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Dominique Robertson

Publications and source records attributed to Dominique Robertson.

6 recordsLinked to original sources
PubMed2002

Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants.

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

Agrobacterium tumefaciens↗
PubMed2002

Geminivirus-based vectors for gene silencing in Arabidopsis.

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.

Animals↗
PubMed2002

Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli.

Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.

Arabidopsis↗
PubMed2002

Host DNA replication is induced by geminivirus infection of differentiated plant cells.

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

Aphidicolin↗
PubMed2002

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.

Base Sequence↗