Although N-acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT) that catalyzes the final step of the hexosamine biosynthetic pathway and is conserved among, organisms, produces UDP-N-acetylglucosamine (UDP-GlcNAc), an essential sugar moiety involved in protein glycosylation and structural polymers, its biological function in plants remains unknown. In this study, two GlcNA.UT genes were characterized in Arabidopsis thaliana. The single mutants glcna.ut1 and glcna.ut2 revealed no obvious phenotype, but their homozygous double mutant was lethal, reflecting the functional redundancy of these genes in being essential for plant growth. Mutant plants, GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2, obtained from an F2-segregating population following reciprocal crosses of glcna.ut1 with glcna.ut2, displayed shorter siliques and fewer seed sets combined with impaired pollen viability and unfertilized ovules. Genetic analyses further demonstrated that the progeny of the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants, but not those of the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants, suffer from the aberrant transmission of (glcna.ut1 glcna.ut2) gametes. In parallel, cell biology analyses revealed a substantial defect in male gametophytes appearing during the late vacuolated or pollen mitosis I stages and that the female gametophyte is arrested during the uninucleate embryo sac stage in GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. Nevertheless, although the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants exhibited a normal transmission of (glcna.ut1 glcna.ut2) gametes and gametophytic development, the development of numerous embryos was arrested during the early globular stage within the embryo sacs. Collectively, despite having overlapping functions, the GlcNA.UT genes play an indispensable role in the unique mediation of gametogenesis and embryogenesis.
Being in the role of both a preceptor and nurse was perceived as a challenge by participants because of heavy workloads and fears of failure. Thus, reducing the preceptor's patient care responsibilities while educating new nurses should be a priority. This study also found cultivating a positive work climate as crucial to support preceptors and new nurses so that preceptors do not feel alienated or overly stressed. A workshop is a useful strategy to introduce preceptors and new nurses to standardized training procedures and documents; this, in turn, can provide a more holistic approach to teaching and learning and reduce pressures on preceptors caused by additional, unfamiliar paperwork.
N-acetylglucosamine (GlcNAc) is the fundamental amino sugar moiety that is essential for protein glycosylation. UDP-GlcNAc, an active form of GlcNAc, is synthesized through the hexosamine biosynthetic pathway (HBP). Arabidopsis N-acetylglucosamine-1-P uridylyltransferases (GlcNAc1pUTs), encoded by GlcNA.UTs, catalyze the last step in the HBP pathway, but their biochemical and molecular functions are less clear. In this study, the GlcNA.UT1 expression was knocked down by the double-stranded RNA interference (dsRNAi) in the glcna.ut2 null mutant background. The RNAi transgenic plants, which are referred to as iU1, displayed the reduced UDP-GlcNAc biosynthesis, altered protein N-glycosylation and induced an unfolded protein response under salt-stressed conditions. Moreover, the iU1 transgenic plants displayed sterility and salt hypersensitivity, including delay of both seed germination and early seedling establishment, which is associated with the induction of ABA biosynthesis and signaling. These salt hypersensitive phenotypes can be rescued by exogenous fluridone, an inhibitor of ABA biosynthesis, and by introducing an ABA-deficient mutant allele nced3 into iU1 transgenic plants. Transcriptomic analyses further supported the upregulated genes that were involved in ABA biosynthesis and signaling networks, and response to salt stress in iU1 plants. Collectively, these data indicated that GlcNAc1pUTs are essential for UDP-GlcNAc biosynthesis, protein N-glycosylation, fertility, and the response of plants to salt stress through ABA signaling pathways during seed germination and early seedling development.
δ‐Crystallin is the major structural protein in avian and reptilian eye lenses, and confers special refractive properties. The protein is a homotetramer arranged as a dimer of dimers. In the present study, the roles of the side chains of Glu267, Lys315, and Glu327, which provide hydrogen bonds at the double dimer interface, were investigated. Hydrophobic side chain substitution led to all mutant proteins having an unstable dimer interface. The E267L/E327L mutant had the greatest sensitivity to temperature, urea and guanidinium hydrochloride denaturation, and the most extensive exposure of hydrophobic patches, as judged by 1‐anilinonaphthalene‐8‐sulfonic acid fluorescence, CD, and tryptophan fluorescence. In contrast, the E267L/K315L/E327L mutant showed higher stability than the E267L/E327L mutant. Some level of the dissociated dimeric form was observed in the K315L mutant, but it was not observed for the K315A and E267L/K315L mutants. The E327L mutant was partially in the dissociated dimeric form, whereas the E267/E327L mutant was predominantly dissociated into dimers. In contrast, the triple mutant of E267L/K315L/E327L retained a tetrameric structure. In the presence of urea, a stable monomeric intermediate with higher stability than the wild type was identified for the K315A mutant. Disruption of interfacial interactions at Glu267 led to polymerization of partly unfolded intermediates in the presence of 3 m urea. However, these polymeric forms were not observed with combinations of the E267L mutation with other mutations. These results indicate that these hydrogen bonds, which are present at different contact surfaces in the dimer–dimer interface, perform distinct functions in double dimer assembly. The coordination of these interactions is critical for the stability and tetramer formation of δ‐crystallin.
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