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. 2015 May 22;116(1):91–99. doi: 10.1093/aob/mcv067

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa

G Bartoli 1,*, L M C Forino 1, M Durante 2, A M Tagliasacchi 1
PMCID: PMC4479754  PMID: 26002256

Abstract

Background and Aims Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved.

Methods Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes.

Key Results Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified.

Conclusions The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells.

Keywords: Aerenchyma differentiation, apoptotic-like bodies, aquatic plant, constitutive aerenchyma, Egeria densa, Hydrocharitaceae, hydrogen peroxide, lysigenous aerenchyma, nitric oxide, programmed cell death, PCD, schizogenous aerenchyma, submergence tolerance, TUNEL, waterweed

INTRODUCTION

The reduced O2 availability in the aquatic environment and sediments, and limitation of inorganic carbon represent some of the main limitations affecting the plant life in flood-prone or chronically submerged habitats. In order to cope with the limitations imposed by submergence, plants develop various adaptive strategies, including the differentiation of an aerenchyma throughout their organs, characterized by wide gas spaces.

Aerenchyma acts as a mediator of internal gas exchange, supports the respiratory demand of plant tissues with the O2 produced by photosynthesis and provides the photosynthesizing cells with the CO2 taken up from the sediment by roots or produced by cell respiration. Many aquatic plants form a constitutive aerenchyma as a part of the normal development of their organs, whereas others, including certain amphibious and land plants, induce aerenchyma differentiation in response to hypoxic or anoxic conditions.

Compared with the inducible aerenchyma, that occurs in mature organs in response to a stimulus and whose degree of differentiation is proportional to the time from induction (Campbell and Drew, 1983; Gunawardena et al., 2001; Jung et al., 2008), the constitutive aerenchyma is very difficult to investigate because it is an intrinsic part of organogenesis.

The main types of cortical aerenchyma in stems and roots can be distinguished as lysigenous and schizogenous on the basis of their mechanism of formation. The schizogenous aerenchyma is usually constitutive and occurs without cell death events. The differential growth of adjacent cells, followed by the hydrolysis of the middle lamella, leads to separation of the cells that maintain their structural and functional integrity. In contrast, lysigenous aerenchyma results from the active and genetically programmed death of specific cells (programmed cell death, PCD), according to a precise and predictable spatial pattern (Rascio, 2002), and shows an array of morphological and biochemical features also shared by animal apoptosis.

The formation of lysigenous gas spaces can be a part of the developmental programme of an aquatic plant or can be induced and regulated by different environmental signals (Armstrong and Armstrong, 1994; Kawai et al., 1998; Rascio, 2002).

The two types of aerenchyma can coexist in the same organ or in different organs of the same plant, and schizogenous aerenchyma may precede or accompany the formation of lysigenous aerenchyma in the same organ (Schussler and Longstreth, 1996; Saego et al., 2005; Thomas et al., 2005, and references therein; Jung et al., 2008), suggesting the occurrence of a spatial patterning of target cells able to respond in a specific way to endogenous or environmental signals (Rascio, 2002).

As soil waterlogging and flooding affect primarily the roots, at present much research has focused on the study of aerenchyma formation in this organ (Steffens et al., 2011, and references therein) mainly in dominant wetland colonizers and in survival crop species of economic significance (Justin and Armstrong, 1987; Shiono et al., 2008). In contrast, the study of aerenchyma differentiation in plant stem has received less attention.

The fresh water macrophyte Egeria densa is an important invasive plant, with a great ability to colonize many and different aquatic habitats, and also offers competitive advantages over others species (Spencer and Bowes, 1990; Becker Rodrigues and Thomaz, 2010). Given its ability to grow in polluted aquatic environments, to produce large amounts of biomass and to bioremediate aquatic ecosystems contaminated by cadmium, arsenic or eutrophic substances (Robinson et al., 2006; Malec et al., 2009; Takayanagi et al., 2012), E. densa offers significant ecological perspectives in terms of environmental remediation.

A well-developed system of internodal gas spaces in its photosynthetic stem is an anatomical trait that contributes to the adaptability, competitiveness and ecological uses of E. densa: consequently, a better understanding of the differentiation of this tissue might provide useful insights for the development of effective management policies to control its invasivity or for its employment in the remediation of polluted aquatic environments.

