Plant Biology ISSN 1435-8603
RESEARCH PAPER
Anatomical alterations of Phaseolus vulgaris L. mature leaves irradiated with X-rays V. De Micco1, C. Arena2 & G. Aronne1 1 Department of Agriculture, University of Naples Federico II, Portici, Italy 2 Department of Biology, University of Naples Federico II, Portici, Italy
Keywords Cell wall; chloroplasts; ionising radiation; leaf anatomy; phenolics; pigment content; space biology; X-rays. Correspondence V. De Micco, Department of Agriculture, University of Naples Federico II, via Universit a 100, Portici, Naples 80055, Italy. E-mail: [email protected] Editor K. Palme Received: 21 January 2013; Accepted: 27 September 2013 doi:10.1111/plb.12125
ABSTRACT The cultivation of higher plants in Space involves not only the development of new agro-technologies for the design of ecologically closed Space greenhouses, but also understanding of the effects of Space factors on biological systems. Among Space factors, ionising radiation is one of the main constraints to the growth of organisms. In this paper, we analyse the effect of low-LET radiation on leaf histology and cytology in Phaseolus vulgaris L. plants subjected to increasing doses of X-rays (0.3, 10, 50, 100 Gy). Leaves irradiated at tissue maturity were compared with not-irradiated controls. Semi-thin sections of leaves were analysed through light and epi-fluorescence microscopy. Digital image analysis was applied to quantify anatomical parameters, with a specific focus on the occurrence of signs of structural damage as well as alterations at subcellular level, such as the accumulation of phenolic compounds and chloroplast size. Results showed that even at high levels of radiation, general anatomical structure was not severely perturbed. Slight changes in mesophyll density and cell enlargement were detected at the highest level of radiation. However, at 100 Gy, higher levels of phenolic compounds accumulated along chloroplast membranes: this accompanied an increase in number of chloroplasts. The reduced content of chlorophylls at high levels of radiation was associated with reduced size of the chloroplasts. All data are discussed in terms of the possible role of cellular modifications in the maintenance of high radioresistance and photosynthetic efficiency.
INTRODUCTION Higher plants are candidates to support long-term human permanence in Space, as key organisms in Bioregenerative Life Support Systems (BLSS; Wheeler et al. 2003; Paradiso et al. 2013). Among Space factors, ionising radiation is one of the main constraints to organism growth because it affects molecular, morpho-anatomical, biochemical and physiological processes (De Micco et al. 2011a). Plants can take advantage of very low levels of radiation, and are very resistant to high doses, which are, instead, detrimental to animals (Casarett 1968; Kumagai et al. 2000; Real et al. 2004). The higher radioresistance of plants is expected for seeds because they are a specific stage of the higher plant life cycle, characterised by peculiar metabolic and structural properties conferring higher resistance to disturbance factors. Other assumptions at the base of higher radioresistance in plants than in animals are the presence of a complex cell wall in the former and differences in behaviour in the case of losses of genetic material (Kovacs et al. 1995; Endo & Gill 1996). Indeed, polyploidy is often present in plants and the presence of multiple wild copies of a gene in a cell can maintain the phenotype unaltered when radiation determines genetic mutations at specific loci (Endo & Gill 1996). Years of extensive experimentation aimed at verifying the effect of ionising radiation on plants have not always produced comparable results because research has been based on differ-
ent irradiation procedures (e.g. radiation type, level and exposure time), various species and cultivars, and differences in parameters measured (not always unambiguously defined) (De Micco et al. 2011a). The effects of ionising radiation on plants at genetic level have been extensively investigated, with a focus on DNA mutations, cell death and chromosomal aberrations (Casarett 1968; Bayonove et al. 1984; Wu et al. 2001; Hase et al. 2002; Kikuchi et al. 2009; Takatsuji et al. 2010; De Micco et al. 2011a). DNA aberrations are intuitively considered responsible for changes in growth and reproduction, which are often the direct consequence of genetic modifications on the resulting phenotype. Indeed, all processes involving mitosis and meiosis can be altered by ionising radiation, thus resulting in lowered germination, increased mortality, altered leaf morphology and reduced reproductive success (Bayonove et al. 1984; Hase et al. 1999; Nagata et al. 1999; Wu & Yu 2001; Komai et al. 2003). However, there is also evidence for a direct effect of ionising radiation on already developed plant structures: phenomena of degradation of cell walls and increased porosity of seed teguments and nutritional layers have been reported (Kovacs et al. 1995, 1997; Hammond et al. 1996). At subcellular level, apart from nuclei, chloroplasts are also a sensitive target of ionising radiation. Alterations in chloroplast structure as well as pigment content have been reported, and are considered responsible for reduced efficiency of the photosynthetic apparatus (Strydom et al. 1991;
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Palamine et al. 2005; Arena et al. 2013, 2014). All observed phenomena on the photosynthetic apparatus have generally been considered either an outcome of genetic modifications or a consequence of oxidative damage due to the high production of reactive oxygen species (ROS) generated by water radiolysis induced by high levels of radiation (Abe et al. 2002; Zaka et al. 2002). The aim of this paper is to analyse the effect of low- linear energy transfer (LET) ionising radiation on mature leaves of Phaseolus vulgaris L. in order to evaluate whether structures already formed at the time of irradiation undergo alterations at anatomical level. The reason for focusing on leaf structure was driven by the awareness that major metabolic and physiological processes are ultimately regulated by the physics of plant structure (Brodribb 2009). MATERIAL AND METHODS Plant material and growth conditions Seeds of dwarf Phaseolus vulgaris L. were germinated in the dark in Petri dishes on filter paper moistened with distilled water. Developing seedlings were transplanted into pots (10cm diameter), filled with 1/1 peat/soil. Plants were cultivated in a growth chamber, with a photoperiod of 12 h, under controlled conditions of temperature (25/20 °C, day/night), relative humidity (60/75%, day/night) and light (90–100 lmolphotonsm 2s 1 at the top of the canopy). Irradiation in the growth chamber was supplied by a series of 58 W fluorescent tubes (Philips TLD 54 and 84) and halogen lamps (Philips HPI-T 400 W). Photosynthetic photon flux density (PPFD) was measured with a quantum sensor (Li-Cor 185; Li-Cor, Lincoln, NE, USA). During the growth period (April–June 2011), plants were irrigated to reintegrate water lost through evapotranspiration. Irradiation procedure X-rays were chosen as low-LET radiation because their effectiveness is similar to low-energy protons that contribute to space radiation, together with other charged particles. Thus, we focused our study on the effects of a wide range of X-ray doses on P. vulgaris leaves to explore the dose range where this species is more sensitive. Plants were irradiated with four total doses of X-rays (0.3, 10, 50 and 100 Gy) 200 kVp, at dose rate of 1 Gymin 1 (Fig. 1). The highest doses (50 and 100 Gy) were established as positive controls: these are radiation treatments
Fig. 1. Scheme indicating the target organ (i.e. dosimetry was calculated on the first trifoliate leaf), X-ray doses and exposure times.
