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. 2021 Feb 3;185(4):1652–1665. doi: 10.1093/plphys/kiab006

Time-course observation of the reconstruction of stem cell niche in the intact root

Meizhi Xu 1,#, Xu Gu 1,#, Qiaozhi Yu 1,#, Yuting Liu 2, Xinxin Bian 1, Renyin Wang 1, Meina Yang 1, Shuang Wu 1,✉,3
PMCID: PMC8133607  PMID: 33599750

Abstract

The stem cell niche (SCN) is critical in maintaining continuous postembryonic growth of the plant root. During their growth in soil, plant roots are often challenged by various biotic or abiotic stresses, resulting in damage to the SCN. This can be repaired by the reconstruction of a functional SCN. Previous studies examining the SCN’s reconstruction often introduce physical damage including laser ablation or surgical excision. In this study, we performed a time-course observation of the SCN reconstruction in pWOX5:icals3m roots, an inducible system that causes non-invasive SCN differentiation upon induction of estradiol on Arabidopsis (Arabidopsis thaliana) root. We found a stage-dependent reconstruction of SCN in pWOX5:icals3m roots, with division-driven anatomic reorganization in the early stage of the SCN recovery, and cell fate specification of new SCN in later stages. During the recovery of the SCN, the local accumulation of auxin was coincident with the cell division pattern, exhibiting a spatial shift in the root tip. In the early stage, division mostly occurred in the neighboring stele to the SCN position, while division in endodermal layers seemed to contribute more in the later stages, when the SCN was specified. The precise re-positioning of SCN seemed to be determined by mutual antagonism between auxin and cytokinin, a conserved mechanism that also regulates damage-induced root regeneration. Our results thus provide time-course information about the reconstruction of SCN in intact Arabidopsis roots, which highlights the stage-dependent re-patterning in response to differentiated quiescent center.

Time course live imaging technique revealed stage-dependent reconstruction patterns of stem cells in the intact Arabidopsis roots in response to differentiated quiescent center.

Introduction

The extraordinary developmental plasticity in plants partly depends on the ever-growing meristems at the root (or shoot) tip. The root apical meristem is derived from the stem cell niche (SCN), which is composed of the mitotically inactive quiescent center (QC) and a group of stem cells that surround the QC (van den Berg et al., 1997; Bennett and Scheres, 2010). Past studies have established the framework in which multiple plant hormones (Ortega-Martinez et al., 2007; Benková and Hejátko, 2009; Ding and Friml, 2010; Zhang et al., 2010; Chen et al., 2011; Lee et al., 2013; Chaiwanon and Wang, 2015), and a number of transcriptional factors including WUSCHEL-RELATED HOMEOBOX 5 (WOX5), SHORT-ROOT (SHR), SCARCROW (SCR), and PLETHORA (PLT) are integrated to regulate SCN maintenance in Arabidopsis (Arabidopsis thaliana) roots (Helariutta et al., 2000; Sabatini et al., 2003; Aida et al., 2004; Sarkar et al., 2007; Dolan, 2009; Forzani et al., 2014; Pi et al., 2015).

Despite all regarded as stem cells, QC as well as neighboring stem cells are quite different in terms of the properties and functions. A recent study suggested that the distinct stemness of stem cells within SCN could derive from the spatially intersected expression of multiple transcriptional regulators (Xu et al., 2019). The early pilot study by Ben Scheres’s group showed that laser ablated QC promoted stem cell differentiation, indicating the potential intercellular signals derived from QC (van den Berg et al., 1997). Further evidence proved that such cell-to-cell communication was transmitted via plasmodesmata (PD; Liu et al., 2017). Mobile regulators including SHR and WOX5 can traffic intercellularly, which could contribute to the spatial patterning of the SCN (Nakajima et al., 2001; Pi et al., 2015; Xu et al., 2019).

As stem cells serve as the source of new cells during the development as well as upon damage, many studies had been focusing on how stem cells were re-specified during organ regeneration. The surgical excision of root-tip or laser ablation of stem cells had been conducted in several species, including pea (Pisum sativum L), maize (Zea mays), and Arabidopsis (Feldman, 1976; Rost and Jones, 1988; Xu et al., 2006). These anatomic and cellular observations have provided the full scenario of the SCN restoration, and also demonstrated the role of the SCN as the patterning organizer during root regeneration (Sena et al., 2009). However, SCN seemed to be dispensable for the early steps of root regeneration as the initiation of regeneration could be clearly observed in plt1/2 or scr4 mutants in which SCN was severely affected (Xu et al., 2006; Sena et al., 2009). A similar regenerative reprogramming was also reported in animal organs, in which the trans-differentiation of specific cell types occurred without the transition to stereotypical stem cells (Thowfeequ et al., 2007; Zhou et al., 2008). Therefore, it remains enigmatic whether a stem cell independent mechanism regulates cell specification and patterning in the meristematic region during the root regeneration.

