Spectral effects of blue and red light on growth, anatomy, and physiology of lettuce.

Characterizing spectral effects of blue and red light ratios on plants could help expand our understanding of factors that regulate growth and development, which is becoming increasingly important as narrowband light-emitting diodes become common for sole-source lighting. Herein we report growth, physiological, and anatomical responses of two lettuce cultivars grown indoors under various blue and red ratios including monochromatic treatments. When used in combination with red, increasing the proportion of blue light generally reduced growth but increased chloroplast abundance and single-leaf photosynthetic efficiency. However, when used as single wavebands, both blue and red light increased leaf area and epidermal cell area, but reduced root dry mass, SPAD index, stomatal density, and leaf thickness compared to dichromatic light. In addition, chloroplast abundance and single-leaf physiological responses were higher in plants grown under monochromatic blue compared to red light, but the opposite trend was measured for shoot biomass. Our results show that spectral effects on morpho-anatomical leaf responses can largely influence plant growth and single-leaf physiological responses. However, a significant blue light reduction in radiation capture ultimately limits growth and productivity of lettuce plants when dichromatic blue and red light is used.

Fiber-optic refractometer for in vivo sugar concentration measurements of low-nectar-producing flowers.

Sugar concentration in floral nectars is an assessment required in several diverse fields of application. The widely used analysis, consisting of nectar extraction with a microcapillary and sugar concentration measurement with a light refractometer, is not reliable when the nectar is secreted in small quantities, unextractable with a microcapillary. Ancillary methods adopted in such cases are destructive, rather complicated and often provide much less precise and accurate results. The microscopic-size, low cost and biocompatibility of optical fibers were exploited to deliver light directly inside the flower with minimal invasiveness and measure instantaneously the refractometric properties of the nectar without extracting it. After comparing the new and old methods using two known nectariferous species, the new approach was validated on Primula palinuri, whose nectar is unextractable with microcapillaries. The fiber-optic probe was able to measure the nectar refractive index in P. palinuri flowers making it possible to highlight a previously undetected significant trend of the sugar concentration throughout the long anthesis of the single flowers. Changes in nectar concentrations are similar in both longistylous and brevistylous flowers. The fiber-optic refractometer is an advancement of light refractometer analysis. Further customization of the laboratory set-up into portable equipment will boost applications.

Drought impact on water use efficiency and intra-annual density fluctuations in Erica arborea on Elba (Italy).

Erica arborea (L) is a widespread Mediterranean species, able to cope with water stress and colonize semiarid environments. The eco-physiological plasticity of this species was evaluated by studying plants growing at two sites with different soil moistures on the island of Elba (Italy), through dendrochronological, wood-anatomical analyses and stable isotopes measurements. Intra-annual density fluctuations (IADFs) were abundant in tree rings, and were identified as the key parameter to understand site-specific plant responses to water stress. Our findings showed that the formation of IADFs is mainly related to the high temperature, precipitation patterns and probably to soil water availability, which differs at the selected study sites. The recorded increase in the (13) C-derived intrinsic water use efficiency at the IADFs level was linked to reduced water loss rather than to increasing C assimilation. The variation in vessel size and the different absolute values of δ(18) O among trees growing at the two study sites underlined possible differences in stomatal control of water loss and possible differences in sources of water uptake. This approach not only helped monitor seasonal environmental differences through tree-ring width, but also added valuable information on E. arborea responses to drought and their ecological implications for Mediterranean vegetation dynamics.

From flower to honey bouquet: possible markers for the botanical origin of Robinia honey.

Flowers are complex structures devoted to pollinator attraction, through visual as well as chemical signals. As bees collect nectar on flowers to produce honey, some aspects of floral chemistry are transferred to honey, making chemical markers an important technique to identify the botanical and geographical origins of honey. We applied a new approach that considers the simultaneous analysis of different floral parts (petals, stamens + pistils, calyxes + nectarines, and nectar) and the corresponding unifloral honey. We collected fresh flowers of Robinia pseudoacacia L. (black locust), selected five samples of Robinia honey from different geographical origins, applied SPME-GC/MS for volatile analyses, and defined the chemical contribution added by different floral parts to the honey final bouquet. Our results show that honey blends products from nectar as well as other flower parts. Comparing honey and flower profiles, we detected compounds coming directly from flower parts but not present in the nectar, such as hotrienol and ß-pinene. These may turn out to be of special interest when selecting floral markers for the botanical origin of honey.