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with doses at which a severe plant reaction is definitely triggered. At the moment of irradiation, each plant presented a mature first trifoliate leaf, while other leaves were still developing. To avoid cumulative effects on the same sample, the doses of X-rays were delivered as one dose per each plant (Kurimoto et al. 2010). Three plants per dose were irradiated, while three control plants were not irradiated. An X-ray generator (Siemens, Forchheim, Germany) was used to apply the total doses of X-rays. Dosimetry was performed by means of an ionising chamber (Harshaw 3500). After each X-ray dose exposure, the irradiated plants were transferred again to the growth chamber and subjected to the same conditions experienced before irradiation. Three days after the irradiation, two leaflets per plant were collected from fully-grown leaves. The leaflets were destined for microscopy analyses and pigment extraction. Irradiated leaves were compared with controls. Since no differences were found in observations between leaves irradiated with 0.3 and 10 Gy, only data on leaves treated with 10 Gy are reported in the results. Microscopy analyses Collected leaflets were fixed in FAA (40% formaldehyde/glacial acetic acid/50% ethanol; 5/5/90 by volume) for several days. Leaflets were cut under a dissection microscope (SZX9; Olympus, Hamburg, Germany) to obtain subsamples of 5 9 5 mm. These subsamples were dehydrated in an ethanol series and embedded in the acrylic resin JB4 (Polysciences, Warrington, PA, USA). Semi-thin cross-sections (5-lm thick) were cut with a rotary microtome. Sections were stained with 0.5% toluidine blue in water (Feder & O’Brien 1968), mounted with Canadian balsam and observed under a light microscope (BX60; Olympus). Unstained sections were mounted in mineral oil for fluorescence observations with an epi-fluorescence microscope (BX60; Olympus) equipped with a mercury lamp, band-pass filter of 330–385 nm, dichromatic mirror of 400 nm and above, and a barrier filter of 420 nm and above. With such filters it was possible to detect the presence of simple phenolics that autofluoresce at such wavelengths (Fukuzawa 1992; Ruzin 1999). Three sections per leaflet were analysed and images were collected with a digital camera (CAMEDIA C4040; Olympus) at various magnifications. Digital image analysis All digital images were analysed with the ANALYSISâ 3.2 (Olympus) software program for image analysis. Lamina thickness and mesophyll density (percentage of tissue occupied by intercellular spaces) were measured. Cell size and shape of upper epidermis, palisade and spongy parenchyma were quantified. More specifically, cell size was quantified by measuring the following parameters: area, maximum, mean and minimum Feret diameters (the measured distance between parallel lines tangential to the cell perimeter). Cell shape was characterised by measuring the following indices: aspect ratio (maximum width/height ratio of a bounding rectangle for the cell, defining how it is elongated), sphericity (roundness of a particle: a spherical particle has a maximum value of 100), and convexity (the fraction of the cell area and the area of its convex surface: when a cell is turgid the convexity value tends to 100, while
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when it is shrunken the convexity value is lowered; Van Buggenhout et al. 2008; De Micco et al. 2008). The cell area occupied by phenolics was measured in a random selection of cells and expressed as a percentage of tissue occupied by phenolic compounds, appearing auto-fluorescent at the above-reported filter settings, over a given surface (De Micco & Aronne 2008). The number of chloroplasts per mm2 of mesophyll surface and chloroplast size (length of main axis) were measured in palisade tissue.
All results were subjected to statistical analysis (ANOVA) using the SPSS statistical package (SPSS, Chicago, IL, USA). Multiple comparison tests were performed with Student–Newman–Keuls and LSD coefficients, with P < 0.05 as the significant level of probability. Data on sphericity, convexity and percentage of phenolics were transformed through an arcsine function before statistical analysis.
Pigment extraction
RESULTS AND DISCUSSION
Pigments were extracted from leaf discs in ice-cold 100% acetone, with a mortar and pestle, and determined using a spectrophotometer (UV-VIS Cary 100; Agilent Technologies, Santa Clara, CA, USA) according to Lichtenthaler (1987).
