Abstract
Background:
Long-term space missions will necessarily require producing viable seeds to be used for plant cultivation over time under altered gravity conditions. Pollen is known to play a key role in determining seed and fruit production over seed-to-seed cycles, but very few studies have evaluated pollen functionality under altered gravity.
Methods:
We performed ground-based experiments to test how simulated microgravity can affect the directional growth of pollen tubes as a potential bottleneck in seed and fruit sets. The effect of clinorotation was assessed in the pollen of Solanum lycopersicum L. cv. ‘Micro-Tom’ and Brassica rapa L. var. silvestris, both eligible for cultivation in space. Pollen tube length and tortuosity were compared under 1g and simulated microgravity with a uniaxial clinostat.
Results:
The main results highlighted that simulated microgravity significantly increased pollen tube length and tortuosity compared to 1g conditions. Further, clinorotation prompted a differential effect on pollen germination between S. lycopersicum and B. rapa. A more in-depth analysis evaluating the effect of gravity on the directional growth of pollen tubes excluded gravitropic responses as responsible for the tube tip position reached after germination.
Discussion:
This research provides new insights into how altered gravity can interfere with plant reproduction and, in particular, microgametophyte functionality. Our findings represent a basis for further studies aimed at understanding the effect of real microgravity on plant reproduction and developing countermeasures to ensure seed-to-seed cultivation in long-term space missions and achieve self-sufficiency in food supplies from Earth.
1 Introduction
Future space exploration requires plant-based bioregenerative life support systems (BLSS) to produce edible biomass and oxygen, recycle water, absorb carbon dioxide, and support astronauts’ psychological health (). Indeed, plants are essential components of BLSS, and for decades, many experiments with plants have been performed to test the feasibility of plant cultivation in space (e.g., ; ; ).
Early efforts to cultivate plants for extended periods in microgravity resulted in reduced growth and complications in the transition to the reproductive stage (; ). In 1982, Arabidopsis thaliana became the first plant species to flower in space and produce seeds (). However, only half of the seeds were viable and germinated in developing plants without growth defects (). Environmental constraints such as low ventilation and high ethylene concentrations have been considered the main causes of interrupted or reduced reproductive success in many early experiments in space (). Recently, the advancement of plant growth systems has led to numerous successful seed sets in space and to a seed-to-seed-to-seed cycle of A. thaliana that was achieved entirely on the International Space Station ().
Overall, plant biology research in space indicates that real and simulated microgravity do not prevent plant reproduction, but they can affect the quality of embryos and the development of seedlings (; ; ; ). Various alterations in seeds produced in space include delayed embryonic development, modification of storage reserves, delayed starch use in cotyledons, and decreased cell number in cotyledons (; ; ). Currently, the identification of mechanisms by which space factors can affect plant reproduction is becoming increasingly critical for plant cultivation in future human settlements on the Moon and Mars (). Indeed, the completion of the seed-to-seed cycle in the BLSSs is essential to producing viable seeds to be used for plant cultivation over time independently of Earth’s supply.
Within the plant reproductive cycle, viable and germinable pollen is needed to trigger the double fertilization responsible for seed formation. Indeed, both pollen viability and germination are positively correlated with fruit and seed set (; ). Therefore, possible effects of space factors on successful seed production can be addressed by defects in pollen viability and germinability. Previous experiments showed that most Arabidopsis and Brassica pollen grains produced under microgravity conditions were viable (; ). Nevertheless, this is not enough to fulfill the reproductive cycle. Once formed, viable pollen needs to be transferred to the stigma, posing some challenges in microgravity, especially for self-incompatible species that require cross-pollination to succeed in seed production (). Furthermore, once it arrives on the stigma surface, pollen must germinate and guide its pollen tube across the ovary into the ovule to trigger double fertilization and seed formation. In the alternation between sporophyte (diploid) and gametophyte (haploid) generation in sexual plant reproduction, pollen represents the male gametophyte and is therefore an independent organism with a very short lifespan whose survival is essential to achieve the full completion of the plant reproductive cycle ().
Pollen is very sensitive to environmental factors that could affect pollen tube development. The earliest stage of pollen development was revealed to be the most sensitive to temperature changes, with a drastic reduction of pollen germination caused by high temperatures during microsporogenesis (). Moreover, pollen germination is strongly influenced by relative humidity and its combination with temperature conditions (; ). Indeed, a drastic loss in pollen viability occurs when pollen is exposed to a combination of high humidity and high temperature, whereas this does not happen when pollen is exposed to the same temperatures but lower humidity levels (). Additionally, the possibility of plants growing and reproducing in space will depend on their capability to accomplish both sporophyte and gametophyte generations under altered microgravity conditions. In this scenario, investigations into how simulated microgravity can affect pollen germination and tube growth might provide useful insights into space-related issues in plant reproduction.
