Long-term missions in space to the moon and beyond will require efficient plant growth and food production in orbit. Several factors, such as light intensity, exposure to pathogens, and air quality, will vary on crewed missions. However, perhaps the most extreme difference is the lack of gravity. On Earth, gravity regulates the direction of plant growth and is important for nutrient, water, and light acquisition.
Life is well-adapted to the 1 g environment of the Earth. Plants use the gravity vector as a cue to establish their orientation; roots grow in the same direction as the gravity vector, while shoots grow in the opposite direction (Kiss, 2000). Deviation from a 1 g environment will have cascading effects on plant physiology. Circadian rhythms are regular oscillations of an organism's physiology with a period of approximately 24 h. This coordination with the Earth's 24 h rotation is regulated by an endogenous timekeeper, the circadian clock. In the model plant,
Circadian systems function through a series of negative feedback loops that are temperature compensated. The circadian clock functions by modulating signaling pathways, and maintaining a period of about 24 h in the absence of external cues such as light and temperature cycles. However, an organism's circadian period is plastic and can be entrained via external signals. For example, circadian-regulated phenotypes in Arabidopsis persist in constant light (CL) and temperature conditions yet the circadian period decreases with increased light intensity, while reduced sugar has the opposite effect (Somers et al., 1998; Haydon et al., 2013).
The plant core circadian clock consists of a series of negative transcription-translation feedback loops. Components of the core clock include MYB-like transcription factors Circadian-clock Associated 1 (
Three key circadian-controlled pathways that may be relevant to root gravitropism are starch metabolism, starch catabolism, and auxin signaling. At the level of the whole leaf, starch metabolism and catabolism are gated by the circadian clock. Two models have been proposed for the regulation of starch homeostasis by the clock. The first suggests that the clock operates as a timekeeper, calculating the appropriate starch degradation rate based on the available starch at dusk and the time until dawn (Seki et al., 2017). The second model proposes that a starch monitoring signal adjusts the phase of starch degradation activity (Webb and Satake, 2015).
The root gravitropic response has been extensively studied. Critical steps in gravity sensing and signaling are well characterized (for recent reviews, see (Sato et al., 2015; Su et al., 2017; Vandenbrink and Kiss, 2019; Muthert et al., 2019; Telewski, 2006). In plant root, gravity perception primarily occurs in specialized columella cells located in the root cap (Blancaflor et al., 1998). The sedimentation of dense, starch-filled organelles called statoliths located within the columella cells triggers a signaling cascade that leads to root curvature in response to gravity. Starch-deficient mutants, which in turn have smaller statoliths, exhibit reduced sensitivity to gravity, while mutants with larger statoliths are hypersensitive to gravity (Kiss et al., 1989, 1996; Fitzelle and Kiss, 2001; Vitha et al., 2007). Although circadian and diel variation controls leaf starch levels, it is unknown if such rhythmic cycling of starch levels also occurs in the statoliths (Lu et al., 2005).
The plant growth regulator auxin is an important player in the gravitropic response. When a plant is placed on its side horizontally, lateral gradients of auxin develop at the root tip and move to the elongation zone resulting in the downward curvature of the root to enable the plant root to reorient to vertical (Band et al., 2012). The formation of the asymmetric auxin gradient is facilitated by proteins involved in auxin influx (AUX and LAX) and efflux (PIN-formed, PINs) (Friml et al., 2002; Band et al., 2012) reviewed in (Sato et al., 2015).
In addition to mediating the differential growth response, an intriguing recent report (Zhang et al., 2019b) suggests that auxin may also play a role in gravity sensing by regulating starch synthesis within the columella cells. Auxin levels at the root tip were correlated with starch accumulation; furthermore, auxin was implicated in the expression of starch synthesis genes via the TIR1/AFB auxin receptor AXR3/IAA17 auxin signaling pathway.
