PhyscomitrellaCyclin-Dependent Kinase A Links Cell Cycle Reactivation to Other Cellular Changes during Reprogramming of Leaf Cells    

Abstract

During regeneration, differentiated plant cells can be reprogrammed to produce stem cells, a process that requires coordination of cell cycle reactivation with acquisition of other cellular characteristics. However, the factors that coordinate the two functions during reprogramming have not been determined. Here, we report a link between cell cycle reactivation and the acquisition of new cell-type characteristics through the activity of cyclin-dependent kinase A (CDKA) during reprogramming in the moss Physcomitrella patens. Excised gametophore leaf cells of P. patens are readily reprogrammed, initiate tip growth, and form chloronema apical cells with stem cell characteristics at their first cell division. We found that leaf cells facing the cut undergo CDK activation along with induction of a D-type cyclin, tip growth, and transcriptional activation of protonema-specific genes. A DNA synthesis inhibitor, aphidicolin, inhibited cell cycle progression but prevented neither tip growth nor protonemal gene expression, indicating that cell cycle progression is not required for acquisition of protonema cell-type characteristics. By contrast, treatment with a CDK inhibitor or induction of dominant-negative CDKA;1 protein inhibited not only cell cycle progression but also tip growth and protonemal gene expression. These findings indicate that cell cycle progression is coordinated with other cellular changes by the concomitant regulation through CDKA;1.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens

“…Fortyeight hours after leaf detachment, expression of the protonemaspecific reporters RM09 and RM55 (Ishikawa et al, 2011) was present in all cells that underwent divisions in both the wild type and the double deletion mutant line Δppwox13lab#186 (Fig. 3D), regardless of whether tip growth was initiated or not.…”

“…In order to investigate PpWOX13L expression during the induced formation of stem cells, we used a detached leaf assay, in which chloronema stem cells are established from reprogrammed differentiated leaf cells (Ishikawa et al, 2011). We observed that whole-leaf PpWOX13LA and PpWOX13LB transcript levels transiently increased after detachment (Fig.…”

“…During stem cell formation from differentiated leaf cells, several cellular changes, including the re-entry into cell cycle and the initiation of tip growth, occur (Ishikawa et al, 2011). Our results indicate that during this process: (1) the expression levels of PpWOX13LA and PpWOX13LB genes are upregulated, (2) both genes are redundantly required to initiate cellular outgrowth but not for entry into the cell cycle, and (3) both genes are required for upregulation of genes predicted to encode cell wall loosening factors.…”

“…In non-seed plants, differentiated cells can generate stem cells without the requirement of exogenously supplied hormones and without forming a callus (Chopra and Kumra, 1988;Raghavan, 1989). In the moss Physcomitrella patens (Physcomitrella), differentiated cells at the wound margin of detached gametophore leaves are reprogrammed into apical stem cells, which subsequently produce the filamentous chloronemata by asymmetric divisions (Prigge and Bezanilla, 2010;Ishikawa et al, 2011). Although several transcription factors involved in the induction of reprogramming have been characterized in flowering plants (Banno et al, 2001;Gordon et al, 2007;Sugimoto et al, 2010;Iwase et al, 2011), no regulators have been reported in non-seed plants.…”

Construction of the intermediate vector pVBG2307 by incorporating vital elements of expression vectors pBI121 and pBI221

“…The absence of NOS terminal (Ishikawa et al, 2011) on the right border of pCAMBIA2300 paved the way for extracting the NOS element from vector pBI221 by the action of EcoRI and SacI and successfully ligated into pCAMBIA2300 (Figure 1).…”