Mechanisms of ROS Regulation of Plant Development and Stress Responses

1It is well known that improving crop yield and productivity requires improved understanding of the coordinated growth of plant tissues and organs. Plant morphogenesis is regulated by both intrinsic genetic programmes and external environmental factors. Reactive oxygen species (ROS) are regarded as by-products of plant aerobic metabolism and are generated in several cellular compartments such as chloroplasts, mitochondria, and peroxisomes. ROS not only cause irreversible DNA damage and cell death, but also function as important signaling molecules that regulate normal plant growth, and responses to stress. This suggests that ROS have a dual role in vivo depending on different levels of reactivity, sites of production and potential to cross biological membranes. From an evolutionary point of view, the emergence of oxygen-releasing photosynthetic life had a profound impact on all living organisms. As the source of all ROS, oxygen (O2) is stable and not very reactive in plants. However, it can be converted into high-energy ROS in several organelles by various processes that affect plant metabolism. As reactive molecules, ROS oxidize and modify some cellular components and prevent them from performing their original functions. Under unfavorable circumstances, plants generate a large number of ROS species involved in regulation of various processes including pathogen defense, programmed cell death (PCD), and stomatal behavior. These reactions exert profound or irreversible effects on development of tissues and organs, often leading to abnormal plant growth or death. Additionally, ROS interplay with epigenetic modifiers and hormones to control plant developmental processes, and stress responses. In general, low ROS levels are necessary for the progression of several basic biological processes, including cellular proliferation and differentiation. At higher levels ROS pose a significant threat that may eventually lead to DNA damage, and incorrect timing of PCD directly.

2In plants, ROS exist in ionic and/or molecular states. Ionic states include hydroxyl radicals (•OH) and superoxide anions, while molecular states mainly include hydrogen peroxide (H2O2), and singlet oxygen (1O2). Each type of ROS has a different oxidative capacity and affects different physiological and biochemical reactions regulated by different genes in plants. As an excited oxygen, singlet oxygen (1O2) is usually generated in chloroplast photosystem II (PSII) and has strong oxidizability. Although 1O2 exists for a very short time and is extremely unstable in cells, once generated, it has great impact on photosynthesis. Superoxide anion is the precursor of various ROS because of its instability and strong oxidation/reducibility. could maintain the stability of plant stem cells. However, excessive also causes increased ROS levels and eventually leads to cell death. In rice, roots, and stems seem to be the main organs of production, which might be related to their adaptation to the aquatic environment. can be produced by photosynthetic electron transport chains, mitochondrial respiratory electron transport chains, and membrane-dependent NADPH oxidase (RESPIRATORY BURST OXIDASE HOMOLOG proteins) systems, which react with hydrogen ions to form oxygen molecules or with superoxide dismutase (SOD) to form H2O2. Among these, H2O2 is considered an important redox molecule, given its specific physical and chemical properties, including a remarkable stability within cells (half life of 10–3 s), and rapid and reversible oxidation of target proteins. H2O2 can be transported by aquaporins localized in the cell membrane, not only causing long-distance oxidative damage, but also participating in cell signaling regulation. H2O2 has been shown to participate in cell differentiation, senescence, PCD, and cell wall formation in plants. Additionally, H2O2 interplays with hormones to regulate plant developmental process and stress responses. •OH can be formed when the O−O double bond in H2O2 cleaves. •OH is active and usually acts very near its production site. Therefore, •OH is the most reactive ROS, and it can react with all biological molecules. It can oxidize the cell wall polysaccharides, resulting in cell wall loosening, and it can also induce DNA single-strand breakage. Under normal conditions, excessive ROS can be scavenged by various antioxidative defense mechanisms. The equilibrium between production and scavenging of ROS may be perturbed by various biotic and abiotic stresses. These disturbances of the equilibrium can cause sudden increases in intracellular ROS levels and significantly damage cell structures. Taken together, plants are obliged to cope with excessive ROS generation in order to maintain cellular redox homeostasis. Accordingly, the augmented ROS levels are sensed and restrictively controlled by a battery of ROS-scavenging systems.

