WUS-RELATED HOMEOBOX 14 boosts de novo plant shoot regeneration

Abstract

WUS-RELATED HOMEOBOX 14 and its putative rice ortholog boost de novo plant shoot regeneration

Dear Editor,

Tissue culture recalcitrance-caused low plant regeneration efficiencies restrict plant transformation and genome editing. Expression of some morphogenic factors, including WUSCHEL (WUS) and BABYBOOM (BBM), improve regeneration efficiency but harbor negative pleiotropic effects when constitutively expressed in various plant species (Boutilier et al., 2002; Lowe et al., 2016; Gordon-Kamm et al., 2019; Maher et al., 2020). Recent studies also show that overexpression of WUS-RELATEDHOMEOBOX5 (WOX5) or the GROWTH-REGULATINGFACTOR(GRF)-GRF-INTERACTING FACTOR (GIF) chimera dramatically enhance regeneration and transformation with less genotype dependence and less growth penalty (Debernardi et al., 2020; Kong et al., 2020; Wang et al., 2022a). Nevertheless, it is still urgent to develop new, generic factors that boost regeneration for a broader range of recalcitrant genotypes and species.

WOX proteins are key regulators implicated in stem cell proliferation and maintenance in different types of meristems (Dolzblasz et al., 2016), thereby serving as promising developmental regulators of plant regeneration. Indeed, WUS and WOX5 and their homologs that promote de novo regeneration of shoots and generation of somatic embryos are exploited to improve regeneration efficiency in various plants (Gallois et al., 2002; Zuo et al., 2002; Lowe et al., 2016; Zhang et al., 2017; Maher et al., 2020; Wang et al., 2022a). Additionally, the wound-inducible WOX13 is required for callus formation and organ reconnection in Arabidopsis (Arabidopsis thaliana) (Ikeuchi et al., 2021). Likewise, the putative moss (Physcomitrium patens) WOX13 ortholog PpWOX13L is involved in cellular reprogramming at moss wound sites (Sakakibara et al., 2014). We previously reported the role of the WOX11 protein and the WOXfunctionally-linkedCLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling peptides in de novo organogenesis (Zhao et al., 2009; Zhou et al., 2017; Kang et al., 2022; Wang et al., 2022b). As a part of our efforts to further understand the roles of WOX proteins in plant regeneration, we here identified WOX14 and its putative rice ortholog as key regulators in promoting shoot regeneration ability.

To determine whether WOX14 plays a role in shoot regeneration, we examined the shoot regenerative capacities of Arabidopsis WOX14 (AtWOX14) gain- and loss-of-function mutants. Three independent AtWOX14 overexpressors driven by the CaMV35S promoter exhibited normal callus formation but drastically higher regenerative capacity (Figure 1A and Supplemental Figures S1 and S2, A and B). In contrast, the shoot regenerative capacity was heavily compromised in two wox14 mutant alleles (Figure 1A and Supplemental Figure S2C). Supporting a role of AtWOX14 in promoting in vitro shoot regenerative capacity, AtWOX14 is essential for the pluripotency acquisition during callus formation (Kim et al., 2018), thus impacting shoot regeneration potential. Regenerated plants from 35S:AtWOX14 callus are viable and fertile, similar to reported overexpression lines (Denis et al., 2017). Thus, our results demonstrate that AtWOX14 positively regulates shoot regenerative capacity with less growth penalty.

Figure 1.

AtWOX14 greatly improves shoot regeneration in Arabidopsis. A, Shoot regeneration capacity of wox14-1 and 35S:AtWOX14 mutants in comparison with WT plants (n ≥ 32). Asterisks indicate statistically significant differences compared with WT determined by a Dunnett's multiple comparison test following one-way ANOVA analysis (***P < 0.001, ****P < 0.0001). Scale bar, 0.2 cm. B, Shoot regeneration capacity of WT and 35S:WOX14 explants (n = 24) in the absence of cytokinin (Student’s t test, ****P < 0.0001). Images on the right panel show representative WT and 35S:WOX14 explants on the cytokinin-free SIM medium. Scale bar, 0.5 cm. In (A) and (B), the horizontal line inside the box represents the median, the box indicates the 25th–75th percentile, and the whiskers show the value range (minimum to maximum). C, AtWOX14 expression in callus tissue incubated on callus induction medium (CIM) and shoot induction medium (SIM) at indicated time points as indicated by the pWOX14:GUS reporter (the first column to the fourth column, scale bar, 50 μm) and the pWOX14:YFP reporter (the last column, scale bar, 25μm).

