DOI: 10.5061/dryad.sn02v6xjp
title: Data from: Autoinhibitory calcium ATPases regulate the calcium gradient required for rapid polarized growth

description: Polarized growth drives the morphogenesis of elongated cellular
 structures. In plants, polarized growth depends on actin and a tip-focused
 ionic calcium gradient. How the calcium gradient is maintained remains
 unclear. We discovered that autoinhibitory calcium ATPases (ACAs)
 redundantly contribute to the steepness of the calcium gradient. ACA1 and
 ACA2 localize to the subapical plasma membrane and ACA5 to the vacuole
 membrane, providing spatial regulation of calcium efflux. Tip-growing
 plant cells also exhibit apical calcium fluctuations. Even though
 Δaca1/2/5 cells have a diminished calcium gradient, they exhibit normal
 fluctuations and actin but have significantly reduced apical secretion.
 Furthermore, cells lacking apical actin retain a strong calcium gradient
 but have reduced apical secretion. Suppression of both the calcium
 gradient and apical actin dramatically impairs growth, supporting a model
 where two independent and parallel processes, the calcium gradient and
 apical actin, promote rapid polarized growth.
description: # Data from: Autoinhibitory calcium ATPases regulate the calcium gradient
 required for rapid polarized growth Dataset DOI:
 [10.5061/dryad.sn02v6xjp](https://doi.org/10.5061/dryad.sn02v6xjp) ##
 Description of the data and file structure Macros developed for Fiji: 1.
 Calcium Gradient Quantification - takes the CFP and YFP images and
 calculates YFP/CFP ratio. Aligns the tips of the growing cell. Measures
 apical YFP/CFP levels. Measures the spatial gradient of YFP/CFP. 2.
 Morphology Macro V3.2 - measures the chloroplast autofluorescence area for
 individual plants 3. PMQuantificationMicrons - used to measure the levels
 of CSLD and actin at the cell tip. R code: 1. Data_Wrangle.Rmd: Aligning
 and normalizing data obtained from PMQuantificationMicrons. Sample data
 files titled LA)CSLD3_1200x_Actin.csv included. 2. Figure2B.Rmd: FDANOVA
 statistics  Data from Figure 2B included - first10min.csv, last10min.csv,
 pretreatment.csv 3. Figure2D.Rmd: FDANOVA statistics Data from Figure 2D
 included - aip.csv, WildType.csv 4. Figure4.Rmd: Kruskal Wallis statistics
 with multiple comparisons Data from Figure 4 included
 - StatisticsMultipleComparisons - Sheet1.csv 5. Figure5.Rmd: FDANOVA
 statistics Data from Figure 5B included - ∆aca1*2*5-28.csv,
 ∆aca1_5acaD464A-5.csv, wildtype.csv 6. Figure6D.Rmd: FDANOVA statistics
 Data from Figure 6D included - Mutant2_6D.csv, Wildtype2_6D.csv 7.
 Figure6E.Rmd: FDANOVA statistics Data from Figure 6E included -
 actinMutant_6E.csv, actinWildtype_6E.csv 8. Figure7B.Rmd: Kruskal Wallis
 statistics with multiple comparisons Data from Figure 7B included
 - StatisticsMultipleComparisons - Sheet1.csv 9. Figure7D.Rmd: FDANOVA
 statistics Data from Figure 7D included - aipFigure7.csv,
 strongQuadFigure7.csv, weakQuadFigure7.csv, WTFigure7.csv 10. Wavelets and
 Timeseries RevisedLW063021-pdf.Rmd: Empirical mode decomposition code ###
 Files and variables #### File:
 Wavelets_and_Timeseries_RevisedLW063021-pdf.R **Description:** Empirical
 mode decomposition code #### File: Data_Wrangle.Rmd **Description:** used
 to align data from PM quantification #### File: LA)CSLD3_12001_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12005_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12007_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12003_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12013_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12011_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12017_Actin.csv
 **Description:** sample file for Data_Wrangle.Rmd ##### Variables * First
 column is pixel position along the line * Second column (value) is the
 measured intensity at that position. #### File: LA)CSLD3_12024_Actin.csv
 **Description:**  ##### Variables * First column is pixel position along
 the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12027_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12025_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12029_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12015_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12019_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12009_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: LA)CSLD3_12021_Actin.csv **Description:** sample file
