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Cell changes during the re-induction of desiccation tolerance in germinated seeds of Sesbania virgata (Cav.) Pers.

Mudanças celulares durante a re-indução da tolerância à dessecação em sementes germinadas de Sesbania virgata (Cav.) Pers.

Abstract

During germination, orthodox seeds lose their ability to tolerate desiccation resembling recalcitrant seeds. This research aimed to investigate the cell changes during the re-induction of the desiccation tolerance (DT) in Sesbania virgata germinated seeds with 1, 3 and 5 mm long radicles. To re-establish DT, germinated seeds were incubated for 72 h in polyethylene glycol (PEG, -2.04 MPa) before dehydration in silica gel (at 10% moisture content) followed by rehydration. Cell viability was assessed through TUNEL test in dry radicles and transmission electron microscopy in both fresh and dry radicles. The positive-TUNEL confirmed the DNA degradation, through the green fluorescence of the cell nuclei from 5 mm radicle length and the ultra structural evaluations detected loss of cellular content integrity in 3 and 5 mm cell radicles that did not survive dehydration to 10%.

Index terms:
cell viability; transmission electron microscopy; TUNEL test

Resumo

Durante a germinação, as sementes ortodoxas perdem sua habilidade de tolerar a dessecação assemelhando-se com as sementes recalcitrantes. Este trabalho objetivou investigar as mudanças celulares que ocorrem durante a re-indução da tolerância à dessecação (TD) em sementes germinadas de Sesbania virgata com 1, 3 e 5 mm de comprimento de radícula. Para restabelecer a TD, as sementes germinadas foram incubadas durante 72 h em solução de polietilenoglicol (PEG, -2,04 MPa), posteriormente foram desidratadas em sílica gel (10% de teor de água) e reidratadas. A viabilidade celular foi avaliada pelo teste de TUNEL e através de microscopia eletrônica de transmissão em radículas que foram submetidas à secagem ou não. O TUNEL-positivo confirmou a degradação do DNA por meio da fluorescência dos núcleos nas células da radícula com cinco mm de comprimento e as avaliações ultraestruturais detectaram a perda da integridade do conteúdo celular nas radículas com 3 e 5 mm de comprimento e que não sobreviveram à desidratação a 10% de teor de água.

Index terms:
viabilidade celular; microscopia eletrônica de transmissão; teste de TUNEL

Introduction

The integrity of DNA and cell structures during severe water loss in seeds is predicated on the seeds survival and their capacity of resume their growth after rehydration, suggesting that eventual DNA degradation is linked to death in seeds (Osborne and Boubriak, 1997OSBORNE, D.J.; BOUBRIAK, I.I. DNA status, replication and repair in desiccation tolerance and germination. Basic and applied aspects of seed biology. Kluwer Academic Publishers, 1997. p. 23-32.; Faria et al., 2005FARIA, J.M.R.; BUITINK, J.; LAMMEREN, A.A.M.; HILHORST, H.W.M. Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds. Journal of Experimental Botany , v.56, n.418, p.2119-2130, 2005. http://jxb.oxfordjournals.org/content/56/418/2119.short
http://jxb.oxfordjournals.org/content/56...
). Studies at cellular level in germinated orthodox seeds during dehydration and following rehydration may help to better understand the mechanisms that control desiccation tolerance (DT) and sensitivity in seeds (Masetto et al., 2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
). Thus, assessment of DNA integrity may also help to explain DT and sensitivity, since DNA stability during dehydration or the ability of its repairment upon rehydration is an essential component of the tolerance mechanisms (Boubriak et al., 1997BOUBRIAK, I.; KARGIOLAK, H.; LYNE, L.; OSBORNE, D. J. The requirement for DNA repair in desiccation tolerance of germinating embryos. Seed Science Researchv.7, n.2, p.97-105, 1997. http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=1293168&fileId=S0960258500003433
http://journals.cambridge.org/action/dis...
). This can be done through techniques such as transmission electron microscopy and TUNEL (Terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling) test, in which modified nucleotides (dUTP) are incorporated to extremities of DNA fragments by the terminal deoxynucleotide transferase enzyme (El-Maarouf-Bouteau et al., 2011EL-MAAROUF-BOUTEAU, H.; MAZUY, C.; CORBINEAU, F.; BAILLY, C. DNA alteration and programmed cell death during ageing of sunflower seed. Journal of Experimental Botany, v.62, n.14, p.5003-5011, 2011. https://jxb.oxfordjournals.org/content/62/14/5003.full
https://jxb.oxfordjournals.org/content/6...
)

It has previously been shown that there was a relationship between the loss of DNA integrity and loss of DT in 3 and 5-mm-long radicles of Sesbania virgata (Cav.) (Pers.) (Fabaceae) - a shrub useful in land reclamation - when dehydrated to 10% moisture content, and the cytological assessment of the radical meristem provided evidence of the occurrence of cell death in the 3 and 5-mm-long radicles, which did not survive dehydration (Masetto et al., 2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
). Although the authors indicated that, this study seeks to understand the nature and extent of damage from intracellular dehydration during the re-establishment of DT in S. virgata germinated seeds.