As early as the 1930s, it was recognized that plant sensitivity to auxin varied according to the time of day (Went and Thimann, 1937). While many transcripts are under circadian control, genes involved in auxin signaling are disproportionately regulated by the clock (Covington and Harmer, 2007). Furthermore, in both diel and constant environmental conditions, transcriptional responses to auxin treatment are more robust at dawn. This differential response culminates in altered growth responses to auxin that depend on the time of treatment. Finally, there is evidence that auxin may feedback into clock outputs, indicating a two-way relationship between auxin and the clock (Covington and Harmer, 2007).
The gating of starch metabolism, starch catabolism, and auxin signaling raises the possibility that the time of the day that a change in the direction or magnitude of the gravity vector occurs could impact the response. Beyond the potential for the circadian influence, in diel conditions, daily changes in light and temperature could also affect the gravitropic response differently throughout the day. The interaction between the timing of the gravitropic stimulus and the response could have important consequences for experimental design and understanding the mechanisms of gravitropic signaling. Therefore, determining how temporal factors such as time of day and the circadian clock influence plant gravity sensing and responses will be necessary for cultivation outside of the typical Earth environment.
The first goal of this work was to characterize the role of the circadian clock on the root gravitropic response. Root bending assays were carried out which involved turning vertically grown seedlings by 90° over a 24-hour time course and recording time-course images.
We analyzed the root phenotype of seedlings that experienced gravistimulation at different times of day in diel and constant environmental conditions and examined genotypes with disrupted circadian clocks.
The second goal of this study was to determine if root growth responses were affected by simulated microgravity. Ground-based microgravity simulators provide cost-effective means to study how microgravity affects plant growth and development. Several simulators are available for use including 2-dimensional (2D) clinostats and Random Positioning Machines (RPM). A 2D clinostat rotates samples around one axis. The RPM is a specialized type of clinostat that rotates samples around two axes and the arms are capable of rotating at different speeds (Kiss et al., 2019). We designed a lighting system that allowed for two separate lighting regimes on the RPM so that direct comparisons can be made between different light conditions on the same RPM run. Our results indicate that root gravitropism is affected by the time of day the experiment is performed and is controlled, at least in part, by the circadian clock.
We selected two time points with significant differences in their root bending angle for further study, ZT12 and ZT22. These time points had different bending angles 24 h post gravistimulation (Tukey posthoc, p-value < 0.05) and showed consistently different responses across multiple replicates (Figure 2A). Although the total root growth over a 24 h period was similar across all time points (Figure S1A), the root growth rate relative to the time of gravistimulation varied for each time point (Figure S1B). To visualize the connection between root growth rate and the rate of bending, we first calculated the progression of the bending angle every 4 h across the 24 h after the plate was rotated horizontally for plants reoriented 90° at ZT12 and ZT22 (Figure 2A).). Plates rotated horizontally at ZT12 appeared to have a slow change in the bending angle until about 12 h post-turn (Figure 2A). In contrast, plates rotated horizontally at ZT22 responded with an increasing bending angle until about 16 h post-turn, but after that there was almost no additional change in the angle. We next examined change in the growth rate throughout the 24 h period post-turn (Figure 2B). The growth rate was measured in 4-hour increments as this is the smallest unit of time where we could consistently detect a change in growth across all 4 h windows in the 24 h day. Plants rotated horizontally at ZT12 were just entering their period of maximal growth, whereas plants rotated horizontally at ZT22 entered a period of slower root growth right after turning. From the bending angle and the 4 h growth rate we determined the adjusted bending angle by calculating the change in the root angle/growth rate (Figure 2C). The rate of bending/growth for plants rotated horizontally at ZT12 and ZT22 was different 8 h and 24 h post-turn (Figure 2C). The early growth rate immediately after turning is steady at ZT22. When the growth rate increases about 16 h later, there is limited reorientation during this period of rapid growth. The opposite is true for ZT12 (Figure S1A). Immediately after turning, the growth of plants reoriented at ZT12 is slow but rapidly increases. The consistent bending rate throughout this period of rapid growth results in a greater overall bending response in ZT12 compared with ZT22 (Figures S1B, 2).
