High Auxin and High Phosphate Impact on RSL2 Expression and ROS-Homeostasis Linked to Root Hair Growth in Arabidopsis thaliana

Abstract

Root hair size determines the surface area/volume ratio of the whole roots exposed to the nutrient and water pools, thereby likely impacting nutrient and water uptake rates. The speed at which they grow is determined both by cell-intrinsic factors like hormones (e.g., auxin) and external environmental signals like nutrient availability in the soil (e.g., phosphate). Overall root hair growth is controlled by the transcription factors RSL4 and RSL2. While high levels of auxin promote root hair growth, high levels of inorganic phosphate (Pi) in the media are able to strongly repress RSL4 and RSL2 expression linked to a decreased polar growth. In this work, we inquired the mechanism used by root hairs to integrate conflicting growth signals like the repressive signal of high Pi levels and a concomitant high auxin exposure that promotes growth and questioned whether these complex signals might activate known molecular players in root hair polar growth. Under these conditions, RSL2 expression (but not RSL4) is activated linked to ROS production and root hair growth. On the other hand, by blocking ROS production derived from the NADPH Oxidase C (or RBOHC for RESPIRATORY BURST OXIDASE HOMOLOG C) and ROS production from Secreted type-III Peroxidases (PERs), it was possible to repress the auxin growth-promoting effect. This study identifies a new layer of complexity between auxin, Pi nutrient availability and RSL2/RSL4 transcription factors all acting on ROS homeostasis and growth at the root hair level.

Keywords: Arabidopsis thaliana; NADPH oxidases; ROS; auxin; peroxidases; phosphate; root hairs.

FIGURE 1

Auxin overcomes high Pi root hair growth repression by enhancing ROS production. (A) Auxin circumvents root hair growth repression imposed by increased levels of Pi by enhancing ROS production. Root hair length (mean ± SD) was measured in Wt Col-0 roots non-treated, treated with 100 nM IAA (indole 3-acetic acid) or with 5 mM inorganic Pi, or with both treatments at the same time (on the left). Total ROS levels generated by oxidation of H₂DCF-DA were measured at the root hair tip in different stages of root hair development in the four different treatments (on the right). (B) Auxin recovery of root hair growth is dependent on ROS production. Root hairs treated with 100 nM IAA in the presence of high levels of Pi and VAS inhibitor fail to recover their normal growth (on the left). ROS levels were downregulated in the root hairs treated with 100 nM IAA in the presence of high levels of Pi and VAS inhibitor (on the right). ND, not detected. P value of one-way ANOVA, (^∗∗) P > 0.001, (^∗) P > 0.01. NS = not significantly different. Error bars indicate ± SD from 3 biological replicates. AU = Arbitrary Units.

FIGURE 2

Auxin fails to overcome high Pi root hair growth repression in a ROS-depleted background. (A) Auxin fails to circumvent root hair growth repression imposed by increased levels of Pi in a ROS-depleted noxc mutant. Root hair length (mean ± SD) was measured in noxc roots treated with 5 mM inorganic Pi with or without 100 nM IAA (indole 3-acetic acid) (on the left). Total ROS levels generated by oxidation of H₂DCF-DA were measured at the root hair tip in different stages of root hair development in the four different treatments (on the right). (B) Auxin fails to rescue root hair growth repression imposed by increased levels of Pi in a ROS-depleted PER environment by the inhibition of SHAM (salicylhydroxamic acid). Root hair length (mean ± SD) was measured in roots in the presence of SHAM and treated with 5 mM inorganic Pi with or without 100 nM IAA (indole 3-acetic acid) (on the left). Total ROS levels generated by oxidation of H₂DCF-DA were measured at the root hair tip in different stages of root hair development in the four different treatments (on the right). (C) High levels of ARF5 and RSL4 expression in root hair cells (EXPANSIN 7 promoter, E7) are able to partially overcome the growth repression imposed by high levels of Pi on cell elongation. Comparisons are made between Wt Col-0 and _E7:ARF5 or _E7:RSL4 lines under high levels of Pi. P value of one-way ANOVA, (^∗∗) P > 0.001, (^∗) P > 0.01. NS = not significantly different. Error bars indicate ± SD from 3 biological replicates.

