Developmental trajectory of pluripotent stem cell establishment in Arabidopsis callus guided by a quiescent center-related gene network

Tissue culture, a widely used plant biotechnology for vegetative propagation, is based on the regenerative abilities of plants (Skoog and Miller, 1957; Ikeuchi et al., 2019). In the two-step tissue culture method of Arabidopsis thaliana, callus tissue forms from the vasculature of detached explants on callus-inducing medium (CIM) in response to a high concentration of auxin. Callus contains pluripotent cells that are competent for de novo organ regeneration, i.e. they can regenerate either roots on root-inducing medium (RIM) in response to a low auxin concentration, or shoots on shoot-inducing medium (SIM) in response to a high cytokinin concentration.

In Arabidopsis, the developmental pathway for vasculature-derived callus formation borrows from the pathways of lateral or adventitious root organogenesis in plants (Sugimoto et al., 2010, 2011; Duclercq et al., 2011; Fan et al., 2012; He et al., 2012; Liu et al., 2014). Therefore, the cellular structure of callus on CIM resembles that of the root primordium (RP) or the root apical meristem (RAM) (Sugimoto et al., 2010; Motte et al., 2014; Hu et al., 2017; Zhai and Xu, 2021). However, different from root organogenesis, in which the division of cells to form a mature root tip is strictly and developmentally controlled, callus exhibits more extensive cell division and it can be kept at the RP/RAM stage for a relatively long time by a high concentration of exogeneous auxin in the CIM.

Marker gene expression during callus formation in Arabidopsis has been extensively studied. Briefly, the regeneration-competent cells, which are vascular adult stem cells such as pericycle, procambium, and some vascular parenchyma cells (Che et al., 2007; Atta et al., 2009; Sugimoto et al., 2010, 2011; Liu et al., 2014; Hu et al., 2017) within detached explants, undergo cell fate transition to become callus founder cells by expressing WUSCHEL-RELATED HOMEOBOX11 (WOX11) in response to a high concentration of auxin in the CIM (Liu et al., 2014; Hu et al., 2017). Then, callus founder cells divide to form the callus and express the RP/RAM identity genes LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16), PLETHORA1 and 2 (PLT1/2), WOX5/7 and SCARECROW (SCR) (Di Laurenzio et al., 1996; Aida et al., 2004; Okushima et al., 2007; Gordon et al., 2007; Atta et al., 2009; Sugimoto et al., 2010; Fan et al., 2012; Liu et al., 2014; Kareem et al., 2015; Hu et al., 2017; Liu et al., 2018; Kim et al., 2018; Zhai and Xu, 2021). In this process, WOX11 is able to directly activate LBD16 and WOX5/7 expression (Hu and Xu, 2016; Sheng et al., 2017). PLT3/5/7 are highly expressed in the callus during all stages of callus formation (Kareem et al., 2015). In addition, many epigenetic factors affect regeneration by regulating the expression of key genes in callus formation (Lee et al., 2018, 2019a, 2021; Kim et al., 2018; Ishihara et al., 2019; Wu et al., 2022).

Recently, we analyzed the single-cell transcriptome atlas in callus on CIM and found that the middle cell layer, which has quiescent center (QC)-like identity, plays a central role in pluripotency acquisition and, subsequently, in organ regeneration (Zhai and Xu, 2021). PLT1/2 and WOX5/7 are highly enriched in the middle cell layer of callus on CIM and promote auxin accumulation and cytokinin hypersensitivity, which are important for root or shoot organogenesis on RIM or SIM, respectively. In this study, we analyzed the developmental landscape of cell layer formation in callus tissue and revealed the gene network related to the QC in regulation of pluripotency acquisition in the middle cell layer.

