Callus Initiation from Root Explants Employs Different Strategies in Rice and Arabidopsis

Callus Initiation from Root Explants Employs Different Strategies in Rice and Arabidopsis Abstract Callus formation in tissue culture follows the rooting pathway, and newly formed callus seems to be a group of root primordium-like cells. However, it is not clear whether there are multiple mechanisms of callus initiation in different species and in different organs. Here we show that the OsIAA11-mediated pathway is specifically and strictly required for callus initiation in the lateral root (LR) formation region of the primary root (PR) but not for callus initiation at the root tip or the stem base in rice. OsIAA11 and its Arabidopsis homolog AtIAA14 are key players in lateral rooting. However, the AtIAA14-mediated pathway is not strictly required for callus initiation in the LR formation region in Arabidopsis. LRs can be initiated through either the AtIAA14-mediated or AtWOX11-mediated pathway in the Arabidopsis PR, therefore providing optional pathways for callus initiation. In contrast, OsIAA11 is strictly required for lateral rooting in the rice PR, meaning that the OsIAA11 pathway is the only choice for callus initiation. Our study suggests that multiple pathways may converge to WOX5 activation during callus formation in different organs and different species. Introduction Plants have powerful regenerative abilities, which have been widely applied in agricultural technologies (Knizewski et al. 2008, Sugimoto et al. 2011, Su and Zhang 2014, Xu and Huang 2014, Ikeuchi et al. 2016, Kareem et al. 2016, Lup et al. 2016, Birnbaum and Roudier 2017). In tissue culture, when stimulated by a high level of auxin, detached explants form a pluripotent callus, from which roots and shoots may regenerate (Sugimoto et al. 2011, Xu and Huang 2014). Recent studies have suggested that callus formation follows the rooting pathway, and newly formed callus cells are likely to be a group of fast-dividing root primordium-like cells (Che et al. 2007, Atta et al. 2009, Sugimoto et al. 2010, Fan et al. 2012, He et al. 2012, Liu et al. 2014, Yu et al. 2017, Liu et al. 2018). The ability to form callus in tissue culture varies in diverse species. For example, mature leaves of the dicot Arabidopsis (Arabidopsis thaliana) readily form callus, while the mature region of leaves in the monocot rice (Oryza sativa) is extremely unresponsive to tissue culture (Hu et al. 2017). Not all somatic cells are competent to form callus; only a group of adult stem cells, i.e. regeneration-competent cells, may initiate regeneration (Che et al. 2007, Atta et al. 2009, Sugimoto et al. 2010, Liu et al. 2014). In Arabidopsis, the procambium and some vascular parenchyma cells in leaves and the xylem-pole pericycle cells in roots may serve as regeneration-competent cells, while in rice the bundle sheath and some immature vascular cells in leaves and the phloem-pole pericycle cells in roots may serve as regeneration-competent cells (Hu et al. 2017). These competent cells are responsible not only for callus initiation but also for root primordium initiation during adventitious root (AR) or lateral root (LR) formation (Liu et al. 2014, Hu and Xu 2016, Hu et al. 2017, Sheng et al. 2017). The different differentiation statuses of these regeneration-competent cells result in diverse regenerative abilities in Arabidopsis and rice (Hu et al. 2017). In Arabidopsis, there are at least two rooting pathways that may contribute to post-embryonic root system formation: the WUSCHEL-RELATED HOMEOBOX11 (AtWOX11)-mediated and AtIAA14-AUXIN RESPONSE FACTOR7/19 (AtIAA14–AtARF7/19)-mediated rooting pathways (Sheng et al. 2017, Ge et al. 2018). The AtWOX11-mediated rooting pathway contributes to AR formation from leaf explants or adventitious LR formation from the primary root (PR) in response to wounding or environmental signals (Sheng et al. 2017, Ge et al. 2018). Using AR formation from leaf explants as an example, auxin first activates AtWOX11 expression during the fate transition from regeneration-competent cells to root founder cells, and then AtWOX11 initiates the fate transition from root founder cells to root primordium cells via activation of WUSCHEL-RELATED HOMEOBOX5 (AtWOX5) and LATERAL ORGAN BOUNDARIES DOMAIN16 (AtLBD16) (Liu et al. 2014, Hu and Xu 2016, Sheng et al. 2017, Xu 2018). Co-expression of AtWOX5 and AtLBD16 may be a specific marker of root primordium cells (Xu 2018). The AtIAA14–AtARF7/19-mediated pathway (also known as the non-AtWOX11-mediated pathway) is required for acropetal LR formation (Ge et al. 2018) following an oscillatory root cap-derived auxin flux signal (Fukaki et al. 2002, Okushima et al. 2007, Lee et al. 2009, Peret et al. 2009, Moreno-Risueno et al. 2010, Goh et al. 2012, Lavenus et al. 2013, Van Norman et al. 2014, Xuan et al. 2015, Xuan et al. 2016). Degradation of AtIAA14 upon auxin signaling releases AtARF7/19 to activate AtLBD16, and probably also AtWOX5, for LR primordium initiation (Fukaki et al. 2002, Okushima et al. 2007, Lee et al. 2009, Goh et al. 2012). In rice, there are also two pathways to initiate roots in the post-embryonic stage, i.e. the OsWOX11-mediated and the OsIAA11-mediated pathways. OsWOX11 is the AtWOX11 homolog in rice and is required for AR (crown root) formation but not LR formation (Zhao et al. 2009). OsIAA11 in rice is the closest homolog of AtIAA14 in Arabidopsis. The Osiaa11 mutant, which has mutations in domain II of the OsIAA11 protein and interrupts the auxin signaling pathway by constitutively suppressing ARF activity, is defective in generating LRs (Zhu et al. 2012). However, ARs (crown roots) could be formed in the Osiaa11 mutant, suggesting that the OsIAA11-mediated pathway is specifically required for LR formation. Callus formation borrows the rooting pathway and newly formed callus seems to be a group of root primordium-like cells which specifically express WOX5 and LBD16 (Sugimoto et al. 2010, Fan et al. 2012, Liu et al. 2014, Hu et al. 2017, Lee et al. 2017, Liu et al. 2018). However, it is not clear whether there are different mechanisms of callus initiation in different species and in different organs. In this study, we show that the OsIAA11-mediated pathway is strictly required for callus initiation from