3ROS scavenging mechanisms can be classified into two types: enzymatic and non-enzymatic antioxidant defense systems, which work synergistically and interactively to neutralize free radicals. The enzymatic systems mainly include SOD, catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX). In rice, most of these genes participating in ROS removal exhibit tissue/organ-specific expression profiles (Table 1). However, their function in ROS homeostasis and regulation of gene expression remain unclear. Among the enzymatic systems, SOD is able to rapidly convert ⋅OH to H2O2, and the generated H2O2 is then converted to water and dioxygen by peroxidase and CAT. The non-enzymatic systems are mainly mediated by low molecular mass antioxidants, such as glutathione, ascorbic acid (AsA) and flavonoids, which are known to remove hydroxyl radicals and singlet oxygen. When ROS levels in the cell exceed the range of the scavenging systems, cells enter the oxidative state, resulting in oxidative modification and cell damage which can lead to death. When ROS levels are low, the cells are in the reduced state, and ROS can be used as second messengers that participate in stem cell maintenance, cell division, and differentiation, organogenesis, and biotic and abiotic responses, etc.. Thus, it is necessary to maintain ROS levels within the right range for plant health. Of course, alterations in ROS levels that are part of the normal function of the plant should not exceed the threshold boundary between cytostatic and cytotoxic levels.

4The appearance of aerobic conditions gave organisms the opportunity to use oxygen as an electron acceptor, while enabling them to harness its reactive properties for metabolism and signaling. It was therefore, inevitable that evolution in an oxygenic environment would necessitate integration of oxidative processes and ROS sensing and signaling into the developmental programs. From seed germination to plant senescence, ROS are dynamically generated or removed, which makes plants regulate their development in order to adapt to different environments. However, the effects of ROS on plant growth and development are more complex due to the temporal and spatial variability of ROS regeneration and interplay between them in plants.

5Emerging evidence indicates that ROS homeostasis shapes plant vegetative apex development (Figure 1). In Arabidopsis thaliana, primarily accumulates in the meristematic zone of the root tip and is required for cell division, whereas H2O2 mainly accumulates in the elongation zone, which confers cell differentiation. These two different ROS micro-environments coincide with the meristematic and the elongation zone, and their distributions are important for localization of the transition zone. Because of the ROS species gradients, cells entering the transition zone can still proliferate. Once the ratio of to H2O2 reaches a certain level, cells stop dividing and begin to elongate. Therefore, the ROS balance in the transition zone is essential. The transcription factor UPB1 (UPBEAT1) is one of the key regulators maintaining this balance. Further studies showed that H2O2 itself affects UPB1 expression, and this regulatory system contains a feedback loop that plays a role in both ROS homeostasis and root growth. In addition, the quiescent center (QC) and distal stem cell (DSC) are required for root apical meristem (RAM) size maintenance. APP1 (Arabidopsis thaliana P-loop NTPase1) affects root stem cell niche (SCN) identity through its control of local ROS homeostasis. Disruption of APP1 is accompanied by a reduction in ROS levels, a rise in the rate of cell division in the QC, and the promotion of root DSC differentiation, suggesting that ROS levels are directly related to RAM size in Arabidopsis. More importantly, ROS, together with hormones and other signal molecules, regulate plant root primary growth. In the RAM, ROS, and auxin signaling are antagonistically regulated to balance root meristem growth. The imbalance of different ROS species or accumulation of ROS induced by high levels of glucose oxidizes active IAA, resulting in its degradation, impairs root meristem activity, and subsequently inhibits root growth through the conserved macroautophagy/autophagy pathway. The findings suggest that autophagy is an essential mechanism for glucose-mediated maintenance of the root meristem by modulating the homeostasis of cellular ROS and promoting the degradation of the oxidatively damaged peroxisomes. Recent studies have shown that Brassinosteroids (BRs) also control root tip stem cell activity through ROS. Binding of BR to receptor kinase BRI1 (BRASSINOSTEROID INSENSITIVE1) increases cellular levels of H2O2, and the increased H2O2 induces oxidative modification of BZR1 (BRASSINAZOLE-RESISTANT1) and BES1 (BRI1-EMSSUPPSSOR1), the key transcription factors in BR signaling. The oxidative modification enhances BZR1 transcriptional activity by promoting its interaction with PIF4 (PHYTOCHROME INTERACTING FACTOR4) and ARF6 (AUXIN RESPONSE FACTOR6), thereby promoting root meristem development. However, at present, there is limited information about the relationship between ROS and cytokinins in regulation of apex growth. Taken together, it is clear that the hormonal and ROS networks can no longer be regarded as independent mechanisms. Rather, they are interconnected in order to trigger physiological and stress adaptation responses. Glutathione reductase (GR) plays a key role in controlling the levels of reduced glutathione in the Arabidopsis RAM. Excessive accumulation of oxidized glutathione in GR2 (Glutathione reductase2) mutants results in root apical cells entering the oxidized state and eventually leads to abnormal growth. Exogenous application of reduced glutathione partially restores the normal root phenotype. Glutathione regulates ROS levels in cells through both auxin/PLETHORA (PLT) dependent and independent pathways, thereby participating in and maintaining ROS homeostasis in the RAM. VTC1, which is a rate-limiting gene affecting the quantity of AsA, as well as several downstream steps, is shown to be a key modulator of H2O2 levels. Knockout of VTC1 resulted in elevated H2O2 levels and numbers of QC cells and periclinal divisions in the RAM. This result revealed the interaction between AsA and H2O2 in maintaining RAM size. Accumulating evidence has shown that the redox state of the cell affects its proliferation/differentiation program. For example, in Arabidopsis embryonic roots, division of cells in G1 phase is accelerated if they are in the oxidized state and slowed if they are in the reduced state. These results suggest that controlled oxidation is a key feature of the early stages of the plant cell cycle.