Effects of altered gravity on growth and morphology in Wolffia globosa implications for bioregenerative life support systems and space-based agriculture.

Understanding the response of plants to varied gravitational conditions is vital for developing effective food production in space bioregenerative life support systems. This study examines the impact of altered gravity conditions on the growth and morphological responses of Wolffia globosa (commonly known as "water lentils" or "duckweed"), assessing its potential as a space crop. Although an experiment testing the effect of simulated microgravity on Wolffia globosa has been previously conducted, for the first time, we investigated the effect of multiple gravity levels on the growth and morphological traits of Wolffia globosa plants. The plant responses to simulated microgravity, simulated partial gravity (Moon), and hypergravity environments were evaluated using random positioning machines and the large-diameter centrifuge. As hypothesized, we observed a slight reaction to different gravitational levels in the growth and morphological traits of Wolffia globosa. The relative growth rates (RGR) of plants subjected to simulated microgravity and partial gravity were reduced when compared to those in other gravity levels. The morphological analysis revealed differences in plant dimensions and frond length-to-width ratios under diverse gravity conditions. Our findings showed that Wolffia globosa is responsive to gravitational changes, with its growth and morphological adaptations being slightly influenced by varying gravitational environments. As for other crop species, growth was reduced by the microgravity conditions; however, RGR remained substantial at 0.33 a day. In conclusion, this study underscores the potential of Wolffia globosa as a space crop and its adaptability to diverse gravitational conditions, contributing to the development of sustainable food production and bioregenerative life support systems for future space exploration missions.

Stochastic changes affect Solanum wild species following autopolyploidization.

Polyploidy is very common within angiosperms, and several studies are in progress to ascertain the effects of early polyploidization at the molecular, physiological, and phenotypic level. Extensive studies are available only in synthetic allopolyploids. By contrast, less is known about the consequences of autopolyploidization. The current study aimed to assess the occurrence and extent of genetic, epigenetic, and anatomical changes occurring after oryzaline-induced polyploidization of Solanum commersonii Dunal and Solanum bulbocastanum Dunal, two diploid (2n=2×=24) potato species widely used in breeding programmes. Microsatellite analysis showed no polymorphisms between synthetic tetraploids and diploid progenitors. By contrast, analysis of DNA methylation levels indicated that subtle alterations at CG and CHG sites were present in tetraploids of both species. However, no change occurred concurrently in all tetraploids analysed with respect to their diploid parent, revealing a stochastic trend in the changes observed. The morpho-anatomical consequences of polyploidization were studied in leaf main veins and stomata. With only a few exceptions, analyses showed no clear superiority of tetraploids in terms of leaf thickness and area, vessel number, lumen size and vessel wall thickness, stomata pore length and width, guard cell width, and stomatal density compared with their diploid progenitors. These results are consistent with the hypothesis that there are no traits systematically associated with autopolyploidy.

Maize root behavior as three-inputs-three-outputs logical gates due to positive gravitropism and nutritropism.