Using the ClearSee assay (Kurihara et al., 2015), we analyzed cell behavior and marker gene expression during callus formation from hypocotyl explants of Arabidopsis (Fig. 1). Here, callus formation is described in four steps, borrowing the concept from root organogenesis (Xu, 2018): (1) the priming step, which involves the fate transition from regeneration-competent cells (i.e. the vascular adult stem cells) to callus founder cells at 1 day on CIM and does not require cell division; (2) the initiation step, which involves the division of callus founder cells to initiate the callus primordium at 2 days on CIM; (3) the patterning step, which involves continuous cell division in the callus primordium to form mature callus with three cell layers (i.e. inner, middle and outer cell layers) at 5 days; and (4) the maintenance of mature callus tissue, with further cell division and callus continuing to grow on CIM.

WOX11 transcripts were present in callus founder cells at 1 day and not in the callus primordium or mature callus from 2 days on CIM (Fig. 1A) (Liu et al., 2014; Hu et al., 2017). LBD16 was expressed in callus founder cells at 1 day and callus primordium at 2 days, and then its expression level gradually decreased as the callus matured and formed three cell layers (Fig. 1B). Transcripts of SCR, WOX5/7 and PLT1/2 were not detected in callus founder cells at 1 day, but were detected in the callus primordium at 2 days (Fig. 1C-E; Fig. S1). High transcript levels of these genes were gradually restricted to the middle cell layer in mature callus at 5 days (Fig. 1C-E; Fig. S1). Lower transcript levels of WOX5/7 and PLT1/2 could be detected in some inner cell layers, but SCR transcripts were detected only in the middle cell layer (Fig. 1C-E; Fig. S1). The lower expression level of PLT1/2 could also be observed in the outer cell layer in mature callus at 5 days (Fig. 1E; Fig. S1B). ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) transcripts were not detected in the callus primordium, and were restricted to the outer cell layer during the formation of mature callus after 4 days (Fig. 1F). The promoter of the GRAS family gene SHORT-ROOT (SHR) (Helariutta et al., 2000) became active at the formation of callus founder cells at 1 day and callus primordium at 2 days, and then its activity was gradually restricted to the inner cell layer of mature callus at 5 days (Fig. 1G). Interestingly, the nuclear-localized SHR proteins were detected in callus founder cells at 1 day and callus primordium at 2 days as well as in the inner and middle cell layers of mature callus at 5 days (Fig. 1H), indicating that SHR proteins can move from the inner cell layer to the middle cell layer in mature callus (Nakajima et al., 2001). WOODEN LEG (WOL) (Mähönen et al., 2000) transcripts were not detected in the callus primordium, but were detected in the inner cell layer from 3 days during the formation of mature callus (Fig. 1I). PLT3 transcripts were detected in many cells from callus founder cells to the three cell layers of mature callus (Fig. 1J). These data indicate that the middle cell layer markers in mature callus (i.e. SCR, WOX5/7 and PLT1/2 genes as well as SHR proteins) are also present in the callus primordium, whereas the outer and inner cell layer markers (ATML1 and WOL genes) are not in the callus primordium and begin their expression during patterning of the callus.

We previously reported that the middle cell layer in mature callus exhibits a QC-like identity and is pluripotent for organ regeneration (Zhai and Xu, 2021). Analysis of previously published single-cell RNA sequencing (scRNA-seq) data (Zhai and Xu, 2021) confirmed that cells co-expressing the QC-related gene network SCR, WOX5 and PLT1/2 were present in the middle cell layer (cell cluster 2 in Fig. 2A). We further tested three QC markers (QC25, QC184 and QC46) (Sabatini et al., 2003) during callus formation. The result showed that the β-glucuronidase (GUS) signals of QC25 and QC184 were highly concentrated in the middle cell layer (Fig. 2B,C) (for QC25 analysis in callus, see also Atta et al., 2009). We did not find a QC46 signal in mature callus (Fig. 2D), and all three GUS markers were absent in the callus primordium. Therefore, the QC-like identity is progressively obtained in the middle cell layer during callus patterning.

