The Ubiquitin Receptor DA1 Regulates Seed and Organ Size by Modulating the Stability of the Ubiquitin-Specific Protease UBP15/SOD2 in Arabidopsis

Abstract

Although the control of organ size is a fundamental question in developmental biology, little is known about the genetic and molecular mechanisms that determine the final size of seeds in plants. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligases DA2 and ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to restrict seed growth in Arabidopsis thaliana. Here, we describe UBIQUITIN-SPECIFIC PROTEASE15 (UBP15), encoded by SUPPRESSOR2 OF DA1 (SOD2), which acts maternally to regulate seed size by promoting cell proliferation in the integuments of ovules and developing seeds. The sod2/ubp15 mutants form small seeds, while overexpression of UBP15 increases seed size of wild-type plants. Genetic analyses indicate that UBP15 functions antagonistically in a common pathway with DA1 to influence seed size, but does so independently of DA2 and EOD1. Further results reveal that DA1 physically associates with UBP15 in vitro and in vivo and modulates the stability of UBP15. Therefore, our findings establish a genetic and molecular framework for the regulation of seed size by four ubiquitin-related proteins DA1, DA2, EOD1, and UBP15 and suggest that they are promising targets for increasing seed size in crops.

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

“…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). We showed that TCP15 and the related TCP22 as well as UBP15 were cleaved by DA1 peptidase activity (Fig.…”

“…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).…”

“…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). …”

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

“…DA1 encodes a ubiquitin receptor that has been proposed to be involved in ubiquitin-mediated protein degradation (Li et al, 2008;Xia et al, 2013;Du et al, 2014). To test whether TCP14/15 protein levels might be affected by proteasome-dependent mechanisms, we treated Arabidopsis TCP14 pro :TCP14-GUS and TCP15 pro :TCP15-GUS transgenic lines (Kieffer et al, 2011;Li et al, 2012) with the proteasome inhibitor MG132.…”