Mechanisms of stress response in the root stem cell niche.

As plants are sessile organisms unable to escape from environmental hazards, they need to adapt for survival. The stem cell niche in the root apical meristem is particularly sensitive to DNA damage induced by environmental stresses such as chilling, flooding, wounding, UV, and irradiation. DNA damage has been proven to cause stem cell death, with stele stem cells being the most vulnerable. Stress also induces the division of quiescent center cells. Both reactions disturb the structure and activity of the root stem cell niche temporarily; however, this preserves root meristem integrity and function in the long term. Plants have evolved many mechanisms that ensure stem cell niche maintenance, recovery, and acclimation, allowing them to survive in a changing environment. Here, we provide an overview of the cellular and molecular aspects of stress responses in the root stem cell niche.

Introductionroot stem cell niche structure and dynamics

The root apical meristem (RAM), consisting of proliferating cells at the root tip, maintains root growth and development throughout the plant life cycle (reviewed by Jiang and Feldman, 2005). The RAM harbors a stem cell niche, the specific microenvironment that provides signals that block cell differentiation (reviewed by Laux, 2003; Stahl and Simon, 2005; Aichinger ; Perilli ). Stem cells (initials) in the root are maintained around a group of mitotically inert quiescent center (QC) cells. The QC of angiosperms varies in size among different plant species, from as few as four cells in Arabidopsis (Arabidopsis thaliana) to hundreds of cells in maize (Zea mays) (Clowes, 1956, 1958; Dolan ).

Mitotically active initials, capable of unlimited self-renewal and giving rise to differentiating descendants, circumscribe the QC. Figure 1 shows the classical stem cell niche architecture of A. thaliana. Stele stem cells (SSCs) located proximally to the QC generate the stele; stem cells lateral to the QC (CESCs) form the endodermis and cortex, with adjacent stem cells forming the epidermis and lateral root cap (ESCs); and columella stem cells (CSCs) are located below the QC (Dolan ). Root stem cells divide asymmetrically (formative cell division), giving rise to a new stem cell plus a daughter cell that differentiates after a limited number of symmetric cell divisions (Stahl and Simon, 2005; De Smet and Beeckman, 2011). CESCs show a division pattern comprising first anticlinal symmetric and second periclinal asymmetric divisions. ESCs undergo two variants of formative cell division: they first divide periclinally, producing a new ESC and a lateral root cell daughter cell; the new ESC then divides anticlinally, producing the epidermis daughter cell (Willemsen ; De Smet and Beeckman, 2011).

Fig. 1.

The root stem cell niche in Arabidopsis.

Experiments with QC laser ablation (Xu ) showed that positional information rather than QC cell identity specifies the location and size of the stem cell niche. Thus, the structure of the stem cell niche changes during root development despite stereotypical cell division patterns. In some plants, such as Sinapis alba, Vicia faba, and Malva sylvestris, the QC is absent from the root apex at germination and is organized later during root growth (Clowes, 1958, 1961, 1978). In other plants, including Arabidopsis, the QC is specified during embryogenesis but can be quickly restored upon injury after germination. The duration of the QC mitotic cycle is long but not infinite (Rahni and Birnbaum, 2019). QC daughter cells usually become CSCs (Cruz-Ramirez et al., 2013); however, clonal analysis shows that QC cells can potentially replace all stem cells in the meristem (Kidner ). Cell divisions in the QC accelerate upon meristem aging, disturbing the stem cell niche structure (Timilsina ; Wein ). It is unclear whether meristem aging is part of an inherent developmental program or is the consequence of accumulating stress.

Specific cellular stress responses in the root stem cell niche

Upon severe or prolonged stress, the stem cell niche shows two specific cellular responses: activation of QC cell divisions and death of root stem cells (Fig. 2A). Clowes described QC activation upon high dosage irradiation in the middle of the 20th century (Clowes, 1959, 1963). However, the specific vulnerability of root stem cells and their programmed cell death (PCD) as a result of various stresses has been discovered only recently (Fulcher and Sablowski, 2009; Furukawa ; Heyman ; Hong ).

