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.

Cytosolic free calcium ([Ca²⁺]_cyt) is a second messenger in plant development, abiotic stress signaling and immunity.^1,2 Stimulus-specific [Ca²⁺]_cyt elevations (“signatures”) are decoded by arrays of Ca²⁺-binding proteins to evoke specific physiological or transcriptional responses.³ It was recently reported that phosphate (P) availability has a significant impact on Arabidopsis thaliana root [Ca²⁺]_cyt signatures.⁴ P deprivation resulted in dampening of the root [Ca²⁺]_cyt signatures evoked by mechanical stress, peroxide, NaCl and its equivalent hyperosmotic stress, and also by exogenous purine nucleotides (eATP and eADP).

Extracellular purine nucleotide signaling is involved in wounding, immunity, growth regulation and guard cell dynamics.⁵ eATP evoked a biphasic [Ca²⁺]_cyt increase in Arabidopsis root tips under full P nutrition (determined using cytosolic aequorin or YC3.6 as genetically encoded [Ca²⁺]_cyt reporters).⁴ The first [Ca²⁺]_cyt increase in response to eATP occurred at the apex and the second [Ca²⁺]_cyt peak was sub-apical. P deprivation resulted in the loss of the second [Ca²⁺]_cyt peak, which correlated with an increased level of cytosolic reactive oxygen species (ROS).⁴ Removing iron (Fe), but not copper, from the growth medium not only restored the second eATP-induced [Ca²⁺]_cyt peak under P deprivation but also restored normal cytosolic ROS in that region. As P deprivation causes abnormal Fe accumulation in roots,⁶ these results are consistent with an Fe overload’s driving abnormal ROS accumulation^6–9 that would be inhibitory to the second eATP-induced [Ca²⁺]_cyt elevation. Fe availability could, therefore, have a significant role to play in [Ca²⁺]_cyt signaling. In this addendum, the effect of Fe deprivation under P-replete conditions on the root eATP-induced [Ca²⁺]_cyt signature has been investigated.

Using the protocol described previously,⁴ root tips (1 cm) were excised from Arabidopsis roots expressing cytosolic aequorin that had either been grown on replete medium (“full P_50 µM Fe”), or P-starved but with normal Fe medium (“zero P_50 µM Fe”) or grown on P-replete medium and Fe-starved (“full P_zero Fe”). No significant differences were found in root response to mechanical stimulation by addition of control medium (“touch response”; Figure 1(a–c)); p ≥ 0.223 for all comparisons). Treatment with 1 mM eATP led to the characteristic [Ca²⁺]_cyt response (touch followed by eATP-induced biphasic increase) in nutrient-replete root tips, which was strongly knocked down in P-starved root tips (zero P_50 µM Fe; Figure 1(d–h)), in agreement with the previous study.⁴ Fe-starved root tips (full P_zero Fe) retained the multiphasic response but with markedly different dynamics. Fe starvation caused a significantly increased touch response and first eATP-induced [Ca²⁺]_cyt increase compared to nutrient-replete root tips (Figure 1(d–f)). In contrast, Fe-starved roots tips had a significantly lower second eATP-induced peak [Ca²⁺]_cyt increase compared to nutrient-replete tips which were also delayed by more than 10 s (Figure 1(d,g)). Although the dynamics of the [Ca²⁺]_cyt response were perturbed by Fe starvation compared to nutrient-replete conditions, the total [Ca²⁺]_cyt mobilized (estimated by the area under the curve) was not (Figure 1(h)).

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).

Root lengths of Fe-starved plants were significantly shorter (3.39 ± 0.07 cm; p < 0.05) than nutrient-replete plants but not P-starved plants,⁴ suggesting that growth was not a determining factor in the [Ca²⁺]_cyt responses. However, analysis of cytosolic ROS accumulation using 20 µM CM-H₂DCFDA⁴ revealed lower levels in Fe-starved roots compared to those grown on nutrient-replete medium (Table 1). The first eATP-induced [Ca²⁺]_cyt elevation occurs in the first apical millimeter of the root tip.⁴ It may be that lower Fe content there from Fe-starvation prevents normal ROS accumulation and any ROS-inhibition of the [Ca²⁺]_cyt increase is lessened. This would be at odds with current models² in which ROS increase [Ca²⁺]_cyt but that paradigm could hold true for the second [Ca²⁺]_cyt peak. If so then the lower sub-apical ROS in Pi-replete but Fe-starved root tips would not support a normal second eATP-induced [Ca²⁺]_cyt elevation, and this was indeed observed (Figure 1(d)).