Among the instruments used to simulate microgravity, clinostats are meant to reduce the effect of constant gravity acceleration on Earth, and they have been largely used to perform ground-based experiments on the effects of simulated microgravity on plant growth and development (; ). Even if clinostats do not reproduce weightlessness, ground-based research in simulated microgravity provides useful understandings to predict plant responses in real microgravity (). Although plants are known to be extremely sensitive to gravity (; ), gravitropism in pollen tubes has not yet been assessed. To date, the microgametophyte (pollen) tube path towards the ovary is known to be guided exclusively by chemotropic stimuli due to specialized pistil tissues (), whereas the possible co-existence with pollen gravitropism has been generally overlooked.
To date, a few studies have shown that simulated microgravity affects several aspects of pollen tube development, such as alterations in sperm cell migration and callose plug formation (; ), but gravitropism in pollen tubes has not yet been assessed. Previous studies were performed under in vitro conditions where the absence of a Ca2+ gradient allowed isolation of the effect of simulated microgravity. Thus, to test the hypothesis that chemotropism always coexists with gravitropism in germinating pollen, in vitro conditions would be ideal to reveal the effect of altered gravity in orientating pollen tubes. In the framework of future space missions with seed-to-seed crop cycles, it is crucial to study how altered gravity could interfere with pollen germination and tube directional growth to achieve fertilization.
Among candidate space crops, Solanum lycopersicum L. cv. ‘Micro-Tom’ and Brassica rapa L. are two promising crops for cultivation on board spacecrafts, mainly for their compact size and short life cycle (; ; ; ). ‘Micro-Tom’ is a macro-thermal fruity crop that has already achieved a seed-to-seed cycle under simulated microgravity, even though fruit setting was almost halved under clinorotation (). B. rapa is a micro-thermal leafy crop that has already been grown from seed to seed under microgravity conditions on the Mir space station in 1997; however, seeds and siliques formed from the second generation of plants on board resulted in compromise (). Brassica spp. are close relatives of A. thaliana, and there is substantial homology between their nuclear genomes (). Particularly, the pollen of B. rapa L. var. sylvestris has already been investigated for aberrant callose plug deposition during tube development under simulated microgravity ().
Research on possible gravitropic responses of pollen is needed to have a better understanding of how altered gravity can limit seed and fruit production over repeated seed-to-seed cycles in candidate space crops. In this context, our study is the first to perform in vitro pollen germination on a clinostat to isolate the effect of simulated microgravity and unravel the possible gravitropism of the pollen tube. Specifically, our aim was to compare the effect of simulated microgravity and Earth gravity on pollen tube orientation in S. lycopersicum L. cv. ‘Micro-Tom’ and Brassica rapa L var. sylvestris as potential bottlenecks in regular seed and fruit development for cultivation in space.
2 Material and methods
2.1 Plant material
The effect of simulated microgravity on pollen germination and tube orientation was assessed in S. lycopersicum L. cv. ‘Micro-Tom’ and B. rapa L var. sylvestris, both recognized as candidate space crops. ‘Micro-Tom’ and Brassica plants were grown from seeds to flower in two different growth chambers. In ‘Micro-Tom’ plants, a photosynthetic photon flux density (PPFD) of 400 ± 15 μmol m−2 s−1 was provided by white LEDs in a 16 h d−1 photoperiod. Relative humidity was kept at 70 ± 5% and the air temperature was set at 22 ± 0.5°C both reported as the optimum for tomatoes (; ). As for Brassica plants, the PPFD from white LEDs was 250 ± 10 μmol m−2 s−1 in a 12 h d−1 photoperiod. Relative humidity and air temperature were kept at 70 ± 5% and 18 ± 0.5°C, respectively. PPFD was measured during each treatment using a spectroradiometer (SS-110; Apogee Instruments Inc., Logan, UT).
2.2 Pollen sampling and viability measurement
Pollen was collected from flowers sampled shortly before anthesis to prevent possible pollen damage once exposed to the external environment. More specifically, undehisced anthers were detached from 200 flower buds per species, distributed in two different Petri dishes, and left to dehisce in a closed container filled with dry silica gel. This method sped up pollen release from anthers and allowed for the production of one bulk sample of pollen per species within a few hours of flower sampling ().
