The Microtubule-Associated Protein CLASP Sustains Cell Proliferation through a Brassinosteroid Signaling Negative Feedback Loop

Abstract

The capacity for sustained cell division within the plant meristem is a critical determinant of organ structure and performance. This capacity is diminished in mutants lacking the microtubule-associated protein CLASP and when brassinosteroid signaling is increased. Here, we discovered that CLASP is both targeted by and promotes activity of the brassinosteroid pathway in Arabidopsis root apical meristems. We show that enhanced brassinosteroid signaling reduces CLASP transcript and protein levels, dramatically shifts microtubule organization, and reduces the number of cells in the meristem. In turn, CLASP, which tethers sorting nexin 1 vesicles to microtubules, sustains brassinosteroid signaling by fostering retrieval of endocytosed BRI1 receptors to the plasma membrane. clasp-1 null mutants have dampened brassinosteroid (BR)-mediated transcriptional activity and responses. Global transcript profiling confirmed the collapse of cell-cycle activity in clasp-1 and identified CLASP-mediated hormone crosstalk. Together, these findings reveal an unprecedented form of negative feedback supporting meristem homeostasis.

The microtubule-associated protein CLASP is translationally-regulated in light-dependent root apical meristem growth

“…When plants were deprived of light and a source of sucrose, the auxin transporter PIN2 and BR receptor BRI1 were less abundant at the plasma membrane and accumulated in the vacuole. This redistribution is consistent with a lack of CLASP, which normally promotes recycling of PIN2 and BRI1 to the plasma membrane through tethering of SNX1 vesicles (Ambrose et al, 2013; Ruan et al, 2018). Cells in the division zone of roots normally have several small vacuoles, whereas an enlarged central vacuole is characteristic of elongating and differentiated cells.…”

“…Previous work has shown that CLASP promotes recycling of the BRI1 receptor (Ruan et al, 2018) and PIN2 transporter (Ambrose et al, 2013) to the plasma membrane. Since CLASP protein levels were depleted in dark-grown seedlings, we hypothesized that the distribution and abundance of BRI1 and PIN2 would be affected in light-versus dark-grown seedlings.…”

“…Our study has identified a very specific and specialized regulatory system that controls CLASP protein levels in response to light/dark conditions, and further underlines the importance of CLASP as a regulator of meristem size in the Arabidopsis root tip (Ambrose et al, 2007). CLASP was previously shown to be transcriptionally downregulated by the BR signalling pathway (Ruan et al, 2018), but regulation at the translational level has not yet been demonstrated. Our results indicate a general dampening of the BR signalling pathway in root meristems under dark conditions, which is consistent with a study showing BZR1 degradation when carbon availability was limited (Zhang et al, 2016), and another study that found DWF4 transcript was reduced in dark-grown root tips (Sakaguchi and Watanabe, 2017).…”

“…The 35S pro : YFP-CLASP reporter was described in (Kirik et al, 2007) and given to us by Dr. Viktor Kirik. Microtubules were visualized with lines expressing UBQ1 pro : GFP-MBD (Ruan et al, 2018). The BRI1 pro : BRI1-GFP was from Dr. Joanne Chory and described in (Geldner et al, 2007).…”

“…The CLASP-SNX1 interaction also promotes recycling of the BR receptor BRI1 to the PM, thus enhancing BR signalling. Ruan et al (2018) identified a negative feedback loop whereby the BR-activated transcription factors BZR1 and BZR2/BES1 bind to the CLASP promoter and repress CLASP gene expression. The downregulation of CLASP , through application of exogenous BR or in mutants with constitutively active BR signalling, is strongly correlated with premature exit of cells from the division zone, producing a smaller meristem phenotype similar to that observed in clasp-1 mutants.…”