Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots

Abstract

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root's mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsisthaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

Keywords: Arabidopsis; FERONIA; calcium; mechano; phosphate; root; skew; touch.

Figure 1

Mechano-stimulated root [Ca²⁺]_cyt increase is impaired in Pi-starved root tips. (a) Schematic of [Ca²⁺]_cyt analysis of aequorin time-course data. Each value was calculated with the average baseline value (i) subtracted. Touch peak was the highest value of the touch response due to mechanical stimulus from the solution application (ii; 35–155 s). Total [Ca²⁺]_cyt accumulation (iii) was estimated by integrating the area under the curve (AUC). (b) Individual root tips (1 cm) of 11-day old Arabidopsis seedlings in ± Pi liquid growth medium were mechanically stimulated by application of full Pi or zero Pi liquid growth medium. There was no significant difference in pre-test baseline [Ca²⁺]_cyt. Time course trace represents mean ± standard error of mean (SEM) from three independent trials, with 17–18 individual root tips averaged per data point. (c) Time course data were analysed for touch maxima relative to the baseline, with each dot representing an individual data point. The thick middle line denotes the median, separating the upper and lower half of the data; the hinges (box outline) denote median of the upper and the lower half of the data, respectively; the bars denote entirety of data excluding outliers. (d) Time course data were analysed for AUC beyond the baseline concentration. Significance levels: *** p < 0.001, Welch two sample t-test.

Figure 2

Excised Pi-starved whole roots have an impaired [Ca²⁺]_cyt touch peak. (a) Individual excised roots of 11-day old seedlings in ± Pi liquid growth medium were mechanically stimulated by application of full Pi or zero Pi liquid growth medium. Time course trace represents mean ± SEM from five independent trials, with 41–61 individual roots averaged per data point. (b) Time course data were analysed for touch maxima, with each dot representing an individual data point. Boxplot middle line denotes median. (c) Time course data were analysed for AUC. Significance levels: *** p < 0.001, Welch two sample t-test.

Figure 3

Pi-starved roots suffered growth inhibition. Arabidopsis Col-0 roots were grown vertically for 11 days on (a) full or (b) zero Pi medium. (c) Primary root length. (d) Lateral root density. Mean ± SEM from three independent trials with 100–106 plants. Significance levels: n.s., not significant; *** p < 0.001, Welch two sample t-test. Scale bar in (a) and (b) = 1 cm. Scans were taken viewed from the back of the plate.

Figure 4

Pi-starved plants have a reduced rightward skewing angle. (a) Absolute skewing angle (value of the skew relative to vertical, regardless of direction) of roots after 11 days of vertical growth on full or zero Pi medium. (b) Relative skewing angle (+ to the right, − to the left, when viewed from the back of the plate). Mean ± SEM from three independent trials shown in Figure 3, with 100–106 plants per growth condition. Significance levels: *** p < 0.001, Welch two sample t-test.

Figure 5

Pi-starved roots do not increase their skewing angle in response to inclined growth. (a) Roots were grown vertically on full or zero Pi medium. (b) Roots were grown at a 45° incline on full or zero Pi medium. Mean ± SEM from three independent trials with 106–121 plants. Significance levels: *** p < 0.001, Welch two sample t-test.