Iron availability modulates the Arabidopsis thaliana root calcium signature evoked by exogenous ATP

Abstract

Plants use changes in cytosolic free Ca²⁺ ("signatures") to encode information from the specific signals generated in development, immunity and stress perception. Phosphate availability has a significant impact on the Arabidopsis thaliana root calcium signatures generated in response to abiotic stress stimuli and exogenous purine nucleotides. In the case of the response to exogenous ATP, the effect of low phosphate availability is linked to abnormal iron and reactive oxygen species accumulation with iron deprivation's restoring normal signature dynamics. Here, the effect of iron deprivation with normal phosphate availability has been examined. Iron deprivation significantly alters the root calcium signature evoked by exogenous ATP and may link to levels of reactive oxygen species and callose deposition.

Keywords: ATP; calcium; callose; iron; phosphate; reactive oxygen species; wave.

Figure 1.

The [Ca²⁺]_cyt response of Fe-starved root tips to exogenous ATP. Col-0 aequorin-expressing seedlings were grown on standard half MS growth medium (“full P_50 µM Fe”; green trace), “zero P_50 µM full Fe” (blue trace) or “full P_zero Fe” (pink trace). Root tips (1 cm) of 11-day-old seedlings were challenged with treatments applied at 35 s, and [Ca²⁺]_cyt was measured for 155 s. (a) Application of control solution (liquid growth medium corresponding to that used for growth); time course trace represents mean ± standard error of the mean (SEM) from 3 to 6 independent trials, with n= 33–35 individual root tips averaged per datapoint. Time course data were analyzed for (b) touch maxima and (c) area under the curve (AUC), all baseline-subtracted, with each dot representing an individual data point.⁴ Boxplot middle line denotes median. (d–h) Responses to 1 mM eATP (3–6 independent trials, n = 33–61 root tips per growth condition). Analysis of variance (ANOVA) with post-hoc Tukey Test was used to assess statistical differences. Significance levels (p-values): *** (<0.001), ** (<0.01), * (<0.05), n.s. (not significant).

Figure 2.

Callose distribution in phosphate- and iron-starved primary root tips. Arabidopsis Col-0 were grown on growth medium with different P and Fe levels: (a) full P_50 µM Fe, (b) zero P_50 µM Fe, (c) zero P_zero Fe, (d) full P_zero Fe. Ten- to 11-day-old seedlings were stained with 0.01 % (w/v) aniline blue before bright field (on the left) and UV fluorescence images (on the right) were captured with a stereomicroscope. (a) Scale bar: 0.5 mm, white and red scale bars indicate regions scored for callose presence. (b) White triangles indicate unspecific background signal. (e). Regions analyzed for the presence of distinct callose spots: 0–500 µm from the root tip, or 1000–1500 µm from the root tip (yellow-dashed boxes). Scale bar: 0.5 mm. F. Heat map (bottom) color-codes percentage of roots scored for the presence of callose depositions (darker color indicates more callose deposition). Three independent trials were conducted, n = 13–16 individual roots per growth condition.