| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
0 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803-1705 USA
Received for publication November 10, 1998. Accepted for publication May 10, 1999.
ABSTRACT
In many wetland species, root aerenchyma is produced by the predictable collapse of root cortex cells, indicating a programmed cell death (PCD). The objective of this study was to characterize the cellular changes that accompany this PCD in the marsh species Sagittaria lancifolia. Structural changes in membranes and organelles were examined during development of root cortex cells to compare with previous examples of PCD. The organization of cortical microtubule (CMT) arrays in root cells from S. lancifolia was also evaluated as a possible predictor of cell lysis. Nuclear fragmentation and condensation were the earliest changes observed in cells undergoing lysis. Breakdown of the tonoplast and other organelles and disruption of the plasma membrane followed. After loss of cytoplasm, cells collapsed to form gas spaces. These results were compared to collapse of root cortical cells of Zea mays and Oryza sativa during aerenchyma development. Changes in the appearance of the cytoplasm of all three species were similar at later stages of aerenchyma development. The relative timing of disintegration of the tonoplast and middle lamella appeared to differ among the three species. Changes in the organization of CMT arrays did not appear to be a predictor of PCD in S. lancifolia. Aerenchyma production in plants involves a type of PCD that is morphologically distinct from PCD described from many animals.
Key Words: aerenchyma • apoptosis • flooding • microtubules • plant • programmed cell death (PCD) • wetland
The stress of low oxygen concentrations in a waterlogged environment is minimized in wetland plants that produce aerenchyma, a tissue characterized by continuous gas spaces. These gas spaces provide a pathway for oxygen transport from shoots to roots. Development of gas spaces in many wetland species is constitutive because this process occurs whether or not plants are growing in aerated or waterlogged soils (Schussler, 1997
). The predictable lysis of root cortex cells suggests aerenchyma production in roots depends on a genetically controlled program of cell death (Jones and Dangl, 1996
; Drew, 1997
; Kawai et al., 1998
). Programmed cell death (PCD) is recognized as an important developmental mechanism in a wide range of plant tissues (Greenberg, 1996
; Jones and Dangl, 1996
). In many cases PCD is characterized by initial changes in the nuclear organization of the cell and an ordered breakdown of the cellular contents (Kerr, Wyllie, and Currie, 1972
). By comparison, necrotic cell death is characterized by initial changes in mitochondria and cell swelling prior to death (Kerr, Wyllie, and Currie, 1972
). In PCD, a variety of other changes in cell structure can follow the initial changes in nuclear structure. The presence and absence of these later changes in cell structure depend on the type of cell death examined (Greenberg, 1996
).
In studies of animal cells, cell shrinkage, condensation of nuclear chromatin (pycnosis), nuclear fragmentation, and exocytosis of cell contents in membrane-bound "apoptotic" bodies characterize a common type of PCD termed apoptosis (Kerr, Wyllie, and Currie, 1972
). PCD in other types of animal cells lacks some of the features of apoptosis and has been termed nonapoptotic PCD (Schwartz et al., 1993
). Clarke (1990)
defined several types of nonapoptotic PCD in animal cells that include distinctive morphological features such as autophagic vacuoles that engulf cell contents and nonlysosomal destruction of organelles.
Several features of apoptosis are absent in plant cells that undergo PCD (Greenberg, 1996
). Tracheary element differentiation in Zinnia elegans (Fukuda, 1997
; Groover et al., 1997
) and the cell degradation that accompanies the hypersensitive response to pathogen attack (Mittler and Lam, 1996
) are two examples of PCD in plants that lack certain features characteristic of apoptosis. We hypothesize that aerenchyma development is also nonapoptotic based on earlier studies of gas space formation in roots of Zea mays (Campbell and Drew, 1983
) and Oryza sativa (Webb and Jackson, 1986
).
The organization of cortical microtubules (CMT) plays a key role in developing plant cells because the orientation of CMT arrays predicts the direction of cell expansion (Barlow and Parker, 1996
). CMT arrays are mostly perpendicular or diagonal to the root axis in expanding cortex cells, and these cells elongate in a direction parallel to the root axis (Williamson, 1991
). Orientation of CMT arrays, however, can be rapidly altered by external signals such as hormones or environmental stress, leading to changes in patterns of cell expansion (Hush and Overall, 1996
; Wymer and Lloyd, 1996
). Ordered arrays of CMTs become disorganized in certain cortex cells of Z. mays roots that have been induced to form aerenchyma (Balu
ka et al., 1993
). This observation suggests that the breakdown of order in CMT arrays may be an early event in cell lysis.
