Spatially patterned hydrogen peroxide orchestrates stomatal development in Arabidopsis

Shi, Wen; Wang, Lingyan; Yao, Lianmei; Hao, Wei; Han, Chao; Fan, Min; Wang, Wenfei; Bai, Ming-Yi

doi:10.1038/s41467-022-32770-7

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Published: 26 August 2022

Spatially patterned hydrogen peroxide orchestrates stomatal development in Arabidopsis

Nature Communications volume 13, Article number: 5040 (2022) Cite this article

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Abstract

Stomatal pores allow gas exchange between plant and atmosphere. Stomatal development is regulated by multiple intrinsic developmental and environmental signals. Here, we show that spatially patterned hydrogen peroxide (H₂O₂) plays an essential role in stomatal development. H₂O₂ is remarkably enriched in meristemoids, which is established by spatial expression patterns of H₂O₂-scavenging enzyme CAT2 and APX1. SPEECHLESS (SPCH), a master regulator of stomatal development, directly binds to the promoters of CAT2 and APX1 to repress their expression in meristemoid cells. Mutations in CAT2 or APX1 result in an increased stomatal index. Ectopic expression of CAT2 driven by SPCH promoter significantly inhibits the stomatal development. Furthermore, H₂O₂ activates the energy sensor SnRK1 by inducing the nuclear localization of the catalytic α-subunit KIN10, which stabilizes SPCH to promote stomatal development. Overall, these results demonstrate that the spatial pattern of H₂O₂ in epidermal leaves is critical for the optimal stomatal development in Arabidopsis.

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Introduction

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere and play pivotal roles in regulating CO₂ uptake for photosynthesis as well as water vaporization through transpiration^1,2,3. In Arabidopsis (Arabidopsis thaliana), stomata arise from a subset of undifferentiated meristemoid mother cells, which undergo an asymmetric cell division to generate a meristemoid and a stomatal lineage ground cell (SLGC) as its smaller and larger daughters, respectively. The meristemoid is the stomatal precursor, which may go through one to three asymmetric divisions to produce guard mother cells^1,2. The guard mother cell undergoes a single symmetric division to eventually generate a pair of guard cells surrounding a microscopic pore⁴. The division and differentiation of stomatal lineage cells are tightly controlled by a suit of closely related and sequentially expressed basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (SPCH), MUTE, and FAMA in Arabidopsis^4,5,6. SPCH is mainly expressed in the meristemoid cells and regulates the initiation and proliferation of stomatal precursors⁵. Several endogenous and environmental signals finely regulate SPCH activity to control stomatal development in response to changing environment^7,8,9,10,11.

Hydrogen peroxide (H₂O₂), an endogenous oxidant present in all aerobic cells, was recently viewed as a key signaling molecule in plant stem cell regulation^12,13. Several studies have reported that H₂O₂ spatial distribution is critical for the plant stem cell activities^14,15,16,17. In the shoot apical meristem (SAM), H₂O₂ is remarkably enriched in the peripheral zone to promote stem cell differentiation¹⁵. The accumulated H₂O₂ induces the reversible protein phase separation of transcription factor TERMINATING FLOWER (TMF) in the SAM of tomato. H₂O₂-induced oxidative modification activates TMF to bind and inhibit the expression of the floral identity gene ANANTHA, thereby directing the stem cell fate for flowering transition¹⁷. In the root apical meristem (RAM), H₂O₂ mainly accumulates in the expanding cells of the elongation zone, whereas O₂^•− mainly accumulates in the dividing cells of meristem zone. The basic helix-loop-helix (bHLH) transcription factor UPBEAT1(UPB1) regulates the expression of a set of peroxidases that are involved in establishing the reactive oxygen species (ROS) gradient pattern in root tips¹⁴. The ROOT MERISTEM GROWTH FACTOR1 (RGF1) peptide also plays a critical role for the ROS gradient pattern in root tips. RGF1 binding to RGF1 Receptor (RGFR) activates the transcription factor RGF1 INDUCIBLE TRANSCRIPTION FACTOR1 (RITF1) to control the ROS distribution and enhance the stability of PLT1/2, thus determining the root meristem size¹⁶. In addition to SAM and RAM, there are other meristems in plants to regulate their own development; however, it remains unclear whether H₂O₂ presents a specific spatial distribution pattern in other meristems to control their activities.

The maintenance of cellular and organismal energy homeostasis is vital for all living organisms. Eukaryotes have evolved a very sophisticated system to modulate the metabolism based on nutrient availability¹⁸. A central component of this system in plants is SUCROSE NON-FERMENTING-1 (SNF1)-RELATED KINASE 1 (SnRK1), which is orthologous to the AMP-ACTIVATED KINASE (AMPK) and SNF1 in mammals and yeast, respectively^19,20,21. SnRK1/AMPK/SNF1 function as cellular energy sensors to induce catabolic reactions and repress energy-consuming anabolic processes when energy supplies become limited^22,23. SnRK1 in Arabidopsis is a heterotrimeric complex that includes a catalytic α-subunit (KIN10, KIN11, and KIN12), two regulatory subunits consisting of three isoforms of KINβ (KINβ1, KINβ2, and KINβ3), and an atypical KINβγ subunit^20,24. Low-energy stress caused by darkness or hypoxia promoted KIN10 nuclear translocation to regulate downstream gene expression²⁵. The myristoylated and membrane-associated SnRK1 β regulatory subunits interact with KIN10 and restrict its nuclear localization, thereby inhibiting its functions^25,26. A recent study showed that KIN10 exhibits the ubiquitous expression pattern in all epidermal cells of Arabidopsis leaves, but is mainly localized to the nucleus of meristemoid cells to phosphorylate and stabilize SPCH, then promoting stomatal development¹¹. However, the molecular mechanism of the specific nuclear localization of KIN10 in meristemoid cells remains unclear. Here, we demonstrated that H₂O₂ is specifically enriched in meristemoids to reduce the interaction between KIN10 and KINβ2, thereby promoting KIN10 nuclear localization and activating SPCH to promote stomatal development.

