Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots^,

Phosphate starvation, but not nitrogen starvation, changes the cytosolic free calcium signatures of Arabidopsis thaliana roots in response to mechanical, salt, osmotic, and oxidative stress as well as to extracellular nucleotides.

Abstract

Phosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca²⁺ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca²⁺]_cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca²⁺]_cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca²⁺]_cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca²⁺]_cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca²⁺]_cyt, Pi starvation strongly affects stress-induced [Ca²⁺]_cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca²⁺ as a signal transducer.

Plant roots foraging in the soil have to sense, transduce, and respond to fluctuations in water and nutrients plus a multitude of stresses they may additionally be subjected to. Plants employ a wide range of signal transducers, with free calcium ion (Ca²⁺) being a common second messenger in the response to stress stimuli. Biotic and abiotic stresses (including mechanical, salt, osmotic, and oxidative stress) trigger rapid and transient modulations in cytosolic and organellar free Ca²⁺ (Knight et al., 1991, 1992, 1997a; Kiegle et al., 2000; Monshausen et al., 2009; Loro et al., 2012, 2016; Bonza et al., 2013; Laohavisit et al., 2013; Behera et al., 2018; Manishankar et al., 2018). These Ca²⁺ signatures are held to be specific to the stimulus and result in stimulus-specific outcomes, enabled by suites of decoding proteins (Whalley et al., 2011; Whalley and Knight, 2013; Liu et al., 2015; Lenzoni et al., 2018).

Recently, Ca²⁺ has been described as also functioning in signaling nutrient status and availability. In the model plant Arabidopsis (Arabidopsis thaliana), nitrate resupply to initially nitrate-starved roots prompted a rapid (within seconds) and monophasic increase in cytosolic free Ca²⁺ ([Ca²⁺]_cyt), followed by an increase in nuclear [Ca²⁺] (Riveras et al., 2015; Liu et al., 2017). Potassium (K⁺) deficiency was found to trigger two distinct [Ca²⁺]_cyt elevations within the Arabidopsis root, the first response occurring within 1 to 4 min and the second response within 18 to 32 h after the onset of K⁺ deficiency (Behera et al., 2017). [Ca²⁺]_cyt signaling was further found to be necessary for vacuolar magnesium (Mg²⁺) detoxification in a high Mg²⁺ environment (Tang et al., 2015).

Another nutrient of great importance for optimal plant growth is inorganic phosphate (Pi). Pi-limited conditions are known to induce profound changes in plant growth and metabolism. On a systemic level, root growth is favored over shoot growth, indicative of maximizing soil exploration and, thus, nutrient uptake (Gruber et al., 2013). In addition, root system architecture is remodeled, which has been well described in many crop species, such as barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and tomato (Solanum lycopersicum), as well as Arabidopsis (Péret et al., 2014). Multicomponent nutrient studies found Pi to be the predominant nutrient controlling primary root length (Gruber et al., 2013; Kellermeier et al., 2014). On a cellular level, metabolism shifts to alternative pathways that consume less Pi (Pratt et al., 2009; Plaxton and Tran, 2011; Nakamura, 2013; Pant et al., 2015). The Pi deficiency response is orchestrated by intricate signaling networks, involving hormones, nutrient cross talk, and transcriptional and translational feedback loops (for review, see Abel, 2017; Chien et al., 2018). Ca²⁺ has been hypothesized to be involved as a signal transducer (Chiou and Lin, 2011; Linn et al., 2017; Chien et al., 2018), but this has not been confirmed to date.

While the involvement of Ca²⁺ as a second messenger in nutrient signaling is now beginning to be explored, few studies have examined the impact of nutrient deficiency on Ca²⁺ signaling per se. Boron-deprived tobacco (Nicotiana tabacum) BY-2 cells (expressing cytosolic aequorin as a luminometric reporter of [Ca²⁺]_cyt) sustained an enhanced [Ca²⁺]_cyt signature when challenged with 3 mM Ca²⁺ compared with boron-replete cells (Koshiba et al., 2010). Boron deficiency caused an increase in steady-state [Ca²⁺]_cyt of Arabidopsis roots, but the consequences for stress-induced Ca²⁺ signatures were not examined (Quiles-Pando et al., 2013).

Thus, we set out to test how Pi starvation of Arabidopsis might influence the root’s use of Ca²⁺ as a signal, employing a range of abiotic stresses known to evoke robust, rapid, and transient [Ca²⁺]_cyt signatures (mechanical, salt, osmotic, and oxidative stress). Additionally, as mechanical stimulation, salt, and osmotic stress increase the accumulation of extracellular ATP (eATP; Weerasinghe et al., 2009; Dark et al., 2011), which in turn transiently increases [Ca²⁺]_cyt (Demidchik et al., 2003; Tanaka et al., 2010a, 2010b; Costa et al., 2013; Choi et al., 2014), we also tested the root response to extracellular purine nucleotides.

We show that Pi starvation, but not nitrogen (N) starvation, strongly altered [Ca²⁺]_cyt signatures in response to all abiotic stresses and extracellular nucleotides tested. Furthermore, Pi-starved root apices of Arabidopsis showed a distinct spatiotemporal [Ca²⁺]_cyt response to eATP. This was remodeled during development and was dependent on iron (Fe) availability and reactive oxygen species (ROS) production but could be reversed by Pi resupply. These data reveal how nutritional status adds another so far unexplored level of complexity to the use of [Ca²⁺]_cyt as a signal transducer and further our understanding of how plants integrate various environmental cues.

