Abstract
The ability to induce Arabidopsis protoplasts to dedifferentiate and divide provides a convenient system to analyze organelle dynamics in plant cells acquiring totipotency. Using peroxisome-targeted fluorescent proteins, we show that during protoplast culture, peroxisomes undergo massive proliferation and disperse uniformly around the cell before cell division. Peroxisome dispersion is influenced by the cytoskeleton, ensuring unbiased segregation during cell division. Considering their role in oxidative metabolism, we also investigated how peroxisomes influence homeostasis of reactive oxygen species (ROS). Protoplast isolation induces an oxidative burst, with mitochondria the likely major ROS producers. Subsequently ROS levels in protoplast cultures decline, correlating with the increase in peroxisomes, suggesting that peroxisome proliferation may also aid restoration of ROS homeostasis. Transcriptional profiling showed up-regulation of several peroxisome-localized antioxidant enzymes, most notably catalase (CAT). Analysis of antioxidant levels, CAT activity and CAT isoform 3 mutants (cat3) indicate that peroxisome-localized CAT plays a major role in restoring ROS homeostasis. Furthermore, protoplast cultures of pex11a, a peroxisome division mutant, and cat3 mutants show reduced induction of cell division. Taken together, the data indicate that peroxisome proliferation and CAT contribute to ROS homeostasis and subsequent protoplast division induction.
Highlights
Plant protoplasts provide an invaluable experimental system to study cellular processes such as signal transduction (Sheen, 2001), cell wall regeneration (Leucci et al, 2007), the role of stress and hormones (Pasternak et al, 2002) and transient gene expression (Yoo et al, 2007)
Given the significance of peroxisomes in plant metabolism, we have investigated the dynamics of peroxisomes in cultured protoplasts acquiring totipotency in relation to proliferation, inheritance and reactive oxygen species (ROS) homeostasis
There was a strong, negative correlation between ROS level and peroxisome number in protoplasts (Figure 4D; Supplementary Figure S3D). These results indicate that ROS levels are perturbed by protoplast isolation, but ROS homeostasis is largely restored before cell division and concomitant with an increase in peroxisome number
Summary
Plant protoplasts provide an invaluable experimental system to study cellular processes such as signal transduction (Sheen, 2001), cell wall regeneration (Leucci et al, 2007), the role of stress and hormones (Pasternak et al, 2002) and transient gene expression (Yoo et al, 2007). Differentiated protoplasts can be induced to acquire totipotency and regenerate (Takebe et al, 1971; Chupeau et al, 2013), providing a convenient system to analyze organelle dynamics and inheritance in plant cells. In this system we have previously examined chloroplasts, the endoplasmic reticulum (ER; Sheahan et al, 2004), mitochondria (Sheahan et al, 2004, 2005), and vacuoles (Sheahan et al, 2007a); in relation to the cytoskeleton. Peroxisomes, ROS and cell regeneration provided insights into the proliferation and inheritance of these organelles and the identification of “massive mitochondrial fusion.” Given the significance of peroxisomes in plant metabolism (del Rio et al, 2006; Palma et al, 2009; Hu et al, 2012), we have investigated the dynamics of peroxisomes in cultured protoplasts acquiring totipotency in relation to proliferation, inheritance and reactive oxygen species (ROS) homeostasis
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