WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis

Abstract

De novo organogenesis is a process through which wounded or detached plant tissues or organs regenerate adventitious roots and shoots. Plant hormones play key roles in de novo organogenesis, whereas the mechanism by which hormonal actions result in the first-step cell fate transition in the whole process is unknown. Using leaf explants of Arabidopsis thaliana, we show that the homeobox genes WUSCHEL RELATED HOMEOBOX11 (WOX11) and WOX12 are involved in de novo root organogenesis. WOX11 directly responds to a wounding-induced auxin maximum in and surrounding the procambium and acts redundantly with its homolog WOX12 to upregulate LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and LBD29, resulting in the first-step cell fate transition from a leaf procambium or its nearby parenchyma cell to a root founder cell. In addition, our results suggest that de novo root organogenesis and callus formation share a similar mechanism at initiation.

Figure 1.

Wound-Induced Auxin Accumulation and Polar Transport Are Essential for Adventitious Root Formation. (A) and (B) Leaf explants at 8 (A) and 12 DAC (B) on B5 medium. (C) The 12-DAC leaf explant on NPA-B5. A total of 30 leaf explants were analyzed, and they all failed to form adventitious roots. (D) Addition of IAA to NPA-B5 could rescue the NPA-caused rooting defect. Bars show sd with three biological repeats. n = 30 in each individual repeat. (E) to (J) GUS staining at time 0 ([E] and [F]), 1 DAC ([G] and [H]), and 2 DAC ([I] and [J]) of DR5_pro:GUS leaf explants cultured on B5 medium. (K) Transverse section through the GUS-staining region of a 2-DAC DR5_pro:GUS leaf explant grown on B5 medium. Note that GUS staining was mainly concentrated in the procambium and the nearby parenchyma cells. See the similar section without toluidine blue staining in Supplemental Figure 1. (L) and (M) GUS staining of a 4-DAC DR5_pro:GUS leaf explant on B5 medium. Arrow in (M) indicates an emerging root primordium. (N) and (O) GUS staining of a 2-DAC DR5_pro:GUS leaf explant cultured on NPA-B5. (F), (H), (J), (M), and (O) are close-ups of the boxed regions in (E), (G), (I), (L), and (N), respectively. rp, root primordium; xy, xylem; xp, xylem parenchyma cell; pc, procambium; ph, phloem. Bars = 1 mm in (A) to (C), 500 μm in (E), (G), (I), (L), and (N), 100 μm in (F), (H), (J), (M), and (O), and 50 μm in (K).

Figure 2.

Callus Formation Resembles Adventitious Root Formation. (A) to (C) Phenotypes of the 8-DAC leaf explants on B5 medium containing 1 μM (A), 0.1 μM (B), and 0.01 μM (C) IAA. (D) and (E) Leaf explants of alf4-1 (D) and clf-50 swn-1 (E) mutants, which are defective in adventitious root formation. A total of 30 leaf explants from each mutant were analyzed, and the results were consistent. (F) and (G) Lateral root formation differs between wild-type Col-0 (F) and arf7-1 arf19-1 (G). Seedlings were grown on half-strength MS medium for14 d. (H) and (I) Leaf explants from the arf7-1 arf19-1 double mutant on B5 medium (H) or B5 medium containing 1 μm IAA (I). Note that seedlings of the arf7-1 arf19-1 double mutant produce no lateral roots but can generate adventitious roots normally without IAA or callus with 1 μM IAA. A total of 30 leaf explants were tested, and the results were consistent. Bars = 1 mm.

Figure 3.

