Rocks in the auxin stream: Wound-induced auxin accumulation and ERF115 expression synergistically drive stem cell regeneration
Edited by Mark Estelle, University of California San Diego, La Jolla, CA, and approved May 27, 2020 (received for review April 14, 2020)
Significance
Plants show remarkable regenerative abilities, allowing them to recover from wounds and organ loss. This regenerative capacity is controlled in part by auxin, the most promiscuous plant hormone controlling organogenesis and tissue patterning. We show that stem cell death diverges the auxin flow, much like rocks in a stream, resulting in an auxin accumulation in the tissues surrounding the wound. We demonstrate that within these tissues, wound-induced expression of the plant-specific transcription factor ERF115 works synergistically with the change in auxin accumulation, thereby specifying stem cell identity in the cells surrounding the damaged stem cells. This gain of stem cell identity drives formative divisions, allowing replacement of the lost stem cells and thus successful regeneration.
Abstract
Plants are known for their outstanding capacity to recover from various wounds and injuries. However, it remains largely unknown how plants sense diverse forms of injury and canalize existing developmental processes into the execution of a correct regenerative response. Auxin, a cardinal plant hormone with morphogen-like properties, has been previously implicated in the recovery from diverse types of wounding and organ loss. Here, through a combination of cellular imaging and in silico modeling, we demonstrate that vascular stem cell death obstructs the polar auxin flux, much alike rocks in a stream, and causes it to accumulate in the endodermis. This in turn grants the endodermal cells the capacity to undergo periclinal cell division to repopulate the vascular stem cell pool. Replenishment of the vasculature by the endodermis depends on the transcription factor ERF115, a wound-inducible regulator of stem cell division. Although not the primary inducer, auxin is required to maintain ERF115 expression. Conversely, ERF115 sensitizes cells to auxin by activating ARF5/MONOPTEROS, an auxin-responsive transcription factor involved in the global auxin response, tissue patterning, and organ formation. Together, the wound-induced auxin accumulation and ERF115 expression grant the endodermal cells stem cell activity. Our work provides a mechanistic model for wound-induced stem cell regeneration in which ERF115 acts as a wound-inducible stem cell organizer that interprets wound-induced auxin maxima.
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Plant growth is controlled predominantly through cell division activity in the apical zones, called the meristems. Within the core of each meristem, a subpopulation of dividing cells can be found that operate as stem cells, collectively forming the stem cell niche (SCN). Within the Arabidopsis root SCN is a cluster of infrequently dividing cells, the quiescent center (QC). The QC is surrounded by a single tier of stem cells that give rise to all cells within the root, composing the columella, lateral root cap, epidermis, cortex, endodermis, and vasculature (1). The QC cells are characteristically marked by the expression of the homeobox gene WOX5, which maintains the quiescent state and helps to keep the SCN in an undifferentiated state (2). The position of the QC is determined by a maximum in auxin, a phytohormone involved in virtually every aspect of plant development, including organ production and tissue patterning (3, 4). Along with maintaining the SCN, auxin is known as a central player governing regenerative processes, such as recovery from wounding, organ loss, and tissue damage. Chemical or genetic perturbations in auxin biosynthesis, transport, and signaling are associated with impairments in de novo root regeneration from leaf explants, regeneration after root tip excision, adventitious rooting, tissue reunion following grafting, and the ability to form callus following wounding (5–10).
The core regulators of auxin signaling belong to three protein families: F-box TRANSPORT INHIBITOR RESPONSE/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) auxin receptors, AUXIN/INDOLE ACETIC ACID (AUX/IAA) transcriptional repressors, and AUXIN RESPONSE FACTOR (ARF) transcription factors (TFs). In the absence of auxin, ARFs are bound by AUX/IAA proteins, repressing their activity. The presence of auxin promotes an interaction between TIR1/AFB receptors and AUX/IAA proteins that targets them for proteasome-mediated degradation. Subsequently, ARFs collectively control the expression of a multitude of downstream target genes to constitute a global auxin response (11). The ARF with perhaps the most prominent role with respect to meristem regulation and stem cell activity is ARF5/MONOPTEROS (MP), which is involved in the formation of embryonic polarity, shoot apical meristem primordia, lateral organs, and vascular tissues (12–17).