Fig. 2.

Pathways of the stem cell niche response to stress. The summary (A) and the details of DNA damage (B) and hormonal (C) stress responses. The processes are in boxes; the proteins are colored blue. SA, salicylic acid; CK, cytokinins; JA, jasmonic acid; ABA, abscisic acid; BR, brassinosteroids; Eth, ethylene; ROS, reactive oxygen species.

DNA damage-mediated death of root stem cells is triggered by UVB and γ irradiation (Furukawa ), X-rays, and radiomimetic drugs such as bleomycin and zeocin (Fulcher and Sablowski, 2009; Heyman ). Notably, the PCD response of the root stem cells is cell type specific. SSCs are especially prone to entering the PCD pathway (Fulcher and Sablowski, 2009; Heyman ; Cahner et al., 2020). In addition to SSCs, high-concentration, long-term (24 h) zeocin treatment kills CSCs and QC cells (Fulcher and Sablowski, 2009); bleomycin also triggers death of CSCs (Cahner et al., 2020). Chilling stress induces root stem cell death; however, in most cases, columella stem cell daughters are sacrificed to ensure survival of stem cells (Hong ).

In contrast to stem cells, QC cells are highly tolerant of DNA-damaging agents, dying only after exposure to acute stress (Fulcher and Sablowski, 2009; Furukawa ). Instead, stress signals activate the cell division machinery in the QC, accelerating the cell cycle (reviewed by Heyman ). Thus, the QC serves as a reservoir of cells able to restore root growth in the case of significant damage. Mitotic activation of the QC occurs following root cap cutting (Jiang ; Ivanov ; Bystrova ), chilling stress (Clowes and Stewart, 1967; Barlow and Rathfelder, 1985), flooding-induced hypoxia (Mira ), lead-induced toxicity stress (Kozhevnikova ), and heat exposure (Clowes and Wadekar, 1989; Kidner ; Heyman ).

Sacrificing root stem cells undergoing PCD allows the RAM to survive severe stress (Fulcher and Sablowski, 2009). A plausible role for the stem cell death response is maintaining the genetic material of rapidly dividing cells undamaged in order to sustain tissue patterning. It is likely that for symplastically growing plant tissues, either slowly dividing QC cells or dedifferentiated tissues are best able to replenish stem cells with compromised DNA.

Below, we discuss the mechanisms that provoke stress-induced changes in stem cell niche activity and help this region withstand unfavorable conditions (Fig. 2).

Mechanisms behind root stem cell susceptibility to DNA damage

Accumulating evidence suggests that severe stress leads to DNA fragmentation in root stem cells and their early descendants (Fulcher and Sablowski, 2009; Furukawa ; Mironova and Xu, 2019). DNA breaks in stem cells cause DNA replication stress and are particularly disruptive when a cell undergoes mitosis, leading to chromosomal aberrations and mutations. The root stem cell-specific DNA replication stress mechanism is associated with DNA topoisomerases (Zhang ) (Fig. 2B). DNA TOPOISOMERASE1 (TOP1) is essential for the survival of SSCs, which appear to be particularly sensitive to torsional stress during DNA replication. DNA topoisomerases relax DNA supercoils by introducing temporary single- or double-strand breaks (Champoux, 2001).

Two cell cycle checkpoint kinases, ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR), transmit DNA damage signals in plant cells (Abraham, 2001; Garcia ; Culligan ). ATM responds to double-strand DNA breaks (Bensimon ), while ATR transmits signals about single-strand DNA breaks (Flynn and Zou, 2011). The NAC family transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) is phosphoactivated by ATM to trigger the DNA damage response (Yoshiyama ). In plant stem cells, SOG1 governs the key cell death pathway induced by high-intensity UVB radiation, X-rays, and radiomimetic drugs (Fulcher and Sablowski, 2009; Furukawa ). The SOG1 downstream cascade that triggers cell death remains largely unknown; however, many direct targets of SOG1 have been discovered recently (Ogita ; Ryu ).