 for Data_Wrangle.Rmd ##### Variables * First column is pixel position
 along the line * Second column (value) is the measured intensity at that
 position. #### File: Figure2B.Rmd **Description:** FDANOVA statistics ####
 File: first10min.csv **Description:** data figure 2B ##### Variables *
 index: pixel position * microns: pixel position translated to microns *
 cell1: intensity at that position for cell 1 * cell2: intensity at that
 position for cell 2 * cell3: intensity at that position for cell 3 *
 cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 average: average intensity of cells 1-9 * standard error: standard error
 of the intensity of cells 1-9 #### File: last10min.csv
 **Description:** data figure 2B ##### Variables * index: pixel position *
 microns: pixel position translated to microns * cell1: intensity at that
 position for cell 1 * cell2: intensity at that position for cell 2 *
 cell3: intensity at that position for cell 3 * cell4: intensity at that
 position for cell 4 * cell5: intensity at that position for cell 5 *
 cell6: intensity at that position for cell 6 * cell7: intensity at that
 position for cell 7 * cell8: intensity at that position for cell 8 *
 cell9: intensity at that position for cell 9 * average: average intensity
 of cells 1-9 * standard error: standard error of the intensity of cells
 1-9 #### File: pretreatment.csv **Description:** data figure 2B #####
 Variables * index: pixel position * microns: pixel position translated to
 microns * cell1: intensity at that position for cell 1 * cell2: intensity
 at that position for cell 2 * cell3: intensity at that position for cell 3
 * cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 average: average intensity of cells 1-9 * standard error: standard error
 of the intensity of cells 1-9 #### File: aip.csv **Description:** data
 figure 2D ##### Variables * index: pixel position * microns: pixel
 position translated to microns * cell1: intensity at that position for
 cell 1 * cell2: intensity at that position for cell 2 * cell3: intensity
 at that position for cell 3 * cell4: intensity at that position for cell 4
 * cell5: intensity at that position for cell 5 * cell6: intensity at that
 position for cell 6 * average: average intensity of cells 1-6 * standard
 error: standard error of the intensity of cells 1-6 #### File:
 Figure2D.Rmd **Description:** FDANOVA statistics #### File: WildType.csv
 **Description:** data figure 2D ##### Variables * index: pixel position *
 microns: pixel position translated to microns * cell1: intensity at that
 position for cell 1 * cell2: intensity at that position for cell 2 *
 cell3: intensity at that position for cell 3 * cell4: intensity at that
 position for cell 4 * cell5: intensity at that position for cell 5 *
 cell6: intensity at that position for cell 6 * average: average intensity
 of cells 1-6 * standard error: standard error of the intensity of cells
 1-6 #### File: Figure4.Rmd **Description:** Kruskall Wallis Statistics
 with multiple comparisons #### File:
 StatisticsMultipleComparisons_-_Sheet1.csv **Description:** Data for
 Figure 4B ##### Variables * YCNLA: area of YCNLA plants * aca1-8: area of
 ∆aca1-8 * aca1-13: area of ∆aca1-13 * aca2-40: area of ∆aca2-40 *
 aca2-d464a-14: area of ∆aca2-D464A-14 * aca5-3: area of ∆aca5-3 *
 aca5-7: area of ∆aca5-7 * aca1/2-2: area of ∆aca1/2-2 * aca1/2-19: area of
 ∆aca1/2-19 * aca1/5-7: area of ∆aca1/5-7 * aca1/5-9: area of ∆aca1/5-9 *
 aca2/5-109: area of ∆aca2/5-109 * aca1/2/5-28: area of ∆aca1/2/5-28 *
 aca1/2/5-36: area of ∆aca1/2/5-36 * trip-5: area of ∆aca1/5/aca2-D464-5 *
 trip-9: area of ∆aca1/5/aca2-D464-9 #### File: ∆aca1_2_5-28.csv
 **Description:** Data for Figure 5B ##### Variables * index: pixel
 position * microns: pixel position translated to microns *
 cell1: intensity at that position for cell 1 * cell2: intensity at that
 position for cell 2 * cell3: intensity at that position for cell 3 *
 cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 cell10: intensity at that position for cell 10 * cell11: intensity at that
 position for cell 11 * average: average intensity of cells 1-11 *
 normalized: normalized average intensity of cells 1-11 * standard error:
 standard error of intensity of cells 1-11 #### File: ∆aca1_5acaD464A-5.csv
 **Description:** Data for Figure 5B ##### Variables * index: pixel
 position * microns: pixel position translated to microns *