While the ultimate source of auxin in plants is local biosynthesis and metabolism, transport of auxin is essential for communication between tissues and various aspects of morphogenesis (18). The combination of local auxin biosynthesis and auxin transport leads to the creation of auxin gradients, which in turn determine the positioning of new organs and maintenance of stem cell identity (19–21). In addition, rapid changes in auxin concentration, caused by environmental factors, can mediate the physiological and molecular responses of plants to external factors, such as nutrient availability, shade avoidance, wounding, and infection (9, 18, 22–27).
Although the bulk transport of auxin to distant tissues occurs passively through vascular tissues, fine-tuning and patterning are achieved by active transport. Such directional transport of auxin is largely mediated by efflux carrier proteins known as PINs that are polarly localized on the cell membranes (18, 28, 29). Consequently, auxin flows in a “reverse-fountain” pattern that is sustained by coordinated PIN activity and local auxin biosynthesis. The reverse-fountain pattern ensures that the auxin that travels downward through the vascular tissue cells is redirected sideways and upward after passing through the SCN and columella. Finally, it is transported back into the meristem and reinforces the auxin maximum. Through mathematical modeling, it has been demonstrated that PIN-based polar auxin transport in combination with local auxin biosynthesis is sufficient to explain such processes as vascular venation patterning, embryo axis formation, and recovery from wounding (30–32).
To obtain the plant’s highly intricate tissue patterns and precisely defined cell identities, phytohormones are known to be in constant cross-talk with other molecular signaling components (33–35). ERF115, a member of the plant ethylene response factor (ERF) transcription factor family, was initially identified as a regulator of QC stem cell division (36). Aside from its role in regulating the division of slowly dividing QC stem cells, ERF115 is also a central regulator of plant regeneration responses (37). Various modes of wounding, such as mechanical removal of root meristems, DNA damage-induced stem cell death, and laser ablation, have been found to trigger a rapid transcriptional activation of ERF115. Expression of ERF115 following meristematic cell death was found to be essential for the generation of new tissue files, which enables replacement of the damaged cells by new cells (37, 38). During recovery from stem cell ablation or root tip excision, ERF115 interacts with the RBR-SCR signaling network to regulate stem cell division and the response to environmental stress (39). In addition, ERF115 activity was found to be controlled through its interaction with a GRAS transcription factor, PAT1. Strikingly, co-overexpression of ERF115-PAT1 results in hyperproliferation, triggering spontaneous callus formation (37).
Along with the identification of ERF115-interacting proteins, several studies have proposed other mechanisms to explain how cell death induces ERF115. A jasmonate signaling network acting in synergy with auxin was reported to cause ERF115 induction upon wounding or nematode infection (39). Furthermore, oxygen species were shown to control the expression of ERF115 for maintaining the stem cell division and differentiation balance (40).
Here we studied the role of ERF115 activation in endodermal cells following DNA damage-induced vascular stem cell death and its potential interplay with auxin. We found that stem cell death triggers an accumulation of auxin around the wound, due mainly to interruption of auxin transport. Although not the initial trigger for ERF115 expression, auxin accumulation is required to sustain ERF115 activity, which confers an organizing center capacity to the endodermal cells, contributing to successful replenishment of the damaged SCN.
Results
The Endodermis Contributes Directly to Replenishment of the Vasculature in an ERF115-Dependent Manner.
It has been previously observed that the killing of vascular cells through the administration of the DNA damage-inducing drug bleomycin (BLM) triggers the transcriptional induction of ERF115 in QC and endodermal cells (36). ERF115 expression was found to correlate with the occurrence of endodermal periclinal cell divisions, indicative of a role of endodermal cells in repopulating the damaged stem cell population. To visualize the contribution of the endodermal formative divisions in the replenishment of the SCN in real time, a lineage tracking tool was used to fluorescently label all endodermal cells and their daughters using a dexamethasone (DEX)-inducible CFP gene driven by the endodermis-specific SCR promoter, pSCR:CRE_GR p35S loxp-tOCS-loxp-CFP (23). Following induction, the CFP signal was restricted to the QC and endodermal cells in both wild type and the dominant-negative line overexpressing ERF115 fused to the SUPERMAN REPRESSOR DOMAIN (35S:ERF115-SRDX) (Fig. 1A). In the absence of stem cell death, the CFP signal expanded only to the young cortex cells, presumably due to labeling of cortex/endodermis initials at the initial induction (SI Appendix, Fig. S1 A and B). In contrast, the seedlings recovering from BLM-induced vascular cell death displayed penetration of the CFP signal within the vascular bundle, indicating that the newly formed vascular cells originate from the endodermis and QC (Fig. 1A). However, CFP penetration was not observed in the 35S:ERF115-SRDX background, and CFP-labeled endodermis/cortex files finally zipped together due to the collapse of the vascular bundle (Fig. 1A).
