The Arabidopsis thaliana seedlings of three different genetic backgrounds (Col-0, nuc1 and nuc2) grown under simulated microgravity (Sim µg, RPM) with a photoperiod regime showed oriented hypocotyls and roots, the same as in 1 g control samples. In other words, since hypocotyls presented positive phototropism and the roots negative phototropism, and the phototropic axis coincided with the gravitropic axis, phototropism by itself was capable of driving the seedling growth in the absence of a gravitropic signal. However, plants in the absence of any tropistic signal to guide their growth, subjected to simulated microgravity and darkness, showed disoriented hypocotyls and roots, confirming the validation of the simulation system.
Several experiments carried out on board the International Space Station (ISS), in real microgravity, have shown that, without the gravitational cue, the position of the light source influences the growth direction of both the hypocotyl and the root. The Arabidopsis thaliana plants grown during APEX01 experiment in ABRS (Advanced Biological Research System) hardware with directional light (as in our experiment) showed hypocotyls and roots with an oriented growth, the same as the corresponding Earth control. In contrast, plants grown in microgravity in the CARA hardware with environmental light, or in darkness, showed hypocotyls and roots with a random growth direction [34, 35].
In addition, different studies performed in space experiments revealed non-random growth directions of seedling organs, when they developed in microgravity. In an experiment performed in the Space Shuttle STS-95 mission, most of rice roots elongated in a constant direction forming a constant angle of about 55° relative to the axis of the caryopsis in the early phase of growth, but later the roots grew in various directions, including away from the agar medium [36]. In the GRAVI-1 experiment carried out in the ISS, lentil roots initially curved strongly away from the cotyledons and then slowly straightened out forming a relatively constant angle. This establishment of growth direction in a stimulus-free environment was termed automorphogenesis [37]. Furthermore, etiolated Arabidopsis seedlings grown in the space shuttle were shown to exhibit a left-handed skewing response [38]. Actually, the deviation of the root growth from the gravity direction, termed root skewing, was repeatedly described [39–41]. It has been demonstrated that it is independent of both the tropic force of gravity and the gravity-induced contact forces between roots and growth media, and, interestingly, it is more intense in the WS ecotype of Arabidopsis thaliana, than in the Col-0 ecotype. However, the molecular mechanisms underlying such growth phenomenon remain unresolved. Various genes and factors have been proposed to regulate skewing. These include a complex network of processes including polar auxin transport and cytoskeletal dynamics [40, 42]. A recent study in the ISS, using two mutants of skewing behavior, affecting different cellular functions, concludes that genes related to skewing could play a prominent role in plant spaceflight adaptation [43].
Interestingly, our results in simulated microgravity and different conditions of light have not revealed any special orientation of roots, but an evident skewing of hypocotyls was revealed in the absence of tropistic cues (darkness and simulated microgravity). This behavior of hypocotyls has not been previously described. In general, hypocotyl skewing has been paid little attention in studies of seedling growth in altered gravity.
The existence of significant differences between the response of the root and the hypocotyl to different conditions of gravity and light is a novel finding of this work. Certainly, it is known a predominant effect of light on tropistic responses in the shoot [1]. Apparently, the presence of a directional light source is sufficient for the shoot to orient its growth towards this source and to develop correctly in microgravity, the same as it does in ground gravity level. Experiments conducted in shoot apical meristem of the Arabidopsis thaliana seedlings have demonstrated that white light is able to rescue the cell cycle arrest in G1/S and G2/M transitions caused by darkness, as well as to promote ribosome biogenesis efficiency [19, 20].
In particular, our study of the ribosome biogenesis using markers of different steps of 45S pre-rRNA transcript processing revealed a different sensitivity of this process to gravity and light in the hypocotyl and in the root. In the aerial part, neither simulated microgravity nor darkness appeared to alter this process in any of the genotypes analyzed (Col-0, nuc1 and nuc2) with respect to the control experiment, in 1 g and photoperiod. However, in root cells, processing of the primary pre-ribosomal transcript was affected under simulated microgravity with photoperiod regime in the wild type and nuc2 mutant, as indicated by the higher accumulation of 5´ETS and 3´ETS ends and 25S rRNA. This suggests an effect on the rate of production of ribosomes. Alterations of ribosome biogenesis induced by both real and simulated microgravity in root meristematic cells and in vitro cell cultures have been reported in previous experiments from our laboratory [25, 44–46]. Different indirect markers of the process have been used in these studies, such as the protein levels and gene expression regulation of nucleolin and fibrillarin, nucleolar proteins known to play a key regulatory role. Furthermore, the size and structure of the nucleolus have been used as faithful markers of the ribosomal biosynthetic activity. Indeed, three structural types of the nucleolus were defined as corresponding to different rates of ribosome biogenesis rate, namely vacuolated (highly active and present mainly in G2 phase of the cell cycle), compact (active and characteristic of the G1 phase) and fibrillar (inactive and present in the GO phase). Under simulated microgravity conditions, the percentage of vacuolated and compact nucleoli decreased significantly, consistently with a lower efficiency of ribosome biogenesis [44, 45]. The results of the present work confirm these alterations and identify specific steps of the mechanism that appear modified due to the environmental change.
