Protein N -glycosylation in the endoplasmic reticulum (ER) and in the Golgi apparatus is an essential process in eukaryotic cells. Although the N -glycosylation pathway in the ER has been shown to regulate protein quality control, salt tolerance, and cellulose biosynthesis in plants, no biological roles have been linked functionally to N -glycan modifications that occur in the Golgi apparatus. Herein, we provide evidence that mutants defective in N -glycan maturation, such as complex glycan 1 ( cgl1 ), are more salt-sensitive than wild type. Salt stress caused growth inhibition, aberrant root-tip morphology, and callose accumulation in cgl1 , which were also observed in an ER oligosaccharyltransferase mutant, staurosporin and temperature sensitive 3a ( stt3a ). Unlike stt3a , cgl1 did not cause constitutive activation of the unfolded protein response. Instead, aberrant modification of the plasma membrane glycoprotein KORRIGAN 1/RADIALLY SWOLLEN 2 (KOR1/RSW2) that is necessary for cellulose biosynthesis occurred in cgl1 and stt3a . Genetic analyses identified specific interactions among rsw2 , stt3a , and cgl1 mutations, indicating that the function of KOR1/RSW2 protein depends on complex N -glycans. Furthermore, cellulose deficient rsw1-1 and rsw2-1 plants were also salt-sensitive. These results establish that plant protein N -glycosylation functions beyond protein folding in the ER and is necessary for sufficient cell-wall formation under salt stress.

We compare three Arabidopsis (Arabidopsis thaliana) complex glycan1 (cgl1) alleles and report on genetic interaction with staurosporin and temperature sensitive3a (stt3a). STT3a encodes a subunit of oligosaccharyltransferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the endoplasmic reticulum; cgl1 mutants lack N-acetyl-glucosaminyltransferase I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-green fluorescent protein fusions in transient assays, we show that the extra N-glycosylation site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the endoplasmic reticulum. Tunicamycin treatment or expression in the stt3a-2 mutant relieved the folding block, and migration to Golgi stacks resumed. Complementation tests with C5-green fluorescent protein and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glycosylation restores activity. Interestingly, CGL1 seems to be functional also as nonglycosylated enzyme. Two other cgl1 alleles showed splicing defects of their transcripts. In cgl1 C6, a point mutation affects the 3# splice site of intron 14, resulting in frame shifts; in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the N-acetyl-glucosaminyltransferase I defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the conditional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glycosylation is important for normal root growth and morphology in Arabidopsis.

Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including −80 °C, 4 °C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, β-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.

In plant cells, glycans attached to asparagine (N) residues of proteins undergo various modifications in the endoplasmic reticulum and the Golgi apparatus. The N-glycan modifications in the Golgi apparatus result in complex N-glycans attached to membrane proteins, secreted proteins and vacuolar proteins. Recently, we have investigated the role of complex N-glycans in plants using a series of Arabidopsis thaliana mutants affected in complex N-glycan biosynthesis. 1 Several mutant plants including complex glycan 1 (cgl1) displayed a salt-sensitive phenotype during their root growth, which was associated with radial swelling and loss of apical dominance. Among the proteins whose N-glycans are affected by the cgl1 mutation is a membrane anchored β1,4-endoglucanase, KORRIGAN1/RADIALLY SWOLLEN 2 (KOR1/ RSW2) involved in cellulose biosynthesis. The cgl1 mutation strongly enhanced the phenotype of a temperature sensitive allele of KOR1/RSW2 (rsw2-1) even at the permissive temperature. This establishes that plant complex N-glycan modification is important for the in vivo function of KOR1/RSW2. Furthermore, rsw2-1 as well as another cellulose biosynthesis mutant rsw1-1 exhibited also a salt-sensitive phenotype at the permissive temperature. Based on these findings, we propose that one of the mechanisms that cause salt-induced root growth arrest is dysfunction of cell wall biosynthesis that induces mitotic arrest in the root apical meristem.

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Despite increasing data from population-wide sequencing studies, the risk for recessive disorders in consanguineous partnerships is still heavily debated. An important aspect that has not sufficiently been investigated theoretically, is the influence of inbreeding on mutation load and incidence rates when the population sizes change. We therefore developed a model to study these dynamics for a wide range of growth and mating conditions. In the phase of population expansion and shortly afterwards, our simulations show that there is a drop of diseased individuals at the expense of an increasing mutation load for random mating, while both parameters remain almost constant in highly consanguineous partnerships. This explains the empirical observation in present times that a high degree of consanguinity is associated with an increased risk of autosomal recessive disorders. However, it also states that the higher frequency of severe recessive disorders with developmental delay in inbred populations is a transient phenomenon before a mutation-selection balance is reached again.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.