RESULTS

Pi, But Not N, Starvation Dampens the [Ca²⁺]_cyt Response to Abiotic Stresses in Arabidopsis Root Tips

To determine if Pi starvation alters stress-induced [Ca²⁺]_cyt signatures, Arabidopsis seedlings ubiquitously expressing cytosolic (apo)aequorin were grown on Pi-replete conditions (0.625 mM KH₂PO₄; full Pi), lowered Pi conditions (0.1 mM KH₂PO₄; medium Pi), or chronically starved of Pi (0 mM KH₂PO₄; zero Pi). As has been reported previously (Williamson et al., 2001; Svistoonoff et al., 2007; Balzergue et al., 2017), Pi starvation for 10 d led to significantly shorter primary roots (mean root length ± se: full Pi, 6.01 ± 0.06 cm; medium Pi, 4.42 ± 0.04 cm; zero Pi, 2.69 ± 0.04 cm [P < 0.001 for all comparisons]; Supplemental Table S1). To account for differences in root growth and architecture induced by Pi growth regime, a fixed length of the primary root (the first 1 cm from the apex) from an 11-d-old seedling was challenged with acute abiotic stress and aequorin luminescence was recorded every 1 s for 155 s (Fig. 1). As the experimental setup necessitates the injection of treatment solutions, control solution treatments were run with every set of experiments to control for the effect of mechanical stimulation.

Figure 1.

Pi-starved root tips have a dampened [Ca²⁺]_cyt response to mechanical, salt, and osmotic stress. Arabidopsis Columbia-0 (Col-0) aequorin-expressing seedlings were grown on full, medium (med), or zero Pi (green, purple, and blue traces, respectively). Individual root tips (1 cm) of 11-d-old seedlings were challenged with treatments applied at 35 s (black arrows), and [Ca²⁺]_cyt was measured for 155 s. A, Mechanical stimulation (control solution). Time-course traces represent means ± se from 18 independent trials, with n = 150 to 155 root tips per growth condition averaged per data point. B and C, Time-course data were analyzed for touch maxima (B) and area under the response curve (AUC; C), both baseline subtracted, with each dot representing an individual data point (see Supplemental Fig. S1 for details). The middle line in the box plot denotes the median. D to F, Responses to 150 mM NaCl (three independent trials, n = 35–36 root tips). G to I, Responses to 280 mM sorbitol (three independent trials, n = 22–24 root tips). ANOVA with posthoc Tukey’s test was used to assess statistical differences; different lowercase letters denote significant differences (P < 0.05).

An immediate and monophasic increase in [Ca²⁺]_cyt was observed upon mechanical stimulation (application of control solution; Fig. 1, A–C), salt stress (150 mM sodium chloride [NaCl]; Fig. 1, D–F), or an equivalent osmotic stress (280 mM d-sorbitol; Fig. 1, G–I). The response to mechanical stimulation was variable in root tips of all Pi growth conditions, likely due to limitations of the experimental setup, but overall, the Pi-starved root tip response was significantly lower. The response to salt and osmotic stress was as immediate as the response to mechanical stimulation, but of much greater amplitude and duration in root tips from all Pi growth conditions. In all cases, Pi starvation significantly dampened the stress-induced [Ca²⁺]_cyt response (as quantified by peak maxima [Fig. 1, B, E, and H] and area under the curve [as a proxy of how much [Ca²⁺]_cyt was mobilized upon stress treatment; Fig. 1, C, F, and I]; see Supplemental Fig. S1 for details). For all stress experiments, Pi-replete root tips showed the strongest [Ca²⁺]_cyt response, medium Pi root tips showed a moderately dampened [Ca²⁺]_cyt response, and Pi-starved root tips showed the most strongly impaired [Ca²⁺]_cyt response.

To test if this dampened [Ca²⁺]_cyt response was specific to Pi nutrition or due to a more general nutrient deficiency response, we assayed root tips starved of another macronutrient, N. Primary root lengths of severely N-starved plants (0 mM N) were comparable to zero Pi-grown roots (zero N, 2.5 ± 0.13 cm [P = 0.203]; Supplemental Table S1). However, N-starved root tips showed a [Ca²⁺]_cyt response to mechanical stimulation, salt, and osmotic stress that was similar to that of nutrient-replete root tips (Supplemental Fig. S2). These results indicate that Pi nutrition specifically alters the [Ca²⁺]_cyt response to abiotic stresses.

Pi Starvation Alters the Root Tip [Ca²⁺]_cyt Response to Extracellular Nucleotides and ROS

Mechanical stimulation, salt, and osmotic stress are known to evoke the accumulation of eATP, which in turn triggers increases in ROS (Kim et al., 2006; Song et al., 2006; Demidchik et al., 2009, 2011; Chen et al., 2017), and ROS such as hydrogen peroxide (H₂O₂) induce [Ca²⁺]_cyt increases (Price et al., 1994; Rentel and Knight, 2004; Demidchik et al., 2007; Richards et al., 2014). Therefore, we next challenged aequorin-expressing root tips from 11-d-old seedlings, grown on full/medium/zero Pi conditions, with 0.1 or 1 mM eATP. eATP treatment evoked robust multiphasic [Ca²⁺]_cyt increases in full Pi-grown root tips (Fig. 2), as reported previously (Demidchik et al., 2003; Tanaka et al., 2010b). The different phases of the response were classified as an immediate touch response (0–6 s after treatment application), followed by peak 1 (7–28 s after treatment application) and subsequent peak 2 (29–120 s after treatment application; see Supplemental Fig. S1 for details). The touch response elicited by eATP was variable but overall not significantly different from the application of control solution alone (Fig. 1A) and was nonresponsive to an increase in eATP concentration (0.1 mM eATP [Fig. 2B] and 1 mM eATP [Fig. 2G]), indicating that this initial response was due to the mechanical stimulation of treatment application rather than to eATP perception.

Figure 2.

