Oxygen and reactive oxygen species-dependent regulation of plant growth and development

Abstract

Oxygen and reactive oxygen species (ROS) have been co-opted during evolution into the regulation of plant growth, development, and differentiation. ROS and oxidative signals arising from metabolism or phytohormone-mediated processes control almost every aspect of plant development from seed and bud dormancy, liberation of meristematic cells from the quiescent state, root and shoot growth, and architecture, to flowering and seed production. Moreover, the phytochrome and phytohormone-dependent transmissions of ROS waves are central to the systemic whole plant signaling pathways that integrate root and shoot growth. The sensing of oxygen availability through the PROTEOLYSIS 6 (PRT6) N-degron pathway functions alongside ROS production and signaling but how these pathways interact in developing organs remains poorly understood. Considerable progress has been made in our understanding of the nature of hydrogen peroxide sensors and the role of thiol-dependent signaling networks in the transmission of ROS signals. Reduction/oxidation (redox) changes in the glutathione (GSH) pool, glutaredoxins (GRXs), and thioredoxins (TRXs) are important in the control of growth mediated by phytohormone pathways. Although, it is clear that the redox states of proteins involved in plant growth and development are controlled by the NAD(P)H thioredoxin reductase (NTR)/TRX and reduced GSH/GRX systems of the cytosol, chloroplasts, mitochondria, and nucleus, we have only scratched the surface of this multilayered control and how redox-regulated processes interact with other cell signaling systems.

Oxygen and reactive oxygen species regulate plant growth, development, and differentiation through multiple interlinked signaling pathways.

Advances

• Developmentally regulated hypoxia and reactive oxygen species (ROS) production are key features of the stem cell niches, providing information about stem cell position, the environment, and metabolic state.

• Protein cysteine oxidation is central to oxygen and ROS signaling. However, S-nitrosylation, S-glutathionylation, S-sulfhydration, and S-sulfenylation modifications can occur on the same cysteine. The influence of each modification on stability, localization, and function remains unknown.

• Numerous intersecting ROS signaling pathways are probable and likely depend on the site of ROS production and the nature of the oxidized receptor protein. ROS sensors such as the hydrogen peroxide (H₂O₂)-INDUCED Ca²⁺ INCREASES 1 (HPCA1) leucine rich receptor kinase translate redox signals into protein modifications to regulate signaling cascades. H₂O₂ perception/transduction is dependent on thiol-dependent mechanisms policed by the ferredoxin/thioredoxin (TRX), NAD(P)H TRX reductase C (NTRC), reduced glutathione (GSH), and glutaredoxin (GRX) systems.

• ROS waves transmit redox signals from cell to cell in the apoplast, and probably through plasmodesmata. Long-distance transport of H₂O₂ and other ROS, therefore, appears to be unnecessary. Similarly, contact sites between organelles allow ROS transfer.

• Convergence points for oxygen and ROS signaling occur on proteins such as ROH OF PLANT 2 (ROP2) GTPase,RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), and TRX-h to regulate meristematic activity via TARGET OF RAPAMYCIN (TOR) kinase activity.

Introduction

Molecular oxygen is the central respiratory substrate and thus the heart of energy metabolism. Moreover, plants and other organisms perceive oxygen as a signal that initiates post-translational modifications (PTMs) that effect transcriptional responses (Masson et al., 2019). The availability of oxygen regulates the half-life of transcription factors via the PROTEOLYSIS 6 (PRT6) N-degron pathway, to control multiple aspects of plant development, acclimation, and stress response processes (Considine et al., 2017; Vicente et al., 2017; Weits et al., 2020). In addition, enzymes and other proteins that facilitate the partial reduction of oxygen to generate superoxide, hydrogen peroxide (H₂O₂), or hydroxyl (^.OH) radicals trigger different signaling pathways that generally involve the oxidation and reduction of protein cysteinyl (Cys) thiols, reversibly changing in protein characteristics and functions. These oxidative signals, which arise from the active metabolic state, are referred to as reactive oxygen species (ROS). Like many other important signaling molecules such as nitric oxide (^.NO) and hydrogen sulfide (H₂S), ROS were originally considered to be detrimental to cells. However, the ROS-induced oxidation of cellular components is an important mechanism that triggers cell signaling.

ROS are formed in all cellular compartments (Figure 1), including the mitochondria, chloroplasts, peroxisomes, and plasma membrane (Noctor et al., 2018). Crucially, they provide information on metabolism and the energy state of the different cell compartments and contrary to early concepts, they are the major pro-survival signals in plants (Mittler, 2017). Oxygen availability and ROS thereby serve as important cellular cues, playing key roles in calibrating plant development with environmental change. There is now an extensive literature on the enzymes involved in ROS production and ROS functions in organs such as roots (see, for example, the review by Eljebbawi et al. [2020]). Our aim in the following discussion is to explore how the oxygen/redox interface controls plant growth and development. We highlight recent advances that have facilitated an evolution of current concepts concerning the respective roles of developmental hypoxia and ROS in nearly every aspect of these processes from meristem maintenance to local and systemic signaling systems that facilitate coordinated changes in shoot and root architecture. In addition, we consider the importance of the different oxygen and ROS signaling pathways, the compartments in which oxygen and reduction/oxidation (redox) signals are generated and how each type of signaling pathway may interact with other signaling hubs to achieve the specific and appropriate regulation of plant growth and development.

Figure 1.