In this research we used an integrated approach, based on both cytohistochemical and molecular assays, to study the differentiation of the extensive network of gas spaces in cortical regions of E. densa stem. Additionally, we investigated whether PCD events might be involved as a regulator of differentiation of the cortical aerenchyma.

MATERIALS AND METHODS

Plant material

Shoots of Egeria densa Planch. Planch. [Hydrocharitaceae; also called Philotria densa Planch. Small & St. John, Elodea densa (Planch) Caspary, Anacharis densa (Planch.) Victorin or commonly known as Brazilian or dense waterweed] were collected from plants living in a pool of the ‘idrofitorio’ of the Botanical Garden of Pisa. To study the cortical aerenchyma differentiation, for each collected shoot, three regions were sampled: the apical region (0–1 mm from the apex), the sub-apical region (1–2 mm from the apex) and the distal region (2–3 mm from the apex). Samples were immediately processed for cyto-histological investigations (on fresh or fixed material) or frozen in liquid nitrogen and stored at −80 °C until DNA extraction.

Histochemistry and quantification of intercellular air spaces

Samples from each shoot region were excised from five E. densa plants (five samples for each region) and were fixed for 24 h in buffered 4 % formalin [in phosphate-buffered saline (PBS), pH 7·4], dehydrated in a graded ethanol series and embedded in LR-White medium Grade (London Resin Company, Reading, Berkshire, UK). Semi-thin sections (3 µm) were stained with different dyes: toluidine blue O (TBO; 0·05 % in 0·1 m benzoate buffer at pH 4·4) staining or haematoxylin and eosin double staining for general cytological investigations (Feder and O’Brien, 1968; Al Hazzaa and Bowen, 1998), and periodic acid–Schiff (PAS)–TBO double staining for detection of non-cellulosic polysaccharides (Jensen, 1962; O’Brien and Mc Cully, 1981). The sections were cleared in xylene, air-dried and mounted in DPX. Histological observations were performed on at least 100 sections randomly selected from the samples belonging to the three shoot regions considered and the cytological details were investigated in serial sections on the same slide.

For quantification of intercellular air spaces, two transverse sections from ten E. densa plants (in total 20 non-consecutive histological sections for each stem region) were considered. The amount of intercellular spaces in each stem region was expressed as the percentage of air cavities on the total tissue cross-sectional area and was determined by the image analysis software SCION IMAGE, release 4.0.2 (Scion Corporation, Frederick, MD, USA).

Viability staining

The cell viability in the three stem regions was assessed by a two-colour fluorescence assay employing fluorescein diacetate (FDA; Sigma-Aldrich, Germany) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)-hexatrienyl) pyridinium dibromide (FM4-64; Sigma-Aldrich), according to Bethke and Jones (2001). FDA enters the cells where esterase cleaves off the acetate residues, leaving fluorescein, which then accumulates and fluoresces, leading to a yellow-green staining of the cytoplasm at 450–490 nm. FM4-64 freely penetrates dead or damaged cells, leading to red fluorescence of the cellular contents (Fath et al., 2001) at 450–490 nm, whereas in intact living cells it can penetrate only via internalization of vesicles containing the dye originating from the plasma membrane.

For quantification of cell death, two samples from ten plants (in total 20 fresh sections for each stem region) were considered. The amount of dead cells in each stem region was calculated as the number of orange-red fluorescing cells and expressed as a percentage of the total cells. Each assay was repeated at least three times.

In situ determination of H2O2 and other peroxides

Two samples for each region were collected from ten plants. The fresh sections were incubated in darkness for 30 min at room temperature with 1 mL of 20 µm H2DCFDA (Molecular Probes, Eugene, OR, USA) in 5 mm MES/KOH buffer (pH 5·6). After washing to remove possible reactive oxygen species (ROS) released by cutting, the sections were collected on a slide, mounted with glycerine and observed: the oxidation of the fluorogenic probe by ROS generates a green fluorescent derivative under blue light excitation (450–490 nm, emission at 515 nm). The amount of green fluorescing cells in each stem region was calculated on the captured images as the number of H2DCFDA-positive cells and expressed as a percentage of the total cells. Each assay was repeated at least three times.