Pi-starved root tips show a dampened [Ca²⁺]_cyt response to eATP. Arabidopsis Col-0 aequorin-expressing seedlings were grown on full, medium (med), or zero Pi (green, purple, and blue traces, respectively). Individual root tips (1 cm) of 11-d-old seedlings were challenged with treatments applied at 35 s (black arrows), and [Ca²⁺]_cyt was measured for 155 s. A, Treatment with 0.1 mM ATP. Time-course traces represent means ± se from six independent trials, with n = 34 to 36 individual root tips averaged per data point. B to E, Time-course data were analyzed for touch maxima (B), peak 1 maxima (C), peak 2 maxima (D), and area under the response curve (AUC; E), all baseline subtracted, with each dot representing an individual data point (see Supplemental Fig. S1 for details). The middle line in the box plot denotes the median. F to J, Responses to 1 mM ATP (five independent trials, n = 27–45 root tips per growth condition). ANOVA with posthoc Tukey’s test was used to assess statistical differences; different lowercase letters denote significant differences (P < 0.05).

In contrast, the subsequent [Ca²⁺]_cyt increases (defined as peak 1 and peak 2) were specific to eATP treatment, and their magnitude was dependent on root Pi status. Peak 1 maxima were similar between full Pi- and medium Pi-grown root tips but significantly dampened in zero Pi-grown root tips in response to 0.1 mM ATP (Fig. 2C) and 1 mM ATP (Fig. 2H). Peak 2 maxima were significantly dampened in medium Pi root tips compared with full Pi root tips, with zero Pi root tips mostly lacking any apparent [Ca²⁺]_cyt increase within that phase (Fig. 2, D and I). Overall, the more Pi starved the roots, the less [Ca²⁺]_cyt was mobilized in response to eATP (based on the area under the response curves; Fig. 2, E and J). While increasing the eATP concentration 10-fold (from 0.1 to 1 mM eATP) significantly increased the [Ca²⁺]_cyt mobilized in full Pi root tips, medium and zero Pi root tips were insensitive to an increase in eATP concentration.

As up to 2 mM Pi could potentially be liberated readily from 1 mM eATP, and this Pi pulse itself might evoke a [Ca²⁺]_cyt response, we treated root tips with a Pi source alone. Application of 2 mM Pi led to a rapid and monophasic increase in [Ca²⁺]_cyt, very similar in shape, duration, and amplitude to control solution treatment across all three Pi growth regimes (Supplemental Fig. S3). This indicated that under the Pi starvation conditions tested, Pi alone did not trigger an increase in [Ca²⁺]_cyt, in contrast to what had been reported for nitrate resupply in a similar setup (Riveras et al., 2015). To further test if ATP hydrolysis played a role in the differing [Ca²⁺]_cyt signatures, ADP and a nonhydrolyzable ATP analog (adenosine 5′-[γ-thio]triphosphate tetralithium [γ-ATP]) were applied to root tips from the different Pi growth regimes. ADP and γ-ATP treatment (1 mM) resulted in [Ca²⁺]_cyt signatures strikingly similar to those observed with 1 mM eATP treatment (Supplemental Figs. S4 and S5), indicating that ATP hydrolysis could not mechanistically explain the altered [Ca²⁺]_cyt signature of Pi-starved roots.

Next, we applied ROS, as 1 and 5 mM H₂O₂, to excised root tips. This treatment induced a rapid increase in [Ca²⁺]_cyt followed by a pronounced secondary increase (Supplemental Fig. S6). Full Pi- and medium Pi-grown root tips showed a similar [Ca²⁺]_cyt response, while zero Pi-grown root apices showed a significantly dampened secondary response to both H₂O₂ concentrations tested (Supplemental Fig. S6). N-starved root apices did not show a dampened response to either 1 mM eATP (Supplemental Fig. S7) or 1 mM H₂O₂ (Supplemental Fig. S8), again indicating that the severely altered [Ca²⁺]_cyt signature observed in Pi-starved roots was not a general response to nutrient starvation but a consequence specific to Pi nutrition.

Pi-Starved Roots Respond to eATP at the Root Apex, But a Secondary [Ca²⁺]_cyt Response in the Distal Region Is Lost

As the aequorin-based [Ca²⁺]_cyt determinations did not allow spatial resolution, we employed ratiometric imaging to map the [Ca²⁺]_cyt response of the root. Arabidopsis Col-0 constitutively expressing the ratiometric [Ca²⁺]_cyt reporter Yellow Cameleon YC3.6 (nuclear export signal [NES]-YC3.6; Krebs et al., 2012) was grown on full Pi or zero Pi, and intact 10-d-old plants were mounted into a custom-built perfusion chamber (Behera and Kudla, 2013) with shoots exposed to air and roots constantly superfused with control imaging solution. This constant superfusion system circumvented mechanical stimulation due to treatment injection (present in aequorin-based assays), excluding any touch response. As eATP application had so far produced the most prominent Pi-dependent [Ca²⁺]_cyt response and was at the crossroads of signaling in other stress pathways, we used it as the standard treatment from here onward.

In Pi-replete roots of NES-YC3.6-expressing seedlings (Fig. 3A), superfusion with 1 mM eATP led to a strong increase in cpVenus/cyan fluorescent protein (CFP) ratio over prestimulus levels in the apex (approximately within the first 1 mm), which was sustained over the period of eATP treatment (3 min; visualized as a representative kymograph in Figure 3B). Approximately 30 to 40 s after this initial response, a secondary increase in ratio occurred in the more distal part of the root tip (1 mm or more from the root apex), which appeared to propagate along the root (Fig. 3B; Supplemental Movie S1). These two distinct increases in [Ca²⁺]_cyt were reminiscent of what had been termed peak 1 and peak 2 in aequorin-based assays (Fig. 2).