Root growth and their interactions can provide valuable information for the development of asynchronous logic systems. Here, maize root behavior due to positive gravitropism and nutritropism is evaluated as three-inputs-three-outputs logical gates. Using plant roots as the element for unconventional computing, the Boolean functions of each root tropism were constructed through arithmetic-logical operations. One gravity gate (rGG) and two nutrient gates (rNG1 and rNG2) were fabricated using additive manufacturing. The rGG platform was oriented with roots directly pulled down by gravity which computes (x, y, z) = (xz + yz, x + y¯z+yz¯, xy + yz), whereas specific output channels in rNG1 and rNG2 were fertigated with high phosphorus concentration resulting in (x, y, z) = (x + y + z, xy + xz, xyz) for rNG1 and (x, y, z) = (xyz, xy¯z+xyz¯, x + y + z) for rNG2. For rGG, rNG1, and rNG2, the symbols x, y, and z pertain to "root presence" in the related channel, whereas top bar on the symbols indicates "root absence". Anatomical traits of roots were evaluated to assess possible differences in vascular tissues due to gravitropic and nutritropic responses. Overall, maize primary roots showed prominent positive gravitropism and nutritropism, and the roots that were most attracted by gravitational or nutritional stimuli showed an increase in the diameter of phloem and xylem. The logic exhibited by roots was dependent on the gravitropic and nutritropic stimuli to which they were exposed in the different logic gates. The responsiveness of maize roots to environmental stimuli such as gravity and nutrients provided valuable information to be used in computational bioelectronics.

Perspectives for plant biology in space and analogue environments.

Advancements in plant space biology are required for the realization of human space exploration missions, where the re-supply of resources from Earth is not feasible. Until a few decades ago, space life science was focused on the impact of the space environment on the human body. More recently, the interest in plant space biology has increased because plants are key organisms in Bioregenerative Life Support Systems (BLSS) for the regeneration of resources and fresh food production. Moreover, plants play an important role in psychological support for astronauts. The definition of cultivation requirements for the design, realization, and successful operation of BLSS must consider the effects of space factors on plants. Altered gravitational fields and radiation exposure are the main space factors inducing changes in gene expression, cell proliferation and differentiation, signalling and physiological processes with possible consequences on tissue organization and organogenesis, thus on the whole plant functioning. Interestingly, the changes at the cellular and molecular levels do not always result in organismic or developmental changes. This apparent paradox is a current research challenge. In this paper, the main findings of gravity- and radiation-related research on higher plants are summarized, highlighting the knowledge gaps that are still necessary to fill. Existing experimental facilities to simulate the effect of space factors, as well as requirements for future facilities for possible experiments to achieve fundamental biology goals are considered. Finally, the need for making synergies among disciplines and for establishing global standard operating procedures for analyses and data collection in space experiments is highlighted.

Applying productivity and phytonutrient profile criteria in modelling species selection of microgreens as Space crops for astronaut consumption.

Introduction: Long-duration missions in outer Space will require technologies to regenerate environmental resources such as air and water and to produce food while recycling consumables and waste. Plants are considered the most promising biological regenerators to accomplish these functions, due to their complementary relationship with humans. Plant cultivation for Space starts with small plant growth units to produce fresh food to supplement stowed food for astronauts' onboard spacecrafts and orbital platforms. The choice of crops must be based on limiting factors such as time, energy, and volume. Consequently, small, fast-growing crops are needed to grow in microgravity and to provide astronauts with fresh food rich in functional compounds. Microgreens are functional food crops recently valued for their color and flavor enhancing properties, their rich phytonutrient content and short production cycle. Candidate species of microgreens to be harvested and eaten fresh by crew members, belong to the families Brassicaceae, Asteraceae, Chenopodiaceae, Lamiaceae, Apiaceae, Amarillydaceae, Amaranthaceae, and Cucurbitaceae. Methods: In this study we developed and applied an algorithm to objectively compare numerous genotypes of microgreens intending to select those with the best productivity and phytonutrient profile for cultivation in Space. The selection process consisted of two subsequent phases. The first selection was based on literature data including 39 genotypes and 25 parameters related to growth, phytonutrients (e.g., tocopherol, phylloquinone, ascorbic acid, polyphenols, lutein, carotenoids, violaxanthin), and mineral elements. Parameters were implemented in a mathematical model with prioritization criteria to generate a ranking list of microgreens. The second phase was based on germination and cultivation tests specifically designed for this study and performed on the six top species resulting from the first ranking list. For the second selection, experimental data on phytonutrients were expressed as metabolite production per day per square meter. Results and discussion: In the final ranking list radish and savoy cabbage resulted with the highest scores based on their productivity and phytonutrient profile. Overall, the algorithm with prioritization criteria allowed us to objectively compare candidate species and obtain a ranking list based on the combination of numerous parameters measured in the different species. This method can be also adapted to new species, parameters, or re-prioritizing the parameters for specific selection purposes.