In these previous explorations, physical damages including surgical excision or laser ablation were employed, which could cause wounding effect. It has been well documented that wound signaling acts as the early trigger of plant regeneration (Chen et al., 2016; Zhang et al., 2019). Re-specification of cell identity was shown to begin within hours of root excision or laser-assisted cell elimination (Xu et al., 2006; Sena et al., 2009). Similar injuries sometimes can also be observed when plant tissues are challenged by adverse environments. Chilling stress was reported to induce cell death in root columella stem cell daughters (CSCDs) to protect QC, which was thought to enhance stress tolerance (Hong et al., 2017). Besides, stele stem cells (SSCs) in roots and their early descendants seemed to be the most vulnerable cells to DNA damage induced by UV light, ionizing radiation, and radiomimetic drugs (Curtis and Hays, 2007; Fulcher and Sablowski, 2009; Furukawa et al., 2010; Heyman et al., 2013). However, physically damaged cells often disrupt root anatomy and can alter mechanical properties of the damaged tissue. In nature, many environmental conditions stress plant tissues without putting on physical damage. A previous study demonstrated that the phosphate limitation led to SCN defects through inducing callose deposition in the SCN which blocked the intercellular communication (Müller et al., 2015).

Compared with the physically damaged tissues in which the expression and distribution pattern of auxin and cytokinin are dramatically disrupted (Müller and Sheen, 2008; Efroni et al., 2016), stressed intact tissues maintain the normal anatomy and positional information within the tissue. Importantly, such stress triggered arrest of SCN seems to be reversible. Recovery in the standard growth conditions often allows SCN activity to be rescued. Therefore, it is important to understand how the SCN is restored when the normal tissue patterning is retained and only the intrinsic properties of stem cells are affected. We want to know, in such scenarios, if SCN can directly dedifferentiate from these affected cells, and how the interaction between auxin and cytokinin is involved.

To address these questions, we employed previously reported the pWOX5:icals3m system, in which root apical stem cells became differentiated upon the precise induction of callose accumulation in QC (Liu et al., 2017). Our time-course observations of the reconstruction of SCN in pWOX5:icals3m roots suggest that the successive but distinct stages occurred, with the division-driven anatomic reorganization in the early stage and cell fate specification of new SCN in later stages. No direct dedifferentiation was observed in pWOX5:icals3m roots, suggesting SCN differentiation is an irreversible process. During the recovery, auxin local accumulation shifted from the center of the root tip to other parts, which was coincident with the division pattern shift. With the contribution of divided cells from stele in the early stage and endodermis in later stages, the anatomy re-patterning and cell fate re-specification ensure the formation of new SCN in the converged center of all cell files. This precise position is likely determined by the mutual antagonism between auxin and cytokinin, a conserved mechanism that could be also used by the damaged-induced root regeneration.

Results

Anatomic center reforms prior to cell fate determination in SCN

In the previous study, the icals3m system in which PD-mediated symplastic communication is blocked was reported to have inducibly compromised SCN (Vatén et al., 2011; Wu et al., 2016; Liu et al., 2017). Upon estradiol (ES) induction, impaired symplastic communication in QC due to specific callose deposition disrupted SCN maintenance, without much impact on other parts of the tissues. In particular, it does not introduce physical damage to the root, allowing for the observation of the cell fate conversion processes in the intact organ and well-patterned tissues.

Using pWOX5:icals3m lines, we performed the time-course observation of the SCN phenotypes. After 48-h induction with ES, most QC cells in pWOX5:icals3m roots became differentiated (about 70%; Figure 1, A and B), and by 3-d treatment, almost all QC cells were differentiated, marked by the presence of starch granules in the cell (Figure 1, A and C). Thus, we decided 3-d ES induction of pWOX5:icals3m lines was the time-point when the stem cells within SCN was entirely removed. We next observed the time-course reconstruction of the compromised SCN. As the SCN phenotype in pWOX5:icals3m roots was derived from the QC-specific callose deposition, we monitored the dynamic change of callose accumulation in pWOX5:icals3m roots. At 3-d ES induction, the accumulation of callose was clearly visible in QC cells (Figure 1, D). But after being transferred to the normal medium, pWOX5:icals3m roots could immediately remove the callose and the aniline blue staining detected no callose after 1-d recovery (Figure 1, E).

Figure 1.

Figure 1

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Time course observation of anatomic reorganization and QC re-formation. A–C, Root mPS-PI staining of WT (A), pWOX5:icals3m after 10 μM ES induction for 2 d (B) or 3 d (C). Numbers in (B) and (C) represent the frequency of presented phenotype in all examined roots. D and E, Aniline blue staining for the callose deposition in pWOX5:icals3m roots after 10 μM ES induction for 3 d (D), and being transferred to the normal medium for 1 d after 3-d ES induction (E). Asterisks note the specific deposition of callose in the QC position. F–I, SCN repatterning in pWOX5:icals3m roots at different time-points post recovery: 1 d (F), 2 d (G), 3 d (H), or 4 d (I) after 3-d ES induction. The insets of (F–I) show the 3-D views. Note the differentiation in the QC cell and columella stem cell (CSC) region in (F–I) (red dots indicate starch grains). J–N, Confocal images of the 2-D and 3-D views in pWOX5:icals3m roots expressing the QC marker pWOX5:erGFP after 3-d ES induction (J), and at different time-points post recovery: 2 d (K), 3 d (L), 5 d (M), and 7 d (N). Numbers in (J–N) represent the frequency of presented phenotype in all examined roots. Yellow arrowheads indicate QC position, blue brackets indicate stele cells, green brackets indicate differentiated columella cells, and yellow dotted boxes indicate enlargements as 3-D views. 2-D indicates two-dimensional view, 3-D indicates three-dimensional views. Scale bars represent 20 μm.