Figure 3.

eATP elicits two spatiotemporally distinct [Ca²⁺]_cyt increases in the root, which are altered by Pi starvation. A, Representative full Pi-grown Arabidopsis Col-0 root expressing NES-YC3.6. The white dashed line in the root micrograph indicates the line used for kymograph extraction. B, Kymograph depicts temporal and spatial changes in [Ca²⁺]_cyt of a representative full Pi-grown root in response to a 3-min 1 mM eATP treatment (purple bar), preceded and followed by superfusion with control imaging solution. C and D, Representative zero Pi-grown root. White triangles indicate secondary increase in [Ca²⁺]_cyt in the full Pi root (B), which is missing in the zero Pi root (marked by the white star; D). Bars = 1 mm.

Pi-starved roots (Fig. 3C) sustained a similar increase of cpVenus/CFP ratio at the root apex in response to 1 mM eATP. In more distal parts of the roots (1 mm or more from the root apex), Pi-starved roots only showed a slight or no ratio increase at all in response to eATP treatment (Fig. 3D, representative kymograph; Supplemental Movie S2). This lack of a secondary response resembled the absence of peak 2 in Pi-starved aequorin-expressing root tips (Fig. 2). Quantifying the ratiometric changes occurring in response to eATP in specific regions of interest (ROIs) along the root (apical 2.5 mm; see micrograph in Fig. 4) corroborated these observations. Pi starvation effectively abolished an eATP-induced [Ca²⁺]_cyt elevation in more distal regions (Fig. 4, A–D) and dampened it nearer to the apex (Fig. 4, E and F). However, at the apex, Pi starvation had no effect on the eATP-induced [Ca²⁺]_cyt elevation (Fig. 4, G and H; n = 7–9 roots per growth condition), indicating that Pi-starved roots were not impaired in perceiving eATP per se.

Figure 4.

Quantification of differential [Ca²⁺]_cyt responses of Pi-starved roots to eATP. Arabidopsis Col-0 expressing NES-YC3.6 was grown on full or zero Pi. In a superfusion chamber, a root of a 10-d-old seedling was superfused with imaging solution before switching to 1 mM eATP (applied 50–230 s after the start of image acquisition; purple shading), followed by washout with imaging solution. On the left, a representative root with annotated ROIs (Roi; white boxes), analyzed and plotted over time in A to H: Roi D (A and B); Roi C (C and D); Roi B (E and F); and Roi A (G and H). A, C, E, and G, Mean Förster resonance energy transfer (FRET) ratio (cpVenus/CFP) ± se, background subtracted. B, D, F, and H, Normalized FRET ratio (ΔR/R₀) ± se of full Pi roots (green traces) and zero Pi roots (blue traces). Data from three independent trials are shown, with n = 7 to 9 individual roots per growth condition. Bar in root image at left = 1 mm.

The Altered [Ca²⁺]_cyt Signature Manifests during Prolonged Pi Starvation and Is Reversed by Pi Resupply

To place the response of Pi-starved root tips into developmental context, we next tested aequorin-expressing Arabidopsis of different ages for an altered [Ca²⁺]_cyt response to eATP. The youngest root material tested (on day 6) did not yet significantly differ in primary root length between full Pi- and zero Pi-grown plants (mean primary root length ± se: full Pi, 1.99 ± 0.08 cm; zero Pi, 1.83 ± 0.04 cm [P = 0.908]). In 7-d-old (or older) material, a significantly shorter primary root was observed in Pi-starved seedlings (Supplemental Table S1).

Assaying 6-, 7-, 8-, and 11-d-old full Pi-grown root tips (1 cm) replicated the characteristic multiphasic [Ca²⁺]_cyt response to 1 mM eATP throughout development (Fig. 5, A–D), as well as eliciting [Ca²⁺]_cyt responses of comparable magnitude (quantified as area under the response curves; Fig. 5E). In contrast, 6-d-old Pi-starved root tips showed a [Ca²⁺]_cyt response comparable in shape to the response of Pi-replete tips, but it was dampened in magnitude (Fig. 5A). Seven- and 8-d-old Pi-starved tips (1 cm) showed a much dampened peak 2, which was completely absent in 11-d-old tips (Fig. 5, B–D). Concomitantly, Pi-starved tips showed a decrease in total mobilized [Ca²⁺]_cyt, with prolonged growth on zero Pi medium (Fig. 5E).

Figure 5.

Pi starvation modulates the root tip [Ca²⁺]_cyt response to eATP during development. Arabidopsis Col-0 aequorin-expressing seedlings were grown on full or zero Pi (green and blue traces, respectively). A to D, Root tips (1 cm) of 6-d-old (A), 7-d-old (B), 8-d-old (C), or 11-d-old (D) seedlings were challenged with 1 mM eATP applied at 35 s (black arrows), and [Ca²⁺]_cyt was measured for 155 s. Time-course traces represents means ± se from three to five independent trials, with n = 13 to 45 individual root tips averaged per data point. E, Time-course data were analyzed for area under the response curve, baseline subtracted, with each dot representing an individual data point (see Supplemental Fig. S1 for details). The middle line in the box plot denotes the median. ANOVA with posthoc Tukey’s test was used to assess statistical differences; different lowercase letters indicate groups of significant statistical difference (P < 0.05), and the same letters indicate no statistical significance (P > 0.05).

To test if the dampened and altered [Ca²⁺]_cyt response of Pi-starved root tips could be rescued by Pi resupply or was irreversibly lost, we grew seedlings on zero Pi medium until the altered [Ca²⁺]_cyt response to eATP would have manifested (day 8). On day 8, seedlings were (1) not transferred, (2) transferred to zero Pi growth medium (representing a transfer control), or (3) transferred to full Pi growth medium and grown for another 2 d. Pi-starved root tips (both not transferred and transferred to zero Pi) showed a dampened [Ca²⁺]_cyt response to 1 mM eATP, with peak 2 being mostly absent. In contrast, seedlings that had been resupplied with Pi (zero Pi-to-full Pi transfer) showed a clear multiphasic [Ca²⁺]_cyt response to eATP (Fig. 6A). Resupply of Pi significantly rescued the amplitude of peak 1 and peak 2 as well as the overall response magnitude (Fig. 6, C–E).

