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The Circadian-clock Regulates the Arabidopsis Gravitropic Response Cover

The Circadian-clock Regulates the Arabidopsis Gravitropic Response

Gravitational and Space Research
Volume 9 (2021): Issue 1 (January 2021)
By: Joseph S. TolsmaORCID,  Kaetlyn T. RyanORCID,  Jacob J. Torres,  Jeffrey T. Richards,  Zach Richardson,  Eric S. LandORCID,  Imara Y. PereraORCID and  Colleen J DohertyORCID  
Open Access
|Dec 2021

Figures & Tables

Figure 1

Gravitropic, root-bending response to a 90° reorientation to gravity at different times of the day. The gray shaded area indicates the 95% confidence intervals. Red triangles represent the mean for a single time point of all plates. Each black dot indicates the mean of a single plate with 15–20 plants per plate. There are at least three plates per time point. ZT times (x-axis) are the time after the last “time cue,” at dawn, in hours at which the vertically oriented plates were turned by 90°. An ANOVA followed by a Tukey posthoc test was performed to identify time-of-day differences between groups (p-value < 0.05). ZT time points with differences in root reorientation angle were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. The drawings of the plant root on the y-axis show a simplified view of the associated bending phenotype; a smaller degree indicates a plant with a greater bend thus a greater gravitropic response.
Gravitropic, root-bending response to a 90° reorientation to gravity at different times of the day. The gray shaded area indicates the 95% confidence intervals. Red triangles represent the mean for a single time point of all plates. Each black dot indicates the mean of a single plate with 15–20 plants per plate. There are at least three plates per time point. ZT times (x-axis) are the time after the last “time cue,” at dawn, in hours at which the vertically oriented plates were turned by 90°. An ANOVA followed by a Tukey posthoc test was performed to identify time-of-day differences between groups (p-value < 0.05). ZT time points with differences in root reorientation angle were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. The drawings of the plant root on the y-axis show a simplified view of the associated bending phenotype; a smaller degree indicates a plant with a greater bend thus a greater gravitropic response.

Figure 2

Root bending dynamics of two time points, ZT12 and ZT22, over 24 h of growth. Shaded areas indicate the confidence interval. Lines are the locally weighted mean, while dots are individual measurements or root angle/growth. Colors differentiate the ZT time comparison (yellow = ZT22 and gray = ZT12). The x-axis indicates the amount of time after gravistimulation. Each dot represents a single root. (A) The unadjusted root curvature angle was measured every 4 h during the 24 h root bending experiment to show how the change in root angle progresses over the 24 h. Plates were rotated horizontally at ZT12 and ZT22, and time point “0” references the time of the plate turn. (B) The root growth over each 4 h period was measured. Every 4 h after the plates were turned the increase in root length over the previous 4 h was measured and plotted. Although plants rotated horizontally at ZT12 and ZT22 had the same cumulative growth over the whole 24 h period, most of the growth occurred in the first 16 h post-turn for ZT12 while for ZT22 most of the growth occurred after 12 h post-turn. The Y-axis indicates root growth in mm over the 4 h prior to the indicated time point. (C) Root curvature angle adjusted by growth rate for plates rotated horizontally at ZT12 and ZT22. The curvature angle was divided by the growth rate over the same 4 h period to adjust the change in curvature by the growth rate. This adjustment is made by calculating the root angle/growth rate to capture the bending relative to the growth.
Root bending dynamics of two time points, ZT12 and ZT22, over 24 h of growth. Shaded areas indicate the confidence interval. Lines are the locally weighted mean, while dots are individual measurements or root angle/growth. Colors differentiate the ZT time comparison (yellow = ZT22 and gray = ZT12). The x-axis indicates the amount of time after gravistimulation. Each dot represents a single root. (A) The unadjusted root curvature angle was measured every 4 h during the 24 h root bending experiment to show how the change in root angle progresses over the 24 h. Plates were rotated horizontally at ZT12 and ZT22, and time point “0” references the time of the plate turn. (B) The root growth over each 4 h period was measured. Every 4 h after the plates were turned the increase in root length over the previous 4 h was measured and plotted. Although plants rotated horizontally at ZT12 and ZT22 had the same cumulative growth over the whole 24 h period, most of the growth occurred in the first 16 h post-turn for ZT12 while for ZT22 most of the growth occurred after 12 h post-turn. The Y-axis indicates root growth in mm over the 4 h prior to the indicated time point. (C) Root curvature angle adjusted by growth rate for plates rotated horizontally at ZT12 and ZT22. The curvature angle was divided by the growth rate over the same 4 h period to adjust the change in curvature by the growth rate. This adjustment is made by calculating the root angle/growth rate to capture the bending relative to the growth.