Results

H₂O₂ induces KIN10 nuclear localization in plants

Our previous study showed that when plants were grown in ½ MS liquid medium containing 1% sucrose under long-day condition, KIN10 is expressed in all epidermal cells of Arabidopsis leaves, but the subcellular localization of KIN10 protein exhibited a specific spatial pattern and was enriched in the nucleus of the stomatal lineage cells and guard cells as well as in the cytoplasm of the pavement cells in the epidermal cells of Arabidopsis cotyledons¹¹. To determine whether KIN10 has such a spatial subcellular localization pattern in plants that are grown under other environmental conditions, we analyzed the subcellular localization of KIN10 in cotyledon epidermal cells of plants grown in ½ MS solid medium with or without 1% sucrose under different light intensities or different photoperiod conditions. The results showed that the nuclear-localized KIN10 proteins were highly enriched in the stomatal lineage cells and guard cells under all examined conditions, and that sucrose enhanced this distribution pattern of KIN10 (Supplementary Fig. 1a–d). To determine whether this spatial subcellular localization pattern of KIN10 exists in other leaves, we analyzed the subcellular localization of KIN10 in the rosette leaves and found a similar distribution pattern as that in cotyledons (Supplementary Fig. 1e, f). These results suggest that the spatial subcellular localization pattern of KIN10 is widespread in plant leaves.

Metabolic stress caused by hypoxia or 3-(3,4-dichlorophenyl)−1,1-dimethylurea (DCMU) treatment has been reported to induce KIN10 nuclear translocation²¹. ROS are the byproducts of aerobic metabolism and play critical roles in plant growth, development, and stress responses^12,13. Therefore, we speculated that metabolic stress may generate ROS to induce KIN10 nuclear localization. To test this possibility, we measured the H₂O₂ content of wild-type plants in the presence or absence of DCMU and/or potassium iodide (KI), a quencher of H₂O_2, using the fluorescent dye 2′,7′- dichlorodihydrofluorescein diacetate (H₂DCFDA). The results showed that DCMU treatment significantly increased the H₂O₂ levels, whereas DCMU and KI cotreatment inhibited H₂O₂ accumulation (Supplementary Figs. 2a, b and 3a, b). Staining with 3,3′-diaminobenzidine (DAB) further confirmed that DCMU treatment resulted in H₂O₂ accumulation in the leaves, whereas KI cotreatment strongly counteracted the effects of DCMU (Supplementary Fig. 2c, d). To examine the effects of DCMU and KI on KIN10 subcellular localization, we analyzed the subcellular localization of KIN10 in the leaf epidermal cells of the pKIN10::KIN10-YFP transgenic plants that were grown on ½ MS solid medium containing or spraying with or without DCMU and/or KI. DCMU treatment significantly increased the nuclear-to-cytoplasmic ratio of KIN10-YFP in the stomatal lineage cells, pavement cells and guard cells, but such effects of DCMU were counteracted by the cotreatments with KI (Fig. 1b–d, Supplementary Figs. 3c, d, and 4a, b). These results indicate that H₂O₂ plays an important role for DCMU-induced KIN10 nuclear localization in plants.

**Fig. 1: H₂O₂ induces the nuclear localization of KIN10.**

To examine the effects of H₂O₂ on KIN10 subcellular localization, we analyzed the relative levels of nuclear and cytoplasmic signals of KIN10-YFP with or without H₂O₂ treatment. The results showed that H₂O₂ treatment significantly increased the nuclear-to-cytoplasmic ratio of KIN10-YFP in meristemoids, stomatal lineage cells, pavement cells, and guard cells (Fig. 1e–g, Supplementary Figs. 4c, d and 7a–f). To determine whether the H₂O₂-induced nuclear localization of KIN10 is due to the biosynthesis of KIN10 protein, we analyzed the H₂O₂-promoted nuclear enrichment of KIN10 in the presence of cycloheximide (CHX), a protein translation inhibitor. The results showed that CHX didn’t affect the H₂O₂-mediated nuclear localization of KIN10 (Fig. 1e–g and Supplementary Fig. 4c, d). To determine whether other types of ROS regulate the nuclear localization of KIN10, we analyzed the subcellular localization of KIN10 in the presence of methyl viologen (MV) or N,N’-dimethylthiourea (DMTU), which are specific superoxide anion (O₂^•−)-generating reagent or the scavenger of free radicals, respectively^27,28. The results showed that MV and DMTU treatment had no significant effects on the subcellular localization of KIN10 in the epidermal cells of Arabidopsis cotyledons (Supplementary Fig. 5a–d). These results suggest H₂O₂ specifically induces the nuclear localization of KIN10 in plants.

