A rapid method for detection of plant gene transcripts from single epidermal, mesophyll and companion cells of intact leaves

Abstract

SummaryA protocol for the detection of gene transcripts from single plant cells in living, undamaged plant tissue is described. Samples of leaf epidermal, mesophyll and companion cells were extracted by using glass microcapillaries and directly subjected to RT±PCR without any puri®cation steps nor time consuming construction of cDNA libraries. The procedure is not restricted to surface cells or outer cell layers. Even cells from the central region of leaves could be harvested. For identi®cation, companion cells were labelled by expression of the green¯uorescent protein under control of a companion cell speci®c promoter. The described method is applicable to a wide range of plants and genes with different expression levels.

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells

“…This work extends that of Karrer et al (1995); Brandt et al (1999) and Koroleva et al (2001) in establishing SC-RT-PCR as a technique applicable to plant cells. Here we have demonstrated that the technique can be applied successfully to identify patterns of expression of the different but related members of a single family of homologous genes, each of which is present at only moderate abundance (Karrer et al 1995).…”

“…Naturally occurring transcripts (in this case RBCS) can be used as both positive and negative controls. This can be further extended using tissue-specific transgenes (Brandt et al 1999). Here we have used a simple PCR detection system to determine whether the level of particular individual transcripts is greater than an as yet not fully quantified detection limit.…”

“…Using a binocular microscope and a silanized glass microcapillary previously sterilized at 200°C, samples of sap were isolated from single cells, or from groups of adjacent cells of the same type, from rosette leaves of intact plants (Tomos et al 1994;Brandt et al 1999). The turgor pressure of each cell was sufficient to drive a proportion of its contents into the capillary upon puncture.…”

“…A fine microcapillary is employed to extract cell sap from the individual cells, after which RT-PCR is performed (Lambolez et al 1992;Karrer et al1995;Brandt et al 1999;Koroleva et al 2001). The critical advantage of single-cell (SC)-RT-PCR is that it enables expression to be measured directly from living tissue at the finest spatial resolution possible, that of the individual cell.…”

A micromethod for high throughput RNA extraction in forest trees

“…1). Moreover, the possibility to extract RNA from specific and small organs would allow the monitoring of gene expression at a higher tissue resolution (Brandt et al, 1999). Consequently, this micro-scale method offers the possibility to study specific gene expression during developmental processes (i.e.…”