Fig. 1.
To determine the endodermal contribution to the replenishment of the SCN, we expressed the dominant-negative ERF115-SRDX fusion from the endodermis-specific EN7 promoter. We verified that the EN7 promoter was not ectopically induced by BLM treatment (SI Appendix, Fig. S1C). Despite showing no effect on total root elongation (SI Appendix, Fig. S1E), specific suppression of ERF115 activity in the endodermis resulted in a substantial reduction in recovery success following BLM treatment (59% in wild type vs. 21% and 28% in two independent EN7:ERF115-SRDX lines) (Fig. 1B). The level of recovery was much like that seen in the 35S:ERF115-SRDX line, suggesting that the endodermal cells are major contributors to the replenishment process under the tested conditions. Interestingly, in contrast to the 35S:ERF115-SRDX line, the EN7:ERF115-SRDX lines displayed a recovery rate following root tip excision similar to that of wild-type plants (Fig. 1C), suggesting that ERF115 activity in the endodermis is not essential for recovery from root tip excision. The erf115-1 T-DNA mutant did not differ from wild type with respect to BLM recovery performance, suggesting functional redundancy among genes closely related to ERF115 (SI Appendix, Fig. S1F).
Wound-Induced Auxin Accumulation Grants the Endodermis at Least Partial QC Identity.
It has been previously reported that on DNA damage-triggered stem cell death, genes associated with QC cell identity, such as WOX5 and AGL42, become ectopically induced around the dead cells (36, 41). In accordance with these reports, we found that the expression of WOX5 was induced upon BLM treatment (Fig. 1 D and E), suggesting a potential ectopic gain of QC identity, predominantly in the endodermis. The ectopic WOX5 induction was strongly suppressed in the 35S:ERF115-SRDX line, illustrating the involvement of ERF115 in attaining QC cell fate. The lack of endodermal expression of QC25:GUS, an independent marker of QC fate, suggests that the endodermal cells might acquire only partial QC identity following BLM treatment (SI Appendix, Fig. S1D).
We reasoned that ectopic WOX5 expression might be indicative of a change in the auxin maximum within the root tip, and thus investigated the changes in auxin dynamics and transcriptional response during BLM recovery. After 24 h of BLM treatment, the DR5:VENUS-NLS reporter revealed activation of the transcriptional auxin response around and above the cell death zone, which became more pronounced after 24 h of recovery on BLM-free medium (Fig. 2A). We next investigated the change in auxin input using the ratiometric R2D2 reporter, for which high and low red:green ratios indicate relative high and low auxin accumulation, respectively (42). Ratiometric quantification obtained from BLM-treated R2D2 seedlings revealed that at 24 h posttreatment, auxin accumulated most pronouncedly in the vasculature and endodermal cells (Fig. 2C and Movies S1 and S2). These results suggest that in addition to the activation of auxin-responsive genes, as indicated by the DR5:VENUS-NLS reporter, there is an increase in auxin itself. Interestingly, the R2D2 data suggest that the greatest fold change in auxin concentration occurred in the endodermal cells in positions 2 to 6 (1 being closest to the QC), corresponding to the immediate vicinity of the dead vascular stem cells (Fig. 2D). Despite only a minor difference in root length (SI Appendix, Fig. S2B), BLM treatment of auxin receptor mutants tir1-1 and tir1-1afb2-3 revealed a significantly lower recovery rate in both mutants (67% and 54%, respectively) compared with wild type (92%), suggesting that receptor-mediated auxin perception is essential during BLM recovery.
Movie S1.
Time-lapse movie of the R2D2 reporter line stained with SR2200 dye during BLM treatment. The seedling was pre-treated with BLM for 8 h before imaging was started. The root tip tracking algorithm used here was described previously (12).
Movie S2.
Time-lapse movie of the PI-stained R2D2 reporter line during BLM treatment. The seedling was pre-treated with BLM for 10 h before imaging was started. The root tip tracking algorithm used here was described previously (12).
Fig. 2.
ERF115 Activation and Auxin Signaling Are Codependent.