To study the cell cycle process during the initiation of the callus primordium from callus founder cells, we treated hypocotyl explants with the S-phase inhibitor hydroxyurea (HU) (Cools et al., 2010) and the M-phase inhibitor nocodazole (NOCO) (Fig. S2). Treatment with either HU or NOCO blocked cell division in callus founder cells (Fig. 3A,B).

WOX11 and LBD16 were expressed normally under HU or NOCO treatment, indicating that the establishment of callus founder cells in the priming step might not be dependent on entry into the cell cycle (Fig. 3A,B). Expression of SCR, WOX5 and PLT1 was blocked by HU treatment, but not by NOCO treatment (Fig. 3A,B). These results indicate that S phase, but not M phase, is essential for the activation of SCR, WOX5 and PLT1 expression and for the fate transition from callus founder cells to callus primordium.

Interestingly, when callus founder cells were arrested at the M phase by NOCO treatment, the nuclei continuously propagated and the cell continuously expanded. However, the cell could not divide, resulting in a huge syncytium-like structure with mixed expression of WOX11 and WOX5 (Fig. 3C). After the NOCO treatment was withdrawn from CIM, new cell plates could form between the nuclei in the syncytium-like structure (Fig. 3D-F). After the formation of new cell plates, decreased WOX11 expression and continuous WOX5 expression were observed in the syncytium-like structure (Fig. 3F). These findings indicate that auxin promotes repeated chromatin replication regardless of M-phase completion in callus founder cells, and that WOX11 expression may cease after the completion of callus founder cell division.

To analyze the role of the SCR-SHR complex in the middle cell layer of callus, we first observed the regeneration phenotypes of shr-2 and scr-6 mutants. Shooting ability was almost lost in the callus of shr-2 or scr-6 when moved to SIM (Fig. 4A-D) (for analysis of SCR in shoot regeneration, see also Kim et al., 2018). Rooting ability was also lost in scr-6 and significantly reduced in shr-2 when callus was moved to RIM (Fig. 4E). Therefore, SCR and SHR may be crucially involved in pluripotency acquisition in the callus.

Both shr-2 and scr-6 calli lost the normal pattern of three cell layers (Fig. 4F-Q). However, calli of the shr-2 and scr-6 mutants showed different phenotypes on CIM. Callus cell division was faster in shr-2 (Fig. 4J-M) but slower in scr-6 (Fig. 4N-Q) compared with Col-0 callus (Fig. 4F-I). Abnormal cell plate formation could be observed in scr-6 callus (Fig. 4N-Q).

Next, we analyzed the expression patterns of marker genes in the calli of shr-2 and scr-6. Compared with their expression patterns in the wild-type background (Figs 1C-E, 2B), WOX5, PLT1 and QC25 showed decreased expression levels and discontinuous expression patterns in the shr-2 mature callus, and SCR expression was barely detected in the shr-2 mature callus (Fig. 4R-U). WOX5 showed a decreased expression level and a discontinuous expression pattern in the scr-6 mature callus, and PLT1 and QC25 were barely detected in the scr-6 mature callus (Fig. 4V-X).

Together, these data indicate that the QC-like identity was partially lost in shr-2 and scr-6 mature calli on CIM, leading to loss of pluripotency for organ regeneration. In addition, SCR and SHR might have additional roles independent of the SCR-SHR complex in the callus middle cell layer.

Our recent study showed that WOX5/7 are involved in pluripotency acquisition in callus via the promotion of auxin accumulation and cytokinin hypersensitivity on CIM (Zhai and Xu, 2021). We found that the QC marker QC25 lost its expression in the wox5-1 wox7-1 callus (Fig. 5A). In addition, RNA-seq data of calli from wox5-1 wox7-1 and Col-0 (Zhai and Xu, 2021) and SCR marker line analysis showed that the SCR expression level was reduced in the middle cell layer of wox5-1 wox7-1 callus compared with Col-0 callus (Fig. S3A,B). Therefore, QC-like identity is partially lost in the middle cell layer of wox5-1 wox7-1 callus.