6In the Arabidopsis shoot apical meristem (SAM), enrichment of in stem cells activates the WUSCHEL gene to maintain stem cell activities, whereas H2O2 accumulation in the peripheral zone (PZ) promotes cell differentiation. Moreover, H2O2 negatively regulates generation in stem cells, and increased H2O2 levels or scavenging leads to the termination of stem cells. These results suggest that ROS mediate the control of plant stem cell fate, and the balance between and H2O2 is essential for shoot stem cell maintenance and differentiation. The expression of transcription factor TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF), which is associated with the cell cycle, is inhibited by higher ROS levels in the SAM. At the same time, glutaredoxin (GRX) reduces ROS levels in the SAM. Lower ROS levels activate TCP and directly regulate the expression of cell cycle-related genes CYCA2;3, and CYCB1;1, thus promoting SAM cell division and maintaining SAM stability. Recently, a novel thioredoxin DCC1 has been shown to determine the shoot regeneration capacity of various Arabidopsis ecotypes. Further studies demonstrated that DCC1 altered the activity of respiratory chain NAD(P)H dehydrogenase complex I. Knock-out of DCC1 triggered production of mitochondrial ROS. The process further regulates shoot regeneration. Meanwhile, six different SNPs (Single Nucleotide Polymorphism) in the DCC1gene sequence were found to be closely related to bud regeneration in different Arabidopsis ecotypes, and ROS levels varied in ecotypes harboring different SNPs. Taken together, these results demonstrate that modulation of ROS homeostasis plays an essential role in many processes from apical meristem maintenance to de novo shoot initiation.