The observed accumulation of auxin around the dead cells, reminiscent of the ERF115 expression pattern, suggests that auxin might be a main trigger for ERF115 induction following vascular cell death. A recent study has shown that methyl jasmonate (MeJA) and auxin work in synergy to induce ERF115 in protoxylem and QC cells in a manner depending on the MeJA receptor CORONATINE INSENSITIVE 1 (COI1) (39). Independently, it has been reported that ERF115 expression can be induced by hydrogen peroxide (H2O2) (40). We observed that in the absence of BLM, ERF115 expression induced by the auxin indole-3-acetic acid (IAA), a combination of IAA and MeJA, or H2O2 treatment remained restricted to the two protoxylem strands and QC cells (SI Appendix, Fig. S3 A–C). These results suggest that auxin, MeJA, or H2O2 by itself is not sufficient to induce ERF115 as broadly as after cell death, which likely also requires an as-yet unidentified signal originating from the dead cells. Although we have confirmed that the protoxylem induction pattern depends on the COI1 receptor, BLM-induced ERF115 expression does not, as the ERF115:GFP-GUS reporter was equally induced around the cell death zone in the wild type and coi1-2 receptor mutant background (SI Appendix, Fig. S3B). This result indicates that COI1-mediated JA perception is not necessary for ERF115 expression on BLM-induced cell death.
In contrast to MeJA, auxin appears to be essential for both the ERF115 expression pattern and the stem cell recovery process. BLM recovery on medium with kynurenine and yucasin, chemical inhibitors of auxin biosynthesis, caused a reduction in ERF115 accumulation and interfered with the root tip regeneration process, resulting in collapsed meristems (Fig. 3 A and B). Inversely, recovery on medium with 1-naphthaleneacetic acid (NAA) caused a clear thickening of the vascular bundle along with a seemingly increased number of ERF115-expressing cells (Fig. 3C). Taken together, these results suggest that while auxin is not the main trigger of ERF115 following BLM-induced cell death, it is necessary to maintain ERF115 expression once induced.
Fig. 3.
To investigate whether ERF115 in turn could be involved in the activation of auxin signaling, we introduced the DR5:GFP reporter into the 35S:ERF115-SRDX background. In this background, following prior treatment for 24 h with BLM, the DR5:GFP signal was strongly reduced during the recovery on BLM-free medium (Fig. 3D) compared with that in wild type plants (Fig. 3E). Recovery on kynurenine and yucasin resulted in a similar absence of auxin signaling (Fig. 3F), suggesting that both ERF115 activity and auxin biosynthesis are necessary for the activation of auxin signaling during stem cell replenishment.
BLM-Induced Stem Cell Death Obstructs Auxin Transport, Causing Auxin Accumulation without Activating Auxin Biosynthesis.
Since treatment with auxin biosynthetic inhibitors impaired DR5:GFP accumulation after BLM treatment and resulted in meristem collapse, we reasoned that auxin biosynthesis might be activated around the dead cells. It was previously shown that the auxin biosynthesis gene YUCCA9 is rapidly induced on root tip excision (43). In contrast, the YUC9:VENUS reporter line revealed no apparent transcriptional induction after 24 h of BLM treatment or 24 h after recovery (Fig. 4A). Similarly, TAA1, a major auxin biosynthetic gene with expression in the SCN, was not induced, as revealed by the TAA1:TAA1-GFP reporter line (Fig. 4B).
Fig. 4.
Similarly, a transcriptome dataset obtained from BLM-treated root tips (Dataset S1) showed no significantly up-regulated transcripts associated with the auxin biosynthesis pathway (Fig. 4C). Furthermore, free and conjugated IAA levels in root tips did not change significantly after BLM treatment (Fig. 4D). This led us to hypothesize that the observed auxin accumulation might be sourced from already established centers of auxin biosynthesis. Interestingly, transcript levels for auxin transport genes PIN1, PIN4, and LAX2 were significantly down-regulated following BLM treatment, pointing to altered auxin transport (Fig. 4C). Imaging of translational reporter lines for PIN efflux proteins revealed a general absence of PIN1 and PIN3 at the position of dead cells, with an additional 30% down-regulation of PIN1 along the entire meristem. The PIN4 signal also showed a 13% decrease; however, owing to the high variability in this specific reporter line, the difference was not significant (SI Appendix, Fig. S4 A–D). PIN2 remained largely unchanged (SI Appendix, Fig. S4B).