In situ detection of NO

Two samples for each region were collected from ten plants. The fresh sections were incubated (for 1 h at room temperature and in darkness) with 1 mL of 20 µm DAF-2DA (Callbiochem, Darmstadt, Germany) in 5 mm MES/KOH (pH 5·6). After washing to remove the fluorescence which originated due to the reaction of the fluorochrome with nitric oxide (NO), possibly released by cutting, the sections were collected on a slide, mounted with glycerine and then observed. In the presence of NO and O2, the probe is converted into a green fluorescent derivative under blue light excitation (450–490 nm, emission at 515 nm). The amount of green fluorescing cells in each stem region was calculated on the captured images as the number of DAF-2DA-positive cells and expressed as a percentage of the total cells. Each assay was repeated at least three times.

In situ detection of DNA fragmentation by TUNEL assay

Samples from the three selected regions of E. densa stems were excised, fixed overnight at 4 °C in 4 % (w/v) buffered paraformaldehyde (in PBS, pH 7·4); then they were dehydrated in ethanol, embedded in Paraplast Plus (Sigma-Aldrich, Milan, Italy) at 60 °C for 48 h, sliced in 10 µm sections and finally collected on polylysine-coated slides. Deparaffinized sections were rehydrated and subjected to TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling] assay, using a TACS•XL Blue Label Kit (Trevigen, Gaithersburg, MD, USA) and counterstained with Nuclear Fast Red, according to the manufacturer’s instructions. Appropriate controls were done: a negative control was included in each experiment, omitting TdT from the reaction mixture; as a positive control, sections were incubated with DNase I (10 U mL–1) for 10 min before TUNEL assay. The sections were air-dried and mounted with DPX. Experiments were repeated three times and each time ten slides were labelled for each stem region. About 100 tissue sections were analysed. Two non-consecutive histological sections from ten plants (in total 20 sections for each stem region) were considered to calculate the amount of TUNEL-positive cells and they were expressed as a percentage of the total cells. Each assay was repeated at least three times.

Microscope observation and image acquisition

Histological sections used for histochemical investigations, for quantification of intercellular air spaces and for the in situ detection of TUNEL-positive cells were observed with a LEITZ DIAPLAN light microscope (Wetzlar, Germany) and then captured using a Leica DFC 420 digital camera (Leica Microsystems, Heerbrugg, Germany).

Histological sections considered for cell viability assessment, for determination of H2O2 and other peroxides in situ (H2DCFDA-positive cells) and for NO in situ detection (DAF-2DA-positive cells) were observed with a LEICA DB LM microscope (Wetzlar), equipped with a filter set for group A (BP-340–380, dichroic mirror 450, barrier filter Lp 430). The images were captured by an Image Leica DC300F digital camera (Leica Microsystems).

DNA extraction and analyses

Intact, high molecular weight DNA was isolated from frozen samples from the three selected stem regions, following the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990). DNA samples were subjected to electrophoresis in a 1 % (w/v) agarose gel at constant voltage (70 V) and then visualized under a UV transilluminator, in order to detect DNA fragmentation patterns. The electrophoretic migrations were captured using a Digi Doc-It imaging System (Ultraviolet Products Limited, Cambridge, UK). A 1000 bp ladder was used as a standard. The assay was repeated three times.

The average DNA fragment lengths and percentages were assessed as reported in Bartoli et al. (2013). Briefly, a densitometric scanning analysis of images of each electrophoretic pattern was performed by Quantity One 4.1.1 software (Bio-Rad, Hercules, CA, USA) and the obtained traces were elaborated. After confirming that the area under the peaks of the densitometer tracings was proportional to the DNA concentration (Bird and Southern, 1978), the fragments as a percentage of total DNA and the average length of DNA fragments were determined: we determined the axis bisecting each peak into equal areas by ‘cutting and weighting’, then we found the corresponding value of the weight average length of DNA by use of a ‘standard curve’ consisting of a semi-log plot of the migration distance of fragments of known size (the 1000 bp ladder used as a standard) subjected to electrophoresis on the same gel.