The remarkable shoot regeneration capacity in 35S:AtWOX14 lines provoked us to investigate whether AtWOX14 overexpression could bypass cytokinin application during the de novo shoot regeneration procedure. To test this hypothesis, callus of 35S:AtWOX14 and wild-type (WT) explants was placed onto a cytokinin-free shoot induction medium (SIM) after callus induction medium (CIM) induction. Whereas WT explants completely failed to regenerate shoots on the cytokinin-free medium, 35S:AtWOX14 overexpressors were able to regenerate shoots (Figure 1B). These data reveal that as a shoot regeneration–promoting factor, in addition to its role of pluripotency acquisition in callus cells (Kim et al., 2018), AtWOX14overexpression could likely affect cytokinin signaling to trigger de novo shoot formation.

In concordance with the shooting phenotype, AtWOX14 expression examined using the pAtWOX14:GUS reporter was hardly detected before CIM incubation, but was strongly induced and expressed in dividing callus cells upon CIM incubation, spreading into the entire callus-forming regions of 2-day and 3-day emerging callus, consistent with pAtWOX14:YFP activity (Figure 1C). Thereafter, AtWOX14 expression largely decreased and was barely present in callus cells upon transferring onto SIM (Figure 1C). Our study of AtWOX14 expression agrees with the single-cell transcriptome analysis in which AtWOX14 expression is restricted to callus founder cells (Zhai and Xu, 2021), thus supporting a specific function of AtWOX14 in promoting the shoot regeneration capacity.

Given the strongly increased shoot regeneration capacities induced by AtWOX14 overexpression in Arabidopsis, we further investigated whether WOX14-enhanced shoot regeneration could be recapitulated in other plant species. Consequently, we examined the regeneration potential of ectopically expressing AtWOX14 heterogeneously driven by the UBIQUITIN promoter in rice (Oryza sativa L.), a monocotyledonous staple food crop. Transgenic rice lines overexpressing AtWOX14 (AtWOX14-OE1 and AtWOX14-OE2) displayed no markedly detrimental pleiotropic effects (Figure 2, A and B and Supplemental Figures S3 and S4); instead, these lines showed increased tiller number with comparable yield per plant but smaller grain size and lower1000-grain weight compared with the untransformed cultivar ZH11 (Figure 2, A and B and Supplemental Figures S3 and S4). Strikingly, transgenic rice lines overexpressing AtWOX14 significantly improved regeneration efficiencies compared to the controls and generated fully fertile transgenic plants (Figure 2, E and F), indicating an interspecific functional conservation of AtWOX14 in promoting shoot regeneration efficiency.

Figure 2.

AtWOX14 and its putative rice ortholog OsWOX13 significantly promote shoot regeneration capacity in rice. A, B, AtWOX14 could complement the oswox13-1 mutant phenotype. A, The growth phenotype of representative rice lines of ZH11, AtWOX14-OE1, oswox13-1, and AtWOX14-OE oswox13-1 (oswox13-1 expressing AtWOX14-OE). B, The tiller number of ZH11, AtWOX14-OE1, AtWOX14-OE2, oswox13-1, and AtWOX14-OE oswox13-1 lines (n = 10). Values are mean ± SD. C, D, OsWOX13 complemented the oswox13-1 mutant phenotype. C, Representative rice lines of oswox13-1 expressing OsWOX13-OE (OsWOX13-OE oswox13-1), ZH11, oswox13-1, and OsWOX13-OE1. D, The tiller number of ZH11, OsWOX13-OE1, OsWOX13-OE2, oswox13-1, and OsWOX13-OE oswox13-1 lines (n = 16). Values are mean ± SD. E, Regeneration phenotypes of callus derived from ZH11, AtWOX14-OE1, AtWOX14-OE2, OsWOX13-OE1, and OsWOX13-OE2 lines. F, Shoot regeneration capacity of AtWOX14-OE1, AtWOX14-OE2, OsWOX13-OE1, OsWOX13-OE2, and oswox13-1 plants in comparison with ZH11 (n = 20). Values are mean ± SD. Asterisks in (B), (D), and (F) indicate statistically significant differences compared with controls determined by a Dunnett's multiple comparison test following one-way ANOVA analysis (* indicates P < 0.05, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates not significant).