Table 1.

Fluorescence intensity values from ROS-reporting CM-H₂DCFDA measured at set distances from the root apex. Plants were grown on medium as indicated and fluorescence quantified as described.⁴ Data for “Full P_50 µM Fe” and “Zero P_50 µM Fe” are derived from published results.⁴ n is the number of roots sampled in three independent trials.

	Fluorescence intensity
	100 µm from apex		1000 µm from apex		2000 µm from apex
Growth condition	Mean	± SEM	Mean	± SEM	Mean	± SEM	n
Full P_50 µM Fe	3.25	0.44	28.30	6.39	13.70	2.57	15
Full P_zero Fe	1.74	0.19	13.95	2.61	3.45	0.85	14
Zero P_50 µM Fe	21.28	14.44	115.93	11.06	42.01	3.45	15

Callose deposition has recently been reported to occur due to P starvation-induced Fe accumulation, and resulting ROS overload, in Arabidopsis primary roots, with the possibility that it impairs symplastic communication^7–9. To investigate if callose might influence the eATP [Ca²⁺]_cyt signature, aniline blue was used to resolve callose deposits as a function of growth regime. This data set included “zero P-zero Fe”, which results in restoration of normal ROS and the second eATP [Ca²⁺]_cyt peak⁴ compared to zero P_50 µM Fe. Fluorescence emission revealed a strong and distinct pattern of callose deposition in most samples (Figure 2). Nutrient-replete roots showed small, but distinct fluorescence emissions, spread out over the apical root tip (0–0.5 mm), and centering more to the stele further up the root (Figure 2(a)). P-starved but Fe-replete root tips showed a signal comparable to nutrient-replete plants (Figure 2(b)). This contradicts a previously published result⁷ of increased callose deposition under P starvation.

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.

Strikingly, the fluorescence emission was much lower in roots that were grown without Fe, regardless of whether P was included or excluded from the medium (Figure 2(c,d)). The single variable correlating with less callose in the root tip, and almost no callose deposits further up the root (1–1.5 mm), was the availability of Fe in the medium. Both full P_zero Fe and zero P_zero Fe roots showed this pattern of low callose occurrence. This is in broad agreement with a study¹⁰ in which low Fe correlated with reduced callose deposition. There is no qualitative correlation between callose deposition, ROS and the magnitude of the second eATP [Ca²⁺]_cyt peak (although it should be noted that callose quantification here has been crude). However, the lower callose at the apex in full P_zero Fe roots does match with lower ROS and a higher first eATP-induced [Ca²⁺]_cyt increase. The recent development of an estradiol-inducible system to overproduce callose in specific tissues is promising,¹¹ and if coupled with a [Ca²⁺]_cyt reporter, could elegantly show if callose deposition affects cell-to-cell dependent [Ca²⁺]_cyt signaling. This would be particularly relevant to understanding the basis of [Ca²⁺]_cyt “waves” that can travel from the apex to signal to shoot tissue.¹²

Overall, a picture emerges of fine control of eATP-induced [Ca²⁺]_cyt elevation by Fe availability. The mechanistic basis of the first [Ca²⁺]_cyt increase at the apex may well differ markedly from that of the second that occurs sub-apically. At the apex under P-replete conditions, Fe-driven ROS may well dampen the [Ca²⁺]_cyt elevation but sub-apically they are required for a normal second [Ca²⁺]_cyt peak. Under Pi-deprivation, too high a level of Fe-driven ROS inhibits both phases of the eATP signature. Dissecting out the components of these signals will be challenging but progress has been made in characterizing the root epidermal plasma membrane Ca²⁺ influx channels that respond to eATP^13–15. It remains to be seen what role Fe, ROS and callose have to play in the modulation of other stress-induced root [Ca²⁺]_cyt signatures.

Funding Statement

Support for this work was from the BBSRC (BB/J0145401/10) and the University of Cambridge Broodbank Trust.

Abbreviation

[Ca²⁺]_cyt

cytosolic free calcium

Fe

iron

P

phosphate

ROS

reactive oxygen species