The mutants so far tested, six in all, can be grouped into two classes, one including male-transmissible (MT) isolates not expressed in the endosperm if their contribution is paternal, and a second class of isolates (NMT) that are permanently lost following paternal transmission. We suggest that the NMT mutations are on a chromosome that carries an intercalary deletion. Assuming linkage between the mutant and the deletion, selection against the deficient chromosome during male gametogenesis would account for the failure to recover Dap seeds in the progeny of Dap/+ male parents. We have obtained genetic evidence supporting this hypothesis. This interpretation, however, does not apply to MT alleles. For these, other mechanisms, such as imprinting and/or dosage effects may be proposed. The mutable pattern in the endosperm to which all Dap mutants give rise is an intriguing phenotype which remains to be clarified. An unexpected finding is that aleuronic and subaleuronic cells corresponding to the colourless areas are abnormal in shape and anthocyanin biosynthesis is blocked in these cells. This finding calls for further investigation in light of a possible connection between flavonoid precursors and cell shape.

The distribution of repetitive DNA in the chromosomes of cultured cells of Drosophila melanogaster

The distribution patterns of different stains (orcein, quinacrine and Giemsa) in an established cell line of Drosophila melanogaster (GM3 WS) were compared. Each chromosome stained both with quinacrine and with Giemsa shows up a specific banding pattern for heterochromatin. The comparison between the two patterns suggests a hypothesis concerning the significance of the fluorescence; moreover it permits the conclusion that heterochromatin in D. melanogaster mitotic chromosomes is all constitutive and that there is a correspondence between repetitive DNA and sections poor in mappable genes.The distribution patterns of different stains (orcein, quinacrine and Giemsa) in an established cell line of Drosophila melanogaster (GM3 WS) were compared. Each chromosome stained both with quinacrine and with Giemsa shows up a specific banding pattern for heterochromatin. The comparison between the two patterns suggests a hypothesis concerning the significance of the fluorescence; moreover it permits the conclusion that heterochromatin in D. melanogaster mitotic chromosomes is all constitutive and that there is a correspondence between repetitive DNA and sections poor in mappable genes.

Analysis of four maize mutants arrested in early embryogenesis reveals an irregular pattern of cell division

The process that leads to embryo formation appears to follow a defined pattern, whose sequential developmental steps—under strict genetic control—can be analysed through the study of mutants affecting embryogenesis. We present the analysis of four embryo-specific (emb) mutants of maize, characterised by abnormal development not overcoming the proembryo or early transition stage, that define three separate genes on the basis of their chromosomal location and complementation pattern. A common feature emerging from histological analysis is that suppression of morphogenesis is accompanied by an uncontrolled pattern of cell division. The block in embryo development is associated with abnormal suspensor proliferation, possibly due to the absence of a signal elaborated by the embryo proper and required for suspensor cell identity maintenance. Mutant endosperm morphogenesis is not impaired, as shown by the formation of the expected domains, i.e. aleurone, starchy endosperm, embryo-surrounding region and basal endosperm transfer layer. The program of cell death appears impaired in the mutants, as expected if this process is essential in determining the shape and morphology of the developing organs. An unexpected result is obtained when mutant embryo rescue is attempted. Immature embryos transferred to a basal medium germinated, yielding small but otherwise normal seedlings, an observation not consistent with the histological evidence of a complete absence of morphogenetic potential. The analysis of emb mutants appears a promising tool to elucidate crucial points of embryo development such as the coupling of cell division with morphogenesis, cell-to-cell interactions, the relationship between embryo and endosperm development, and the interaction between embryo proper and suspensor.

Light inducibility and tissue specificity of theR gene family in maize

The red and purple anthocyanin pigments of plants are visible genetic markers and their spatial and temporal accumulation is strictly regulated. Anthocyanin biosynthesis is also modulated by environmental factors such as light and temperature. Thus this process represents an appealing model system for the study of gene regulation, as well as for studying developmental biology. In maize, the pattern of pigmentation of the plant is controlled by theR, Sn andB genes, a small family of HLH transcription factors. Here we report the pattern of light induction and tissue specific expression of the regulatory and structural genes involved in this biosynthesis. TwoSn alleles differing in their light response have been considered and analyzed by Northern andin situ hybridization experiments. An unusual phenomenon of interaction between the homologousR andSn genes leading to a partial gene silencing is reported. We hypothesize a model in which silencing is achieved through methylation of specific sites.