Sources and compartmentation of ROS in plants. The photosynthetic electron transport chain in the chloroplasts is composed of two photosystems (PSs): PSI and PSII. Water is split to molecular oxygen at the water splitting site of PSII located in the thylakoid lumen. Molecular oxygen can be reduced to superoxide at several sites in the electron transport chain, but the highest level of superoxide production is on the reducing side of PSI. In addition, energy can be transferred directly from the excited triplet state of chlorophyll to ground state triplet oxygen to generate ¹O₂ at PSII. The fixation of carbon dioxide in the Calvin cycle occurs through the carboxylation of ribulose-1,5-bisphosphate (RuBP), which produces 3-phosphoglycerate (PGA) in the chloroplasts. PGA is converted to triose phosphates (Sugar-P) that are exported from the chloroplasts, retained in the Calvin cycle, or used for the synthesis of starch. Triose phosphates are used in the cytosol to drive various processes, including the generation of reducing power in the form of NAD(P)H used to drive the plasma membrane-localized NADPH oxidases (RBOH), which generate superoxide from molecular oxygen in the apoplast. Reducing power in the form of NADH is used in the mitochondria to drive the respiratory electron transport chain (RETC). Like the photosynthetic electron transport chain, the RETC can reduce molecular oxygen to superoxide. Photorespiration is initiated in the chloroplasts by oxygenation (rather than carboxylation) of RuBP. The oxygenation reaction generates phosphoglycolate, from which glycolate is generated in the chloroplasts and exported to the peroxisomes. Here, the enzyme glycolate oxidase converts glycolate to glyoxylate, which—through further reactions—is converted to glycine. The glycolate oxidase reaction reduces molecular oxygen to H₂O₂ during the conversion of glycolate to glyoxylate. Glycine is imported into the mitochondria, where it is converted to serine in reactions that also generate NADH. Serine is exported from the mitochondria back to the peroxisomes, where it is converted to glycerate. Glycerate is shipped back to the chloroplasts for conversion to PGA, which re-enters that Calvin cycle.

ROS signaling

Superoxide is produced by the reduction of molecular oxygen with a single electron. It has no reactivity to most biological molecules, except for ascorbate, plastocyanin, and ^.NO. It is, however, able to inactivate several enzymes that are important in energy production and amino acid metabolism, such as aconitase in the mitochondrial matrix (Scandroglio et al., 2014). Superoxide radicals are potent inhibitors of many iron (Fe)–S cluster-containing proteins because they can oxidize the [4 Fe–4 S] clusters, causing a release of Fe. The superoxide-dependent inactivation of aconitase leads to enhanced glycolysis relative to oxidative phosphorylation in cellular energy generation. This may occur through the mitochondria to nucleus signaling pathways that activate the expression of a specific set of genes called mitochondrial dysfunction stimulon genes, including those encoding the alternative oxidases, the cytosolic sulfotransferase12 and the NAC DOMAIN-CONTAINING PROTEIN (ANAC)013, through the action of the ANAC013 and ANAC017 transcription factors (Shapiguzov et al., 2019).

The chemical or enzymatic dismutation of superoxide generates H₂O₂, which in the presence of free transition metals can give rise to the highly reactive hydroxyl radical. While H₂O₂ can be removed by enzymes such as ascorbate peroxidase and 2-Cys peroxiredoxins, Cys residues are highly susceptible sites to oxidation by H₂O₂ and reactive nitrogen species (RNS; Akter et al., 2015). Most Cys thiol oxidation takes place through nonenzymatic reactions with ROS/RNS (Yang et al., 2016). Exposure of the redox-sensitive Cys thiols to ROS leads to reversible sulfenic acid (–SOH) formation. The highly reactive –SOH is stabilized by forming a disulfide bond (S–S) with a nearby thiol or a mixed S–S with glutathione (GSH; Zaffagnini et al., 2012). Thiol/disulfide exchange reactions regulate many important redox proteins such as the plasma membrane H₂O₂ receptor, H₂O₂-INDUCED Ca²⁺ INCREASES 1 (HPCA1; Wu et al., 2020). In addition, RNS-mediated S-nitrosylation of proteins such as RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) occurs through a covalent attachment of ^.NO to a Cys residue to form S-nitrosothiol. While S-nitrosylation typically inhibits protein functions, S-sulfhydration through interaction with HS⁻ or H₂S to form a persulfide residue, can also activate proteins (Aroca et al., 2015).

The reversibility of Cys thiol redox PTMs allows Cys to act as a regulatory switch that can alter the interactome by changing enzyme activity, conformational integrity, and protein stability in response to cellular redox state changes (Foyer et al., 2020). H₂O₂ generation and the resultant oxidation of protein thiol groups is thought to be the central mechanism by which redox signals are integrated into cellular signal transduction pathways (Foyer and Noctor, 2016; Noctor et al., 2018). Such thiol-based signal transduction mechanisms are crucial to energy-generating organelles, such as mitochondria and chloroplasts that experience fluctuating energy inputs with the consequent generation of strong oxidants and reductants. While chloroplasts are the major source of ROS in photosynthesizing cells, ROS are also produced in mitochondria and other cellular compartments (Figure 1). ROS production in these organelles is crucial for the transfer of information concerning the redox and energy state of each compartment to the nucleus. Moreover, ROS signals are specifically generated in the apoplast by the activation of RBOH NADPH oxidases, resulting from the recognition of any physical or chemical change in the cellular environment that is perceived by the plasma membrane (Gilroy et al., 2014). Plant RBOH, which catalyze apoplastic superoxide formation by transferring an electron to oxygen, have a catalytic subunit that is homologous to mammalian NADPH oxidases. Activation of these enzymes depends on phosphorylation and calcium-binding to the EF-hands domains in the N-terminal region (Kadota et al., 2015). Class III heme peroxidases are also potentially important sources of superoxide and H₂O₂ in the apoplast (Janku et al., 2019). Although the chemical dismutation reaction alone is rapid, superoxide generated by NADPH oxidases is also converted to H₂O₂ by apoplastic superoxide dismutases. The regulation of spatiotemporal patterns of ROS-metabolizing enzymes may thus be important in establishing gradients of superoxide and H₂O₂.

H₂O₂ produced on the apoplastic face of the plasma membrane can be directly sensed by membrane receptor kinases such as HPCA1 that trigger an influx of calcium ions into the cell, leading to the activation of mitogen-activated protein kinases (MAPK) and other signaling pathways (Wu et al., 2020). HPCA1, which was also recently called CANNOTRESPONDTODMBQ1 (CARD1), is involved in quinone as well as H₂O₂ sensing. The CARD1 pathway triggers defense-related gene expression by elevating cytoplasmic Ca²⁺ and activating MAPK signaling pathways (Laohavisit et al., 2020). HPCA1/CARD1 recognizes quinone molecules at its apoplastic ectodomain through the oxidation of Cys residues through a similar mechanism to the recognition of H₂O₂, triggering immunity against bacterial pathogens (Laohavisit et al., 2020). These observations suggest that the sensing of extracellular quinones and H₂O₂ is mechanistically related.

