Limited phosphate resources are efficiently allocated to each organ by the PHR1-EIN3/EIL1 module to coordinate their establishment

Abstract

Phosphate (Pi) is an essential nutrient that frequently limits plant growth because of its low availability in soils, especially during early seedling development in Arabidopsis. Under Pi-deficient conditions, etiolated seedlings exhibit elongated hypocotyls, shortened roots, and small, pale cotyledons-a morphological adaptation that enhances light-foraging capacity. However, how Pi is strategically distributed among these organs to optimize seedling establishment remains unclear. We here identify a PHR1-EIN3/EIL1 regulatory module that directs minimal Pi resources to suppress root elongation and cotyledon expansion or to accelerate greening, promoting Pi conservation. This mechanism prioritizes hypocotyl elongation to improve light acquisition. In contrast, under sufficient Pi supply, hypocotyl and root growth is promoted along with cotyledon enlargement, while greening is delayed. Thus, the PHR1-EIN3/EIL1 module enables efficient allocation of limited Pi to maximize hypocotyl elongation for light foraging during early seedling establishment while strategically restricting Pi usage in other organs to enhance overall survival.

Fig. 1.. Pi positively regulates hypocotyl/root length and cotyledon area and negatively modulates cotyledon greening.

(A) Representative images of 7-day-old wild-type (Col-0) etiolated seedlings after 10 hours of “normal” white light irradiation on a solid MS medium containing low Pi (Pi−), medium Pi (Pi+), or high Pi (Pi++). Scale bars, 2 mm. (See Materials and Methods for Pi levels.) (B) Cotyledon greening of seedlings described in (A). Scale bar, 0.1 mm. (C) IOSA (18) of etiolated seedlings shown in (A). Scale bars, 2 mm. (Refer to Materials and Methods for Pi distribution/accumulation details.) (D) Hypocotyl length (mm) of seedlings in (A). (E) Root length (mm) of seedlings in (A). (F) Pi content in seedlings shown in (C). (G) Cotyledon greening rate from seedlings in (A) and (B). (H) Cotyledon area from seedlings in (B). The cotyledon area in Pi− was set to 1.0. Data are the means ± SD. Very obvious differences were determined by a two-way ANOVA [n = 52 for (D), (E), (G), and (H); n = 3 for (F); for (F), Pi was assayed in three replicate samples of 50 seedlings, as in (2)]. Tukey’s post hoc test was used; different lowercase letters indicate very obvious differences at **P < 0.01 [(D) to (H)].

Fig. 2.. PHR1 and EIN3/EIL1 negatively regulate hypocotyl or root elongation.

(A) Representative images of etiolated seedlings of 7-day-old wild-type (Col-0) and indicated lines after 10 hours of “normal” white light irradiation on a solid MS medium with low Pi (Pi−). Scale bars, 5 mm. [Note that cotyledon greening is distinct from cotyledon opening; e.g., cop1-4 has inhibited greening but promoted opening (19)]. Experiments repeated three times independently with similar results. (B) Hypocotyl length (mm) of seedlings in (A). (C) Root length (mm) of seedlings in (A). Very obvious differences were determined by a two-way ANOVA [n = 50 for (B) and (C)]. Tukey’s post hoc test was used; different lowercase letters indicate very obvious differences at **P < 0.01 [(B) and (C)].

Fig. 3.. PHR1 and EIN3/EIL1 positively regulate cotyledon greening.

(A) Representative images of 7-day-old wild-type (Col-0) etiolated seedlings after 10 hours of “normal” white light irradiation on a solid MS medium with low Pi (Pi−). Scale bars, 0.1 mm. Experiments repeated three times independently with similar results. (B) Cotyledon greening rate of seedlings in (A). (C) Cotyledon area of seedlings in (A). The cotyledon area in the wild type was set to 1.0. (D) Pi content in cotyledons of seedlings in (A). Very obvious differences were determined by a two-way ANOVA [n = 53 for (B) and (C); n = 3 for (D); for (D), Pi levels were measured in three replicates of 42 seedlings, as in (2)]. Tukey’s post hoc test was used; different lowercase letters indicate very obvious differences at **P < 0.01 [(B) to (D)].

Fig. 4.. PHR1 directly binds to the EIL1 promoter to elevate EIL1 expression.

(A) EIL1 gene expression in 7-day-old wild-type, phr1-1 (SALK_067629), and phr1 (CS2110006) etiolated seedlings after 10 hours of “normal” white light irradiation on a solid MS medium with low Pi (Pi−). (B) Quantitative analysis of luminescence intensity. pGreen-mEIL1 intensity represents arbitrary units (~10). Other expressions quantified using Adobe Photoshop CS. (C) Schematic of the EIL1 promoter loci and amplicon for ChIP-qPCR analysis. (D) ChIP-qPCR analysis. Chromatin regions within the EIL1 promoter were enriched using an anti-GFP antibody and detected by real-time qPCR. ChIP materials: 7-day-old PHR1ox-GFP seedlings grown on a Pi− MS medium. The coding sequence (CDS) used as the control. E2 quantification in qRT-PCR set to 1.0. (E) EMSA showing the unlabeled EIL1 promoter used as the competitor to determine the specific interaction of EIL1 DNA with the PHR1 protein in vitro. GST: negative control. (F) EMSA using a biotin-unlabeled mutant version of the E2 fragment in the EIL1 promoter with PHR1 polypeptides. [(E) and (F)] Asterisk: free probe; arrows: PHR1-probe complexes. Very obvious differences were determined by a two-way ANOVA [n = 3 for (A), (B), and (D)]. Tukey’s post hoc test was applied; distinct lowercase letters denote very obvious differences at P < 0.01 [(A), (B), and (D)].