To track the SCN re-patterning, we performed the long-term 2-D and 3-D observations of pWOX5:icals3m roots with mPS-PI staining. In the early stage of the SCN recovery, we detected a group of undifferentiated cells in the position of the SCN. This group of new cells started to form in the SCN position within 2 d (Figure 1, F and G). By 3–4 d, there were a number of undifferentiated cells locating at the converged point of all cell files (Figure 1, H and I). To observe the identity reacquisition of QC cells, we then performed Lugol’s staining in pWOX5:icals3m roots with the QC-specific markers QC25. In WT, QC25 marker was specifically expressed in QC cells (Supplemental Figure S1, A), but this expression was mostly abolished after 3-d ES induction in pWOX5:icals3m roots (Supplemental Figure S1, B). Despite the new undifferentiated cells form in the SCN position, QC25 expression stayed at fairly low level until the fourth day of the recovery (Supplemental Figure S1, C–E), indicating the delayed reinstatement of QC identity. At the seventh day of the recovery, the starch staining in CSCs was abolished (Supplemental Figure S1, F and G), indicating the newly formed QC started to play roles. To test if the re-established QC cells gain functions, we visualized the 2-D and 3-D expression of WOX5, a QC-specific gene that is indispensable for the QC-mediated repression of stem cell differentiation (Sarkar et al., 2007; Pi et al., 2015). The expression of pWOX5:erGFP was almost abolished in the QC after 3-d ES induction in pWOX5:icals3m roots, but became surprisingly expanded to other cells surrounding QC (Figure 1, J). During the recovery, the pWOX5:erGFP expression was mostly excluded from the central undifferentiated cells in SCN, with the low level in neighboring cells (Figure 1, K–M). At this point, we found the adjacent CSC cell still stayed differentiated (Supplemental Figure S1, H–K). The centered-expression pattern of pWOX5:erGFP was regained until 7 d after the recovery (Figure 1, N). Importantly, no starch accumulation was observed in the CSC position (Supplemental Figure S1, L) suggesting the repression of CSC differentiation by the reformed functional QC. These observations indicate that the division-driven anatomic reorganization occurred prior to the reacquisition of QC identity.

The division in endodermal layers contributes to SCN reconstruction

Our time-course observations do not support that the new QC re-forms through a direct conversion or de-differentiation from the differentiated QC cells in pWOX5:icals3m roots. The cell division must be reactivated to generate a group of undifferentiated cells before any cell identity could be specified. The newly formed cells could be easily recognized based on their size, relative position to the existing cell files, and the differentiation status. In most cases, the newly divided cells interdigitated with the differentiated cells, often forming the protruding patchy (Figure 2, A and B). To examine the spatial distribution of dividing cells and differentiated cells within the previous SCN position during the recovery, we employed a marker line-Q0608 that is expressed in differentiated columella cells, as well as mPS-PI staining that clearly shows the starch granules in differentiated cells. Based on the observation of 2-D and 3-D reconstruction, we divided the dynamic pattern of the division in SCN into four forms: model-a in which the dividing cells are mainly located on the upper side of the differentiated cells (Figure 2, C and D); model-b in which the dividing cells are located on both sides of the root tip with the differentiated cells in the middle (Figure 2, E and F); model-c showing an opposite distribution to model b, with the dividing cells in the middle and differentiated cells in both sides (Figure 2, G and H); and model-d in which the dividing cells and the differentiated cells are located on opposite sides of the root tip (Figure 2, I and J). To determine how the anatomic patterning changes over the recovery, we quantified the frequency of each model. The results revealed that the model-d was the most dominant type during the early stage of SCN regeneration, while mode-a took an increasing proportion in later stages (Figure 2, K). Model c, however, exhibited the lowest frequency in all stages, indicating the higher mitotic competency in outer cell layers during the SCN reconstruction (Figure 2, K).

Figure 2.

Figure 2

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The spatial distribution of dividing cells and differentiated cells during the SCN reconstruction. A–J, Expression of differentiated columella cells marker Q0608 in WT (A, B) and pWOX5:icals3m (C–J) roots during the recovery. The right panels in (A, C, E, G, and I) show the same root with 3-D reconstruction. Phenotypes were analyzed by modified pseudo‐Schiff PI (mPS‐PI) staining in (B, D, F, H, and J). The below panels in (B, D, F, H, and J) show the same roots with 3-D reconstruction. The schematic models of the dynamic division patterns in the SCN position. Red colored cells indicate differentiated cells; green colored cells represent dividing cells. Dotted boxes indicate the position of GFP expression, blue arrow heads indicate the differentiated QC position. 2-D indicates two-dimensional view, 3-D indicates three-dimensional views. Scale bars represent 20 μm. K, The heatmap showing the quantified frequency of each patterning model during the recovery. Quantification was based on ES 3d (n = 30), recovery 2d (n = 32), recovery 3d (n = 30), and recovery 4d (n = 35); “n” represents the total number of roots used for the quantification.

