The root meristem is an interesting cellular model system. Meristematic tissues, which are exclusive of plants, constitute a permanent reserve of undifferentiated totipotent cells, which serve as the source of differentiated cells for plant development, as well as for the plant response to environmental stimuli.
The function of meristematic cells is a continuous cycle of growth and division, which is called the cell cycle. Mitotic cell division gives rise to the formation of two daughter cells, which grow through interphase in the two gap phases (G1 and G2), which are separated by S phase characterized by DNA replication. All the process is carefully regulated, specifically at two checkpoints occurring in the G1/S and G2/M transitions, respectively. Meristematic cell function stands on a strict coordination between the rates of cell proliferation and cell growth, which is called “meristematic competence”.

The Cell Cycle
The Cell Cycle and its checkpoints


A correlation has been described between the cell cycle periods, the rate of ribosome synthesis and the structure and morphometrical features of the nucleolus (Sáez-Vásquez and Medina 2008). Regarding cell cycle progression, expressed as the rate of cell proliferation, abiotic stresses, such as thermal shock (heat or cold), drought or saline stress, cause alterations in its regulation, eventually leading to the arrest of the cell cycle.

What happens to root meristematic cells in microgravity?

On October 18, 2003 a Russian Soyuz TMA-3 capsule was launched to the ISS from the Baikonur cosmodrome in Kazakhstan. On board the spaceship, the Spanish ESA astronaut Pedro Duque was about to start the “Cervantes Mission” (Spanish Soyuz Mission) during a stay of 10 days in the ISS, by developing a program of 25 European experiments in physical and biological sciences, Earth observation, education and technology. From these experiments, seven were designed by Spanish scientists, among which the “Root” experiment, the first European experiment on Plant Biology carried out in the ISS. In this experiment, seeds of the plant model species Arabidopsis thaliana were germinated in orbit, seedlings grew for 4 days and they were finally preserved by chemical fixation for their further analysis in the lab by microscopical methods (light and electron microscopy).

"It will be a while before astronauts are self-sufficient, using seeds actually grown in space",

declared Dr. F.J. Medina to ESA at the time of the Cervantes Mission.

Pedro Duque
Pedro Duque and the NASA astronaut Michael Foale in the ISS during the "Cervantes" Mission (2003)

The analysis paid special attention to the root meristematic cells, by examining parameters reporting the status of meristematic competence. When samples grown in space were compared with control samples grown on ground, a striking increase of the cell proliferation rate in the root meristems of space samples, accompanied by a decrease of the rate of cell growth, expressed as production of pre-ribosomal precursors in the nucleolus, was found (Matía et al. 2010). 
The space experiment was followed by complementary assays performed in facilities for microgravity simulation on ground, namely the clinostat, the random positioning machine (RPM) and the magnetic levitation, as well as in hypergravity. In these experiments, different times of incubation and different conditions of illumination were tested and different genotypes, corresponding to either gene reporter lines or mutants affecting genes of interest, were used. The uncoupling of cell proliferation and ribosome biogenesis induced by microgravity was confirmed and it was shown that this environmental condition causes a noticeable stress, specifically for plant proliferating cells (Herranz and Medina 2014).
The reason for this malfunction should be found in alterations in the cell cycle regulation. The use of in vitro cultured cells synchronized in their progression through the cell cycle, grown under microgravity simulation, showed an acceleration of the cell cycle, which was the result of a shortened G2 phase and a slightly longer G1 phase. Relevant genes playing regulatory roles in cell cycle checkpoints were found altered in their expression. At the same time, the rate of ribosome biogenesis was lower under simulated microgravity, as shown by the levels of marker proteins, the expression of relevant genes and the structure of the nucleolus (Kamal et al. 2019). 
Meristematic competence is physiologically connected with gravity sensing by means of the phytohormone auxin, which is a major driver of the meristematic activity by regulating cell growth and cell proliferation. Gravity sensing at the root tip is transduced to the entire root by affecting the lateral balance of auxin in the root. The different distribution of auxin in the external layers of cells cause skews and bends in the root whose consequence is the loss of orientation and the alteration of gravitropism. However, this role of auxin does not explain the effects found in cell cultures, in which neither “professional cells” specialized in gravity sensing, nor the polar transport of auxin through the root are present. The existence of gravity sensors at the cellular level, probably located at the cell wall, as well as of intracellular mechanisms of signal transduction, has been postulated (Herranz and Medina 2014). 

In search for countermeasures. The role of light

The adaptation and survival of plants in space could greatly benefit from the substitution of gravity by another external cue, which could play the same or a similar role in driving plant growth and development as gravity does on Earth. Light is a good candidate to be one of such these cues.  
Light is indeed a tropic stimulus. Phototropism complements gravitropism under normal ground conditions with the objective of optimizing the efficiency of the capture of nutrients. In addition, illumination, especially by red light, is sensed and mediated by phytochromes to produce changes in the regulation of auxin responsive genes and many growth coordinators (Vandenbrink et al. 2014). The culture of seedlings in simulated microgravity under a photoperiod regime, was capable of reverting many of the alterations found on etiolated seedlings incubated in the same facility for microgravity simulation (Manzano et al. 2020).
We have attempted to know to what extent light can act as a signal capable of counteracting the effects caused by the lack of gravity. For this purpose, the series of experiments termed “The Seedling Growth Project” was conducted in the ISS. The global objectives of this project were to understand how gravity and light responses influence each other, to discriminate the cellular signaling mechanisms involved in plant tropisms and to determine the combined influence of light and gravity on plant development by paying special attention to the effect of these cues on the root meristem.
The Seedling Growth Project was carried out in the ISS in three experiments performed respectively in 2013, 2014 and 2017. The project was the result of the cooperation of NASA and ESA, using a European incubator combined with an American culture chamber for seed germination and growth of seedlings, and a Spanish novel device for preservation of samples (Manzano et al. 2020). Different collections of mutants of Arabidopsis thaliana, affecting phytochromes, nucleolar proteins and auxin responsive genes, were used. Seeds germinated in flight and grew for six days under different regimes of illumination and gravity. In addition to microgravity, seedlings were subjected to different levels of gravity between 0g and 1g, including the Moon and Mars gravity levels, which were produced by a centrifuge installed in the incubator. 

Seedling Growth in ISS
The “Seedling Growth” experiment, performed in the International Space Station. A) An astronaut operating the culture chambers in which seedlings have grown, in order for their processing and preservation to make possible the post-flight analysis upon their return to Earth. B) Seedlings grown in space. µg: Microgravity. 1g: Control ground gravity, obtained in space by means of a centrifuge. Note the great differences in orientation of seedlings. 


The experiments of this project have identified new phototropic responses to blue light in space, which complement previous findings (Vandenbrink et al. 2016). A global transcriptomic study provided a very clear differential transcriptional response to each gravity level from microgravity to 1g. In the case of the microgravity exposed plants, functions associated with light sensing and response, such as photosynthesis and related factors, appeared downregulated with respect to 1g controls, suggesting that the growth is not following the phototropic environmental cue in the absence of the gravitropic one (Vandenbrink et al. 2019). A similar analysis performed under different levels of gravity, including those corresponding to the Moon and Mars, showed that the effects induced by microgravity were gradually removed by increasing g-load, and that different functions appeared affected at different g-levels. (Herranz et al. 2019). Furthermore, a positive effect of red light in counteracting the stress caused by microgravity on cell growth and proliferation in the root meristem has been found (Valbuena et al. 2018). 
All these results will represent a substantial advance in our knowledge on the procedures to succeed in the culture of plants as part of the Life Support Systems for space exploration.