To corroborate these pharmacological data, we analyzed the effects of H₂O₂ on KIN10 subcellular localization in the catalase overexpressing plants (p35S::CAT2-Myc), which accumulate less H₂O₂ in plants. The results showed that the nuclear signals of KIN10-YFP in the stomatal lineage cells, pavement cells and guard cells were significantly reduced in the p35S::CAT2-Myc plants compared with those in the wild-type background, and such reduction effects were counteracted by H₂O₂ treatment (Fig. 1h–l and Supplementary Fig. 6a, b). These results suggest H₂O₂ plays an important role in the KIN10 nuclear localization in plants.

H₂O₂ specifically accumulates in meristemoids

Considering the important role of H₂O₂ in KIN10 nuclear localization in plants, it is speculated that the meristemoid cells may contain high levels of H₂O₂. To test this hypothesis, we examined the H₂O₂ distribution pattern in cotyledon epidermal cells of wild-type plants that were grown on ½ MS solid medium containing 1% sucrose under long-day conditions using the H₂DCFDA and BES-H₂O₂-Ac fluorescent dyes. The reduced non-fluorescent compound H₂DCFDA can be oxidized and converted into fluorescent 2′, 7′-dichlorofluorescein (DCF) by intracellular H₂O₂. BES-H₂O₂-Ac is a highly selective fluorescent probe for H₂O₂, and exhibits a higher H₂O₂ specificity but a lower fluorescent signal than H₂DCFDA²⁹. The results showed that the fluorescent signals of H₂DCFDA and BES-H₂O₂-Ac were specifically enriched in the meristemoid cells and guard cells, but less distributed in pavement cells of Arabidopsis leaves on the abaxial side. (Fig. 2a–h and Supplementary Fig. 8a–d). HyPer is a genetically encoded probe, in which the H₂O₂-sensitive transcription factor OxyR is incorporated into circularly permuted yellow fluorescent protein³⁰. It has been shown that Hyper is highly sensitive to H₂O₂ and not to other ROS³⁰. The plants expressing the HyPer have been used to analyze the real-time production of H₂O₂ in plants. In the pHyPer transgenic plants, H₂O₂ content is measured by determining the ratio of fluorescent signals at an excitation wavelength of 488 nm relative to that at 405 nm³¹. In the abaxial epidermal cells of wild-type leaves, the ratio of fluorescent signals was higher in the meristemoid cells and guard cells, but lower in the pavement cells (Fig. 2i-l and Supplementary Fig. 8e, f). DAB staining was also used to analyze the H₂O₂ distribution pattern in the leaves and found that H₂O₂ was more abundant in the abaxial meristemoid cells and guard cells than in the pavement cells (Fig. 2m). To further verify whether H₂O₂ is enriched in meristemoids, we examined the co-localization of the fluorescent signals of H₂DCFDA and pSPCH::SPCH-RFP. The results showed that the fluorescent signals of H₂DCFDA and SPCH-RFP were present simultaneously in the meristemoid cells (Fig. 2n, o). These results indicate that H₂O₂ is highly enriched in the abaxial meristemoids and guard cells in the epidermal cells of Arabidopsis leaves.

**Fig. 2: H₂O₂ specifically accumulates in meristemoid cells.**

To verify the enrichment of H₂O₂ in meristemoids and guard cells, we also analyzed the H₂O₂ distribution pattern in cat2 cat3, upb1, p35S::CAT2-Myc, and p35S::UPB1 plants^14,32. CAT2 and CAT3 are key catalase enzymes that scavenge H₂O₂ in plants^33,34. UPB1 has been reported to repress a set of peroxidases to regulate the meristem activity in the plants^14,15. The H₂DCFDA fluorescent signals were stronger in the cat2 cat3 and p35S::UPB1 plants than those in the wild-type plants, whereas the distribution pattern of H₂O₂ was similar to that in wild-type plants. The H₂DCFDA fluorescent signals were weaker in the upb1 and p35S::CAT2-Myc plants than those in wild-type plants, and H₂DCFDA fluorescent signals were observed only in the abaxial guard cells, but very weak in meristemoid and pavement cells (Supplementary Fig. 9a–d). These results indicate that catalase and UPB1 play important roles in the H₂O₂ distribution pattern in epidermal cells of leaves.

To determine whether the spatial pattern of H₂O₂ in the epidermal cells is widespread, we analyzed the H₂O₂ distribution pattern in the cotyledon epidermal cells of plants grown on ½ MS solid medium with or without 1% sucrose under different light intensities or different photoperiod conditions. The results showed that H₂O₂ specifically accumulated in the meristemoid cells under all conditions examined (Supplementary Fig. 10a–d). We also analyzed the H₂O₂ distribution pattern in the rosette leaves, and found that H₂O₂ was highly enriched in the meristemoid cells (Supplementary Figs. 10e, f and 11a–d). These results suggest that the spatial pattern of H₂O₂ is widespread in the epidermal cell of Arabidopsis leaves.

To determine whether other types of ROS display the specific distribution pattern in the epidermal cells of leaves, we analyzed the O₂^•− distribution pattern using the fluorescent dye dihydroethidium (DHE), which specifically detects O₂^•−. The results showed that O₂^•− was evenly distributed in the abaxial epidermal cells of leaves and did not exhibit a specific distribution pattern (Supplementary Fig. 12a–f).