To further evaluate the spatiotemporal distribution of the mitosis, we introduced the cell cycle marker CYCB1;1-GUS into pWOX5:icals3m lines (Supplemental Figure S2, A–E). In the early stage of the recovery, pWOX5:icals3m roots exhibited higher mitotic index in the inner part of tissues (Supplemental Figure S2, C and F). To verify this, we visualized the expression of CYCB1;1-GFP in pWOX5:icals3m lines. The results showed the higher frequency of CYCB1;1-GFP expression in stele during the early recovery (Supplemental Figure S2, G and H). It was previously reported that local auxin accumulation relates to restorative division during root regeneration (Hoermayer et al., 2020). To observe the potential changes in auxin signaling, we examined the expression pattern of R2D2, a previously reported semi-quantitative auxin marker (Liao et al., 2015), in pWOX5:icals3m roots (Supplemental Figure S3, A–D). We found the auxin level in stele adjacent to QC was markedly elevated at 2-d of the recovery (Supplemental Figure S3, C and E), which was coincided with the division pattern in stele at the early stage. Interestingly, both auxin level and division frequency became reduced in stele cells after this stage (Supplemental Figures S2, F, S3, E). In contrast, the ground tissues showed an increasing trend of division rate during the recovery, and became the dominant region of mitosis after 3-d of recovery (Supplemental Figure S2, D–F). In contrast, the outer cell layers including epidermis and lateral root cap displayed low division frequency throughout the entire recovery in pWOX5:icals3m roots (Supplemental Figure S2, F). This spatial change of division hot zone could be an indication of the distinct contribution of cell types to the reconstitution of SCN.

To further examine this, we tracked the expression of different cell identity markers during the regeneration of SCN. Consistent with the anatomic observation, the expression of pWOL:erGFP was expanded to the dividing cells in SCN in the early stage of recovery but was excluded in later stages (Figure 3, A). The expression of the epidermal marker, pWER:H2B-GFP, was not detected in the SCN position throughout the entire regeneration (Figure 3, B). As ground tissues seemed to contribute cells to the SCN reconstruction, we observed reporter lines of both CO2 (the cortex cells marker) and EN7 (endodermal cell marker; Heidstra et al., 2004). Surprisingly, the pCO2:H2B-GFP was not detected in the SCN position during the regeneration (Figure 3, C). But pEN7:H2B-GFP showed an opposite trend to pWOL:erGFP, with no expression in SCN in the early stage but high expression in all dividing cells in SCN in the later stages (Figure 3, D). Together our results suggest that the division in the neighboring vascular cells could be the direct response to the disrupted SCN, but the eventual contribution of the SCN reconstruction may derive from the endodermal cells.

Figure 3.

Figure 3

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Dynamic change of cell identity markers during the recovery. A, Expression of the stele marker pWOL:erGFP in pWOX5:icals3m roots during the recovery after 3-d ES induction. The inset shows the weakening GFP in the consecutively dividing cells in the SCN position. Yellow arrowhead marks the strongest fluorescence and the blue arrowheads mark the weakening fluorescence in the dividing cells. B, Expression of epidermal marker pWER:H2B-GFP in pWOX5:icals3m roots during the recovery after 3-d ES induction. C, Expression of the cortex marker pCO2:H2B-GFP in pWOX5:icals3m roots during the recovery after 3-d ES induction. D, Expression of the endodermal marker pEN7:H2B-GFP in pWOX5:icals3m roots during the recovery after 3-d ES induction. The insets show the expression of GFP in dividing cells in the SCN position during the later stages of the recovery. Yellow arrowhead marks the strongest fluorescence and the blue arrowheads mark the weakening fluorescence in the dividing cells. Scale bars represent 20 μm.

Auxin re-distribution is coincident with cell division pattern during SCN regeneration

Auxin has been shown to position the SCN, and be critical for the root regeneration (Xu et al., 2006). Laser-induced wounding was proposed to disrupt the auxin flow and subsequently stabilize auxin distribution in the renewed root tips (Xu et al., 2006). To investigate the role of auxin in the SCN reconstruction in pWOX5:icals3m roots, we performed the live-imaging to monitor the change of spatial auxin level in the root tip.

In line with the previous report (Liu et al., 2017), DR5:mCherry level became substantially decreased in the SCN after 3-d ES induction in pWOX5:icals3m roots (Figure 4, A and B). In the early stage of the recovery (2-d), auxin response shown by DR5:mCherry appeared to shift to one side of the root tips (Figure 4, C). The auxin level was slightly increased in the later recovery stages (3–4 d; Figure 4, D and E), indicating the potential enhanced auxin flow or local biosynthesis. Further observation of the marker of DR5:mCherry verified this spatial shift of auxin level (Figure 4, F). We next observed the time-course change of R2D2 (Figure 4, G). We found the fluorescence intensity of DII of R2D2 often markedly declined only on one side of the root tip after 2–3 d recovery (Figure 4, H and I), indicating the risen auxin level on this side. The unbalanced spatial distribution of auxin was coincident with the distribution pattern of the dividing cells in the recovery of SCN. It is possible auxin acts as the possible spatial signaling directing the spatial organization of the cell division and differentiation during the SCN reconstruction. It was widely accepted that auxin and the pathway mediated by AP2-domain transcription factor PLETHORA (PLT) act as the core module in root stem cell maintenance (Galinha et al., 2007; Mähönen et al., 2014). It was also reported that PLT2 played the dosage-dependent role in root tip regeneration (Durgaprasad et al., 2019). We then observed the expression of PLT1/2 in pWOX5:icals3m roots during the SCN restoration. In consistent with the previous report (Liu et al., 2017), we found a dramatic reduction of the expression level of pPLT1:erCFP and pPLT2:erCFP in the SCN position after 3-d ES induction in pWOX5:icals3m roots (Supplemental Figure S4, A–D). Similar to the change of auxin level, the expression of both PLT1 and PLT2 was gradually restored in the SCN region during the recovery (Supplemental Figure S4, A–D).

