The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis

Abstract

ORCID ID: 0000-0002-0025-4444 (Y.L.).Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.

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

“…These data indicated that BB may influence leaf final size at earlier stages of growth than DA1. We demonstrated previously that DA1 and DA2 physically interact (Xia et al 2013). Pull-down experiments showed that GST-tagged DA1 also interacted with HIS-tagged BB but not with HIStagged BBR (BB-related; At3g19910), a close homolog of BB ( Fig.…”

“…We previously identified genetic interactions between the da1-1 allele of DA1 and genes encoding the RING E3 ligases BB (Li et al 2008) and DA2 (Xia et al 2013) that led to synergistic increases in seed and organ sizes. In this study, we used the da1-1-enhancing allele of BB called eod1-2 (Li et al 2008) and refer to the mutant version as bbeod1-2 and the wild-type version as BB.…”

“…The RING E3 ligases Big Brother (BB) (Disch et al 2006) and DA2 (Xia et al 2013) limit the duration of cell proliferation during organ growth. 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.…”

“…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).…”

Networks controlling seed size in Arabidopsis

“…DA2 447 and enhancer of DA1 (EOD1) encode proteins with E3 448 ubiquitin ligase activity and are also negative regulators of 449 seed size, as shown by the enlarged seeds of single mutants 450 da2-1 and eod1. They may act synergistically with DA1,as 451 observed by the enhanced seed size of da1-1 da2-1 and 452 da1-1 eod1 double mutants in comparison with da1 mutant 453 (Xia et al 2013). EOD3 encodes cytochrome P450 454 CYP78A6.…”