We next asked whether the putative rice WOX14 ortholog also influences shoot regeneration. AtWOX14 is a member of the most ancestral WOX13 subclade of the WOX protein family (Dolzblasz et al., 2016). We found that OsWOX13 (Os01g60270), which was named previously (Nardmann et al., 2009; Sakakibara et al., 2014), was the putative rice ortholog of AtWOX14 with the highest sequence similarity and most similar domain organization. AtWOX14 complemented the decreased tiller number phenotype of the oswox13-1 mutant in which one base pair deletion was introduced through CRISPR/Cas9-mediated gene editing (Figure 2, A and B and Supplemental Figure S5), supporting that OsWOX13 and AtWOX14 are indeed functionally conserved counterparts. The oswox13-1 mutant exhibited decreased tiller number that also could be complemented by introducing the OsWOX13 gene under the control of the UBIQUITIN promoter (OsWOX13-OE) (Figure 2, C and D and Supplemental Figure S5). In line with the reduced tiller number and grain length of oswox13-1 mutants (Figure 2, C and D and Supplemental Figures S5 and S6), their yield per plant and 1000-grain weight were significantly lower than that of the ZH11 rice (Supplemental Figure S6). Conversely, OsWOX13-OE rice lines (OsWOX13-OE1 and OsWOX13-OE2) were fertile and showed significantly decreased rice grain length and width, resulting in a substantial decrease in 1000-grain weight (Supplemental Figure S6). However, the yield per plant of OsWOX13-OE rice lines is comparable to that of the controls given the increased tiller number of OsWOX13-OE rice lines (Figure 2, C and D and Supplemental Figure S6). We then tested whether OsWOX13 exerts a similar role in improving shoot regeneration in rice. In callus overexpressing OsWOX13, a substantial increase of regeneration efficiency leading to a significantly enhanced number of regenerated seedlings compared to the controls was observed (Figure 2, E and F). However, loss of OsWOX13 strikingly reduced rice regeneration efficiency (Figure 2F). Similar to AtWOX14, our results demonstrate that OsWOX13, the functional rice counterpart of AtWOX14, is highly effective in enhancing shoot regeneration in an agronomically important monocotyledonous species. Taken together, we conclude that WOX14 and its putative ortholog boost the regeneration abilities in both dicot and monocot species.

In conclusion, the cytokinin-free plant regeneration of WOX14 overexpressors and the cross-species shooting promotion stimulated by WOX14 and its putative rice ortholog OsWOX13 indicate functional conservation of WOX14 in boosting shoot regeneration across diverse plant species. However, the detailed molecular mechanism(s) enhancing shoot regeneration ability requires further investigation. In addition, the possibility that WOX14 promotes shoot regeneration by affecting cytokinin signaling awaits future validation. The excellent performance of WOX14 and its putative ortholog OsWOX13 in both monocot rice and dicot Arabidopsis highlights their broad application potential in circumventing low plant regeneration efficiency. Nevertheless, it will be intriguing to evaluate the shoot regeneration potential of WOX14 in some recalcitrant plant species such as cereal crops including wheat (Triticum aestivum L.), maize (Zea mays L.), and barley (Hordeum vulgare L.). By contrast to known regeneration regulators WUS and BBM, WOX14 and its putatively orthologous genes boost plant regeneration without obvious negative effects on plant fertility and without needing further laborious and time-consuming excision. Thus, WOX14 and its orthologs may represent exceptionally good candidates as regeneration-promoting factors to facilitate shoot regeneration, potentially in diverse recalcitrant plant species and cultivars.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1 . The callus formation in AtWOX14 overexpression lines.

Supplemental Figure S2 . Phenotypic characterization of additional loss- and gain-of-function mutants of AtWOX14 and its expression levels in the overexpression lines.

Supplemental Figure S3 . Phenotypic characterization of one additional AtWOX14 overexpression rice line (AtWOX14-OE2) and the expression level of AtWOX14 in rice overexpression lines.

Supplemental Figure S4 . Comparison of agronomic traits of seeds between cultivar ZH11 and AtWOX14 overexpression transgenic rice lines.

Supplemental Figure S5 . Generation of gain- and loss-of-function of OsWOX13 rice mutants.

Supplemental Figure S6 . Agronomic performance of OsWOX13 gain- and loss-of-function rice mutants.

Funding

This work is supported by the National Natural Science Foundation of China (31771556 to G.W., 31701294 to C.L., 31970806 to Y.Z., and 32000609 to Y.H.Z.), the Natural Science Basic Research Program of Shaanxi for Distinguished Young Scholars (2020JC-29 to G.W.), the Fundamental Research Funds for the Central Universities (GK202002008 to G.W.), the Natural Science Foundation of Shaanxi (2020JM-284 to A.H. and 2023-JC-ZD-09 to G.W.), and the Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302 to C.L.).

Data availability

The data supporting the findings of this study have been provided in the text and in the supplementary data files and are available upon request.