It has been shown with light microscopy that root aerenchyma is produced by the predictable lysis of cortex cells in the marsh species Sagittaria lancifolia L. (Alismataceae) (Schussler and Longstreth, 1996
). The primary objective of the present research was to determine the sequence of changes in membrane and organelle structure that occur during cell lysis. Different stages of root aerenchyma development were examined by transmission electron microscopy (TEM) to meet this objective. For comparison, the structure of root cortex cells was also examined at late stages of aerenchyma development in Z. mays and O. sativa roots. A second objective of this research was to determine whether a change in the organization of CMTs is an early stage in the lysis of root cortex cells in S. lancifolia. Microtubules in expanding root cells were labeled with a fluorescent probe and examined by confocal microscopy. Our results show aerenchyma development involves many changes in cell structure that are associated with PCD in general but not specifically apoptosis. While CMT orientation was unusual in some cortex cells, a change in the organization of CMT arrays was not a predictor of cell lysis in S. lancifolia.
MATERIALS AND METHODS
Plant culture
Sagittaria lancifolia plants (Horticultural Systems, Parrish, Florida, USA) were grown in aerated, one-quarter strength Hoagland's solution (Epstein, 1972
) under controlled temperatures and photoperiod in a growth chamber (Environmental Growth Chambers, Chagrin Falls, Ohio, USA). Seeds of Zea mays and Oryza sativa cv. Labelle were germinated in moist vermiculite in a growth chamber, and 2-wk old seedlings were transferred to nutrient solutions optimized for each species (Yoshida et al., 1976
; Drew, Jackson, and Giffard, 1979
). All plants were grown at a 12-h photoperiod with 100–150 µmol photons · m-2 · s-1 photosynthetically active radiation at plant height and at day and night temperatures of 32°C and 25°C, respectively. Plants were grown in hydroponic solutions that were either constantly aerated (producing an O2 concentration from 9 to 10 ppm) or bubbled with N2 gas twice daily for 30 min (producing an O2 concentration of 3 to 5 ppm) for at least 1 wk prior to sampling.
Preparation for analysis of cell structure
Sagittaria lancifolia roots, 6–7 cm in length, were sampled at four different distances behind the tip, representing different stages of root development. Only tissue with fully developed aerenchyma was sampled in Z. mays and O. sativa roots. Tissue pieces were fixed in 1% para-formaldehyde, 2% glutaraldehyde, 0.05 mol/L sodium cacodylate buffer (pH 7.2), 0.05% calcium chloride, and 2% sucrose, under vacuum, overnight at room temperature. The tissue was rinsed in sodium cacodylate buffer with sucrose and postfixed in 4% osmium tetraoxide. The tissue was rinsed in sodium cacodylate buffer without sucrose, stained in 0.5% uranyl acetate, and dehydrated in a graded ethanol series. LR White medium grade resin (Electron Microscopy Sciences, Fort Washington, Pennsylvania, USA) was substituted for the ethanol through a graded resin-ethanol series. Polymerization was at 58°–60°C for 24 h.
Sections (1 µm thick) were cut perpendicular to the root axis with a microtome (Sorvall MT-2 Porter Blum Ultra-Microtome, Norwalk, Connecticut, USA) and placed on collodion-coated grids. These cross sections were stained with lead citrate and cortex cells examined with a transmission electron microscope (JEOL JEM 100 CX).
Orientation of CMT arrays in S. lancifolia
Sagittaria lancifolia plants were grown as described above. Sections of roots (3–4 mm long) representing different stages of root development were fixed overnight at 4°C in 3.7% formaldehyde in PHEM (60 mmol/L Pipes, 25 mmol/L Hepes, 2 mmol/L MgCl2, 10 mmol/L EGTA, pH 6.9) buffer with 5% dimethylsulfoxide (DMSO). After rinsing in the buffer, longitudinal sections (70 µm thick) were cut with a vibratome. The sections were placed on a coverslip and covered with an agarose-gelatin film (Brown and Lemmon, 1995
). Cell walls were initially digested in an aqueous solution of 1% cellulase, 1.5% ß-glucuronidase, and 1% glucose for 25 min, and then permeabilized in 1% Triton X-100 in PHEM for 5 min. Sections were incubated in an antitubulin primary antibody (rat antiyeast tubulin, Accurate Chemical and Scientific Corporation, Westbury, New York, USA) for 1 h. Sections were then incubated overnight, in the dark, with a secondary antibody (goat antirat IgG conjugated to FITC (fluorescein-5-isothiocyanate), Accurate Chemical and Scientific Corporation, Westbury, New York, USA). Sections were counterstained with 0.01% propidium iodide for 10 s and mounted with 20% Mowiol 4.88 with the anti-fade agent p-phenylenediamine. Sections were examined with a Bio-Rad MRC-600 confocal laser scanning microscope (Bio-Rad Laboratories, Hercules, California, USA).
