Abscisic acid promotes auxin biosynthesis to inhibit primary root elongation in rice

Abstract

Soil compaction is a global problem causing inadequate rooting and poor yield in crops. Accumulating evidence indicates that phytohormones coordinately regulate root growth via regulating specific growth processes in distinct tissues. However, how abscisic acid (ABA) signaling translates into auxin production to control root growth during adaptation to different soil environments is still unclear. In this study, we report that ABA has biphasic effects on primary root growth in rice (Oryza sativa) through an auxin biosynthesis-mediated process, causing suppression of root elongation and promotion of root swelling in response to soil compaction. We found that ABA treatment induced the expression of auxin biosynthesis genes and auxin accumulation in roots. Conversely, blocking auxin biosynthesis reduced ABA sensitivity in roots, showing longer and thinner primary roots with larger root meristem size and smaller root diameter. Further investigation revealed that the transcription factor basic region and leucine zipper 46 (OsbZIP46), involved in ABA signaling, can directly bind to the YUCCA8/rice ethylene-insensitive 7 (OsYUC8/REIN7) promoter to activate its expression, and genetic analysis revealed that OsYUC8/REIN7 is located downstream of OsbZIP46. Moreover, roots of mutants defective in ABA or auxin biosynthesis displayed the enhanced ability to penetrate compacted soil. Thus, our results disclose the mechanism in which ABA employs auxin as a downstream signal to modify root elongation and radial expansion, resulting in short and swollen roots impaired in their ability to penetrate compacted soil. These findings provide avenues for breeders to select crops resilient to soil compaction.

Figure 1

ABA is involved in soil compaction-modulated root growth. (A and B) Morphology (A) and primary root length (B) of 4-d-old wild-type (WT) seedlings grown in uncompacted or compacted soil. Bars = 1 cm. Data are means ± SD (n ≥ 30 independent seedlings). The individual images in (A) were digitally extracted for comparison. C, Representative propidium iodide staining of longitudinal sections of root tips of 4-d-old WT seedlings grown in uncompacted or compacted soil. White arrows indicate the proximal end of the root meristem. White rectangle insets are an enlargement (three times magnification) of the regions at the proximal end of the root meristem. Bars = 100 μm. (D and E) Length (D) and cortical cell number (E) of the root meristem zones of the corresponding seedlings indicated in panel (C). Data are means ± SD (n ≥ 10 independent seedlings). F, Representative propidium iodide staining of radial sections of root elongation zone of 4-d-old WT seedlings grown in uncompacted or compacted soil. Bars = 100 μm. G, Root diameter of the corresponding seedlings indicated in panel (F). Data are means ± SD (n ≥ 10 independent seedlings). (H and I) Morphology (H) and primary root length (I) of 4-d-old Nip and mhz5 seedlings grown in uncompacted or compacted soil. Bars = 1 cm. Data are means ± SD (n ≥ 30 independent seedlings). The individual images in (H) were digitally extracted for comparison. Different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's test). Asterisks in (B), (D), (E) and (G) indicate significant differences compared with uncompacted values at *P < 0.05 and **P < 0.01 (Student's t-test).

Figure 2

ABA promotes auxin accumulation to inhibit root growth. A, Representative images of DR5-GUS expression in root with or without ABA treatment. Seedlings of 4-d-old transgenic lines containing DR5-GUS were treated with or without 1 μM ABA for 12 h before GUS activity was assayed. Bar = 1 cm. B, Representative longitudinal sections of meristematic zone (MZ), elongation zone (EZ) and differentiation zone (DZ) in panel (A). Bar = 100 μm. C, Expression of YUCs, TAA1 and TAR1 in the roots of 4-d-old wild-type seedlings treated with or without 1 μM ABA for 4 h. The data are shown as mean ± SD; n = 3 biological replicates. D, Indole-3-acetic acid (IAA) contents in 4-d-old wild-type seedlings treated with or without 1 μM ABA for 24 h. The data are shown as mean ± SD; n = 3 biological replicates. (E and F) Morphology (E) and primary root length (F) of 4-d-old wild-type seedlings grown in the absence or presence of 0.5 μM ABA, with or without supplementation of 5 μM yucasin or L-Kyn. Bar = 1 cm. Data are means ± SD (n ≥ 30 independent seedlings). The individual images in (E) were digitally extracted for comparison. For (C), (D) and (F), asterisks indicate significant differences compared with mock at *P < 0.05 and **P < 0.01 (Student's t-test).

Figure 3

Mutation in TAA1 weakens ABA response in roots. A, Phenotypes of the primary roots of 4-d-old Zhonghua 11 (ZH11, a wild-type strain) and taa1 seedlings with or without 0.5 μM ABA treatment. Bar = 1 cm. The individual images were digitally extracted for comparison. B, Primary root length of 4-d-old ZH11 and taa1 seedlings treated with various concentrations of ABA. The data are shown as mean ± SD, n ≥ 30 independent seedlings. C, Representative propidium iodide staining of longitudinal sections of root tips of 4-d-old ZH11 and taa1 seedlings with or without 0.5 μM ABA treatment. White arrows indicate the proximal end of the root meristem. White rectangle insets are an enlargement (three times magnification) of the regions at the proximal end of the root meristem. Bars = 100 μm. (D and E) Length (D) and cortical cell number (E) of the root meristem zones of the corresponding seedlings indicated in panel (C). Data are means ± SD (n ≥ 10 independent seedlings). F, Representative propidium iodide staining of radial sections of root elongation zone of 4-d-old ZH11 and taa1 seedlings with or without 0.5 μM ABA treatment. Bars = 100 μm. G, Root diameter of the corresponding seedlings indicated in panel (F). Data are means ± SD (n ≥ 10 independent seedlings). For (B), (D), (E) and (G), different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's test).