To test whether the disruption of PIN-mediated auxin transport routes caused by cell death could explain the auxin accumulation in the vasculature and endodermal cells, we used an in silico model of polar auxin transport. Ten individual cell death patterns inferred from confocal images of BLM-treated roots were mimicked in silico by removing all auxin flows into and from the dead cells (SI Appendix, Fig. S5B). The model parameters were also adjusted for PIN1 down-regulation. Accumulation of auxin above and around the dead cells was predicted by the computational model in all different cell death patterns. Interestingly, the exact pattern of auxin accumulation tended to differ slightly among varying cell death patterns (SI Appendix, Fig. S4). Averaging the data obtained from 10 simulated cell death patterns revealed the greatest auxin accumulation in the vasculature and endodermis (Fig. 4E). The fold changes in the predicted auxin concentrations were in striking agreement with the auxin accumulation data obtained from the in vivo R2D2 reporter line (Fig. 2 C and D).
To study the contribution of the down-regulation in PINs, we recalculated the steady-state solutions by assuming that PIN levels were unchanged. Interestingly, the pattern of auxin accumulation was very similar to that obtained by assuming PIN down-regulation, but the fold changes in auxin ratios were reduced (SI Appendix, Fig. S6 A and B). Our modeling results indicate that simply omitting the dead cells as components of auxin flux is sufficient to recreate the experimentally observed pattern of auxin accumulation. Down-regulation of PIN-mediated rootward auxin transport likely contributes to the increased intensity of auxin accumulation. Taken together, these results suggest that disruption of auxin transport is likely one of the major dynamics contributing to the auxin accumulation observed after stem cell death.
ERF115 Boosts the Ability of Auxin Accumulation to Grant Stem Cell Identity and Results in Abundant Formative Cell Divisions.
To study the effect of ERF115 on auxin accumulation, seedlings were grown on medium supplemented with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) and analyzed at 5 and 14 d after germination. In addition to the appearance of QC markers in the endodermis, germination on NPA was reported to cause the expansion of the columella fate domain along the root cap, as indicated by accumulation of starch granules (44). After 5 d of growth on NPA, expansion of the starch granule region was not changed in the 35S:ERF115-SRDX plants compared with the wild type, but was greatly enhanced in the 35S:ERF115 plants (Fig. 5A). Specific expression of ERF115 in the endodermis (EN7:ERF115) was sufficient to mimic this phenotype (Fig. 5A and SI Appendix, Fig. S7A), suggesting that ERF115 enhances the effect of auxin accumulation in the endodermis to induce stem cell divisions in the surrounding cell files. NPA treatment for 2 wk caused the formation of thick stubby roots in the 35S:ERF115 line (Fig. 5D) compared with the wild type (Fig. 5C). Images from transverse microtome sections revealed massive cell proliferation in the 35S:ERF115 line and to a lesser extent in the EN7:ERF115 line compared with the wild type (Fig. 5B). In contrast, the 35S:ERF115-SRDX line responded to NPA treatment similarly to wild type (Fig. 5 A and B). These results suggest that ERF115 expression increases the number of formative divisions triggered as a result of auxin accumulation. ERF115 expression in the endodermis is sufficient to enhance its ability to organize stem cell fates in the surrounding cell files on auxin accumulation (Fig. 5 A and B). The similar response to NPA treatment in the 35S:ERF115-SRDX line and wild-type plants might indicate that ERF115 signaling is not required for the lateral root cap cells to acquire starch granules.
Fig. 5.
To understand how ERF115 might regulate the response to auxin accumulation caused by DNA damage-induced cell death, we looked at the components of auxin signaling differentially regulated in our BLM transcriptome dataset. Among these were several members of the AUX/IAA (IAA1, 5, 13, 19, 20, 29, 33) and AUXIN RESPONSE FACTOR (ARF3, 5, 8) gene families that regulate TIR1/AFB receptor- mediated auxin signaling (Fig. 6 A and B) (11). Notably, ARF5/MP was the only ARF significantly induced by both BLM treatment (fold change = 1.62; FDR = 1,44e-02) and ERF115 overexpression (fold change = 1.53; P = 4,5e-03) (Dataset S1). Bioinformatic analysis based on previously published DNA affinity purification sequencing (DAP-seq) data (45) suggested that the upstream genomic regions of BLM-induced and -repressed genes (Dataset S1) are significantly enriched and depleted for ARF5/MP-binding regions, respectively (SI Appendix, Table S3). Therefore, we reasoned that the activation of auxin signaling on auxin accumulation after BLM treatment and during recovery might be regulated, at least in part, by ARF5/MP. The DAP-seq data indicated two general AP2/ERF-binding regions (−2,753 to −2,543 bp and −2,293 to −2,093 bp upstream of the ARF5/MP start codon) that overlapped with a DNase hypersensitive (DHS) site (−2,793 to −2,643 bp). This DHS region also coincided with one of two putative ERF115-binding regions (−2,950 to −2,487 bp) annotated by TChAP-seq (Fig. 6C) (45, 46).
