The Ubiquitin Receptors DA1, DAR1, and DAR2 Redundantly Regulate Endoreduplication by Modulating the Stability of TCP14/15 in Arabidopsis

Abstract

Organ growth involves the coordination of cell proliferation and cell growth with differentiation. Endoreduplication is correlated with the onset of cell differentiation and with cell and organ size, but little is known about the molecular mechanisms linking cell and organ growth with endoreduplication. We have previously demonstrated that the ubiquitin receptor DA1 influences organ growth by restricting cell proliferation. Here, we show that DA1 and its close family members DAR1 and DAR2 are redundantly required for endoreduplication during leaf development. DA1, DAR1, and DAR2 physically interact with the transcription factors TCP14 and TCP15, which repress endoreduplication by directly regulating the expression of cell-cycle genes. We also show that DA1, DAR1, and DAR2 modulate the stability of TCP14 and TCP15 proteins in Arabidopsis thaliana. Genetic analyses demonstrate that DA1, DAR1, and DAR2 function in a common pathway with TCP14/15 to regulate endoreduplication. Thus, our findings define an important genetic and molecular mechanism involving the ubiquitin receptors DA1, DAR1, and DAR2 and the transcription factors TCP14 and TCP15 that links endoreduplication with cell and organ growth.

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

“…7B), but we could not consistently detect TCP14 cleavage by DA1 or DAR1. These data show that DA1 peptidase activity can cleave UBP15, which promotes cell proliferation, and TCP15, which inhibits endoreduplication (Du et al 2014;Peng et al 2015).…”

“…We showed previously that TCP14 and TCP15 function downstream from DA1 and other family members in controlling organ size in Arabidopsis, and reduced function of DA1 family members led to increased TCP14 and TCP15 protein levels (Peng et al 2015). Similarly, levels of UBP15 protein, which promotes cell proliferation (Liu et al 2008) and also functions downstream from DA1, were increased in the da1-1 reduced-function mutant (Du et al 2014).…”

“…These patterns of ubiquitylation are shown in Figure 2D. DA1 and four other family members have multiple ubiquitin interaction motifs (UIMs) that interact with ubiquitin (Li et al 2008;Peng et al 2015). UIMs are part of a larger class of ubiquitin-binding domains (UBDs) formed from a single α helix that is often found in multiple arrays (Hicke et al 2005;Husnjak and Dikic 2012).…”

“…The increased levels of UBP15 (Du et al 2014), TCP14, and TCP15 proteins (Peng et al 2015) observed in the da1-1 mutant suggested that DA1 activity may reduce the stability of these proteins by peptidase-mediated cleavage. Figure 7A shows that DA1 peptidase cleaved UBP15 close to its C terminus when transiently expressed together in da1-ko1 dar1-1 protoplasts.…”

“…Members of the DA1 family also limit cell proliferation (Li et al 2008), and loss-offunction mutations in BB and DA2 interact synergistically with the da1-1 allele of DA1 to increase organ and seed size in Arabidopsis (Li et al 2008;Xia et al 2013), suggesting that one of their growth-limiting activities is mediated by enhancing the growth-repressive activity of DA1 family members. Genetic analyses showed that DA1 reduced the stability of both UBP15 (Du et al 2014), a deubiquitylation enzyme promoting cell proliferation (Liu et al 2008), and TEOSINTE BRANCED 1/CYCLOI-DEA/PCF 14 (TCP14) and TCP15 proteins (Peng et al 2015), which repress endoreduplication by transcriptional control of RETINOBLASTOMA-RELATED1 (RBR1) and CYCLIN A2;3 (CYCA2;3) gene expression (Li et al 2012). …”