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Redundant function of two Arabidopsis COPII components, AtSec24B and AtSec24C, is essential for male and female gametogenesis

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Abstract

Anterograde vesicle transport from the endoplasmic reticulum to the Golgi apparatus is the start of protein transport through the secretory pathway, in which the transport is mediated by coat protein complex II (COPII)-coated vesicles. Therefore, most proteins synthesized on the endoplasmic reticulum are loaded as cargo into COPII vesicles. The COPII is composed of the small GTPase Sar1 and two types of protein complexes (Sec23/24 and Sec13/31). Of these five COPII components, Sec24 is thought to recognize cargo that is incorporated into COPII vesicles by directly interacting with the cargo. The Arabidopsis genome encodes three types of Sec24 homologs (AtSec24A, AtSec24B, and AtSec24C). The subcellular dynamics and function of AtSec24A have been characterized. The intracellular distributions and functions of other AtSec24 proteins are not known, and the functional differences among the three AtSec24s remain unclear. Here, we found that all three AtSec24s were expressed in similar parts of the plant body and showed the same subcellular localization pattern. AtSec24B knockout plant, but not AtSec24C knockdown plant, showed mild male sterility with reduction of pollen germination. Significant decrease of AtSec24B and AtSec24C expression affected male and female gametogenesis in Arabidopsis thaliana. Our results suggested that the redundant function of AtSec24B and AtSec24C is crucial for the development of plant reproductive cells. We propose that the COPII transport is involved in male and female gametogenesis in planta.

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Abbreviations

CaMV:

Cauliflower mosaic virus

CDS:

Coding sequence

CLSM:

Confocal laser scanning microscopy

COPII:

Coat protein complex II

DAPI:

4′,6-diamidino-2-phenylindole

ER:

Endoplasmic reticulum

ERES:

ER export site

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

mRFP:

Monomeric red fluorescent protein

PMII:

Pollen mitosis II

SEM:

Scanning electron microscopy

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Acknowledgments

We thank Dr. Takashi Ueda (The University of Tokyo, Bunkyo-ku, Japan) for the gift of the SYP31 and ST-GFP clones, Dr. Shoji Mano (National Institute for Basic Biology, Okazaki, Japan) for the gift of pRbcsTP221, the RIKEN for distributing the full-length AtSec24A clone (pda09694), and the ABRC for distributing the A. thaliana T-DNA insertion lines (SALK_013076 and SALK_001648) and the qut1 mutant (CS8846). This work was supported by a Research Fellowship (24-2609 to YT) from the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research (24570052 to TN) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Correspondence to Tsuyoshi Nakagawa.

Electronic supplementary material

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Online Resource 1. Oligonucleotides used in this study (PDF 12 kb)

425_2013_1913_MOESM2_ESM.pdf

Online Resource 2. Multiple alignment of the amino acid sequences of ScSec24p, ScIss1p, ScLst1p, AtSec24A, AtSec24B, and AtSec24C. The amino acid sequences including Sec24 family members from A. thaliana and S. cerevisiae were aligned using ClustalW version 1.83 (http://clustalw.ddbj.nig.ac.jp/). Accession nos.: AtSec24A (A. thaliana, NP_187366), AtSec24B (A. thaliana, NP_566869), AtSec24C (A. thaliana, BAM76809), ScSec24p (S. cerevisiae, YIL109C), ScIss1p (S. cerevisiae, YNL049C), and ScLst1p (S. cerevisiae, YHR098C). To find the sequences corresponding to the five domains, the ScSec24p amino acid sequence and the motif database Pfam (http://pfam.sanger.ac.uk/) were used: zinc finger domain (rose), ‘trunk’ domain (yellow),β-barrel domain (green), all-helical region (light blue), and gelsolin-like domain (blue). The red letters indicate the essential amino acid residues for binding of cargo in budding yeast Sec24 family members reported by Miller et al. (2003). These amino acid residues are conserved in AtSec24s. The blue letters indicate the newly added 12 amino acids of AtSec24C determined in this study (PDF 124 kb)

Online Resource 3. Colocalization of SYP31-mRFP and ST-GFP in A. thaliana epidermal cells. Confocal images of A. thaliana leaf epidermal cells co-expressing SYP31-mRFP and the Golgi marker ST-GFP. ST-GFP (CaMV35S promoter:ST-GFP) and pUGW54-SYP31 plasmids were mixed at ratio of 1:1 and introduced into leaf epidermal cells of wild-type plants by the method described in the experimental procedures. The left and middle panels show GFP (green) and RFP fluorescence (red), respectively, and are merged in the right panel. Golgi apparatus labeled by ST-GFP colocalized with the structures labeled by SYP31-mRFP, as indicated by yellow punctate structures in the merged image. The sale bar = 10 µm (PDF 24 kb)

Online Resource 4. Movement of punctate structures labeled with AtSec24s-GFP together with Golgi labeled with SYP31-mRFP in A. thaliana epidermal cells. Time-lapse confocal microscopy of A. thaliana leaf epidermal cells co-expressing the Golgi marker SYP31-mRFP and AtSec24B-GFP (Online Resource 4) or AtSec24C-GFP (Online Resource 5). pUGW51-AtSec24B, pUGW51-AtSec24C, and pUGW54-SYP31 were introduced into leaf epidermal cells by particle bombardment as described in the experimental procedures. Fluorescence was observed in epidermal cells on midrib of leaf. GFP and RFP fluorescence are shown in green and red, respectively. The punctate structures of AtSec24B-GFP and AtSec24C-GFP move in association with SYP31-mRFP in the cytoplasm (MPEG 1370 kb)