Before clinorotation, the pollen viability of the bulk samples was assessed through the diaminobenzidine (DAB) reaction () to ensure that the pollen used for the experiment was of high quality. We evaluated pollen viability on five pollen samples per species taken from the respective bulk. Each pollen sample was spread into a 10 μl droplet of water previously placed on a slide. One droplet of 10 μl of DAB reagent was added to each pollen sample, and slides were gently warmed on a heating plate and mounted. Pollen grains were scored as viable when stained dark/brown and as not viable when they remained faint/colorless. Each slide mounting a single pollen sample was photographed under a light microscope, and pollen viability was scored through image analyses on six photos per slide. The viability percentage was measured by counting at least 100 pollen grains per photo on a total of 30 photos per species (two species × five slides × six photos).
2.3 Pollen germination and gravity treatments
To test the effect of simulated microgravity on pollen germination, Petri dishes with germinating pollen were split between a control (1g) and a clinostat treatment (cl). In the control treatment, pollen germinated on agar germination medium inside Petri dishes vertically positioned according to the 1g gravity vector. Simulated microgravity treatment was performed on a uniaxial clinostat consisting of two metallic supports and two rotating dishes joined by a rigid shaft. Petri dishes with germinating pollen were fixed on the rotating shaft at a specific distance from the rotating axis to obtain ineffective centrifuge acceleration on pollen, according to a spin velocity of the rigid shaft set at two revolutions per minute. In vitro pollen germination was tested using two different agar media optimized for ‘Micro-Tom’ and B. rapa (; ). In more detail, pollen from the bulk samples was placed in 10 Petri dishes per species lined with an agar medium made of 0.9% agar and 15% sucrose (w/v) for ‘Micro-Tom’ pollen and 0.9% agar and 20% sucrose (w/v) for B. rapa.
2.4 Microscopy and image analysis of germinating pollen
The effect of clinorotation on pollen germination and tube development in agar media was assessed through image analyses comparing pollen germinability, tube length, and tortuosity index between control (1g) and clinostat (simulated microgravity) treatments. Image analysis of germinating pollen was performed with ImageJ v1.53 software on photos taken with a digital camera (EOS 60D, Canon) mounted on a light microscope (Olympus® BX-60). Each photo was taken keeping track of the Petri dish orientation with respect to the direction of the gravity vector. Germinating pollen was photographed after 24 and 48 h, and germination scoring was performed through image analyses on 400 photos (two species × two gravity treatments × five Petri dishes × two germination times × 10 photos). Germination percentages were assessed by considering as germinated grains those with a pollen tube longer than the grain diameter. Pollen tube length (L) was assessed on 600 pollen tubes and scored on 200 photos tracing the whole length of the pollen tube from the exine apertures to the pollen tube tip. On the same photos, the pollen tortuosity index was measured to estimate bending/directionality throughout the pollen tube path (Figure 1). The tortuosity index (T) was calculated using the following equation:
Figure 1
where L (length) is the total length of the pollen tube and C (chord) is the distance between the pollen exine aperture and the pollen tube tip. The reported tortuosity index can vary from 1 (L = C) to +∞ (L > C). More specifically, tortuosity values close to 1 indicate that the pollen tube developed straight from the exine to the tube tip, whereas increasing tortuosity values indicate an increase in direction changes during elongation of the pollen tube.
Furthermore, to detect a possible gravitropic response of pollen tubes, we investigated the direction of tube elongation in the control and clinostat treatments. For this purpose, we divided the images of each germinated pollen grain into four quadrants with respect to the direction of the gravity vector, scored the direction of 300 pollen tubes inside the agar medium, and calculated the percentage of pollen tube tips reaching each quadrant (Figure 2).
Figure 2
We hypothesized that the expected frequencies of pollen tube tip location would be equally distributed across the Petri dish containing the germination medium (25% in each quadrant). In the case of P <0.05, the observed frequencies would differ from those expected, therefore showing a preferential growth direction of pollen tube tips. Specifically, in the control treatment, a positive or negative gravitropic response would result if pollen tube tips were preferentially located in quadrants I or III, respectively.
2.5 Data analysis
Data were analyzed with IBM® SPSS Statistics. Shapiro–Wilk’s test and Levene’s test were respectively used to assess the normality and homogeneity of the variances of the datasets. Differences in pollen germination, tube length, and tortuosity between ground and clinostat treatments were tested using ANOVA (P <0.05) and the Brown–Forsythe robust test for equality of means. The observed and expected frequencies of pollen tube tip location in the different quadrants under control and simulated microgravity treatments were compared through a Chi-square test.
