How the endoplasmic reticulum (ER) and the Golgi apparatus maintain their morphological and functional identity while working in concert to ensure the production of biomolecules necessary for the cell's survival is a fundamental question in plant biology. Here, we isolated and characterized an Arabidopsis thaliana mutant that partially accumulates Golgi membrane markers and a soluble secretory marker in globular structures composed of a mass of convoluted ER tubules that maintain a connection with the bulk ER. We established that the aberrant phenotype was due to a missense recessive mutation in sec24A, one of the three Arabidopsis isoforms encoding the coat protomer complex II (COPII) protein Sec24, and that the mutation affects the distribution of this critical component at ER export sites. By contrast, total loss of sec24A function was lethal, suggesting that Arabidopsis sec24A is an essential gene. These results produce important insights into the functional diversification of plant COPII coat components and the role of these proteins in maintaining the dynamic identity of organelles of the early plant secretory pathway.

Background: Accumulation of the plant hormone jasmonoyl-L-isoleucine (JA-Ile) is tightly controlled to prevent overactivation of defense responses. Results: Cytochrome P450 94s (CYP94s) with distinct tissue expression patterns localize to ER and oxidize JA-Ile to a dicarboxy derivative that fails to assemble JA-Ile co-receptor complexes. Conclusion: Sequential CYP94-catalyzed oxidations block receptor activation and signaling. Significance: P450s inactivate fatty acid-derived signals in both plants and animals.

SUMMARYPectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell-wall stiffness, cell-to-cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)-like proteins and an unrelated plant-specific protein, cotton Golgi-related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss-of-function mutants and over-expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell-wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over-expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell-wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.

SUMMARYMixed-linkage glucan (MLG) is a significant cell wall carbohydrate in grasses and an important carbon source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of membrane spanning glucan synthases encoded by the CSLH and CSLF cellulose synthase-like gene families. CSLF proteins are the best characterized to date but relatively little information is known about their topology with respect to the biosynthetic membranes. In this study, we report on the topology of CSLF6 protein derived from the model grass species Brachypodium distachyon (BdCSLF6) when it is expressed in heterologous systems. Using live cell imaging and immuno-electron microscopy analyses of tobacco epidermal cells expressing BdCSLF6, we demonstrate that a functional yellow fluorescent protein (YFP) fusion of BdCSLF6 is localized to the Golgi apparatus and that the Golgi localization of BdCSLF6 is sufficient for MLG biosynthesis. By implementing protease protection assays of BdCSLF6 expressed in the yeast Pichia pastoris, we also demonstrate that the catalytic domain, the N-terminus and the C-terminus of the protein are exposed in the cytosol. Furthermore, we found that BdCSLF6 is capable of producing MLG not only in tobacco cells but also in Pichia, which generally does not produce MLG. Together, these results support the conclusion that BdCSLF6 can produce both of the linkages present in the (1,3;1,4)-b-D-glucan chain of MLG and that the product is channelled at the Golgi into the secretory pathway for deposition into the cell wall.