The time of day-dependent variation in the root bending response to gravity could be due to either the effects of light and dark conditions the plants were in at the time of root reorientation or the endogenous circadian clock. To evaluate the potential for circadian control of the root-bending response, we repeated the root bending experiment in clock mutants and after WT plants were shifted to growth in constant environmental conditions. Clock mutants were grown in the same conditions as WT plants above. For constant condition experiments, plants were grown in 12 h light:12 h dark for 5 days. Two days before the reorientation event, the lights were left on at night until the completion of the experiment. Thus, the plants were in CL for 48 h before the reorientation and remained in continuous light after turning. Starting at ZT48, reorientation was performed every 2 h on a set of plates. Each plate of vertically grown Arabidopsis seedlings was turned by 90°. An image was taken 1 h before turning and every 15 min after turning for 24 h. Root angles and growth rate were measured after 24 h. The bending angle was compared between the different ZT time points when reorientation occurred (Figure 3). The difference in reorientation response at ZT12 and 22 observed in 12 h light:12 h dark cycles persisted in constant environmental conditions suggesting that the circadian clock mediates this differential response.
To further evaluate the role of the circadian clock in the gravitropic response, we compared WT plants and plants with an altered circadian clock using a two-dimensional (2D) clinostat (Wang et al., 2016). Root tip positions were compared between pre- and post-clinorotation to capture the differences in root response. The final root positions relative to the position before clinorotation were plotted (Figure 4A, B). Generally, plants grown on a clinostat exhibit random skewed root growth with significant deviation from vertical. We observed different root growth patterns post-clinorotation according to genotype. In 12 h light:12 h dark conditions, differences between genotypes were primarily noted between plants with constitutively high CCA1 expression (CCA1 OX) and all other genotypes (Figure 4A). In most genotypes, individual roots deviated on either side of the vertical axis with an even distribution. CCA1 OX and
When placed on the 2D clinostat for 24 h in CL conditions (Figure 4B), VGI and Lc/L were not significantly different to 12 h light: 12 h dark conditions for WT roots (p-value < 0.05) indicating the deviation from vertical was similar to 12 h light:12 h dark conditions. However. CL conditions significantly affected how the
In fact, when comparing genotypes, CCA1 OX went from growing straighter in 12 h light:12 h dark on the 2D clinostat (Lc/L 0.98) with a larger VGI (0.92) than WT (Lc/L 0.94; VGI 0.82) to being less straight (Lc/L 0.90) with a smaller VGI (0.71).
We observed that differences in root growth between the genotypes could affect the deviation from vertical (Figures 4, 5). Therefore, vertical root growth was measured in all genotypes in both 12 h light:12 h dark and CL conditions after 10 days of growth (Figure 6). CCA1 OX had reduced growth compared to WT in 12 h light: 12 h dark, which could explain the apparent insensitivity of CCA1 OX plants to the altered gravity in 12 h light:12 h dark (Figure 4A and 5). However, root growth of CCA1 OX in CL is showed no further reduction from the12 h light:12 h dark root growth, yet in CL, CCA1 OX showed more deviation from vertical, surpassing the deviation level observed in WT (Figure 4B, 5). Therefore, this suggests that the growth rate alone does not account for the differences in response observed on the 2D clinostat. Additionally, WT plants have significantly reduced root growth in CL, approaching the length of CCA1 OX plants, yet maintaining similar VGI and Lc/L values in both conditions (Figure 5), and in the root reorientation assays, the response waveform is similar in both 12 h light: 12 h dark (Figure 1) and CL (Figure 3). This also suggests that the variation in growth rate does not completely explain the variation in the gravitropic response. Consistent with its known circadian phenotype (Covington et al., 2001; Chou and Yang, 1999),
Amyloplasts are important for gravity sensing. Starch levels in aerial tissues (leaves) vary throughout the day (Li et al., 1992). However, it is not known if this diel variation in total starch levels impacts the amyloplast. Therefore, we asked whether daily fluctuations in amyloplast starch occur and could explain the observed variation in gravity response throughout the day (Figures 1–3). Roots were harvested from WT, CCA1 OX, and
In contrast to WT,
Seedlings were transferred at two different time points (ZT12 and ZT22) to a RPM, Airbus Defense & Space, to examine if the time of day affected the response to simulated microgravity. Two sets of seedlings were entrained to time points shifted by 10 h so that the RPM treatment was carried out simultaneously on the same RPM for both time points. After 48 h, seedlings were removed from the RPM and imaged. Controls were grown vertically under identical conditions. The roots of seedlings grown on the RPM exhibited disorganized growth patterns compared to the vertical controls, growing in various directions, including out of the plate and into the media (Figure 8A). Our single plane images could not capture the overall growth and response in three dimensions (Figure 8A). Therefore, we calculated the variation only for roots that grew laterally in a single plane on the agar surface. Despite this limitation, we observed that seedlings grown on the RPM starting at ZT12 exhibited more dramatic skewing or slanted growth phenotypes than plants started at ZT22 (Figures 8B, S2). This trend was consistent between four independent RPM experiments.
