Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation

Environmental cues profoundly modulate cell proliferation and cell elongation to inform and direct plant growth and development. External phosphate (Pi) limitation inhibits primary root growth in many plant species. However, the underlying Pi sensory mechanisms are unknown. Here we genetically uncouple two Pi sensing pathways in the root apex of Arabidopsis thaliana. First, the rapid inhibition of cell elongation in the transition zone is controlled by transcription factor STOP1, by its direct target, ALMT1, encoding a malate channel, and by ferroxidase LPR1, which together mediate Fe and peroxidase-dependent cell wall stiffening. Second, during the subsequent slow inhibition of cell proliferation in the apical meristem, which is mediated by LPR1-dependent, but largely STOP1–ALMT1-independent, Fe and callose accumulate in the stem cell niche, leading to meristem reduction. Our work uncovers STOP1 and ALMT1 as a signalling pathway of low Pi availability and exuded malate as an unexpected apoplastic inhibitor of root cell wall expansion.

(a) Under +Pi, exogenous malate does not inhibit primary root growth. WT and almt1 seedlings grown under +Pi for 2 days were transferred for 6 days to +Pi with or without 200 M malate (mean +/-s.d., n = 7-10 seedlings per condition).
(b) Gene expression analysis. Seedlings of the indicated genotypes were grown for 5 days under +Pi and transferred for 24 h to +Pi or -Pi medium and roots collected for RNA extraction followed by qRT-PCR on STOP1, ALMT1 and MATE genes (mean +/-s.d., n = 3 independent experiments, each with three technical replicates).
Expression levels are normalized relatively to the WT (Col er105 ) control grown under +Pi.
(c) Analysis of the mate KO lines under -Pi.
Top: Primary root length of the mate KO  but not for the response to -Pi. Left: seedlings were grown five days in 0 or 4 M Al 3+ (n = 10-18). Right: seedlings were grown six days in + or -Pi (n = 33-39). Mean +/s.d.; two-tailed t-test, **** P < 0.0001; *** P < 0.001; NS, not significant. The experiment was performed twice with consistent results; one experiment is shown.
(e) Complementation of almt1 32 mutant with the pUBQ::ALMT1-GFP construct (five independent transgenic lines). Seedlings were grown for 5 days under -Pi and the primary root lengths measured (mean +/-s.d., n = 11-16 seedlings per line and condition). The experiment was performed twice with consistent results; one experiment is shown.
(g) Yeast one-hybrid assay of STOP1 and pALMT1. Coloured bars represent parts of pALMT1 fused to LacZ used in the assay; they are numbered with respect to first ATG of the ALMT1 coding sequence. -Gal activity is calculated relatively to the density of cells (see Methods) (mean +/-s.d., n = 3 independent experiments, each with two biological replicates per condition). The sequence in green indicates the STOP1-binding site found previously 28 .  Four-day old pALMT1::GUS #2 seedlings grown under +Pi at pH 7.1 were transferred 48 h in + or -Pi at the indicated pH before GUS staining. Two independent experiments were performed with consistent results and one representative experiment is shown.

Supplementary
(b) At pH 7.1, the constitutive expression of ALMT1 does not promote a short root under -Pi. The UBQ::ALMT1-GFP construct has been introduced in the almt1 32 and Stop1 48 mutants and five independent transgenic lines selected. Homozygous seedlings were sown on -Pi+Fe plates at pH 5.8 or 7.1 and the primary root lengths measured after 6 days (mean +/-s.d., n = 8-15). Note that at pH 5.8, the UBQ::ALMT1-GFP construct complements both the almt1 32 and Stop1 48 mutants, but it does not confer a short primary root at pH 7.1.  Seedlings were grown for 6 days to +Pi or -Pi medium and the primary root lengths measured (mean +/-s.d., n = 10-15 seedlings per line and condition).
(c) GFP-fluorescence of GFP-STOP1 in the primary root tip. Five day-old stop1 KO ;pSTOP1::GFP-STOP1 (1 line, independent from that in Fig. 1g) and stop1 KO ;pUBQ::GFP-STOP1 (2 independent lines) seedlings grown under +Pi were transferred for 24 h +Pi or -Pi plates and GFP-fluorescence pictured with confocal microscope. All pictures were taken with same microscope and camera settings as in (c) Effect of neutral pH on the peroxidase activity in the primary root tip. Four-day-old seedlings of the indicated genotypes were transferred for 48 h to +Pi or -Pi, pH 7.1 plates prior staining with 4-chloro-1-naphtol. The same lines grown at pH 5.8 are shown in Fig. 4k. The experiment was performed three times with consistent results; one representative experiment is shown. Scale bar, 100 m. (c) Seedlings of the indicated genotypes were grown for 6 days under -Pi and the primary root length measured. Respectively 5 and 7 independent transgenic lines for the pSCR::Stop1 48 and pCo2::Stop1 48 constructs (in the WT Col er105 background) are shown. The experiment has been performed three times with consistent results; one representative experiment is shown (mean +/-s.d., n = 12-15 seedlings per line; one-way ANOVA; **** P<0.0001; NS, not significant).