Biology and crop production in Space environments: Challenges and opportunities.

Long-term manned space-exploration missions and the permanence of human colonies on orbital stations or planetary habitats will require the regeneration of resources onboard or in-situ. Bioregenerative Life Support Systems (BLSSs) are artificial environments where different compartments, involving both living organisms and physical-chemical processes, are integrated to achieve a safe, self-regulating, and chemically balanced Earth-like environment to support human life. Higher plants are key elements of such systems and Space greenhouses represent the producers' compartment. Growing plants in Space requires the knowledge of their growth responses not only to all environmental factors acting on Earth, but also to specific Space constraints such as altered gravity, ionizing radiations and confined volume. Moreover, cultivation techniques need to be adjusted considering such limitations. The type and intensity of environmental factors to be taken into account depend on the mission scenarios. Here, we summarize constraints and opportunities of cultivating higher plants in Space to regenerate resources and produce fresh food onboard. Both biological and agro-technological issues are considered briefly going through experiments both ground-based on Earth and in Space.

Xylogenesis reveals the genesis and ecological signal of IADFs in Pinus pinea L. and Arbutus unedo L.

Background and Aims: Mediterranean trees have patterns of cambial activity with one or more pauses per year, leading to intra-annual density fluctuations (IADFs) in tree rings. We analysed xylogenesis (January 2015-January 2016) in Pinus pinea L. and Arbutus unedo L., co-occurring at a site on Mt. Vesuvius (southern Italy), to identify the cambial productivity and timing of IADF formation. Methods: Dendrochronological methods and quantitative wood anatomy were applied and enabled IADF identification and classification. Key Results: We showed that cambium in P. pinea was productive throughout the calendar year. From January to March 2015, post-cambial (enlarging) earlywood-like tracheids were observed, which were similar to transition tracheids. The beginning of the tree ring was therefore not marked by a sharp boundary between latewood of the previous year and the new xylem produced. True earlywood tracheids were formed in April. L-IADFs were formed in autumn, with earlywood-like cells in latewood. In A. unedo, a double pause in cell production was observed, in summer and winter, leading to L-IADFs in autumn as well. Moreover, the formation of more than one IADF was observed in A. unedo. Conclusions: Despite having completely different wood formation models and different life strategies, the production of earlywood, latewood and IADF cells was strongly controlled by climatic factors in the two species. Such cambial production patterns need to be taken into account in dendroecological studies to interpret climatic signals in wood from Mediterranean trees.

Reproducing under a warming climate: long winter flowering and extended flower longevity in the only Mediterranean and maritime Primula.

Under the pressure of global warming, general expectations of species migration and evolution of adaptive traits should always be confirmed with species-specific studies. Within this framework, some species can be used as study systems to predict possible consequences of global warming also on other relatives. Unlike its mountain congeneric, Primula palinuri Petagn. has endured all the climatic fluctuations since the Pleistocene, while surviving on Mediterranean coastal cliffs. The aim of this work was to investigate the possible evolution of reproductive biological and ecological traits in P. palinuri adaptation to a warmer environment. Data showed that flowering starts in mid-winter; single flowers remain open for over a month, changing from pendulous to erect. The number of insects visiting flowers of P. palinuri increases during the flowering season, and pollination reduces flower longevity. Overall, the best pollen performances, in terms of viability and germinability, occur at winter temperatures, while pollinator activity prolongs flowering until spring. Moreover, extended longevity of single flowers optimises reproductive success. Both phenotypic plasticity and selective processes might have occurred in P. palinuri. However, we found that reproductive traits of the only Mediterranean Primula remain more associated with cold mountain habitats than warm coastal cliffs. Given the rapid trend of climate warming, migration and new adaptive processes in P. palinuri are unlikely. Response to past climate warming of P. palinuri provides useful indications for future scenarios in other Primula species.

Leaf anatomy and photochemical behaviour of Solanum lycopersicum L. plants from seeds irradiated with low-LET ionising radiation.

Plants can be exposed to ionising radiation not only in Space but also on Earth, due to specific technological applications or after nuclear disasters. The response of plants to ionising radiation depends on radiation quality/quantity and/or plant characteristics. In this paper, we analyse some growth traits, leaf anatomy, and ecophysiological features of plants of Solanum lycopersicum L. "Microtom" grown from seeds irradiated with increasing doses of X-rays (0.3, 10, 20, 50, and 100 Gy). Both juvenile and compound leaves from plants developed from irradiated and control seeds were analysed through light and epifluorescence microscopy. Digital image analysis allowed quantifying anatomical parameters to detect the occurrence of signs of structural damage. Fluorescence parameters and total photosynthetic pigment content were analysed to evaluate the functioning of the photosynthetic machinery. Radiation did not affect percentage and rate of seed germination. Plants from irradiated seeds accomplished the crop cycle and showed a more compact habitus. Dose-depended tendencies of variations occurred in phenolic content, while other leaf anatomical parameters did not show distinct trends after irradiation. The sporadic perturbations of leaf structure, observed during the vegetative phase, after high levels of radiation were not so severe as to induce any significant alterations in photosynthetic efficiency.