Statistical analyses

The data (expressed as means ± s.e.) were analysed by one-way analysis of variance (ANOVA) and Student–Newman–Keuls post-hoc test with P-values <0·01 sufficient to reject the null hypothesis. Statistical analyses were performed using the statistical package Primers of Biostatistics (S.A. Glantz, Statistical Software Program Version 6.0, McGraw Hill 2005). Microphotographs were representative of at least 20 samples yielding similar results.

RESULTS

Stem aerenchyma differentiation: morpho-anatomical and cyto-histochemical characterization

Aerenchyma differentiation proceeded basipetally and centripetally in the stem (Fig. 1A). The cells of the stem apex appeared hexagonally packed, with no significant intercellular gas spaces between neighbouring cells (Fig. 1B); the first sign of differentiation of gas spaces occurred at the base of the apical region of the stem (Fig. 1C) in the outer portions of the cortical parenchyma. The cells around these gas spaces had round-shaped nuclei with chromatin organized in chromocentres, one or two vacuolated nucleoli, dense cytoplasm and intact walls (Figs 1C and 2D, G). In the sub-apical region, gas spaces delimited by a few cell layers were detectable (Fig. 1D). In the distal region, cortical gas spaces were organized in concentric rings, in which neighbouring gas spaces were delimited by only a cell monolayer (Fig. 1E), around a central cylinder without gas spaces (Fig. 1A). The cell monolayers consisted of both barrel-shaped cells, arranged in columns and delimiting two neighbouring gas spaces, and polygonal cells, also arranged in columns, connecting three adjacent gas spaces. The fully differentiated aerenchyma showed a honeycomb arrangement (Fig. 1E).

Fig. 1.

Fig. 1.

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Egeria densa stem. (A) Medial longitudinal section (assembled by combining serial sections of the same sample) showing the progressive differentiation of the aerenchyma in the cortical cylinder. The three regions considered in this study are detailed. (TBO; scale bar = 500 µm). (B–E) Cross-sections showing the cell arrangement of the meristematic apex (upper apical region; B), the early differentiation of gas spaces (arrows) in the outer part of the cortical cylinder (lower apical region; C), the gas spaces delimited by more cell layers (sub-apical region; D) and the fully differentiated aerenchyma (distal region; E). (Haematoxylin and eosin staining; scale bar = 50 µm.)

Fig. 2.

Fig. 2.

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Cell death, PCD hallmarks and detection of H2O2 and NO in the cortical aerenchyma of E. densa stem. (A–C) Fresh cross-sections showing living cells (green fluorescence) and dead cells (orange-red fluorescence) in the apical region (A), in the sub-apical region (B) and in the distal region (C) of the stem. (FDA–FM4-64 double staining, scale bar = 50 µm). (D–F) Longitudinal sections showing early differentiated gas spaces in the apical region with cells showing intact and round nuclei and well-defined nucleoli (D), gas spaces in the sub-apical region delimited by cells showing shrunken nuclei with condensed chromatin (E) and wall breakdown (F) (TBO; scale bar = 50 µm). (G–I) Cross sections showing the cells delimiting early differentiated gas spaces in the apical region (G), the cells delimiting gas spaces in the sub-apical region with intact chloroplasts and signs of plasmolysis (arrow) (H) (haematoxylin and eosin staining; scale bar = 20 µm) and some round PAS-positive bodies (I) (arrows) (PAS–TBO double staining; scale bar = 50 µm). (J, K) Fresh cross-sections showing localization of H2O2 and peroxides in samples from the apical region (J) and from the sub-apical region (K) using the fluorescent probe H2DCFDA: green fluorescence indicates the occurrence of peroxides, and red fluorescence derives from chlorophylls (scale bar = 50 µm). (L, M) Fresh cross-sections of cortical aerenchyma, showing NO localization in samples from the apical region (L) and the sub-apical region (M) using the fluorescent probe DAF-2DA: green fluorescence indicates the occurrence of NO. and red fluorescence derives from chlorophylls (scale bar = 50 µm). In the insets, the most significant cyto-histological details are shown (× 70 magnification compared with the corresponding image).

A progressive increase in the amount of gas spaces occurred in E. densa stem: the increase in the percentage of aerenchyma observed in the sub-apical region and especially in the distal region was particularly significant, with gas spaces reaching 27 % of the total surface area of the stem in transverse section (Table 1).

