A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate

Abstract

Phosphate (P) is an essential macronutrient for plant growth. Roots employ adaptive mechanisms to forage for P in soil. Root hair elongation is particularly important since P is immobile. Here we report that auxin plays a critical role promoting root hair growth in Arabidopsis in response to low external P. Mutants disrupting auxin synthesis (taa1) and transport (aux1) attenuate the low P root hair response. Conversely, targeting AUX1 expression in lateral root cap and epidermal cells rescues this low P response in aux1. Hence auxin transport from the root apex to differentiation zone promotes auxin-dependent hair response to low P. Low external P results in induction of root hair expressed auxin-inducible transcription factors ARF19, RSL2, and RSL4. Mutants lacking these genes disrupt the low P root hair response. We conclude auxin synthesis, transport and response pathway components play critical roles regulating this low P root adaptive response.

Fig. 1

Root hair elongation is a conserved response to low P in Arabidopsis and results in IAA accumulation in the root tip. a Representative images of 9 day old seedlings grown in 3 µM (LP) or 312 µM P (HP). Scale bar = 0.5 mm. b IAA abundance was measured in 9 days old root tips (tip) and root hair region (RH) in Arabidopsis seedlings grown under low (LP) and high P (HP). Error bars represent standard error, N = 4. * Indicates significant difference (q-value < 0.05) judged by Student’s t-test. c–h. Confocal images of 9 day old Arabidopsis roots expressing auxin response reporter DR5:VENUS (yellow) under low (LP) and high P (HP) showing median (c, d) and surface (e, f) views. g and h are maximum projections. Propidium iodide was used for counter staining (red). Scale bar = 50 µm

Fig. 2

Auxin homeostasis is crucial for root hair elongation under low P. a Genes regulating Auxin biosynthesis (TAA1) and auxin degradation (DAO1, DAO2) are upregulated under low P (LP) conditions. *Indicate significant difference (q-value < 0.0001) judged by Student’s t-test and after Benjamini and Hochberg false discovery rate correction. b, c Representative images (b) and boxplot (c) showing root hair length in the root hair zone under low (LP) or high P (HP) in Columbia (Col), aux1 (aux1-22) and taa1 mutant seedlings. N = 3, scale bar = 0.5 mm. *, **, and *** indicate significant difference (q-value < 0.005, 0.0001 and 0.00001, respectively) judged by Student’s t-test. d, e Representative images (d) and boxplot (e) showing root hair length under low (LP) or high (HP) P in Columbia (Col) and dao1 (dao1.2 & dao1.2D) mutant seedlings. N = 3, scale bar 0.5 mm. *, **, and *** indicate significant difference (q-value < 0.005, 0.0001, and 0.00001, respectively) judged by Student’s t-test

Fig. 3

Expressing AUX1 in LRC and epidermal cells can restore aux1 root hair defect under low P. a The schematic diagram of the Arabidopsis root apex. b Confocal image showing AUX1 expression in lateral root cap and epidermal cells in aux1-22J0951»AUX1 line. Scale bar = 20 µm. c Representative images showing root hair growth in the driver line aux1-22J0951 and aux1-22J0951»AUX1 lines under low (LP) or high P (HP). N = 3, scale bar = 0.5 mm

Fig. 4

Auxin Response Factor ARF19 regulates root hair elongation under low P in Arabidopsis. a Cartoon showing positions of two different arf19 mutant alleles used in this study (top) and Reverse Transcription-PCR results showing that arf19-a and arf19-b are null alleles. b Representative images showing root hair growth of Columbia (Col), arf19-a and arf19-b seedlings under low P (LP) or high P (HP) conditions. N = 3, scale bar = 0.5 mm. c Boxplot showing root hair growth of Columbia (Col), arf19-a and arf19-b seedlings under low P (LP) or high P (HP) conditions. *, **, and *** indicate significant difference (q-value < 0.005, 0.0001, and 0.00001, respectively) judged by Student’s t-test. d Expression profiling studies showing ARF19 expression under low P (LP) or high P (HP) conditions. *Indicates significant difference (q-value < 0.05) judged by Student’s t-test and after Benjamini and Hochberg false discovery rate correction). e Fluorescence intensity of pARF19::3XVENUS-NLS seedlings grown under low P (LP) and high P (HP). N = 3, scale bar = 100 µm

Fig. 5

bHLH transcription factors RSL2 and RSL4 are involved in low P responsive RH elongation in Arabidopsis. a, b Representative images (a) and boxplot (b) showing root hair growth of Columbia (Col), rsl1, rsl2, and rsl4 mutant seedlings under low P (LP) or high P (HP) conditions. *, **, and *** indicate significant difference q-value < 0.005, 0.0001, and 0.00001, respectively, judged by Student’s t-test. N = 3, scale bar = 0.5 mm. c. Fluorescence intensity of pRSL2::GFP-RSL2 and pRSL4::GFP-RSL4 seedlings grown under low P (LP) and high P (HP). N = 3, scale bar = 100 µm. d Measured raw integrated fluorescence intensities of pRSL2::GFP-RSL2 and pRSL4::GFP-RSL4 grown for 5 days under low (LP, empty bars) and high (HP, black bars) P conditions. *Indicates significant difference (q value < 0.05), judged by Student’s t-test

Fig. 6

Model for root hair elongation under low P. Low P elevates IAA levels in the root apex facilitated by TAA1 mediated auxin synthesis and AUX1 dependent auxin transport. The resulting increase in IAA levels leads to the induction of ARF19 in the root apex resulting in RSL2 and RSL4 induction in the elongation and differentiation zones, respectively, promoting root hair elongation