 cell1: intensity at that position for cell 1 * cell2: intensity at that
 position for cell 2 * cell3: intensity at that position for cell 3 *
 cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 cell10: intensity at that position for cell 10 * cell11: intensity at that
 position for cell 11 * cell12: intensity at that position for cell 12 *
 cell13: intensity at that position for cell 13 * cell14: intensity at that
 position for cell 14 * cell15: intensity at that position for cell 15 *
 cell16: intensity at that position for cell 16 * cell17: intensity at that
 position for cell 17 * cell18: intensity at that position for cell 18 *
 cell19: intensity at that position for cell 19 * cell20: intensity at that
 position for cell 20 * cell21: intensity at that position for cell 21 *
 cell22: intensity at that position for cell 22 * average: average
 intensity of cells 1-22 * normalized: normalized average intensity of
 cells 1-22 * standard error: standard error of intensity of cells 1-22
 #### File: Figure5.Rmd **Description:** FDANOVA statistics #### File:
 Figure6D.Rmd **Description:** FDANOVA statistics #### File: Mutant2_6D.csv
 **Description:** Data for Figure 6D ##### Variables * index: pixel
 position * microns: pixel position translated to microns *
 cell1: intensity at that position for cell 1 * cell2: intensity at that
 position for cell 2 * cell3: intensity at that position for cell 3 *
 cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 cell10: intensity at that position for cell 10 * cell11: intensity at that
 position for cell 11 * cell12: intensity at that position for cell 12 *
 cell13: intensity at that position for cell 13 * cell14: intensity at that
 position for cell 14 * cell15: intensity at that position for cell 15 *
 cell16: intensity at that position for cell 16 * cell17: intensity at that
 position for cell 17 * cell18: intensity at that position for cell 18 *
 cell19: intensity at that position for cell 19 * cell20: intensity at that
 position for cell 20 * cell21: intensity at that position for cell 21 *
 cell22: intensity at that position for cell 22 * average: average
 intensity of cells 1-22 * normalized: normalized average intensity of
 cells 1-22 * standard error: standard error of intensity of cells 1-22
 #### File: Wildtype2_6D.csv **Description:** Data for Figure 6D #####
 Variables * microns: position in microns * cell1: intensity at that
 position for cell 1 * cell2: intensity at that position for cell 2 *
 cell3: intensity at that position for cell 3 * cell4: intensity at that
 position for cell 4 * cell5: intensity at that position for cell 5 *
 cell6: intensity at that position for cell 6 * cell7: intensity at that
 position for cell 7 * cell8: intensity at that position for cell 8 *
 cell9: intensity at that position for cell 9 * cell10: intensity at that
 position for cell 10 * cell11: intensity at that position for cell 11 *
 cell12: intensity at that position for cell 12 * cell13: intensity at that
 position for cell 13 * average: average intensity of cells 1-13 * standard
 error: standard error of intensity of cells 1-13 #### File:
 actinMutant_6E.csv **Description:** Data for Figure 6E ##### Variables *
 microns: position in microns * cell1: intensity at that position for cell
 1 * cell2: intensity at that position for cell 2 * cell3: intensity at
 that position for cell 3 * cell4: intensity at that position for cell 4 *
 cell5: intensity at that position for cell 5 * cell6: intensity at that
 position for cell 6 * cell7: intensity at that position for cell 7 *
 cell8: intensity at that position for cell 8 * cell9: intensity at that
 position for cell 9 * cell10: intensity at that position for cell 10 *
 cell11: intensity at that position for cell 11 * cell12: intensity at that
 position for cell 12 * cell13: intensity at that position for cell 13 *
 cell14: intensity at that position for cell 14 * cell15: intensity at that
 position for cell 15 * cell16: intensity at that position for cell 16 *
 cell17: intensity at that position for cell 17 * cell18: intensity at that
 position for cell 18 * cell19: intensity at that position for cell 19 *
 cell20: intensity at that position for cell 20 * cell21: intensity at that
 position for cell 21 * cell22: intensity at that position for cell 22 *
 average: average intensity of cells 1-22 * Standard error: standard error
 of intensity of cells 1-22 #### File: actinWildtype_6E.csv