RESULTS
Cell structure in S. lancifolia
Structural features of aerenchyma formation in roots of S. lancifolia were the same whether plants were grown in hydroponic solutions bubbled with air or with nitrogen. Just behind the root tip, cortex cells of S. lancifolia possessed normal nuclei with intact membranes, smooth surfaces, and dispersed chromatin. These cells also had distinct and apparently normal ribosomes, dictyosomes, and mitochondria (Fig. 1). Cortex cells from just behind the root tip had larger amounts of cytoplasm and smaller vacuoles than cells 10 mm or more behind the root tip where large vacuoles restricted cytoplasm to the cell periphery (Fig. 2). The earliest divergence between normal and lysing cells was the appearance of unusual-looking nuclei. Deterioration of nuclear membranes (Fig. 3), general nuclear fragmentation (Figs. 4, 5), and the apparent mixing of nuclei and vacuoles were also observed as lysis progressed (Fig. 6). These changes in nuclear structure were observed in cortex cells found just behind the root tip to as far as 2 cm behind the root tip (depending on the root examined).
|
|
|
|
|
Orientation of CMT arrays in S. lancifolia
CMT arrays were oriented in various directions in cells very close to the root tip (Fig. 21). Farther behind the root tip, the orientation of the CMT arrays in most cortex cells was perpendicular to the root axis (Fig. 22). At 2–3 cm behind the root tip, cortex cells had elongated significantly and the CMT arrays of these cells were oriented diagonally (Fig. 23). The organization of CMT arrays was maintained in cortex cells even at late stages of aerenchyma development.
|
) were separated axially by several layers of cortex cells. The diaphragm cells were alive in the mature aerenchyma tissue. These cells possess thin, cellular extensions that were in contact with extensions from adjacent diaphragm cells (Fig. 24). The CMT arrays of these diaphragm cells were largely oriented parallel to the root axis, as compared to the diagonal orientations seen in other cortex cells at maturity (Fig. 25).
|
Several features of root aerenchyma formation in S. lancifolia are consistent with PCD—specifically, the targeting of certain cortex cells for death (Schussler and Longstreth, 1996
), the obligate production of aerenchyma under a variety of environmental conditions (Schussler, 1997
), and the early changes in nuclear structure found in this study. These changes in nuclear structure included clumping of the chromatin, fragmentation, disruption of the nuclear membrane, and apparent engulfment by the vacuole. These changes to the nucleus were followed by the plasma membrane becoming crenulated, formation of electron-lucent regions in the cytoplasm, tonoplast disintegration, organellar swelling and disruption, the loss of cytoplasmic contents, and collapse of the cell. These observations are not consistent with cell necrosis where initial changes are commonly destruction of mitochondria followed by swelling of the cell (Kerr, Wyllie, and Currie, 1972
).
The changes in nuclear structure, condensation of the cytoplasm, and maintenance of organelle integrity until late in the cell death process are structural features of apoptosis (Kerr, Wyllie, and Currie, 1972
). However, other characteristics of apoptosis such as pycnosis of the nucleus, plasma membrane blebbing, and the subsequent production of apoptotic bodies were not observed in lysing cells of S. lancifolia. Since the death of cortex cells in S. lancifolia lacks several features of apoptosis (especially the formation of apoptotic bodies), PCD in S. lancifolia should be classified as nonapoptotic PCD. There is not currently a system to classify nonapoptotic PCD in plants as there is in animals (Clarke, 1990
).
Previous studies of Z. mays (Campbell and Drew, 1983
) and O. sativa (Webb and Jackson, 1986
) have shown that many aspects of aerenchyma formation are similar in these two species. During the process that leads to cell collapse, tonoplast disruption precedes breakdown of the plasma membrane. A similar sequence of events was reported for S. lancifolia in this study. There does appear to be differences among species in the timing of middle lamella breakdown and tonoplast disruption. Webb and Jackson (1986)
concluded that cell wall degradation followed tonoplast disruption in Z. mays, while in O. sativa middle lamella dissolution preceded breakdown of the tonoplast. In S. lancifolia, middle lamella dissolution occurred at about the same time as the tonoplast began to disintegrate, and well before the plasma membrane breakdown.