3 Results
The DAB test for pollen viability showed that pollen was well formed and functional in both species. Moreover, the viability of all pollen samples taken from the bulk of each species was high and uniform, ranging between 87%–91% in ‘Micro-Tom’ and 86%–94% in B. rapa, and no significant differences were detected among pollen samples intra-species (Figure 3).
Figure 3
Data on pollen germination showed clear differences between control and clinostat treatments (Figure 4). More specifically, the two-way ANOVA showed a significant (P <0.05) effect of germination time, gravity, and their interaction. The two species reacted differently to gravity since simulated microgravity reduced pollen germination in ‘Micro-Tom’ but increased pollen germination in B. rapa compared to the control condition. However, in both species, simulated microgravity significantly increased pollen germination when prolonging pollen incubation from 24 to 48 h, whereas no differences in pollen germination were found between 24 and 48 h of incubation under the 1g condition.
Figure 4
Thereafter, the pairwise comparison of pollen tube length between control and clinostat treatments highlighted a similar response in ‘Micro-Tom’ and B. rapa. Despite the great difference in tube length, pollen of both species developed longer tubes in simulated microgravity than in control conditions (Figure 5). Considering pollen tube length and pollen germination data, results highlighted that simulated microgravity promoted pollen tube length in both species despite reducing the germination rate in B. rapa.
Figure 5
As far as pollen tube path in germinated grains, a first visual overview of pollen tubes showed a clear difference between control and clinostat treatments, especially in ‘Micro-Tom’ (Figure 6). More in detail, control pollen developed straighter tubes compared to the simulated microgravity condition, in which tube tips changed their direction more frequently throughout the pollen tube path. The straightness/curviness of pollen tube paths was compared between control and clinostat by measuring the tortuosity index in 300 pollen tubes per species. Results showed that simulated microgravity significantly increased pollen tube tortuosity compared to control. Moreover, ANOVA reported that the difference in tortuosity between control and simulated microgravity was more significant in ‘Micro-Tom’ (P <0.001, Figure 7A) than in B. rapa (P <0.05, Figure 7B), which is also highlighted in the box plots showing the distribution of tortuosity values in the two gravity treatments (Figure 7). Indeed, in ‘Micro-Tom,’ most of the values of the tortuosity index are distributed consistently around the median in the control treatment, whereas a much wider distribution with increasing tortuosity values results in the clinostat treatment. Differently, the tortuosity index of B. rapa pollen tubes were similarly distributed around the median both in control and in simulated microgravity, but still had significantly higher values in the clinostat treatment.
Figure 6
Figure 7
Finally, the evaluation of possible gravitropic responses in pollen tubes showed no preferential distribution of pollen tube tips across the Petri dishes, both in control and simulated microgravity treatments (Figure 8). Specifically, we analyzed data assuming no gravitropic response of pollen tubes of ‘Micro-Tom’ and B. rapa, therefore hypothesizing an equal distribution (25% in each quadrant) of tube tips across the Petri dish in both control and simulated microgravity. The Chi-square test revealed no significant differences between observed and expected frequencies of pollen tube tips in the four quadrants, independently of the gravity treatments and the species tested (Table S1, P <0.05). Therefore, we confirmed the null hypothesis that the orientation of pollen tubes of ‘Micro-Tom’ and B. rapa was not subjected to gravitropic responses.
Figure 8
4 Discussion
Results on initial pollen viability revealed that growing conditions were optimally set to avoid any interference of abiotic factors with reproductive features. Indeed, pollen samples of both species reported viability over 80%, and no significant differences intraspecies between pollen samples proved high uniformity in pollen grains taken from the same bulk (Figure 3). Therefore, the two bulk pollen samples (one bulk per species) represented a proper starting point to perform further analyses and compare results intra- and inter-species concerning further pollen traits under simulated microgravity conditions.
The viability of pollen grains is not sufficient to predict pollen’s capability to elongate a pollen tube and achieve fertilization (; ). Our results also demonstrated that pollen with high viability can germinate under simulated microgravity with a significantly different ratio according to the species tested (Figure 4). Considering this, pollen germination rate may be applied as an inclusion criterion in ranking space crops for seed-to-seed cultivation in space (). Both pollen viability and germination are strongly correlated with fruit and seed set (; ) and are therefore essential to ensuring repeated seed-to-seed cycles on board a spacecraft.