7As signaling components, ROS are distributed in all plant tissues, especially in metabolically active tissues. Maintenance of ROS homeostasis and ROS generation regulates seed germination through GA and/or ABA metabolism and signaling in Arabidopsis and barley, respectively. Late embryogenesis abundant protein OsLEA5 interacted with zinc finger transcription factor ZFP36 to co-regulate ABA-inhibited seed germination by controlling the expression of APX OsAPX1 in rice. Phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) played a critical role in coordinating ROS homeostasis and cotyledon development in etiolated Arabidopsis seedlings. The results revealed that cytosolic Phe played a critical role during the transition of seedlings from heterotrophy to autotrophy by protecting the cells from oxidative damage, and by providing substrates for defense. ROS are also thought to play essential roles in leaf development, senescence and organ dormancy. For instance, expression of CAT2 (Catalase 2) is reduced in the leaves of Arabidopsis upon bolting. This results in accumulation of H2O2 which promotes the expression of the WRKY53 gene, which is required for leaf senescence. Loss of function of mitochondrial ATP-dependent protease FtSH4 increases ROS, and is involved in leaf senescence via regulation of WRKY-dependent salicylic acid (SA) accumulation and signaling. Potato tuber dormancy is a complicated physiological process. When levels are inhibited, tuber sprouting is delayed. When treated with exogenous H2O2, potato tuber dormancy is released. This indicates that different ROS play different roles during the progression of potato tuber dormancy. Similarly, the concentration of H2O2 also dynamically changes in olives as they progress from bud formation through fertilization, which indicates that the difference in H2O2 concentrations has important physiological significance for the development of different organs. During the process of Arabidopsis vernalization and flowering, the content of ROS initially increases and then decreases. This suggests that from the initiation of floral buds to maturation of sexual organs, ROS levels might play different roles. In rice, homeobox gene MADS3 has been proved to be essential for stamen formation during early floral development. However, at later stages of anther development, MADS3 regulates ROS homeostasis, and abnormal expression of MADS3 causes the accumulation of and pollen sterility. PEROXIDASE9 and PEROXIDASE40, which catalyze the oxidation of various substrates by H2O2, are genetically redundant and essential for proper anther and pollen development in Arabidopsis, likely through their extensin cross-linking activity. OsCIPK31 perceives the response to stresses and regulates ROS accumulation and IAA distribution in the panicle. Excess IAA might lead to ROS accumulation in the apical spikelet, which ultimately leads to cell death in rice panicles. H2O2 and superoxide are formed during lateral root (LR) development, and contribute to the elongation of LRs but, intriguingly, not to the initiation of LR primordia. In addition, ROS are also essential for the development of crown roots (CRs) in rice. WOX11, a WUSCHEL-related homeobox transcription factor, is required in crown root development. In wox11 mutants, significant alteration is observed in the expression of many genes involved in the regulation of ROS homeostasis. This suggests that ROS might be involved in crown root development controlled by WOX11. Accumulation of ethylene in rice CRs promotes the production of ROS under flooding. ROS together with other signals trigger epidermal cell death, thus promoting crown root emergence and elongation. SA inhibits the expression of ROS scavenging-related genes, which increases ROS levels and promotes root meristem activity. However, decreased ROS levels in the ABNORMAL INFLORESCENCE MERISTEM (AIM1) mutant, which participates in SA synthesis, resulted in inhibition of rice crown root growth. Exogenous application of SA or H2O2 could partially restore root development. These results further support the interplay between SA, ethylene and ROS in rice crown root development. RSL4 (ROOT HAIR DEFECTIVE SIXLIKE 4) is a member of the auxin-responsive factor family. In Arabidopsis, auxin promotes the expression of a series of ROS-related genes by activating the expression of RSL4, thereby regulating the elongation of root hair cells, indicating that ROS also play an important role in root hair development. These findings establish a molecular link between auxin and ROS-mediated polar root hair growth.