Several mechanisms that restrict uncontrolled stem cell death exist. The MEDIATOR (MED) complex subunit (MED18) protects root meristem cells from DNA damage-mediated cell death; med18 mutants show spontaneous death of vascular initials and their daughters (Raya-González ). Besides cell death, the SOG1-dependent pathway also mediates stem cell survival following stress-induced DNA damage by inhibiting cell cycle progression at the G2/M checkpoint (Furukawa ; Yoshiyama ). Temporal cell cycle arrest prevents the mitotic catastrophe that might happen if DNA is left unrepaired. ATR regulates cell cycle arrest at the G2/M checkpoint in response to irradiation (Culligan ) and aluminum (Rounds and Larsen, 2008; Sjogren ). Temporal inhibition of CSC division occurs upon chilling stress; when it finally divides, the CSC daughter tends to undergo PCD (Hong ).

SOG1 targets numerous genes responsible for cell cycle regulation at the G2/M transition, including cyclin-dependent kinase (CDK) inhibitor genes KIP-RELATED PROTEIN 6 (KRP6), SIAMESE-RELATED (SMR) SMR4, 5, 7, and WEE1 (Ogita ). KRP and SMR bind to CDK–cyclin complexes and inhibit their kinase activity (Van Leene ; Yi ). WEE1 kinase phosphorylates and inactivates the CDKs that mediate the G2/M phase cell cycle transition (De Schutter ). Direct targets of SOG1, homologous transcription factor-coding genes ANAC044 and ANAC085, play a crucial role in G2/M cell cycle arrest upon DNA damage response (Takahashi ). Similar to sog1 mutants, the stem cell niche of anac044 and anac085 mutant plants is tolerant to DNA-damaging agents. However, the increased tolerance of anac044/085 mutants is associated with G2/M checkpoint control, rather than through increased DNA repair. ANAC044/ANAC085 are essential in regulating protein accumulation of the R1R2R3-type Myb transcription factors (MYB3R), which mediate G2/M-specific genes expression both positively and negatively (Okumura ). Notably, myb3r3 and myb3r5 mutants, which cannot induce G2/M arrest upon DNA damage response, also show less zeocin-induced stem cell death (Chen ). Interestingly, ANAC044/ANAC085 have been implicated in the regulation of a stress-specific function, mediating heat stress, but not osmotic stress-induced G2/M arrest (Takahashi ).

DNA damage-activated backup plan: QC cell divisions

DNA damage-induced cell death activates regeneration programs in the stem cell niche, consequently inducing divisions in the QC (Heyman , 2014). The transcription factor ETHYLENE RESPONSE FACTOR 115 (ERF115) is the master regulator of damage-induced regenerative processes. Under non-stress conditions, ERF115 is only expressed in dividing QC cells, serving as a rate-limiting regulator of divisions in the QC (Heyman ). Under stress, ERF115 is activated in the QC, around dead cells, and in the endodermis. This activation induces restorative cell divisions (Heyman ; Zhou ; Canher ). ERF115-dependent activation of QC cell division is detected in response to heat stress, wounding, and nematode infection (Heyman ; Zhou ).

