Agredano-Moreno, Segura-Valdez, Jiménez-Ramírez, and Jiménez-García: Lacandonia granules are present in the cell nucleus of Welwitschia mirabilis



Lacandonia schismatica E. Martínez & C. H. Ramos (Triuridaceae) is a plant from the Lacandona forest in Chiapas, Mexico. It is a plant displaying distribution of gynoecium surrounding the androecium (Márquez-Guzmán et al. 1989, Martínez & Ramos 1989). The cell nucleus of this species is characterized by the presence of granules of 32 nm in diameter called Lacandonia granules (Jiménez-García et al. 1992). These particles are ribonucleoproteins (RNPs), intermediate in size and distribution between interchromatin and perichromatin granules present in mammalian cell nucleus. Lacandonia granules contain SR proteins and poly-A RNA tail as perichromatin granules (Agredano-Moreno & Jiménez-García 2000). In addition, the morphology of these particles was previously analyzed using atomic force microscopy (Fragoso-Soriano et al. 2009).

Lacandonia granules are also present in the related Triuris brevistylis Donn.Sm., also a member of the order Pandanales as L. schismatica (Stevens 2017). The observation of Lacandonia granules in the nuclei of the gymnosperm tree Ginkgo biloba L., (Jiménez-Ramírez et al. 2002), suggests that granules are present in other groups and may display a general function. In the present work we report the presence of Lacandonia granules in the nuclei of Welwitschia mirabilis Hook.f., a member of Welwitschiaceae (order Gnetales; Stevens 2017).

Materials and methods

Plants. W. mirabilis seeds were disinfected with 1 % captain for 1 hour and washed with deionized water. Seeds were cultivated in tepojal (small volcanic grain covered with clay), substrate in a germination chamber Lab-Line at 26 °C, 60 % relative humidity and photoperiod (light/dark): 16/8.

Transmission Electron Microscopy. 1 mm3 fragments of young leaves were processed following the standard protocol for electron microscopy (Jiménez-García & Segura-Valdez 2004). Briefly, fragments were fixed overnight at room temperature in a mixture of 6 % glutaraldehyde and 4 % paraformaldehyde, in PBS buffer (pH 7.2). Post-fixation was performed with 2 % osmic acid overnight. Samples were subsequently dehydrated in a graded series of ethanol and embedded in an epoxy resin at 60 °C for 48 h following the standard protocol for electron microscopy. Thin sections were placed on copper grids covered with formvar. Contrast was conducted with 5 % uranyl acetate and 0.5 % lead citrate. Grids were observed with a transmission electron microscope (JEOL, JEM 1010, Peabody, MA) working at 80 kV. Images were obtained with a charge-coupled device camera coupled to the microscope. The diameter of the nuclear granules was determined on 100,000x electron micrographs of thin sections stained with Bernhard´s EDTA technique (Bernhard 1969).

Light microscopy. Thin sections were stained with toluidine blue and 100x pictures were taken in brightfield illumination with an optical microscope (Nikon, Eclipse E800).

Atomic force microscopy. Atomic force microscopy was conducted as previously described (Jiménez-García & Segura-Valdez 2004, Segura-Valdez et al. 2010). Briefly, semithin sections (about 250 nm thickness) were mounted on glass slides and observed with an atomic force microscope (model BioScope, Digital Instruments, Santa Barbara CA, USA) working in contact mode. The scan size was from 30 to 100 µm at a scan rate of 2.1 Hz. Images were produced with the NanoScope IIIa control system. The AFM tips were silicon nitride tips with a curvature radius of 20-60 nm (model NP).

EDTA staining for RNPs. Thin sections of samples fixed with 6 % glutaraldehyde and 4 % paraformaldehyde without osmium tetroxide were used for Bernhard's EDTA technique, for preferential staining of ribonucleoproteins (RNPs) (Bernhard 1969). Basically, 5 % uranyl acetate was used for 3 min, followed by treatment with EDTA for 13 min and 0.5 % lead citrate for 3 min.

Osmium amine. Specific staining of DNA in the cell nucleus of W. mirabilis was performed according to Vázquez-Nin et al. (1995) with modifications. Briefly, 60-90 nm sections mounted in gold grids without formvar were floated on a drop of 5N HCl for 1 hour at room temperature (acid hydrolysis). Grids were rinsed with deionized water and incubated in a wet chamber containing a drop of osmium amine solution. Finally, grids were washed and observed with an electron microscope without additional staining.

Results

The cell nuclei of Welwitschia mirabilis were observed with light, atomic force, and transmission electron microscopy.