FIGURE 3

Auxin triggers changes in _cytH₂O₂ levels detected with HyPer under high Pi levels. _cytH₂O₂ levels Wt Col-0 root hairs expressing HyPer sensor treated with 5 mM Pi and 100 nM IAA. _cytH₂O₂ levels are based on the ratio 488/405 nm of HyPer biosensor at the root hair tip over 200 s. On the right, selected kymographs resulting of this analysis, only for root hairs of >200 μm in length. Scale bar = 5 μm. P value of one-way ANOVA, (^∗∗) P > 0.001, (^∗) P > 0.01. NS = not significantly different. Error bars indicate ± SD from 3 biological replicates. AU = Arbitrary Units.

FIGURE 4

RSL2 but not RSL4 mediates the auxin mediated growth recovery response in the presence of high Pi levels. (A) Root hair length (mean ± SD) was measured in Wt Col-0, rsl4 and rsl2 roots non-treated, treated with 100 nM IAA (indole 3-acetic acid) or with 5 mM inorganic Pi, or with both treatments at the same time. ROS levels were partially recovered in Wt but not in rsl2 when treated with 100 nM IAA in the presence of high levels of Pi. No ROS was detected in rsl4 under high levels of Pi. Only root hairs <200 μm in length were analyzed. (B) Under high levels of Pi, auxin triggers the expression of both RSL4 and RSL2. Levels of RSL4 and RSL2 expression in Wt Col-0 roots non-treated, treated with 100 nM IAA (indole 3-acetic acid) or with 5 mM inorganic Pi, or with both treatments at the same time (on the left). Levels of RSL4 and RSL2 expression in rsl2 and rsl4, respectively, non-treated, treated with 100 nM IAA (indole 3-acetic acid) or with 5 mM inorganic Pi, or with both treatments at the same time (on the left). To simplify the graphs, Col 0 and the rsl2- and rsl4- mutants are shown separately although the experiments were performed at the same time. Each experiment, with the three genotypes together (Wt Col-0, rsl2- and rsl4-) was performed in triplicate, and qRT-PCR reaction was repeated three times using independent preparations of RNA. Comparisons are made for the same genetic background between levels of gene expression (of RSL2 or RSL4) in non-treated samples (no IAA/no Pi) versus treated with IAA or Pi. In addition, Pi-treated samples are compared only to Pi+IAA-treated roots. P-value of one-way ANOVA, (^∗∗) P > 0.001, (^∗) P > 0.01. NS = not significantly different. Error bars indicate ± SD from 3 biological replicates. AU = Arbitrary Units.

FIGURE 5

Proposed models of auxin-RSL2/RSL4 regulation of ROS-mediated polar root hair growth in the absence and in the presence of high Pi. Transcriptional responses under high level of auxin in the absence of Pi (A) and in the presence of high Pi together with high levels of auxin are shown. This proposed model is based on evidences shown in (1) Bhosale et al., 2018; (2) Giri et al., 2018; (3) Mangano et al. (2017); (4) current work. (A) Low levels of Pi in the soil (media) triggers auxin biosynthesis (involving TAA1) and transport (by AUX1) from the root cap and epidermis cells to the root hair cells. High levels of auxin then induces the activation of several Auxin Response Factors (ARFs; ARF19 and ARF5,7,8). The bHLH transcription factor RSL4 as well as RSL2 are both transcriptionally activated by high levels of auxin (IAA) and its expression is directly regulated by these ARFs. Through RSL4 and possibly RSL2, auxin activates the expression of two RBOHs (RBOHC,J) and four PERs (PER1,44,60,73) that together regulate ROS homeostasis in the apoplast (in combination with RBOHH). (B) High levels of Pi in the presence of high levels of auxin are able to activate the expression of RSL2 and control ROS homeostasis derived from RBOHs and PERs activities. Solid lines indicate transcriptional activation or metabolite production. The main reactive oxygen species (ROS) highlighted is hydrogen peroxide (H₂O₂).