the rice LR formation region of PR explants, whereas the AtIAA14-mediated pathway is not strictly required for callus initiation in the LR formation region in Arabidopsis. Thus, rice and Arabidopsis employ different strategies for callus initiation from root explants. Results and Discussion Callus initiation strictly requires the OsIAA11-mediated pathway in the lateral root formation region of rice root explants To analyze the mechanism of callus initiation from rice root explants, we first carried out phenotype analysis using PRs from 7-day-old wild-type Kasalath and Osiaa11 mutant plants (Zhu et al. 2012). The 7-day-old PRs were at the growing stage, and almost no LRs could be observed by eye in the wild type (Fig. 1A). We cultured the detached PRs on callus-inducing medium (CIM) to induce callus formation and on N6 medium without exogenous hormones as a control. At 14 d after culture (DAC) on N6 medium, the wild-type PRs formed many LRs (Fig. 1B), while the Osiaa11 PRs had no LRs (Fig. 1F, G), which was consistent with previous reports that OsIAA11 is required for LR formation (Zhu et al. 2012). At 14 DAC on CIM, the wild-type PR explants formed callus in both the LR formation region and the root tip region (Fig. 1C–E), while the root explants from the Osiaa11 mutant PR formed callus only in the root tip region, not in the LR formation region (Fig. 1H–J). No callus was found in the Osiaa11 LR formation region during a prolonged culture period at 20 DAC (Supplementary Fig. S1). Therefore, callus formation strictly required the OsIAA11-mediated lateral rooting pathway in the LR formation region, but not in the root tip region. Fig. 1 View largeDownload slide Callus formation from rice primary roots. (A) PR from 7-day-old wild-type rice cultured on MS medium before tissue culture. (B) LR formation from wild-type rice after 14 d on N6 medium. (C) Callus formation from wild-type rice PRs after 14 d on CIM. The PR explants were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type rice PRs. (F) PR from 7-day-old Osiaa11 rice cultured on MS medium before tissue culture. (G) No LR formation from Osiaa11 PRs cultured on N6 medium for 14 d. (H) Callus formation in the root tip region but not in the LR formation region from Osiaa11 PRs cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Osiaa11 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of OsWOX5 in the wild type and Osiaa11 in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 5 mm in (A–C, F–H), 50 μm in (D, E, I, J). Fig. 1 View largeDownload slide Callus formation from rice primary roots. (A) PR from 7-day-old wild-type rice cultured on MS medium before tissue culture. (B) LR formation from wild-type rice after 14 d on N6 medium. (C) Callus formation from wild-type rice PRs after 14 d on CIM. The PR explants were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type rice PRs. (F) PR from 7-day-old Osiaa11 rice cultured on MS medium before tissue culture. (G) No LR formation from Osiaa11 PRs cultured on N6 medium for 14 d. (H) Callus formation in the root tip region but not in the LR formation region from Osiaa11 PRs cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Osiaa11 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of OsWOX5 in the wild type and Osiaa11 in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 5 mm in (A–C, F–H), 50 μm in (D, E, I, J). We then tested expression levels of OsWOX5, which is a marker gene of newly formed callus, in root explants from the wild type and the Osiaa11 mutant. OsWOX5 was highly up-regulated in both the LR formation region and the root tip region in wild-type root explants at 2 DAC on CIM, while it was up-regulated in the root tip region but not in the LR formation region of the Osiaa11 root explants (Fig. 1K, L; Supplementary Fig. S2). This suggests that the establishment of the callus cell fate is strictly dependent on the OsIAA11-mediated pathway in the LR formation region but not in the root tip region.Quantitative reverse transcription–PCR (qRT–PCR) results showed that OsWOX11 was up-regulated in the root tip region of PRs in rice (Supplementary Fig. S3). OsWOX11 was also up-regulated in the LR formation region of PRs from Osiaa11 in rice (Supplementary Fig. S4). However, the up-regulation of OsWOX11 was not sufficient to induce callus initiation in the LR formation region in the Osiaa11 background. The AtIAA14-mediated pathway is not strictly required for callus initiation in the lateral root formation region of Arabidopsis root explants We next analyzed the role of AtIAA14 in callus initiation from Arabidopsis root explants. We cultured PRs from 7-day-old wild-type Col-0 and Atiaa14 mutant plants. The 7-day-old PRs had almost no LRs in the wild type or Atiaa14 at this stage (Fig. 2A, F). The PRs were cultured on CIM to induce callus formation and on N6 medium without exogenous hormones as a control. The wild-type PRs developed many LRs on N6 medium at 14 DAC (Fig. 2B), while the Atiaa14 PRs did not produce LRs (Fig. 2G), suggesting that AtIAA14, like its rice homolog OsIAA11, is critical for LR initiation (Fukaki et al. 2002). Interestingly, both the wild-type and Atiaa14 root explants formed callus in the LR formation region and the root tip region, although the callus mass was relatively smaller in Atiaa14 than in the wild type (Fig. 2C, H). A prolonged culture period led to better growth of the callus mass at 20 DAC (Supplementary Fig. S5). AtWOX11 was up-regulated in the LR formation region of both Col-0 and Atiaa14 PRs (Supplementary Fig. S6). Taken together, this suggested that, in contrast to the case in rice, the AtIAA14-mediated lateral rooting pathway is not strictly required for callus initiation in the LR formation region of Arabidopsis. Fig. 2 View largeDownload slide Callus formation from Arabidopsis primary roots. (A) PR from 7-day-old wild-type Arabidopsis cultured on MS medium before tissue culture. (B, C) LR formation after 14 d on N6 medium (B) and callus formation after 14 d on CIM (C) from wild-type Arabidopsis PRs. The PR explants in (C) were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type Arabidopsis PRs. (F) PR from a 7-day-old Atiaa14 mutant cultured on MS medium before tissue culture. (G) No LR formation after 14 d on N6 medium. (H) Callus formation