Figure 4.

Figure 4

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The spatially decentered auxin distribution during the SCN reestablishment. A–E, Expression of DR5:mCherry in WT (A), pWOX5:icals3m after 3-d ES induction (B), and pWOX5:icals3m during the recovery after 3-d ES induction (C–E). F, Quantified fluorescent intensity of DR5:mCherry in the SCN position of pWOX5:icals3m during the recovery. Data represented are mean SD of 20–23 roots for each sample. G–I, Expression of R2D2 in WT (G), pWOX5:icals3m with 2-d (H), or 3-d (I) recovery after 3-d ES induction. The dotted circles indicate unbalanced GFP fluorescence with stronger level on one side than the other in the root tip during the recovery. Scale bars represent 20 μm.

An auxin distribution pattern is mainly determined by auxin transportation, local biosynthesis, or both. To address how these events change over recovery stages, we first observed PIN1-GFP and PIN3-GFP, two well-characterized auxin efflux transporters. We found the overall level of PIN1-GFP and PIN3-GFP had only moderate change during the recovery (Figure 5, A and B). However, the PIN7-GFP level became greatly reduced after 3-d ES induction (Figure 5, C). During the recovery, PIN7-GFP seemed be restored only in columella cells (Figure 5, C and D). In line with this, PIN3-GFP seemed to be also increased in the columella cells (Figure 5, B). To visualize the auxin biosynthesis in the root tip, we examined the expression of pTAA1:GFP and only detected a slight increment during the recovery (Figure 5, E and F), indicating the limited contribution of TAA1-medited auxin biosynthesis to the auxin re-distribution. We further examined YUC1:GUS in pWOX5:icals3m roots during the SCN restoration. Similar to the auxin uneven distribution, the YUC1:GUS was also often accumulated on one side of the root tip in the early recovery stage (Figure 5, G). Thus, both auxin local biosynthesis and polar transport may contribute to the shift of auxin distribution for the SCN re-establishment.

Figure 5.

Figure 5

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Re-establishment of auxin polar transportation and local biosynthesis during the SCN regeneration. A–C, Expression of PIN1:PIN1-GFP (A), PIN3:PIN3-GFP (B), and PIN7:PIN7-GFP (C) in pWOX5:icals3m roots during the recovery after 3-d ES induction. The yellow arrowheads point to the fluorescence in the columella cells. D, Quantified fluorescence of PIN7:PIN7-GFP in the columella cells of pWOX5:icals3m roots during the recovery after 3-d ES induction. Error bars show mean SD of three biological replicates. E, Expression of TAA1:GFP in pWOX5:icals3m roots during the recovery after 3-d ES induction. F, Quantified fluorescence of TAA1:GFP in the SCN position of pWOX5:icals3m roots during the recovery after 3-d ES induction. Data represented are mean SD of 14–20 roots for each sample. G, Expression of YUC1:GUS in pWOX5:icals3m roots after 2–3 d recovery. Yellow brackets indicate the auxin accumulation on one side of the root. Scale bars represent 20 μm.

Auxin–cytokinin interaction guides the establishment of the new SCN

The interaction between auxin and cytokinin controls many developmental processes (Schaller et al., 2015). A previous report also demonstrated the importance of synergic interaction between auxin and cytokinin in root regeneration (Efroni et al., 2016). To follow the dynamics of cytokinin level, we visualized the cytokinin response reporter, TCS:GFP (Figure 6, A; Zurcher et al., 2013). After 3-d ES induction, we failed to detect the change of TCS:GFP level in the root cap, but saw a slight increase of TCS:GFP fluorescence in the stele (Figure 6, B). Surprisingly, the TCS:GFP became considerably declined in pWOX5:icals3m root cap in the early recovery stage (2d; Figure 6, C). Even more surprisingly, this downward trend of TCS:GFP was not retained, but instead the GFP signal gradually became enhanced, resulting in the WT level in the later stages (Figure 6, D–F).

Figure 6.