The current study identified an additional difference among Z. mays, O. sativa, and S. lancifolia in the way aerenchyma was formed. Concentric circles of membranes were observed in lysing cells of Z. mays and O. sativa, but not in S. lancifolia. Similar membrane structures have been observed in Pisum sativum roots growing at low O2 concentrations (Davies et al., 1987
). Davies et al. (1987)
hypothesized that these membranes are endoplasmic reticulum that has become reorganized at energy charge values below that found in cells with normal-appearing endoplasmic reticulum.
The orientation of CMT arrays in most of the cortex cells in S. lancifolia roots was consistent with previous reports from other species (Williamson, 1991
). CMT arrays were perpendicular to the root axis in cells close to the root tip, but the CMT arrays became diagonal as cells expanded. Baluka et al. (1993)
found ethylene treatment could produce randomization of CMTs in root cortex cells of Zea mays. The degree of randomization was greater in the inner cortex cells, and these cells then collapsed to form aerenchyma. In S. lancifolia, CMTs remained organized at late stages of cell lysis, as far as 5–6 cm behind the root tip. A transition from organized to disorganized CMT arrays did not appear to be an early stage of aerenchyma formation in S. lancifolia.
The diaphragm cells of S. lancifolia roots possess CMT arrays oriented parallel to the root axis, an orientation that is rare in expanding root cells (Williamson, 1991
). This orientation predicts expansion of diaphragm cells that could produce a separation of radial files of cells (perhaps via elongation of the projections seen in Fig. 24) rather than an elongation of cells parallel to the root axis. Radial files of root cortex cells separate prior to lysis during the formation of root aerenchyma in S. lancifolia (Schussler and Longstreth, 1996
). Separation of the files of diaphragm cells by growth of the extensions between cells could be the mechanism that separates files of cortex cells prior to cell lysis. This growth of the extensions would be consistent with the orientation of CMT arrays in diaphragm cells. Diaphragm cells are common in roots of many wetland monocots (Tomlinson, 1982
), but not in Z. mays and O. sativa.
Structural changes of cells during lysis to form root aerenchyma in S. lancifolia are consistent with a type of nonapoptotic PCD. The formation of aerenchyma in roots of Z. mays and O. sativa also appears to be nonapoptotic. In all these species there is alteration or disappearance of the nucleus at early developmental stages, followed by disruption of the membranes and cytoplasmic structure, and finally collapse of the cell. Comparisons of cell lysis in S. lancifolia, Z. mays, and O. sativa, however, show differences, particularly in relation to the timing and manner of membrane disruption. While aerenchyma formation is apparently an example of PCD, there are clearly differences among plant species in the mechanisms that produce gas spaces. Studies of aerenchyma development in more wetland plant species are needed to understand the range of mechanisms that produce this important adaptation to flooded soils.
FOOTNOTES
1 The authors thank Drs. Roy Brown and Betty Lemmon for technical assistance with the cortical microtubule data, helpful discussions, and comments on the manuscript; Dr. Olga Borkhsenious for assistance with the electron microscopy; Mr. Ron Bouchard for assistance with preparation of figures; and Drs. Malcolm Drew and Robert Kaul for helpful comments on the manuscript during the review process. 
2 Author for correspondence (phone: 225-388-8559, FAX: 225-388-4638, e-mail: btlong{at}lsu.edu
).
LITERATURE CITED
Baluka, F., R. W. Brailsford, M. Hauskrecht, M. B. Jackson, and P. W. Barlow. 1993 Cellular dimorphism in the maize root cortex: involvement of microtubules, ethylene and gibberellin in the differentiation of cellular behavior in postmitotic growth zones. Botanica Acta 106: 394–403.
[ISI]
Barlow, P. W., and J. S. Parker. 1996 Microtubular cytoskeleton and root morphogenesis. Plant and Soil 187: 23–36. [CrossRef][ISI]
Brown, R. C., and B. E. Lemmon. 1995 Methods in plant immunolight microscopy. Methods in Cell Biology 49: 85–107. [ISI][Medline]
Campbell, R., and M. C. Drew. 1983 Electron microscopy of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to oxygen shortage. Planta 157: 350–357. [CrossRef][ISI]
Clarke, P. G. H. 1990 Developmental cell death: morphological diversity and multiple mechanisms. Anatomy and Embryology 181: 195–213. [Medline]
Davies, D. D., P. Kenworthy, B. Mocquot, and K. Roberts. 1987 The effects of anoxia on the ultrastructure of pea roots. In R. M. M. Crawford [ed.], Plant life in aquatic and amphibious habitats, 265–277. Blackwell Scientific Publications, Oxford, UK.