in the LR formation region and the root tip region from Atiaa14 PR explants cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Atiaa14 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of AtWOX5 in wild-type Arabidopsis and the Atiaa14 mutant in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 3 mm in (A–C, F–H), 50 μm in (D, E, I, J). Fig. 2 View largeDownload slide Callus formation from Arabidopsis primary roots. (A) PR from 7-day-old wild-type Arabidopsis cultured on MS medium before tissue culture. (B, C) LR formation after 14 d on N6 medium (B) and callus formation after 14 d on CIM (C) from wild-type Arabidopsis PRs. The PR explants in (C) were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type Arabidopsis PRs. (F) PR from a 7-day-old Atiaa14 mutant cultured on MS medium before tissue culture. (G) No LR formation after 14 d on N6 medium. (H) Callus formation in the LR formation region and the root tip region from Atiaa14 PR explants cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Atiaa14 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of AtWOX5 in wild-type Arabidopsis and the Atiaa14 mutant in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 3 mm in (A–C, F–H), 50 μm in (D, E, I, J). To confirm this finding at the molecular level, we analyzed the expression levels of the callus marker gene AtWOX5 in root explants from the wild type and the Atiaa14 mutant. AtWOX5 was highly up-regulated in both the LR formation region and the root tip region in the wild type and the Atiaa14 mutant at 2 DAC on CIM (Fig. 2K, L). Therefore, the cell fate transition to callus could be accomplished in both the wild type and the Atiaa14 mutant. Overall, the major difference between OsIAA11 and AtIAA14 is their roles in callus formation in the LR formation region, suggesting that callus formation in the LR formation region is controlled by different mechanisms in rice and Arabidopsis. Different rooting capacities of rice and Arabidopsis primary roots upon wounding We thought that the different strategies of callus initiation in the LR formation region in rice and Arabidopsis might be due to the different LR formation mechanisms in the two species. Our previous study suggested that the Arabidopsis PR can produce two types of LRs, i.e. acropetal LRs and adventitious LRs (Sheng et al. 2017, Ge et al. 2018). When Arabidopsis is grown vertically on the medium, LR formation usually follows the AtIAA14–AtARF7/19 pathway to initiate the acropetal LR primordium; however, when Arabidopsis PRs are wounded, the AtWOX11-mediated rooting pathway can be used for adventitious LR initiation. We tested whether the AtWOX11-mediated rooting pathway functioned in the Atiaa14 mutant by cutting the PRs and culturing the wounded PRs on N6 medium. The results showed that the wounded Atiaa14 PRs could produce adventitious LRs upon wounding (Fig. 3A, B). WOX11 expression was induced in the wound site at 1 d after excision (DAE) (Fig. 3C, D). Fig. 3 View largeDownload slide Rooting abilities of primary roots of Arabidopsis and rice. (A, B) Formation of adventitious LRs on a wounded PR (arrow) of Atiaa14 at 14 DAE. (B)A close-up of the wound site in (A). (C, D) GUS staining of AtWOX11pro:GUS in intact (C) or wounded (D) PRs of Atiaa14 at 1 DAE. (E, F) No LR formation was found on wounded primary roots (arrow) of Osiaa11 at 5 DAE (E) and 14 DAE (F). Scale bars = 5 mm in (A), 50 μm in (C, D) and 1 mm in (B, E, F). Fig. 3 View largeDownload slide Rooting abilities of primary roots of Arabidopsis and rice. (A, B) Formation of adventitious LRs on a wounded PR (arrow) of Atiaa14 at 14 DAE. (B)A close-up of the wound site in (A). (C, D) GUS staining of AtWOX11pro:GUS in intact (C) or wounded (D) PRs of Atiaa14 at 1 DAE. (E, F) No LR formation was found on wounded primary roots (arrow) of Osiaa11 at 5 DAE (E) and 14 DAE (F). Scale bars = 5 mm in (A), 50 μm in (C, D) and 1 mm in (B, E, F). We then tested whether the OsWOX11-mediated rooting pathway could be used in rice PRs to produce LRs. We cut Osiaa11 PRs and cultured the wounded PRs on N6 medium. The results showed that the wounded Osiaa11 PRs were not able to produce roots at the wound sites (Fig. 3E, F). Therefore, OsIAA11 is the sole pathway for LR initiation in rice PRs. This can explain why callus formation is strictly dependent on OsIAA11 in the LR formation region of rice. Callus formation at the stem base of the Osiaa11 mutant Although the Osiaa11 mutant was defective in LR initiation, it had the ability for AR (crown root) formation (Fig. 4A, B, D, E) (Zhu et al. 2012). We then tested the callus formation ability in the stem base of Osiaa11. When the excised shoots were cultured on CIM for 14 d, callus was produced in the crown root initiation region on the stem bases of both the wild type (Fig. 4C) and Osiaa11 (Fig. 4F). Fig. 4 View largeDownload slide Callus formation at the stem base in rice. (A) The explant of wild-type rice stem base from the 3-day-old seedling grown in the dark. (B) AR formation from the wild-type rice stem base after 1 d on N6 medium. (C) Callus formation from the wild-type rice stem base after 14 d culture on CIM. (D) The explant of the Osiaa11 rice stem base from the 3-day-old seedling grown in the dark. (E) AR formation from the Osiaa11 rice stem base after 1 d on N6 medium. (F) Callus formation from the Osiaa11 rice stem base after 14 d culture on CIM. (G) Relative transcript levels of OsWOX5 and OsWOX11 in the stem base region of the wild type or Osiaa11. Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) were arbitrarily fixed at 1.0. (H–M) Longitudinal (H, I, K, L) and transverse (J, M) sections of the wild-type stem base, showing in situ hybridization of OsWOX5. Seedlings grown in the dark for 3 d were used to observe AR primordium formation (H–J). Those seedlings were then cultured on CIM for 4 d to observe callus formation (K–M). (H) and (K) are sense controls. Scale bars = 5 mm in (A–F), 1 mm in (H, K) and 500 μm in (I, J, L, M). Fig. 4 View largeDownload slide Callus formation at the stem base in rice. (A) The explant of wild-type rice stem base from the 3-day-old seedling grown in the dark. (B) AR formation from the wild-type rice stem base after 1 d on N6 medium. (C) Callus formation from the wild-type rice stem base after 14 d culture on CIM. (D) The explant of the Osiaa11 rice stem base from the 3-day-old seedling grown in the dark. (E) AR formation from the Osiaa11 rice stem base after 1 d on N6 medium. (F) Callus formation from the Osiaa11 rice stem base after 14 d culture on CIM. (G) Relative transcript levels of OsWOX5 and OsWOX11 in the stem base region of the wild type or Osiaa11. Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) were arbitrarily fixed at 1.0. (H–M) Longitudinal (H, I, K, L) and transverse (J, M) sections of the wild-type stem base, showing in situ hybridization of OsWOX5. Seedlings grown in the dark for 3 d were used to observe AR primordium formation (H–J). Those seedlings were then cultured on CIM for 4 d to observe callus formation (K–M). (H) and (K) are sense controls. Scale bars = 5 mm in (A–F), 1 mm in (H, K) and 500 μm in (I, J, L, M). The qRT–PCR results showed that the expression levels of OsWOX5 and OsWOX11 increased significantly in the stem bases of the wild type and Osiaa11 cultured on CIM for 1 d (Fig. 4G). In situ hybridization results showed that OsWOX5 expression was detected in the crown root primordium and callus (Fig. 4H–M). These data indicated that the mutation in OsIAA11 does not affect the AR and callus initiation in the stem base of rice. Conclusion Overall, the strategies for callus initiation in the LR formation region are different: in rice, OsIAA11 is strictly required for callus initiation, but AtIAA14 is not indispensable in Arabidopsis. This is due to differences in LR initiation between rice and Arabidopsis. In Arabidopsis, LR formation can occur via two alternative pathways: the AtIAA14–AtARF7/19-mediated pathway for acropetal LRs developing from PRs and the AtWOX11-mediated pathway for adventitious LRs initiated upon wounding (Ge et al. 2018). In rice, LR formation from PRs can only occur via the OsIAA11-mediated pathway. The different rooting mechanisms in the LR formation region of PRs result in different callus initiation strategies in Arabidopsis and rice (see the model in Fig. 5). However, because Osiaa11 and Atiaa14 are both gain-of-function mutants, currently we cannot exclude the possibility that the repression of auxin signaling by mutated AtIAA14 is weaker than that of OsIAA11. Further analysis to reveal why OsWOX11 cannot function to produce LRs in rice PRs could improve our understanding of the evolution of root system formation and regenerative abilities in dicots and monocots. Fig. 5 View largeDownload slide Model of root and callus formation in Arabidopsis and rice. Fig. 5 View largeDownload slide Model of root and callus formation in Arabidopsis and rice. Materials and Methods Plant material Kasalath (Oryza sativa L. ssp. indica) was used as the rice wild type, and Columbia-0 (Col-0) was used as the Arabidopsis wild type. The Osiaa11 mutant (in the Kasalath background) was previously described (Zhu et al. 2012). Atiaa14 and AtWOX11pro:GUS were also described previously (Fukaki et al. 2002, Liu et al. 2014, Shang et al. 2016). Tissue culture and in situ hybridization For root-derived callus induction, sterile rice seeds were grown on Murashige and Skoog (MS) medium in a growth chamber with a 16 h light, 28°C/8 h dark, 24°C cycle. Sterilized seeds of Arabidopsis were sown on B5 medium (Gamborg B5 basal medium with 0.5 g l–1 MES, 3% sucrose and 0.8% agar, pH 5.7) and grown at 24°C under a 16 h light/8 h dark photoperiod. PRs were used for incubation of LRs or callus. MS medium and N6 medium were described previously (Murashige and Skoog 1962, Chu et al. 1975). Callus induction was performed on CIM (N6 basal medium with 10 μM 2,4-D, 0.5 g l–1 MES, 3% sucrose and 0.4% phytagel, pH 5.8). In situ hybridization was performed according to our previous method (Hu et al. 2017). qRT–PCR RNA extraction and qRT–PCR were performed as previously described (Guo et al. 2016), using the following gene-specific primers: 5′-ACCGGCTCATGACATGCTAC-3′ and 5′-ATACCGGACCTTGTCCACCT-3′ for OsWOX5; 5′-ACCACTTCGACCGCCACTACT-3′ and 5′-ACGCCTAAGCCTGCTGGTT-3′ for OsUbiqutin; 5′-ATGTTTGGGCAGGACGTGAT-3′ and 5′-GGAAGTAGCTCTCGCCCATC-3′ for OsWOX11; 5′-ACAATAACGGAGGAACGGGG-3′ and 5′-TGTTGGAGTTCTAAGACCGGC-3′ for AtWOX5; and 5′-TGAGCCTTCCTTGATGATGCT-3′, 5′-GCACTTGCGGCAAATCATCT-3′ for AtUbiqutin. The qRT–PCR results are shown as relative expression levels normalized against the expression of OsUbiqutin and AtUbiqutin. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [grant Nos. 31771776, 31630007 and 31422005)]; the China Agriculture Research System [grant CARS-05]; the National Basic Research Program of China [973 Program, grant No. 2014CB943500]; the Chinese Academy of Science (CAS) [Key Research Program (grant No. QYZDB-SSW-SMC010) and the Strategic Priority Research Program ‘Molecular Mechanism of Plant Growth and Development’ (grant No. XDPB0403)]. Acknowledgments We thank Dr. Chuanzao Mao (Zhejiang University, China) and Dr. Yuxin Hu (Institute of Botany, Chinese Academy of Sciences, China) for providing plant materials, and Hua Wang for providing technical assistance in the in situ hybridization experiment. F.G., H.Z. and W.L. performed most of the experiments; F.G., X.H., H.B. and L.X. designed the experiments and analyzed the data; N.H. and Q.Q. supervised the experiments; H.B. and L.X. conceived the project and wrote the article with contributions of all the authors; H.B. supervised and complemented the writing. Disclosures The authors have no conflicts of interest to declare. References Atta R., Laurens L., Boucheron-Dubuisson E., Guivarc’h A., Carnero E., Giraudat-Pautot V., et al.  . ( 2009) Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. Plant J . 57: 626– 644. Google Scholar CrossRef Search ADS PubMed  Birnbaum K.D., Roudier F. ( 2017) Epigenetic memory and cell fate reprogramming in plants. Regeneration (Oxf)  4: 15– 20. Google Scholar CrossRef Search ADS PubMed  Che P., Lall S., Howell S.H. ( 2007) Developmental steps in acquiring competence for shoot development in Arabidopsis tissue culture. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations AR adventitious root ARF7/19 AUXIN RESPONSE FACTOR7/19 CIM callus-inducing medium DAC days after culture DAE days after excision LBD16 LATERAL ORGAN BOUNDARIES DOMAIN16 LR lateral root PR primary root qRT–PCR quantitative reverse transcription–PCR WOX5 WUSCHEL-RELATED HOMEOBOX5 WOX11 WUSCHEL-RELATED HOMEOBOX11 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Callus Initiation from Root Explants Employs Different Strategies in Rice and Arabidopsis