Soilless cultivation of soybean for Bioregenerative Life-Support Systems: a literature review and the experience of the MELiSSA Project - Food characterisation Phase I.

Higher plants play a key role in Bioregenerative Life-Support Systems (BLSS) for long-term missions in space, by regenerating air through photosynthetic CO2 absorption and O2 emission, recovering water through transpiration and recycling waste products through mineral nutrition. In addition, plants could provide fresh food to integrate into the crew diet and help to preserve astronauts' wellbeing. The ESA programme Micro-Ecological Life-Support System Alternative (MELiSSA) aims to conceive an artificial bioregenerative ecosystem for resources regeneration, based on both microorganisms and higher plants. Soybean [Glycine max (L.) Merr.] is one of the four candidate species studied for soilless (hydroponic) cultivation in MELiSSA, because of the high nutritional value of the seeds. Within the MELiSSA programme - Food characterisation Phase I, the aim of the research carried out on soybean at the University of Naples was to select the most suitable European cultivars for cultivation in BLSS. In this context, a concise review on the state-of-the-art of soybean cultivation in space-oriented experiments and a summary of research activity for the preliminary theoretical selection and subsequent agronomical evaluation of four cultivars will be presented in this paper.

Microgravity effects on different stages of higher plant life cycle and completion of the seed-to-seed cycle.

Human inhabitation of Space requires the efficient realisation of crop cultivation in bioregenerative life-support systems (BLSS). It is well known that plants can grow under Space conditions; however, perturbations of many biological phenomena have been highlighted due to the effect of altered gravity and its possible interactions with other factors. The mechanisms priming plant responses to Space factors, as well as the consequences of such alterations on crop productivity, have not been completely elucidated. These perturbations can occur at different stages of plant life and are potentially responsible for failure of the completion of the seed-to-seed cycle. After brief consideration of the main constraints found in the most recent experiments aiming to produce seeds in Space, we focus on two developmental phases in which the plant life cycle can be interrupted more easily than in others also on Earth. The first regards seedling development and establishment; we discuss reasons for slow development at the seedling stage that often occurs under microgravity conditions and can reduce successful establishment. The second stage comprises gametogenesis and pollination; we focus on male gamete formation, also identifying potential constraints to subsequent fertilisation. We finally highlight how similar alterations at cytological level can not only be common to different processes occurring at different life stages, but can be primed by different stress factors; such alterations can be interpreted within the model of 'stress-induced morphogenic response' (SIMR). We conclude by suggesting that a systematic analysis of all growth and reproductive phases during the plant life cycle is needed to optimise resource use in plant-based BLSS.

Anatomical alterations of Phaseolus vulgaris L. mature leaves irradiated with X-rays.

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.

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls.

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.

Effects of simulated microgravity on male gametophyte of Prunus, Pyrus, and Brassica species.

In this study we evaluated the effect of simulated microgravity on pollen germination of both herbaceous and woody species in order to investigate the possibility of applying gametophytic selection for plant growth in the space environment. The behaviour of gametophytes exposed to the stress of clinostat rotation could be used to screen the degree of tolerance of the sporophyte to simulated microgravity. The use of male gametophyte selection overcomes the problems generally encountered by sporophytic selection in space especially for woody plants: the large size of plants and their long juvenile phase. In this experiment, pollen collected from just bloomed flowers of Prunus persica, P. avium, P. domestica, Pyrus communis, and Brassica rapa was subjected to tests assessing its viability by techniques such as fluorochromatic reaction. Once pollen viability was ascertained by fluorescence microscopy, pollen was placed on the growth medium in petri dishes both at 1 g and on the clinostat. After incubating for 1 day at room temperature, pollen was observed under a light microscope in order to detect parameters such as the percentage of germination and the growth direction. Then histochemical analyses were performed in order to verify the presence and distribution of nuclei, cytoplasm, and storage substances. Moreover, the presence, size, and morphology of callose plugs were observed. Results showed that the response of gametophytes to simulated microgravity is dependent on the species, some showing altered metabolism, others being unaffected.

Effects of relative humidity and temperature conditions on pollen fluorochromatic reaction of Rosmarinus officinalis L. (Lamiaceae).