ERF115 is not a direct target of SOG1; rather, it is induced in the stem cell niche as a result of SOG1-dependent PCD (Johnson ). ERF109, a close homolog of ERF115, is rapidly induced after cell ablation and triggers ectopic ERF115 expression around the dead cells, activating regeneration processes in the meristem (Heyman ; Zhou ). ERF115 recruits the regulatory circuit SCARECROW (SCR)–SHORT ROOT (SHR)–RETINOBLASTOMA-RELATED (RBR), which guides asymmetric cell division of root stem cells (Paquette and Benfey, 2005; Cruz-Ramírez , 2013). ERF115 binds to and inhibits RBR activity (Zhou ). This blocks RBR–SCR interaction and allows QC cell division. ERF115 also forms a heterodimer with PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1) to mediate restorative cell divisions, for example stem cell niche recovery upon root tip excision (Heyman ). One putative PAT1–ERF115 target is WOUND INDUCED DEDIFFERENTIATION1 (WIND1), a key factor promoting plant cell dedifferentiation (Iwase ; Heyman ). Co-expression of PAT1 with ERF115 hyperinduces WIND1. Another potential target of ERF115 is the AUXIN RESPONSE FACTOR 5 (ARF5), a major regulator of auxin signaling in root development (Canher ). The ARF5 upstream region possesses ERF115-binding sites, and its expression corresponds with the level of ERF115.

Not merely a by-product: the crucial role of ROS in the stem cell niche

Stress-induced changes in stem cell niche activity also rely on changes in the distribution of reactive oxygen species (ROS). A by-product of aerobic metabolism, ROS are highly reactive molecules that can induce DNA damage, protein oxidation, and lipid peroxidation (reviewed by Gill and Tuteja, 2010; Huang ). ROS exist in ionic and molecular states: ionic forms include hydroxyl radicals (OH·) and superoxide anions (O2· –); molecular forms include hydrogen peroxide (H2O2) and singlet oxygen (1O2). An antioxidant system consists of ROS scavenger enzymes and non-enzymatic low molecular weight metabolites [ascorbic acid (ASC), reduced glutathione (GSH), carotenoids, flavonoids, and proline] that counteract uncontrolled oxidation (Schafer and Buettner, 2001; Conklin and Barth, 2004; reviewed in Jiang and Feldman, 2005). Cellular redox potential is determined by the contribution of different redox couples and ROS, and is controlled by a delicate balance between ROS production and scavenging (reviewed by Lee ).

Cellular redox potential plays a critical role in regulating cell proliferation. In the stem cell niche, QC cells have a more highly oxidized status than surrounding stem cells (Jiang ; Jiang and Feldman, 2005), which is essential for maintenance of QC dormancy (reviewed by Huang ; Eljebbawi ). Indeed, miao mutants deficient in the plastid-localized GR2 enzyme, which is part of the plant antioxidant system, exhibit a partial loss of QC identity mediated by a perturbed auxin maximum (Yu ). Knockout of VITAMIN C DEFECTIVE 1 (VTC1), a rate-limiting gene affecting the quantity of ascorbic acid, results in elevated H2O2 levels that increase the number of QC cells and periclinal divisions in the root meristem (Kka ). It is noteworthy that both reducing the oxidative status of the QC and treating roots with exogenous H2O2 lead to QC activation (Jiang ; Kong ).

Stress conditions such as heat, cold, drought, heavy metals, and pathogens rapidly disturb the redox balance by inducing ROS accumulation in plant tissues (Lee ; Kawarazaki ; Kim and Hwang, 2014; Zhao ). While ROS bursts under severe stress conditions can cause intense oxidative stress, sometimes leading to whole-organ death, under moderate stress conditions ROS activate signaling pathways that trigger adaptive stress response programs. A well-described example of ROS-mediated damage and adaptation in the stem cell niche is flooding-induced hypoxia. Maintaining well-balanced, low levels of ROS is crucial for root meristem survival under hypoxic conditions (Sasidharan ). Hypoxia-induced accumulation of ROS and nitric oxide (NO) causes QC cell division and death of meristematic root cells (Mira ). A decline in either O2 or NO leads to expression of core hypoxia genes and hypoxia acclimation (Gibbs , 2018).

Pivotal role of auxin in root stem cell niche maintenance

The auxin concentration maximum defines QC identity and maintains stem cell niche integrity (Jiang and Feldman, 2005). Auxin biosynthesis, conjugation, oxidation, and, most critically, transportation networks work together to generate and support the auxin maximum in the stem cell niche. Maintaining a dynamic balance in auxin patterning helps plants withstand the rigors of environmental stress.