Fig. 6.
Next, we scanned the ARF5/MP upstream region with the position weight matrix for ERF115 (SI Appendix, Fig. S7B) according to O’Malley et al. (47), which revealed two partially overlapping ERF115-binding regions at −2,753 to −2,737 bp (P = 3.47e-05) and −2,745 to −2,729 bp (P = 8.73e-05) upstream (Fig. 6D). These data suggested that ARF5/MP might be a direct transcriptional target of ERF115; therefore, we asked whether the absence of auxin signaling activation in BLM-treated 35S:ERF115-SRDX seedlings could be explained by a reduction in ARF5/MP levels. qRT-PCR analysis revealed lower ARF5/MP expression under control conditions in 35S:ERF115-SRDX seedlings, and no induction was observed on BLM treatment (Fig. 6D). Confocal imaging of an MP:MP-GFP translational reporter revealed greatly enhanced ARF5/MP accumulation in the 35S:ERF115 background under control conditions, after BLM treatment, and after 24 h of recovery. Reciprocally, the MP:MP-GFP signal was reduced in the 35S:ERF115-SRDX line (Fig. 6E). Furthermore, there was a significant decrease in NPA-induced starch granule accumulation among seedlings hemizygous for the mpB4149 mutation compared with wild-type seedlings, pointing to the need for MP/ARF5 in the response to auxin accumulation (Fig. 6F). Taken together, these results suggest that the activation of auxin signaling on BLM-induced cell death might be regulated, at least in part, by ARF5/MP in an ERF115-dependent manner.
Discussion
ERF115 has previously been identified as a rate-limiting factor for the proliferation of QC stem cells, which are marked by an auxin maximum (36). In addition, ERF115 activity was found to be induced within the root meristem on wounding by tip excision or DNA damage and to be essential for the root regeneration process following such wounding (37). Here we report that induction of ERF115 by cell death acts as a modulator within regions of developmental or wound-induced auxin accumulation to confer the formative capacity necessary for regeneration.
Using a lineage tracking tool, we showed that the replenishment of vascular stem cells that have undergone DNA damage-induced cell death is driven predominantly by endodermal cells undergoing formative divisions. However, since the SCR promoter used in the lineage tracking line also labels the QC cells, a contribution from the QC cells cannot be excluded. In the ERF115-SRDX dominant-negative background, the CFP-labeled endodermis/cortex cell files were zipped together following collapse of the damaged vascular cells. Furthermore, suppression of ERF115 activity specifically in the endodermis impaired recovery, suggesting that endodermal ERF115-dependent formative divisions are essential for successful replenishment of vascular cells. This regenerative program is likely linked to the type of cell death. With the vascular stem cells characteristically more sensitive to DNA damage, resulting in severe vascular cell death, endodermal cells appear to be the most likely tissue to initiate the recovery of vascular cell files. In contrast, Efroni et al. (23) reported that on removal of the SCN by root tip excision, an inside-out mode of regeneration occurs, in which the new SCN is formed mainly by the surviving vascular cells. In this mode of regeneration, the endodermal cells contribute mostly to the epidermis and lateral root cap. Similarly, in the case of targeted cell elimination by laser ablation, the dead cells were shown to be replaced exclusively by their inward neighbors (38). Taken together, these findings suggest that the mode of regeneration can adapt to meet the specific regenerative requirements created by different types and intensities of injury.