Plant and microbial science and technology as cornerstones to Bioregenerative Life Support Systems in space.

Long-term human space exploration missions require environmental control and closed Life Support Systems (LSS) capable of producing and recycling resources, thus fulfilling all the essential metabolic needs for human survival in harsh space environments, both during travel and on orbital/planetary stations. This will become increasingly necessary as missions reach farther away from Earth, thereby limiting the technical and economic feasibility of resupplying resources from Earth. Further incorporation of biological elements into state-of-the-art (mostly abiotic) LSS, leading to bioregenerative LSS (BLSS), is needed for additional resource recovery, food production, and waste treatment solutions, and to enable more self-sustainable missions to the Moon and Mars. There is a whole suite of functions crucial to sustain human presence in Low Earth Orbit (LEO) and successful settlement on Moon or Mars such as environmental control, air regeneration, waste management, water supply, food production, cabin/habitat pressurization, radiation protection, energy supply, and means for transportation, communication, and recreation. In this paper, we focus on air, water and food production, and waste management, and address some aspects of radiation protection and recreation. We briefly discuss existing knowledge, highlight open gaps, and propose possible future experiments in the short-, medium-, and long-term to achieve the targets of crewed space exploration also leading to possible benefits on Earth.

Occurrence of morphological and anatomical adaptive traits in young and adult plants of the rare Mediterranean cliff species Primula palinuri Petagna.

Cliffs worldwide are known to be reservoirs of relict biodiversity. Despite the presence of harsh abiotic conditions, large endemic floras live in such environments. Primula palinuri Petagna is a rare endemic plant species, surviving on cliff sites along a few kilometres of the Tyrrhenian coast in southern Italy. This species is declared at risk of extinction due to human impact on the coastal areas in question. Population surveys have shown that most of the plants are old individuals, while seedlings and plants at early stages of development are rare. We followed the growth of P. palinuri plants from seed germination to the adult phase and analysed the morphoanatomical traits of plants at all stages of development. Our results showed that the pressure of cliff environmental factors has been selected for seasonal habitus and structural adaptive traits in this species. The main morphoanatomical modifications are suberized cell layers and accumulation of phenolic compounds in cell structures. These features are strictly related to regulation of water uptake and storage as well as defence from predation. However, we found them well established only in adult plants and not in juvenile individuals. These findings contribute to explain the rare recruitment of the present relict populations, identifying some of the biological traits which result in species vulnerability.

Morphofunctional traits and pollination mechanisms of Coronilla emerus L. flowers (Fabaceae).

It is accepted that the papilionaceous corolla of the Fabaceae evolved under the selective pressure of bee pollinators. Morphology and function of different parts of Coronilla emerus L. flowers were related to their role in the pollination mechanism. The corolla has a vexillum with red nectar lines, a keel hiding stamens and pistil, and two wing petals fasten to the keel with two notched folds. Pollinators land on the complex of keel and wings, trigger the protrusion of pollen and finally of the stigma from the keel tip. Data on pollen viability and stigma receptivity prove that flowers are proterandrous. The results of hand-pollination experiments confirmed that insects are fundamental to set seed. Interaction with pollinators allows not only the transport of pollen but also the rupture of the stigmatic cuticle, necessary to achieve both allogamy and autogamy. Field observations showed that Hymenoptera, Lepidoptera, and Diptera visited the flowers. Only some of the Hymenoptera landed on the flowers from the front and elicited pollination mechanisms. Most of the insects sucked the nectar from the back without any pollen transfer. Finally, morphological and functional characteristics of C. emerus flowers are discussed in terms of floral larceny and reduction in pollination efficiency.

High temperatures during microsporogenesis fatally shorten pollen lifespan.