Figure 3

Gravitropic response at two time points in CL conditions. (A) Time of day variation in root-bending response to a 90° reorientation with respect to the gravity vector. The gray shaded area indicates the 95% confidence interval. Triangles represent the mean for a single time point of all plates. Each black dot indicates the mean of a single plate with 15–20 plants per plate. There are at least four plates per time point. Root bending angles (Y-axis) were measured as described in materials and methods. ZT times (x-axis) are the time after the last time the lights were turned on (dawn) at which the vertically oriented plates were turned by 90°. ANOVA and Tukey posthoc tests were performed to identify time-of-day differences between groups (p-value < 0.05). ZT time points with differences in root reorientation angle were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. The drawings of the plant root on the y-axis show a simplified view of the associated bending phenotype; a smaller degree indicates a plant with a greater bend thus a greater gravitropic response. (B) Boxplot of the root reorientation angles for plants turned at either ZT12, just before the lights are turned off, or ZT22, just before dawn, for WT plants and the CCA1 OX genotype. CL, constant light; ZT, Zeitgeber time.
Gravitropic response at two time points in CL conditions. (A) Time of day variation in root-bending response to a 90° reorientation with respect to the gravity vector. The gray shaded area indicates the 95% confidence interval. Triangles represent the mean for a single time point of all plates. Each black dot indicates the mean of a single plate with 15–20 plants per plate. There are at least four plates per time point. Root bending angles (Y-axis) were measured as described in materials and methods. ZT times (x-axis) are the time after the last time the lights were turned on (dawn) at which the vertically oriented plates were turned by 90°. ANOVA and Tukey posthoc tests were performed to identify time-of-day differences between groups (p-value < 0.05). ZT time points with differences in root reorientation angle were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. The drawings of the plant root on the y-axis show a simplified view of the associated bending phenotype; a smaller degree indicates a plant with a greater bend thus a greater gravitropic response. (B) Boxplot of the root reorientation angles for plants turned at either ZT12, just before the lights are turned off, or ZT22, just before dawn, for WT plants and the CCA1 OX genotype. CL, constant light; ZT, Zeitgeber time.

Figure 4

Genotype differences in response to growth on a 2D clinostat. Dots indicate root growth trajectories after clinorotation. Each root was assigned coordinates by zeroing root location at t = 0 to (0,0). Root growth was measured after clinorotation and plotted. (A) Root response for plants grown under 12 h light: 12 h dark conditions. (B) Root response for plants grown in continuous light. Colors correspond to genotype (green = WT, yellow = CCA1 OX, blue = prr7, gray = elf3-2).
Genotype differences in response to growth on a 2D clinostat. Dots indicate root growth trajectories after clinorotation. Each root was assigned coordinates by zeroing root location at t = 0 to (0,0). Root growth was measured after clinorotation and plotted. (A) Root response for plants grown under 12 h light: 12 h dark conditions. (B) Root response for plants grown in continuous light. Colors correspond to genotype (green = WT, yellow = CCA1 OX, blue = prr7, gray = elf3-2).

Figure 5

Skewing metrics of the VGI (Grabov et al., 2005b), and straightness, Lc/L (Vaughn and Masson, 2011), from plants grown for 24 h on the 2D clinostat (Figure 4). (A) Cartoon of the calculations for each metric Lc/L and VGI= Ly/L. The root tip position at t=0, before the plants were placed on the clinostat, is the (0,0) position for the measurements. The three examples show the inputs for different levels of response. When the root tip was above the starting point (middle image) the absolute value of Ly was used. Image created using Biorender. com (B) The average of Lc/L skewing metric is shown for each genotype and condition. (C) The average VGI is shown for each genotype and condition. Error bars represent standard deviation. 12:12 = 12 h light: 12 h dark, CL = continuous light. An ANOVA followed by a Tukey posthoc test was performed to identify differences between groups (p-value < 0.05). ZT time points with differences in vertical root growth were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. VGI, vertical growth index.
Skewing metrics of the VGI (Grabov et al., 2005b), and straightness, Lc/L (Vaughn and Masson, 2011), from plants grown for 24 h on the 2D clinostat (Figure 4). (A) Cartoon of the calculations for each metric Lc/L and VGI= Ly/L. The root tip position at t=0, before the plants were placed on the clinostat, is the (0,0) position for the measurements. The three examples show the inputs for different levels of response. When the root tip was above the starting point (middle image) the absolute value of Ly was used. Image created using Biorender. com (B) The average of Lc/L skewing metric is shown for each genotype and condition. (C) The average VGI is shown for each genotype and condition. Error bars represent standard deviation. 12:12 = 12 h light: 12 h dark, CL = continuous light. An ANOVA followed by a Tukey posthoc test was performed to identify differences between groups (p-value < 0.05). ZT time points with differences in vertical root growth were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. VGI, vertical growth index.

Figure 6

Root growth comparisons between genotypes. Total root growth (mm) was measured after 10 days of growth and compared between genotypes and conditions. Colors indicate genotype (green=WT, yellow=CCA1 OX, blue=prr7, grey=elf3-2). Shading indicates lighting conditions for growth experiments (solid= 12:12, growth in 12 h light: 12 h dark; shaded= CL, growth in continuous light). An ANOVA followed by a Tukey posthoc test was performed to identify differences between groups (p-value < 0.05). ZT time points with differences in root growth were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. ZT, Zeitgeber time.
Root growth comparisons between genotypes. Total root growth (mm) was measured after 10 days of growth and compared between genotypes and conditions. Colors indicate genotype (green=WT, yellow=CCA1 OX, blue=prr7, grey=elf3-2). Shading indicates lighting conditions for growth experiments (solid= 12:12, growth in 12 h light: 12 h dark; shaded= CL, growth in continuous light). An ANOVA followed by a Tukey posthoc test was performed to identify differences between groups (p-value < 0.05). ZT time points with differences in root growth were identified and grouped using a Tukey posthoc test with a Bonferroni correction (α = 0.05). Letters indicate significant groups. ZT, Zeitgeber time.