WOX11 and WOX5 Are Differentially Expressed during Adventitious Root Formation. (A) The level of epigenetic marker H3K27me3 at the WOX11 locus was dramatically reduced in the 20-DAC leaf explants that produce callus (brown) compared with that in the time-0 leaf explants (green). Note that the high level of H3K27me3 at a locus usually marks repression of the corresponding gene (Schatlowski et al., 2008). (B) WOX11 expression was upregulated in the 20-DAC leaf explants cultured on CIM compared with that in the time-0 explants. The data in (A) and (B) were generated from previous ChIP-chip and microarray analyses, respectively (He et al., 2012). (C) qRT-PCR analysis of WOX11 expression in the 1-DAC leaf explants on B5 media without IAA or with 2 μM IAA. The value of time-0 leaf explants was arbitrarily fixed at 1.0. Bars show se with three technical repeats. **P < 0.01 in two-sample t test compared with time-0 leaf explants. (D) to (G) GUS staining of time-0 ([D] and [E]) and 2-DAC ([F] and [G]) leaf explants from WOX11_pro:GUS plants, cultured on B5 medium. (H) Transverse section of a 2-DAC WOX11_pro:GUS leaf explant grown on B5 medium. Note that GUS staining appeared mainly in the procambium cells and some xylem parenchyma cells. See the similar section without toluidine blue staining in Supplemental Figure 1. (I) and (J) GUS staining of a 4-DAC WOX11_pro:GUS leaf explant cultured on B5 medium. (K) to (N) GUS staining of 2-DAC ([K] and [L]) and 4-DAC ([M] and [N]) WOX5_pro:GUS leaf explants cultured on B5 medium. (E), (G), (J), (L), and (N) are close-ups of the boxed regions in (D), (F), (I), (K), and (M), respectively. rf, root founder cell; rp, root primordium; xy, xylem; xp, xylem parenchyma cell; pc, procambium; ph, phloem. Asterisks indicate the dividing cells that are in the process of forming a root primordium. Bars = 500 μm in (D), (F), (I), (K), and (M) and 50 μm in (E), (G), (H), (J), (L), and (N).

Figure 4.

Auxin Directly Induces WOX11 Expression. (A) Diagram of structures of the WOX11_pro:GUS and mWOX11_pro:GUS constructs. (B) to (E) GUS staining of WOX11_pro:GUS ([B] and [C]) and mWOX11_pro:GUS ([D] and [E]) leaf explants after 6 h cultured on B5 medium containing 1 μM IAA. Two independent WOX11_pro:GUS or mWOX11_pro:GUS lines were analyzed and the results were consistent. (F) and (G) GUS staining was undetectable in 2-DAC leaf plants from WOX11_pro:GUS plants cultured on NPA-B5. (C), (E), and (G) are close-ups of the boxed regions in (B), (D), and (F), respectively. Bars = 500 μm in (B), (D), and (F) and 100 μm in (C), (E), and (G).

Figure 5.

WOX11 and WOX5 Expression Patterns in Callus Formation. (A) to (C) GUS staining of the 2-DAC (A) and 4-DAC ([B] and [C]) leaf explants from WOX11_pro:GUS plants on CIM. GUS staining was found in the vascular tissues near wounds in 2-DAC leaf explants (A) and became stronger at 4 DAC ([B] and [C]). However, GUS staining was absent in the rapidly proliferating callus cells in the 4-DAC explants (C). (D) to (F) GUS staining of the 2-DAC (D) and 4-DAC ([E] and [F]) WOX5_pro:GUS leaf explants cultured on CIM. GUS staining of WOX5_pro:GUS was not detected in the 2-DAC leaf explants (D) but was found in the proliferating callus cells in the 4-DAC explants ([E] and [F]). (C) and (F) are close-ups of the boxed regions in (B) and (E), respectively. Bars = 100 μm.

Figure 6.