Table 1.

Percentages of intercellular air spaces and of cells stained by FM4-64, H2DCFDA, DAF-2DA and TUNEL in the three stem regions

Parameters Apical region Sub-apical region Distal region
Intercellular air spaces (%) 1·82 ± 0·28a 7·97 ± 0·31b 27·07 ± 0·9c
FM4-64-stained cells (%) 3·13 ± 0·22a 38·8 ± 0·57c 4·67 ± 0·64b
H2DCFDA-stained cells (%) 2·09 ± 0·12a 45·2 ± 0·23c 8·53 ± 0·52b
DAF-2DA-stained cells (%) 5·46 ± 0·34a 48·3 ± 0·57c 10·02 ± 0·24b
TUNEL+ cells (%) 2·58 ± 0·42a 34 ± 1·56b 4·23 ± 0·44a
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Values are given as the mean ± s.e., n = 10.

Values in rows marked with different letters are significantly different at P < 0·01.

During aerenchyma differentiation, significant cell death events occurred mainly in the sub-apical region of the stem (Table 1), where the cells which were positive for the fluorescent probe FM4-64 reached 38·8 % of the total scored cells. Dead cells were mainly localized in the inner cell layer delimiting the gas spaces in the sub-apical region and occurred asynchronously in the same layer (Fig. 2B). A low number of FM4-64-positive cells was detected in the apical region of the stem (Table 1; Fig. 2B) and also in the distal region (Table 1; Fig. 2C) where FM4-64-positive cells can be detected in cell monolayers, between neighbouring gas spaces.

With respect to the parenchymatic cells of the apical region (Fig. 2D, G), characterized by spherical nuclei with a large vacuolated nucleolus or many small nucleoli, the cortical cells around air cavities in the sub-apical region showed significant signs of degeneration. The nucleoli disappeared and the nuclei showed an irregular shape with chromatin disorganization and fragmentation (Fig. 2E). Cell wall disintegration events (Fig. 2F), membrane plasmolysis (Fig. 2H) and PAS-positive spherical bodies in the gas spaces (Fig. 2I) were also detectable. Additionally, considerable increases in H2O2 (Fig. 2K; Table 1) and NO (Fig. 2M; Table 1) were demonstrated histologically around gas spaces in the cortical parenchyma of the sub-apical region; in contrast, low amounts of both H2O2 and NO were observed in the other regions of the stem (Fig. 2J and L, respectively; Table 1).

Determination of DNA fragmentation

No significant TUNEL-positive nuclei were found in samples from the apical and the distal regions of the stem (Fig. 3A and C, respectively; Table 1). A significant increase in TUNEL-positive nuclei occurred in samples from the sub-apical region (Table 1) and they were mainly localized in the inner layer of cells delimiting an aerenchymatic lacuna (Fig. 3B); TUNEL-positive nuclei were sometimes detectable in the cell monolayer, separating two neighbouring gas spaces. TUNEL-positive nuclei appeared asynchronously in the same cell layer.

Fig. 3.

Fig. 3.

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DNA fragmentation during aerenchyma differentiation detected by TUNEL assay and DNA electrophoresis. (A–C) TUNEL assay carried out in the apical, sub-apical and distal regions; TUNEL-positive cells, with blue-stained nuclei, occurred mainly in the sub-apical region (B) around aerenchymatic gas spaces and only a few in the distal region; no TUNEL-positive cells were seen in samples from the apical region (A) (scale bar = 50 µm). (D) Electrophoresis of DNAs extracted from samples of the apical region (lane 1), the sub-apical region (lane 2) and the distal region (lane 3) of the E. densa stem. In lane 4, the DNA marker is shown (1 kb).

Electrophoresis assays on genomic DNA from the three stem regions showed that DNA fragmentation events occurred mainly in the sub-apical region: a unique band of undegraded DNA with a molecular complexity up to 20 kb (also detectable in electrophoretic migrations of DNA from the apical and the distal regions of the stem) was accompanied by a slight smear, mainly constituted by fragments having a molecular complexity of 3·1 kb (Fig. 3D; Table 2).

Table 2.