Many crop species are cultivated to produce seeds and/or fruits and therefore need reproductive success to occur. Previous studies proved that high temperature on mature pollen at anther dehiscence reduce viability and germinability therefore decreasing crop productivity. We hypothesized that high temperature might affect pollen functionality even if the heat treatment is exerted only during the microsporogenesis. Experimental data on Solanum lycopersicum 'Micro-Tom' confirmed our hypothesis. Microsporogenesis successfully occurred at both high (30 °C) and optimal (22 °C) temperature. After the anthesis, viability and germinability of the pollen developed at optimal temperature gradually decreased and the reduction was slightly higher when pollen was incubated at 30 °C. Conversely, temperature effect was eagerly enhanced in pollen developed at high temperature. In this case, a drastic reduction of viability and a drop-off to zero of germinability occurred not only when pollen was incubated at 30 °C but also at 22 °C. Further ontogenetic analyses disclosed that high temperature significantly speeded-up the microsporogenesis and the early microgametogenesis (from vacuolated stage to bi-cellular pollen); therefore, gametophytes result already senescent at flower anthesis. Our work contributes to unravel the effects of heat stress on pollen revealing that high temperature conditions during microsporogenesis prime a fatal shortening of the male gametophyte lifespan.

A Machine-Learning Method to Assess Growth Patterns in Plants of the Family Lemnaceae.

Numerous new technologies have been implemented in image analysis methods that help researchers draw scientific conclusions from biological phenomena. Plants of the family Lemnaceae (duckweeds) are the smallest flowering plants in the world, and biometric measurements of single plants and their growth rate are highly challenging. Although the use of software for digital image analysis has changed the way scientists extract phenomenological data (also for studies on duckweeds), the procedure is often not wholly automated and sometimes relies on the intervention of a human operator. Such a constraint can limit the objectivity of the measurements and generally slows down the time required to produce scientific data. Herein lies the need to implement image analysis software with artificial intelligence that can substitute the human operator. In this paper, we present a new method to study the growth rates of the plants of the Lemnaceae family based on the application of machine-learning procedures to digital image analysis. The method is compared to existing analogical and computer-operated procedures. The results showed that our method drastically reduces the time consumption of the human operator while retaining a high correlation in the growth rates measured with other procedures. As expected, machine-learning methods applied to digital image analysis can overcome the constraints of measuring growth rates of very small plants and might help duckweeds gain worldwide attention thanks to their strong nutritional qualities and biological plasticity.

A novel device to study altered gravity and light interactions in seedling tropisms.

Long-duration space missions will need to rely on the use of plants in bio-regenerative life support systems (BLSSs) because these systems can produce fresh food and oxygen, reduce carbon dioxide levels, recycle metabolic waste, and purify water. In this scenario, the need for new experiments on the effects of altered gravity conditions on plant biological processes is increasing, and significant efforts should be devoted to new ideas aimed at increasing the scientific output and lowering the experimental costs. Here, we report the design of an easy-to-produce and inexpensive device conceived to analyze the effect of interaction between gravity and light on root tropisms. Each unit consisted of a polystyrene multi-slot rack with light-emitting diodes (LEDs), capable of holding Petri dishes and assembled with a particular filter-paper folding. The device was successfully used for the ROOTROPS (for root tropisms) experiment performed in the Large Diameter Centrifuge (LDC) and Random Positioning Machine (RPM) at ESA's European Space Research and Technology centre (ESTEC). During the experiments, four light treatments and six gravity conditions were factorially combined to study their effects on root orientation of Brassica oleracea seedlings. Light treatments (red, blue, and white) and a dark condition were tested under four hypergravity levels (20 g, 15 g, 10 g, 5 g), a 1 g control, and a simulated microgravity (RPM) condition. Results of validation tests showed that after 24 h, the assembled system remained unaltered, no slipping or displacement of seedlings occurred at any hypergravity treatment or on the RPM, and seedlings exhibited robust growth. Overall, the device was effective and reliable in achieving scientific goals, suggesting that it can be used for ground-based research on phototropism-gravitropism interactions. Moreover, the concepts developed can be further expanded for use in future spaceflight experiments with plants.

The World Smallest Plants (Wolffia Sp.) as Potential Species for Bioregenerative Life Support Systems in Space.