Figure 6.

Resupply of Pi to Pi-starved seedlings rescues the dampened root tip [Ca²⁺]_cyt response to eATP. Arabidopsis Col-0 expressing aequorin was grown on zero Pi for 8 d, when plants were (1) not transferred (zero Pi no transfer), (2) transferred to zero Pi growth medium (zero Pi to zero Pi), or (3) transferred to full Pi growth medium (zero Pi to full Pi). After 2 d, individual excised root tips (1 cm) were challenged with treatments applied at 35 s (black arrow), and [Ca²⁺]_cyt was measured for 155 s. A, Application of 1 mM ATP. Time-course traces represents means ± se from three independent trials, with n = 15 to 28 individual root tips averaged per data point. B to E, Time-course data were analyzed for touch maxima (B), peak 1 maxima (C), peak 2 maxima (D), and area under the response curve (AUC; E), all baseline subtracted, with each dot representing an individual data point (see Supplemental Fig. S1 for details). The middle line in the box plot denotes the median. ANOVA with posthoc Tukey’s test was used to assess statistical differences; different lowercase letters indicate significant differences (P < 0.05).

Fe Exclusion Restores the eATP-Induced [Ca²⁺]_cyt Signature in Pi-Starved Root Tips

Pi starvation causes Fe accumulation in Arabidopsis root tips, and exclusion of Fe from the growth medium (as well as Pi) restores primary root growth (Svistoonoff et al., 2007; Ward et al., 2008; Ticconi et al., 2009; Müller et al., 2015; Balzergue et al., 2017). To test if Fe availability influences the Pi starvation effect on the eATP-induced [Ca²⁺]_cyt signature, we again grew aequorin-expressing plants on varied Pi levels (full Pi, 0.625 mM Pi; zero Pi, 0 mM Pi) while additionally varying Fe levels (full Fe, 50 µM Fe; low Fe, 10 µM Fe; zero Fe, 0 µM Fe). As expected, Fe exclusion in a zero Pi background rescued primary root growth (Supplemental Table S1). Strikingly, this growth condition (zero Pi-zero Fe) also rescued the altered root tip [Ca²⁺]_cyt response to 1 mM eATP induced by Pi starvation alone (zero Pi-full Fe; Fig. 7A). Zero Pi-zero Fe-grown roots supported an eATP-induced [Ca²⁺]_cyt signature similar to those grown in nutrient-replete (full Pi-full Fe) conditions (Fig. 7, B–E).

Figure 7.

Fe levels modify the [Ca²⁺]_cyt response of Pi-starved root apices to eATP. Arabidopsis Col-0 aequorin-expressing seedlings were grown on standard one-half-strength Murashige and Skoog (MS) growth medium, full Pi-full Fe (green trace), zero Pi-full Fe (blue trace), zero Pi-low Fe (pink trace), or zero Pi-zero Fe (orange trace). Excised root apices (1 cm) of 11-d-old seedlings were challenged with treatments applied at 35 s (black arrow), and [Ca²⁺]_cyt was measured for 155 s. A, Application of 1 mM eATP. Time-course traces represent means ± se from three to six independent trials, with n = 24 to 61 individual root tips averaged per data point. B to E, Time-course data were analyzed for touch maxima (B), peak 1 maxima (C), peak 2 maxima (D), and area under the response curve (AUC; E), all baseline subtracted, with each dot representing an individual data point (see Supplemental Fig. S1 for details). The middle line in the box plot denotes the median. ANOVA with posthoc Tukey’s test was used to assess statistical differences; different lowercase letters indicate significant differences (P < 0.05).

An intermediate Fe level in a Pi-deplete background (zero Pi-low Fe) led to an intermediate [Ca²⁺]_cyt response to 1 mM eATP for all parameters quantified (touch response [Fig. 7B], peak 1 maxima [Fig. 7C], peak 2 maxima [Fig. 7D], and area under the response curve [Fig. 7E]). This was particularly interesting, as zero Pi-low Fe-grown plants had longer root lengths than full Pi-full Fe-grown plants (Supplemental Table S1), indicating that long primary roots alone could not explain the rescued, altered [Ca²⁺]_cyt signature. As a test of Fe specificity, copper (also a micronutrient transition metal) was excluded from the zero Pi growth medium. This treatment rescued neither primary root growth nor the eATP-induced [Ca²⁺]_cyt signature (Supplemental Table S1; Supplemental Fig. S9).

Pi and Fe Availability Influences Root Cellular ROS Level

Pi-dependent Fe accumulation has been linked to hotspots of ROS (Müller et al., 2015; Balzergue et al., 2017), implying a link between cellular redox status and aberrant [Ca²⁺]_cyt response to eATP of Pi-starved root tips. Using the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; which reports intracellular ROS), nutrient-replete roots showed low intracellular ROS levels along the root tip (Fig. 8, A and B). In Pi-starved root tips, we observed overall higher ROS levels, with a particular ROS hotspot localized at approximately 1 mm from the root apex (Fig. 8, C, D, and blue trace in I). Excluding Fe (in zero Pi background) reversed the higher ROS load back to nutrient-replete low ROS levels (Fig. 8, E and F). Thus, root tips sustaining a low ROS load qualitatively correlated with root tips capable of producing a multiphasic [Ca²⁺]_cyt response to 1 mM eATP (compare with Fig. 7). Root tips showing a high ROS load correlated with root tips exhibiting a much dampened [Ca²⁺]_cyt response to 1 mM eATP.

Figure 8.