The extracellular location of ROS production and the nature of the receptor may provide specificity to the oxidative signal, as discussed previously (Noctor et al., 2018). The movement of apoplastic H₂O₂ to the cytoplasm is thought to be facilitated by aquaporins (Rodrigues et al., 2017). Moreover, in addition to the local effects of ROS production in the apoplast, such signals can be propagated throughout the plant, in a process that is called the “ROS wave” (refer to section Root/shoot (systemic) signaling herein; Choudhury et al., 2018; Devireddy et al., 2018).

In contrast to other ROS forms, singlet oxygen (¹O₂) is generated by the direct transfer of energy from photodynamic pigments to ground state molecular oxygen. ¹O₂ activates specific signaling pathways that change the expression of nuclear genes. Depending on the site of generation and the amount produced, ¹O₂ signals can lead to acclimation or programmed cell death (Ramel et al., 2013). The site of ¹O₂ generation also dictates the nature of the signaling pathway that is activated. Within membranes, ¹O₂ leads to changes in gene expression through the production of carotenoid breakdown products such as β-CC and dhA which act as signaling molecules that elicit an increase in stress tolerance (Ramel et al., 2012). However, β-CC tends to decrease the expression of genes related to cell growth and development. β-CC induces the SCARECROW LIKE14-controlled xenobiotic detoxification pathway (D’Alessandro and Havaux, 2019). The β-CC signaling pathway is independent of the EXECUTER (EX)-dependent signaling pathway, which is triggered when ¹O₂ is generated at the membrane surface or in the stroma or cytosol (Ramel et al., 2013; Shumbe et al., 2016). Moreover, β-CC signaling may involve several pathways, at least one of which involves the METHYLENE BLUE SENSITIVITY zinc finger proteins 1 and 2 (Shumbe et al., 2017). Although much of the ¹O₂ in plants is produced by photosynthesis, ¹O₂ generation has also been reported in roots, particularly in response to stress (Mor et al., 2014; Chen and Fluhr, 2018).

Oxygen sensing and signaling

Cysteine oxidation is also a fundamental feature of plant oxygen signaling, which operates by an oxygen-dependent N-degron mechanism (van Dongen and Licausi, 2015; Holdsworth et al., 2020). The N-degron pathways operate in conjunction with the ubiquitin proteome system in plants (Holdsworth et al., 2020). N-degrons and C-degrons are degradation signals, which are determined respectively, by the N- and C-terminal residues of proteins. The PRT6 N-degron substrates include the transcription factors of the group VII ETHYLENE RESPONSE FACTOR (ERF-VII) and LITTLE ZIPPER 2 (ZPR2), as well as the VERNALIZATION 2 component of the Polycomb Repressive Complex 2. The ERF-VIIs function to regulate gene expression of conserved cellular and metabolic processes under low oxygen conditions. In normoxic conditions, the N-terminal methionine is excised, exposing a terminal cysteine that is subsequently converted to a Cys-sulfinic acid (-SO₂H) by a PLANT CYSTEINE OXIDASE (PCO), allowing its arginylation by tRNA-ARGINYL-TRANSFERASE (ATE). The PCO irreversibly oxidize Cys residues to -SO₂H and sulfonic acid. Such irreversible PTM of Cys residues deactivate protein functions and play a role in protein turnover. The N-terminal arginylated ERF-VIIs are thus targeted for degradation by the PRT6. At low oxygen levels, the process is inhibited resulting in stable ERF-VIIs. Mutants that are defective in ERF-VIIs are unable to sense oxygen levels and behave as if they are in constant hypoxia (Abbas et al., 2015). Many facets of the operation of plant oxygen signaling remain to be elucidated. In particular, it has been consistently shown that ^.NO as well as low oxygen are required for the N-degron degradation in vivo (Gibbs et al., 2014); however, in vitro analysis shows that oxidation by the PCOs does not require ^.NO (Weits et al., 2014; White et al., 2017). How these dependencies operate in vivo, therefore, remains unknown. In addition, both molecular oxygen (hypoxia) and ROS metabolism are known to be important in cell identity and fate in plants, and are known to act coordinately in animal cells (Movafagh et al., 2015; Smith et al., 2017). Yet, to date, the direct relationship between oxygen signaling and ROS signaling in plants remains elusive. Stress-induced hypoxia such as that caused by flooding results in a large increase in cellular oxidation because of an accumulation of ROS (GonzaLi et al., 2015; Yao et al., 2017). The stress-induced production of ROS is at least in part mediated by the activation of plasma membrane-bound RBOH (Yeung et al., 2018). Of the hypoxia-inducible RBOH genes in Arabidopsis (Arabidopsis thaliana), RBOHD is considered to be the most important for survival flooding or hypoxia/anoxia stress (Chen et al., 2015; Yeung et al., 2018). However, RBOHF may also play a key role in low oxygen tolerance (Chen et al., 2015; Liu et al., 2017). The RBOH proteins provide localized ROS bursts to regulate growth and developmental processes, as well as stress responses (Chapman et al., 2019). The stress-regulated expression of RBOHD is modulated by ERF-VII transcription factors (ERF74/RAP2.12; Yao et al., 2017). There may be a reciprocal effect of RBOH on ERF-VII expression (ERF73/HRE1; Yang and Hong, 2015). RBOH proteins also play a key role in developmental hypoxia, as illustrated in Figure 2. One feature of crosstalk between hypoxia and ROS signaling is the role of ethylene, which in flooded conditions accumulates quickly and serves to stabilize ERF-VII levels, expediting the hypoxia response (Hartman et al., 2019). The accumulation and impact of ethylene is less likely to occur in developmental contexts where, for example, localized hypoxia is established by respiratory demand.

Figure 2.