The transcript levels of the VASCULAR-RELATED NAC-DOMAIN (VND) genes VND3 and VND5 were higher in wox5-1 wox7-1 callus than in wild-type callus (Fig. 5B,C) (Zhai and Xu, 2021). In addition, WOX5 was able to repress the expression of VND3 and VND5 in the in vitro protoplast assay (Fig. 5D,E). The VND3 and VND4 marker lines (VND3_pro:NLS-3×eGFP and VND4_pro:NLS-3×eGFP) showed that VND3/4 were ectopically expressed in the middle and inner cell layers of wox5-1 wox7-1 callus compared with wild-type callus (Fig. 5F,G). VND genes are involved in the differentiation of xylem vessel elements (Kubo et al., 2005). We observed that ectopic xylem formation occurred in the callus of wox5-1 wox7-1 when it was moved to SIM or RIM, but not when it was on CIM (Fig. 5H-M). We hypothesize that a high level of auxin in CIM might prevent xylem differentiation although VND genes were ectopically expressed in wox5-1 wox7-1 callus (Lee et al., 2019b). Induced overexpression of VND3 or VND4 fused with the repression domain SRDX (Hiratsu et al., 2003) (pER8:gVND3-SRDX or pER8:gVND4-SRDX) on CIM could repress the ectopic xylem formation in the wox5-1 wox7-1 callus (Fig. S3C).

We also observed that the SHR promoter, which was active in the inner cell layer of wild-type callus (Fig. 1G), was ectopically activated in the middle cell layer of the wox5-1 wox7-1 callus on CIM (Fig. 5N). The inner cell layer of the callus has vascular initial identity (Zhai and Xu, 2021), and the SHR promoter is active in the vasculature of root tips (Helariutta et al., 2000; Nakajima et al., 2001).

Together, these data suggest that WOX5/7 may maintain the identity of the middle cell layer and prevent cell differentiation into vasculature.

We previously showed that PLT1/2 could form a protein complex with WOX5/7 to promote auxin biosynthesis in the middle cell layer of callus on CIM (Zhai and Xu, 2021). Further analyses of the RNA-seq data from Col-0 and plt1-21 plt2-21 calli on CIM revealed that JKD, which encodes a zinc finger protein, was downregulated in plt1-21 plt2-21 callus compared with Col-0 callus (Zhai and Xu, 2021) (Fig. 6A,B). Gene ontology (GO) analysis indicates that the expression levels of genes involved in asymmetric cell division, root cap development, regulation of meristem growth, auxin biosynthetic process, and gibberellin biosynthetic process were downregulated in plt1-21 plt2-21 callus, and JKD was annotated to be involved in asymmetric cell division and regulation of meristem growth (Welch et al., 2007) (Fig. 6B). qRT-PCR confirmed that JKD was downregulated in plt1-21 plt2-21 callus compared with Col-0 callus (Fig. 6C). PLT2-YFP was able to directly bind to the JKD locus (Fig. 6D) in ChIP-seq analysis (Santuari et al., 2016). We also showed that PLT1 was able to activate JKD expression in the in vitro protoplast assay (Fig. 6E).

Analysis of the JKD marker line (JKD_pro:JKD-eYFP) showed that JKD expression started in the callus primordium at 2 days of culture, and a high expression level was detected in the middle cell layer of mature callus at 5 days on CIM (Fig. 6F). A lower expression level of JKD could also be detected in the outer cell layer of mature callus at 5 days (Fig. 6F). The JKD_pro:JKD-eYFP marker line indicated that JKD was also present in the middle cell layer of plt1-21 plt2-21 mature callus on CIM (Fig. 6G), but its expression level was lower in the middle cell layer of plt1-21 plt2-21 callus compared with Col-0 callus (Fig. 6G,H).