WOX11 and WOX12 Are Involved in Adventitious Root and Callus Formation. (A) Overexpression of WOX11 accelerated adventitious root formation on B5 medium. Bars show sd with three biological repeats. n = 30 in each repeat. (B) The 8-DAC leaf explant of 35S_pro:WOX11, showing five regenerated adventitious roots on B5 medium. (C) The 8-DAC leaf explant of 35S_pro:WOX11, showing callus formation (arrowhead) on B5 medium. (D) Defective adventitious root formation in 10% of leaf explants (three out of 30 from two independent 35S_pro:WOX11 lines) on NPA-B5 could be rescued by overexpression of WOX11. Shown is a 12-DAC leaf explant. (E) and (F) Leaf explants from wild-type (E) and 35S_pro:WOX11 (F) plants were cultured on CIM. Note that the callus formation in the 35S_pro:WOX11 leaf explants was markedly accelerated compared with that in the wild-type leaf explants. A total of 30 leaf explants from two independent 35S_pro:WOX11 lines were tested. (G) WOX11 and WOX12 functions are involved in de novo root organogenesis. Both wox11-2 and wox12-1 displayed delayed adventitious root formation, and the wox11-2 wox12-1 double mutant showed even slower rooting on B5 medium. Bars show sd with three biological repeats. n = 30 in each repeat. (H) Quantitative analyses of the adventitious root number per 12-DAC leaf explant grown on B5 medium. Bars show sd with three biological repeats. n = 30 in each repeat. (I) The 12-DAC leaf explant of the 35S_pro:WOX11-SRDX plant Line 2, showing rooting defect on B5 medium. (J) Quantitative analyses of the adventitious root formation using leaf explants from two independent 35S_pro:WOX11-SRDX transgenic lines, Line 1 and Line 2. A total of 30 leaf explants from each line were analyzed. (K) qRT-PCR analyses of WOX11-SRDX expression levels in Line 1 and Line 2 of the 35S_pro:WOX11-SRDX transgenic plants. Note that the higher WOX11-SRDX expression level in Line 2 explants is consistent with its stronger defect in rooting. (L) qRT-PCR analysis showed that WOX5 was only weakly expressed in 4-DAC leaf explants of Line 2, compared with that in wild-type Col-0. In (K) and (L), the value of wild-type leaf explants was arbitrarily fixed at 1.0, and bars show se from three technical repeats. (M) Quantitative analyses of the lateral root number per 1 cm in length of the primary root from 12-d-old Line 1 and Line 2 grown on half-strength MS medium. Bars show sd from three biological repeats. n = 10 seedlings in each individual repeat. Bars = 2 mm in (B) and 1 mm in (C) to (F) and (I).

Figure 7.

WOX11 Upregulates LBD16 and LBD29. (A) qRT-PCR analyses showed that expression levels of the analyzed LBD genes were elevated during the culture of leaf explants on B5 medium. (B) qRT-PCR analyses of LBD and WOX11 expression using rosette leaves of 12-d-old 35S_pro:WOX11 seedlings. Two independent 35S_pro:WOX11 lines were analyzed, and the results were consistent. Values of time-0 leaf explants in (A) and wild-type leaves in (B) were arbitrarily fixed at 1.0. Bars show se from three technical repeats. **P < 0.01 in two-sample t test comparing with time-0 leaf explants (A) or wild-type leaves (B). (C) The 12-DAC 35S_pro:LBD29 leaf explant on B5 medium, showing multiple regenerated adventitious roots. (D) The 8-DAC 35S_pro:LBD29 leaf explant cultured on B5 medium, showing callus formation (arrowhead). A total of 40 leaf explants from two independent 35S_pro:LBD29 lines were analyzed on B5 medium. Among them, eight showed callus growth (D), while the rest regenerated multiple adventitious roots (C). (E) The 8-DAC leaf explant of 35S_pro:LBD29 cultured on CIM, showing robust callus growth. A total of 30 leaf explants from two independent 35S_pro:LBD29 lines were tested, and all of them showed similar phenotypes. (F) The 12-DAC leaf explant of pER8:LBD29-SRDX, showing defective adventitious root formation on B5 medium. A total of 70 leaf explants from three independent lines were tested, and 47 of them showed the regeneration defect. (G) The 10-DAC leaf explant of 35S_pro:LBD29/35S_pro:WOX11-SRDX, in which the regeneration defect caused by 35S_pro:WOX11-SRDX was partly rescued. The 35S_pro:LBD29/35S_pro:WOX11-SRDX plants were constructed by crossing a phenotypically tested 35S_pro:LBD29 line to Line 1 of 35S_pro:WOX11-SRDX. The F1 seedlings, after PCR verification, were used for regeneration analysis. Among a total of 16 leaf explants analyzed, 12 formed adventitious roots or callus on B5 medium. (H) qRT-PCR analyses of WOX11 and LBD29 expression using rosette leaves from 12-d-old 35S_pro:LBD29 seedlings. Two independent 35S_pro:LBD29 lines were analyzed, and the results were consistent. Values of wild-type Col-0 leaves were arbitrarily fixed at 1.0, and bars show se from three technical repeats. **P < 0.01 in two-sample t test comparing with wild-type leaves. Bars = 1 mm in (C) to (G).

Figure 8.

A Model for de Novo Root Organogenesis. A cellular and molecular framework of de novo root organogenesis from Arabidopsis leaf explants is shown in the model. Note that there are two steps of cell fate transition during adventitious root formation.