Material and Methods

The seed collection and processing was carried out according to Masetto et al. (2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
) from about 40 seed-trees, in Ijaci (21º10'S, 44º54'W), South of Minas Gerais State, Brazil. The moisture content of the seeds was assessed in four replications of 2 g each, by oven-drying at 103 ºC for 17 hours (Brasil, 2009BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 395p. http://www.agricultura.gov.br/arq_editor/file/2946_regras_analise__sementes.pdf
http://www.agricultura.gov.br/arq_editor...
). The seeds showed 10% moisture content. For the obtainment of germinated seeds, initially the seeds were scarificated with concentrated sulphuric acid, for 40 min, washed in running water for 10 min, disinfested with sodium hypochlorite at 2% for 2 min, washed again and sown on moistened filter paper in germination boxes. The test was carried out in incubators at 25 ºC and constant white light (Brasil, 2009BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 395p. http://www.agricultura.gov.br/arq_editor/file/2946_regras_analise__sementes.pdf
http://www.agricultura.gov.br/arq_editor...
). The evaluations after re-induction of desiccation tolerance were carried out with germinated seeds with 1, 3 and 5 mm long radicles which were chosen for cell investigations according to the results of the previous experiments of Masetto et al. (2015)MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
as follows: Germinated seeds with 1, 3 and 5 mm radicle length were put in Petri dishes with a filter paper on the bottom, moistened with 20 mL of PEG 8000 solutions (380 g dissolved in 1 L water, according to Michel and Kaufmann, 1973MICHEL, B.E.; KAUFMANN, M.R. The osmotic potential of polyethylene glycol 6000. Plant Physiology, v.51, n.5, p.914-916, 1973. http://www.plantphysiol.org/content/51/5/914.short
http://www.plantphysiol.org/content/51/5...
) at 5 ºC for 72 h, which provides an osmotic potential of -2.04 MPa. Germinated seeds were washed in running water to remove the PEG solution residues and superficially dried on paper towel, for 10 min, following the seeds were dehydrated in silica gel at 20 ºC/60% RH. Samples were dried to the original seed moisture content (10%), pre-humidified (100% UR/ 24 h/ 25 ºC) and rehydrated as described previously. Four independent experiments with 25 germinated seeds of each radicle length were carried out.

The Terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction is used to evaluate the DNA fragmentation by the 3'-OH extremities detection of the DNA strand, by the action of the Terminal deoxynucleotide transferase enzyme, through the green fluorescence (APO-BrdUTM TUNEL Assay Kit, Invitrogen, Molecular Probes, with Alexa fluor). Radicles of germinated seeds (1, 3 and 5 mm long) dehydrated in silica to 10% MC after PEG-treatment, were fixed in paraformaldehyde 1% for 12 h. After this period, radicles were dehydrated in alcohol gradient for 1 h each (30%, 50%, 70%, 90% and 100%), fixed in Steedman's wax 37%, using one series of wax:ethanol (v:v) (50:50%, 70:30%, 90:10%) and 100% of wax every 1 h. The radicles were sectioned longitudinally with 10 μm width using microtome and reactions were prepared according to the manufacturer. The images were observed with an epifluorescence microscope, utilizing 500 nm wave length (Olympus BX60).

For the cellular morphology analysis through electron microscopy transmission, the 1, 3 and 5 mm long radicles from both fresh germinated seeds and after PEG treatment, dehydration in silica gel and rehydration, were analyzed (five germinated seeds per treatment). Samples were fixed in the modified Karnovsky's solution (glutaraldehyde 2.5%; formaldehyde 2% in a cacodylate sodium buffer 0.05M; CaCl2 0.001M, pH 7.2), washed three times (10 min each) in cacodylate buffer 0.05M, fixed in an aqueous solution of osmium tetroxide 1% for 2 h, at room temperature and washed in Milli-Q water for 15 min. Later, samples were contrasted in uranyl acetate (0.5%) for 12 h, at 4 ºC, and dehydrated in an acetone gradient (25%, 50%, 75%, 90% and 100% three times). Afterwards the material was embedded in an increasing gradient of spurr/acetone solutions 30%/8 h; 70%/12 h and 100% twice, for 24 h each. Samples were then transferred to silicon molds and polymerized in oven at 70 ºC, for 48 h. Sections were made in the meristematic region of the radicles, using a Reichart-Jung ultramicrotome, with 100 nm thickness and contrasted with uranyl acetate, followed by lead citrate (three min). Samples were evaluated on transmission electron microscope (Zeiss EM 109, Carl Zeiss, Jena, Germany), at 80 kV.

