NAI2 is an endoplasmic reticulum body component that enables ER body formation in Arabidopsis thaliana - PubMed (original) (raw)

NAI2 is an endoplasmic reticulum body component that enables ER body formation in Arabidopsis thaliana

Kenji Yamada et al. Plant Cell. 2008 Sep.

Abstract

Plants develop various endoplasmic reticulum (ER)-derived structures, each of which has specific functions. The ER body found in Arabidopsis thaliana is a spindle-shaped structure that specifically accumulates high levels of PYK10/BGLU23, a beta-glucosidase that bears an ER-retention signal. The molecular mechanisms underlying the formation of the ER body remain obscure. We isolated an ER body-deficient mutant in Arabidopsis seedlings that we termed nai2. The NAI2 gene (At3g15950) encodes a member of a unique protein family that is only found in the Brassicaceae. NAI2 localizes to the ER body, and a reduction in NAI2 gene expression elongates ER bodies and reduces their numbers. NAI2 deficiency does not affect PYK10 mRNA levels but reduces the level of PYK10 protein, which becomes uniformly diffused throughout the ER. NAI1, a transcription factor responsible for ER body formation, regulates NAI2 gene expression. These observations indicate that NAI2 is a key factor that enables ER body formation and the accumulation of PYK10 in ER bodies of Arabidopsis. Interestingly, ER body-like structures are also restricted to the Brassicales, including the Brassicaceae. NAI2 homologs may have evolved specifically in Brassicales for the purpose of producing ER body-like structures.

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Figures

Figure 1.

Figure 1.

The nai2-1 Mutant Lacks ER Bodies. Epidermal cells of 5-d-old seedlings and 19-d-old mature plants were inspected with a confocal laser scanning microscope. Bars = 10 μm.

Figure 2.

Figure 2.

The NAI2 Gene Is At3g15950. (A) The structure of the genome region around the NAI2 gene. The black boxes indicate the coding regions of each gene. The nai2-1 mutant has a putative large genome deletion indicated by the region marked by dashes. The blue bar indicates the genomic region that was used for the complementation testing of the nai2-1 mutant. (B) The exon/intron structure of the NAI2 gene. The black boxes indicate the protein-coding regions. The white boxes indicate the untranslated regions. The triangles indicate the T-DNA insertion sites in the nai2-2 and nai2-3 mutants. (C) Test of allelism between the nai2 mutants. Cotyledon epidermal cells of nai2-1 × nai2-2 or nai2-1 × nai2-3 F1 progeny were inspected with a confocal laser scanning microscope. Bars = 10 μm. (D) Complementation of the nai2-1 phenotype by the introduction of a genome fragment carrying the NAI2 gene. Cotyledon epidermal cells from 6-d-old nai2-1 seedlings were bombarded with gold particles coated with plasmids containing the NAI2 genome fragment shown in (A) plus the tdTomato gene (NAI2gDNA + tdTom; left panels) or the plasmid carrying the tdTomato gene only (right panels). After germination for 2 d, the cells were inspected with a confocal laser scanning microscope. The florescence of tdTomato indicates cells that were transfected with plasmids. The filamentous structures observed by tdTomato are cytoplasmic strands. Bars = 10 μm.

Figure 3.

Figure 3.

Structure of NAI2. (A) The deduced amino acid sequence of NAI2. The numbers show the amino acid residue positions. NAI2 contains a signal peptide, 10 EFE repeats, and the NAI2 domain. The boldface letters show the conserved amino acids within the EFE repeats. (B) Hydrophobicity index of NAI2. The structure of NAI2 is shown below. SP, signal peptide.

Figure 4.

Figure 4.

NAI2 Localizes in ER Bodies. (A) Immunoblot analysis of 7-d-old seedlings from the indicated strains using antibody against NAI2/ΔSP (without signal peptide). Arrowheads indicate the bands corresponding to the NAI2 polypeptide: closed, major bands; open, minor bands. (B) Immunofluorescence analysis of NAI2 in 7-d-old GFP-h seedlings. Left panels, the ER-targeted GFP signals; middle panels, the NAI2 (top) and GFP (bottom) signals, which were detected by antibodies against NAI2/ΔSP and GFP, and Cy3-labeled second antibodies, respectively; right panels, the merged images. Bars = 10 μm. (C) Immunoblot analysis of the subcellular fractions enriched in ER bodies (P1) or ER networks (P100) using antibodies against BiP, GFP (GFP-HDEL), PYK10, and NAI2/ΔSP (NAI2).

Figure 5.

Figure 5.

Reduction of NAI2 Reduces the Number of ER Bodies and Elongates Their Shape. (A) Extracts from 8-d-old seedlings of GFP-h, two independent NAI2 RNAi lines, and nai2-1 were subjected to immunoblot analysis with anti-NAI2/C antibodies. (B) ER bodies in 8-d-old cotyledons. Bars = 5 μm. (C) Quantification of ER bodies. The GFP fluorescent spots in a 5.29 × 104 μm2 image area were automatically counted after shutting out weak ER fluorescence. The iNAI2 plants had significantly fewer ER bodies than GFP-h (* P < 0.05, Welch's t test). The error bars indicate

sd

(n = 4 to 6). ** The lower number of GFP fluorescent spots in nai2-1 shows that ER fluorescence was removed during counting analysis. (D) Quantification of ER body length. Average ER body length was calculated from a single image. The error bars indicate

sd

of the averages (n = 4 to 6). The length of the ER bodies in nai2-1 was not measured, since so few ER bodies were detected (n.d., not determined).

Figure 6.

Figure 6.

NAI2, PYK10, and NAI1 mRNA Levels in the nai2 and nai1 Mutants. Total RNAs from 7-d-old seedlings were subjected to quantitative RT-PCR analysis. The data were normalized with respect to actin8 mRNA levels. The relative quantity of each mRNA was calibrated with the amounts in wild-type plants (Columbia [Col] accession). The relative quantity was calculated by the 2−ΔΔCt method (Levak and Schmittgen, 2001). Error bars indicate

se

of the threshold cycle (Ct) values, which are calculated using the formula 2−ΔΔCt±se. The data represent the results of three independent biological replications.

Figure 7.

Figure 7.

PYK10, BiP, and PBP1 Levels in nai2 Mutants. Total proteins from 7-d-old seedlings were subjected to immunoblot analysis using antibodies against PYK10, BiP, or PBP1. Coomassie blue staining shows the ribulose-1,5-bis-phosphate carboxylase/oxygenase large subunit (RbcL), which served as a loading control.

Figure 8.

Figure 8.

Localization of GFP-PYK10 in nai2 Mutants. Six-day-old wild-type (Columbia [Col] accession), nai2-2, and nai2-3 seedlings were bombarded with gold particles coated with plasmids carrying the GFP-PYK10 gene and germinated for 1 d. Cotyledon epidermal cells were inspected with a confocal laser scanning microscope to observe the distribution of GFP-PYK10. Bars = 10 μm.

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