Fragment percentage and sizes of the DNAs extracted from the three stem regions

Parameters Apical region Sub-apical region Distal region
Fragment percentage 7·09 ± 0·18a 44·49 ± 0·41c 26·45 ± 0·57b
Fragment size (kb) 6·1 ± 0·03c 3·1 ± 0·04a 3·9 ± 0·09b
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Values are given as the mean ± s.e., n = 10.

Values in rows marked with different letters are significantly different at P < 0·01.

DISCUSSION

The extensive honeycomb-arranged aerenchyma of the cortical region of Egeria densa stem differentiates basipetally and centripetally, following a schizo-lysigenic developmental pattern. This tissue allows the stem to act as an ‘aerial organ’ (Rascio et al., 1991), essential for survival in submerged environments, and sustains the metabolic requirements of submersed organs, providing the photosynthetic O2 for the respiratory demand, as well the CO2 derived from cell respiration for photosynthesis. Moreover, the aerenchyma improves light availability for the photosynthetic process in turbid waters (Boeger and Poulson, 2003), allowing the whole plant to float near the water surface (Becker Rodrigues and Thomaz, 2010).

The aerenchyma formation pattern and volume of the gas spaces may change in relation to plant type and plant adaptive strategies to flooded or totally submerged conditions. This can explain why different developmental patterns can exist to produce aerenchyma in plant organs: schizogeny, lysigeny or, as seen in E. densa, schizo-lysigeny. The co-occurrence of schizogeny and lysigeny in the aerenchymal ontogenesis was also observed in the totally submerged roots of the wetland plant Sagittaria lancifolia (Schussler and Longstreth, 1996). In this plant, slight schizogenous gaps appear between radial files of large cells that then undergo lyses and generate radial strands connecting the living outer and inner cortical cells. From the anatomical point of view, however, the aerenchyma of the E. densa shoot is more similar to that of the petiole of S. lancifolia, a partly submerged organ, which develops a honeycomb aerenchyma, derived exclusively by schizogeny and with a centrifugal differentiation pattern (Schussler and Longstreth, 1996). Given that the main part of submerged roots showed aerenchyma derived from lysigeny, and that E. densa shoot is a totally submerged organ, we can hypothesize that the submergence might be one of the factors determining the lysigenic differentiation model of the aerenchyma.

Our results showed that, in E. densa, the formation of gas spaces starts very early during stem development. The aerenchyma differentiated from a pre-aerenchymatic tissue consisting of hexagonally packed cells and resulted in a conspicuous network of gas spaces, spatially arranged in a honeycomb pattern (Fig. 1B and E, respectively). Differentiation of gas spaces in root is strictly linked to both cortical cell configuration type and mechanical stress levels (Justin and Armstrong, 1987; Drew et al., 2000). Where mechanical stresses are relatively low, a cubic cell packing is the main cell configuration type in root cortical tissues that subsequently will be transformed in various ways into aerenchyma, as in many wetland or intermediate plants. In contrast, in tissues experiencing high mechanical impedance and bending, a hexagonal cell packing occurs and rare or at best poorly developed aerenchyma is formed (Justin and Armstrong, 1987; Evans, 2003). Similarly to what was proposed for aerenchyma formation in roots, we can hypothesize that the unusual origin of gas spaces in E. densa stem from a tissue with hexagonally packed cells probably reflects a compromise between the metabolic requirements imposed by submergence and the need to cope with mechanical stresses to which the stem, although submerged, can be subjected.

The differentiation of cortical aerenchyma in the stem of E. densa appeared as a tightly regulated process, dependent on the distance from the shoot apex: small aerenchymatic spaces occurred in the outer layers of cortical parenchyma at about 1 mm from the apex (Fig. 1C) and then differentiated basipetally and centripetally to form gas spaces, delimited, at first, by a few cell layers at 1–2 mm from the apex (Fig. 1D) and then by only a monolayer of living and metabolically active cells in the fully developed aerenchyma, at 2–3 mm from the apex (Fig. 1E).

The final honeycomb architecture of the cortical aerenchyma derived from the co-operation of two distinct processes: an early schizogenous differentiation mechanism, as previously morpho-anatomically documented by Hulbary (1944), with which a lysigenous PCD-dependent mechanism overlapped.

