assays/AutophagicActivityAssay/README.md

+
+# Autophagic Activity Assay
+
+## Movie S3. Application of the RoPod for time-lapse imaging of pHusion-ATG8 in roots treated with autophagy modulators.
+
+Representative movies of WT seedlings expressing the autophagosomal marker pHusion-ATG8
+grown in a RoPod chamber and treated with vehicle (0.01% DMSO), 500 nM Concanamycin A (ConA)
+or 500 nM AZD8055 (AZD). ConA blocks the final step of autophagy, i.e. degradation of the
+autophagic bodies in the vacuole, causing massive accumulation of the pHusion-ATG8-positive
+puncta in the vacuolar lumen. AZD8055 induces autophagic activity, causing incorporation of the
+ATG8a-based reporter into autophagosomes followed by its delivery to the vacuole and
+degradation. Note decrease of the fluorescent signal after ca 4h of treatment. The magenta and
+green lines indicate respectively hair and non-hair cell files used for quantification. Scale bar, 50
+µm.
+
+![41598_2024_63226_MOESM3_ESM.mp4](dataset/41598_2024_63226_MOESM3_ESM.mp4)
+
+
+## Figure S1. Dynamic changes in the basal autophagic activity detected in root hair and non-hair
+cells.
+
+Quantification of pHusion-ATG8-positive puncta per area in the epidermal root cells of WT seedlings
+grown in RoPods and treated with 500 nM ConA. The data represents 0h, 4h and 12h time points of
+the time-lapse assays shown in the Figure 5 and Movie S3. T-test, p-value<0.01.
+
+![FigS1.png](dataset/FigS1.png)
+
+
+
+## Figure S2. Root hair cells accumulate less autophagic bodies than non-hair cells under prolonged ConA treatment.
+
+Arabidopsis seedlings co-expressing autophagy reporter (GFP-ATG8) with vacuolar marker (spL-
+mRFP) were grown in a RoPod v23.4 and treated with 0.5uM ConA overnight prior to imaging
+using confocal microscope. (a) A representative maximal projection of a tiled z-tack encompassing
+complete cells with vacuoles was used to segment hair (yellow outline) and non-hair (blue outline)
+cells; scale bar, 100 µm. (b) illustrates segmented non-hair (left) and hair (right) cells outlined in
+(a). Scale bars, 20 µm.
+The number of GFP-positive puncta was quantified per µm3 (c) and per a cell (d). Charts in c and d
+represent data from one out of four independent experiments. Four roots were analyzed,
+selecting five cells for each cell type for each root, n= 20. OneWay Anova, p-value <0.01 for both
+charts.
+
+![FigS2.png](dataset/FigS2.png)
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assays/ComparisonOfAutophagicActivityInRoots/README.md

+
+# Comparison Of Autophagic Activity In Roots
+
+## Figure 2
+
+Prolonged incubation of Arabidopsis seedlings on microscopy glass upregulates autophagy in the epidermal root cells. (a) Arabidopsis seedlings expressing fluorescent marker for autophagy (pHusion-ATG8) in the wild-type (WT) or autophagy-deficient (atg5/7 KO) backgrounds were grown on standard Petri plates or in the RoPod v23. 5 days-old seedlings grown on a Petri plate were mounted between a standard microscopy-grade sample slide and a cover slip in a liquid 0.5xMS medium and incubated on the bench for the designated amount of time prior to imaging using confocal microscope. Seedlings of the same genotypes grown in the RoPod were imaged using the same settings (left panel). (b) Quantification of pHusion-positive puncta in the root cells illustrated in (a) reveals gradual upregulation of autophagic activity in the roots mounted on microscopy glass. The chart comprises representative data from one out of three individual experiments. Two-tailed t-test with unequal variants, n = 35; *, p-value < 0.05.
+
+![41598_2024_63226_Fig2_HTML.webp](dataset/41598_2024_63226_Fig2_HTML.webp)
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assays/FluoresceinDiffusionAssay/README.md

