Abstract
In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed ACTIN (ACT) genes. Of the eight expressed isoforms, ACT2, ACT7, and ACT8 are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in act7 mutants, but not act2-1 or act8-2. ACT7 expression was upregulated during protoplast culture whereas ACT2 and ACT8 expression did not substantially change. In act2-1, ACT7 expression increased in response to loss of ACT2, whereas in act7-1, neither ACT2 nor ACT8 expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in act7-1 was only two-thirds that of wild-type or act2-1 after 96 h culture. Over-expression of ACT2 and ACT8 under control of ACT7 regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in act7 mutants, rather than strong functional specialization of the ACT7 isoform.
Highlights
The actin cytoskeleton facilitates numerous cellular processes required for the correct functioning and development of multicellular eukaryotes
Prior to the first cell division in cultured tobacco mesophyll protoplasts, chloroplasts move via protoplast culture, this did not happen in the act7 mutants and total actin levels remained low
We confirmed that a similar relocation and perinuclear clustering of clustering, this indicates that the loss of chloroplast clustering in the act7 mutants is probably due to chloroplasts occurred in cultured Arabidopsis mesophyll protoplasts (Figure 1)
Summary
The actin cytoskeleton facilitates numerous cellular processes required for the correct functioning and development of multicellular eukaryotes. In Arabidopsis thaliana (Arabidopsis), eight expressed actin isoforms exist [2] Based on their phylogenetic relationship and expression pattern, Arabidopsis actins are classified as either vegetative or reproductive, with each class being expressed predominantly in vegetative or reproductive tissues, respectively. The sequence divergence between individual actin isoforms in plants is greater than the divergence between non-muscle and muscle isoforms of actin in animals, suggesting the potential for plant actin isoforms to perform discrete functions within the cell [3,4]. Such divergence may reflect developmental rather than intracellular specialization. While the number of actin genes varies dramatically between different plant species, with the eudicot Medicago truncatula containing only four actin genes but with the monocot Zea mays containing 21 separate genes, the division of actins into vegetative and reproductive classes is broadly conserved across plant species [4,5]
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