Drew, M. C. 1997 Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia. Annual Review of Plant Physiology and Plant Molecular Biology 48: 223–250. [CrossRef][ISI][Medline]
———, M. B. Jackson, and S. Giffard. 1979 Ethylene-promoted adventitious rooting and development of cortical air spaces (aerenchyma) in roots may be adaptive responses to flooding in Zea mays L. Planta 147: 83–88. [CrossRef][ISI]
Epstein, E. 1972 Mineral nutrition of plants: principles and perspectives. John Wiley & Sons, New York, New York, USA.
Fukuda, H. 1997 Tracheary element differentiation. Plant Cell 9: 1147–1156. [CrossRef][ISI][Medline]
Greenberg, J. T. 1996 Programmed cell death: a way of life for plants. Proceedings of the National Academy of Sciences, USA 93: 12094–12097.
Groover, A., N. Dewitt, A. Heidel, and A. Jones. 1997 Programmed cell death of plant tracheary elements differentiating in vitro. Protoplasma 196: 197–211.
Hush, J. M., and R. L. Overall. 1996 Cortical microtubule reorientations in higher plants: dynamics and regulation. Journal of Microscopy 181: 129–139.
Jones, A. M., and J. L. Dangl. 1996 Logjam at the styx: programmed cell death in plants. Trends in Plant Science 1: 114–119. [CrossRef][ISI]
Kawai, M., P. K. Samarajewwa, R. A. Barrero, M. Nishiguchi, and H. Uchimiya. 1998 Cellular dissection of the degradation pattern of cortical cell death during aerenchyma formation of rice roots. Planta 204: 277–287. [CrossRef][ISI]
Kerr, J. F. R., A. H. Wyllie, and A. R. Currie. 1972 Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26: 239–257. [ISI][Medline]
Mittler, R., and E. Lam. 1996 Sacrifice in the face of foes: pathogen-induced programmed cell death in plants. Trends in Microbiology 4: 10–15. [CrossRef][ISI][Medline]
Schussler, E. E. 1997 Aerenchyma development in the freshwater marsh species Sagittaria lancifolia L. Ph.D. dissertation. Louisiana State University, Baton Rouge, Louisiana, USA.
———, and D. J. Longstreth. 1996 Aerenchyma develops by cell lysis in roots and cell separation in leaf petioles in Sagittaria lancifolia (Alismataceae). American Journal of Botany 83: 1266–1273. [CrossRef][ISI]
Schwartz, L. M., S. W. Smith, M. E. E. Jones, and B. A. Osborne. 1993 Do all programmed cell deaths occur via apoptosis? Proceedings of the National Academy of Sciences, USA 90: 980–984.
Tomlinson, P. B. 1982 Anatomy of the monocotyledons, VII. Helobiae (Alismatidae). In C. R. Metcalfe [ed.], 43–86. Clarendon Press, Oxford, UK.
Webb, J., and M. B. Jackson. 1986 A transmission and cryo-scanning electron microscopy study of the formation of aerenchyma (cortical gas-filled space) in adventitious roots of rice (Oryza sativa). Journal of Experimental Botany 37: 832–841.
Williamson, R. E. 1991 Orientation of cortical microtubules in interphase plant cells. International Review of Cytology 129: 135–206. [CrossRef]
Wymer, C., and C. Lloyd. 1996 Dynamic microtubules: implications for cell wall patterns. Trends in Plant Science 1: 222–227. [CrossRef][ISI]
Yoshida, S., D. A. Forno, J. H. Cock, and K. A. Gomez. 1976 Laboratory manual for physiological studies of rice, 3rd ed. International Rice Research Institute, Los Banos, Laguna, Philippines.
This article has been cited by other articles:
|
|
 |
|
  P. Sarkar, T. Niki, and D. K. Gladish Changes in cell wall ultrastructure induced by sudden flooding at 25{degrees}C in Pisum sativum (Fabaceae) primary roots Am. J. Botany, July 1, 2008; 95(7): 782 - 792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K. GLADISH, J. XU, and T. NIKI Apoptosis-like Programmed Cell Death Occurs in Procambium and Ground Meristem of Pea (Pisum sativum) Root Tips Exposed to Sudden Flooding Ann. Bot., May 1, 2006; 97(5): 895 - 902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. THOMAS, S. M. C. GUERREIRO, and L. SODEK Aerenchyma Formation and Recovery from Hypoxia of the Flooded Root System of Nodulated Soybean Ann. Bot., December 1, 2005; 96(7): 1191 - 1198. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