The results obtained in the nuc1 mutant are of particular interest. In a previous paper, the effects of simulated microgravity on this mutant line were described by looking at the ultrastructure of the nucleolus, which was shown severely damaged, containing the so-called “nucleolar peri-chromatin-like granules”, which are the structural expression of a bad processing of pre-rRNA [46]. This effect actually represents an intensification, caused by simulated microgravity, of the defective pre-rRNA processing that occurs naturally in this mutant under control 1 g gravity conditions [16]. In the present experiment, the higher accumulation of 25S rRNA indicates an unbalanced processing of 45S primary transcript pre-rRNA, and the lower accumulation of the intermediate 5´ETS and 3´ETS pre-rRNA ends expresses an acceleration of the processing mechanism. Interestingly, despite these severe alterations, the nuc1 mutant is viable, not only when grown under control 1 g gravity conditions, but also when subjected to simulated microgravity.
The status of meristematic competence has been assayed in roots of seedlings (Col-0, nuc1 and nuc2) grown under simulated microgravity and light (photoperiod) by means of the estimation of cellular parameters (cells/mm ratio in meristematic cell layers, cyclin B1 expression in situ and nucleolar area immuno-labelled by fibrillarin). Our results indicate that seedlings maintain the coordination of growth and proliferation in meristematic cells at equivalent levels to the 1 g control. This means that the microgravity stress can be compensated at the cellular level by means of light. In other words, phototropism can counteract the lack of gravitropism, at least to a certain extent. These results are in agreement with a previous experiment carried out in a clinostat [46]. In general, there is increasing evidence that light is an important regulator of stress acclimation processes [47].
However, when comparing the general response to microgravity at the molecular level (Fig. 8), nuc1 shows a differential pattern of expression of marker genes, not only for the genes affecting ribosome biogenesis, but also for genes related to auxin transport and cell cycle regulation. It appears that the alterations inherent to this mutant, that reduce its efficiency in the production of ribosomes with respect to the wild type, also alter its response to gravitational stress in other physiological processes somehow related to ribosome biogenesis and meristematic competence. Therefore, the nuc1 mutant would have a reduced ability to counteract the lack of gravity, while the wild type and nuc2 mutant share a common profile (but gene fold ratios are smaller in the case of nuc2). It should be noticed that NUC2 protein is a stress-related variant of nucleolin [18]. Therefore, the similar, but attenuated response to gravitational stress in this mutant could be interpreted as a sign of resilience to an adverse environmental condition.
In particular, we have found expression changes in marker genes of cell proliferation and auxin transport in the root meristem. Whereas a decrease in the EIR (PIN2) gene expression was recorded in the three plant genotypes (Col-0, nuc1 and nuc2), this expression change was not accompanied by any alteration of the auxin distribution pattern observed in the DII-VENUS line between the samples grown in microgravity and the 1 g control. In two space experiments, namely CARA (with environmental light from Destiny module of ISS) and APEX03-2 (with directional light supplied by the Vegetable Production System hardware), no differences in the root meristem fluorescence pattern of pDR5r::GFP line were observed (Ferl y Paul 2016). Therefore, the expression changes in a particular auxin transport gene (EIR) suggest that the process is sensitive to microgravity, but, in the presence of light, the overall efficiency of the process would not be significantly altered. The functional redundancy between PIN proteins [48–50] could be capable of counteracting the observed alterations of gene expression.
The most severe alteration observed concerned the expression levels of the CYCB1;2 gene, which showed an important decrease in simulated microgravity with respect to ground control (1 g) for WT, nuc1 and nuc2 plants, even under photoperiod. Previous observations reported by our laboratory in real microgravity and in ground-based facilities, have consistently shown a decrease of CYCB1 gene expression. This included histochemical measurements of GUS staining in Arabidopsis thaliana reporter lines (Manzano et al. 2009; Matía et al. 2009), as well as RT-qPCR experiments in a cell culture exposed for 3 h to simulated microgravity. In the latter case, the observed shortening of the G2 phase of cell cycle did not produce any change of cell size between the two experimental conditions [45]. As in the present case, a molecular alteration was not correlated with any change at the cellular level.