Compact chromatin. In the cell nuclei of Welwitschia mirabilis dense strands are observed between areas of low density (Figure 1A, B and C). The three dimensional arrangement of the strands is evident when they are observed with light (Figure 1A) and atomic force microscopy (Figure 1B). Conventional staining for transmission electron microscopy shows the strands heavily stained with uranyl acetate and lead citrate (Figure 1C). To determine whether the dense strands observed in the nuclei of W. mirabilis correspond to DNA, we used the osmium ammine technique specific for DNA. The strands were densely stained indicating that they correspond to DNA. Little or no DNA is present in the nucleoplasm and nucleolus and no staining is observed in the cytoplasm (Figure 2).

Figure 1

Welwitschia mirabilis cell nuclei show a reticulated pattern of compact chromatin. (A) Bright field of a cell nucleus stained with toluidine blue. (B) Atomic force microscopy of this organelle showing three dimensional arrangement of intranuclear strands. (C) Conventional staining for transmission electron microscopy showing strands of compact chromatin (c). Nucleus (N), cytoplasm (cyt), cell wall (cw), Nucleolar Organizer Region (NOR), nucleolus (nu).

2007-4476-bs-96-04-678-gf1.jpg

Figure 2

Nucleus of W. mirabilis stained with osmium amine specific for DNA. Dense strands (c), are stained with this technique. Nucleolus (nu) and cytoplasm (cyt) are not stained.

2007-4476-bs-96-04-678-gf2.jpg

Nuclear particles. The nucleoplasm of W. mirabilis is composed of a fibrogranular environment among the strands of chromatin, with electrodense particles about 32 ± 1.7 nm in diameter. Granules are associated or interconnected to electron dense fibers (Figures 3-4), both, in interchromatin and perichromatin areas (Figure 4). The EDTA regressive technique for ribonucleoproteins bleached the compact chromatin strands and granules and fibers are heavily stained (Figure 5). The association of W. mirabilis extranucleolar granules to reticulated chromatin, their location, abundance and ribonucleoproteic nature indicate that these particles correspond to Lacandonia granules reported in the species L. schismatica, T. brevystilis (Jiménez-García et al. 1992) and Ginkgo biloba (Jiménez-Ramírez et al. 2002).

Figure 3

Nucleus of W. mirabilis stained with uranyl acetate and lead citrate. Abundant granules (large arrows), bigger than ribosomes (small arrows), are observed in the nucleoplasm. Cytoplasm (cyt). Chromatin (c), nuclear envelope (ne).

2007-4476-bs-96-04-678-gf3.jpg

Figure 4

Intranuclear granules in a cell of W. mirabilis (large arrows). Ribosomes (small arrows) are indicated in the cytoplasm (cyt), compact chromatin (c), nuclear envelope (ne).

2007-4476-bs-96-04-678-gf4.jpg

Figure 5

EDTA regressive staining of W. mirabilis cell nucleus. Granules (thick arrows) and associated fibers (thin arrow) are stained. The chromatin is bleached (c). Cytoplasm (cyt), ribosomes (r).

2007-4476-bs-96-04-678-gf5.jpg

Discussion

Compact chromatin. Optical microscopy showed strands that stain with toluidine blue distributed in the nuclei of cells of Welwitschia mirabilis (Figure 1A). Thin sections of samples prepared for transmission electron microscopy and observed with the electron microscope corroborated the reticulated pattern of the strands (Figure 1B) and atomic force microscopy showed the three dimensional arrangement of the strands in the nucleoplasm. The strands are also positive for the osmium amine technique specific for DNA. Therefore, these strands correspond to the reticulated pattern reported previously using also results from atomic force microscopy (Jiménez-Ramírez et al. 2002).

Nuclear particles. In previous studies, the presence of ribonucleoprotein granules has been described in nuclei that have a reticulated pattern such as Lacandonia schismatica and G. biloba (Agredano-Moreno et al. 1994, Agredano-Moreno et al. 2000, Fragoso-Soriano et al. 2009, Jiménez-García et al. 1992, Jiménez-García & Fragoso-Soriano 2000, Jiménez-Ramírez et al. 2002). Therefore, we searched for Lacandonia granules in cell nuclei of W. mirabilis. Using standard transmission electron microscopy, we observed granules in the nucleoplasm, about 32 nm in diameter, associated to fibers in the periphery of chromatin. These granules were stained after the EDTA regressive staining preferential for ribonucleoproteins, while compact chromatin is observed with low contrast. These particles also may correspond to similar although scarce, particles observed in some members of bryophytes (Alonso-Murillo & Jiménez-García 2015).

Therefore, the size, distribution, shape, positivity to the EDTA technique, and fibrogranular arrangement of the nuclear particles in W. mirabilis reported here, correspond to Lacandonia granules described previously in the plant Lacandonia schismatica (Jiménez-García et al. 1992, Agredano-Moreno & Jiménez-García 2000, Jiménez-García & Fragoso-Soriano 2000, Jiménez-Ramírez et al. 2002, Fragoso-Soriano & Jiménez-García 2009).