Figure 7

Starch staining of amyloplasts at ZT12 and ZT22. (A) Representative images of root tips from WT, elf3-2, CCA1 OX, and pgm plants stained for starch. We stained roots that were harvested and fixed at ZT12 or ZT22. The starchless pgm-1 genotype was used as a control for background staining levels. Scale bar represents 25 μm. (B) The average percent area was calculated for WT and elf3-2 plants at both time points, ZT12 (solid), ZT22 (striped). Error bars indicate standard deviation. *** indicates p-value < 0.001, two-sided t-test comparing ZT22 to ZT12. n for each genotype and time point is shown at the bottom of the bar. ZT, Zeitgeber time.
Starch staining of amyloplasts at ZT12 and ZT22. (A) Representative images of root tips from WT, elf3-2, CCA1 OX, and pgm plants stained for starch. We stained roots that were harvested and fixed at ZT12 or ZT22. The starchless pgm-1 genotype was used as a control for background staining levels. Scale bar represents 25 μm. (B) The average percent area was calculated for WT and elf3-2 plants at both time points, ZT12 (solid), ZT22 (striped). Error bars indicate standard deviation. *** indicates p-value < 0.001, two-sided t-test comparing ZT22 to ZT12. n for each genotype and time point is shown at the bottom of the bar. ZT, Zeitgeber time.

Figure 8

Root orientation after 48 h on the RPM when started at ZT12 or ZT22. (A) Images of Arabidopsis roots after 48 h on the RPM. Plants were grown in alternating light/dark cycles so that when each set of two plates was moved to the RPM one set of plants was at ZT12 and the other was at ZT22. Plants were kept in the same light cycle for 48 h. The arrowheads show the root tip position before placing it on the RPM. Only the red arrowheads were analyzed; white arrowheads indicate roots with no visible growth and blue arrowheads indicate roots that grew <2 mm. The red line traces the growth of the root. The top and bottom indicate two separate RPM runs. (B) Quantification of the root bending angle for plants placed on the RPM at ZT12 or ZT22. Roots from four RPM runs, two plates each, were analyzed. Only roots that showed >2 mm growth after 48 h and did not grow into or out of the agar were counted. RPM, Random Positioning Machine; ZT, Zeitgeber Time.
Root orientation after 48 h on the RPM when started at ZT12 or ZT22. (A) Images of Arabidopsis roots after 48 h on the RPM. Plants were grown in alternating light/dark cycles so that when each set of two plates was moved to the RPM one set of plants was at ZT12 and the other was at ZT22. Plants were kept in the same light cycle for 48 h. The arrowheads show the root tip position before placing it on the RPM. Only the red arrowheads were analyzed; white arrowheads indicate roots with no visible growth and blue arrowheads indicate roots that grew <2 mm. The red line traces the growth of the root. The top and bottom indicate two separate RPM runs. (B) Quantification of the root bending angle for plants placed on the RPM at ZT12 or ZT22. Roots from four RPM runs, two plates each, were analyzed. Only roots that showed >2 mm growth after 48 h and did not grow into or out of the agar were counted. RPM, Random Positioning Machine; ZT, Zeitgeber Time.

Genotypes and sources of the plants used in this study_

GenotypeDescriptionABRC Accession numberReference
CCA1 OXConstitutive expression of CCA1 in Col-0 backgroundCS67793(Wang and Tobin, 1998)
elf3-2Knockout of ELF3 (1- to 2-kb deletion) in Col-0 backgroundCS3788(Hicks et al., 2001)
prr7-3t-DNA insertion disrupting PRR7 in Col-0 backgroundCS862938(Michael et al., 2003)
pgm-1Starchless mutantCS210(Caspar and Pickard, 1989)
DOI: https://doi.org/10.2478/gsr-2021-0014 | Journal eISSN: 2332-7774
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Language: English
Page range: 171 - 186
Published on: Dec 31, 2021
Published by: American Society for Gravitational and Space Research
In partnership with: Paradigm Publishing Services
Publication frequency: 2 issues per year
Keywords:
Gravitropism,
circadian rhythms,
simulated microgravity
Related subjects:
Life sciences,
Life sciences, other,
Materials sciences,
Materials sciences, other,
Physics,
Physics, other

© 2021 Joseph S. Tolsma, Kaetlyn T. Ryan, Jacob J. Torres, Jeffrey T. Richards, Zach Richardson, Eric S. Land, Imara Y. Perera, Colleen J Doherty, published by American Society for Gravitational and Space Research
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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