Figure 6

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Dynamic change of cytokinin distribution during the SCN regeneration. A–E, Expression of the cytokinin marker TCS:GFP during the recovery after 3-d ES induction in pWOX5:icals3m roots. F, Quantification of TCS:GFP fluorescence in pWOX5:icals3m roots during the recovery after 3-d ES induction. Error bars indicate the mean SD of three biological replicates. G–J, Representative patterns of TCS:GFP distribution in the root tip of pWOX5:icals3m during the recovery. Dotted box showing the GFP fluorescence in the middle part of the root tip. K, The heatmap showing the quantified frequency of four different models of TCS:GFP distribution during the recovery. Quantification was based on ES 3-d (n = 29), recovery 2-d (n = 37), recovery 3-d (n = 34), and recovery 4-d (n = 28). “n” represents the total number of roots used for the quantification. Scale bars represent 20 μm.

The balance between auxin and cytokinin influences cell division and cell fate determination in many developmental events (Müller and Sheen, 2008; Ruzicka et al., 2009). In root regeneration, auxin was shown to associate with the cell division (Sena et al., 2009; Efroni et al., 2016). Our observation also confirmed the similar distribution pattern between auxin level and dividing cells. We then examined how TCS:GFP level was dynamically changed during the recovery. Our observations also showed four different patterns for TCS:GFP distribution in the root tip of pWOX5:icals3m lines during the recovery: model-a in which TCS:GFP was mostly expressed in the distal region of the root tip (Figure 6, G); model-b with TCS:GFP accumulated in the middle part of the root tip (Figure 6, H); model-c with TCS:GFP located on both sides of root tip (Figure 6, I); and model-d in which TCS:GFP was only on one side of the root tip (Figure 6, J). The quantification of the percentage of each model showed that the model a and model d were the major forms of TCS:GFP distribution in the early stage of SCN regeneration (Figure 6, K). Such patterns were gradually switched to the model a dominancy in later stages, suggesting a potential role of cytokinin in SCN reconstruction.

In the previous elegant study, Birnbaum group uncovered the vital role of the transient overlap between auxin and cytokinin in determining the position of newly formed SCN in the root regeneration after the surgical excision (Efroni et al., 2016). Here, we found the dynamic changes of the distribution pattern of auxin and cytokinin were similar to these in the excised roots, suggesting a common mechanism might also be used by the SCN reconstruction in the intact roots. To further verify this possibility, we performed the live-imaging in the roots with both auxin and cytokinin markers. Different from the excised root regeneration, our time-course observation showed that there was no overlapped zone between DR5:mcherry and TCS:GFP in the SCN region during the reconstruction in pWOX5:icals3m roots (Supplemental Figure S5, A–D). To further verify this dynamic process, we performed the time-lapse observation of the single root with both DR5:mcherry and TCS:GFP markers. Both markers became dramatically decreased after the ES induction (Figure 7, A–C). The distribution domain of both markers seemed to be quite separated during the recovery and they often seemed to take up opposite side of the root (Figure 7, D–G). In later stages, auxin domain intruded into the cytokinin domain in the root columella, which is very similar to the pattern in WT roots (Figure 7, H).

Figure 7.

Figure 7

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Time-course observation of interaction between auxin and cytokinin in the single root. A–C, Confocal time-lapse observation of the same single root after ES induction. Both auxin-responsive reporter DR5:mCherry and the cytokinin reporter TCS:GFP were introduced into pWOX5:icals3m roots. The root was induced by ES for 2-d (A), 3-d (B), and 4-d (C). D–G, Confocal time-lapse observation of the same single roots during the SCN regeneration. pWOX5:icals3m roots expressing both DR5:mCherry and TCS:GFP at 2-d (D), 3-d (E), 4-d (F), and 5-d (G) recovery after 4-d ES induction. H, Cartoons showing the temporal change of the interaction between auxin and cytokinin during the recovery. The left panels of (A–G) show the overlay between DR5:mCherry and TCS:GFP; the middle panels of (A–G) show DR5:mCherry; the right panels of (A–G) show TCS:GFP. Yellow brackets indicate the absence of GFP in the position. Scale bars represent 20 μm.

Our results suggest functionally disrupted SCN can activate the mitotic activity in the neighboring tissues, which promotes the reorganization of the anatomic structure of the root tip. The pattern of auxin local enrichment experiences the spatial shift, which may associate with the stage-specific cell division pattern during the SCN recovery. The mutual antagonism between auxin and cytokinin may eventually help to position the QC cells and confer the specific cell identity, a similar mechanism that could be also used by the damaged-induced root regeneration.

Discussion

In the root tip, the enriched auxin in the SCN is essential for stem cell maintenance (Xu et al., 2006). The auxin polar transport is disturbed following the ablation, which contributes to the formation of the auxin maxima around the damaged area (Xu et al., 2006). As early as 3 h following the QC laser ablation, the auxin response maxima shifts quickly to the stele and cortex/endodermis stem cells and their daughter cells (Xu et al., 2006). Despite the complete removal of the distal auxin maximum, auxin signaling was rapidly induced in the stele region that was close to the cutting site (Efroni et al., 2016). The combination of auxin polar transport and physical barrier in the region of damage possibly generates the spot of auxin accumulation. This forced local auxin buildup interacts with cytokinin to position the newly formed SCN during the root regeneration (Efroni et al., 2016). Opposite to such wound-induced regeneration processes, the auxin level became substantially decreased in the SCN of pWOX5:icals3m roots after 3-d ES induction. In addition, the unbalanced spatial auxin distribution was observed in the neighboring meristematic region during the early stage of the recovery. The pWOX5:icals3m system provides a non-intrusive method to generate an intact root with the differentiated SCN, which permits the observation of the re-establishment of SCN within the context of well-patterned tissues. This system also avoids the potential physical barrier of auxin polar flux which usually occurred in either laser ablation experiments or root excisions.