Acknowledgments

This work was partially supported by Dirección General de Asuntos del Personal Académico-Universidad Nacional Autónoma de México-Programas institucionales de apoyo e Impulso a la investigación y a la docencia (DGAPA-UNAM-PAPIIT IN217917, PAPIME PE213916).

The authors thank María Eugenia Muñiz Díaz de León [Department of Comparative Biology, Faculty of Sciences, National Autonomous University of Mexico (UNAM)], for her expertise in the cultivation of Welwitschia mirabilis.

Literature cited

1

Agredano-Moreno LT, Jiménez-García LF, Echeverría OM, Martínez E, Ramos C, Vázquez-Nin GH. 1994. Cytochemical and immunocytochemical study of structures of Lacandonia schismatica. Biology of Cell 82: 177-184. DOI: https://doi.org/10.1016/S0248-4900(94)80020-0

LT Agredano-Moreno LF Jiménez-García OM Echeverría E Martínez C Ramos GH Vázquez-Nin 1994Cytochemical and immunocytochemical study of structures of Lacandonia schismaticaBiology of Cell8217718410.1016/S0248-4900(94)80020-0

2

Agredano-Moreno LT, Jiménez-García LF. 2000. New evidence that Lacandonia granules are ultrastructurally related to perichromatin and Balbiani ring granules. Biology of the Cell 92: 71-78. DOI: https://doi.org/10.1016/S0248-4900(00)88765-1

LT Agredano-Moreno LF Jiménez-García 2000New evidence that Lacandonia granules are ultrastructurally related to perichromatin and Balbiani ring granulesBiology of the Cell92717810.1016/S0248-4900(00)88765-1

3

Alonso-Murillo CD, Jiménez-García LF. 2015. Plants related to early evolutionary events (Bryophytes) contain Lacandonia granules previously discovered in flowering plants. Acta Microscopica 24: 152-158.

CD Alonso-Murillo LF Jiménez-García 2015Plants related to early evolutionary events (Bryophytes) contain Lacandonia granules previously discovered in flowering plantsActa Microscopica24152158

4

Bernhard W. 1969. A new staining procedure for electron microscopical cytology. Journal of Ultrastructural Research 27: 250-265. DOI: https://doi.org/10.1016/S0022-5320(69)80016-X

W Bernhard 1969A new staining procedure for electron microscopical cytologyJournal of Ultrastructural Research2725026510.1016/S0022-5320(69)80016-X

5

Fragoso-Soriano RJ, Vázquez-López C, Pérez-García CB, Jiménez-García LF. 2009. Atomic Force Microscopy Imaging of Thin Sections of Lacandonia Granules. Journal of Scanning Probe Microscopy 4: 1-5. DOI: https://doi.org/10.1166/jspm.2009.1011

RJ Fragoso-Soriano C Vázquez-López CB Pérez-García LF Jiménez-García 2009Atomic Force Microscopy Imaging of Thin Sections of Lacandonia GranulesJournal of Scanning Probe Microscopy41510.1166/jspm.2009.1011

6

Jiménez-García LF, Agredano-Moreno LT, Segura-Valdez ML, Echeverría O, Martínez E, Ramos CH, Vázquez-Nin GH. 1992. The ultrastructural study of the interphase cell nucleus of Lacandonia schismatica (Lacandoniaceae:Triuridales) reveals a non-typical extranucleolar particle. Biology of the Cell 75: 101-110. DOI: https://doi.org/10.1016/0248-4900(92)90129-O

LF Jiménez-García LT Agredano-Moreno ML Segura-Valdez O Echeverría E Martínez CH Ramos GH Vázquez-Nin 1992The ultrastructural study of the interphase cell nucleus of Lacandonia schismatica (Lacandoniaceae:Triuridales) reveals a non-typical extranucleolar particleBiology of the Cell7510111010.1016/0248-4900(92)90129-O

7

Jiménez-García LF, Fragoso-Soriano R. 2000. Atomic force microscopy of the cell nucleus. Journal of Structural Biology 129: 218-222. DOI: https://doi.org/10.1006/jsbi.2000.4233

LF Jiménez-García R Fragoso-Soriano 2000Atomic force microscopy of the cell nucleusJournal of Structural Biology12921822210.1006/jsbi.2000.4233