Mediterranean ecosystems are characterized by seasonal and annual fluctuations in humidity and temperature which are considered limiting factors for plant growth and might have played a key role in the selection of species that compose the present vegetation. After anther release, pollen is generally exposed to various changes of temperature and humidity conditions, therefore its viability and consequently successful fruit set are strongly affected by these environmental parameters. The aim of this research was to study the effect of different combinations of humidity and temperature on pollen membrane integrity of Rosmarinus officinalis L. in order to investigate possible relations between pollen features and climatic conditions during flowering. This species is an evergreen perennial shrub, occurring among the sclerophyllous vegetation of Mediterranean maquis. In many areas of Southern Italy, it shows a flowering period starting from the beginning of winter and spreading throughout spring months. The analysis of data showed that duration of pollen viability of R. officinalis is enhanced by the co-occurrence of low temperature and high humidity. Moreover, these conditions are able to newly raise the viability of pollen kept at higher temperatures and lower humidity. These observations indicate that reactivation of the pollen membrane depends on low temperature apart from high humidity. Therefore "vernalization" of rosemary pollen enhances its viability, supporting that pollen behavior is adapted to winter condition and allows flowering in winter and early spring.

The effect of simulated microgravity on seed germination and seedling anatomy of Phaseolus vulgaris L.

Seed germination and root anatomy were investigated in seedlings of Phaseolus vulgaris L. developed on a slowly rotating bi-dimensional clinostat and in 1g. Germination time, percent germination, curvature and anatomy of developing root apexes were monitored on the clinostat and compared with the control. Interesting differences were found in germination and root features of the seeds developed on the clinostat compared with 1g ones: the main being germination time, root cap formation, the quantity and distribution of amyloplasts in statocytes. The use of a software to quantitatively analyse root cap anatomy allowed us to detect some differences otherwise unlikely to highlight. Our results showed that prolonged rotation on a bi-dimensional clinostat has an effect on some aspects of germination and on the statocytes that continuously perceives gravity from ever-changing directions.

Classification and counting of fluorescent pollen using an image analysis system.

Pollen viability is commonly assessed by fluorochromatic reaction (FCR) because of the high correlation between positive fluorescence of the pollen grains and their ability to germinate. One of the advantages of this method is its simplicity. An experiment to test FCR analysis for reproducibility, however, showed that results are affected by subjectivity. There is little consistency between analysts, and assessment by the same analyst may differ for the same pollen sample image examined at different times. These problems were solved by a computerized image analysis system that provides a method for classifying positive and negative fluorescent pollen and automatic counting of the grains in each class. The computerized image analysis system does not change the biochemistry of the FCR test, but avoids some experimental errors owing to the subjectivity of the analyst. Microscope images of the pollen after FCR were digitized and later analyzed by specially designed software, "Plant Meter." This software deletes the dark background of the image to isolate the grains, and subsequently counts positive and negative fluorescent pollen grains. An experiment was carried out to validate software output and it showed reliable results. Moreover, the software is user friendly and very little training is necessary for analysts to achieve reliable results.

First evidence of myrmecochory in fleshy-fruited shrubs of the Mediterranean region.

In the mediterranean ecosystems of South Africa and Australia, the role of ants in the dispersal of seeds is well known and antagonistic interactions as well as myrmecochory occur. The high prevalence of seed dispersal by ants, relative so that by vertebrates, is viewed as evidence of adaptation to the nutrient poor soils typical of these environments. Ant dispersal has been considered unimportant in the Mediterranean region and, until now, no systematic study of ant-seed interaction has been undertaken. In the Mediterranean shrubland of Southern Italy the seed content of functioning nests of the ant Messor minor (André) has been quantitatively analyzed. The existence in the nest of seeds from shrub species, as well as those of the Fabaceae and Poaceae, suggest that ant dispersal may be much more widespread in the Mediterranean environments than has been realized. A large proportion of the seeds came from fleshy-fruited shrub species previously reported to be bird dispersed, e.g. Rhamnus alaternus L., Myrtus communis L., Smilax aspera L., Pistacia lentiscus L. and Phillyrea latifolia L. Analysis of nest mounds showed that, although many seeds bad been partially predated by the ants, a very large number had been collected, transported to the nest and subsequently removed to the mound without damage. Examination of the structure of the fruits and seeds of R. alaternus and M. communis, and summer fruiting phenology of R. alaternus, provided further evidence of specific adaptation to ant dispersal. The presence of an elaiosome on the seed is reported for the first time in both specific. Feeding trials showed that ants selectively remove specific diaspores, preferentially collecting the seeds with the elaiosome.