Plant cell death is an integral part of plant growth and development and can also occur as a reaction to wounding or pathogen attack [1–3]. A well-known example of regulated cell death during normal plant development takes place during the maturation of xylem cells . Plant cell death can also be seen during anther development , in lateral root cap cells at the end of the lateral root cap , and in many other tissue types .
Not surprisingly, mutations that lead to the premature death of certain plant cells have a detrimental effect on plant development. In the Arabidopsis mosaic death1 (mod1) mutant, for example, patches of cells within different organs die prematurely, leading to severe growth defects . The Arabidopsis tornado (trn) mutant shows misspecification of some root epidermal cells as lateral root cap cells. These cells die in the elongation zone, as lateral root cap cells would normally do, and this results in the formation of gaps in the epidermis. As a consequence, trn roots are impaired in growth .
To study plant development, it is essential to have simple means to identify non-viable cells within living plant tissue. Although different modes of plant cell death exist in plants , one common characteristic of cell death is that the plasma membrane of dying cells ceases to function as a selective barrier. Hence, some dyes that cannot penetrate through the plasma membrane of living cells can be used to stain internal components of non-viable cells (= dye exclusion method). For example, trypan blue has been used to stain non-viable cells during pathogen-induced cell death (e.g. ). Non-viable cells were stained blue and could be observed with light microscopy. However, as fluorescence microscopy becomes a more and more important tool for the study of plant development, there is also a need for fluorescent stains to visualise non-viable cells.
The fluorescent stain SYTOX green exhibits bright fluorescence when bound to DNA, but cannot penetrate the plasma membrane of viable plant cells. These properties make the dye a useful tool for the detection of membrane permeabilisation in unicellular organisms [10, 11]. In higher plants, however, SYTOX green is most commonly used to stain the nuclei of dead cells in fixed tissues . We have previously used SYTOX green to identify non-viable cells in embryos of mutant plants . Here we show that this dye and its spectral variants, SYTOX blue and SYTOX orange, can be easily used to identify non-viable cells within plant tissue. Moreover, the spectral properties of the SYTOX dyes allows them to be used in combination with other fluorescent stains or in plants expressing fluorescent proteins, such as the gene for GREEN FLUORESCENT PROTEIN (GFP).