The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis.

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.

The stochastic silencing phenotype of Arabidopsis morc6 mutants reveals a role in efficient RNA-directed DNA methylation.

The RNA-directed DNA methylation (RdDM) pathway is of central importance to the initiation and maintenance of transcriptional gene silencing in plants. DNA methylation is directed to target sequences by a mechanism that involves production of small RNAs by RNA polymerase IV and long non-coding RNAs by RNA polymerase V. DNA methylation then leads to recruitment of histone-modifying enzymes, followed by establishment of a silenced chromatin state. Recently MORC6, a member of the microrchidia (MORC) family of adenosine triphosphatases (ATPases), has been shown to be involved in transcriptional gene silencing. However, reports differ regarding whether MORC6 is involved in RdDM itself or acts downstream of DNA methylation to enable formation of higher-order chromatin structure. Here we demonstrate that MORC6 is required for efficient RdDM at some target loci, and, using a GFP reporter system, we found that morc6 mutants show a stochastic silencing phenotype. By using cell sorting to separate silenced and unsilenced cells, we show that release of silencing at this locus is associated with a loss of DNA methylation. Thus our data support a view that MORC6 influences RdDM and that it is not acting downstream of DNA methylation. For some loci, efficient initiation or maintenance of DNA methylation may depend on the ability to form higher-order chromatin structure.