Multiple pathways connect plant oxygen signaling to the TARGET OF RAPAMYCIN (TOR) kinase. Under hypoxic conditions, degradation of ERF-VII transcription factors by the PRT6 N-degron pathway (N-deg) is uncoupled. Accumulation of ERF-VII leads to an increase in starch catabolism that, depending on context, may lead to increased glucose (Glc) and/or decline in trehalose-6-phosphate (T6P). Glc directly regulates the TOR kinase in apical meristems and positively regulates cell division by other pathways. The decline in T6P, meanwhile, would de-repress SNF1-RELATED PROTEIN KINASE 1 (SnRK1), which in turn negatively regulates the TOR kinase, both directly and indirectly, by promoting abscisic acid (ABA) signaling. A decline in T6P levels and an increase in SnRK1 activity have been shown in hypoxic conditions. ABA and TOR, meanwhile have a reciprocal antagonistic relationship. Among the transcriptional targets of ERF-VIIs under hypoxia are HYPOXIA RESPONSIVE UNIVERSAL STRESS RESPONSIVE PROTEIN 1 (HRU1) and RBOHD. Several RBOH genes are induced under hypoxia. HRU1 can enhance the activity of RBOHD directly and indirectly via ROH OF PLANT 2 (ROP2), and also positively regulates TRX-h. RBOH generates H₂O₂, which—depending on context—may trigger cell division or cell death. ROP2 directly regulates TOR kinase, providing a point of convergence between oxygen, ROS, and energy signaling. In addition, SnRK1 is regulated by redox status, while the relationship to ABA may depend on the physiological context (∼). Finally, ERF-VII may regulate TOR kinase by other pathways yet to be characterized (?). Dashed arrows indicate indirect relationships.

Cell cycle

An important premise of multicellular life is that cells must faithfully duplicate and segregate their genetic information to daughter cells. Direct interaction of DNA with ROS can result in single- and double-strand breaks, as well as the formation of DNA adducts and cross-links (Bray and West, 2005). Oxygen and ROS signaling thus form important features of cell division and identity. Indeed, it is argued that fluctuations in oxygen availability and redox status in the environment constrained the evolution of multicellular life (Lyons et al., 2014), including meiosis (Hörandl and Hadacek, 2013).

Cell division by all eukaryotes is driven by cyclin-dependent kinases (CDKs), which are regulated by phase-specific partnership with cyclins and other interacting proteins (Inzé and De Veylder, 2006; De Veylder et al., 2007). These latter proteins integrate metabolic status and stress signals with the regulation of the cell cycle (Xiong and Sheen, 2014). The net activity of the CDK complexes oscillates to bring about phase-specific events that comprise the cell cycle, that is: G1 (Gap 1), S (Synthesis), G2 (Gap 2), and M (Mitosis). Quality control is executed at checkpoints in the G1/S and G2/M transitions (Cools and De Veylder, 2009). Proteolysis through the anaphase-promoting complex and the Skp1/Cullin/F-box-related E3 complexes ensures the directionality of the cell cycle, as well as enabling cell cycle exit to differentiation, and transition to the endocycle.

Evidence for a regulated redox rhythm in the cell cycle in animal cells emerged nearly a century ago (reviewed by Menon and Goswami, 2007). The abundance of nonprotein thiols was tightly coupled with the phases of mitosis, and a transient increase in oxidants was a requisite for the G1/S phase transition (Menon et al., 2003). In plants, differential gradients of oxygen availability and redox status have been related to meristem function and fate (Considine et al., 2017), and depletion of the major soluble antioxidants results in pronounced meristem phenotypes, which we elaborate on further below. In plant cell cultures, the abundance and oxidation state of ascorbate and GSH is coupled to the phasic nature of the cell cycle. The uptake of oxidized ascorbate (dehydroascorbate) to tobacco (Nicotiana tabacum) BY-2 cells was maximal during the M phase and M/G1 transition (Horemans et al., 2003), while the chemical inhibition of GSH synthesis (Vernoux et al., 2000), or addition of the oxidant menadione (Reichheld et al., 1999) resulted in G1 arrest. The establishment of leaf protoplasts has also been shown to depend on transient oxidation (Pasternak et al., 2007).

It is widely presumed that ROS are not actively generated in the nucleus; however, evidence shows that H₂O₂ can accumulate in the nucleus (Ashtamker et al., 2007). In addition, the nucleolus was shown to accumulate high levels of Fe, enabling the potential generation of ROS by Fenton reactions (Roschzttardtz et al., 2011). As the nucleolus loses its integrity during mitosis, this may also represent an important feature of redox rhythms key stages of mitosis.

Evidence shows that cytosolic GSH preferentially shuttles to the nucleus early during the cell cycle, resulting in a redox differential across the nuclear membrane that is mitotically rhythmic (Diaz-Vivancos et al., 2010a, 2010b). In addition, several GSH-dependent TRXs and GRX have been shown or predicted to localize to the nucleus (Martins et al., 2018), as well as a GSH reductase 1 (GR1), indicating a capacity to reduce oxidized GSH (GSSG; Delorme-Hinoux et al., 2016). A pea TRX, PsTrxo1 was recently shown to bind to the PROLIFERATING CELLULAR NUCLEAR ANTIGEN (PCNA), and tobacco BY-2 cells over-expressing the PsTrxo1 showed a greater rate of cell division, increased abundance of the PCNA, and a decline in cellular GSH (Calderón et al., 2017). A computational analysis of the canonical and accessory cell cycle proteins of Arabidopsis showed that the majority have between 1 and 3 exposed Cys residues, with less than one-fifth having none, or only inaccessible Cys (Foyer et al., 2018). However, to date, few mechanistic relationships with redox activities and canonical cell cycle proteins have been established.