A mutation in JKD resulted in significantly reduced shoot or root regeneration from callus on SIM or RIM, respectively (Fig. 6I,J) (for analysis of JKD in shoot regeneration, see also Wu et al., 2022). In addition, JKD overexpression could partially rescue the root and shoot regeneration defects of plt1-21 plt2-21 callus (Fig. S4).

Together, these data indicate that PLT1/2 might directly activate JKD in the middle cell layer for organ regeneration.

Studies from animals show that pluripotent stem cells (PSCs) are able to self-renew and differentiate into all cell types found in the adult organism, whereas multipotent or unipotent stem cells can only differentiate into specific cell type(s) in tissues (Robinton and Daley, 2012; De Los Angeles et al., 2015; Sang et al., 2018). We propose that PSCs exist in the middle cell layer of mature callus in tissue culture on the basis of the following evidence: (1) those cells are able to self-renew, as indicated by their maintenance in a pluripotent state on CIM; and (2) those cells can form the stem cell niche of either the RAM on RIM or the shoot apical meristem on SIM, thereby producing almost all cell types found in the adult plant. However, plant PSCs in callus tissue do not exhibit embryonic features, and this differs from PSCs in animals (Robinton and Daley, 2012; De Los Angeles et al., 2015).

PSCs in the middle cell layer of callus have a QC-like identity to some extent, because the transcriptome and marker gene expression in the middle cell layer resemble that of the QC in the root apical meristem (Zhai and Xu, 2021). In addition, the gene network that is essential for QC identity is also required for PSC establishment in the middle cell layer. However, PSCs are not equivalent to the QC. There are many differences in the transcriptome between the two types of cells, and cell division is highly activated in PSCs of callus but not in the QC (Zhai and Xu, 2021).

In this study, we examine in detail the cell lineage during establishment of three cell layers in callus on CIM (summarized in Fig. 7). In the priming step, the regeneration-competent cell undergoes cell fate transition to form the callus founder cell at around 1 day on CIM by expressing WOX11 and LBD16. In the initiation step, the long, bar-shaped callus founder cell mainly undergoes anticlinal cell divisions to form the callus primordium with small, square-shaped cells. In this step, SCR, SHR (both mRNA and nuclear protein), WOX5/7, PLT1/2 and JKD are expressed in the callus primordium, and the upregulation of SCR, WOX5 and PLT1 is dependent on the callus founder cell entering S phase. WOX11 expression decreases dependent on the completion of callus founder cell division. LBD16 is also highly expressed in the callus primordium independently of S phase. In the patterning step, the cells of the callus primordium further undergo rapid cell division with pattern establishment of marker genes to form mature callus with three cell layers. Periclinal cell divisions lead to the separation of the inner cell layer expressing the marker genes SHR (mRNA) and WOL. Some cells in the inner cell layer also show relatively lower expression of WOX5/7 and PLT1/2. Then, periclinal cell divisions further lead to separation of the outer cell layer expressing the marker gene ATML1. Some cells in the outer cell layer also show relatively lower expression of JKD and PLT1/2. The middle cell layer acquires QC-like identity by high expression levels of SCR, WOX5/7, PLT1/2 and JKD, as well as the SHR proteins.

By molecular analysis, we show that QC-related genes form a regulatory network to promote middle cell layer establishment on CIM. SCR and WOX5 might form a positive-activation loop in the middle cell layer (Shimotohno et al., 2018). SHR-SCR promotes the expression of PLT1. WOX5/7 prevent cell differentiation by repressing the expression of VND genes, which are involved in xylem differentiation. PLT1/2 directly promote JKD expression. Further identification of key genes and exploration of the gene regulatory network in regulation of PSCs in the middle cell layer of callus will be important to understand pluripotency acquisition and organ regeneration in tissue culture.