Results and Discussion

Images of cells after TUNEL test are presented on Figure 1. Germinated seeds with 1 and 3 mm long radicles showed TUNEL-negative (Figure 1A and Figure 1B, respectively). The green fluorescent-colored nuclei indicate TUNEL-positive cells as revealed by bright green fluorescent spots (Figure 1C), which means that numerous nuclei were labelled. This corresponds to the total loss of integrity in the genetic material of PEG-treated 5 mm long radicles which did not survived dehydration and was unable to produce normal seedlings, as demonstrated by Masetto et al. (2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
). TUNEL analysis was applied to detect a possible collapse of DNA in situ, since both programmed and necrotic cell death are characterized by the degradation of nuclear DNA and can be visualized with the TUNEL assay that labels the DNA breaks (Gavrieli et al., 1992GAVRIELI, Y.; SHERMAN, Y.; BEN-SASSON, S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology, v.119, n.3, p.493-501, 1992. http://jcb.rupress.org/content/119/3/493.short
http://jcb.rupress.org/content/119/3/493...
; El-Maarouf-Bouteau et al., 2011).

Figure 1
Epifluorescence micrographs of TUNEL assays performed on cells from 1 (A), 3 (B) and 5 (C) mm long radicles of Sesbania virgata germinated seeds, after incubation in PEG (-2.04 MPa), dehydration in silica gel to 10% MC and rehydration. Bright green spots correspond to the TUNEL-positive nuclei.

According to the cytological factors influencing the re-establishment of DT, the transition from fresh germinated radicles to dehydrated and rehydrated ones was accomplished by structural changes in the cells of radicle meristem (Figure 2). In fresh 1, 3 and 5 mm long radicles (Figures 2A, 2C and 2E, respectively) there was a perfect delimitation of the cell wall, small vacuole and presence of lipid bodies and starch granules. After dehydration and rehydration of 3 and 5 mm long radicles (Figure 2D and Figure 2F, respectively) there was loss of cell wall integrity and cellular content, and disappearance of starch granules. Thus, cytosolic constituents that spill into extracellular space through damaged plasma membrane may provoke injury responses (Proskuryakov et al., 2003PROSKURYAKOV, S.Y.A.; KONOPLYANNIKOV, A.G.; GABAI, V.L. Necrosis: a specific form of programmed cell death? Experimental Cell Research, v.283, p.1-16, 2003. http://www.sciencedirect.com/science/article/pii/S0014482702000277
http://www.sciencedirect.com/science/art...
).

In despite of these results, only 5 mm long radicles showed Tunel-positive (Figure 1 C), indicating the total loss of nuclei viability provoking 100% of dead seedlings (Masetto et al., 2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
). Possibly, the loss of cell compartmentalization in 3 mm long radicles indicates low cellular repair activities in the desiccated state (Figure 2D) but still not lead to a total accumulation of damage to DNA (Figure 1B), suggesting ability at least, partially, to resume their growth after dehydration following rehydration, as demonstrated in 47% seedlings (Masetto et al., 2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
).