Intracellular ROS are modified by Pi and Fe availability and influence root [Ca²⁺]_cyt response to eATP. A to H, Ten- to 11-d-old Arabidopsis Col-0 grown on full Pi-full Fe (A and B), zero Pi-full Fe (C and D), and zero Pi-zero Fe (E and F) were stained for intracellular ROS using 20 µM CM-H₂DCFDA. G and H represent the nonstained control root. A, C, E, and G, Representative bright-field images. B, D, F, and H, Representative false-colored fluorescence images. Bars = 1 mm. I, Fluorescence intensity was quantified, background subtracted, and averaged along the root length. Mean values (colored lines) ± se (gray shading) are shown. Data are from three independent trials, with n = 14 to 16 roots analyzed per growth condition.

DISCUSSION

[Ca²⁺]_cyt is a seemingly ubiquitous second messenger in plant abiotic stress responses, with roots responding to such stresses with cell-specific Ca²⁺ signatures (Kiegle et al., 2000; Martí et al., 2013; Wilkins et al., 2016). Few studies have addressed the impact of nutrient status on Ca²⁺ signatures (Koshiba et al., 2010; Quiles-Pando et al., 2013). Here, we show that Pi but not N starvation could significantly affect the root tip [Ca²⁺]_cyt response to a range of acute abiotic stressors and intermediate signaling agents (extracellular purine nucleotides and H₂O₂). Pi and Ca²⁺ have a particularly interesting relationship, as they can form undissociated complexes (Cole et al., 1953; Verkhratsky and Parpura, 2014; Edel and Kudla, 2015). In animals, Ca²⁺-Pi complexes play significant structural roles (Plattner and Verkhratsky, 2015), but in plants, these have only recently been discovered in trichomes of a variety of plant species, including Arabidopsis (Ensikat et al., 2016; Mustafa et al., 2018; Weigend et al., 2018). Pi and Ca²⁺ have even been shown to be stored in different plant cell types, presumably to avoid complexation (Conn and Gilliham, 2010). Cellular Pi levels have favored the evolution of a highly efficient and regulated Ca²⁺ flux apparatus to maintain low [Ca²⁺]_cyt and prevent cytotoxicity (Verkhratsky and Parpura, 2014; Edel and Kudla, 2015). While Pi deficiency causes lower cellular and cytosolic Pi levels (Duff et al., 1989; Pratt et al., 2009), our results here show that [Ca²⁺]_cyt signatures still proceed but in altered forms.

The impact of Pi depletion on the eATP-induced [Ca²⁺]_cyt signature was evident in 6-d-old root tips, at a stage where a Pi-dependent inhibition of primary root growth was not yet detectable. This suggests that changes in Ca²⁺ transport and possibly signaling systems are an early consequence of Pi deprivation. The observed dampening of [Ca²⁺]_cyt signatures under Pi deprivation may have several causes. Pi deficiency causes remodeling of membranes such that phospholipids are replaced by glycolipids and sulfolipids (Andersson et al., 2005; Tjellström et al., 2010; Nakamura, 2013; Okazaki et al., 2013), which can be envisaged to have an impact on membrane-based signaling. Additionally, many studies have reported the effect of Pi starvation on gene expression and protein composition (Misson et al., 2005; Lin et al., 2011; Lan et al., 2012; Kellermeier et al., 2014; Hoehenwarter et al., 2016; Wang et al., 2018). However, these studies do not report an enrichment of (down-regulated) Ca²⁺-associated transport and signaling components to help explain dampening of the [Ca²⁺]_cyt signature. As phosphorylated metabolites decrease and phosphorylation patterns reportedly change under Pi starvation (Duan et al., 2013; Pant et al., 2015), it could be envisaged that posttranslational modifications and an altered physicochemical cellular environment strongly affect the activity of the channels involved in generating the signatures.

Therefore, it is likely that as Pi starvation advances, there is a progressive remodeling of Ca²⁺ signaling machinery, affecting the transporters engaged in generating [Ca²⁺]_cyt signatures. Our results show that this is not a determinate effect but is reversible by Pi resupply. These findings have implications for the downstream signaling events and responses, which may change under Pi deprivation. For example, Pi deprivation dampened the mechano-induced [Ca²⁺]_cyt signature, which may have consequences for root penetration of Pi-deplete compacted soil. It also dampened the NaCl-induced [Ca²⁺]_cyt signature, which may have consequences for regulation of the Ca²⁺-dependent SOS pathway (Quintero et al., 2011; Manishankar et al., 2018) and the observation that Pi starvation alleviated the inhibitory effect of low salt concentrations on root growth (Kawa et al., 2016).

The extracellular purine nucleotides ATP and ADP induce root [Ca²⁺]_cyt increases, potentially for regulation of growth, stress responses, and defense (Demidchik et al., 2003, 2009; Rincón-Zachary et al., 2010; Tanaka et al., 2010a; Dark et al., 2011; Loro et al., 2012, 2016; Choi et al., 2014). In common with root [Ca²⁺]_cyt imaging reports (Loro et al., 2012, 2016; Waadt et al., 2017), eATP was used here as a reliable stimulus of a robust [Ca²⁺]_cyt signature as well as an agent of root signal transduction. Under Pi-replete conditions, the temporal biphasic eATP-induced [Ca²⁺]_cyt response found using aequorin mapped well to a spatial biphasic response found using YC3.6 as well as agreed with what has recently been reported using a range of other [Ca²⁺]_cyt reporters (Waadt et al., 2017). This spatiotemporal pattern has been hypothesized to constitute a Ca²⁺ wave, propagating from the meristematic zone toward the elongation zone and into the mature zone (Rincón-Zachary et al., 2010; Loro et al., 2012; Costa et al., 2013).