SPCH directly represses the expression of CAT2 and APX1 in meristemoid cells

To elucidate the mechanism underlying H₂O₂-specific accumulation in meristemoid cells, we analyzed the expression patterns of ROS-related genes in the published cell-type-specific transcriptomic data of Arabidopsis leaves. The expression levels of several ROS-scavenging genes, such as CAT2 and ASCORBATE PEROXIDASE 1 (APX1), were lower in the meristemoid cells than in other types of cells³⁵. To test the bioinformatic analysis results, the pCAT2::GFP and pAPX1::GFP transgenic plants were generated to examine the expression patterns of CAT2 and APX1 in the epidermal cells of leaves. The fluorescent signals of pCAT2::GFP and pAPX1::GFP were clearly observed in the pavement cells, but were difficult to detect in the smaller cells and guard cells (Fig. 3a–d and Supplementary Fig. 13a–c). Generally, the expression patterns of CAT2 and APX1 revealed a positive relationship between the intensity of GFP and the size of epidermal cells (Supplementary Fig. 14a–d). Co-localization analysis with pCAT2::GFP and pSPCH::SPCH-RFP revealed that pCAT2::GFP exhibited very weak signals in the meristemoid cells labeled with pSPCH::SPCH-RFP (Fig. 3e–g). These results indicate that CAT2 and APX1 have low expression levels in meristemoids.

**Fig. 3: SPCH directly represses the expression of *CAT2*.**

The published ChIP-Seq data showed that SPCH binds to the promoters of CAT2 and APX1³⁶. To verify this result, we performed chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and DNA-protein pull-down assays. The results confirmed that SPCH directly binds to the promoters of CAT2 and APX1 (Fig. 3h, i and Supplementary Fig. 13d, e). Quantitative RT-PCR analysis showed that the expression levels of CAT2 and APX1 were increased in spch-4 mutant but decreased in p35S::SPCH-Myc transgenic plants (Fig. 3j, and Supplementary Fig. 13f). These results indicate that SPCH directly binds to the promoters of the ROS-scavenging gene CAT2 and APX1 to repress their expression.

H₂O₂ promotes stomatal development

To address the biological function of enriched H₂O₂ in meristemoids, we examined the stomatal phenotypes of wild-type plants, cat2, cat3, cat2 cat3, cat1 cat2 cat3 mutants, and p35S::CAT2-Myc plants. The amount of meristemoid cells and GMC in the abaxial epidermis at 5 days after germination (DAG) was significantly increased in cat2, cat3, cat2 cat3 and cat1 cat2 cat3 mutants compared with that in the wild type, but was decreased in p35S::CAT2-Myc transgenic plants (Fig. 4a, b). As a result, at 8 DAG, the cat2 and cat3 single mutants exhibited a slightly increased stomatal index, which refers to the number of stomata relative to the total number of epidermal cells, and the cat2 cat3 double mutants and cat1 cat2 cat3 triple mutants showed a significantly increased stomatal index (Supplementary Fig. 15a). Overexpression of CAT2 dramatically reduced the stomatal index, and H₂O₂ treatment partially suppressed the decreased stomatal index of CAT2 overexpression plants (Supplementary Fig. 15a, b). To examine the role of spatial H₂O₂ distribution in stomatal development, we manipulated CAT2 activity in the stomatal lineage cells by expressing CAT2 under SPCH promoter. The results showed that the expression of CAT2 in stomatal lineage cells significantly inhibited stomatal initiation and reduced the stomatal index, and H₂O₂ treatment also partially suppressed the decreased stomatal index of pSPCH::CAT2-RFP transgenic plants (Fig. 4a, b and Supplementary Fig. 15a, b). Consistent with this, the apx1 mutant and p35S::UPB1 transgenic plants exhibited the significant increased stomatal index, whereas the upb1 mutant exhibited the decreased stomatal index (Supplementary Fig. 15c). These results indicate that H₂O₂ plays an important role in stomatal development.

To further determine H₂O₂ effects on stomatal development, we analyzed the stomatal phenotypes of wild-type and kin10 mutants that were grown on ½ MS solid medium containing various concentrations of H₂O₂. The results showed that H₂O₂ increased the stomatal index at low concentrations, but such effects decreased in the presence of high concentrations of H₂O₂ (Fig. 4c). The kin10 mutant displayed a low stomatal index, and H₂O₂ treatment had no significant effects on stomatal development of kin10 mutant (Fig. 4c). Consistent with this, H₂O₂ failed to increase the stomatal index of pSPCH::SPCH^S4A-RFP/spch-4 transgenic plants, in which SPCH^S4A could not be phosphorylated by KIN10. H₂O₂ treatment increased SPCH protein levels, but had no significant effects on SPCH^S4A (Supplementary Fig. 16a-c). These results suggest that KIN10 contributes to H₂O₂-induced stomatal development.

To determine the effects of CAT-mediated H₂O₂ scavenging on the stomatal lineage cell fate transition, we compared the fluorescent signals of cell-type-specific marker lines pSPCH::nucGFP and pSPCH::SPCH-GFP in wild-type and cat2 cat3 mutant background. The marker line pSPCH::nucGFP is mainly expressed in the stomatal lineage cells and the marker line pSPCH::SPCH-GFP is mainly expressed in the meristemoids and MMCs. We observed that mutation of CAT2 and CAT3 markedly increased the number of cells marked by pSPCH::nucGFP and pSPCH::SPCH-GFP compared with those in the wild-type background, whereas KI treatment substantially suppressed this increase (Fig. 4d-g). These results indicate that H₂O₂ increases the frequency of stomata-producing division in Arabidopsis leaves.

