Depletion of Gibberellin Signaling Up-Regulates LBD16 Transcription and Promotes Adventitious Root Formation in Arabidopsis Leaf Explants

Abstract

Adventitious root (AR) formation in plants originates from non-root organs such as leaves and hypocotyls. Auxin signaling is essential for AR formation, but the roles of other phytohormones are less clear. In Arabidopsis, at least two distinct mechanisms can produce ARs, either from hypocotyls as part of the general root architecture or from wounded organs during de novo root regeneration (DNRR). In previous reports, gibberellin acid (GA) appeared to play reverse roles in both types of ARs, since GA treatment blocks etiolation-induced AR formation from hypocotyls, whereas GA synthesis and signaling mutants apparently displayed reduced DNRR from detached leaves. In order to clarify this contradiction, we employed the GA biosynthesis inhibitor paclobutrazol (PBZ) and found that PBZ had positive effects on both types of AR formation in Arabidopsis. Consistently, GA treatment had negative effects on both AR formation mechanisms, while loss of GA synthesis and signaling promoted DNRR under our conditions. Our results show that PBZ treatment can rescue declined AR formation in difficult-to-root leaf explants such as erecta receptor mutants. Furthermore, transcriptional profiling revealed that PBZ treatment altered GA, brassinosteroids, and auxin responses, which included the up-regulation of LBD16 that is well known for its pivotal role in AR initiation.

Keywords: LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16); LATERAL ROOT PRIMORDIUM 1 (LRP1); adventitious roots; blocking of gibberellin biosynthesis; de novo root regeneration (DNRR); paclobutrazol (PBZ); rescuing difficult-to-root mutant leaf explants.

Figure 1

Effects of GA and PBZ treatments on rooting rate of AR formation from hypocotyls and DNRR. (A,B) AR formation from hypocotyls. (A) Rooting phenotype of Col-0 seedlings under mock, GA, and PBZ treatments. Scale bar = 10 mm. (B) ARs per hypocotyls (Col-0) under mock, GA, and PBZ treatments 7 days after shift from dark to LD, 10 DAG. (C–H) DNRR from Col-0 explants under mock, GA, and PBZ treatments (LD and dark conditions). (C) Rooting rate of Col-0 leaf explants under mock, GA, and PBZ treatments 9 DAC (LD). (D,E,H) Phenotype of Col-0 leaf explants under mock, GA, and PBZ treatments 9 DAC (LD). Arrow indicates the absence of an AR (E). Note that roots are thicker after PBZ treatment in comparison to mock treatment (arrow heads in (D,H)). Photos were taken using Nikon (SMZ25) microscope. Scale bar = 500 µm. (G) Dark conditions accelerate rooting additive to PBZ treatment. (B,C,F,G) Average values are shown, ± SEM. Color letters ((B,C); One way ANOVA, p < 0.05) and asterisks ((F,G); Student’s t-test: ** p < 0.01; *** p < 0.001) indicate significant changes.

Figure 2

Effects of different hormone treatments on rooting rate (time courses) and rooting capacity in wild-type (Col-0) leaf explants in darkness. (A–C) Single treatments with IAA, NPA, GA, and PBZ. (A) Rooting phenotype of Col-0 leaf explants. Scale bar = 10 mm. (B) Rooting rate. (C) Rooting capacity. Note that rooting capacity was decreased by PBZ, while IAA treatment increased rooting rate and rooting capacity. (D,E) Treatments with NPA, PBZ, and NPA+PBZ. Note that rooting is completely abolished by NPA treatment even in presence of PBZ, indicating that polar auxin transport is essential for accelerating effects of PBZ on DNRR. (F,G) Treatments with IAA, PBZ, and IAA + PBZ. Note that accelerating effects on rooting rates by IAA is epistatic to PBZ treatment (no additive effect), but PBZ significantly reduced rooting capacity even in presence of IAA. Treatments: 0.1 µM IAA, 1 µM NPA, 1 µM GA, and 5 µM PBZ. (B–G) Average values are shown, ± SEM. Asterisks indicate significant changes (Student’s t-test: * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Reanalysis of GA biosynthesis and GA signaling mutants reveals (i) negative effects of GA and (ii) positive effects of ER signaling on DNRR in darkness. (A) Rooting rate time course: rooting rate of three GA mutants, ga1-4, ga5-1, and gai-1, are significantly higher than those in wild type (Ler-0). (B) Rooting rate time course: in comparison to wild-type Col-0, rooting was almost abolished in leaf explants of both er-1 (Ler-0) and er-105 (Col-0) mutants. (C) Treatment with 5 µM PBZ can recue low rooting rates of both er-1 (Ler-0) and er-105 (Col-0) mutants (9 DAC). (A–C) Average values are shown, ± SEM. Asterisks indicate significant changes (Student’s t-test: * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Expression changes in response to PBZ treatment 24 HAC. (A) Number of genes with significantly decreased (down) and increased (up) expression rates. (B) Percentage of GA/BR- and auxin-regulated genes and their overlaps. (C) Conceptional model of potential pathways explaining how PBZ could increase LBD16 expression. Dotted lines represent known interactions that seem to be covered during DNRR with PBZ. Broken lines represent putative, probable indirect interactions. Note that most arrows indicate transcriptional regulation, but DELLA proteins bind directly to BZR1, PIF, and ARF6 proteins, PBZ blocks GA synthesis, and PIN1 is auxin (IAA) transporter. (D) Expression changes in response to PBZ treatment (24 HAC) of genes that can theoretically increase LBD16 and LRP1 expression levels. Relative expression in PBZ-treated er-105 leaf explants compared to mock treatment using CPM values in Supplementary Tables S1 and S2; ±SEM; n = 4. Asterisks indicate significant changes (Student’s t-test: * p < 0.05; ** p < 0.01). (E) Selection of genes with significantly decreased (down-regulated) and increased (up-regulated) expression rates. Regulation by GA/BR, IAA, and/or wounding as well as promoter binding by PIF4, BZR1, and/or ARF6 is indicated.

Figure 5

Time courses of LBD16::GUS expression in response to mock, PBZ, PBZ + GA, and PBZ + NPA treatments. HAC, hours after cultivation (induction). (A–C) Mock treatment. Note that LBD16::GUS expression is evidently visible at future rooting site after 48 HAC (C,C’). (D,E) PBZ treatment. Note that LBD16::GUS expression is stronger after PBZ treatment (48 HAC, E,E’). (D,E) PBZ+GA treatment. Note that LBD16::GUS expression is strongly reduced by GA (48 HAC, G,G’). (F,G) PBZ + GA treatment. Note that LBD16::GUS expression is strongly reduced by GA (48 HAC, G,G’). (H,I) PBZ + NPA treatment. Note that LBD16::GUS expression is fully quenched by NPA (48 HAC, I,I’). White arrow heads indicate the LBD16::GUS staining, its changes (C’,E’,G’) and/or its absence (I’). Photos were taken using Olympus (CX43) microscope. Scale bar = 250 µm.