Seed dormancy directly affects the phenotype of pre-harvest sprouting, and ultimately affects the quality and yield of rice seeds. Although many genes controlling seed dormancy have been cloned from cereals, the regulatory mechanisms controlling this process are complex, and much remains unknown. The MAPK cascade is involved in many signal transduction pathways. Recently, MKK3 has been reported to be involved in the regulation of seed dormancy, but its mechanism of action is unclear. We found that MKKK62-overexpressing rice lines (OE) lost seed dormancy. Further analyses showed that the abscisic acid (ABA) sensitivity of OE lines was decreased. In yeast two-hybrid experiments, MKKK62 interacted with MKK3, and MKK3 interacted with MAPK7 and MAPK14. Knock-out experiments confirmed that MKK3, MAPK7, and MAPK14 were involved in the regulation of seed dormancy. The OE lines showed decreased transcript levels of OsMFT, a homolog of a gene that controls seed dormancy in wheat. The up-regulation of OsMFT in MKK3-knockout lines (OE/mkk3) and MAPK7/14-knockout lines (OE/mapk7/mapk14) indicated that the MKKK62-MKK3-MAPK7/MAPK14 system controlled seed dormancy by regulating the transcription of OsMFT. Our results showed that MKKK62 negatively controls seed dormancy in rice, and that during the germination stage and the late stage of seed maturation, ABA sensitivity and OsMFT transcription are negatively controlled by MKKK62. Our results have clarified the entire MAPK cascade controlling seed dormancy in rice. Together, these results indicate that protein modification by phosphorylation plays a key role in controlling seed dormancy.

Endoplasmic reticulum-associated degradation (ERAD) is a unique mechanism to degrade misfolded proteins via complexes containing several highly-conserved ER-anchored ubiquitin ligases such as HMG-CoA reductase degradation1 (Hrd1). Arabidopsis has a similar Hrd1-containing ERAD machinery; however, our knowledge of this complex is limited. Here we report two closely-related Arabidopsis proteins, Protein Associated With Hrd1-1 (PAWH1) and PAWH2, which share a conserved domain with yeast Altered Inheritance of Mitochondria24. PAWH1 and PAWH2 localize to the ER membrane and associate with Hrd1 via EMS-mutagenized Bri1 Suppressor7 (EBS7), a plant-specific component of the Hrd1 complex. Simultaneously elimination of two PAWHs constitutively activates the unfolded protein response and compromises stress tolerance. Importantly, the pawh1 pawh2 double mutation reduces the protein abundance of EBS7 and Hrd1 and inhibits degradation of several ERAD substrates. Our study not only discovers additional plant-specific components of the Arabidopsis Hrd1 complex but also reveals a distinct mechanism for regulating the Hrd1 stability.

Bri1-301 is a temperature-sensitive misfolded brassinosteroid receptor that requires kinase activity to promote growth and is rapidly degraded in the endoplasmi

Brassinosteroids (BRs) play vital roles in the plant life cycle and synthetic BRs are widely used to increase crop yield and plant stress tolerance. Among them are 24R-methyl-epibrassinolide (24-EBL) and 24S-ethyl-28-homobrassinolide (28-HBL), which differ from brassinolide (BL, the most active BR) at the C-24 position. Although it is well known that 24-EBL is 10% active as BL, there is no consensus on the bioactivity of 28-HBL. A recent outpouring of research interest in 28-HBL on major crops accompanied with a surge of industrial-scale synthesis that produces mixtures of active (22R,23R)-28-HBL and inactive (22S,23S)-28HBL, demands a standardized assay system capable of analyzing different synthetic “28-HBL” products. In this study, the relative bioactivity of 28-HBL to BL and 24-EBL, including its capacity to induce the well-established BR responses at molecular, biochemical, and physiological levels, was systematically analyzed using the whole seedlings of the wild-type and BR-deficient mutant of Arabidopsis thaliana. These multi-level bioassays consistently showed that 28-HBL exhibits a much stronger bioactivity than 24-EBL and is almost as active as BL in rescuing the short hypocotyl phenotype of the dark-grown det2 mutant. These results are consistent with the previously established structure–activity relationship of BRs, proving that this multi-level whole seedling bioassay system could be used to analyze different batches of industrially produced 28-HBL or other BL analogs to ensure the full potential of BRs in modern agriculture.

The unfolded protein response (UPR) signaling pathway is activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and stimulates the production of ER chaperones to restore ER proteostasis. However, how UPR-induced proteins return to their pre-stress levels upon removal of ER stress remains unknown. TUNICAMYCIN-INDUCED 1 (TIN1) is an Arabidopsis (Arabidopsis thaliana) protein that is normally expressed in pollen but is rapidly induced by ER stress in vegetative tissues. Here, we show that the ER-stress-induced TIN1 is rapidly degraded in the UPR recovery phase. We found that TIN1 degradation depends on its asparagine-linked glycans and requires both EMS-mutagenized bri1 suppressor 5 (EBS5) and EBS6 for its recruitment to the ER-associated degradation (ERAD) complex. Loss-of-function mutations in the core component of this Arabidopsis ERAD complex greatly stabilize TIN1. Interestingly, 2 other UPR-induced proteins that are coexpressed with TIN1 remained stable upon ER-stress removal, suggesting that rapid degradation during the stress-recovery phase likely applies to a subset of UPR-induced proteins. Further investigation is needed to uncover the mechanisms by which the ERAD machinery selectively degrades UPR-induced ER proteins.