After irradiation, mature organs of P. vulgaris did not show any external signs of stress, while stem elongation and growth of young and new leaves were inhibited at 50 and 100 Gy. The analysis of histological and cytological features of mature leaves
Statistical analysis
A
E
B
F
C
G
D
H
Fig. 2. Light (A–D) and epifluorescence (E–H) microscopy views of cross-sections of Phaseolus vulgaris leaves from control (A, E) and irradiated plants given different doses of X-rays: 10 Gy (B, F), 50 Gy (C, G) and 100 Gy (D, H). Phenolic compounds along chloroplast membranes are auto-fluorescent (E–H). Images are at the same magnification. Bar = 50 lm. Plant Biology 16 (Suppl. 1) (2014) 187–193 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands
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A
Fig. 3. Lamina thickness of Phaseolus vulgaris leaves of control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
irradiated with different levels of X-rays showed that there were no structural modifications preventing further growth and plant survival. However, some alterations, clearer at the higher levels of irradiation, may have impaired photosynthetic efficiency. Microscopy analyses showed that mature leaves of P. vulgaris are characterised by a typical dorsiventral structure that is not subject to disruptive structural alterations after irradiation with X-rays (Fig. 2). Leaves irradiated with up to 10 Gy did not show significant differences when compared with control leaves (Fig. 2A, B, E and F). At 50 and 100 Gy, some perturbations occurred in the mesophyll structure due to the presence of more loose parenchyma cells in both palisade and spongy tissues (Fig. 2C, D, G and H). Lamina of leaves irradiated at 50 and 100 Gy became thicker than those of controls and lowdose-irradiated leaves (Figs 2 and 3). An increase in lamina thickness also occurs in natural conditions in leaves exposed to high solar radiation; indeed, sun leaves are typically thicker than shade leaves (Terashima et al. 2006). Moreover, UV-B radiation can induce leaf thickening, which is often linked to other morpho-anatomical traits including changes in cell size and in distribution of chlorophyll in the mesophyll (Jansen et al. 1998). The increase in lamina thickness after irradiation of 50 Gy and above was related to larger cell size in both palisade and spongy tissues. The percentage of tissue occupied by intercellular spaces was not significantly different between controls (44.13 1.57%) and leaves irradiated at 10 Gy (48.81 2.36%). At 50 Gy, it was possible to observe a decrease in intercellular spaces (40.88 3.17%), which became significant at 100 Gy (33.80 5.17%). This indicated the presence of denser mesophyll at high levels of X-rays. Regarding cell size, cell area showed a tendency to increase after irradiation treatments in almost all leaf tissues (Fig. 4). In the upper epidermis, cell area was significantly larger in leaves irradiated with the highest level of X-rays, while leaves treated with 50 Gy showed intermediate values between controls and 100 Gy (Fig. 4A). In palisade parenchyma, cell area was larger in all irradiated leaves than in controls, with maximum areas after irradiation at 50 Gy (Fig. 4B). In spongy parenchyma, cell area showed the same trend as in palisade parenchyma, but with lower, not always significant, values (Fig. 4C). Maximum, mean and minimum Feret diameters showed the same trends 190
B
C
Fig. 4. Cell area of epidermis (A), palisade (B) and spongy (C) parenchyma of Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
of variation as cell size in all tissues, thus indicating that strong changes in cell shape did not occur (Table 1). This was confirmed by values of aspect ratio and sphericity, which were similar among treatments (Table 1). Values of convexity were always very high, suggesting high turgidity in all tissues (Table 1). The overall analysis of cell size and shape suggests that cells were characterised by high turgidity, which probably favoured turgor-driven cell enlargement in irradiated leaves. We hypothesise that increased size of cells in irradiated leaves was due to a loosening of cell walls caused by the radiation. Thus the degradation of polysaccharides, pectins and other
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Table 1. Size and shape of epidermis, palisade and spongy parenchyma cells in control and irradiated leaves. Mean values (X) and significance of ANOVA are reported. Different letters correspond to significantly different values.
epidermis maximum Feret diameter (lm) mean Feret diameter (lm) minimum Feret diameter (lm) aspect ratio sphericity convexity ealisade parenchyma maximum Feret diameter (lm) mean Feret diameter (lm) minimum Feret diameter (lm) aspect ratio sphericity convexity spongy parenchyma maximum Feret diameter (lm) mean Feret diameter (lm) minimum Feret diameter (lm) aspect ratio sphericity convexity
C X
10 Gy X
50 Gy X
100 Gy X
33.23a 26.43a 17.33a 1.86a 34.41a 92.53ab
31.37a 25.22a 16.78a 1.78a 37.19a 92.93a
36.57ab 29.11ab 18.80a 1.95a 31.42a 90.61b
43.13b 34.11b 21.56b 1.97a 31.71a 90.76b
42.34a 32.92a 18.21a 2.36a 13.79a 83.65a
43.48a 34.44ab 21.59bc 2.02b 21.27b 88.59b
53.59c 41.25c 22.10c 2.45a 15.10a 87.01b
48.48b 37.40b 20.07b 2.45a 14.92a 86.24ab
25.58a 21.39a 16.34a 1.53a 43.11ab 91.21ab
27.64a 22.53a 15.93a 1.74b 36.26a 91.46ab
31.82b 26.06b 18.54b 1.69ab 34.27a 90.12a
25.93a 21.10a 17.05ab 1.53a 47.37b 92.51b
matrix material, reported as a response to ionising radiation (Glenn & Poovaiah 1990; Kovacs et al. 1997; Kovacs & Keresztes 2002), would have determined a lowering of mechanical resistance of cell walls, which in normal conditions constrain cell enlargement in adult tissues. The relation between cell wall loosening and cell expansion is quite complex: during cell growth, walls must combine strength and pliancy to allow both resistance to mechanical forces (arising from turgor pressure) and controlled polymer ‘creep’ that expands the wall, creating enough volume for the enlarging protoplast (Cosgrove 1998). Turgor-driven cell enlargement is ascribed to the hydrolysis of matrix polysaccharides and to the action of expansins, which act by locally loosening the attachment of cellulose microfibrils to the matrix (Cosgrove 2000). Increased cell size is reported in either protoplasts or cells in actively growing organs as a consequence of cell wall loosening and altered cellulose microfibril distribution ascribed to the action of Space factors (Skagen & Iversen 1999; De Micco et al. 2008). Degradation of middle lamellae material occurring after irradiation (Kovacs et al. 1995) could also explain the separation of cells in the mesophyll of irradiated leaves, which results in a loosened structure. Use of UV microscopy allowed detection of autofluorescence of phenolic compounds, which were localised mainly along cell membranes in both control and irradiated leaves (Fig. 2E–H). Their distribution did not change after the irradiation treatments: they were nearly always associated with chloroplasts membranes. The percentage of phenolic compounds was higher only in the mesophyll of leaves irradiated at 100 Gy (Fig. 5). The increased content of phenolic compounds can be considered in part as a direct effect of the increase in number of chloroplasts per mm2 (Fig. 6A). Indeed, the increase in phenolic compounds measured in 100 Gy-irradiated leaves was more than proportional to the increase in number of chlo-
Fig. 5. Percentage (%) of mesophyll occupied by phenolic compounds in Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
A
B
Fig. 6. Number (N.) of chloroplasts per mm2 (A) and chloroplast size (measured as length of main axis) (B) in Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
roplasts, also considering that the chloroplasts were smaller than in other treatments and controls (Fig. 6B). Phenolic compounds can be considered as natural screens against high levels of radiation; indeed, also in natural environments with high levels of solar radiation, plants tend to accumulate high amounts of phenolics in organs exposed to light in order to
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Fig. 7. Content of chlorophyll a and b in Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