Environmental cues commonly affect root growth plasticity by influencing auxin biosynthesis, transport, and signaling (Pierik and Testerik, 2014; Korver ). Despite different stresses having specific targets in these auxin pathways, sometimes outside the meristem, all of them potentially influence stem cell niche activity to some extent. Stress-induced messages that affect the shoots are delivered to the root meristem by long-distance auxin transport; short-distance auxin transport consequently alters auxin levels in the root stem cell niche. For example, iron deficiency decreases auxin transport from shoots to roots in rice (Sun ). Mathematical modeling suggests that the rate of auxin inflow into the root meristem is a critical parameter affecting the maintenance of the auxin maximum (Mironova ).

QC activation and root meristem exhaustion upon severe stress often correspond to depletion of auxin in the stem cell niche (Fig. 3). A decrease in activity of the auxin response marker DR5 might also indicate the maladaptive status and vulnerability of the stem cell niche to stress. Low-potassium (K+) conditions slightly decrease DR5 signal in the QC, corresponding to an acceleration of QC cell division compared to control conditions; this phenotype is greatly enhanced in the kup9 mutant defective in K+ and auxin efflux from the endoplasmic reticulum (Zhang ). Chilling stress causes a decrease in DR5 activity in the QC, contributing to induction of CSC division and CSC daughter death (Hong ) (Fig. 3A, B). Generally, a decrease in QC-localized DR5 signal corresponds strongly to misexpression of auxin transporters from PIN-FORMED or AUX/LAX families.

Fig. 3.

Auxin dynamics in the stem cell niche in response to stress. (A) Auxin maximum in the QC maintains stem cell niche integrity. (B) Auxin levels in the meristem are depleted in response to different stresses (e.g. chilling stress at 4 °C; Hong ), resulting in loss of QC identity and precocious divisions in the stem cell niche. (C) Re-establishment of the auxin maximum in the QC occurs after chilling stress-specific CSC daughter death (Hong ). (D) Bleomycin-induced cell death of SSCs and their daughters causes auxin accumulation around the wound, activating restorative cell divisions (Canher ).

Local auxin biosynthesis in the stem cell niche has less influence on QC maintenance than PIN-mediated transport, but helps the plant to rapidly enhance auxin levels in the root tip upon stress. Expression of the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) gene encoding an auxin biosynthesis enzyme is enhanced upon Al exposure (L. Yang ), leading to auxin accumulation in the root tip. Auxin biosynthesis via TAA/TAR enzymes is essential for the root meristem response to the stress hormone ethylene (Brumos ). Enhanced auxin biosynthesis rates are also observed at the site of root tip injury (Matosevich ).

Dead cells affect auxin patterning via disruption of PIN-mediated auxin transport routes in the meristem (Canher ). Bleomycin-mediated death of SSCs leads to rapid accumulation of auxin around the dead cells without activating auxin biosynthesis (Fig. 3D). Auxin accumulation in the endodermis promotes replenishment of SSCs via an ERF115-dependent pathway. As another example, DNA damage-induced CSC daughter death partially blocks lateral auxin redistribution in the columella, leading to auxin accumulation in the QC upon chilling stress (Hong ). Intriguingly, plants with sacrificed CSC daughters and boosted auxin levels in the QC not only recover faster from chilling stress but also withstand accompanying freezing, drought, and even genotoxic zeocin treatments better than those plants not sacrificing these cells. Moreover, auxin protects stem cells against zeocin-induced cell death (Hong ).