It has been previously shown that spatially altering auxin maxima through the application of polar auxin transport inhibitors triggers the QC fate gain in the endodermis and in turn creates stem cell patterns in the surrounding cell layers (44, 49). Based on these findings, a model has been proposed stating that meristematic endodermal cells are competent to become QC stem cells, and that the location of the actual QC is determined by that of the auxin maximum. In turn, the QC cells maintain a functional SCN around them that is essential for indeterminate growth. Our work indicates that the model proposed by Sabatini et al. (44, 49) can be extended from developmental conditions to regeneration processes. At least in the context of BLM-induced vascular cell death, we showed that auxin accumulates in the vascular and endodermal cells after BLM treatment. The auxin receptor mutants tir1-1 and tir1-1afb2-3 have significantly decreased BLM recovery rates, highlighting the importance of receptor-mediated perception of the auxin accumulation following BLM treatment. This auxin accumulation is correlated with ERF115-dependent ectopic expression of WOX5:GFP predominantly in the endodermal cell file, but not of QC25:GUS, suggesting at least a partial gain of QC cell identity of the endodermal cells positioned directly next to the dead vascular cells. Reciprocally, mimicking auxin accumulation in undamaged roots by NPA treatment in combination with ubiquitous or endodermis-specific ERF115 overexpression results in extensive formative divisions in the vasculature and ground tissue and expansion of the columella cell fate region.
These observations suggest that the ectopic (partial) gain of the QC fate by the endodermis observed during BLM recovery is a direct result of the combined ERF115 activation and auxin accumulation in the endodermis. This in turn confers a formative capacity to the endodermis that is necessary for successful regeneration from DNA damage-induced stem cell death. The accumulation of auxin in the endodermis is likely temporary, since the regeneration process reestablishes the auxin flow. This temporary auxin accumulation is insufficient for the full realization of QC attributes, such as the ectopic formation of columella starch granules in the epidermis observed after continuous auxin accumulation by NPA treatment.
Activation of auxin biosynthesis during the recovery from root tip excision and de novo root organogenesis has been reported previously (43, 50, 51). However, BLM-induced stem cell death does not cause up-regulation of auxin biosynthetic genes, as suggested by reporter lines and transcriptome data. Nevertheless, chemical inhibition of auxin biosynthesis impairs the accumulation of auxin and ERF115 expression after cell death. This suggests that preestablished sites of auxin biosynthesis likely provide the auxin that accumulates around the dead cells. Therefore, we reason that a perturbed polar auxin transport might be responsible for the observed auxin accumulation. In line with earlier reports (10), an obstruction in auxin transport was suggested by the disappearance of PIN1 and PIN3 at the positions of dead cells following laser ablation of QC cells or root tip excision. Indeed, simulating stem cell death in an established in silico model of auxin transport predicted an accumulation of auxin around the dead cells, in line with the observed in vivo data. Taken together, these data suggest that on DNA damage, the dead stem cells might obstruct the normal auxin flow, causing auxin accumulation in the surrounding tissues.
This raises a very interesting question: how does the root know when to activate auxin biosynthesis? We hypothesize that the key difference might be the degree to which auxin transport is blocked. Throughout development, organ formation occurs at sites of persistent auxin accumulation formed by polar auxin transport, such as shoot primordia, floral meristems, and lateral roots (52–56). In this hypothesis, auxin biosynthesis is induced only in the case of de novo organ formation, such as de novo root regeneration from an excised root or cotyledon. On root tip excision, the flow of auxin through the vasculature is abruptly and completely blocked, which causes auxin to accumulate persistently at the terminal point of the remaining vasculature. This abrupt accumulation could be the cue for de novo generation of the new tip by reactivation of local auxin biosynthesis. This would lead to determination of a new maximum and regeneration of the root tip, the major structure redirecting auxin flow upward to the epidermis and then back into the meristem. In contrast, DNA damage-induced stem cell death causes a partial and gradual obstruction of auxin transport as stem cells die in a progressive manner (Movie S2), with accumulation below the threshold for triggering de novo biosynthesis. As long as the auxin accumulation intensity is below the necessary threshold, de novo creation of a stem cell niche and root tip is not triggered; instead, replenishment from the surviving nearby tissues by formative divisions is sufficient to restore the auxin flux.