As a result of these processes, starting from the outer parts of the cortical cylinder in the sub-apical region of the stem, specific cells survived, while others died, following a predictable model and showing some typical apoptotic hallmarks: nuclei with irregular shape, disappearance of nucleoli, chromatin disorganization (Fig. 2E) and DNA fragmentation (Fig. 3B, D). As evidenced by TUNEL assay, the DNA fragmentation process occurred asynchronously in the cells delimiting the same gas space (Fig. 3B), according to the cell death pattern shown by the viability test. Given the high heterogeneity of cortical tissue in terms of cell phases, types and timing of death, the total DNA extracted from the sub-apical region of the stem generated a diffuse pattern of fragmentation (Fig. 3D; Table 2), analogously to some cases of PCD that affects small groups of cells buried in non-symptomatic tissues or cells belonging to complex and highly heterogeneous tissues (Wang et al., 1996; Bethke et al., 1999; Gunawardena et al., 2004; Bartoli et al., 2013, and references therein). The final removal of the cells of these layers is anticipated by specific events at the cellular level, such as partial cell wall breakdown, membrane plasmolysis and intracellular increases of H2O2 and NO (Fig. 2F, H, K, M). Interestingly, these last two signalling molecules have been proposed to play an active role in regulating metabolic changes occurring during plant development and in response to stress (Neill et al., 2002; Hoeberichts and Woltering, 2003; Lamattina et al., 2003; Foyer and Noctor, 2005; De Pinto et al., 2006; Bartoli et al., 2013). Furthermore, interactions between H2O2 and NO are considered of primary importance for the control and the realization of several types of cell death (Delledonne 2005; Zago et al., 2006; Gadjev et al. 2008).

During aerenchyma differentiation, spherical bodies, containing high amounts of carbohydrates, appeared in gas spaces close to the walls of the cells facing the cavity (Fig. 2I). Given that these structures were no longer present in the fully differentiated aerenchyma, they could be degraded and then reabsorbed by adjacent cells. The observed spherical bodies are reminiscent of the ‘apoptotic bodies’ occurring during the final phases of animal apoptosis. Because of the cytological features of the plant cell, only rare examples of apoptotic-like bodies have been documented in plants (Levine et al., 1996; Wang et al., 1996; Gunawardena et al., 2001), as well as cases of reabsorption of low molecular weight solutes deriving from dismantling of the cell and wall dissolution during lysigenic aerenchyma formation (van der Weele et al., 1996). In E. densa stem, the cells surrounding the gas spaces might profitably exploit the substances deriving from lysigenic dismantling. Recycling of substances can be a very useful event, mainly in aquatic environments where the availability for submerged macrophytes of both micro- and macronutrients is generally rather low (Rascio, 2002). This hypothesis seems to be supported by the observation that in the fully developed aerenchyma, the surviving cells of the monolayers showed signs of an intense metabolic activity, as suggested by their large size, by the presence of lobed nuclei with easily detectable nucleoli and by numerous chloroplasts (Fig. 1E).

In light of the results of this study, we can conclude that a peculiar and greatly regulated balance occurs between cell differentiation and cell death events during the differentiation of the cortical aerenchyma in the stem of E. densa. The observed PCD events, sharing significant similarities with animal apoptosis and other cases of plant PCD, have relevant morphogenetic, metabolic and functional consequences for E. densa plants: (1) the PCD remodels and adjusts the architecture of the gas spaces previously originated following a schizogenous model; (2) the circulation of gases utilized for photosynthetic and respiratory processes is improved by the increase of intercellular gas spaces derived from the removal of specific cells; and (3) PCD allows both reduction of O2-consuming cells and a convenient recycling of substances derived from the lysogenic dismantling of the cell.

As a result of these events, the stem of E. densa enhances its metabolic efficiency and reaches optimal adaptation levels to submerged habitats, as suggested by the wide, and sometimes undesired, diffusion of this plant in natural and non-natural aquifers (Camargo and Esteves, 1995). The increased metabolic efficiency, putatively related to PCD events, might be one of the causes determining the high ecological efficiency of these plants, in terms of nutrient dynamics in aquatic ecosystems (Van Donk et al., 1993) as well as for phytoremediation purposes.

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