Findings concerning the loss of desiccation tolerance during the seeds germination have already been reported in germinated seeds of Medicago truncatula (Buitink et al., 2003BUITINK, J.; VU, B. L.; SATOUR, P.; LEPRINCE, O. The re-establishment of desiccation tolerance in germinated radicles of Medicago truncatula Gaertn. seeds. Seed Science Research v.13, n.4, p.273-286, 2003. http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=704856&fileId=S0960258503000278
http://journals.cambridge.org/action/dis...
; Faria et al., 2005FARIA, J.M.R.; BUITINK, J.; LAMMEREN, A.A.M.; HILHORST, H.W.M. Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds. Journal of Experimental Botany , v.56, n.418, p.2119-2130, 2005. http://jxb.oxfordjournals.org/content/56/418/2119.short
http://jxb.oxfordjournals.org/content/56...
), Tabebuia impetiginosa (Vieira et al., 2010VIEIRA, C.V.; SILVA, E.A.A.; ALVARENGA, A.A.; CASTRO, E. M.; TOOROP, P.E. Stress-associated factors increase after desiccation of germinated seeds of Tabebuia impetiginosa Mart. Plant Growth Regulation , v.62, n.3, p.257-263, 2010. http://link.springer.com/article/10.1007/s10725-010-9496-3#page-1
http://link.springer.com/article/10.1007...
) and Cedrela fissilis (Masetto et al., 2014MASETTO, T.E.; FARIA, J.M.; FRAIZ, A.C.R. Re-induction of desiccation tolerance after germination of Cedrela fissilis Vell. seeds. Anais da Academia Brasileira de Ciências , v.86, n.3, p.1273-1286, 2014. http://www.scielo.br/scielo.php?pid=S0001-37652014005030164&script=sci_arttext&tlng=pt
http://www.scielo.br/scielo.php?pid=S000...
), where DT could be re-established applying a mild osmotic shock with polyethylene glycol. Mature seeds of S. virgata are desiccation tolerant (survive the loss of most of their water content), but they become desiccation sensitive while progressing to germination. In any way, the maintenance of DNA integrity and the cell compartments of the root tissue seems to be important prerogatives to re-establish the normal seedlings development after the primary root dehydration. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organism (Kraner et al., 2011KRANER, I.; CHEN, H.; PRITCHARD, H.W.; PEARCE, S.R.; BIRTIC, S. Inter-nucleosomal DNA fragmentation and loss of RNA integrity during seed ageing. Plant Growth Regulation, v.63, n.1, p.63-72, 2011. http://link.springer.com/article/10.1007/s10725-010-9512-7#page-1
http://link.springer.com/article/10.1007...
; Atale et al., 2014ATALE, N.; GUPTA, S.; YADAV, U. C. S.; RANI, V. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. Journal of Microscopy, v.255, n1, p.7-19, 2014. http://onlinelibrary.wiley.com/doi/10.1111/jmi.12133/full
http://onlinelibrary.wiley.com/doi/10.11...
; Dresch et al., 2015DRESCH, D.M.; MASETTO, T.E.; SCALON, S.P.Q. Campomanesia adamantium (Cambess.) O. Berg seed desiccation: influence on vigor and nucleic acids. Anais da Academia Brasileira de Ciências, v.87, n.4, p.2217-2228, 2015. http://www.scielo.br/scielo.php?pid=S0001-37652015000502217&script=sci_arttext
http://www.scielo.br/scielo.php?pid=S000...
).

The germinated seeds of S. virgata restrict their resumption of growth according to the PEG treatment, the radicle length and the moisture content attained. According to Masetto et al. (2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
) the occurrence of a non-programmed event of the cell death in 3 and 5 mm long radicle of S. virgata dehydrated at 10% MC may be a regulated cellular response to stress. This behaviour indicates that the maintenance of genetic information is fundamental to DT and cell survival after dehydration and rehydration.

Desiccation sensitivity of PEG-treated 5 mm long radicles has clearly induced the cellular death and indicates that this may be an important mechanism of intolerance to water stress in S. virgata germinated seeds. Considering the results of cytological assays, the data obtained from TUNEL are strongly related with the DNA degradation, as seen in Masetto et al. (2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
) and with the structural changes (Figure 2F). Cellular death in the primary root of Stenocereus gummosus and Pachycereus pringlei was also detected by TUNEL (Shishkova and Dubrovsky, 2005SHISHKOVA, S.; DUBROVSKY, J.G. Developmental programmed cell death in primary roots of sonoran desert cactaceae. American Journal of Botanyv.92, n.9, p.1590-1594, 2005. http://www.amjbot.org/content/92/9/1590.short
http://www.amjbot.org/content/92/9/1590....
). Although the nuclei TUNEL-positive may be considered a cell death indicator in S. virgata germinated seeds, other cytological factors may be assessed, such as the ultra-structural characterization of cellular death in plants, which indicates a progressive loss of cellular compartmentalization and organization (DeBono and Greenwood, 2006DEBONO, A.G.; GREENWOOD, J.S. Characterization of programmed cell death in the endosperm cells of tomato seed: two distinct death programs. Canadian Journal of Botany, v. 84, n.5, p.791-804, 2006. http://www.nrcresearchpress.com/doi/abs/10.113 9/b06-034
http://www.nrcresearchpress.com/doi/abs/...
).

