No abstract

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), a-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and b-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a transGolgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

For important food crops such as wheat and rice, grain yield depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for yield and encodes an E3 RING ligase that negatively regulates grain size. Wheat (Triticum aestivum) has TaGW2 homologues in the A, B, and D genomes, and polymorphisms in TaGW2-A were associated with grain width. Here, to investigate TaGW2 function, RNA interference (RNAi) was used to down-regulate TaGW2 transcript levels. Transgenic wheat lines showed significantly decreased grain size-related dimensions compared with controls. Furthermore, TaGW2 knockdown also caused a significant reduction in endosperm cell number. These results indicate that TaGW2 regulates grain size in wheat, possibly by controlling endosperm cell number. Wheat and rice GW2 genes thus seem to have divergent functions, with rice GW2 negatively regulating grain size and TaGW2 positively regulating grain size. Analysis of transcription of TaGW2 homoeologues in developing grains suggested that TaGW2-A and -D act in both the division and late grain-filling phases. Furthermore, biochemical and molecular analyses revealed that TaGW2-A is a functional E3 RING ubiquitin ligase with nucleocytoplasmic subcellular partitioning. A functional nuclear export sequence responsible for TaGW2-A export from the nucleus to the cytosol and retention in the nucleolus was identified. Therefore, these results show that TaGW2 acts in the regulation of grain size and may provide an important tool for enhancement of grain yield.