This study shows that the plant response to gravistimulation depends on the time of day the stimulation occurs. There were substantial differences between the reorientation response at the end of the day and the end of the night. This altered response persists in CL conditions suggesting a role for the circadian clock in the perception of the gravistimulus or downstream signaling pathways. These findings suggest that attention to time can be a critical component in experimental design for gravitropic experiments. Moreover, the variation between different time points could be exploited to understand how the same stimulus in the same genotype manifests in altered responses. These findings encourage further investigation to evaluate if the time of day exacerbates or mitigates the effects of a change in the magnitude of the gravity vector in other tissues and other species, including humans during spaceflight.
We observed the significant influence of the time of day and the circadian clock on the plant response to a change in gravity using three different methods of altering gravity. First, root reorientation in response to gravistimulation was greatest 12 h after dawn and weakest 2 h before dawn (Figure 1). This pattern persisted in the absence of day/night cues and was lost in CCA1 OX plants suggesting that this variation is circadian regulated.
Root growth responses on a 2D clinostat were also disrupted in plants with an altered circadian clock. CCA1 OX plants showed less deviation from vertical growth than wild-type plants in entraining condition but was more responsive in constant environmental conditions (Figure 3). The
Root length also varied between these genotypes. For example, when the plants were grown in light-dark cycles, CCA1 OX plants had reduced root growth compared to wild-type (12:12, Fig 6). At first glance, this seems to explain the difference in root orientation; with less growth in CCA1 OX plants, there is less variation in their growth on the 2D clinostat. However, when grown in CL, CCA1 OX root growth remained similar to 12 h light:12 h dark levels, at about 55% WT root growth in 12 h light: 12 h dark (Figure 6). Even with this reduced growth, the CCA1 OX gravitropic responses seemed to surpass WT levels in either 12 h light: 12 h dark or CL (Figure 5). WT root growth in CL was significantly reduced (Figure 6), but there was no significant difference in the growth responses in the two conditions on the 2D clinostat as measured by VGI or Lc/L (Figure 5). These results suggest interactions between light cycles, root growth, and the gravitropic response. The connection between auxin signaling, which is gated by the circadian clock (Covington and Harmer, 2007; Rawat et al., 2009), and growth could be one mechanism controlling this interaction.
Growth on a RPM indicated that the time of day the plants first perceive a loss of 1 g gravity vector affects their response even 48 h later (Figure 8). While a full, detailed quantification of the changes on the RPM will require monitoring the root growth in three dimensions, in this first evaluation, we observe differences in the root orientation after 48 h depending on what time of day the plants were transferred to the RPM. The plants that first sensed simulated microgravity at ZT12 bent more than those that first perceived it at ZT22.