Arabidopsis Col-0 was used as wild type in this study. WOX11_pro:H2B-eGFP (Zhai and Xu, 2021), WOX5_pro:NLS-3×eGFP (Zhai and Xu, 2021), PLT1_pro:tdTomato-N7 (Zhai and Xu, 2021), ATML1_pro:eGFP-ER (Gordon et al., 2007), SHR_pro:NLS-mCherry (Marquès-Bueno et al., 2016), SHR_pro:SHR-eGFP (Wu et al., 2014), WOL_pro:eGFP-ER (Gordon et al., 2009), PLT3_pro:PLT3-eYFP (Kareem et al., 2015), JKD_pro:JKD-eYFP (Wu et al., 2022), SCR_pro:SCR-eGFP/scr-4 (Ws background) (Gallagher et al., 2004), QC25, QC46, QC184 (Sabatini et al., 2003), wox5-1 wox7-1 (Hu and Xu, 2016), plt1-21 plt2-21 (Zhai and Xu, 2021), jkd-4 (Welch et al., 2007) and shr-2 (Nakajima et al., 2001) have been described previously. For construction of LBD16_pro:LBD16-Venus, the 4.8-kb promoter and the gene body of LBD16 fused with Venus were cloned into pBI101 to replace the GUS gene. For construction of WOX7_pro:NLS-3×eGFP, VDN3_pro:NLS-3×eGFP, VDN4_pro:NLS-3×eGFP, WOX5_pro:H2B-tdTomato and PLT2_pro:tdTomato-N7, the 3.5-kb promoter of WOX7, 2.9-kb promoter of VDN3, 3.3-kb promoter of VDN4, 4.5-kb promoter of WOX5 and 5.8-kb promoter of PLT2 were cloned into pBI101-NLS-3×eGFP, pBI101-H2B-tdTomato and pBI101-tdTomato-N7 (modified from pBI101, Clontech). For construction of pER8:gVND3-SRDX, pER8:gVND4-SRDX and pER8:JKD, the genomic gene bodies encoding VND3 and VND4 fused with SRDX and the cDNA encoding JKD were each cloned into pER8 (Zuo et al., 2000).

Tissue culture was performed as previously described using hypocotyls as explants (Zhai and Xu, 2021). The cell cycle inhibitors were HU (H8627, Sigma-Aldrich) and NOCO (ab120630, Abcam). The role of NOCO treatment was confirmed by the Cytrap line (Yin et al., 2014) (Fig. S2).

qRT-PCR and GUS staining were carried out as previously described (He et al., 2012). The qRT-PCR results are presented as relative transcript levels, normalized against that of ACTIN.

To construct firefly luciferase reporter vectors, the 2.9-kb promoter of VND3, the 3.2 kb-promoter of VND5 and the 5-kb promoter of JKD were cloned into pAB287 containing the LUC coding sequence. For the in vitro protoplast assay, UBQ10_pro:WOX5-Venus and 35S_pro:PLT1-HA have been described previously (Zhai and Xu, 2021).

The ClearSee assay was performed following previously reported protocols (Kurihara et al., 2015; Zhang et al., 2019; Zhai and Xu, 2021). The Nikon C2 software NIS-Element was used to export 8-bit gray images. ImageJ was used to convert grayscale images to color images by means of a lookup table. This allowed for the comparison of fluorescence levels within but not between images. For marker gene expression, two biological repeats were analyzed and showed similar results.

The primers used for molecular cloning and PCR are listed in Table S1.

Gene sequences were accessed from the Arabidopsis Genome Initiative (https://www.arabidopsis.org/): WOX11 (AT3G03660), LBD16 (AT2G42430), WOX5 (AT3G11260), WOX7 (AT5G05770), PLT1 (AT3G20840), PLT2 (AT1G51190), PLT3 (AT5G10510), SCR (AT3G54220), SHR (AT4G37650), JKD (AT5G03150), ATML1 (AT4G21750) and WOL (AT2G01830).

We thank L. Sheng and Y. Ge for technical assistance in this research. We thank B. Scheres, S. Wu, J.-W. Wang, and ABRC for Arabidopsis seeds used in this work.