protect cellular structures (Lattanzio et al. 2008; De Micco et al. 2011b). Experiments with plants grown in Space found that the phenylpropanoid pathway, leading to the formation of simple and complex phenolics, is altered (Keresztes & Kovacs 1991; Levine et al. 2001a,b). Changes in content and distribution of phenolics among organs have been reported in response to Space factors (Levine et al. 2001b; De Micco & Aronne 2008) and an increase in such compounds is a positive plant response to protect cell structures from oxidative damage. The localisation of simple phenolics along membranes of chloroplasts could play a key role in the radio-protection of the photosynthetic apparatus. However, the photosynthetic pigment analysis showed strong differences between controls and leaves irradiated at 50 and 100 Gy (Fig. 7). At X-rays doses of 50 and 100 Gy, chlorophyll a and b content was significantly lower than when measured in controls and 10 Gy-irradiated leaves. Perturbations in the photosynthetic machinery have been observed in plants subjected to ionising radiation, with photosynthetic impairment detected at different steps of the photosynthetic pathway (De Micco et al. 2011a). The number of chloroplasts per cell, chloroplast ultrastructure, and pigment content have all been reported to be sensitive to irradiation (Keresztes & Kovacs 1991; Martınez-Solano et al. 2005; Kim et al. 2011). However, many modifications seem to depend on the age of leaves at the time of irradiation. Kim et al. (2011) recently analysed the effect of ionising radiation on leaves irradiated at two different moments after sowing, and found opposite responses. More specifically, leaves of younger irradiated plants were characterised by a decrease in the number of chloroplasts per cell and in pigment content, while leaves of older irradiated plants showed an increased number of chloroplasts per cell and augmented pigment content. However, our finding of decreased chlorophyll content in leaves irradiated with high levels of radiation is in agreement with the inhibition of chlorophyll and carotenoid synthesis ascribed to exposure to high REFERENCES Abe T., Matsuyama T., Sekido S., Yamaguchi I., Yoshida S., Kameya T. (2002) Chlorophyll-
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levels of X-rays (Al-Enezi & Al-Khayri 2012). Severe changes in chlorophylls content can be considered responsible for lowering photosynthetic efficiency and are likely the consequence of the high levels of ROS produced after water radiolysis (Zaka et al. 2002). Nevertheless, different responses of photosynthetic pigment content after exposure to ionising radiation have been reported in different plant species and cultivars (Kim et al. 2004). All discrepancies found so far depend not only on differences in plant material used, but also on differences in type of radiation and protocols. Given that opportunities for conducting experiments in Space or with high-LET radiation are limited and costly, experiments with other types of radiation, such as X-rays, are basic to gain preliminary information on plant sensitivity to high levels of radiation. Such simulation approach has also been successfully applied for microgravity experiments, where altered gravity is extensively simulated in preliminary ground-based studies by means of clinostats and other facilities. CONCLUSIONS Mature leaves of P. vulgaris showed significant perturbations in their histological and cytological traits only at high levels of irradiation with X-rays. The main alterations at anatomical level concerned formation of larger cells, likely due to a loosening of cell walls that reduced mechanical constraints to cell enlargement. Such a phenomenon can be considered positive because it favours cell growth, provided that high turgidity is maintained. Another positive change, primed by radiation at cytological level, involved the accumulation of phenolic compounds along membranes, which increased at high X-ray doses. The increased amount of such compounds can confer high radio-resistance to this species since phenolics are natural screens that lower oxidative phenomena. However, alterations at chloroplast level and the decrease in pigments indicate that some impairment of photosynthesis can occur at high levels of radiation. The evidenced different responses of mature leaves to different levels of radiation are interesting because they show the high resistance of plant tissues to low-LET radiation: potential null or even positive effects can have important technical consequences for the design of Space greenhouses. In further research, to obtain a more comprehensive understanding of the resistance of plant tissues to radiation for both space-oriented and radioecology issues, it will be interesting to also analyse the response of the same plant system to more Space-like, highLET radiation, as well as to compare the resistance of P. vulgaris with other candidate species for cultivation in BLSSs. ACKNOWLEDGEMENTS The authors are grateful to Nicola D’Ambrosio (Dept. Biology, University of Naples Federico II) for providing plants used in the experiment, to Mariagabriella Pugliese (Dept. Physics, University of Naples Federico II) for plant irradiation, and Vincenzo Barra for technical support in anatomical analyses.
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Nagata T., Todoriki S., Hayashi T., Shibata Y., Mori M., Kanegae H., Kikuchi S. (1999) c-Radiation induces leaf trichome formation in Arabidopsis. Plant Physiology, 120, 113–119. Palamine M.T., Cureg R.G.A., Marbella L.J., Lapade A.G., Domingo Z.B., Deocaris C.C. (2005) Some biophysical changes in the chloroplasts of a Dracaena radiation-mutant. Philippine Journal of Science, 134, 121. Paradiso R., De Micco V., Buonomo R., Aronne G., Barbieri G., De Pascale S. (2013) Soilless cultivation of soybean for Bioregenerative Life Support Systems (BLSSs): a literature review and the experience of the MELiSSA Project - Food characterization Phase I. Plant Biology, doi: 10.1111/plb.12056. Real A., Sundell-Bergman S., Knowles J.F., Woodhead D.S., Zinger I. (2004) Effects of ionising radiation exposure on plants, fish and mammals: relevant data for environmental radiation protection. Journal of Radiological Protection, 24, A123–A137. Ruzin S.E. (1999) Plant microtechnique and microscopy. Oxford University Press, New York, NY, pp 322. Skagen E.B., Iversen T.-H. (1999) Simulated weightlessness and hyper-g results in opposite effects on the regeneration of the cortical microtubule array in protoplasts from Brassica napus hypocotyls. Physiologia Plantarum, 106, 318–325. Strydom G.J., Staden J.V., Smith M.T. (1991) The effect of gamma radiation on the ultrastructure of the peel of banana fruits. Environmental Botany, 31, 43–49. Takatsuji T., Takayanagi H., Morishita K., Nojima K., Furusawa Y., Nakazawa Y., Matsuse M., Akamatsu S., Hirano N., Hirashima N., Hotokezaka S., Ijichi T., Kakimoto C., Kanemaru T., Koshitake M., Moriuchi A., Yamamoto K., Yoshikawa I. (2010) Induction of micronuclei in germinating onion seed root tip cells irradiated with high energy heavy ions. Journal of Radiation Research, 51, 315–323. Terashima I., Hanba Y.T., Tazoe Y., Vyas P., Yano S. (2006) Irradiance and phenotype: comparative eco-development of sun and shade leaves in relation to photosynthetic CO2 diffusion. Journal of Experimental Botany, 57, 343–354. Van Buggenhout S., Grauwet T., Van Loey A., Hendrickx M. (2008) Structure/processing relation of vacuum infused strawberry tissue frozen under different conditions. European Food Research and Technology, 226, 437–448. Wheeler R.M., Sager J.C., Prince R.P., Knott W.M. (2003) Crop production for advanced life support systems – observation from the Kennedy Space Center Breadboard Project. NASA TM-2003-211184, NASA Kennedy Space Center, Florida, USA. Wu L., Yu Z. (2001) Radiobiological effects of a low-energy ion beam on wheat. Radiation and Environmental Biophysics, 40, 53–57. Wu H., George K., Kawata T., Willingham V., Cucinotta F.A. (2001) Comparisons of F ratios generated from interphase and metaphase chromosome damage induced by high doses of low- and high-LET radiation. Radiation Research, 155, 57–62. Zaka R., Vendecasteele C.M., Misset M.T. (2002) Effects of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae). Journal of Experimental Botany, 53, 1979–1987.