The TARGET OF RAPAMYCIN (TOR) kinase is a master regulator of cell division, which activates the S-phase transcription factor E2Fa to enable the G1/S transition (Xiong and Sheen, 2014). The TOR kinase perceives energy and stress signals directly and indirectly via the SNF1-RELATED KINASE 1 (SnRK1; Robaglia et al., 2012), enabling acclimation at the level of cell division. Converging but as yet unresolved lines of evidence relate oxygen availability and ROS, together with glucose (Glc) and auxin to the direct regulation of the TOR kinase (Considine, 2017) and SnRK1 (Cho et al., 2019; Figure 2). First, under hypoxic conditions, the ERF-VIIs will be stabilized, leading to increased expression of conserved metabolic responses including increased starch hydrolysis and sugar catabolism (António et al., 2016; Paul et al., 2016). The resulting increase in Glc and decline in trehalose-6-phosphate (T6P) may be perceived differentially through the TOR kinase and SnRK1; Glc positively regulates TOR kinase, while T6P negatively regulates SnRK1. Among the transcriptional targets of the ERF-VIIs are a HYPOXIA RESPONSIVE UNIVERSAL STRESS PROTEIN1 (HRU1; GonzaLi et al., 2015) and RBOHD (Yao et al., 2017). The HRU1 directly interacts with RBOHD and TRX-h, positively regulating ROS synthesis and signaling, as well as a ROH OF PLANT 2 GTPase (ROP2; GonzaLi et al., 2015). The ROP2 positively regulates TOR kinase in response to light and sugar cues (Schepetilnikov et al., 2017). Hypothetically, therefore, cellular changes perceived through oxygen- and ROS-signaling pathways should positively regulate the TOR kinase complex and promote meristematic activity. The observation that cell proliferation in crown gall or clubroot requires hypoxia, and is dependent on N-degron signaling supports this notion (Gravot et al., 2016; Kerpen et al., 2019). However, other phenotypes known to depend on the ERF-VIIs are less consistent with it, including the promotion of seed dormancy and reduced growth (Graciet et al., 2009; Paul et al., 2016). Moreover, the SnRK1 appears to be positively regulated by hypoxia (Cho et al., 2016). Further relationships described in Figure 2 remain to be put in a developmental context of hypoxia and/or ROS signaling. For example, a reciprocal antagonistic relationship between abscisic acid (ABA) and TOR kinase was recently described (Wang et al., 2018b), and the SnRK1 subunit AKIN10 was shown to be regulated by redox status (Wurzinger et al., 2017). Taken together, these insights reveal an intricate balance of signaling governed by oxygen availability and ROS that interact to direct the balance of quiescence and proliferation.

Seed germination

The success of seed germination depends on phytohormone signaling pathways. In particular, the relationships between gibberellins (GAs) and ABA control seed germination/seed dormancy. The spatial and temporal localization of ROS plays a pivotal role in regulating ABA/GA control, and in the cell‐to‐cell communication and the breakage of hydrolytic bonds between polysaccharides in the cell wall of endosperm (Tuan et al., 2018).

During maturation and the onset of dormancy, orthodox seeds become desiccated and devoid of ascorbate (Tommasi et al., 2001), and protein thiols of the endosperm and embryo become predominantly oxidized (Alkhalfioui et al., 2007). The rapid synthesis of ascorbate and reduction of storage and regulatory proteins upon the availability of free water is crucial to enable the successful transition (de Simone et al., 2017). Evidence for the role of peroxidases, RBOH, and TRX in modulating the degree of protein carbonylation and Cys–thiol redox regulation have been summarized recently (Bailly, 2019). More recently, the temporal coordination of protein redox status and adenylate charge was illustrated during the acute stages of imbibition in Arabidopsis (Nietzel et al., 2020). Dynamic changes in the adenylate energy charge ([ATP] + 1/2 [ADP])/([ATP] + [ADP] + [AMP]), cyanide-sensitive oxygen consumption and tricarboxylic acid metabolism were reported within 1 h of imbibition. This indicates a remarkably rapid resumption of mitochondrial oxidative phosphorylation, which could only be enabled by the activation of extant enzymes. Changes in the redox states of Cys residues in several proteins were observed in isolated mitochondria, with pronounced shifts in the reduction states of GR2, NAD(P)H thioredoxin reductase (NTR) A/B, and TRX-o1 (Nietzel et al., 2020). Mutant seeds individually lacking these proteins showed higher rates of respiration upon imbibition, and impaired tolerance of aging (Nietzel et al., 2020). This neatly illustrates the fine control of energy and developmental functions by Cys redox regulation during imbibition and germination.

Two recent studies suggest that Ca²⁺-dependent ROS signaling is also involved in germination. The GLUTAMATE RECEPTOR HOMOLOG 3.5 (GLR3.5) is predominantly expressed during germination and acts to increase Ca²⁺ levels, which antagonize ABA-dependent repression of germination (Kong et al., 2015). The GLR3.5 promotes the levels of ROS in the embryonic axes of germinating seeds (Ju et al., 2020).

Oxygen signaling via the N-degron pathway is also implicated in dormancy and germination control. The germination of Arabidopsis or barley seeds lacking the E3 ligase N-recognin PRT6 or the ATEs (ATE1 and ATE2), were less sensitive to ethylene, but still sensitive to GA and chilling (Holman et al., 2009; Gibbs et al., 2014; Wang et al., 2018c). The ERF-VIIs directly bind and transcriptionally regulate the ABSCISIC ACID INSENSITIVE 5 (ABI5) transcription factor, which is a positive regulator of ABA signaling (Gibbs et al., 2014).

After the successful radicle protrusion, the ROS-induced hypersensitive reaction and systemic acquired resistance protect the newly emerged seedling from pathogens (Kadota et al., 2015). Germinating seeds (i.e. in the dark and under the soil) in a skotomorphogenic and heterotrophic state rely on embryonic reserves. Upon germination, the radicle emerges and the root and shoot systems begin to grow. The shoot produces an elongated hypocotyl, a closed apical hook, and yellow cotyledons in the absence of illumination. While oxygen is abundant in air, tissue gradients, combined with metabolic control result in niches of hypoxia. Plant organs such as the apical hook are highly sensitive to available oxygen levels, showing a gradual response (Abbas et al., 2015). Apical hook opening in etiolated seedlings is highly dependent on available oxygen, with even small changes in available oxygen concentrations promoting a different response.