The stem cell niche does not live by auxin alone: other plant hormones

Although a change in auxin patterning precedes division of the QC cells (Jiang ), the QC is activated by exposure to ethylene (Ortega-Martinez et al., 2007), jasmonic acid (JA) (Zhou ), salicylic acid (SA) (Pasternak ), cytokinin (Zhang ), and brassinosteroids (BRs) (Lozano-Elena ) (Fig. 2C). Cytokinin negatively regulates the auxin influx carrier LAX2 in the meristem, with the lax2 mutant showing reduced auxin levels in the stem cell niche and ectopic divisions in the QC (Zhang ). The morphogenetic role of low-level exogenous SA is determined by its dose-dependent control of auxin transport and biosynthesis (Pasternak ). Exposure to low-level SA leads to stem cell niche enlargement via activation of PIN1 and TAA1 and inhibition of PIN2 and PIN7. Another explanation for hormone-induced division of QC cells is precocious RAM aging. Prolonged treatments with relatively high concentrations of exogenous hormones are typically used to induce division of QC cells, which might be stressful for the stem cell niche. QC cell divisions occur more frequently in aging plants than in younger plants (Timilsina ).

The response and acclimation of the root stem cell niche to stress also rely on hormone-specific effects that are independent of auxin. Restricted ethylene diffusion in compacted soil or upon flooding leads to ethylene accumulation in the root tip, which helps the meristem to adapt to the stress (Hartman ; Pandey ). Ethylene signaling activates NO scavenging by PHYTOGLOBIN1 (PGB1), which is essential for acclimating the meristem to flooding-induced hypoxia (Hartman ). PGB1 reduces NO levels and stabilizes ERFVII transcription factors, which help the stem cell niche to withstand hypoxia. PGB1 is also essential for adaptation to water deficit (Mira ).

Abscisic acid (ABA) has a specific role in the stem cell niche, namely maintaining the stem cell niche in a juvenile state and ensuring QC dormancy (Zhang ). ABA treatment suppresses cell division in the meristem for long periods without loss of meristem function. At least partially, ABA exerts its role on meristematic activity by modulating auxin transportation and signaling (Zhang ; Rowe ; Promchuea ). However, ABA-mediated production of ROS in mitochondria is also crucial for maintaining stem cell niche activity and the auxin response maximum (Z.B. Yang ). Furthermore, SA promotes ROS accumulation in the stem cell niche (Wang ).

It is noteworthy that salt stress initiates an increase in ABA and a decrease in BR signaling in the inner tissues; these events are followed by activation of JA and derepression of BR pathways (Geng ). These observations indirectly support the idea that the QC is temporally protected under early stress response but later on its cells divide to replenish the damaged cells. BRs recruit the BRI1-EMS-SUPPRESSOR 1 (BES1)–BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER (BRAVO)–ERF115 signaling module to control QC cell divisions (Vilarrasa-Blasi ).

Recent studies demonstrated that JA plays a pivotal role in stem cell niche regeneration (Zhou ). Wounding leads to JA accumulation that rapidly induces transcription of ERF109. ERF109 stimulates CYCD6;1 expression in the endodermis and QC, and triggers ERF115 expression in the stele. Methyl jasmonate pre-treatment to induce ERF115 expression before cell ablation promotes faster replenishment of dead cells. JA and auxin synergistically activate the SCR–SHR–RBR pathway to guide restorative cell divisions when roots are cut, penetrate the soil, or are infected with nematodes.

Conclusion

Figure 2 summarizes major pathways of stress-induced responses in the root stem cell niche. This roadmap is certainly incomplete, missing multiple condition-specific crosstalk and feedback routes between the major pathways. For example, H2O2 treatment activates ERF115-mediated QC cell division independently of cell death signaling (Kong ); ERF115 enhances auxin signaling via the ARF5/MP transcription factor, and ARF5/MP, in turn, promotes the ERF115 pathway (Canher ); and reduction of the QC oxidation status corresponds to auxin depletion (Jiang ). DNA damage response, ROS, auxin distribution, the ERF115-mediated cascade, and hormonal signaling are all interconnected, facilitating plant adaptation to numerous adverse conditions. Identifying key components of the root stem cell niche response to stress will help scientists to sustain, select, or bioengineer plants that effectively tolerate particular stresses, thus widening the scope of sustainable agriculture.