Root growth and orientation are fundamentally overlapping responses. Roots can only change in orientation through new growth. In the root reorientation experiments, the total root growth after 24 h was the same for all plants, independent of when they were rotated horizontally (Figure S1A). However, previous work has shown that this growth is not even across the 24 h-cycle (Yazdanbakhsh and Fisahn, 2009, 2011; Yazdanbakhsh et al., 2011). Root growth rate peaks in the night and slows just after dawn. This timing coincides with starch utilization with the roots growing as the plant breaks down starch and growth cessation during the day as starch is accumulated. The observed reorientation responses were consistent with this growth pattern. Plants rotated horizontally at ZT12, just before the maximal growth rate, had the largest response, while those at ZT22, just before the period of reduced growth, had a significantly reduced response. This suggests that the timing between the window of graviperception and the growth cycle is important. The plants only respond at their maximum if the change in gravity vector occurs in the window before the period of rapid growth. If the change in gravity happens outside that window, the response is not as robust even though the plants still go through the same rapid growth period the following night.
Plant roots perceive a change in gravity, at least in part through the amyloplasts (Hashiguchi et al., 2013). Gross differences in starch accumulation have been observed previously in gravitropic mutants, such as the extreme starch reduction in the
The
We have identified time of day-dependent differences in the root gravitropic response that persists through three different methods of measuring the plants’ responses to gravity. These results highlight the importance of paying attention to the time of day experiments are performed. The results presented here have implications for spaceflight and suggest that the transition to different gravity levels could be optimally timed.
This work also indicates that the circadian clock is an upstream regulator of the gravitropic response. Such clock-based control has also been observed in
Arabidopsis seeds of WT (Columbia-0), CCA1 OX,
Genotypes and sources of the plants used in this study.
CCA1 OX | Constitutive expression of CCA1 in Col-0 background | CS67793 | (Wang and Tobin, 1998) |
Knockout of |
CS3788 | (Hicks et al., 2001) | |
t-DNA insertion disrupting |
CS862938 | (Michael et al., 2003) | |
Starchless mutant | CS210 | (Caspar and Pickard, 1989) |
Plants were grown in 12 h light:12 h dark cycles at a constant 22 °C. For experiments conducted on plants grown under continuous light, chamber settings were adjusted to CL conditions two days before the experimental period so that the plants were entirely in constant environmental “free-run” conditions with no exogenous light cues.
Light intensity using fluorescent lights in the chamber was measured as 150um/m2/second at the top of the plates. Plates were placed vertically in 3D-printed petri dish holders (Supplementary File STL_PLate holder) and grown for 7 days in a plant growth chamber (Thermo Scientific). After 7 days, seedlings were reoriented by turning the plates by 90° and imaged for 24 h.
Imaging was performed using Raspberry Pi computers connected to IR-enabled cameras capturing an image every 15 min (Supplementary File). An IR light was used for imaging during dark time periods to avoid the effects of light at night on growth or bending responses. IR wavelengths used were >890 nm to avoid the stimulation of plant light signaling responses. Detailed scripts for capture and image analysis are available (Supplementary File 1) and at
Seedlings grown in 12 h light:12 h dark were subjected to a 90° reorientation with respect to the gravity vector. For each plate, this 90° turn was performed at a specific time after dawn. The time the plate was rotated horizontally is listed with reference to the time at dawn, the last ZT. For example, the plates rotated horizontally 2 h after dawn are denoted as ZT2. At ZT0 and every 2 h after, until ZT22, a set of plates with at least 15–20 plants on each plate were reoriented 90°s, or perpendicular to the gravity vector. For all experiments, there were at least three plates replicated in three separate experiments.
Images of the plates were taken every 15 min, beginning 2 h before the plates were rotated horizontally. Root angles were measured using three points: the root shoot boundary, the root tip position when the turn was initiated, and the root tip at the time of evaluation. A line was drawn from the root: shoot boundary to the root tip position when the turn was initiated to establish a straight line for the root growth before gravistimulation. Next, an intersecting line was drawn from the location of the root tip at the turn initiation to the root tip at the time of evaluation. The shootward angle on the side of bending created by these two intersecting lines was recorded as the bending angle. Time-of-day dependent differences in root angles were identified using ANOVA. Images were analyzed using ImageJ software (imagej.nih.gov) and standard measurement tools included in the base package (angle and line tools). Grid lines on the plates were used as a calibration reference to measure differences (Tolsma et al., 2021).
