Abstract
Nuclear lamina organization is similar in metazoan and plants though the latter lack orthologs of lamins, the main components of the metazoan lamina. Current evidence suggests that Nuclear Matrix Constituent Proteins (NMCPs) are the lamin analogues in plants as these proteins share several key features: higher-order secondary structure and domain layout, subnuclear distribution, and involvement in the regulation of nuclear shape and size, as well as in higher-order chromatin organization. Previously, we studied the NMCP family in flowering plants (angiosperms), in which it comprises two phylogenetic groups: NMCP1 and NMCP2. At present, in silico information about NMCP proteins in embryophytes is relatively advanced, though very few proteins, most of them of the NMCP1 type, have been extensively studied in vivo. We previously characterized the NCMP1 protein in the monocot Allium cepa. Here, we report the key features of a second protein of this species NMCP2, which presents a conserved sequence and domain layout. Immunofluorescence and immunoelectronmicroscopy evidence co-localization of endogenous AcNMCP2 and AcNMCP1 in the lamina, while Western blotting and immunoconfocal microscopy reveal a similar pattern of expression and distribution of both NMCP proteins in different root tissues. Our results provide novel insight about endogenous NMCP2-type proteins and complete the characterization of the NMCP family in A. cepa, thus advancing the current understanding of these structural proteins constituting the plant lamina.
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Acknowledgements
We thank M. Carnota for expert technical assistance.
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This work was supported by the Spanish Ministry of Science and Innovation (BFU2010-15900) and the Spanish National Research Council (CSIC) (PIE 201020E019).
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Supplementary Table 1
Accession data for sequences used for phylogenetic analysis. (PDF 112 kb)
Fig. S1
Sequence alignment of selected NMCPs. The sequences of NMCPs of Allium cepa AcNMCP1 (BAM10996) and AcNMCP2 (LC150620) were aligned with NMCP2 proteins from Ananas comosus (Anco2), Oryza sativa (Osa2) and NMCP from Marchantia polymorpha (Mpo) using the MUSCLE tool and edited in Jalview. The coiled-coil segments predicted using MARCOIL (Delorenzi and Speed 2002) are shaded in gray, the predicted NLS in green, the RYNLR conserved region in blue, the stretch of acidic amino acids in red and the conserved region at the C-terminus in orange. The degree of conservation is noted by yellow and brown bars beneath the alignment (generated by Jalview). The region of AcNMCP2 used for antibody production is highlighted by a red dotted box (GIF 850 kb)
Fig. S2
a Positive control of the anti-NMCP2 antibody. Western blots of nuclear proteins (N) and increasing amounts of the peptide used for immunization. b to c″ Negative controls of the CLSM immunolabeling analysis of nuclei and NSKs performed omitting incubation of nuclei (b to b″) and NSK (c to c″) with the primary antibodies. Red channel (b, c), green channel (b′, c′), DAPI (b″) and DIC (c″) images of the corresponding fields. d Negative control of the pre-embedding immunogold-labeling omitting incubation with the primary antibodies that shows no gold particles (GIF 394 kb)
Fig. S3
Co-localization of AcNMCP2 (green channel) and AcNMCP1 (red channel) in NSKs extracted from proliferating meristems, along selected regions of interest (ROIs) and across selected confocal z sections. The line intensity profiles of the green and red channels across the selected lines clearly demonstrate co-localization of both proteins at the lamina (GIF 242 kb)
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Ciska, M., Masuda, K. & Moreno Díaz de la Espina, S. Characterization of the lamin analogue NMCP2 in the monocot Allium cepa . Chromosoma 127, 103–113 (2018). https://doi.org/10.1007/s00412-017-0649-2
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DOI: https://doi.org/10.1007/s00412-017-0649-2