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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10.1093/pcp/pcy095
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Abstract

Abstract Callus formation in tissue culture follows the rooting pathway, and newly formed callus seems to be a group of root primordium-like cells. However, it is not clear whether there are multiple mechanisms of callus initiation in different species and in different organs. Here we show that the OsIAA11-mediated pathway is specifically and strictly required for callus initiation in the lateral root (LR) formation region of the primary root (PR) but not for callus initiation at the root tip or the stem base in rice. OsIAA11 and its Arabidopsis homolog AtIAA14 are key players in lateral rooting. However, the AtIAA14-mediated pathway is not strictly required for callus initiation in the LR formation region in Arabidopsis. LRs can be initiated through either the AtIAA14-mediated or AtWOX11-mediated pathway in the Arabidopsis PR, therefore providing optional pathways for callus initiation. In contrast, OsIAA11 is strictly required for lateral rooting in the rice PR, meaning that the OsIAA11 pathway is the only choice for callus initiation. Our study suggests that multiple pathways may converge to WOX5 activation during callus formation in different organs and different species. Introduction Plants have powerful regenerative abilities, which have been widely applied in agricultural technologies (Knizewski et al. 2008, Sugimoto et al. 2011, Su and Zhang 2014, Xu and Huang 2014, Ikeuchi et al. 2016, Kareem et al. 2016, Lup et al. 2016, Birnbaum and Roudier 2017). In tissue culture, when stimulated by a high level of auxin, detached explants form a pluripotent callus, from which roots and shoots may regenerate (Sugimoto et al. 2011, Xu and Huang 2014). Recent studies have suggested that callus formation follows the rooting pathway, and newly formed callus cells are likely to be a group of fast-dividing root primordium-like cells (Che et al. 2007, Atta et al. 2009, Sugimoto et al. 2010, Fan et al. 2012, He et al. 2012, Liu et al. 2014, Yu et al. 2017, Liu et al. 2018). The ability to form callus in tissue culture varies in diverse species. For example, mature leaves of the dicot Arabidopsis (Arabidopsis thaliana) readily form callus, while the mature region of leaves in the monocot rice (Oryza sativa) is extremely unresponsive to tissue culture (Hu et al. 2017). Not all somatic cells are competent to form callus; only a group of adult stem cells, i.e. regeneration-competent cells, may initiate regeneration (Che et al. 2007, Atta et al. 2009, Sugimoto et al. 2010, Liu et al. 2014). In Arabidopsis, the procambium and some vascular parenchyma cells in leaves and the xylem-pole pericycle cells in roots may serve as regeneration-competent cells, while in rice the bundle sheath and some immature vascular cells in leaves and the phloem-pole pericycle cells in roots may serve as regeneration-competent cells (Hu et al. 2017). These competent cells are responsible not only for callus initiation but also for root primordium initiation during adventitious root (AR) or lateral root (LR) formation (Liu et al. 2014, Hu and Xu 2016, Hu et al. 2017, Sheng et al. 2017). The different differentiation statuses of these regeneration-competent cells result in diverse regenerative abilities in Arabidopsis and rice (Hu et al. 2017). In Arabidopsis, there are at least two rooting pathways that may contribute to post-embryonic root system formation: the WUSCHEL-RELATED HOMEOBOX11 (AtWOX11)-mediated and AtIAA14-AUXIN RESPONSE FACTOR7/19 (AtIAA14–AtARF7/19)-mediated rooting pathways (Sheng et al. 2017, Ge et al. 2018). The AtWOX11-mediated rooting pathway contributes to AR formation from leaf explants or adventitious LR formation from the primary root (PR) in response to wounding or environmental signals (Sheng et al. 2017, Ge et al. 2018). Using AR formation from leaf explants as an example, auxin first activates AtWOX11 expression during the fate transition from regeneration-competent cells to root founder cells, and then AtWOX11 initiates the fate transition from root founder cells to root primordium cells via activation of WUSCHEL-RELATED HOMEOBOX5 (AtWOX5) and LATERAL ORGAN BOUNDARIES DOMAIN16 (AtLBD16) (Liu et al. 2014, Hu and Xu 2016, Sheng et al. 2017, Xu 2018). Co-expression of AtWOX5 and AtLBD16 may be a specific marker of root primordium cells (Xu 2018). The AtIAA14–AtARF7/19-mediated pathway (also known as the non-AtWOX11-mediated pathway) is required for acropetal LR formation (Ge et al. 2018) following an oscillatory root cap-derived auxin flux signal (Fukaki et al. 2002, Okushima et al. 2007, Lee et al. 2009, Peret et al. 2009, Moreno-Risueno et al. 2010, Goh et al. 2012, Lavenus et al. 2013, Van Norman et al. 2014, Xuan et al. 2015, Xuan et al. 2016). Degradation of AtIAA14 upon auxin signaling releases AtARF7/19 to activate AtLBD16, and probably also AtWOX5, for LR primordium initiation (Fukaki et al. 2002, Okushima et al. 2007, Lee et al. 2009, Goh et al. 2012). In rice, there are also two pathways to initiate roots in the post-embryonic stage, i.e. the OsWOX11-mediated and the OsIAA11-mediated pathways. OsWOX11 is the AtWOX11 homolog in rice and is required for AR (crown root) formation but not LR formation (Zhao et al. 2009). OsIAA11 in rice is the closest homolog of AtIAA14 in Arabidopsis. The Osiaa11 mutant, which has mutations in domain II of the OsIAA11 protein and interrupts the auxin signaling pathway by constitutively suppressing ARF activity, is defective in generating LRs (Zhu et al. 2012). However, ARs (crown roots) could be formed in the Osiaa11 mutant, suggesting that the OsIAA11-mediated pathway is specifically required for LR formation. Callus formation borrows the rooting pathway and newly formed callus seems to be a group of root primordium-like cells which specifically express WOX5 and LBD16 (Sugimoto et al. 2010, Fan et al. 2012, Liu et al. 2014, Hu et al. 2017, Lee et al. 2017, Liu