+
+# Fluorescein Diffusion Assay
+
+## Figure 3
+
+Diffusion of chemical compounds in the RoPod. (a) Representative time lapse images of roots treated with fluorescein in the RoPod. Arabidopsis seedlings were grown in RoPods using the described protocol. Liquid MS medium containing 2 μg.ml-1 of fluorescein dye was added to the chambers immediately prior to the start of imaging. Maximum intensity projections of the fluorescent signal are shown on the top row, corresponding transmitted light images are shown in the bottom row. The circles indicate three types of regions of interest (ROI) analyzed for each root hair to produce the data presented in (b). Magenta circle, root hair tip; green circle, root hair base; white circle, background. (b) Quantification of the data illustrated in (a). Diffusion rate of fluorescein in the RoPod chamber demonstrates that chemical compounds reach root hair cells within 15 min of application. Data for two independent experiments is plotted for each type of analyzed ROI. (c) Dynamics of fluorescence accumulation at the bottom coverslip of RoPod. In this experiment liquid MS medium containing 140 μg.ml−1 fluorescein was added to RoPods containing only growth medium. The fluorescence was recorded in four separate chambers, with 2 to 3 fields of view chosen at random positions in each RoPod.
+
+![41598_2024_63226_Fig3_HTML.webp](dataset/41598_2024_63226_Fig3_HTML.webp)
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assays/PlantGrowthAndImageAnalysisOfSucroseTreatmentAssay/README.md

+
+# Plant Growth And Image Analysis Of Sucrose Treatment Assay
+
+## Movie S2. Time-lapse video of root hair growth response to sucrose treatment.
+
+Representative video of Col-0 WT seedlings grown in RoPod5 on a vertical microscope and treated
+with 0.5x MS (control – left panel) or 0.5x MS supplemented with 1 % sucrose (right panel).
+
+![41598_2024_63226_MOESM2_ESM.mp4](dataset/41598_2024_63226_MOESM2_ESM.mp4)
+
+## Figure 4
+
+Monitoring sucrose effect on root hair elongation illustrates applicability of RoPod for long-term time-lapse imaging combined with treatments. Arabidopsis Col-0 WT seedlings were grown for one week in a RoPod5 using the described protocol. The growth of root hairs was firstly recorded for 4 h under control conditions. After that, the chambers were flooded with liquid 0.5 × MS (control treatment) or liquid 0.5 × MS supplemented with 1% of sucrose (sucrose treatment). (a) Representative images showing roots at 0 h and 8 h of treatment. Each panel is a z-projection covering 240 µm. (b,c) Result of root hair tip tracking for one representative root for control (b) and for sucrose (c) treatments. The final root hair length (d), the root hair growth rate (e) and the growth duration (f) were calculated as described in the Supplementary Methods. (d–f) The sample size used for each measurement is shown in the tables below the charts. A Dunn’s multiple comparison test, different letters designate significantly different groups, p < 0.01. The dot in the violin plots is the median of the distribution.
+
+![41598_2024_63226_Fig4_HTML.webp](dataset/41598_2024_63226_Fig4_HTML.webp)
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assays/RootLengthMeasurement/README.md

+
+# Root Length Measurement
+
+## Movie S1. Arabidopsis seedlings grown in a RoPod chamber have normal phenotype.
+
+The time-lapse movie of root growth illustrated and quantified in the Fig.1g-i. Seed germination and
+root elongation of seedling grown in the RoPod v24.3 are comparable to the growth under or on
+top of the growth medium in a Petri plate. Additionally, similar growth of wild-type WT) plants
+corroborates that RoPod provides favourable growth conditions for Arabidopsis plants. Images
+were acquired using SPIRO.
+
+
+![41598_2024_63226_MOESM1_ESM.mp4](assays/RootLengthMeasurement/dataset/41598_2024_63226_MOESM1_ESM.mp4)
+
+## Figure 1g-i
+
+RoPod, a toolkit comprising plant growth protocol and microscopy chamber optimized for long-term time-lapse imaging of Arabidopsis roots.
+
+(g–i) Growth conditions in RoPod chambers are equivalent to standard growing conditions on Petri plates, as demonstrated by comparing root growth on the top of the medium, under the medium inside the standard Petri plate and in RoPod chambers. Seeds of wild-type (WT) and autophagy-deficient atg5-1 Arabidopsis plants expressing GFP-ATG8 were sown on the agar medium in Petri plates or RoPods. Plates and chambers were placed on SPIRO under long day conditions and imaged for a week. (h) Zoomed-in insets shown in (g). (i) The chart represents data from two independent experiments. The root lengths were measured on the 7th day after seed plating. One-Way ANOVA test revealed no difference in root length of seedlings for the WT seedlings grown in RoPods and on Petri plates, significance level of 0.05, n = 103.
+
+![Figure1g-i.png](dataset/Figure1g-i.png)