The spatial resolution afforded by YC3.6 revealed that while Pi starvation had no effect on the eATP-induced [Ca²⁺]_cyt increase at the apex (Fig. 4, G and H), it caused the progressive diminution of the signal in increasingly distal regions in the mature zone (Fig. 4, A–F). Root accumulation of intracellular ROS under Pi deficiency was linked to Fe availability (Fig. 8), consistent with previous reports (Müller et al., 2015; Balzergue et al., 2017). Fe depletion under Pi starvation not only lowered ROS accumulation to that found under nutrient-replete conditions but also restored the second peak of the biphasic eATP-induced [Ca²⁺]_cyt signature (with good spatial coincidence of both phenomena). It is therefore reasonable to conclude that under Pi deprivation alone, aberrant Fe accumulation leads to intracellular ROS accumulation and that the greater oxidative state of that region helps suppress the second eATP-induced [Ca²⁺]_cyt increase. Abiotic stress has been shown previously to cause an increase in root ROS accumulation, with NADPH oxidases implicated in their generation (Foreman et al., 2003; Demidchik et al., 2009). However, the activity of NADPH oxidases is usually linked to amplification of a [Ca²⁺]_cyt increase through activation of Ca²⁺ influx across the plasma membrane (Foreman et al., 2003; Demidchik et al., 2009; Laohavisit et al., 2012; Demidchik, 2018). This is seemingly at odds with the loss of the second eATP-induced [Ca²⁺]_cyt increase under Pi starvation, and the paradigm of the ROS/Ca²⁺ hub in signaling may not hold under Pi deprivation or in general conditions of high baseline ROS. The origin of the intracellular ROS under Pi deprivation may well include leakage from mitochondria, which increase their ROS production under stress (Gleason et al., 2011). It is feasible that the restoration of normal ROS levels with growth on zero Pi and zero Fe medium reflects the impaired mitochondrial function that occurs on chronic deprivation of Fe (Vigani and Briat, 2016), possibly leading to lower ROS production.

Overall, our results reveal how nutritional status adds another layer of complexity to Ca²⁺ signaling, allowing plants to integrate various cues such as nutritional status and environmental changes. While [Ca²⁺]_cyt does not appear to be a second messenger in the sensing of Pi in either Pi-replete Arabidopsis roots (Demidchik et al., 2003) or Pi-starved roots (this study), its use is altered when Pi supply is limited. In addition to elucidating the mechanistic basis of these altered signatures in response to abiotic stress and determining downstream consequences for signaling, it is also now appropriate to investigate the impact of nutritional status on Ca²⁺ signaling in biotic interactions.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All plant material used was in the Arabidopsis (Arabidopsis thaliana) ecotype Col-0 background, stably transformed with constitutively expressed cytosolic (apo)aequorin (pMAQ2; Knight et al., 1991) or the cytosolic sensor NES-YC3.6 (Krebs et al., 2012). Surface-sterilized seeds were sown aseptically on one-half-strength MS growth medium including vitamins (Duchefa), with pH adjusted to 5.6 using KOH, and solidified using 0.8% (w/v) agar (Bacto agar; BD Biosciences). Plates (12 cm × 12 cm; Greiner Bio-One) were sealed using micropore tape (3M) to allow for gas exchange. All seeds were stratified at 4°C and in darkness for 2 to 3 d prior to placing plates vertically into long-day conditions (16 h of light/8 h of dark) in a growth chamber with 78 µmol m⁻² s⁻¹ light intensity at 23°C (CLF Plant Climatics).

Standard one-half-strength MS comprised the full Pi and full N growth conditions. A custom-made MS medium without Pi was used for zero Pi conditions (Duchefa; DU1072) or without N for zero N conditions (PhytoTechnology Laboratories; M531). KCl was used to substitute for missing K⁺ whenever Pi (KH₂PO₄) or N (KNO₃) was excluded. As the N-free medium was not available including vitamins, MS vitamin × 1,000 stock solution (Sigma-Aldrich; M7150) was added to zero N medium to the same final concentration. For all growth conditions requiring modified Fe or copper content, one-half-strength MS medium was prepared from stock solutions and vitamins were supplied from the MS vitamin × 1,000 stock. For transfer experiments, 8-d-old seedlings were transferred to fresh growth medium plates, containing full/zero Pi growth medium (as described in the text), and grown for an additional 2 d.

Quantification of Primary Root Length

Plates containing seedlings were scanned using a Perfection V300 Photo scanner (Epson) with 300-dpi resolution, saving the images in tiff format. The software ImageJ (Abràmoff et al., 2004) and plugin NeuronJ (Meijering et al., 2004) were used to trace primary root lengths.

Aequorin-Based [Ca²⁺]_cyt Measurements

Nutrient growth conditions were maintained throughout the experiments (i.e. all incubation and treatment solutions were prepared in the respective liquid one-half-strength MS medium, including 1.175 mM MES, adjusted to pH 5.6 using Tris). Excised primary root tips of 11-d-old Arabidopsis expressing (apo)aequorin were used for luminescence-based quantification of [Ca²⁺]_cyt dynamics, unless stated otherwise. Reconstitution of aequorin with coelenterazine in vivo was modified after Knight et al. (1997b). In short, an excised tip was placed individually in a well of a white 96-well plate (Greiner Bio-One), incubated in 10 µM coelenterazine (NanoLight Technology) overnight, in darkness and at room temperature. A FLUOstar OPTIMA plate reader (BMG Labtech) was used to record baseline luminescence for 35 s, before injecting 100 µL of different treatment solutions with an injection speed of 150 µL s⁻¹. Changes in luminescence signal were monitored for 120 s, before injecting 100 µL of discharge solution (final concentration, 10% [v/v] ethanol and 1 M CaCl₂) and monitoring for another 45 s. Concentrations of [Ca²⁺]_cyt were calculated as described (Knight et al., 1997b). Treatments included the following: ATP disodium salt trihydrate (ATP; Melford), ADP disodium salt dehydrate (ADP; Melford), nonhydrolyzable ATP-analog γ-ATP (Sigma), phosphoric acid (Thermo Fisher Scientific), NaCl (Thermo Fisher Scientific), osmotic control for NaCl treatments, d-sorbitol (Sigma-Aldrich), and H₂O₂ (Sigma). The accompanying ions (Na⁺ for ATP and ADP; Li⁺ for γ-ATP) were previously shown in our laboratory not to confound the response (Demidchik et al., 2009). Test treatments were pH adjusted to 5.6 using Tris and prepared at double strength, as in the well; a 1:2 dilution led to the desired final concentration. A Vapro5520 osmometer (Wescor) was used to check the osmolality of the NaCl and d-sorbitol treatment solutions.