Figure 4

Auxin-deficient mutants display better root penetration ability than wild type in compacted soil. A, Phenotypes of the primary roots of 4-d-old Zhonghua 11 (ZH11, a wild-type strain), taa1, Kitaake (KT, a wild-type strain) and rein7-1 grown in uncompacted and compacted soil. Bar = 1 cm. B, Primary root length of 4-d-old ZH11, taa1, KT and rein7-1 grown in uncompacted and compacted soil. The data are shown as mean ± SD, n ≥ 30 independent seedlings. Different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's test).

Figure 5

OsbZIP46 positively regulates ABA-modulated root elongation and root swelling. (A and B) Representative propidium iodide staining of longitudinal sections of root tips (A) and radial sections of root elongation zone (B) of 4-d-old Nip, OsbZIP46 knockout and overexpression seedlings with or without 0.5 μM ABA treatment. White arrows indicate the proximal end of the root meristem. White rectangle insets are an enlargement (three times magnification) of the regions at the proximal end of the root meristem. Bars = 100 μm. (C–E) Length (C) and cortical cell number (D) of the root meristem zones and root diameter (E) of the corresponding seedlings indicated in panels (A) and (B). Data are means ± SD (n ≥ 10 independent seedlings). Different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's test).

Figure 6

OsbZIP46 directly binds to the promoter of YUC8/REIN7 to activate its expression. A, Expression of YUC8/REIN7 in the roots of 4-d-old wild type, OsbZIP46 knockout and overexpression seedlings, and OsbZIP46-CA1 overexpression seedlings. The data are shown as mean ± SD; n = 3 biological replicates. Asterisks indicate significant differences compared with Nip or ZH11 at *P < 0.05 and **P < 0.01 (Student's t-test). B, Schematic diagram of G-box (5′-CACGTG-3′) in the YUC8/REIN7 promoter. Black boxes indicate the positions of the G-box. P1–P3 are YUC8/REIN7 promoter fragments. C, The enrichments of YUC8/REIN7 promoter analyzed by ChIP-qPCR using 35S:OsbZIP46-myc transgenic rice plants. Wild-type plants were used as a negative control. The data are shown as mean ± SD, n = 3 biological replicates. Asterisks indicate significant differences compared with Nip values at * P < 0.05 and ** P < 0.01 (Student's t-test). D, EMSA of OsbZIP46 binding to YUC8/REIN7 promoter region containing the G-box. Normal G-box and the mutated form (5′-AAAAAA-3′) are shown. Competition was done by adding an excess of unlabeled probe (Competitor), and for specificity with labeled mutant probe. Three biological replicates were performed, with similar results. E, Dual-LUC assay results from transient transformation of rice mesophyll protoplasts with constructs constitutively expressing OsbZIP46 or OsbZIP46-CA1 and the LUC reporter gene under control of the intact YUC8/REIN7 promoter. The data are shown as mean ± SD, n = 3 biological replicates. “EV” represents empty vector. The asterisks indicate significant differences by Student's t-test between two samples (**P < 0.01).

Figure 7

YUC8/REIN7 acts downstream of OsbZIP46 to regulate ABA-modulated root elongation and root swelling. (A and B) Representative propidium iodide staining of longitudinal sections of root tips (A) and radial sections of root elongation zone (B) of 4-d-old Nip, and combinations of YUC8/REIN7 and OsbZIP46 knockout and overexpression seedlings with or without 0.5 μM ABA treatment. White arrows indicate the proximal end of the root meristem. White rectangle insets are an enlargement (three times magnification) of the regions at the proximal end of the root meristem. Bars = 100 μm. (C–E) Length (C) and cortical cell number (D) of the root meristem zones and root diameter (E) of the corresponding seedlings indicated in panels (A) and (B). Data are means ± SD (n ≥ 10 independent seedlings). Different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's test).

Figure 8

The proposed model of root responses in uncompacted soil and compacted soil. Compacted soil condition promotes higher ethylene response due to restricted diffusion of ethylene, leading to the accumulation of OsEIL1 in the roots. OsEIL1 directly activates YUC8 to promote auxin biosynthesis in epidermal cells, thus inhibiting root elongation. In parallel, OsEIL1 activates ABA biosynthesis in cortical cells, causing radial expansion of root cortical cells and root swelling. Increased ABA mediating OsbZIP46 activates the expression of YUC8 to promoting auxin biosynthesis, which further inhibits epidermal cell elongation and root elongation, ultimately resulting in short and swollen roots. The solid lines indicate direct interactions, the dashed lines indicate indirect interactions, the blue lines indicate regulatory relationships reported in previous studies, the red lines indicate regulatory relationships reported in this study, and the thickness of the lines indicates the strength of regulation.