Meristems, branching, and architecture

Plants exhibit indeterminate growth, which allows the continuous development of new organs such as leaves, stems, and roots because of specialized meristematic tissues, in which continuous cell division occurs. Apical meristems are located at the tips of stems and roots, while lateral meristems facilitate changes in shoot and root architecture as well as thickness. The Arabidopsis primary root contains four types of stem cells (initials) in the root tip. The quiescent center (QC) resides in the middle of the stem cells and is characterized by low mitotic activity. The QC maintains the specification of the stem cell niche. The meristematic zone (MZ), which is characterized by a high mitotic activity is located in the distal part of the apical root. Lateral roots (LRs) develop from the pericycle cells adjacent to xylem cells, which have acquired founder cell status via oscillating endogenous developmental signals and environmental triggers. The founder cells divide to produce an LR primordium (LRP) that passes through eight developmental stages in the differentiation zone to produce the mature organ (Vilches-Barro and Maizel, 2015).

The availability of oxygen and the accumulation of superoxide and H₂O₂ is important for the development of the different zones in roots and shoots. Together with light and sugars, regulated hypoxia plays a key role in meristem activation at the post-embryonic stages of development (Considine, 2017; Signorelli et al., 2018). Oxygen levels can fall ˂5% in the shoot apices and in the LRP of plants grown in the air, leading to the expression of hypoxia-dependent genes (Shukla et al., 2019). In contrast, hypoxia-responsive genes are not induced in the root apical meristem (RAM) under similar conditions (Eysholdt-Derzsó et al., 2017; Shukla et al., 2019). Such findings suggest that oxygen-dependent regulation is different in the LRP and RAM. However, hypoxia might occur in the QC of the RAM to control the balance between proliferation and quiescence (Considine et al., 2017), which is essential to prevent consumption of the stem cells. The oxygen-dependent stability of the ERF-VIIs fulfills important roles in shaping root architecture (Eysholdt-Derzsó et al., 2017; Shukla et al., 2019). For example, RAP2.12 interacts with AUXIN RESPONSE FACTOR 7 (ARF7) in the LRP to repress the expression of meristematic genes such as LBD16, LBD18, IAA19, and PUCHI, allowing LR establishment to occur (Shukla et al., 2019). Oxygen gradients are likely to occur in the RAM, particularly because the QC cells are deficient in antioxidants such as ascorbate and GSH (Jiang et al., 2010), but they either do not induce hypoxia-responsive genes, or local RAM-specific repressors prevent the expression of these hypoxia-marker genes. Flooding-induced hypoxic stress alters auxin flow and distribution in roots in a manner that can shift the redox state of the QC toward a more reduced environment (Mira et al., 2020). This leads to the activation of the QC cells and degradation of the meristem, followed by loss of root functions (Mira et al., 2020).

A developmentally regulated low oxygen niche envelops the stem cells in the shoot apical meristem (SAM) that are responsible for the production of new leaves and flowers. Low oxygen levels promote the stability of the ZPR2 protein that is responsible for cell proliferation and differentiation, whereas superoxide accumulation activates WUSCHEL, which regulates stem cell maintenance. High superoxide levels have been reported in the RAM MZs while H₂O₂ accumulates in the elongation and differentiation tissues (Dunand et al., 2007). The UPBEAT1 (UPB1) transcription factor, which modulates the expression of peroxidase genes, is important in the control of H₂O₂ accumulation in the roots (Tsukagoshi et al., 2010). Loss of UPB1 functions leads to an increase in the size of the root meristem and in primary root length. Conversely, overexpression of UPB1 leads to increased H₂O₂ accumulation and a decrease in the meristem size. Transcriptomic studies have shown that the differential expression of ROS-related genes in developing roots is induced by auxin (Orman-Ligeza et al., 2016; Tognetti et al., 2017; Mhamdi and Van Breusegem, 2018). Auxin-induced and RBOH-mediated ROS production is important in the control of root and shoot architecture, as well as the establishment of the gravitropic curvature response in roots. Spatiotemporal regulation of the patterns of RBOH expression leads to ROS accumulation in the apoplast of the middle lamella regions of cells at pre-branch sites and LRP during emergence, facilitating LR outgrowth by promoting cell wall remodeling of overlying parental tissues (Orman-Ligeza et al., 2016).

Other hormones such as ABA are important in the control of apoplastic ROS production. For example, ABA increases ROS accumulation in the root tip and decreases the growth of the primary root, as well as reducing the number of meristematic cells (Yang et al., 2014). The tip-growth of root hairs also involves RBOH-mediated ROS production, which promotes cell elongation (Foreman et al., 2003). LR and root hair growth are modulated in response to different environmental factors, such as water, nitrogen, and phosphorus. ROS production by RBOH is required for LRP emergence (Manzano et al., 2014; Orman-Ligeza et al., 2016). Superoxide and H₂O₂ generation during LRP development is important in root responses to nutrient deprivation. For example, phosphate deficiency leads to the intracellular accumulation of Fe³⁺ produced by the activities of ferroxidases LOW PHOSPHATE RESONSE (LPR1 and LPR2) that alter the Fe and phosphorous balance within the root cells. This leads to ROS production and callose deposition within the MZ that decreases the primary root growth and induces the elongation of the root hairs (Müller et al., 2015; Ham et al., 2018). Such external signals trigger auxin and ARFs that activate the bHLH transcription factor called ROOT HAIR-DEFECTIVE SIX-LIKE 4, which directly targets the expression of genes encoding RBOH and secreted type III peroxidases, increasing oxidation of the apoplast and stimulating root hair cell elongation (Mangano et al., 2017). Downstream auxin signaling is sustained by an oscillatory feedback loop involving calcium ions (Ca²⁺) as well as ROS. High levels of cytoplasmic Ca²⁺ (_cytCa²⁺) activate RBOHs, while high levels of ROS in the apoplast activate as yet unknown Ca²⁺-permeable channels that promote Ca²⁺ influx into the cytoplasm (Foreman et al., 2003).