8A great deal of evidence has shown that environmental factors such as heat, cold, drought, Al toxicity, organic pollutants (OPs) and pathogens could induce ROS generation in plant cells (Table 2). ROS, acting as signaling molecules, trigger signal transduction pathways in response to those stresses. On the other hand, ROS cause irreversible cellular damage through their strong oxidative properties, which promote alterations in plant morphological structures that enhance resistance. Because of the existence of many interconvertible ROS, it is very difficult to distinguish between the cytotoxic and signaling events that are induced by a particular ROS. It should be pointed out that although ROS cause cell death, it is a necessary process to confer resistance to stress. Altogether, stress-induced ROS-activating responses have to occur rapidly with the appearance of the stress and should decay when the stress disappears. Plants lacking AtFtSH4, an ATP-dependent mitochondrial protease, exhibited an intriguing phenotype of precocious cessation of growth at both the SAM and RAM when grown at elevated temperature (LD 31°C). This was associated with accumulation of internal oxidative stress and progressive mitochondrial dysfunction. These results reveal that maintaining mitochondrial functionality within the SAM and RAM, which depends on AtFtSH4, is vital to preserve stem cell activity and adaptation to temperature stress throughout development. Ethylene accumulation induces the expression of RBOHH, a member of the NADPH oxidase gene family. Knock out of RBOHH by CRISPR/Cas9 reduces ROS accumulation and inducible aerenchyma formation in rice roots, which is essential for rice to adapt to flooding and other oxygen-deficient conditions. Under drought conditions, ABA prevents H2O2 accumulation through induction of CAT OsCATB expression and protects cells against ROS oxidative damage. When wheat E3 ubiquitin ligase TaPUB1 (The Plant U-Box Proteins 1) is transfected into tobacco, less accumulation of ROS and stronger antioxidant capacity are detected in the transgenic plants, thereby improving the survival rate of transgenic tobacco in drought stress. In Arabidopsis, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor RRTF1 (Redox Responsive Transcription Factor 1) is a component of the core redox signaling network. Its expression is rapidly and transiently stimulated by various ROS generated by biotic and abiotic signals. Elevated RRTF1 levels in plants causes ROS accumulation, which suggests that RRTF1 amplifies ROS formation in response to stresses. Stimulation of ROS production by RRTF1 might be important for a rapid and transient establishment of local ROS maxima to induce appropriate downstream responses. When pathogens invade, plants stimulate ROS production, which is rapidly triggered following detection of a pathogen and may synergistically activate the hypersensitive response (HR). In plant disease resistance, ROS play a positive role and directly kill invading bacteria, and at the same time enhance thickening of adjacent cell walls to prevent spread of invading pathogens. The rapid production of the ROS burst is a conserved signaling output in immunity across kingdoms. To protect against infection by fungal pathogens, plants have developed the pattern-recognition receptor (PRRs) for chitin perception, which triggers the intracellular activation of mitogen-activated protein kinase (MAPK) cascades for the rapid production of ROS. In Arabidopsis, PRR-Associated Kinase BIK1 directly phosphorylates NADPH oxidase RBOHD and causes the PAMP-induced ROS burst and antibacterial immunity. Additionally, environmental pollution by OPs also induces accumulation of both H2O2 and nitric oxide (NO) in root tips, resulting in increased malondialdehyde (MDA) content, an indicator of membrane lipid peroxidation, and abnormal root growth. Plant growth and stress tolerance regulator 24-epibrassinolide (EBR) induces non-enzymatic and enzymatic antioxidant defense systems in cucumber, and increases the content of antioxidants such as SOD, CAT, and GSH, that maintains the homeostasis of ROS in cells, consequently enhancing the resistance of cucumber to OPs.