Online Resource 5. Movement of punctate structures labeled with AtSec24s-GFP together with Golgi labeled with SYP31-mRFP in A. thaliana epidermal cells. Time-lapse confocal microscopy of A. thaliana leaf epidermal cells co-expressing the Golgi marker SYP31-mRFP and AtSec24B-GFP (Online Resource 4) or AtSec24C-GFP (Online Resource 5). pUGW51-AtSec24B, pUGW51-AtSec24C, and pUGW54-SYP31 were introduced into leaf epidermal cells by particle bombardment as described in the experimental procedures. Fluorescence was observed in epidermal cells on midrib of leaf. GFP and RFP fluorescence are shown in green and red, respectively. The punctate structures of AtSec24B-GFP and AtSec24C-GFP move in association with SYP31-mRFP in the cytoplasm (MPEG 1368 kb)

Online Resource 6. Subcellular localization of Arabidopsis Sec24 homologs co-expressed with chloroplast marker in A. thaliana leaf epidermal cells. Confocal images of A. thaliana leaf epidermal cells co-expressing cyan fluorescent protein (CFP) fused AtSec24s and the chloroplast marker. For transient expression of mRFP linked to chloroplast transit peptide of ribulose 1,5-bisphosphate carboxylase/oxylase small subunit (RBCS) 1A under control of CaMV35S promoter as a chloroplast marker, pRbcsTP221 donated by Dr. Shoji Mano (National Institute for Basic Biology) was introduced into pUGW54 (Nakagawa et al. 2007) by LR reaction, which generated pUGW54-RbcsTP. To generate clones for expressing AtSec24s fused CFP under control of CaMV35S promoter (pUGW44-AtSec24s), pDONR201-AtSec24s entry clones were introduced into pUGW44 (Nakagawa et al. 2007) by the LR reaction. pUGW44-AtSec24s and pUGW54-RbcsTP plasmids were mixed at ratio of 8:1 and introduced into leaf epidermal cells of wild-type plants by the method described in the experimental procedures. The cells were viewed with a TCS SP5 CLSM (Leica Microsystems) using an HCX PL APO CS 63.0x1.20 WATER UV objective lens. The CFP was exited with the argon laser line (458 nm) and the CFP fluorescence was detected at 465–510 nm. The RFP fluorescence was obtained under same condition described in the experimental procedures. Images were processed with Photoshop CS6 (Adobe Systems). The left and middle panels show CFP (cyan) and RFP fluorescence (red), respectively, and are merged in the right panel. Arrowheads and arrows indicate punctate structures and plastids in Arabidopsis leaf epidermal cells, respectively. As in case of AtSec24s-GFP, CFP fused AtSec24s were localized in the cytoplasm with bright punctate structures. AtSec24B-CFP (b) and AtSec24C-CFP (c), as well as AtSec24A-CFP (a), did not show the subcellular localization pattern as plastids labeled by RbcsTP-mRFP. Sale bars = 10 µm (PDF 107 kb)

Online Resource 7. Schematic diagram of genomic AtSec24B and AtSec24C genes and RT-PCR analysis in atsec24b-1 and atsec24c-1 plants. a Schematic diagram of T-DNA insertion sites in genomic AtSec24B and AtSec24C genes. The white and black boxes indicate exons and untranslated regions, respectively. The solid lines show introns. ATG and TGA indicate initiation codons and termination codons in AtSec24B and AtSec24C, respectively. The arrows with a broken line indicate T-DNA and the direction it is inserted in each AtSec24 gene. b RT-PCR analysis using atsec24b-1 and atsec24c-1 plants. Total RNAs were isolated from wild-type plants (WT) and each mutant grown on Jiffy-7 (Jiffy Preforma Production K.K, Yokohama, Japan). cDNAs were synthesized with an oligo-dT primer using ReverTraAce (TOYOBO, Osaka, Japan). To amplify AtSec24A, AtSec24B, and Actin2, PCR was performed using the conditions described in the experimental procedures. To specifically detect disruption of AtSec24C, PCR was performed with 30 cycles using cDNA isolated from each plant as a template and the AtSec24C-PCRd-F and AtSec24C-PCRd-R primers (Online Resource 1) (PDF 50 kb)

Online Resource 8. High-resolution images of a DAPI-stained microspore tetrad from atsec24bc (+/24b, +/24c, qrt1-2/qrt1-2) plant at pollen mitosis II stage. A series of continuous confocal images captured along the z-stack of a DAPI-stained microspore tetrad during pollen mitosis II stage in the atsec24bc (+/24b, +/24c, qrt1-2/qrt1-2) plant. Although three of the four microspores in this microspore tetrad have synchronous development, one of them contains two nuclei and appears to be developmentally delayed at a stage preceding pollen mitosis II (MPEG 1126 kb)

Online Resource 9. A series of confocal images captured along the z-stack of ovules with an immature embryo sac in atsec24bc (+/24b, +/24c) flowers. All ovules were isolated from atsec24bc (+/24b, +/24c) flowers at floral stage 14. a Ovule containing an immature embryo sac arrested at the two-nucleate stage. b Ovule containing an immature embryo sac arrested at the four-nucleate stage. c Ovule containing an immature embryo sac with unfused polar nuclei. AN: antipodal cell nucleus, CN: chalazal nucleus, CV: central vacuole, EN: egg nucleus, LSN: left synergid cell nucleus, MN: micropylar nucleus, PN: polar nucleus, RSN: right synergid cell nucleus. Scale bars = 25 µm (PDF 349 kb)

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Tanaka, Y., Nishimura, K., Kawamukai, M. et al. Redundant function of two Arabidopsis COPII components, AtSec24B and AtSec24C, is essential for male and female gametogenesis. Planta 238, 561–575 (2013). https://doi.org/10.1007/s00425-013-1913-1

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Keywords

  • Arabidopsis
  • Embryo sac
  • Pollen
  • Secretory pathway
  • Vesicle transport