Rino Cella

Global Analysis of Arabidopsis Gene Expression Uncovers a Complex Array of Changes Impacting Pathogen Response and Cell Cycle during Geminivirus Infection

Geminiviruses are small DNA viruses that use plant replication machinery to amplify their genomes. Microarray analysis of the Arabidopsis (Arabidopsis thaliana) transcriptome in response to cabbage leaf curl virus (CaLCuV) infection uncovered 5,365 genes (false discovery rate <0.005) differentially expressed in infected rosette leaves at 12 d postinoculation. Data mining revealed that CaLCuV triggers a pathogen response via the salicylic acid pathway and induces expression of genes involved in programmed cell death, genotoxic stress, and DNA repair. CaLCuV also altered expression of cell cycle-associated genes, preferentially activating genes expressed during S and G2 and inhibiting genes active in G1 and M. A limited set of core cell cycle genes associated with cell cycle reentry, late G1, S, and early G2 had increased RNA levels, while core cell cycle genes linked to early G1 and late G2 had reduced transcripts. Fluorescence-activated cell sorting of nuclei from infected leaves revealed a depletion of the 4C population and an increase in 8C, 16C, and 32C nuclei. Infectivity studies of transgenic Arabidopsis showed that overexpression of CYCD3;1 or E2FB, both of which promote the mitotic cell cycle, strongly impaired CaLCuV infection. In contrast, overexpression of E2FA or E2FC, which can facilitate the endocycle, had no apparent effect. These results showed that geminiviruses and RNA viruses interface with the host pathogen response via a common mechanism, and that geminiviruses modulate plant cell cycle status by differentially impacting the CYCD/retinoblastoma-related protein/E2F regulatory network and facilitating progression into the endocycle.

INTERPLAY BETWEEN ARABIDOPSIS ACTIVATING FACTORS E2FB AND E2FA IN CELL CYCLE PROGRESSION AND DEVELOPMENT

Eukaryotic E2Fs are conserved transcription factors playing crucial and antagonistic roles in several pathways related to cell division, DNA repair, and differentiation. In plants, these processes are strictly intermingled at the growing zone to produce postembryonic development in response to internal signals and environmental cues. Of the six AtE2F proteins found in Arabidopsis (Arabidopsis thaliana), only AtE2Fa and AtE2Fb have been clearly indicated as activators of E2F-responsive genes. AtE2Fa activity was shown to induce S phase and endoreduplication, whereas the function of AtE2Fb and the interrelationship between these two transcription factors was unclear. We have investigated the role played by the AtE2Fb gene during cell cycle and development performing in situ RNA hybridization, immunolocalization of the AtE2Fb protein in planta, and analysis of AtE2Fb promoter activity in transgenic plants. Overexpression of AtE2Fb in transgenic Arabidopsis plants led to striking modifications of the morphology of roots, cotyledons, and leaves that can be ascribed to stimulation of cell division. The accumulation of the AtE2Fb protein in these lines was paralleled by an increased expression of E2F-responsive G1/S and G2/M marker genes. These results suggest that AtE2Fa and AtE2Fb have specific expression patterns and play similar but distinct roles during cell cycle progression.

Two E2F Elements Regulate the Proliferating Cell Nuclear Antigen Promoter Differently during Leaf Development

E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.

Two E2F Sites in the Arabidopsis MCM3 Promoter Have Different Roles in Cell Cycle Activation and Meristematic Expression

The commitment to DNA replication is a key step in cell division control. The Arabidopsis MCM3 homologue forms part of the mini chromosome maintenance (MCM) complex involved in the initiation of DNA replication at the transition G1/S. Consistent with its role at the G1/S transition we show that the AtMCM3 gene is transcriptionally regulated at S phase. The 5′ region of this gene contains several E2F consensus binding sites, two of which match the human consensus closely and whose roles have been studied here. The identity of the two sequences as E2F binding sites has been confirmed by electrophoretic mobility shift assay analyses. Furthermore the promoter is activated by AtE2F-a and AtDP-a factors in transient expression studies. One of the E2F binding sites is shown to be responsible for the G2-specific repression of the promoter in synchronized cell suspension cultures. In contrast, the second E2F binding site has a role in meristem-specific expression in planta as deletion of this site eliminates the expression of a reporter gene in root and apical meristems. Thus two highly similar E2F binding sites in the promoter of the MCM3 gene are responsible for different cell cycle regulation or developmental expression patterns depending on the cellular environment.

AtE2F-a and AtDP-a, members of the E2F family of transcription factors, induce Arabidopsis leaf cells to re-enter S phase.

Abstract. In eukaryotes, transcription factors of the E2F family, in addition to having a role in cell proliferation, participate in regulating apoptosis, differentiation and development. In Arabidopsis thaliana, eight gene sequences have been identified as encoding E2F or DP homologues. DP proteins form heterodimers with E2Fs. The aim of this work was to characterize the functions of three of these factors: AtE2F-a, AtE2F-b and AtDP-a. Here we report that AtE2F-a and AtE2F-b transactivate a reporter gene via an E2F consensus cis-acting element in Arabidopsis protoplasts. AtE2F-a is a more potent activator than AtE2F-b. Furthermore, co-expression of the E2F partner AtDP-a, or the DNA binding protein AtPurα, modulates the activation of AtE2F-a. Taken together, these results suggest that AtE2F-a, AtE2F-b and AtDP-a share features characteristic of members of the E2F family of transcription factors. Moreover, over-expression of AtE2F-a and AtDP-a can induce differentiated, non-dividing, leaf cells to re-enter S-phase. We conclude that the transcription factor AtE2F-a plays an important role in progression into S phase, which probably correlates with its capacity to stimulate transcription.