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RESEARCH PAPER
Anatomical alterations of Phaseolus vulgaris L. mature leaves irradiated with X-rays V. De Micco1, C. Arena2 & G. Aronne1 1 Department of Agriculture, University of Naples Federico II, Portici, Italy 2 Department of Biology, University of Naples Federico II, Portici, Italy
Keywords Cell wall; chloroplasts; ionising radiation; leaf anatomy; phenolics; pigment content; space biology; X-rays. Correspondence V. De Micco, Department of Agriculture, University of Naples Federico II, via Universit a 100, Portici, Naples 80055, Italy. E-mail: [email protected] Editor K. Palme Received: 21 January 2013; Accepted: 27 September 2013 doi:10.1111/plb.12125
ABSTRACT The cultivation of higher plants in Space involves not only the development of new agro-technologies for the design of ecologically closed Space greenhouses, but also understanding of the effects of Space factors on biological systems. Among Space factors, ionising radiation is one of the main constraints to the growth of organisms. In this paper, we analyse the effect of low-LET radiation on leaf histology and cytology in Phaseolus vulgaris L. plants subjected to increasing doses of X-rays (0.3, 10, 50, 100 Gy). Leaves irradiated at tissue maturity were compared with not-irradiated controls. Semi-thin sections of leaves were analysed through light and epi-fluorescence microscopy. Digital image analysis was applied to quantify anatomical parameters, with a specific focus on the occurrence of signs of structural damage as well as alterations at subcellular level, such as the accumulation of phenolic compounds and chloroplast size. Results showed that even at high levels of radiation, general anatomical structure was not severely perturbed. Slight changes in mesophyll density and cell enlargement were detected at the highest level of radiation. However, at 100 Gy, higher levels of phenolic compounds accumulated along chloroplast membranes: this accompanied an increase in number of chloroplasts. The reduced content of chlorophylls at high levels of radiation was associated with reduced size of the chloroplasts. All data are discussed in terms of the possible role of cellular modifications in the maintenance of high radioresistance and photosynthetic efficiency.
INTRODUCTION Higher plants are candidates to support long-term human permanence in Space, as key organisms in Bioregenerative Life Support Systems (BLSS; Wheeler et al. 2003; Paradiso et al. 2013). Among Space factors, ionising radiation is one of the main constraints to organism growth because it affects molecular, morpho-anatomical, biochemical and physiological processes (De Micco et al. 2011a). Plants can take advantage of very low levels of radiation, and are very resistant to high doses, which are, instead, detrimental to animals (Casarett 1968; Kumagai et al. 2000; Real et al. 2004). The higher radioresistance of plants is expected for seeds because they are a specific stage of the higher plant life cycle, characterised by peculiar metabolic and structural properties conferring higher resistance to disturbance factors. Other assumptions at the base of higher radioresistance in plants than in animals are the presence of a complex cell wall in the former and differences in behaviour in the case of losses of genetic material (Kovacs et al. 1995; Endo & Gill 1996). Indeed, polyploidy is often present in plants and the presence of multiple wild copies of a gene in a cell can maintain the phenotype unaltered when radiation determines genetic mutations at specific loci (Endo & Gill 1996). Years of extensive experimentation aimed at verifying the effect of ionising radiation on plants have not always produced comparable results because research has been based on differ-
ent irradiation procedures (e.g. radiation type, level and exposure time), various species and cultivars, and differences in parameters measured (not always unambiguously defined) (De Micco et al. 2011a). The effects of ionising radiation on plants at genetic level have been extensively investigated, with a focus on DNA mutations, cell death and chromosomal aberrations (Casarett 1968; Bayonove et al. 1984; Wu et al. 2001; Hase et al. 2002; Kikuchi et al. 2009; Takatsuji et al. 2010; De Micco et al. 2011a). DNA aberrations are intuitively considered responsible for changes in growth and reproduction, which are often the direct consequence of genetic modifications on the resulting phenotype. Indeed, all processes involving mitosis and meiosis can be altered by ionising radiation, thus resulting in lowered germination, increased mortality, altered leaf morphology and reduced reproductive success (Bayonove et al. 1984; Hase et al. 1999; Nagata et al. 1999; Wu & Yu 2001; Komai et al. 2003). However, there is also evidence for a direct effect of ionising radiation on already developed plant structures: phenomena of degradation of cell walls and increased porosity of seed teguments and nutritional layers have been reported (Kovacs et al. 1995, 1997; Hammond et al. 1996). At subcellular level, apart from nuclei, chloroplasts are also a sensitive target of ionising radiation. Alterations in chloroplast structure as well as pigment content have been reported, and are considered responsible for reduced efficiency of the photosynthetic apparatus (Strydom et al. 1991;