8

Jiménez-García LF, Segura-Valdez M de L. 2004. Visualizing nuclear structure in situ by atomic force microscopy. In: Braga PC, & Ricci D. eds. Atomic Force Microscopy: Methods and Protocols in Biomedical Applications. Methods in Molecular Medicine, New Jersey, USA: Humana Press, 242: 191-199. DOI: https://doi.org/10.15406/mojap.2017.03.00109. ISBN 978-1-61779-105-5

LF Jiménez-García L Segura-Valdez M de 2004Visualizing nuclear structure in situ by atomic force microscopy PC Braga D Ricci Atomic Force Microscopy: Methods and Protocols in Biomedical Applications. Methods in Molecular MedicineNew Jersey, USAHumana Press24219119910.15406/mojap.2017.03.00109978-1-61779-105-5

9

Jiménez-Ramírez J, Agredano-Moreno LT, Segura-Valdez ML, Jiménez-García LF. 2002. Lacandonia granules are present in Ginkgo biloba cell nuclei. Biology of the Cell 94: 511-518. DOI: https://doi.org/10.1016/S0248-4900(02)00019-9

J Jiménez-Ramírez LT Agredano-Moreno ML Segura-Valdez LF Jiménez-García 2002Lacandonia granules are present in Ginkgo biloba cell nucleiBiology of the Cell9451151810.1016/S0248-4900(02)00019-9

10

Márquez-Guzmán J, Engleman M, Martínez-Mena A, Martínez E, Ramos C. 1989. Anatomía reproductiva de Lacandonia schismatica (Lacandoniaceae). Annals of the Missouri Botanical Garden 76: 124-127. DOI: https://doi.org/10.2307/2399345

J Márquez-Guzmán M Engleman A Martínez-Mena E Martínez C Ramos 1989Anatomía reproductiva de Lacandonia schismatica (Lacandoniaceae)Annals of the Missouri Botanical Garden7612412710.2307/2399345

11

Martínez E, Ramos CH. 1989. Lacandoniaceae (Triuridales): una nueva familia de México. Annals of the Missouri Botanical Garden 76: 128-135. DOI: https://doi.org/10.2307/2399346

E Martínez CH Ramos 1989Lacandoniaceae (Triuridales): una nueva familia de MéxicoAnnals of the Missouri Botanical Garden7612813510.2307/2399346

12

Segura-Valdez M L, Zamora-Cura A, Gutiérrez-Quintanar N, Villalobos Nájera E, Rodríguez-Vázquez JB, Galván-Arrieta TC, Jiménez-Rodríguez D, Agredano-Moreno LT, Lara-Martínez R, Jiménez-García LF. 2010. In: Méndez-Vilas A, &Díaz J, eds. Visualization of Cell Structure in Situ by Atomic Force Microscopy. Badajoz, Spain: Microscopy: Science, Technology, Applications and Education pp. 441-448. Formatex,

L Segura-Valdez M A Zamora-Cura N Gutiérrez-Quintanar E Villalobos Nájera JB Rodríguez-Vázquez TC Galván-Arrieta D Jiménez-Rodríguez LT Agredano-Moreno R Lara-Martínez LF Jiménez-García 2010 A Méndez-Vilas J Díaz Visualization of Cell Structure in Situ by Atomic Force MicroscopyBadajoz, SpainMicroscopy: Science, Technology, Applications and Education441448

13

Stevens PF. 2001 (onwards). Angiosperm Phylogeny Website, Version 14. < http://www.mobot.org/MOBOT/research/APweb/ >. (accessed July, 2017).

PF Stevens 2001onwardsAngiosperm Phylogeny Website14 http://www.mobot.org/MOBOT/research/APweb/ July, 2017

14

Vázquez-Nin GH, Biggiogera M, Echeverría OM. 1995. Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA. European Journal of Histochemistry. 39: 101-106

GH Vázquez-Nin M Biggiogera OM Echeverría 1995Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNAEuropean Journal of Histochemistry39101106

Notas

1 Associated editor: Salvador Arias



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Botanical Sciences is an international peer-reviewed journal that publishes scientific papers in plant sciences. The arguments, figures / schemes / photographs, quality and the general contents of this publication are full responsibility of the authors, and not commit the Editor- in-Chief or the Sociedad Botánica de México.

Botanical Sciences year 8, Vol. 97, No. 1, January-March 2019. Quarterly publication edited and published by Sociedad Botánica de México A.C. (www.socbot.mx). Editor in Chief Salvador Arias, Jardín Botánico, Instituto de Biología, 3er Circuito s/n, Ciudad Universitaria, Delegación Coyoacán, C.P. 04510. Reserves of Rights to the Exclusive Use No. 04-2017-040716054100-203, digital-ISSN 2007-4476, both granted by the Instituto Nacional del Derecho de Autor. Responsible for updating the page Pedro López, email: plopez@escire.mx, eScire. Last update March 11, 2019.

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