Submitted to: New Phytologist
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/19/2009
Publication Date: 1/1/2010
Citation: Kuepper, H., Kochian, L.V. 2010. Transcriptional regulation of metal transport genes and mineral nutrition during acclimation to cadium and zinc in the Cd/Zn hyperaccumulator, Thlaspi caerulescens (Ganges population). New Phytologist. 185(1):114-129. 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 which cell types the transport genes are functioning. 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: We investigated changes in mineral nutrient uptake and cellular expression levels of metal transporter genes using the Cd/Zn hyperaccumulator Thlaspi caerulescens. We analyzed those changes genesis under different long-term (one year) treatments of the plants with zinc and cadmium using quantitative mRNA in situ hybridization (QISH), combined with ICP-AES measurements. Our experiments revealed that heavy metal transporter gene expression (ZNT1, ZNT5 and MTP1=ZTP1˜ZAT investigated here) drastically changes not only as a function of plant metal nutrition/toxicity, but even more depending on the plant and leaf age. ZNT1 mRNA was abundant mainly in mature leaves of young plants, while ZNT5 mRNA was abundant mainly in young leaves, and MTP1 was expressed equally in young leaves of both young and mature plants. Further, significantly different cellular expression patterns were found for ZNT1 and ZNT5, which are closely related members of the ZIP (ZRT- and IRT-related protein) family of micronutrient/heavy metal transporters. While ZNT1 was expressed most strongly in mesophyll and bundle sheath cells, ZNT5 was highly expressed in the non-photosynthetic epidermal metal storage cells and bundle sheath cells. Plant exposure to cadmium inhibited the uptake of Zn, Fe and Mn, which could be due to competition for the same transporters, but also could be due to Cd-induced alteration in metal transporter gene expression. The changes in cellular expression levels of ZNT1, ZNT5 and MTP1 could also be part of a long-term acclimation to Cd toxicity. Finally, defence against Cd toxicity was confirmed to involve enhanced uptake of Mg, Ca and S, which is discussed in detail.