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Palamine et al. 2005; Arena et al. 2013, 2014). All observed phenomena on the photosynthetic apparatus have generally been considered either an outcome of genetic modifications or a consequence of oxidative damage due to the high production of reactive oxygen species (ROS) generated by water radiolysis induced by high levels of radiation (Abe et al. 2002; Zaka et al. 2002). The aim of this paper is to analyse the effect of low- linear energy transfer (LET) ionising radiation on mature leaves of Phaseolus vulgaris L. in order to evaluate whether structures already formed at the time of irradiation undergo alterations at anatomical level. The reason for focusing on leaf structure was driven by the awareness that major metabolic and physiological processes are ultimately regulated by the physics of plant structure (Brodribb 2009). MATERIAL AND METHODS Plant material and growth conditions Seeds of dwarf Phaseolus vulgaris L. were germinated in the dark in Petri dishes on filter paper moistened with distilled water. Developing seedlings were transplanted into pots (10cm diameter), filled with 1/1 peat/soil. Plants were cultivated in a growth chamber, with a photoperiod of 12 h, under controlled conditions of temperature (25/20 °C, day/night), relative humidity (60/75%, day/night) and light (90–100 lmolphotonsm 2s 1 at the top of the canopy). Irradiation in the growth chamber was supplied by a series of 58 W fluorescent tubes (Philips TLD 54 and 84) and halogen lamps (Philips HPI-T 400 W). Photosynthetic photon flux density (PPFD) was measured with a quantum sensor (Li-Cor 185; Li-Cor, Lincoln, NE, USA). During the growth period (April–June 2011), plants were irrigated to reintegrate water lost through evapotranspiration. Irradiation procedure X-rays were chosen as low-LET radiation because their effectiveness is similar to low-energy protons that contribute to space radiation, together with other charged particles. Thus, we focused our study on the effects of a wide range of X-ray doses on P. vulgaris leaves to explore the dose range where this species is more sensitive. Plants were irradiated with four total doses of X-rays (0.3, 10, 50 and 100 Gy) 200 kVp, at dose rate of 1 Gymin 1 (Fig. 1). The highest doses (50 and 100 Gy) were established as positive controls: these are radiation treatments
Fig. 1. Scheme indicating the target organ (i.e. dosimetry was calculated on the first trifoliate leaf), X-ray doses and exposure times.
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with doses at which a severe plant reaction is definitely triggered. At the moment of irradiation, each plant presented a mature first trifoliate leaf, while other leaves were still developing. To avoid cumulative effects on the same sample, the doses of X-rays were delivered as one dose per each plant (Kurimoto et al. 2010). Three plants per dose were irradiated, while three control plants were not irradiated. An X-ray generator (Siemens, Forchheim, Germany) was used to apply the total doses of X-rays. Dosimetry was performed by means of an ionising chamber (Harshaw 3500). After each X-ray dose exposure, the irradiated plants were transferred again to the growth chamber and subjected to the same conditions experienced before irradiation. Three days after the irradiation, two leaflets per plant were collected from fully-grown leaves. The leaflets were destined for microscopy analyses and pigment extraction. Irradiated leaves were compared with controls. Since no differences were found in observations between leaves irradiated with 0.3 and 10 Gy, only data on leaves treated with 10 Gy are reported in the results. Microscopy analyses Collected leaflets were fixed in FAA (40% formaldehyde/glacial acetic acid/50% ethanol; 5/5/90 by volume) for several days. Leaflets were cut under a dissection microscope (SZX9; Olympus, Hamburg, Germany) to obtain subsamples of 5 9 5 mm. These subsamples were dehydrated in an ethanol series and embedded in the acrylic resin JB4 (Polysciences, Warrington, PA, USA). Semi-thin cross-sections (5-lm thick) were cut with a rotary microtome. Sections were stained with 0.5% toluidine blue in water (Feder & O’Brien 1968), mounted with Canadian balsam and observed under a light microscope (BX60; Olympus). Unstained sections were mounted in mineral oil for fluorescence observations with an epi-fluorescence microscope (BX60; Olympus) equipped with a mercury lamp, band-pass filter of 330–385 nm, dichromatic mirror of 400 nm and above, and a barrier filter of 420 nm and above. With such filters it was possible to detect the presence of simple phenolics that autofluoresce at such wavelengths (Fukuzawa 1992; Ruzin 1999). Three sections per leaflet were analysed and images were collected with a digital camera (CAMEDIA C4040; Olympus) at various magnifications. Digital image analysis All digital images were analysed with the ANALYSISâ 3.2 (Olympus) software program for image analysis. Lamina thickness and mesophyll density (percentage of tissue occupied by intercellular spaces) were measured. Cell size and shape of upper epidermis, palisade and spongy parenchyma were quantified. More specifically, cell size was quantified by measuring the following parameters: area, maximum, mean and minimum Feret diameters (the measured distance between parallel lines tangential to the cell perimeter). Cell shape was characterised by measuring the following indices: aspect ratio (maximum width/height ratio of a bounding rectangle for the cell, defining how it is elongated), sphericity (roundness of a particle: a spherical particle has a maximum value of 100), and convexity (the fraction of the cell area and the area of its convex surface: when a cell is turgid the convexity value tends to 100, while
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when it is shrunken the convexity value is lowered; Van Buggenhout et al. 2008; De Micco et al. 2008). The cell area occupied by phenolics was measured in a random selection of cells and expressed as a percentage of tissue occupied by phenolic compounds, appearing auto-fluorescent at the above-reported filter settings, over a given surface (De Micco & Aronne 2008). The number of chloroplasts per mm2 of mesophyll surface and chloroplast size (length of main axis) were measured in palisade tissue.
All results were subjected to statistical analysis (ANOVA) using the SPSS statistical package (SPSS, Chicago, IL, USA). Multiple comparison tests were performed with Student–Newman–Keuls and LSD coefficients, with P < 0.05 as the significant level of probability. Data on sphericity, convexity and percentage of phenolics were transformed through an arcsine function before statistical analysis.