Ratiometric [Ca²⁺]_cyt Measurements

Ten-day-old Arabidopsis seedlings expressing NES-YC3.6 were mounted into a custom-built superfusion chamber (Behera and Kudla, 2013), stabilized with wetted cotton wool, and continuously superfused with imaging solution (IS; 5 mM KCl, 10 mM CaCl₂, and 10 mM MES, set to pH 5.8 using Tris; Loro et al., 2016) using an EconoPump system (Bio-Rad) with a tube diameter of 0.8 mm and a speed of 0.9 mL min⁻¹. Seedlings were left to acclimatize to constant superfusion for 10 to 15 min before starting an experiment. At the start of an experiment, seedlings were imaged for 2 min while superfusing IS. Extracellular ATP treatment (1 mM ATP, in IS background, pH 5.8) was then superfused over the roots for 3 min before changing back to IS without ATP. Images were captured using a Ti-E wide-field inverted fluorescence microscope (Nikon) with a Nikon Plan Fluor 4× 0.13 dry objective. The samples were excited at 440 nm using a Prior Lumen 200 PRO fluorescent light source (Prior Scientific). Images were collected with an ORCA-D2 Dual CCD camera (Hamamatsu) every 5 s for up to 30 min. NIS Elements AR 4.0 software (Nikon) was used to control the microscope, light source, and camera. ImageJ Fiji software was used to process the cpVenus and CFP fluorescence intensities. Using the Roi Manager tool, each root sample was individually fitted with comparable ROIs. The z axis profiles were plotted for each channel, individually background subtracted, and used to calculate FRET raw ratios (cpVenus/CFP). Normalization of data was carried out by taking into account differences in prestimulus baseline (ΔR/R₀ = R – R₀/R₀, with R – cpVenus/CFP ratio, R₀ – averaged cpVenus/CFP prestimulus baseline ratio, after Loro et al., 2016).

ROS Imaging

The membrane-permeable dye CM-H₂DCFDA (Thermo Fisher Scientific) was used at a final concentration of 20 µM, in assay medium (0.1 mM KCl, 0.1 mM CaCl₂, and 1.175 mM MES, set to pH 6 using Tris; adapted from Foreman et al., 2003). Ten- to 11-d-old seedlings were incubated for 1 h (dark, 4°C), gently washed in fresh assay medium without dye, and placed on plates containing growth medium maintaining previous growth conditions for 1 h (light, room temperature) to acclimatize, before imaging primary root tips with a stereomicroscope, M205 FA (Leica), with a DFC365FX camera (Leica) and a Sola SE365 light source (Lumencor). Excitation at 470/40 nm was used and a GFP-ET filter collected emission at 525/50 nm, with a 400-ms exposure time, 70% light intensity, and a gain of 2 and 50× magnification. LAS X software (Leica) was used to control the microscope, light source, and camera. Image analysis was done using Fiji ImageJ software, tracing each root using the line tool (line width, 10) in combination with the plot profile function, which reports signal intensity along the root (Reyt et al., 2015). For each root, three lines were drawn (from root apex shootward through the center of the root, from root apex shootward along the upper side of the root, and from root apex shootward along the lower part of the root), and the intensity profiles were averaged per root (Reyt et al., 2015).

Data Analysis

Data analysis and all statistical tests were performed using the open-source software R (www.r-project.org; version 3.5.1) in an R studio environment. The package MESS was used to calculate area under the response curve. ANOVA and Tukey’s honestly significant difference posthoc test were employed to determine differences among the groups. A 95% family-wise confidence level was applied.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers M11394.1 (aequorin) and AB178712 (YC3.6).

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Schematic representation of aequorin-based [Ca²⁺]_cyt time-course analyses.

• Supplemental Figure S2. The [Ca²⁺]_cyt response of N-starved root tips to salt and osmotic stress.

• Supplemental Figure S3. The [Ca²⁺]_cyt response of Pi-starved root tips to mechanical stimulation and a Pi source.

• Supplemental Figure S4. The [Ca²⁺]_cyt response of Pi-starved root tips to extracellular ADP.

• Supplemental Figure S5. The [Ca²⁺]_cyt response of Pi-starved root tips to a nonhydrolyzable ATP analog.

• Supplemental Figure S6. The [Ca²⁺]_cyt response of Pi-starved root tips to oxidative stress.

• Supplemental Figure S7. The [Ca²⁺]_cyt response of N-starved root tips to eATP.

• Supplemental Figure S8. The [Ca²⁺]_cyt response of N-starved root tips to oxidative stress.

• Supplemental Figure S9. The [Ca²⁺]_cyt response of copper- and Pi-starved root tips to eATP.

• Supplemental Table S1. Mean primary root lengths of Arabidopsis plants used in this study.

• Supplemental Movie S1. Ratiometric false-color movie from a representative time series of a Pi-replete Col-0 root expressing NES-YC3.6, response to 1 mM eATP.

• Supplemental Movie S2. Ratiometric false-color movie from a representative time series of a Pi-starved Col-0 root expressing NES-YC3.6, response to 1 mM eATP.