Volume 11, Issue 5, Pages 678-690 (May 2018) Trimming of N-Glycans by the Golgi-Localized α-1,2-Mannosidases, MNS1 and MNS2, Is Crucial for Maintaining RSW2 Protein Abundance during Salt Stress in Arabidopsis  Chuanfa Liu, Guanting Niu, Huchen Zhang, Yafei Sun, Shubin Sun, Fugen Yu, Shan Lu, Yonghua Yang, Jianming Li, Zhi Hong  Molecular Plant  Volume 11, Issue 5, Pages 678-690 (May 2018) DOI: 10.1016/j.molp.2018.01.006 Copyright © 2018 The Author Terms and Conditions

Figure 1 The mns1 mns2 Double Mutant Displays a Salt-Hypersensitivity Phenotype. (A) Root phenotype of 9-day-old seedlings grown vertically on ½ MS medium supplemented with or without 160 mM NaCl. WT, wild-type. Scale bar, 1.0 cm. (B) Relative primary root length of mutants to WT. Primary root lengths of seedlings treated in (A) were individually measured. The average root length of the non-treated WT seedlings was set as 100. Three biological replicates (>20 seedlings/each) were performed, and error bars indicate SE. (C) Photographic (left) and microscopic (right) observations of root tips of seedlings shown in (A). Scale bars represent 1 mm (vertical white bar) and 100 μm (horizontal black bar). Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 2 Golgi MNSI Activity Plays a Key Role in Salt Hypersensitivity. (A) Immunoblot analysis of complex N-glycans. Total proteins were separated by SDS–PAGE and analyzed by CBB staining (left) or immunoblotting (right) with an anti-horseradish peroxidase (α-HRP) antibody capable of detecting the β-1,2-xylose and α-1,3-fucose residues, which are the characteristic features of plant complex N-glycans. The asterisk denotes non-specific bands. (B) Salt-sensitive phenotype of the mns1 mns2 mutant was complemented by the introduction of a MNSI genomic transgene, gMNS1. The MNS1 transcript in WT and mns1 mns2 was detected by RT–PCR (the middle panel) with Actin2 as an internal control (bottom panel). (C) Root growth phenotype of seedlings grown on 160 mM NaCl-containing agar medium supplemented with or without 10 μM Kif. Scale bars =0.5 cm. (D) Quantitative analysis of primary root length of seedlings sampled in (C). The average root lengths are presented as the values relative to the average primary root length of WT seedlings treated with NaCl alone. Each presented value is the average of three replicates (>20 seedlings each). Error bars indicate SE; statistically significant difference at *P < 0.05 and **P < 0.01 using Student's t-test. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 3 Arabidopsis Mutants Lacking the C Branch of N-Glycans Are Resistant to Kif-Triggered Salt Hypersensitivity. (A) Predicted structures of the predominant oligomannosidic N-glycans in indicated Arabidopsis mutants. Black boxes and circles denote GlcNAc and Man residues, respectively. (B) Immunoblot analysis of N-glycan structures on the ER-residential glycoprotein PDI. The predicted N-glycan structures in different mutants were evaluated with the different mobility of PDI using western immunoblot analysis. RbcS stained with CBB is shown as the loading control. (C) Images of seedlings grown on basal MS medium (Mock) or MS medium supplemented with or without 10 μM Kif and/or 160 mM NaCl. The seedlings were photographed after 9 days of growth. Vertical scale bars represent 0.5 cm. (D) Quantitative analysis of relative primary root length. The data points on the graph are presented as the values relative to the average root length of the WT seedlings grown on the NaCl-containing medium. Three biological replicates (>20 seedlings each) were performed for each data point with the error bars indicating SE. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 4 The ebs3 Mutation Suppressed the Salt-Sensitive Root Phenotype of mns1 mns2. (A) Images of 9-day-old seedlings grown vertically on regular MS medium (Mock) or MS medium supplemented with 160 mM NaCl (NaCl) or 160 mM NaCl and 10 μM Kif (NaCl + Kif). Scale bars = 0.5 cm. (B) Quantitative analysis of primary root lengths of seedlings sampled in (A). Error bars indicate SE, n > 20; significant difference at **P < 0.01 with Student's t-test. Three replicates were performed. (C) Schematic representations of the major N-glycan structures of TGG1 in different mutants. Black boxes and circles denote GlcNAc and Man residues, respectively. (D) Immunoblot analysis of TGG1. The N-glycan structures of TGG1 in different mutant backgrounds were evaluated by the mobility shift of TGG1, a vacuolar protein decorated with oligomannosidic N-glycans. The TGG1 mobility differences were eliminated after the Endo-Hf digestion. CBB-stained RbcS served as the loading control. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 5 Cellulose Biosynthesis Is Compromised in the Salt-Stressed mns1 mns2 Mutant. (A) Confocal imaging of calcofluor-stained root tips. The top panel shows the images of the root maturation zones and the lower panel shows images of the apical meristem region. The white arrow indicates fluorescent patches of cellulose deposition. Scale bars, 30 μm. (B) Microscopic images of cross-sections of salt-stressed root tips. The root tips of 2-week-old seedlings grown on medium supplemented with 160 mM NaCl were embedded in Spurr resin and sectioned (7 μm thick). Scale bars, 20 μm. (C) Quantitative analysis of cellulose content. The data points shown represent cellulose content relative to that of the non-treated WT (Mock) seedlings. Error bars denote SE; statistical significance at **P < 0.01 by Student's t-test. Three replicates were performed. (D) Comparison of the root growth curves of WT, N-glycosylation mutants, and rsw2-1 grown on MS medium supplemented without (Mock) or with 160 mM NaCl. DAG, days after germination. Error bars indicate SE. Three replicates were performed for each growth curve. (E) Over-expression of RSW2 partially rescues the root phenotype of mns1 mns2 under salt stress. Images of 9-day-old seedlings of WT, mns1 mns2, and two gRSW2 mns1 mns2 transgenic lines grown vertically on MS medium supplemented without (Mock) or with 160 mM NaCl. Scale bars, 0.5 cm. (F) Quantitative analysis of salt stress-induced root growth inhibition. The data points are the lengths of primary roots relative to those of the mock-treated WT seedlings. Three replicates (>20 seedlings per assay) were performed, and error bars represent SE. (G) Immunoblot analysis of RSW2 protein abundance. Total proteins extracted from seedlings sampled in (E) were separated by SDS–PAGE and analyzed by immunoblotting with anti-RSW2 antibody. CBB-stained RbcS served as the loading control, and the numbers shown below the filter strips indicate RSW2 abundance relative to that of the mock-treated WT seedlings after normalization with the signal intensity of RbcS. Measurement of signal intensity of both RSW2 and RbcS was performed by ImageJ. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 6 N-Glycan Structures Affect RSW2 Abundance under Salt Stress. (A) Real-time qRT–PCR analysis of the RSW2 transcripts. Total RNAs isolated from seedlings treated with or without 160 mM NaCl for 4 h were converted to cDNAs and used as templates for PCR analysis. The RSW2 transcript abundance is presented as the value relative to that of the mock-treated WT seedlings after normalization with ACTIN2 transcript levels. Error bars represent SE. Three biological replicates were performed. (B) Immunoblot analysis of RSW2 protein stability. Twelve-day-old Arabidopsis seedlings grown on ½ MS medium were treated with 160 mM salt supplemented with 50 μM CHX (CHX + NaCl) or 50 μM CHX plus 10 μM Kif (CHX + NaCl + Kif) and collected at different time points to extract total proteins that were separated by SDS–PAGE and analyzed by immunoblotting or CBB staining. The signal intensities of RSW2 and RbcS bands were measured by ImageJ and used to calculate the relative RSW2 abundance (the numbers shown below the RbcS gel strip) after normalization based on the abundance of RbcS. (C and D) Degradation curves of the RSW2 protein. The fitting curves of RSW2 degradation were obtained using the relative RSW2 abundance values shown in (B). Error bars indicate SE. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 7 Eliminating Golgi MNSI Activity Synergistically Enhances the Growth Defects of the rsw2-1 Mutant. (A) Images of a 4-week-old mns1 mns2 rsw2-1 triple mutant (left, photographed with a regular camera; right, enlarged after visualization under a stereomicroscope). (B) Immunoblot analysis of RSW2 protein abundance. The CBB-stained RbcS bands are used as loading controls, revealing the overloading required for detection of the mutant rsw2-1 protein. (C) Root phenotypes of WT, mns1 mns2, cgl1-1, and rsw2-1 seedlings grown on ½ MS medium containing 10 μM Kif for 9 days. Scale bar, 0.5 cm. (D) Root phenotypes of transgenic Arabidopsis lines overexpressing the mutant rsw2-1 protein. Ten-day-old seedlings were transferred to a 30°C growth chamber for 2 days, returned to a 22°C growth room for additional growth for 2 days, and photographed. (E) Immunoblot analysis of the RSW2 and rsw2-1 abundance. Total proteins extracted from 2-week-old seedlings were incubated without (−) or with (+) Endo-Hf, separated by SDS–PAGE, and analyzed by immunoblotting with the anti-RSW2 antibody. RbcS was stained with CBB as the loading control. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions

Figure 8 The ebs3 Mutation Partially Suppresses the rsw2-1 Phenotype and Increases RSW2 Protein Abundance. (A) Phenotypes of 2-week-old seedlings grown on ½ MS medium. Scale bars, 1.0 cm. (B) Quantitative analysis of primary root lengths of seedlings sampled in (A). The average root length of WT was set as 100%. Error bars represent SE; statistical significance **P < 0.01 by Student's t-test. Three replicates were performed. (C) Phenotypes of 3-week-old seedlings treated with Kif. Seeds were germinated on ½ MS medium and subsequently transferred to ½ MS medium containing 10 μM Kif for continued growth. Scale bar, 0.5 cm. (D) Phenotypes of 2-week-old mns1 mns2 rsw2-1 triple mutant and ebs3 mns1 mns2 rsw2-1 quadruple mutant. Scale bar, 0.5 cm. (E) Immunoblot analysis of RSW2 and rsw2-1 protein abundance in WT, rsw2-1, and ebs3 rsw2-1 seedlings treated without (−) or with (+) 10 μM Kif for 24 h. Asterisks denote non-specific bands, and the CBB-stained RbcS bands were used as the loading control. (F) Immunoblot analysis of RSW2 and rsw2-1 protein levels. Total protein extracts were incubated without (−) or with (+) Endo-Hf for deglycosylation of N-glycans. Asterisks denote non-specific bands, and the CBB-stained RbcS is shown as the loading control. (G) Schematic of MNS1/MNS2-mediated N-glycan trimming that might regulate the stability and/or trafficking of RSW2/rsw2-1 under salt stress. TGN indicates the trans-Golgi network and PM denotes the plasma membrane. Molecular Plant 2018 11, 678-690DOI: (10.1016/j.molp.2018.01.006) Copyright © 2018 The Author Terms and Conditions