A NOVEL METHOD FOR QUANTITATIVE IN SITU HYBRIDISATION IN PLANTS REVEALS REGULATION OF A ZINC TRANSPORTER IN THE CD/ZN HYPERACCUMULATOR THLASPI CAERULESCENS (GANGES)

Authors: KUPPER, HENDRIK - CORNELL UNIVERSITY; SEIB, LAURA - CORNELL UNIVERSITY; Kochian, Leon; SIVAGURU, MAYANDI - UNIV. OF ILLINOIS; Hoekenga, Owen

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2006
Publication Date: 4/4/2007
Citation: Kupper, H., Seib, L.O., Kochian, L.V. A novel method for quantitative in situ hybridisation in plants reveals regulation of a zinc transporter in the cd/zn hyperaccumulator thlaspi caerulescens (ganges). Plant Journal. 50:159-175.

Interpretive Summary: As our understanding of the molecular biology of plant micronutrient and heavy metal transport advances, researchers are finding that the genes encoding these metal transporters are organized into families of related transporter genes and the proteins they encode. It appears that members of these transporter gene families might have the same function, but are believed to operate in different cell and/or tissues. Thus, one of the most important research approaches in studying these transporters is to determine what cells the transport genes are functioning in. Unfortunately, this research has been stymied by the lack of a straight-forward method for determining the cell-specific localization of transporter gene function. In this paper a new microscopic technique is described that allows researchers to easily visualize in which cells a particular gene is turned on (expressed). The technique is based on the fact that when a gene is expressed, the cell makes a nucleotide sequence that is a mirror image of the gene DNA sequence. This nucleotide, known as messenger RNA (mRNA), is the template for the protein that is subsequently synthesized. The new technique involves the synthesis in a test tube of a synthetic nucleotide that is a mirror image of a portion of the mRNA molecule. This synthesized molecule will bind tightly to the mRNA molecule and because it is labeled with a fluorescent compound, the cells where the gene are expressed will give off fluorescent light which can be detected with a special microscope. Thus we have been able to quickly determine in which cells of the plant root or shoot a particular micronutrient or heavy metal transporter gene is expressed, which is yielding valuable information about the function of this transporter in the plant.

Technical Abstract: A novel quantitative in situ hybridisation technique (QISH) for plants is described and used to elucidate expression patterns for a Zn transporter in the Zn/Cd hyperaccumulator Thlaspi caerulescens. The technique employs direct hybridisation of fluorescently labelled gene-specific oligonucleotides in large tissue pieces combined with optical sectioning. It dramatically increases the throughput compared to conventional antibody and microtome-based in situ mRNA hybridisation methods, while simultaneously eliminating artifact-prone preparation steps that prevent reliable quantification in conventional techniques. The key feature of this technique is the quantification of gene expression using housekeeping genes (cytosolic GAPDH and 18s rRNA) as internal standards. In this paper, we use the technique to investigate cellular aspects of plant heavy metal uptake, which is a topic important for both plant stress physiology and human nutrition. This initial study focused on ZNT1 mRNA levels in leaves, the site of Cd/Zn hyperaccumulation in T. caerulescens. ZNT1 belongs to the ZIP family of micronutrient transporters and appears to mediate Zn uptake into leaves and roots. In young leaves of low Zn-grown plants, ZNT1 mRNA was abundant mainly in the bundle sheath of the veins as well as in adjacent mesophyll cells. In mature leaves, its abundance was rather homogeneous in all photosynthetically active cell types. In response to high zinc levels, ZNT1 transcript abundance in the spongy mesophyll cells was strongly reduced, but was less affected in other cell types. Additionally, under severe Cd2+ toxicity stress, ZNT1 transcript abundance increased in large epidermal cells that are known to be an important sink for heavy metals in leaves of T. caerulescens.