A seed-specific Brassica napus oleosin promoter interacts with a G-box-specific protein and may be bi-directional

In Brassica napus, oleosins are expressed at high levels in the seed during the latter stages of embryo development. The cis-acting regulatory properties of an 872 bp promoter fragment of a B. napus oleosin gene were examined by analysis of β-glucuronidase (GUS) expression in transgenic tobacco plants containing an oleosin promoter-GUS transcriptional fusion. The reporter gene was expressed at high levels only in seeds, specifically in embryo and endosperm tissue and regulated throughout seed development. These data demonstrate that oleosin gene transcription is regulated in a tissue-specific and temporally regulated manner and clearly indicate that oleosin protein expression is co-ordinated primarily at the transcriptional level. Oleosin mRNA was shown to be abscisic acid (ABA) inducible and an ABA-response element in the oleosin promoter was shown to be bound by a protein factor in a sequence-specific manner. Sequence analysis of the oleosin promoter has identified several other putative cis-acting sequences which may direct oleosin gene expression. The presence of a large open reading frame in the bottom strand of the oleosin promoter (ORF2) which encodes a polypeptide similar to the ethylene-induced E4 gene of tomato is reported. A PCR-generated DNA probe containing the ORF2 sequence hybridised with a 1.4 kb transcript in total RNA extracts of a variety of tissues, including leaves and germinated seed cotyledons. This finding suggests that the oleosin gene promoter directs transcription in both directions. It is the first report of a bi-directional nuclear gene promoter in plants.In Brassica napus, oleosins are expressed at high levels in the seed during the latter stages of embryo development. The cis-acting regulatory properties of an 872 bp promoter fragment of a B. napus oleosin gene were examined by analysis of β-glucuronidase (GUS) expression in transgenic tobacco plants containing an oleosin promoter-GUS transcriptional fusion. The reporter gene was expressed at high levels only in seeds, specifically in embryo and endosperm tissue and regulated throughout seed development. These data demonstrate that oleosin gene transcription is regulated in a tissue-specific and temporally regulated manner and clearly indicate that oleosin protein expression is co-ordinated primarily at the transcriptional level. Oleosin mRNA was shown to be abscisic acid (ABA) inducible and an ABA-response element in the oleosin promoter was shown to be bound by a protein factor in a sequence-specific manner. Sequence analysis of the oleosin promoter has identified several other putative cis-acting sequences which may direct oleosin gene expression. The presence of a large open reading frame in the bottom strand of the oleosin promoter (ORF2) which encodes a polypeptide similar to the ethylene-induced E4 gene of tomato is reported. A PCR-generated DNA probe containing the ORF2 sequence hybridised with a 1.4 kb transcript in total RNA extracts of a variety of tissues, including leaves and germinated seed cotyledons. This finding suggests that the oleosin gene promoter directs transcription in both directions. It is the first report of a bi-directional nuclear gene promoter in plants.

Multiple transcription start sites of the carrot dihydrofolate reductase-thymidylate synthase gene, and sub-cellular localization of the bifunctional protein

The analysis of clones obtained by rapid amplification of the 5′ endand by primer extension of the mRNA for carrot bifunctionaldihydrofolate reductase-thymidylate synthase showed transcripts ofdiffering lengths that belonged to two sub-populations. The longertranscripts were found to contain a translation start site 147 ntupstream of, and in frame with, the one which is present in the shortertranscripts. The ORF that begins at this ATG codes for a protein of64714 Da, which is much larger than mature DHFR-TS subunit. TheN-terminusregion of this polypeptide shows features typical of plant transitpeptides. Immunogold labelling studies and immunorecognition of theplastid-containing sub-cellular fraction suggested a plastidiallocalisation of the bifunctional protein. Although plant cells wereshown to contain folate pools in plastids, in mitochondria and in thecytosol, few enzymes of the folate pathway have been associated with anysub-cellular compartment. Thus, this is the first indication for thepresence of an enzyme of the folate biosynthetic pathway in plastids.The longer transcripts revealed the presence of a TC microsatellite atthe 5′-untranslated end.

Genetic markers in cultured plant cells: Differential sensitivities to amethopterin, azetidine-2-carboxylic acid and hydroxyurea

Abstract The effects of three growth inhibitors, amethopterin, L-azetidine-2-carboxylic acid and hydroxyurea, have been tested on cell cultures from 6 plant species. The results show that naturally-occurring differences in drug sensitivity are present and can be easily demonstrated by assaying the effects of the drugs on the growth of cells in suspension culture under strictly-controlled experimental conditions. Similar differences are also evident in cells of the plants from which the cultures were derived. In order to determine the usefulness of these different sensitivities as selective tools, a culture flask was devised in which the effect of the drugs on a mixed culture of two cell species can be determined.

Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.

Characterization of a carrot cell line resistant to azetidine-2-carboxylic acid

Abstract The mechanism of resistance of a carrot cell line to azetidine-2-carboxylic acid (AZCA) was investigated. Two main alterations were observed in the resistant cells: an over-production of proline and of some other amino acids and a reduced rate of uptake either of AZCA or of other amino acids tested. This latter phenomenon appears to be a secondary effect due to the increased internal amino acid concentration, which inhibits the activity of the amino acid carrier(s).