Fig. 5.. EIN3 expression in roots suppresses Pi accumulation to establish early seedlings.

(A and B) Representative images of 7-day-old EBS-GUS (A) and PBS-GUS (B) etiolated seedlings after 20 hours of “normal” white light irradiation on a solid MS medium with middle Pi (Pi+). Scale bars, 2 mm. (C) Immunoblot of EIN3-3XFLAG in 7-day-old etiolated seedlings [as in (A)] after 10 hours of light irradiation on a Pi+ MS medium using an anti-FLAG antibody. Tubulin: loading control. (D) EIN3 expression in indicated organs (normalized to UBQ5). Mock quantification set to 1.0. (E) Representative images of 7-day-old EIN3-3XFLAG (iER) etiolated seedlings. Roots of 3-day-old iER seedlings were treated with 15 μM estradiol or water (mock) every 2 days. (F) Pi content in seedlings shown in (E). (G) Representative images of 7-day-old iER etiolated seedlings [as in (E)]. Scale bars, 5 mm. (H) Cotyledon greening of seedlings shown in (G). Scale bars, 0.25 mm. (I to L) Cotyledon area (I), greening seedling rate (J), hypocotyl length (K), and root length (L) of seedlings in (G). Data are the means ± SD. Very obvious differences were determined by a two-way ANOVA [n = 3 for (D) and (F); n = 44 for (I) to (L); for (D) and (F), Pi was assayed in three replicate samples of 15 seedlings]. Tukey’s post hoc test was used; different lowercase letters indicate very obvious differences at **P < 0.01 [(D), (F), and (I) to (L)].

Fig. 6.. Root removal suppresses Pi accumulation in hypocotyls and cotyledons, inhibiting hypocotyl elongation/cotyledon enlargement and promoting cotyledon greening.

(A) Representative images and IOSA staining (18) of 7-day-old wild-type (Col-0) etiolated seedlings after 10 hours of light irradiation on a Pi+ MS medium. Roots were removed from 3-day-old seedlings. Scale bars, 2 mm. Experiments repeated three times independently with similar results. (B) Pi content in hypocotyls and cotyledons of seedlings shown in (A). (C) Representative images of seedlings described in (A) (without staining). Scale bars, 5 mm. Experiments repeated three times independently with similar results. (D) Cotyledons of seedlings described in (A) (without staining). Scale bars, 0.1 mm. Experiments repeated three times independently with similar results. (E) Cotyledon area of seedlings in (C). (F) Greening seedling rate of seedlings in (C). (G) Hypocotyl length of seedlings in (C). Very obvious differences were determined by a two-way ANOVA [n = 3 for (B); n = 42 for (E) to (G); for (B), Pi was assayed in three replicate samples of 25 seedlings, as in (2)]. Tukey’s post hoc test was used; different lowercase letters indicate very obvious differences at **P < 0.01 [(B) and (E) to (G)].

Fig. 7.. Model illustrating the coordinated regulation of Arabidopsis early seedling establishment by Pi status within each organ of an etiolated seedling.

Understanding how EIN3 and PHR1 regulate internal Pi distribution is key. Interorgan nutrient transport is dynamic, with source organs supplying nutrients to sinks and sinks potentially exporting nutrients under certain conditions (36, 37), making source-sink designations relative. In this study, EIN3 and PHR1 exhibit higher transcriptional activity/stability and associated Pi transport activity in hypocotyls compared to cotyledons or roots (Fig. 5 and fig. S7). Furthermore, these transcription factors negatively regulate Pi distribution and uptake in the darkness (Figs. 2 and 3). Consequently, the Pi transport capacity is greater from cotyledons or roots to hypocotyls than in the reverse direction. This directional bias is supported by ³³P tracing data showing the highest Pi accumulation in hypocotyls (table S1). Thus, hypocotyls accumulate higher Pi levels than cotyledons or roots. Elevated Pi levels promote hypocotyl and root elongation as well as cotyledon enlargement but inversely correlate with cotyledon greening (Fig. 1). Collectively, this results in the etiolated seedling phenotype: two small, pale cotyledons, a short root, and a very long hypocotyl, maximizing its chances of pushing the cotyledons toward the light, which is spatially controlled by the PHR1-EIN3 module–mediated Pi availability. Greater Pi import activities are depicted as white thick lines. Bars represent negative regulation.