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Protein ubiquitination is a dynamic and reversible post‐translational modification that controls diverse cellular processes in eukaryotes. Ubiquitin‐dependent internalization, recycling, and degradation are important mechanisms that regulate the activity and the abundance of plasma membrane (PM)‐localized proteins. In plants, although several ubiquitin ligases are implicated in these processes, no deubiquitinating enzymes (DUBs), have been identified that directly remove ubiquitin from membrane proteins and limit their vacuolar degradation. Here, we discover two DUB proteins, UBP12 and UBP13, that directly target the PM‐localized brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) in Arabidopsis. BRI1 protein abundance is decreased in the ubp12i/ubp13 double mutant that displayed severe growth defects and reduced sensitivity to BRs. UBP13 directly interacts with and effectively removes K63‐linked polyubiquitin chains from BRI1, thereby negatively modulating its vacuolar targeting and degradation. Our study reveals that UBP12 and UBP13 play crucial roles in governing BRI1 abundance and BR signaling activity to regulate plant growth. Synopsis: The deubiquitinating enzymes UBP12 and UBP13 act as negative regulators of brassinosteroid (BR) receptor BRI1 degradation in the vacuole through the deubiquitination of BRI1, thus playing positive roles in BR‐responsive plant growth. Loss of UBP12 and UBP13 (ubp12i/ubp13) results in severe growth defects, and UBP12 and UBP13 positively regulate BR signaling.UBP12 and UBP13 are essential to maintain BRI1 protein abundance.UBP13 interacts with BRI1 and cleaves K63‐linked polyubiquitin chains from BRI1 to limit its internalization and vacuolar targeting. [ABSTRACT FROM AUTHOR]

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)−1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone. The trans-Golgi network (TGN) serves as a platform to sort and transport proteins to their final destinations. Here the authors show that the TGN of Arabidopsis consists of spatially and temporally distinct subregions and propose that these zones may sort cargo to different destinations. [ABSTRACT FROM AUTHOR]

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors. [ABSTRACT FROM AUTHOR]

Plant vacuoles are multifunctional organelles. On the one hand, most vegetative tissues develop lytic vacuoles that have a role in degradation. On the other hand, seed cells have two types of storage vacuoles: protein storage vacuoles (PSVs) in endosperm and embryonic cells and metabolite storage vacuoles in seed coats. Vacuolar proteins and metabolites are synthesized on the endoplasmic reticulum and then transported to the vacuoles via Golgi-dependent and Golgi-independent pathways. Proprotein precursors delivered to the vacuoles are converted into their respective mature forms by vacuolar processing enzyme, which also regulates various kinds of programmed cell death in plants. We summarize two types of vacuolar membrane dynamics that occur during defense responses: vacuolar membrane collapse to attack viral pathogens and fusion of vacuolar and plasma membranes to attack bacterial pathogens. We also describe the chemical defense against herbivores brought about by the presence of PSVs in the idioblast myrosin cell. [ABSTRACT FROM AUTHOR]

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi-enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non-exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter-mediated pathway and the vesicle trafficking-mediated pathway. No molecules involved in the vesicle trafficking-mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan-colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis-sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome-like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9-mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles. [ABSTRACT FROM AUTHOR]

Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana, MAIGO 2 ( MAG2) is involved in protein transport between the endoplasmic reticulum ( ER) and the Golgi apparatus via its association with the ER-localized t- SNARE components SYP81/AtUfe1 and SEC20. To characterize the molecular machinery of MAG2-mediated protein transport, we explored MAG2-interacting proteins using transgenic A. thaliana plants expressing TAP-tagged MAG2. We identified three proteins, which were designated as MAG2- INTERACTING PROTEIN 1-3 [ MIP1 (At2g32900), MIP2 (At5g24350) and MIP3 (At2g42700)]. Both MIP1 and MAG2 localized to the ER membrane. All of the mag2, mip1, mip2 and mip3 mutants exhibited a defect in storage protein maturation, and developed abnormal storage protein body ( MAG body) structures in the ER of seed cells. These observations suggest that MIPs are closely associated with MAG2 and function in protein transport between the ER and Golgi apparatus. MIP1 and MIP2 contain a Zeste-White 10 ( ZW10) domain and a Sec39 domain, respectively, but have low sequence identities (21% and 23%) with respective human orthologs. These results suggest that the plant MAG2- MIP1- MIP2 complex is a counterpart of the triple-subunit tethering complexes in yeast (Tip20p-Dsl1p-Sec39p) and humans ( RINT1- ZW10- NAG). Surprisingly, the plant complex also contained a fourth member ( MIP3) with a Sec1 domain. There have been no previous reports showing that a Sec1-containing protein is a subunit of ER-localized tethering complexes. Our results suggest that MAG2 and the three MIP proteins form a unique complex on the ER that is responsible for efficient transport of seed storage proteins. [ABSTRACT FROM AUTHOR]

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER ; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana , named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16 , which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A–green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover. [ABSTRACT FROM AUTHOR]

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) in a precursor form and then are transported to protein storage vacuoles (PSVs) where they are converted to their mature form. To understand the mechanisms by which storage proteins are transported, we screened Arabidopsis maigo mutants to identify those that abnormally accumulate storage protein precursors. Here we describe a new maigo mutant, maigo 4 (mag4), that abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag4 seed cells abnormally develop a large number of novel structures that exhibit a highly electron-dense core. Some of these structures were surrounded by ribosomes. Immunogold analysis suggests that the electron-dense core is an aggregate of 2S albumin precursors and that 12S globulins are localized around the core. The MAG4 gene was identified as At3g27530, and the MAG4 protein has domains homologous to those found in bovine vesicular transport factor p115. MAG4 molecules were concentrated at cis-Golgi stacks. Our findings suggest that MAG4 functions in the transport of storage protein precursors from the ER to the Golgi complex in plants. In addition, the mag4 mutant exhibits a dwarf phenotype, suggesting that MAG4 is involved in both the transport of storage proteins and in plant growth and development. [ABSTRACT FROM PUBLISHER]