Pigment extraction
RESULTS AND DISCUSSION
Pigments were extracted from leaf discs in ice-cold 100% acetone, with a mortar and pestle, and determined using a spectrophotometer (UV-VIS Cary 100; Agilent Technologies, Santa Clara, CA, USA) according to Lichtenthaler (1987).
After irradiation, mature organs of P. vulgaris did not show any external signs of stress, while stem elongation and growth of young and new leaves were inhibited at 50 and 100 Gy. The analysis of histological and cytological features of mature leaves
Statistical analysis
A
E
B
F
C
G
D
H
Fig. 2. Light (A–D) and epifluorescence (E–H) microscopy views of cross-sections of Phaseolus vulgaris leaves from control (A, E) and irradiated plants given different doses of X-rays: 10 Gy (B, F), 50 Gy (C, G) and 100 Gy (D, H). Phenolic compounds along chloroplast membranes are auto-fluorescent (E–H). Images are at the same magnification. Bar = 50 lm. Plant Biology 16 (Suppl. 1) (2014) 187–193 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands
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A
Fig. 3. Lamina thickness of Phaseolus vulgaris leaves of control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
irradiated with different levels of X-rays showed that there were no structural modifications preventing further growth and plant survival. However, some alterations, clearer at the higher levels of irradiation, may have impaired photosynthetic efficiency. Microscopy analyses showed that mature leaves of P. vulgaris are characterised by a typical dorsiventral structure that is not subject to disruptive structural alterations after irradiation with X-rays (Fig. 2). Leaves irradiated with up to 10 Gy did not show significant differences when compared with control leaves (Fig. 2A, B, E and F). At 50 and 100 Gy, some perturbations occurred in the mesophyll structure due to the presence of more loose parenchyma cells in both palisade and spongy tissues (Fig. 2C, D, G and H). Lamina of leaves irradiated at 50 and 100 Gy became thicker than those of controls and lowdose-irradiated leaves (Figs 2 and 3). An increase in lamina thickness also occurs in natural conditions in leaves exposed to high solar radiation; indeed, sun leaves are typically thicker than shade leaves (Terashima et al. 2006). Moreover, UV-B radiation can induce leaf thickening, which is often linked to other morpho-anatomical traits including changes in cell size and in distribution of chlorophyll in the mesophyll (Jansen et al. 1998). The increase in lamina thickness after irradiation of 50 Gy and above was related to larger cell size in both palisade and spongy tissues. The percentage of tissue occupied by intercellular spaces was not significantly different between controls (44.13 1.57%) and leaves irradiated at 10 Gy (48.81 2.36%). At 50 Gy, it was possible to observe a decrease in intercellular spaces (40.88 3.17%), which became significant at 100 Gy (33.80 5.17%). This indicated the presence of denser mesophyll at high levels of X-rays. Regarding cell size, cell area showed a tendency to increase after irradiation treatments in almost all leaf tissues (Fig. 4). In the upper epidermis, cell area was significantly larger in leaves irradiated with the highest level of X-rays, while leaves treated with 50 Gy showed intermediate values between controls and 100 Gy (Fig. 4A). In palisade parenchyma, cell area was larger in all irradiated leaves than in controls, with maximum areas after irradiation at 50 Gy (Fig. 4B). In spongy parenchyma, cell area showed the same trend as in palisade parenchyma, but with lower, not always significant, values (Fig. 4C). Maximum, mean and minimum Feret diameters showed the same trends 190
B
C
Fig. 4. Cell area of epidermis (A), palisade (B) and spongy (C) parenchyma of Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
of variation as cell size in all tissues, thus indicating that strong changes in cell shape did not occur (Table 1). This was confirmed by values of aspect ratio and sphericity, which were similar among treatments (Table 1). Values of convexity were always very high, suggesting high turgidity in all tissues (Table 1). The overall analysis of cell size and shape suggests that cells were characterised by high turgidity, which probably favoured turgor-driven cell enlargement in irradiated leaves. We hypothesise that increased size of cells in irradiated leaves was due to a loosening of cell walls caused by the radiation. Thus the degradation of polysaccharides, pectins and other
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Table 1. Size and shape of epidermis, palisade and spongy parenchyma cells in control and irradiated leaves. Mean values (X) and significance of ANOVA are reported. Different letters correspond to significantly different values.
epidermis maximum Feret diameter (lm) mean Feret diameter (lm) minimum Feret diameter (lm) aspect ratio sphericity convexity ealisade parenchyma maximum Feret diameter (lm) mean Feret diameter (lm) minimum Feret diameter (lm) aspect ratio sphericity convexity spongy parenchyma maximum Feret diameter (lm) mean Feret diameter (lm) minimum Feret diameter (lm) aspect ratio sphericity convexity
C X
10 Gy X
50 Gy X
100 Gy X
33.23a 26.43a 17.33a 1.86a 34.41a 92.53ab
31.37a 25.22a 16.78a 1.78a 37.19a 92.93a
36.57ab 29.11ab 18.80a 1.95a 31.42a 90.61b
43.13b 34.11b 21.56b 1.97a 31.71a 90.76b
42.34a 32.92a 18.21a 2.36a 13.79a 83.65a
43.48a 34.44ab 21.59bc 2.02b 21.27b 88.59b
53.59c 41.25c 22.10c 2.45a 15.10a 87.01b
48.48b 37.40b 20.07b 2.45a 14.92a 86.24ab
25.58a 21.39a 16.34a 1.53a 43.11ab 91.21ab
27.64a 22.53a 15.93a 1.74b 36.26a 91.46ab
31.82b 26.06b 18.54b 1.69ab 34.27a 90.12a
25.93a 21.10a 17.05ab 1.53a 47.37b 92.51b
matrix material, reported as a response to ionising radiation (Glenn & Poovaiah 1990; Kovacs et al. 1997; Kovacs & Keresztes 2002), would have determined a lowering of mechanical resistance of cell walls, which in normal conditions constrain cell enlargement in adult tissues. The relation between cell wall loosening and cell expansion is quite complex: during cell growth, walls must combine strength and pliancy to allow both resistance to mechanical forces (arising from turgor pressure) and controlled polymer ‘creep’ that expands the wall, creating enough volume for the enlarging protoplast (Cosgrove 1998). Turgor-driven cell enlargement is ascribed to the hydrolysis of matrix polysaccharides and to the action of expansins, which act by locally loosening the attachment of cellulose microfibrils to the matrix (Cosgrove 2000). Increased cell size is reported in either protoplasts or cells in actively growing organs as a consequence of cell wall loosening and altered cellulose microfibril distribution ascribed to the action of Space factors (Skagen & Iversen 1999; De Micco et al. 2008). Degradation of middle lamellae material occurring after irradiation (Kovacs et al. 1995) could also explain the separation of cells in the mesophyll of irradiated leaves, which results in a loosened structure. Use of UV microscopy allowed detection of autofluorescence of phenolic compounds, which were localised mainly along cell membranes in both control and irradiated leaves (Fig. 2E–H). Their distribution did not change after the irradiation treatments: they were nearly always associated with chloroplasts membranes. The percentage of phenolic compounds was higher only in the mesophyll of leaves irradiated at 100 Gy (Fig. 5). The increased content of phenolic compounds can be considered in part as a direct effect of the increase in number of chloroplasts per mm2 (Fig. 6A). Indeed, the increase in phenolic compounds measured in 100 Gy-irradiated leaves was more than proportional to the increase in number of chlo-
Fig. 5. Percentage (%) of mesophyll occupied by phenolic compounds in Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
A
B
Fig. 6. Number (N.) of chloroplasts per mm2 (A) and chloroplast size (measured as length of main axis) (B) in Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
roplasts, also considering that the chloroplasts were smaller than in other treatments and controls (Fig. 6B). Phenolic compounds can be considered as natural screens against high levels of radiation; indeed, also in natural environments with high levels of solar radiation, plants tend to accumulate high amounts of phenolics in organs exposed to light in order to
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Fig. 7. Content of chlorophyll a and b in Phaseolus vulgaris leaves from control and irradiated plants given different doses of X-rays. Mean and SE are shown. Different letters correspond to significantly different values according to multiple comparison tests (P < 0.05).