Once formed in the embryo, QC usually stays in a quiescent state and is not replaced until the physical damage or other stresses. A previous study has shown abiotic stresses like phosphate-deficiency triggered callose accumulation in root SCN, resulting in differentiated stem cells (Müller et al., 2015). In their observation, QC did not undergo cell death, but rather displayed the cell division phenotype (Müller et al., 2015). During the ever-changing environments, plant roots are often challenged by stress effects; some might be permanent while some other might be temporary. In many conditions, SCN could respond by merely promoting the QC division or differentiation, rather than the severe cell death. Considering the importance of SCN during the root adaptation to different conditions, it is important to understand how the status of SCN is changed when only the intrinsic properties of stem cells are affected in the intact tissues. Since no physical damage or mechanical stresses is enforced, the SCN could be reconstructed directly from the cell fate conversion or de-differentiation, in which the differentiated QC or other stem cells could be converted back to stem cells when the normal growth condition is restored. Alternatively, plants could activate the mitosis in the less differentiated cells nearby to create a group of new cells before the cell fate is specified. In this study, our observations support the second possibility. Interestingly, the reconstruction of SCN seemed to be a slow process with dimorphic stages: the early stage devoted to re-pattern the tissue, forming the structural center; and the later stage committed to specify the cell fates in the SCN position.

Despite the cell lineage-dominant mechanism of cell fate determination in animals, positional information is thought to be the deciding factor in plants. However, it has been difficult to evaluate the lineage contribution to the cell fate specification in plants due to the lack of the in vitro system. The previous evidence supporting the position-determined cell-fate in plants was mostly derived from the early surgical experiments and clonal analyses (Kidner et al., 2000; Scheres, 2001; Scheres et al., 2002; Kim and Zambryski, 2005). Our understanding of cell-fate regulation in Arabidopsis mostly relies on the histological studies or the static imaging of cell-specific marker. A recent study using live-imaging technique provides evidence supporting the lineage contribution (Yu et al., 2017). In the root tip, the cell-fate conversion seemed to be a slow readout of developmental instruction, in which the lineage inheritance seemed to be dominant in the early stage while positional information played decisive roles in later stages. Despite the eventual recovery program could be conserved in both pWOX5:icals3m roots and physically damaged system, the major difference could lie in the early stage of the recovery. The roots with intact anatomical structure allow the examination of the possibility of trans-differentiation or de-differentiation, which is different from the physically damaging system. During the recovery of SCN in pWOX5:icals3m roots, differentiated QC cells were not able to convert back to stem cells, although they still localize in the converged center of cell files. Thus, this is another example against the absolute dominance of position-dependent mechanism. However, the position information still seemed to be the essential regulator during the SCN reconstruction. All newly divided cells need to be re-patterned well and only the cells locating in the central region could proceed to re-gain the QC identity.

In the whole stages of SCN reconstruction in pWOX5:icals3m roots, local auxin level seemed to play a critical role to direct different developmental events. The successive cell division and cell fate determination during the SCN reconstruction seemed to associate tightly with the changing local auxin level. In addition, the shift of dividing cells in the root tip was also coincident with the shift of auxin accumulation. It is still unclear why auxin was often enriched on one side of the root tip in the early stage of the recovery. Such biased auxin enrichment seemed to depend on multiple pathways including the YUC1-mediated biosynthesis and PIN-mediated polar transport.

The antagonism between auxin and cytokinin controls a plethora of developmental processes. Recently, Birnbaum group has shown by elegant live-imaging technique that the manipulation of both hormones altered the position of new SCN during the root regeneration. In pWOX5:icals3m system, auxin level was greatly decreased while cytokinin only showed a slight reduction after 3-d ES induction, which results in the breakup of balance between these two hormones. In the early stage of the recovery, auxin and cytokinin seemed to have opposite trend, possibly to restore the auxin dominant micro-environment in the SCN position. This could create an optimal auxin:cytokinin ratio for cell division, a common mechanism regulating the morphogenesis in cultured tissues. However, the declining tendency of cytokinin in the early stage was not a sustained process. The cytokinin level became gradually increased in later stages of the SCN regeneration and eventually reached the normal distribution pattern in undisturbed roots. Thus, the different dominant stages of these two hormones as well as spatially separated distribution domains imply the different roles of auxin and cytokinin in distinct recovery stages, and antagonism between these two hormones.

Materials and methods

Plant materials and growth condition

Arabidopsis thaliana lines were Columbia ecotype (Col-0) background in this study. Transgenic lines including pWOX5:icals3m, pWOX5:erGFP, QC25:GUS, Q0608, CYCB:GUS, CYCB:GFP, pWOL:erGFP, pWER:H2B-GFP, pCO2:H2B-GFP, pEN7:H2B-YFP, DR5:mCherry, R2D2, pPLT1:erCFP, pPLT2:erCFP, PIN1:PIN1-GFP, PIN3:PIN3-GFP, PIN7:PIN7-GFP, TCS:GFP, TAA1:GFP, and YUC1:GUS have been described previously.