GSH and GRXs are important in the transmission of ROS signals that control plant development. Loss-of-function mutants in the GSH1 gene are unable to produce γ-glutamyl cysteine or GSH and show impaired embryo development. In contrast, mutants that cannot produce GSH are able to undergo embryogenesis but the seeds cannot germinate (Cairns et al., 2006; Pasternak et al., 2008). Several weak alleles in the GSH1 gene accumulate only a small proportion of the GSH present in the wild type (Parisy et al., 2007). The root meristemless1 mutant (rml1), which has only 3% of the wild-type GSH shows impaired primary root growth (Vernoux et al., 2000). Other mutants such as phytoalexin deficient2 (pad2-1) and cadmium-sensitive (cad2-1) produce ∼30% of the total GSH found in the wild-type (Cobbett et al., 1998) and have minor changes in root architecture. However, mutants that are defective in the plastid-localized GR2 show defective root growth (Yu et al., 2013). GRX, which uses GSH as a source of reducing power, plays a key role in root development (Belin et al., 2015). For example, GRXS17 is critical for primary root growth and meristem activity. GRXS17 interacts with Nuclear Factor Y Subunit C11/Negative Cofactor 2a (NF-YC11/NC2a) and BolA2 that are involved in abiotic stress tolerance and Fe-S cluster formation, respectively (Couturier et al., 2014; Knuesting et al., 2015). Similarly, the GRXS3/S4/S5/S8 cluster plays a negative role in the control of root responses to nitrogen (Patterson et al., 2016). GRXS8 was recently shown to be a major regulator of the root system architecture that prevents transcriptional and developmental responses to nitrate response, possibly by interfering with the activity of the TGA1 and TGA4 transcription factors (Ehrary et al., 2020). Recent evidence suggests that GSH is required for the conversion of indole butyric acid into indole acetic acid, providing a mechanism for GSH-dependent regulation of the auxin pathway in root development (Trujillo-Hernandez et al., 2020). Links between strigolactone and GSH signaling in the control of root system architecture have also been demonstrated (Marquez-Garcia et al., 2014).

Arabidopsis mutants lacking chloroplast-localized GSH peroxidases show higher LR densities than the wild-type (Passaia et al., 2014). The root NTR/TRX and GSH/GRX systems interact closely in the control of root development. For example, Arabidopsis mutants lacking NTRC show defective LR development, a phenotype that was rescued by the expression of NTRC in leaves but not in roots (Kirchsteiger et al., 2012). Mutants lacking NTRA, NTRB, and GSH synthesis capacity (ntrantrbcad2) had significant impairment in the primary root growth, decreased LR development, and altered auxin transport (Bashandy et al., 2010). Mutants lacking the cytosolic form of GR are viable because of back-up from the NTR/TRX (Marty et al., 2009). The presence of multiple backup systems serves to protect the growth and development of the embryo (Marty et al., 2019).

Flowering and pollen development

The environmental sensitivity of reproduction is linked to the loss of control of ROS production and processing. Germinal cell specification, male meiosis, and pollen development occur in the anthers, which have to accommodate hypoxia. In maize anthers, ROS is limited by diverting carbon away from mitochondrial respiration into alternative pathways (Kelliher and Walbot, 2012, 2014). Moreover, hypoxia triggers the expression of a GRX called Male Sterile Converted Anther 1 in maize, which triggers germinal cell initiation in maize anther primordia. Pollen grains that had not been stimulated to germinate are significantly more oxidized than the germinated pollen grains (García-Quirós et al., 2020). The transition from the oxidized quiescent state to the metabolically active germinated state, therefore, requires a large change in cellular redox homeostasis.

ROS play a major role in the fusion of male and female gametophytes, which ensures the correct fertilization and seed germination. Pollen grains that land on the stigma adhere to the surface and undergo hydration, a process that initiates metabolism from the quiescent state. The viability of mature tomato and Arabidopsis pollen was shown to be related to ROS accumulation, with two types of pollen populations identified as “low‐ROS” and “high-ROS” relating to low or high metabolic activity, respectively (Luria et al., 2019). While the high‐ROS pollen germinated more frequently than the low‐ROS pollen, exposure to stresses such as high temperatures increases pollen ROS levels further resulting in a decrease in germination. The development of the metabolically active high-ROS pollen requires effective ROS processing by systems such as the GSH/GRX pathways. The presence of a highly reduced GSH pool is essential for pollen germination and tube growth (Zechmann et al., 2011; García-Quirós et al., 2020).

The stigma appears to be more oxidized in the unreceptive state (Zafra et al., 2016). A more reduced state in the pollen and stigma is required for pollen development. FERONIA (FER), RAC/ROP GTPases, and NADPH oxidases are the keys to pollen tube development toward the micropyle, as well as pollen tube burst. The fusion of the sperm cell involves ROS and NO/RNS signaling, as well as Ca²⁺/K⁺ influxes. The FER/LORELEI (LRE) signaling complex controls ROS production during pollen tube arrival (Duan et al., 2014). ROS induce bursting of the pollen tube in a Ca²⁺-dependent manner involving the activation of Ca²⁺ channels. The FER signaling pathway is required for induction and maintenance of the Ca²⁺ responses (Ngo et al., 2014) and an Rho GTPase-based signaling mechanism likely acts directly downstream of FER/LRE activation (Li et al., 2015). While ROS are also implicated in incompatibility mechanisms, there is little consensus regarding the mechanism involve (Zafra et al., 2016).

Root/shoot (systemic) signaling

The growth and development of shoots and roots are intimately interconnected through long-distance communication pathways that are dependent on the vascular system for the transport of RNAs, peptides, and phytohormones that control whole-plant growth and resource allocation, as well as defense responses (Ko and Helariutta, 2017). Systemic signal transduction also involves waves of ROS, Ca²⁺ and electrical signals in and around the vascular cells that are largely achieved through the mediation of phytohormones (Gilroy et al., 2014; Zandalinas et al., 2020b). Systemic, whole plant ROS signaling has long been known to be important in plant responses to abiotic stresses, a process that was termed “systemic acquired acclimation” (SAA; Kollist et al., 2019). Such studies have shown that ROS production in the apoplast is able to stimulate auto-propagation of a ROS wave throughout the plant, coordinating systemic responses (Choudhury et al., 2018; Devireddy et al., 2018), as well as triggering SAA (Zandalinas et al., 2019, 2020a). The initiation and propagation of the ROS wave are dependent on the RBOHD and RBOHF (Mittler et al., 2011; Zandalinas et al., 2020b). It is thought that RBOHD activation is triggered post-transcriptionally via changes in calcium levels and/or phosphorylation of RBOHD (Fichman and Mittler, 2020). In all cases, the ROS production in the apoplast may be detected at a cellular level by specific H₂O₂ sensors such as HPCA1 (Wu et al., 2020) or by other similar thiol-modulated proteins, or even by the movement of H₂O₂ into the cells (Noctor et al., 2018).