To understand how ERF115 might regulate the auxin response, we analyzed the expression of core regulators of auxin signaling on BLM treatment. Interestingly, the only ARF induced by BLM treatment was ARF5/MP, a major regulator of embryonic polarity, shoot apical meristem primordia, lateral organ, and vascular tissue development (13–17, 52, 53). BLM-induced genes identified via RNA-seq were significantly enriched in ARF5/MP-binding regions, suggesting that ARF5/MP might play an essential role in the activation of auxin signaling. Moreover, expression of ARF5/MP on BLM treatment was dependent on ERF115 activity, and the promoter region of ARF5/MP contains a putative ERF115-binding region according to two independent datasets (45, 46). These data suggest that ERF115 is a wound-inducible modulator of auxin signaling, possibly by acting upstream of ARF5/MP, feeding wounding input into auxin-mediated developmental processes, such as tissue patterning and organ formation. Since our data also show that ERF115 expression is dependent on the presence of auxin, cell death-induced auxin accumulation by obstruction of polar transport simultaneously maintains ERF115 expression and induces regenerative divisions in a synergistic manner.
Materials and Methods
Plant Materials and Growth Conditions.
pSCR:CRE-GR p35S loxp-tOCS-loxp-CFP seeds were kindly provided by Kenneth Birnbaum, New York University (23). DR5:GFP, PIN1:PIN1-GFP, PIN2:PIN2-GFP, and PIN3:PIN3-GFP reporter lines were obtained from the Arabidopsis Biological Resource Center. The R2D2 reporter line has been described previously (42). For the EN7:ERF115 and EN7:ERF115-SRDX lines, the genomic ERF115 sequence with or without an SRDX domain was cloned with the EN7 promoter into the pB7m24GW,3 destination vector and selected for Basta resistance. The WOX5-NLS/GFP-GUS and ERF115:NLS/GFP-GUS reporter lines have been described previously (36, 37). Plants were grown under a long-day regime (16-h light/8-h dark) on agar-solidified culture medium (Murashige and Skoog medium, 10 g/L saccharose, 4.3 g/L 2-(N-morpholino) ethanesulfonic acid, and 0.8% plant tissue culture agar) at 21 °C.
Study protocols are described more detail in SI Appendix.
Data Availability Statement.
RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE139715) (57). All seeds and vectors are freely available from the corresponding author on request. Protocol details are described in the SI Appendix.
Data Availability
Data deposition: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (accession no. GSE139715).
Acknowledgments
We thank Annick Bleys for help with manuscript preparation, Sevgi Öden and Tim Willems for technical assistance, and Veronique Storme for biostatistical analysis and guidance. This work was supported by a grant from the Research Foundation-Flanders (G007218N). J.H. is indebted to the Research Foundation-Flanders for a postdoctoral fellowship. Mathematical modeling was supported by the Russian Science Foundation (18-74-10008), and sequence analysis was supported by the Ministry of Science and Higher Education of the Russian Federation (0324–2019-0040-С-01). RNA-seq library preparation and sequencing were performed by the VIB Nucleomics Core (www.nucleomics.be).
Supporting Information
Appendix (PDF)
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Dataset_S01 (XLSX)
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Movie S1.
Time-lapse movie of the R2D2 reporter line stained with SR2200 dye during BLM treatment. The seedling was pre-treated with BLM for 8 h before imaging was started. The root tip tracking algorithm used here was described previously (12).
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- 1.56 MB
Movie S2.
Time-lapse movie of the PI-stained R2D2 reporter line during BLM treatment. The seedling was pre-treated with BLM for 10 h before imaging was started. The root tip tracking algorithm used here was described previously (12).
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- 2.14 MB
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© 2020. Published under the PNAS license.
Data Availability
Data deposition: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (accession no. GSE139715).
Submission history
Published online: June 29, 2020
Published in issue: July 14, 2020
Keywords
Acknowledgments
We thank Annick Bleys for help with manuscript preparation, Sevgi Öden and Tim Willems for technical assistance, and Veronique Storme for biostatistical analysis and guidance. This work was supported by a grant from the Research Foundation-Flanders (G007218N). J.H. is indebted to the Research Foundation-Flanders for a postdoctoral fellowship. Mathematical modeling was supported by the Russian Science Foundation (18-74-10008), and sequence analysis was supported by the Ministry of Science and Higher Education of the Russian Federation (0324–2019-0040-С-01). RNA-seq library preparation and sequencing were performed by the VIB Nucleomics Core (www.nucleomics.be).
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This article is a PNAS Direct Submission.
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The authors declare no competing interest.
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Rocks in the auxin stream: Wound-induced auxin accumulation and ERF115 expression synergistically drive stem cell regeneration, Proc. Natl. Acad. Sci. U.S.A.
117 (28) 16667-16677,
https://doi.org/10.1073/pnas.2006620117
(2020).
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