Seeds were planted on 0.5 Murashige and Skoog medium containing 0.05% (w/v) MES (pH 5.8), 1.0% (w/v) sucrose, and 1.0% (w/v) agar and grown vertically under a 16-h light/8-h darkness cycle, at 23°C and 70% relative humidity. After sterilization, the seeds were germinated darkly after being incubated for 2 d at 4°C. Plants were imaged at 4–6 d post germination (dpg) unless otherwise stated.

Aniline blue staining

Aniline blue staining was performed as previously described (Wu et al., 2016; Liu et al., 2017). Briefly, roots were immersed in the staining solution (67 mM K3PO4, 1% Aniline blue, and 1‰ Silwet), and incubated in the dark for 30–60 min. Roots were mounted in water and imaged under the confocal.

GUS staining

β-Glucuronidase (GUS) staining was performed as previously described (Weijers et al., 2001). Seedlings were incubated in the GUS (0.5 mg/mL) staining solution to at 37°C overnight. The seedlings were cleared in chloral hydrate solution (HCG) for microscopy analysis on Nikon ECLIPSE Ni-U microscope

mPS-propidium iodide staining

mPS-propidium iodide (PI) staining was performed as described (Xu et al., 2019). Whole seedlings were fixed in the fixative (50% methanol and 10% acetic acid) for 3 h at 4°C. Then the seedings were rinsed with water and transferred to the 1% periodic acid for 40 min. Next the seedings were rinsed with water again, and incubated in Schiff reagent (100 mM sodium metabisulfite and 0.15 N HCl) with PI (freshly added to a final concentration of 100 μg/mL) for 30 min to 1 h until plants were visibly stained. The plants were mounted in HCG for visualization.

Chemical treatments

For β-ES, treatments were performed as described (Li et al., 2018). Five dpg old seedling were transferred to 0.5 MS medium supplemented with 10 μM β-ES and incubated for 2–4 d. For the recovery, 5 dpg old seedlings (for 3-d ES induction) were transferred to 0.5 MS medium for time course analyses.

Confocal microscopy and imaging

Time-lapse live imaging of a single root was performed as described previously (Wu and Gallagher, 2015; Rahni and Birnbaum, 2019). Roots were mounted in 0.01 μg/mL PI in water. Confocal laser scanning microscopy was performed on a Zeiss LSM880 with dual-channel setting of GFP and PI. Excitation and detection parameters were set as follows: GFP 488 nm, 493–548 nm; PI 561 nm, 566–718 nm. Pictures were taken with a 40× water immersion objectives. To quantify the fluorescence intensity of R2D2, DR5:mCherry, PLT1:erGFP, PLT2:erGFP, and TCS:GFP, we used the polygon selection function to define a region of interest, and then calculated the average intensity of GFP or RFP by ROI manager. For PINs analysis, we used the rectangular selections function to define a region just on the membrane, and then calculated the relative fluorescence intensity. Image quantification was performed using ImageJ 1.4.3 software. Representative images were collected from 10 to 25 roots with three biological replicates.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers WOX5: AT3G11260; CYCB1: AT4G37490; WOL: AT2G01830; WER: AT5G14750; CO2: AT1G62500; EN7: AT4G28100; PLT1: AT3G20840; PLT2: AT1G51190; PIN1: AT1G73590; PIN3: AT1G70940; PIN7: AT1G23080; TAA1: AT1G70560; and YUC1: AT4G32540.

Supplemental data

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

Supplemental Figure S1. Expression of QC25:GUS and pWOX5:erGFP in pWOX5:icals3m roots during the recovery.

Supplemental Figure S2. The spatial cell division distribution during the recovery.

Supplemental Figure S3. The spatial shift of auxin accumulation coincided with the cell division pattern during the recovery.

Supplemental Figure S4. The recovery of PLT1/2 expression during SCN reconstruction.

Supplemental Figure S5. The interaction pattern between auxin and cytokinin during the recovery.

Supplementary Material

kiab006_Supplementary_Data
Click here for additional data file. (1.3MB, pdf)

Acknowledgments

The authors thank Elliot Meyerowitz, Dolf Weijers, Jiří Friml, and Zhaojun Ding for sharing materials. They also thank Jian Xu for critical comments.

Funding

This work was supported by the National Key Research and Development Program of China (2016YFD0100700) and the National Natural Science Foundation of China (31722006). M.X. is supported by the Scientific Research Foundation of Graduate School of Fujian Agriculture and Forestry University (grant 324-1122yb075). Y.L. is partially supported by the National Natural Science Foundation of China (31701293).

Conflict of interest statement. The authors declare that they have no conflict of interest.

 

S.W., X.G., and Q.Y. conceived the research. M.X., X.G., Q.Y., and X.B. performed most experiments and analyzed the data. M.Y. drew the graphic images. S.W., M.X., X.G., R.W., and Y.L. wrote the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Shuang Wu (wus@fafu.edu.cn).

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Associated Data

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Supplementary Materials

kiab006_Supplementary_Data
Click here for additional data file. (1.3MB, pdf)

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