In the shoot, light intensity and quality are key activators of these systemic signals. Phytochromes, which are synthesized in inactive, red (R) light-absorbing Pr forms, are converted upon absorption of light to their physiologically active far-red light-absorbing Pfr forms. The light-activated Pfr forms are then translocated into the nucleus, where they interact with many different transcriptional regulators to control a range of processes. In addition, some phytochromes such as phyB are involved in systemic responses to a local perception of biotic and abiotic stresses. The perception of light by leaves in tomato is mediated by phyB and auxin via a ROS wave to regulate photosynthesis and other physiological responses in lower canopy leaves (Guo et al., 2016; Wang et al., 2018a). phyB is essential for the ROS wave initiation and local and systemic stomatal regulation (Devireddy et al., 2020). Moreover, phyB regulates light-induced root growth during shoot to root signaling (Lee et al., 2016) and shoot-induced ABA synthesis and ROS production in roots (Ha et al., 2018). phyB activates the expression of the light-regulated basic leucine zipper transcription factor called LONG HYPOCOTYL 5 (HY5), which moves from the shoots to the roots (Chen et al., 2016) to regulate root growth by regulating auxin and brassinosteroid levels (Burko et al., 2020). However, it is possible that the HY5 protein activates other signals in the shoots, which travel to the roots to regulate root growth or activate root-specific signals, possibly including local transcription of HY5.

Like light, information concerning high atmospheric carbon dioxide (eCO₂) concentrations perceived by tomato leaves is transferred to the roots to promote symbiosis with arbuscular mycorrhizal fungi (AMF), modifying plant nutrient acquisition and cycling (Zhou et al., 2019). The perception of eCO₂ triggered apoplastic ROS-dependent auxin production in the shoots followed by systemic signaling, leading to increased strigolactone synthesis in the roots. This redox–auxin–strigolactone systemic signaling cascade facilitates eCO₂-induced AMF symbiosis and phosphate utilization, and is likely also to regulate root architecture, but such a mechanism remains to be explored.

A systemic root/shoot signaling loop, which integrates electrical, ROS, and jasmonate (JA) signals, was shown to enhance resistance against root–knot nematodes in tomato. The perception of nematode infestation induced the systemic transmission of electrical and ROS signals that activates the MAPK 1/2 pathway to induce JA synthesis in leaves, improving the plant defense against nematodes (Wang et al., 2019).

Discussion

Plants optimize their growth in fluctuating environmental conditions by using information acquired by different organs. Environment-induced changes in tissue oxygen availability and ROS production serve to activate plant stress responses (Sasidharan et al., 2020) as well as regulating growth, probably through common signaling pathways. Stress-induced hypoxia and oxidation not only induces local changes in gene expression but also triggers systemic signals that are transmitted throughout the plant. The local and long-distance signaling systems involve many components including hormones, ROS burst and waves, electrical signals, and also mobile RNAs and proteins. While relatively few of these signals have been fully characterized and our knowledge of the mechanisms that facilitate long-distance signaling in plants remains incomplete, the concept that ROS waves allow rapid transmission of information between cells and organs is now widely accepted, allowing co-ordination of stress and developmental signals that facilitate whole-plant responses to the environment.

Together with ROS, the relative oxygen concentrations in the stem cell niches of the SAM and the RAM provide information about cell position, environmental conditions, and metabolic state. Low oxygen concentrations are associated with quiescence, while ROS are signals that are only generated by metabolically active cells. ROS accumulate in and around the apoplast of cells at the dynamic interface between quiescence and active growth to allow directional growth and development, as illustrated in Figure 3. The relative contributions of ROS generated in the different cell compartments, particularly the plasma membrane and mitochondria, are likely to engage the different signaling pathways that transmit the ROS signals, through downstream kinases that exert transcriptional and post-transcriptional controls. We have only started to scratch the surface of the complex interplay between oxygen and ROS signaling in the control of plant growth and development. Although many questions remain to be answered (see Outstanding questions box), it is certain that there will be multiple points of reciprocal control that facilitate appropriate responses to environmental as well as developmental cues.

Figure 3.

Schematic model for the oxygen and ROS-dependent regulation of directional growth in meristems. Quiescent cells produce low levels of metabolic ROS, for example, by the RETC, because of limitations in the availability of molecular oxygen. In contrast, ROS are produced by RBOH on the cell surface, where directional growth is to be initiated. The growth-promoting hormone (auxin/ethylene)-induced activation of RBOH and associated processes. Together with apoplastic peroxidases, RBOH regulates localized ROS accumulation in the apoplast/cell wall compartments, particularly in the middle lamella, stimulating directional growth by altering cell wall permeability and related metabolism. The resultant ROS wave passes from cell to cell, stimulating proliferation followed by cell expansion in the direction of growth. The apoplastic ROS wave leads to ROS perception within the target cell. Apoplastic oxidative signals coupled to metabolic ROS signals from the mitochondria and other intracellular compartments lead to redox regulation of the cell cycle (de Simone et al., 2017).

Outstanding questions

• How are hypoxia and the accumulation of ROS co-regulated in stem cell niches?

• How are ROS and oxygen signaling through the N-degron pathway coordinated?

• How does the local accumulation of ROS prime cell differentiation allowing differential spatial localization of different oxidants?

• How are the gradients of superoxide and hydrogen peroxide (H₂O₂) regulated?

• Do ROS assist in the formation of biomolecular condensates and micron-scale membraneless compartments?

• Does ROS-mediated liquid–liquid phase separation serve to confine proteins and constrain chemical reactions to specific locations?

• What precise cysteine modifications in redox-regulated proteins transmit ROS signals?

• What protein–protein interactions dictate the specificity of H₂O₂-mediated oxidation and signaling?

• How is the specificity of intercompartmental signaling achieved?

 

C.H.F. initiated the manuscript. M.J.C. and C.H.F. co-wrote the manuscript and designed the figures.

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: Christine H. Foyer (C.H.Foyer@bham.ac.uk).