Since most plant breeding research has been conducted through the sporophyte, the influence of the gametophytic phase, including pollen tube development, has been generally overlooked. However, the possibility of applying gametophytic selection in plant breeding programs on Earth had already been supported by , who highlighted the overlap in the genetic expression between the sporophyte and the gametophyte in relation to different abiotic stresses (; ; ). Therefore, the selection of space candidate species and cultivars with the highest germination rate of pollen developed under altered gravity conditions can be considered a potential application of gametophytic selection in a space environment. Since it can be performed in a small environment and over a short period, this breeding procedure could be conducted by astronauts with the aim of screening and selecting species and cultivars that best adapt to real microgravity ().
To date, research has shown that simulated microgravity affects several aspects of pollen tube development (; ), but how the gravity vector could affect pollen tube movements is still unknown. Despite this study proving a clear effect of simulated microgravity on the tortuosity of pollen tube movements (Figure 6), our results excluded gravitropism as a pollen tube response affecting the final position of tube tips in the germination medium (Figure 8).
How clinorotation affect the tortuosity of pollen tubes might be addressed by the key role of the cytoskeleton in coordinating vesicle trafficking responsible for pollen tube elongation (). More specifically, clinorotation may have altered the production of metabolites related to pollen tube development, such as starch and callose, involved in the same ultrastructural changes occurring in the actin cytoskeleton of pollen tubes during the self-incompatibility reactions (; ). Indeed, the actin filaments located in the sub-apical portion of elongating pollen tubes are reported to be involved in the vesicle trafficking responsible for the formation and maintenance of the tube apex (; ; ; ). Actin filaments of the cytoskeleton have a crucial role in tube development and growth direction, but how altered gravity can regulate the directionality of pollen tubes remains to be determined ().
To date, the microgametophyte (pollen) tube path towards the ovary is known to be mainly driven by chemotropic stimuli related to the Ca2+ gradient throughout the style (; ), whereas the possible co-existence with pollen gravitropism has been generally overlooked. Despite in vitro conditions being assumed to mimic the natural state of pollen during pollen tube elongation, functional differences with in vivo or in semi-vivo conditions might occur. For instance, pollen tubes elongate faster, tubes live longer, and sperm cells (in bicellular species) form sooner and more reliably in vivo than in vitro (; ). Pollen tube growth in vivo involves interactions with pistil tissues, especially at critical stages such as tube reorientation and the cessation of growth. Thus, considering the role of chemotropism in the directional growth of pollen tubes, we performed the experiment in vitro to isolate the effect of the gravity vector on pollen tube directionality and exclude interferences related to chemotropic stimuli occurring in vivo.
Overall, our analyses investigating male gametophyte functionality in simulated microgravity suggested that pollen germination and tube directional growth are affected by changes in gravity and highlighted different responses between ‘Micro-Tom’ and B. rapa. To date, research has never considered gravitropism as a driver for pollen tube movement. Our in vitro germination tests represented the best first approach to exclude possible chemotropic interferences and isolate the effect of the altered gravity vector on the directional growth of pollen tubes.
Even though our results excluded gravitropism in pollen tubes, further tests are necessary to confirm our results in real microgravity conditions and in other candidate space crops. In conclusion, a better understanding of how gravity can interfere with plant reproduction is needed to ensure seed and fruit production over repeated seed-to-seed-to-seed cycles, and it is a first step towards self-sufficiency from Earth supplies in long-term space missions.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Author contributions
MI: Conceptualization, methodology, investigation, visualization, and writing—original draft preparation. LI: Investigation, visualization, and writing—review & editing. LR: Writing—review & editing. GA: Conceptualization, methodology, writing—review & editing, and funding acquisition. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.
Funding
This research has been financially supported by the project In-situ REsource Bio-Utilization for life Support system (ReBUS), unique project code (CUP) F74I16000000005 financed by the Italian Space Agency.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1186967/full#supplementary-material
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Summary
Keywords
simulated microgravity, pollen tube, pollen germination, clinostat, space crops, Micro-Tom, Brassica rapa, seed-to-seed
Citation
Iovane M, Izzo LG, Romano LE and Aronne G (2023) Simulated microgravity affects directional growth of pollen tubes in candidate space crops. Front. Plant Sci. 14:1186967. doi: 10.3389/fpls.2023.1186967
Received
15 March 2023
Accepted
09 May 2023
Published
02 June 2023
Volume
14 - 2023
Edited by
Maurice Bosch, Aberystwyth University, United Kingdom
Reviewed by
Ioannis-Dimosthenis S. Adamakis, National and Kapodistrian University of Athens, Greece; Sarah E. Wyatt, Ohio University, United States
Updates
Copyright
© 2023 Iovane, Izzo, Romano and Aronne.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Maurizio Iovane, maurizio.iovane@unina.it; Luigi Gennaro Izzo, luigigennaro.izzo@unina.it
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