To further determine whether other types of ROS regulate stomatal development, we analyzed the stomatal phenotype of plants treated with DMTU, the scavenger of free radicals. DMTU treatment did not alter the expression patterns of pSPCH::nucGFP and pSPCH::SPCH-GFP, and had no significant effects on the stomatal index (Supplementary Fig. 17a-e). Moreover, the ndufs4 mutant, in which accumulate less O₂^•− in plants by affecting the NADH dehydrogenase subunits^37,38, showed a similar stomatal index as wide type (Supplementary Fig. 17f). To further test the biological function of O₂^•− in stomatal development, we treated plants with Diethyldithiocarbamic acid (DDC) and MV, which increased the O₂^•− content in plants, and found that DDC and MV had no significant effects on stomatal development (Supplementary Fig. 17g, h). The published transcriptomic data indicated that the expression of O₂^•− biosynthesis and metabolism-related genes did not display cell-type-specific pattern³⁵. Collectively, these results suggest that O₂^•− is not involved in stomatal development in Arabidopsis.

H₂O₂ reduces the interaction between KIN10 and SnRK1 β subunits

A recent study showed that the membrane-associated SnRK1 regulator β subunits interact with KIN10 to restrict KIN10 nuclear localization²⁵. Our results indicated that H₂O₂ promotes the nuclear localization of KIN10 to induce stomatal development. Therefore, we speculate that H₂O₂ may regulate KIN10 subcellular localization by regulating the interaction between KIN10 and SnRK1 β subunits. To test this hypothesis, we analyzed H₂O₂ effects on the binding affinity of KIN10 to KINβ2, which plays major roles in the inhibition of KIN10 nuclear localization²⁵. The pull-down assays revealed that glutathione-S-transferase (GST)-KIN10 interacted with maltose-binding protein (MBP)-KINβ2, but not MBP alone, and H₂O₂ treatment markedly inhibited the interaction between KIN10 and KINβ2 in a dose-dependent manner (Fig. 5a, b and Supplementary Fig. 18). The co-immunoprecipitation assays confirmed that KIN10 interacted with KINβ2 in plants, but this interaction was significantly reduced by H₂O₂ treatment (Fig. 5c, d). To determine whether H₂O₂ regulates the nuclear localization of KIN10 through SnRK1 β subunits, we expressed KIN10 and KINβ2 in the epidermal cells of the tobacco leaves with or without H₂O₂ treatment. The results showed that KIN10-YFP distributed in the nucleus and cytoplasm, co-expression with KINβ2 resulted in the exclusion of KIN10-YFP from the nucleus, and H₂O₂ treatment promoted the nuclear localization of KIN10 (Fig. 5e, f). Consistent with this, the ratio of nuclear-to-cytoplasmic KIN10 protein was significantly decreased in p35S::KINβ2-RFP transgenic plants, but increased in kinβ2 mutant, and H₂O₂ treatment significantly increased the nuclear-to-cytoplasmic ratio of KIN10 in plants (Fig. 5g, h).

**Fig. 5: H₂O₂ induces KIN10 nuclear translocation through reducing the interaction between KIN10 and KINβ2.**

To understand the function of SnRK1 β subunits in the stomatal development, we analyzed the stomatal development phenotypes of wild type, SnRK1 β subunit loss-of-function and overexpression plants. The results showed that the kinβ2 single loss-of-function mutants displayed the slightly increased stomatal index, but simultaneous mutation of KINβ1 and KINβ2 resulted in the significantly increased stomatal index (Fig. 5i, j). Overexpression of KINβ2 exhibited the decreased stomatal index (Fig. 5i, j). These results indicate that SnRK1 β subunits negatively regulate the stomatal development.

Discussion

As the major epidermal structure that facilitates gas exchange between plants and the atmosphere, the formation of stomata is controlled by multiple developmental and environmental signals. Here, we showed that the spatially patterned redox signal, H₂O_2, orchestrates stomatal development in Arabidopsis. SPCH directly binds to the promoters of CAT2 and APX1 to reduce their expression and increase H₂O₂ levels in meristemoid cells. H₂O₂ reduces the interaction between KIN10 and KINβ2, thereby promoting the nuclear localization of KIN10. Nuclear-localized KIN10 phosphorylates and stabilizes SPCH, and subsequently promotes stomatal development. Our results provide a mechanistic framework for H₂O₂-mediated control of epidermal cell differentiation in Arabidopsis leaves (Fig. 5k).

The fine-controlled gradient distribution patterns of H₂O₂ and O₂^•− are critical for the balance between plant stem cell maintenance, proliferation, and differentation¹³. H₂O₂ promotes plant stem cell differentiation, and O₂^•− is the key signaling molecule for plant stem cell maintenance. In the shoot apex, O₂^•− highly accumulates in the central zone to maintain stemness, and H₂O₂ accumulates in the peripheral zone to promote stem cell differentiation¹⁵. In the Arabidopsis root tip, H₂O₂ mainly accumulates in the expanding cells of the elongation zone, whereas O₂^•− mainly accumulates in the dividing cells of meristem zone¹⁴. However, in the present study, we only observed the specific accumulation of H₂O₂ in meristemoid cells, and no specific spatial distribution pattern of O₂^•− in the leaf epidermal cells. One possibility for the different distribution patterns of H₂O₂ and O₂^•− in the leaf epidermal cells is that there are different organizational structure and different regulatory mechanisms in the leaf epidermal stem cells compared with shoot and root apical meristems. The shoot or root apical meristems are organized with the slowly dividing cell zones, such as the central zone at shoot and the quiescent center at root, and the differentiated amplifying cell zones such as the peripheral zone of shoot and the division zone of root. In the epidermal cells of leaves, there is no specific organ that functions similar to the central zone or quiescent center with slow cell division, resulting in no specific enrichment of O₂^•− in leaf epidermal cells.