9Epigenetic modifications, including both post-translational modifications of histone proteins and chemical modifications of DNA, often help regulate the expression of genes in specific redox pathways. Conversely, ROS have impacts on the epigenetic mechanisms of gene regulation. In mammals, histone deacetylases (HDACs) function in epigenetic regulation in connection with oxidative stress. HDACs can change conformation, consequently diminishing their catalytic activity or altering their cellular localization under oxidative stress. On the other hand, increases in ROS result in increases in various histone modifications such as H3K4me2/3, H3K79me3, H3k27me3, and H3K9me2, due to inhibition of histone demethylases. Growing evidence reveals a close link between ROS metabolism and epigenetic regulation during plant growth and environmental acclimation. Four distinct DNA demethylases, REPRESSOR OF SILENCING 1 (ROS1), DEMETER (DME), DME-like 2 (DML2), and DML3 catalyzed the active removal of 5-methylcytosine from DNA. Recent studies demonstrate that ROS1 and DME interact directly with the Fe–S cluster assembly machinery, which is highly susceptible to oxidation by ROS. Their activity can consequently be altered by stress-derived oxidative conditions. This result reveals a connection between DNA methylation and ROS metabolism. Rice plants overexpressing OsSRT1, a SILENT INFORMATION REGULATOR2 (SIR2)-related HDAC gene, have shown an enhanced tolerance to oxidative stress, while OsSRT1 RNAi induces H2O2 overproduction, DNA fragmentation, and cell death. Recent studies also showed that OsSRT1 not only inhibits the “moonlighting” transcriptional activation activity of glyceraldehyde-3-phosphatedehydrogenase (GAPDH),which binds the promoters of glycolytic genes and stimulates their expression, but also reduces the acetylation of GAPDH lysine residues and its nuclear accumulation that are otherwise enhanced by oxidative stress in rice seedlings. Cellular oxidation could reduce HDA19 and HDA9 activity, thereby enhancing histone acetylation and transcription of stress-responsive genes in Arabidopsis. Recent studies also showed that changes in ROS levels caused apparent epigenetic modifications such as acylation, which in turn regulated the activity of ROS related proteins in rice leaves. This suggests that the interplay between ROS and acylation might play important roles in the PTMs (post-translational modifications) of leaf proteins that have key metabolic functions. We also found that the expression of some ROS-related genes was linked to changes in their acetylation modification during crown root development in rice. This implies there is close cooperation between ROS and epigenetic regulation of gene expression during rice crown root development. However, further studies are needed regarding how interactions between ROS and epigenetic modifications regulate gene expression. Histone demethylation is catalyzed by two different classes of enzymes: the jumonji C (JmjC) demethylases, which are Fe (II)- and 2-oxoglutarate-dependent dioxygenases, and FAD-dependent amino oxidases, including lysine-specific demethylase 1 (LSD1). There is a clear connection between histone methylation, energy metabolism, and cell redox balance in animals and yeast cells. Many JmjC proteins have been reported to respond to plant exposure to stress by modulating the expression of stress-related genes, probably acting together with ROS generated during stressful conditions, leading to the establishment of a complex network of defense responses. However, it is not clear whether the activity of JmjC proteins is directly altered by ROS. In mammalian cells, glutathione (GSH) seems to be a new post-translational modifier of the histone code, capable of modulating the chromatin structure. Glutathionylation of histone H3 affects nucleosome stability leading to a more open chromatin structure. This reveals a new connection between epigenetic control and cellular redox homeostasis. In addition, GSH can influence epigenetic processes, inhibiting the activity of the enzymes involved in the synthesis of S-adenosyl-methionine (SAM), which is used by DNA methyltransferases (DNMTs) and HMTs as a substrate for DNA and histone methylation, respectively. Therefore, the modulation of GSH metabolism may control oxidative stress and epigenetic mechanisms. However, this still needs to be extensively investigated in plants.

10During recent years, sources of ROS, mechanisms of production and removal, and key antioxidant molecules and enzymes that scavenge ROS have been reported in plants. However, much of our current knowledge about ROS remains unclear. Firstly, most ROS have short half-lives and are prone to chemical reactions to produce water or secondary ROS. It is still difficult to accurately study how various ROS lead to signaling and drive plant growth and development in a strictly localized and timely manner. Secondly, we do not understand the interplay between the spatio-temporal production of various ROS and their activities. In certain cases, it is very difficult to distinguish whether oxidative stress is the cause or an effect of cellular damage. This restricts our further understanding of their roles in plants. Thirdly, the abnormal accumulation of ROS also leads to oxidative modification of some microRNA and proteins. The mismatch between oxidized miRNAs and proteins might be involved in the initiation of apoptosis, that eventually leads to the cell death. These findings shed new light on our current understanding of the significance of ROS functions. It will be interesting to characterize whether oxidatively modified miRNA or proteins are involved in plant growth and development. Finally, recent studies showed that interplay between ROS levels and epigenetic modifications had important roles in plant development and stress responses, and biotic and abiotic stresses greatly affected plant growth and redox states. However, the regulatory mechanism remains unknown. It will be important to explore the cross-talk between ROS and epigenetic modifications, which will contribute to understanding the mechanisms whereby ROS homeostasis, epigenetics and plant adaptation and tolerance are mutually regulated.

11YZ wrote and revised the manuscript. HH collected all the materials and wrote the draft. FU and D-XZ revised the manuscript. MY gave some suggestion and advice.

12The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.