As we noted that these and other tonoplast proteins move in distinct cell types via distinct pathways to the tonoplast, we developed a protocol to efficiently produce and transiently transform protoplasts from petunia ( Petunia hybrida) petals and compared their features with those of the widely used leaf mesophyll protoplasts and of cells in intact tissue. We recently discovered a new pathway involved in the acidification of the vacuole in epidermal cells and identified via mutants several key components such as the tonoplast H + P-ATPase PH5 ( Verweij et al., 2008b) and a novel tonoplast pump encoded by PH1 (F. The opinion of most researchers about protoplasts falls in two opposite categories: “any type of protoplasts are fine for me as long as they work (and leaf protoplasts are the easiest)” or “protoplasts are just not reliable, so you should not use them.” Here, we try to convince researchers that both views are wrong and that protoplasts can give highly reliable results, if used in an appropriate way based on a proper understanding of their features. No specific studies define whether tissue specificity is retained within the time frame required for isolation, transformation, and transient expression analysis.
This is possibly a consequence of the little attention paid so far to the biological state of freshly made protoplasts or the kinetics by which cell identity changes. Therefore, protoplasts are generally considered to lose their identity and to be comparable with cells from suspension cultures, which would make them unsuitable to investigate cell-type or tissue-specific processes. Protoplasts cultured over a period of weeks can regenerate entire plants, indicating that they undergo dedifferentiation. In contrast to animal cells, plant cells can easily change their identity when taken out of their environment, and when cell lineages are disrupted, and the position of cells is altered, they rapidly change identity according to their new position ( van den Berg et al., 1995). It is assumed that the observations done in protoplasts from this tissue can be extended to (any) intact tissue, but no reports have been published in which this aspect has been studied in detail. Geldner et al., 2009) as characterized by their expression in leaf cells (intact tissue or protoplasts). Several collections of markers for endocellular compartments and structures are available (e.g. They are generally believed to provide information about the cellular function of proteins normally expressed in other cell types. The most used sources of protoplasts are leaf mesophyll ( Sheen, 2001) as universal system for transient expression of plant genes ( Yoo et al., 2007). Protoplasts represent a very convenient system as they can efficiently be transformed with several DNA constructs at the same time to study the colocalization and/or interactions of differently labeled proteins ( Walter et al., 2004 Chen et al., 2006), and because they allow better imaging (higher resolution) compared to cells in an intact tissue. Transient transformation of protoplasts isolated from an adult tissue has been successfully used for developmental studies ( Sheen, 2001), biochemical analysis ( Sheahan et al., 2007), to examine the influence of hormones and stress factors ( Meyer et al., 1984 Pasternak et al., 2002), to investigate cell wall regeneration ( Leucci et al., 2007), to determine the subcellular localization of tagged proteins ( Goodman et al., 2004), and to analyze protein interactions ( Walter et al., 2004). Here, we show that protoplasts are a very reliable experimental system, as long as we carefully chose their source. Plant protoplasts are often used as experimental material without paying attention to the tissue they are isolated from (a protoplast is a protoplast), whereas in other cases, they are considered not sufficiently able to reproduce the in planta situation.
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