The spatial distribution of H₂O₂ in meristems is essential for the stem cell differentiation in plants^14,15,39,40. Here, we showed that H₂O₂ is abundantly enriched in meristemoid cells to promote stomatal development. The Arabidopsis seeds contain a lot of triacylglycerols, which are hydrolyzed during seed germination to provide carbon skeletons and energy for early seedling growth, whereas a large amount of H₂O₂ is generated as a by-product of fatty acid β-oxidation^41,42,43,44. In addition, the plant shoot apical meristem is embedded in a low-oxygen niche and new leaves are produced under the hypoxic condition. When the new leaves emerge from the stem, they encounter an aerobic environment, thereby producing massive amounts of H₂O₂. Catalases and ascorbate peroxidase are key H₂O₂ scavenging enzymes in plants, and play important roles in plant antioxidative and detoxification processes that are closely correlated with ROS generation during plant growth and stress responses. Our results showed that CAT2 and APX1 are widely expressed in most cells of leaf epidermis except meristemoid cells. SPCH directly binds to the promoters of CAT2 and APX1 to repress their expression in meristemoid cells, resulting in the reduced H₂O₂ content in meristemoid cells. UPB1 was previously identified as a key transcription factor to repress a set of peroxidase expression in root. The accumulated H₂O₂ in meristemoids induces the nuclear localization of KIN10, which phosphorylates and stabilizes SPCH to promote stomatal development, thereby forming a positive feedback loop to control stomatal development.

H₂O₂ has been reported to promote stomatal closure by increasing the concentrations of cytosolic free calcium. The plant stress hormone abscisic acid (ABA) as well as diverse biotic and abiotic stress stimulated H₂O₂ accumulation, and subsequently induced stomatal closure, which suggests that H₂O₂ acts as a negative regulator for stomata to inhibit their function on gas exchange between plants and the atmosphere. In the present study, we showed that H₂O₂ positively regulates stomatal development. This opposite regulation on stomatal functions by H₂O₂ may be due to the different effects of different H₂O₂ contents on stomatal development and stomatal movement. The content of H₂O₂ in guard cells is approximately 10 times higher than that in meristemoid cells when plants were grown under normal conditions. ABA treatment significantly increase in the H₂O₂ content in the guard cells, suggesting that the concentrations of H₂O₂ in meristemoid cells that is required to promote stomatal development is at least 10 times lower than that in guard cells, which are responsible for promoting stomatal closure. Consistent with this, our results showed exogenous H₂O₂ treatment promoted stomatal development, but such promoting effects decreased as the increasing H₂O₂ concentrations. Furthermore, our previous study showed that H₂O₂ promoted stomatal opening in the intact leaves at the low concentrations⁴⁵. The rbohd rbohf mutant and CATALASE2 overexpression transgenic plants exhibited low H₂O₂ concentrations in guard cells and failed to open stomata normally⁴⁵. These results indicate that H₂O₂ promotes the stomatal development and stomatal opening at low concentrations, thereby inducing efficient CO₂ uptake and the photosynthesis under the normal growth conditions; whereas H₂O₂ promotes stomatal closure at high concentrations, then reducing water loss and improving stress resistance of plant under stress conditions.

A recent study reported that plastoquinone oxidation inhibits stomatal development by negatively regulating SPCH and MUTE expression⁴⁶. DCMU is a photosynthesis inhibitor, and can induce the oxidation of plastoquinone. DCMU treatment quickly induced the phosphorylation of MPK6 to activate MPK6. Activated MPK6 interacted and phosphorylated the epidermal specific expressed HD-ZIP transcription factor HDG2, and then inhibited the transcriptional activity of HDG2 and reduced the expression of SPCH and MUTE, thereby inhibiting stomatal development. In the present study, we showed that short-term DCMU treatment induced the nuclear localization of KIN10, while the long-term DCMU treatment led to KIN10 protein degradation. DCMU treatment induced the accumulation of H₂O₂ that reduced the interaction between KIN10 and KINβ2 and promoted KIN10 nuclear localization. Cotreatment of KI and DCMU counteracted the promoting effects of DCMU on KIN10 nuclear localization, suggesting H₂O₂ is required for DCMU-induced nuclear localization of KIN10. However, DCMU also has been reported to have no significant effects on H₂O₂ accumulation^46,47,48. One possibility for this inconsistency among different studies maybe the different inhibitory effects of DCMU on photosynthesis in different studies. Plastoquinone functions not only an essential component of photosynthesis to transport electrons, but also acts as a potent antioxidant to regulate the state transitions and gene expression⁴⁹. Treatment with short-term and low-dose DCMU leads to plastoquinone oxidation, while long-term and high-dose DCMU treatment leads to plastoquinone oxidation and H₂O₂ generation, and much high-dose DCMU treatment leads to higher H₂O₂ accumulation and programmed cell death.