Figure 2
Images of meristematic cells obtained by Transmission Electron Microscopy of S. virgata germinated seeds with 1 (A-B), 3 (C-D) and 5 (E-F) mm long radicle before dehydration (A, C, E) and after dehydration and rehydration (B, D, F). Emphasis on the cell wall (CW) with normal aspect before dehydration (A, E). The arrows in the Figures D and F show the loss of cytoplasm integrity after dehydration and rehydration (D, F). LB = lipid bodies; SG = starch granule.

Many morphological and biochemical changes have been found in vegetal cells that suffer stress and show cellular and nuclear phenotypes that result from DNA damage, among them, cytoplasm and nucleus condensation and shrinkage, as seen in Figures 2D and 2F. The cytoplasm and plasmatic membrane presented morphological aberrations. The cytoplasm becomes extremely vesicular and vacuolar, with floccose appearance and deformed (Gunawardena et al., 2001GUNAWARDENA, A.H.A.L.N.; PEARCE, D.M.; JACKSON, M.B.; HAWES, C.R.; EVANS, D.E. Characterization of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of mayze (Zea mays L.). Planta, v.212, n.2, p.205-214, 2001. http://link.springer.com/article/10.1007/s004250000381
http://link.springer.com/article/10.1007...
; Errakhi et al., 2008ERRAKHI, R.; MEIMOUN, P.; LEHNER, A.; VIDAL, G.; BRIAND, G.; CORBINEAU, F.; RONA, J.P.; BOUTEAU, F. Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid. Journal of Experimental Botany, v.59, n.11, p.3121-3129, 2008. http://jxb.oxfordjournals.org/content/59/11/3121.short
http://jxb.oxfordjournals.org/content/59...
). The loss of cell compartmentalization and green fluorescent-colored nuclei assert the desiccation sensitivity in 5 mm long radicles of S. virgata.

According to the Figures 2D and 2F, possibly, in the germinated seeds with 3 and 5 mm long radicles, respectively, the cells die and release their content, which include highly harmful lysosomal proteolytic enzymes that affect other cells and start a cascade of cell-death event, in the surroundings (Atale et al., 2014ATALE, N.; GUPTA, S.; YADAV, U. C. S.; RANI, V. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. Journal of Microscopy, v.255, n1, p.7-19, 2014. http://onlinelibrary.wiley.com/doi/10.1111/jmi.12133/full
http://onlinelibrary.wiley.com/doi/10.11...
), clearly marking the method of cell death as necrosis, according demonstrated in an agarose gel by Masetto et al. (2015MASETTO, T.E.; FARIA, J.M.R.; FRAIZ, A.C.R. Loss and re-establishment of desiccation tolerance in the germinated seeds of Sesbania virgata (Cav.) (Pers.). Acta Scientiarum Agronomy, v.37, n.3, p.313 -320, 2015. http://www.scielo.br/scielo.php?pid=S1807-86212015000300313 &script=sci_arttext
http://www.scielo.br/scielo.php?pid=S180...
).

The underlying mechanisms of orthodox germinated seeds death are less understood than the empirical description of the radicle length and the moisture content attained. In any way the ability of orthodox germinated seeds to withstand severe desiccation generally depends on the integrity of nucleic acids. However, the loss of nucleic acid integrity together with other cell deteriorative phenomena may also be interpreted as the cause for further non overcoming severe dehydration and eventually death of germinated seeds.

Conclusions

The nuclei TUNEL-positive may be considered a cell death indicator in S. virgata germinated seeds, besides the progressive loss of cellular compartmentalization and organization evidence the occurrence of a non-programmed cell death.

Acknowledgements

We are thankful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the support by the Project PPM-00470-14.

References

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    » http://onlinelibrary.wiley.com/doi/10.1111/jmi.12133/full
  • BOUBRIAK, I.; KARGIOLAK, H.; LYNE, L.; OSBORNE, D. J. The requirement for DNA repair in desiccation tolerance of germinating embryos. Seed Science Researchv.7, n.2, p.97-105, 1997. http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=1293168&fileId=S0960258500003433
    » http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=1293168&fileId=S0960258500003433
  • BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 395p. http://www.agricultura.gov.br/arq_editor/file/2946_regras_analise__sementes.pdf
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Publication Dates

  • Publication in this collection
    22 Aug 2016
  • Date of issue
    Jul-Sep 2016

History

  • Received
    14 Mar 2016
  • Accepted
    23 June 2016
ABRATES - Associação Brasileira de Tecnologia de Sementes Av. Juscelino Kubitschek, 1400 - 3° Andar, sala 31 - Centro,, CEP 86020-000 Londrina/PR - Londrina - PR - Brazil
E-mail: jss@abrates.org.br