Root tips were harvested and stained to evaluate gross differences in amyloplasts and quantity throughout the day. Arabidopsis seedlings were grown under 12 h light;12 h dark cycles for 10–11 days. Seedlings were harvested at ZT12 and ZT22 to visualize amyloplasts by staining for starch (Kiss et al., 1996). Harvested seedlings were placed in fixative (4% formaldehyde, 25% EtOH, and 5% Acetic Acid) for ~18 h before staining with Lugol's I2/KI solution (Sigma Aldrich Cat # 62650). Root tips were stained for 1 min, followed by two quick rinses in a modified Clear See solution (25% Urea, 10% Sorbitol, 5% Sodium deoxycholate, for 20 s) followed by dIH2O (1 min each). Root tips were visualized using a Nikon Optiphot microscope under a bright field with a 40× objective and images captured with the Leica Application Suite (LAS 4.4). Images were analyzed ImageJ to determine relative staining intensity. First, a white balance was applied to the image to normalize all images. These corrected images were converted into red, green, blue (RGB) stacks resulting in three black and white images. The red wavelength was used further because it provided the most detail and least noise. Next, the adjust threshold tool was used to identify and highlight regions of the image with the darkest staining. The threshold was adjusted to highlight only the amyloplasts. A measurement region of the same size for each image was drawn around the columella cells and the percentage of the measured above the threshold was recorded.
Plants were grown for 2D clinostat assays using fluorescent lights in the chamber with light intensity measured as 150um/m2/second at the top of the plates. When the plants were 7 d old, each plate of plants was imaged and then moved to the 2D clinostat located inside the chamber at ZT4.5. The plates were removed 24 h later and imaged immediately. In CL conditions, the plants were moved to CL for 48 h before the plants were transferred to the 2D clinostat.
Root growth differences on the 2D clinostat were analyzed by two different methods. First, the root tip positions were compared pre-and post-clinorotation from the images using Image J. The final root positions relative to the position before clinorotation were plotted (Figure 4). Secondly, deviations from the vertical growth and skewing were determined using metrics from Grabov et al. (Grabov et al., 2005a; Vaughn and Masson, 2011) (Figure 5). Lc is defined as a straight line connecting the root tip to the base of the root, while L is the total length of the root. Calculating Lc/L describes how much the root varied from a linear pathway to its current location. Ly represents the ordinate location of the root tip (location in the y axis). VGI=Ly/L and describes how much the root deviates from straight, gravitropic growth.
In addition to the 2D clinostat, the effects of simulated microgravity were also evaluated using a RPM 2.0 at Kennedy Space Center's Microgravity Simulation Support Facility (KSC_MSSF). The RPM was controlled by Airbus software that measured and recorded the x, y, and z axes movement and calculated the force of gravity experienced during the run. The program reached 0 g within 10–15 min of starting the RPM. The light periods and temperature were as described above. To enable growth at the two lighting regimes (ZT12 and ZT22, offset by 10 h) on a single RPM, we utilized LED lights and a light barrier to prevent leakage. The lights were set so that the light intensity at the top of the plate was 150um/m2/second, to match the light intensity of the fluorescent lights in the previous experiments. Lights were controlled via timed power strips connected to variable voltage power sources. Light leak between sides of the RPM was minimal and less than 1um/m2/second. To further protect the roots from excess light exposure, the square plates were placed in black “sleeves,” covering the plate up to the level at which seeds were placed. Because of this, light from the sides was blocked. However, the light was unimpeded from the top. Before being placed on the RPM, plants were grown under the same LED lights in vertically oriented racks. The lighting schedules were timed so that when the plants were transferred to simulated microgravity, it was at ZT12 and 22 (Figure S3). Plants were grown in microgravity conditions for 48 h. Images were captured before and after growth on the RPM. Before starting microgravity simulation, the image before placing the plants on the RPM was imaged under IR lights (>890 nm) for the plates in the dark at the start of microgravity simulation. After 48 h on in the RPM, all plants were immediately imaged in the light.