SnRK1 is a central metabolic regulator of energy homeostasis and plays important roles in regulating plant growth and development in response to energy status^19,20. Our previous work showed that when plants were grown under mild energy starvation conditions, such as short-day photoperiod or liquid cultures, sucrose supply induced the protein accumulation of KIN10, which phosphorylated and stabilized SPCH to promote stomatal development¹¹. Such promoting effects of sucrose and KIN10 on stomatal development were significantly reduced when plants were grown under high light irradiance or long-term photoperiod conditions compared to plants grown under low light irradiance or short-term photoperiod conditions, suggesting the low light irradiance or short-term photoperiod specifically activate KIN10 through an unknown mechanism¹¹. In a recent study, we showed that sucrose dynamically regulates stomatal development through fine-tuning KIN10 activity. Sucrose induced the total KIN10 protein accumulation at all tested concentrations, but only induced the phosphorylated KIN10 protein accumulation at the low concentrations. Under high sucrose concentrations, sucrose supply triggers the accumulation of trehalose-6-phophate, which represses KIN10 activity by reducing the interaction between KIN10 and its upstream kinase SnAK1/2. Consistent with this, sucrose promoted stomatal development at low concentrations, but such promoting activity decreased with the increasing sucrose concentrations, suggesting phosphorylation is an important regulatory mechanism for KIN10-mediated stomatal development⁵⁰. Here, in this study, we showed that H₂O₂ induced the nuclear localization of KIN10 by reducing the interaction between KIN10 and KINβ2. In the epidermal cells of Arabidopsis leaves, H₂O₂ specifically accumulated in the meristemoid cells, which induces the nuclear localization of KIN10. Disruption of the specific spatial distribution of H₂O₂ in the epidermal cells using SPCH promoter to drive CAT2 expression significantly inhibits stomatal development, suggesting H₂O₂-mediated KIN10 nuclear localization plays critical roles for KIN10-mediated stomatal development. Taken together, sucrose-mediated KIN10 protein accumulation, H₂O₂-mediated KIN10 nuclear localization, and KIN10 phosphorylation, these three regulator mechanisms finely regulate KIN10 activity to optimize stomatal development in response to diverse environmental and developmental signals.

Methods

Plant materials and growth conditions

All wild-type, various mutants, and transgenic plants in this study are in Col-0 ecotype background. Mutants and transgenic plants used in this study include p35S::KIN10-Myc¹¹, pKIN10::KIN10-Myc¹¹, pKIN10::KIN10-YFP¹¹, kin10¹¹, pCAT2::GFP, pAPX1::GFP, cat2, cat2 cat3, cat1 cat2 cat3³², p35S::CAT2-Myc, pSPCH::nuc-GFP⁵, pSPCH::SPCH-GFP⁵, pSPCH::SPCH-RFP¹¹, pSPCH::SPCH-RFP/spch-4 (SPCH-8#)¹¹, pSPCH::SPCH^S4A-RFP/spch-4 (4A-2#)¹¹, p35S::SPCH-Myc¹¹, p35S::SPCH-4A-Myc¹¹, pSPCH::CAT2-RFP, p35S::UPB1, pKINβ2::KINβ2-YFP, and p35S::KINβ2-YFP. The T-DNA insertion mutants of SALK_076998 (cat2), SALK_092911 (cat3), SALK_000249 (apx1), SALK_115536 (upb1), SALK_008325 (kinβ1-1), and SALK_052521 (kinβ2) were ordered from the Arabidopsis Biological Resource Center. Plants were grown in a greenhouse with white light at 100 μMol/m²/s under a 16 h light/8 h dark photoperiod at 22 °C for general growth. For chemical treatment, the 4-day-old general growth seedlings of the indicated plants treated with or without DMSO (Mock), 50 μM DCMU and/or 1 mM KI for 12 h.

Plasmid constructs and transgenic plants

Full-length coding region of KINβ2 and CAT2 without stop codon were amplified by PCR and cloned into pENTR^TM/SD/D-TOPO^TM vectors (Thermo Fisher), and then recombined with destination vector pX-YFP (p35S::X-YFP), p1390-MH (p35S::X-Myc-His), and pAL-1296R (Native promoter::RFP), and pMAL2CGW (N-MBP). The promoters of CAT2 and APX1, the promoter and genomic DNA of KINβ2, were amplified by PCR and cloned into pENTR^TM/SD/D-TOPO^TM vector (Thermo Fisher), and then recombined with destination vector pEG-TW1 (Native promoter::YFP) to generate pCAT2::GFP and pAPX1::GFP, respectively. HyPer is a genetically encoded yellow fluorescent protein (YFP)-based H₂O₂ sensor, which is highly sensitive to H₂O₂ and not to other ROS in bacteria and animal cells³⁰. To investigate H₂O₂ metabolism in plants, we amplified the HyPer from the pHyper-cyto plasmid (Evrogen, FP941) and cloned into pENTR^TM/SD/D-TOPO^TM vectors, and then recombined with destination vector pEARLY 100 (p35S::X). This construct was introduced into Agrobacterium tumefaciens (strain GV3101), and transformed into Col-0 plants to generate p35S::HyPer transgenic plants. Fluorescence intensity of pHyPer transgenic plants was determined over regions of interest, at 530 nm after excitation at 488 nm or 405 nm. Emission was collected at 530 nm with a bandpass of 30 nm. Image analysis was performed with an ImageJ software. A fluorescence ratio was calculated as 488/405. Oligo primers used in this study are listed in Supplementary Data 1. All binary vector constructs were introduced into Agrobacterium tumefaciens (strain GV3101), and transformed into Col-0 plants by the floral dipping method⁵¹.