 **Description:** Data for Figure 6E ##### Variables * microns: position in
 microns * cell1: intensity at that position for cell 1 * cell2: intensity
 at that position for cell 2 * cell3: intensity at that position for cell 3
 * cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 cell10: intensity at that position for cell 10 * cell11: intensity at that
 position for cell 11 * cell12: intensity at that position for cell 12 *
 cell13: intensity at that position for cell 13 * average: average
 intensity of cells 1-13 * standard error: standard error of intensity of
 cells 1-13 #### File: Figure6E.Rmd **Description:** FDANOVA statistics
 #### File: Figure7B.Rmd **Description:** Kruskal Wallis Statistics with
 multiple comparisons #### File: aipFigure7.csv **Description:** Data for
 Figure 7D ##### Variables * index: pixel position * microns: pixel
 position translated to microns * cell1: intensity at that position for
 cell 1 * cell2: intensity at that position for cell 2 * cell3: intensity
 at that position for cell 3 * cell4: intensity at that position for cell 4
 * cell5: intensity at that position for cell 5 * cell6: intensity at that
 position for cell 6 * cell7: intensity at that position for cell 7 *
 cell8: intensity at that position for cell 8 * cell9: intensity at that
 position for cell 9 * cell10: intensity at that position for cell 10 *
 cell11: intensity at that position for cell 11 * cell12: intensity at that
 position for cell 12 * Average: average intensity of cells 1-12 * standard
 error: standard error of intensity of cells 1-12 #### File: Figure7D.Rmd
 **Description:** FDANOVA statistics #### File: strongQuadFigure7.csv
 **Description:** Data for Figure 7D ##### Variables * micron: pixel
 position translated to microns * Index: pixel position * cell1: intensity
 at that position for cell 1 * cell2: intensity at that position for cell 2
 * cell3: intensity at that position for cell 3 * cell4: intensity at that
 position for cell 4 * cell5: intensity at that position for cell 5 *
 cell6: intensity at that position for cell 6 * average: average intensity
 of cells 1-6 * standard error: standard error of intensity of cells 1-6
 #### File: weakQuadFigure7.csv **Description:** Data for Figure 7D #####
 Variables * index: pixel position * micron: pixel position translated to
 microns * cell1: intensity at that position for cell 1 * cell2: intensity
 at that position for cell 2 * cell3: intensity at that position for cell 3
 * cell4: intensity at that position for cell 4 * cell5: intensity at that
 position for cell 5 * cell6: intensity at that position for cell 6 *
 cell7: intensity at that position for cell 7 * cell8: intensity at that
 position for cell 8 * cell9: intensity at that position for cell 9 *
 cell10: intensity at that position for cell 10 * average: average
 intensity of cells 1-10 * standard error: standard error of intensity of
 cells 1-10 #### File: WTFigure7.csv **Description:** Data for Figure 7D
 ##### Variables * index: pixel position * micron: pixel position
 translated to microns * cell1: intensity at that position for cell 1 *
 cell2: intensity at that position for cell 2 * cell3: intensity at that
 position for cell 3 * cell4: intensity at that position for cell 4 *
 cell5: intensity at that position for cell 5 * cell6: intensity at that
 position for cell 6 * cell7: intensity at that position for cell 7 *
 cell8: intensity at that position for cell 8 * cell9: intensity at that
 position for cell 9 * cell10: intensity at that position for cell 10 *
 cell11: intensity at that position for cell 11 * cell12: intensity at that
 position for cell 12 * cell13: intensity at that position for cell 13 *
 cell14: intensity at that position for cell 14 * cell15: intensity at that
 position for cell 15 * Average: average intensity of cells 1-15 * Standard
 Error: standard error of intensity of cells 1-15 #### File:
 CalciumGradientQuantification.ijm **Description:** Fiji Macro to generate
 calcium ratio image and to measure calcium gradient #### File:
 MorphologyMacroV3.2.ijm **Description:** Fiji Macro to quantify plant area
 based on chloroplast autofluorescence #### File:
 PMQuantificationMicrons_.ijm **Description:** Fiji Macro to quantify
 fluorescence at the cell tip ## Code/software Fiji Macros run in (Fiji is
 Just) ImageJ 2.14.0/1.54f:Java 1.8.0_202 [64bit] R code runs in R Studio
 Version 2025.05.1+513 (2025.05.1+513)