To colonise other planets, self-sufficiency of space missions is mandatory. To date, the most promising technology to support long-duration missions is the bioregenerative life support system (BLSS), in which plants as autotrophs play a crucial role in recycling wastes and producing food and oxygen. We reviewed the scientific literature on duckweed (Lemnaceae) and reported available information on plant biological traits, nutritional features, biomass production, and space applications, especially of the genus Wolffia. Results confirmed that the smallest existing higher plants are the best candidate for space BLSS. We discussed needs for further research before criticalities to be addressed to finalise the adoption of Wolffia species for space missions.

Root Tropisms: New Insights Leading the Growth Direction of the Hidden Half.

Tropisms are essential responses of plants, orienting growth according to a wide range of stimuli. Recently, considerable attention has been paid to root tropisms, not only to improve cultivation systems, such as those developed for plant-based life support systems for future space programs, but also to increase the efficiency of root apparatus in water and nutrient uptake in crops on Earth. To date, the Cholodny-Went theory of differential auxin distribution remains the principal tropistic mechanism, but recent findings suggest that it is not generally applicable to all root tropisms, and new molecular pathways are under discussion. Therefore, an in-depth understanding of the mechanisms and functions underlying root tropisms is needed. Contributions to this special issue aimed to embrace reviews and research articles that deepen molecular, physiological, and anatomical processes orchestrating root tropisms from perception of the stimulus to bending. The new insights will help in elucidating plant-environment interactions, providing potential applications to improve plant growth on Earth and in space where microgravity diminishes or nullifies the gravitropism dominance.

Building and degradation of secondary cell walls: are there common patterns of lamellar assembly of cellulose microfibrils and cell wall delamination?

During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments.

Variations of vessel diameter and δ13C in false rings of Arbutus unedo L. reflect different environmental conditions.

Woody species in Mediterranean ecosystems form intra-annual density fluctuations (IADFs) in tree rings in response to changes in environmental conditions, especially water availability. Dendrochronology, quantitative wood anatomy and high-resolution isotopic analysis (using a laser ablation technique) were used to characterize IADFs in Arbutus unedo shrubs grown on two sites with different water availability on the island of Elba (Italy). Our findings show that IADF characterization can provide information about the relationship between environmental factors and tree growth at the seasonal level. At the more xeric site, IADFs mainly located in the early and middle parts of the annual ring, showed a decrease in vessel size and an increase in δ(13) C as a result of drought deficit. Opposite trends were found at the more mesic site, with IADFs located at the end of the ring and associated with a lower δ(13) C. Moreover, at the first site, IADFs are induced by drought deficit, while at the second site IADFs are linked with the regrowth in the last part of the growing season triggered by favourable wet conditions. This combined approach is a promising way for dating problematic wood samples and interpreting the phenomena that trigger the formation of IADFs in the Mediterranean environment.

Subsequent inclusion/exclusion criteria to select the best species for an experiment performed on the ISS in a refurbished hardware.

The interest in re-using flown hardware for new and different space biology experiments is increasing. To match the constraints of the flown hardware with the requirements of the new biological system, innovative methodological approaches are necessary. MULTITROP was a successful plant biology experiment that was performed on the ISS to investigate multiple-tropism interactions during the early stage of seedling growth. We used the hardware designed and flown for the IFOAM experiment in 2009. The main challenge was to implement seeds of a crop species in a growth chamber conceived for yeast culture and to grow the seedlings in microgravity condition but activating seed germination on ground before the launch. Our approach was to adapt the biological system to the hardware constraints and also to the experiment timing and the environmental factors expected during the prelaunch, launch and flight operations. We looked for an objective and repeatable method to effectively select the best suited species. Innovatively, we applied the method of inclusion/exclusion criteria to adapt a new biological system to a reused hardware. The list and the consecutive order of the specific inclusive/exclusive criteria turned out to be a valid support to guide the science team in objectively choosing the most suitable species for the experiment. Among the 50 initial food species, the carrot seeds resulted as the best in satisfying all technical requirements and post-flight data confirmed the expectations.