et al. 2018). However, it is not clear whether there are different mechanisms of callus initiation in different species and in different organs. In this study, we show that the OsIAA11-mediated pathway is strictly required for callus initiation from the rice LR formation region of PR explants, whereas the AtIAA14-mediated pathway is not strictly required for callus initiation in the LR formation region in Arabidopsis. Thus, rice and Arabidopsis employ different strategies for callus initiation from root explants. Results and Discussion Callus initiation strictly requires the OsIAA11-mediated pathway in the lateral root formation region of rice root explants To analyze the mechanism of callus initiation from rice root explants, we first carried out phenotype analysis using PRs from 7-day-old wild-type Kasalath and Osiaa11 mutant plants (Zhu et al. 2012). The 7-day-old PRs were at the growing stage, and almost no LRs could be observed by eye in the wild type (Fig. 1A). We cultured the detached PRs on callus-inducing medium (CIM) to induce callus formation and on N6 medium without exogenous hormones as a control. At 14 d after culture (DAC) on N6 medium, the wild-type PRs formed many LRs (Fig. 1B), while the Osiaa11 PRs had no LRs (Fig. 1F, G), which was consistent with previous reports that OsIAA11 is required for LR formation (Zhu et al. 2012). At 14 DAC on CIM, the wild-type PR explants formed callus in both the LR formation region and the root tip region (Fig. 1C–E), while the root explants from the Osiaa11 mutant PR formed callus only in the root tip region, not in the LR formation region (Fig. 1H–J). No callus was found in the Osiaa11 LR formation region during a prolonged culture period at 20 DAC (Supplementary Fig. S1). Therefore, callus formation strictly required the OsIAA11-mediated lateral rooting pathway in the LR formation region, but not in the root tip region. Fig. 1 View largeDownload slide Callus formation from rice primary roots. (A) PR from 7-day-old wild-type rice cultured on MS medium before tissue culture. (B) LR formation from wild-type rice after 14 d on N6 medium. (C) Callus formation from wild-type rice PRs after 14 d on CIM. The PR explants were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type rice PRs. (F) PR from 7-day-old Osiaa11 rice cultured on MS medium before tissue culture. (G) No LR formation from Osiaa11 PRs cultured on N6 medium for 14 d. (H) Callus formation in the root tip region but not in the LR formation region from Osiaa11 PRs cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Osiaa11 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of OsWOX5 in the wild type and Osiaa11 in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 5 mm in (A–C, F–H), 50 μm in (D, E, I, J). Fig. 1 View largeDownload slide Callus formation from rice primary roots. (A) PR from 7-day-old wild-type rice cultured on MS medium before tissue culture. (B) LR formation from wild-type rice after 14 d on N6 medium. (C) Callus formation from wild-type rice PRs after 14 d on CIM. The PR explants were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type rice PRs. (F) PR from 7-day-old Osiaa11 rice cultured on MS medium before tissue culture. (G) No LR formation from Osiaa11 PRs cultured on N6 medium for 14 d. (H) Callus formation in the root tip region but not in the LR formation region from Osiaa11 PRs cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Osiaa11 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of OsWOX5 in the wild type and Osiaa11 in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 5 mm in (A–C, F–H), 50 μm in (D, E, I, J). We then tested expression levels of OsWOX5, which is a marker gene of newly formed callus, in root explants from the wild type and the Osiaa11 mutant. OsWOX5 was highly up-regulated in both the LR formation region and the root tip region in wild-type root explants at 2 DAC on CIM, while it was up-regulated in the root tip region but not in the LR formation region of the Osiaa11 root explants (Fig. 1K, L; Supplementary Fig. S2). This suggests that the establishment of the callus cell fate is strictly dependent on the OsIAA11-mediated pathway in the LR formation region but not in the root tip region.Quantitative reverse transcription–PCR (qRT–PCR) results showed that OsWOX11 was up-regulated in the root tip region of PRs in rice (Supplementary Fig. S3). OsWOX11 was also up-regulated in the LR formation region of PRs from Osiaa11 in rice (Supplementary Fig. S4). However, the up-regulation of OsWOX11 was not sufficient to induce callus initiation in the LR formation region in the Osiaa11 background. The AtIAA14-mediated pathway is not strictly required for callus initiation in the lateral root formation region of Arabidopsis root explants We next analyzed the role of AtIAA14 in callus initiation from Arabidopsis root explants. We cultured PRs from 7-day-old wild-type Col-0 and Atiaa14 mutant plants. The 7-day-old PRs had almost no LRs in the wild type or Atiaa14 at this stage (Fig. 2A, F). The PRs were cultured on CIM to induce callus formation and on N6 medium without exogenous hormones as a control. The wild-type PRs developed many LRs on N6 medium at 14 DAC (Fig. 2B), while the Atiaa14 PRs did not produce LRs (Fig. 2G), suggesting that AtIAA14, like its rice homolog OsIAA11, is critical for LR initiation (Fukaki et al. 2002). Interestingly, both the wild-type and Atiaa14 root explants formed callus in the LR formation region and the root tip region, although the callus mass was relatively smaller in Atiaa14 than in the wild type (Fig. 2C, H). A prolonged culture period led to better growth of the callus mass at 20 DAC (Supplementary Fig. S5). AtWOX11 was up-regulated in the LR formation region of both Col-0 and Atiaa14 PRs (Supplementary Fig. S6). Taken together, this suggested that, in contrast to the case in rice, the AtIAA14-mediated lateral rooting pathway is not strictly required for callus initiation in the LR formation region of Arabidopsis. Fig. 2 View largeDownload slide Callus formation from Arabidopsis primary roots. (A) PR from 7-day-old wild-type Arabidopsis cultured on MS medium before tissue culture. (B, C) LR formation after 14 d on N6 medium (B) and callus formation after 14 d on CIM (C) from wild-type Arabidopsis PRs. The