protect cellular structures (Lattanzio et al. 2008; De Micco et al. 2011b). Experiments with plants grown in Space found that the phenylpropanoid pathway, leading to the formation of simple and complex phenolics, is altered (Keresztes & Kovacs 1991; Levine et al. 2001a,b). Changes in content and distribution of phenolics among organs have been reported in response to Space factors (Levine et al. 2001b; De Micco & Aronne 2008) and an increase in such compounds is a positive plant response to protect cell structures from oxidative damage. The localisation of simple phenolics along membranes of chloroplasts could play a key role in the radio-protection of the photosynthetic apparatus. However, the photosynthetic pigment analysis showed strong differences between controls and leaves irradiated at 50 and 100 Gy (Fig. 7). At X-rays doses of 50 and 100 Gy, chlorophyll a and b content was significantly lower than when measured in controls and 10 Gy-irradiated leaves. Perturbations in the photosynthetic machinery have been observed in plants subjected to ionising radiation, with photosynthetic impairment detected at different steps of the photosynthetic pathway (De Micco et al. 2011a). The number of chloroplasts per cell, chloroplast ultrastructure, and pigment content have all been reported to be sensitive to irradiation (Keresztes & Kovacs 1991; Martınez-Solano et al. 2005; Kim et al. 2011). However, many modifications seem to depend on the age of leaves at the time of irradiation. Kim et al. (2011) recently analysed the effect of ionising radiation on leaves irradiated at two different moments after sowing, and found opposite responses. More specifically, leaves of younger irradiated plants were characterised by a decrease in the number of chloroplasts per cell and in pigment content, while leaves of older irradiated plants showed an increased number of chloroplasts per cell and augmented pigment content. However, our finding of decreased chlorophyll content in leaves irradiated with high levels of radiation is in agreement with the inhibition of chlorophyll and carotenoid synthesis ascribed to exposure to high REFERENCES Abe T., Matsuyama T., Sekido S., Yamaguchi I., Yoshida S., Kameya T. (2002) Chlorophyll-
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levels of X-rays (Al-Enezi & Al-Khayri 2012). Severe changes in chlorophylls content can be considered responsible for lowering photosynthetic efficiency and are likely the consequence of the high levels of ROS produced after water radiolysis (Zaka et al. 2002). Nevertheless, different responses of photosynthetic pigment content after exposure to ionising radiation have been reported in different plant species and cultivars (Kim et al. 2004). All discrepancies found so far depend not only on differences in plant material used, but also on differences in type of radiation and protocols. Given that opportunities for conducting experiments in Space or with high-LET radiation are limited and costly, experiments with other types of radiation, such as X-rays, are basic to gain preliminary information on plant sensitivity to high levels of radiation. Such simulation approach has also been successfully applied for microgravity experiments, where altered gravity is extensively simulated in preliminary ground-based studies by means of clinostats and other facilities. CONCLUSIONS Mature leaves of P. vulgaris showed significant perturbations in their histological and cytological traits only at high levels of irradiation with X-rays. The main alterations at anatomical level concerned formation of larger cells, likely due to a loosening of cell walls that reduced mechanical constraints to cell enlargement. Such a phenomenon can be considered positive because it favours cell growth, provided that high turgidity is maintained. Another positive change, primed by radiation at cytological level, involved the accumulation of phenolic compounds along membranes, which increased at high X-ray doses. The increased amount of such compounds can confer high radio-resistance to this species since phenolics are natural screens that lower oxidative phenomena. However, alterations at chloroplast level and the decrease in pigments indicate that some impairment of photosynthesis can occur at high levels of radiation. The evidenced different responses of mature leaves to different levels of radiation are interesting because they show the high resistance of plant tissues to low-LET radiation: potential null or even positive effects can have important technical consequences for the design of Space greenhouses. In further research, to obtain a more comprehensive understanding of the resistance of plant tissues to radiation for both space-oriented and radioecology issues, it will be interesting to also analyse the response of the same plant system to more Space-like, highLET radiation, as well as to compare the resistance of P. vulgaris with other candidate species for cultivation in BLSSs. ACKNOWLEDGEMENTS The authors are grateful to Nicola D’Ambrosio (Dept. Biology, University of Naples Federico II) for providing plants used in the experiment, to Mariagabriella Pugliese (Dept. Physics, University of Naples Federico II) for plant irradiation, and Vincenzo Barra for technical support in anatomical analyses.
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