PR explants in (C) were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type Arabidopsis PRs. (F) PR from a 7-day-old Atiaa14 mutant cultured on MS medium before tissue culture. (G) No LR formation after 14 d on N6 medium. (H) Callus formation in the LR formation region and the root tip region from Atiaa14 PR explants cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Atiaa14 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of AtWOX5 in wild-type Arabidopsis and the Atiaa14 mutant in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 3 mm in (A–C, F–H), 50 μm in (D, E, I, J). Fig. 2 View largeDownload slide Callus formation from Arabidopsis primary roots. (A) PR from 7-day-old wild-type Arabidopsis cultured on MS medium before tissue culture. (B, C) LR formation after 14 d on N6 medium (B) and callus formation after 14 d on CIM (C) from wild-type Arabidopsis PRs. The PR explants in (C) were cut into approximately 5 mm pieces before culture. (D, E) Close-up of callus formation on CIM at 14 DAC in the LR formation region (D) and the root tip region (E) of wild-type Arabidopsis PRs. (F) PR from a 7-day-old Atiaa14 mutant cultured on MS medium before tissue culture. (G) No LR formation after 14 d on N6 medium. (H) Callus formation in the LR formation region and the root tip region from Atiaa14 PR explants cultured on CIM for 14 d. The PR explants were cut into approximately 5 mm pieces before culture. (I, J) Close-up of Atiaa14 PR explants on CIM at 14 DAC in the LR formation region (I) and the root tip region (J). (K, L) Relative transcript levels of AtWOX5 in wild-type Arabidopsis and the Atiaa14 mutant in the LR formation region (K) and root tip region (L). Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) PRs were arbitrarily fixed at 1.0. Scale bars = 3 mm in (A–C, F–H), 50 μm in (D, E, I, J). To confirm this finding at the molecular level, we analyzed the expression levels of the callus marker gene AtWOX5 in root explants from the wild type and the Atiaa14 mutant. AtWOX5 was highly up-regulated in both the LR formation region and the root tip region in the wild type and the Atiaa14 mutant at 2 DAC on CIM (Fig. 2K, L). Therefore, the cell fate transition to callus could be accomplished in both the wild type and the Atiaa14 mutant. Overall, the major difference between OsIAA11 and AtIAA14 is their roles in callus formation in the LR formation region, suggesting that callus formation in the LR formation region is controlled by different mechanisms in rice and Arabidopsis. Different rooting capacities of rice and Arabidopsis primary roots upon wounding We thought that the different strategies of callus initiation in the LR formation region in rice and Arabidopsis might be due to the different LR formation mechanisms in the two species. Our previous study suggested that the Arabidopsis PR can produce two types of LRs, i.e. acropetal LRs and adventitious LRs (Sheng et al. 2017, Ge et al. 2018). When Arabidopsis is grown vertically on the medium, LR formation usually follows the AtIAA14–AtARF7/19 pathway to initiate the acropetal LR primordium; however, when Arabidopsis PRs are wounded, the AtWOX11-mediated rooting pathway can be used for adventitious LR initiation. We tested whether the AtWOX11-mediated rooting pathway functioned in the Atiaa14 mutant by cutting the PRs and culturing the wounded PRs on N6 medium. The results showed that the wounded Atiaa14 PRs could produce adventitious LRs upon wounding (Fig. 3A, B). WOX11 expression was induced in the wound site at 1 d after excision (DAE) (Fig. 3C, D). Fig. 3 View largeDownload slide Rooting abilities of primary roots of Arabidopsis and rice. (A, B) Formation of adventitious LRs on a wounded PR (arrow) of Atiaa14 at 14 DAE. (B)A close-up of the wound site in (A). (C, D) GUS staining of AtWOX11pro:GUS in intact (C) or wounded (D) PRs of Atiaa14 at 1 DAE. (E, F) No LR formation was found on wounded primary roots (arrow) of Osiaa11 at 5 DAE (E) and 14 DAE (F). Scale bars = 5 mm in (A), 50 μm in (C, D) and 1 mm in (B, E, F). Fig. 3 View largeDownload slide Rooting abilities of primary roots of Arabidopsis and rice. (A, B) Formation of adventitious LRs on a wounded PR (arrow) of Atiaa14 at 14 DAE. (B)A close-up of the wound site in (A). (C, D) GUS staining of AtWOX11pro:GUS in intact (C) or wounded (D) PRs of Atiaa14 at 1 DAE. (E, F) No LR formation was found on wounded primary roots (arrow) of Osiaa11 at 5 DAE (E) and 14 DAE (F). Scale bars = 5 mm in (A), 50 μm in (C, D) and 1 mm in (B, E, F). We then tested whether the OsWOX11-mediated rooting pathway could be used in rice PRs to produce LRs. We cut Osiaa11 PRs and cultured the wounded PRs on N6 medium. The results showed that the wounded Osiaa11 PRs were not able to produce roots at the wound sites (Fig. 3E, F). Therefore, OsIAA11 is the sole pathway for LR initiation in rice PRs. This can explain why callus formation is strictly dependent on OsIAA11 in the LR formation region of rice. Callus formation at the stem base of the Osiaa11 mutant Although the Osiaa11 mutant was defective in LR initiation, it had the ability for AR (crown root) formation (Fig. 4A, B, D, E) (Zhu et al. 2012). We then tested the callus formation ability in the stem base of Osiaa11. When the excised shoots were cultured on CIM for 14 d, callus was produced in the crown root initiation region on the stem bases of both the wild type (Fig. 4C) and Osiaa11 (Fig. 4F). Fig. 4 View largeDownload slide Callus formation at the stem base in rice. (A) The explant of wild-type rice stem base from the 3-day-old seedling grown in the dark. (B) AR formation from the wild-type rice stem base after 1 d on N6 medium. (C) Callus formation from the wild-type rice stem base after 14 d culture on CIM. (D) The explant of the Osiaa11 rice stem base from the 3-day-old seedling grown in the dark. (E) AR formation from the Osiaa11 rice stem base after 1 d on N6 medium. (F) Callus formation from the Osiaa11 rice stem base after 14 d culture on CIM. (G) Relative transcript levels of OsWOX5 and OsWOX11 in the stem base region of the wild type or Osiaa11. Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) were arbitrarily fixed at 1.0. (H–M) Longitudinal (H, I, K, L) and transverse (J, M) sections of the wild-type stem base, showing in situ hybridization of OsWOX5. Seedlings grown in the dark for 3 d were used to observe AR primordium formation (H–J). Those seedlings were then cultured on CIM for 4 d to observe callus formation (K–M). (H) and (K) are sense controls. Scale bars = 5 mm in (A–F), 1 mm in (H, K) and 500 μm in (I, J, L, M). Fig. 4 View largeDownload slide Callus formation at the stem base in rice. (A) The explant of wild-type rice stem base from the 3-day-old seedling grown in the dark. (B) AR formation from the wild-type rice stem base after 1 d on N6 medium. (C) Callus formation from the wild-type rice stem base after 14 d culture on CIM. (D) The explant of the Osiaa11 rice stem base from the 3-day-old seedling grown in the dark. (E) AR formation from the Osiaa11 rice stem base after 1 d on N6 medium. (F) Callus formation from the Osiaa11 rice stem base after 14 d culture on CIM. (G) Relative transcript levels of OsWOX5 and OsWOX11 in the stem base region of the wild type or Osiaa11. Bars indicate the SE from three biological replicates. Two asterisks indicate significant differences (Student’s test, P < 0.01). Values from time 0 (0 D) were arbitrarily fixed at 1.0. (H–M) Longitudinal (H, I, K, L) and transverse (J, M) sections of the wild-type stem base, showing in situ hybridization of OsWOX5. Seedlings grown in the dark for 3 d were used to observe AR primordium formation (H–J). Those seedlings were then cultured on CIM for 4 d to observe callus formation (K–M). (H) and (K) are sense controls. Scale bars = 5 mm in (A–F), 1 mm in (H, K) and 500 μm in (I, J, L, M). The qRT–PCR results showed that the expression levels of OsWOX5 and OsWOX11 increased significantly in the stem bases of the wild type and Osiaa11 cultured on CIM for 1 d (Fig. 4G). In situ hybridization results showed that OsWOX5 expression was detected in the crown root primordium and callus (Fig. 4H–M). These data indicated that the mutation in OsIAA11 does not affect the AR and callus initiation in the stem base of rice. Conclusion Overall, the strategies for callus initiation in the LR formation region are different: in rice, OsIAA11 is strictly required for callus initiation, but AtIAA14 is not indispensable in Arabidopsis. This is due to differences in LR initiation between rice and Arabidopsis. In Arabidopsis, LR formation can occur via two alternative pathways: the AtIAA14–AtARF7/19-mediated pathway for acropetal LRs developing from PRs and the AtWOX11-mediated pathway for adventitious LRs initiated upon wounding (Ge et al. 2018). In rice, LR formation from PRs can only occur via the OsIAA11-mediated pathway. The different rooting mechanisms in the LR formation region of PRs result in different callus initiation strategies in Arabidopsis and rice (see the model in Fig. 5). However, because Osiaa11 and Atiaa14 are both gain-of-function mutants, currently we cannot exclude the possibility that the repression of auxin signaling by mutated AtIAA14 is weaker than that of OsIAA11. Further analysis to reveal why OsWOX11 cannot function to produce LRs in rice PRs could improve our understanding of the evolution of root system formation and regenerative abilities in dicots and monocots. Fig. 5 View largeDownload slide Model of root and callus formation in Arabidopsis and rice. Fig. 5 View largeDownload slide Model of root and callus formation in Arabidopsis and rice. Materials and Methods Plant material Kasalath (Oryza sativa L. ssp. indica) was used as the rice wild type, and Columbia-0 (Col-0) was used as the Arabidopsis wild type. The Osiaa11 mutant (in the Kasalath background) was previously described (Zhu et al. 2012). Atiaa14 and AtWOX11pro:GUS were also described previously (Fukaki et al. 2002, Liu et al. 2014, Shang et al. 2016). Tissue culture and in situ hybridization For root-derived callus induction, sterile rice seeds were grown on Murashige and Skoog (MS) medium in a growth chamber with a 16 h light, 28°C/8 h dark, 24°C cycle. Sterilized seeds of Arabidopsis were sown on B5 medium (Gamborg B5 basal medium with 0.5 g l–1 MES, 3% sucrose and 0.8% agar, pH 5.7) and grown at 24°C under a 16 h light/8 h dark photoperiod. PRs were used for incubation of LRs or callus. MS medium and N6 medium were described previously (Murashige and Skoog 1962, Chu et al. 1975). Callus induction was performed on CIM (N6 basal medium with 10 μM 2,4-D, 0.5 g l–1 MES, 3% sucrose and 0.4% phytagel, pH 5.8). In situ hybridization was performed according to our previous method (Hu et al. 2017). qRT–PCR RNA extraction and qRT–PCR were performed as previously described (Guo et al. 2016), using the following gene-specific primers: 5′-ACCGGCTCATGACATGCTAC-3′ and 5′-ATACCGGACCTTGTCCACCT-3′ for OsWOX5; 5′-ACCACTTCGACCGCCACTACT-3′ and 5′-ACGCCTAAGCCTGCTGGTT-3′ for OsUbiqutin; 5′-ATGTTTGGGCAGGACGTGAT-3′ and 5′-GGAAGTAGCTCTCGCCCATC-3′ for OsWOX11; 5′-ACAATAACGGAGGAACGGGG-3′ and 5′-TGTTGGAGTTCTAAGACCGGC-3′ for AtWOX5; and 5′-TGAGCCTTCCTTGATGATGCT-3′, 5′-GCACTTGCGGCAAATCATCT-3′ for AtUbiqutin. The qRT–PCR results are shown as relative expression levels normalized against the expression of OsUbiqutin and AtUbiqutin. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [grant Nos. 31771776, 31630007 and 31422005)]; the China Agriculture Research System [grant CARS-05]; the National Basic Research Program of China [973 Program, grant No. 2014CB943500]; the Chinese Academy of Science (CAS) [Key Research Program (grant No. QYZDB-SSW-SMC010) and the Strategic Priority Research Program ‘Molecular Mechanism of Plant Growth and Development’ (grant No. XDPB0403)]. Acknowledgments We thank Dr. Chuanzao Mao (Zhejiang University, China) and Dr. Yuxin Hu (Institute of Botany, Chinese Academy of Sciences, China) for providing plant materials, and Hua Wang for providing technical assistance in the in situ hybridization experiment. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations AR adventitious root ARF7/19 AUXIN RESPONSE FACTOR7/19 CIM callus-inducing medium DAC days after culture DAE days after excision LBD16 LATERAL ORGAN BOUNDARIES DOMAIN16 LR lateral root PR primary root qRT–PCR quantitative reverse transcription–PCR WOX5 WUSCHEL-RELATED HOMEOBOX5 WOX11 WUSCHEL-RELATED HOMEOBOX11 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: May 16, 2018

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