Fluorescence imaging of ROS

The H₂DCFDA staining assay was performed slightly modified as described previously⁵². Briefly, seedlings were incubated in 10 μM H₂DCFDA in 10 mM Tris-HCl (pH 7.2) for 20 min in the dark. For BES-H₂O₂-Ac staining, seedlings were infiltrated with BES-H₂O₂-Ac solution (50 μM BES-H₂O₂-Ac in ½ MS liquid medium) and incubated at room temperature for 1 h in the dark. Excess H₂DCFDA and BES-H₂O₂-Ac were removed by washing 5 times with 10 mM Tris-HCl (pH 7.2) and ½ MS liquid medium respectively. H₂DCFDA, BES-H₂O₂-Ac and Propidium Iodide (PI) staining images were captured on a Zeiss LSM700 laser scanning confocal microscope.

DAB staining

2 mg/ml 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich) dissolved in 10 mM disodium hydrogen phosphate (pH 6.5) and were incubated in the dark until a reddish-brown precipitate was observed. The staining was terminated by transferring the plants into the solution (ethanol: glycerin: acetic acid = 3:1:1). The material was then imaged under a light microscope. The midleaf region of the cotyledon was used to analyze the H₂O₂ content by Scion Image software.

Quantitative analysis of fluorescent signals of KIN10-YFP

To capture the fluorescent signal, a series of Z-stack confocal images covering the entire epidermal cells (7 layers) were subjected to surface rendering in green channel to capture pKIN10::KIN10-YFP-expressing nuclei and cytoplasm in the three-dimensional space (for each time point). The Z-stack confocal images were taken using LSM880 microscope from Zeiss. A cut-off value of 0.82 was set for sphericity, which effectively removed objects with non-specific signals. Quantitative analyses of KIN10-YFP nuclear/cytoplasmic ratio were determined along a line drawn on the confocal images using ImageJ software and nuclear and cytoplasmic KIN10-YFP signals from more than 200 stomatal lineage cells in 10 cotyledons were analyzed. All graphs and all statistical analysis were generated using GraphPad Prism 9.0.

Stomatal quantification

Arabidopsis seedlings for stomatal quantification were cleared by decolor solution (75% ethanol: 25% acetic acid) until the leaf were no longer green. Cotyledons were dried on absorbent paper, then submerged into Hoyer’s solution until cotyledon turned transparent completely for microscope observation. Images were captured per cotyledon from central regions of abaxial leaves. Guard cells were counted for stoma index calculation.

Protein-protein pull-down assays

KIN10 fused to GST were purified from bacteria using glutathione beads (GE Healthcare). KINβ2 fused to MBP were purified using amylose resin (New England Biolabs). Glutathione beads containing 1 μg of GST-KIN10 were incubated with 1 µg MBP or MBP-KINβ2 in pull-down buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM EDTA), with or without 2 mM H₂O₂ at room temperature for 1 h and the beads were washed 4 times with wash buffer (20 mM Tris-HCl, pH 7.5; 300 mM NaCl; 1 mM EDTA; 0.5% Triton X-100). The eluted proteins were analyzed by immunoblot analysis with an anti-MBP antibody (TransGen Biotech, Cat: HT701-01,1:5,000 dilution) and anti-GST antibodies (TransGen Biotech, Cat: HT601-01,1:5,000 dilution).

DNA-protein pull-down assays

The recombinant proteins MBP and MBP-SPCH were expressed and affinity purified from Escherichia coli using amylose resin(TransGen Biotech, Cat: HT701-01,1:5000 dilution). The fragments of CAT2 and APX1 promoter were amplified by PCR using the biotin-labeled primers (Supplementary Data 1). The DNA and proteins were incubated, and then DNA-binding proteins were pull down using streptavidin-agarose beads and immunoblotted with anti-MBP antibodies(TransGen Biotech, Cat: HT701-01,1:5000 dilution).

Co-immunoprecipitation assays

Arabidopsis seedlings expressing pKIN10::KIN10-Myc only or co-expressing pKIN10::KIN10-Myc and pKINβ2::KINβ2-YFP were grown on ½ MS solid medium supplemented with 1% sucrose for 10 days and then treated with or without 2 mM H₂O₂ for 3 h. Plant materials were harvested and ground in liquid nitrogen and then extracted in lysis buffer containing 20 mM HEPES-KOH, pH 7.5; 40 mM KCl; 1 mM EDTA; 0.5% Triton X-100; 1 mM PMSF and 1× protease inhibitor cocktail (Sigma-Aldrich). After centrifugation at 4 °C, 12,000 × g for 10 min, the supernatant was incubated with GFP-Trap agarose beads (Chromotek) at 4 °C for 1 h, and the beads were washed 4 times using wash buffer (20 mM HEPES-KOH, pH 7.5; 40 mM KCl; 1 mM EDTA; 300 mM NaCl; and 1% Triton X-100). The proteins were eluted from the beads by boiling with 2× SDS sample buffer, analyzed by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with anti-GFP (TransGen Biotech, Cat: N20610,1:5000 dilution) and anti-Myc (Sigma-Aldrich, Cat: M4439, 1:5000 dilution) antibodies.

Chromatin immunoprecipitation (ChIP) Assay

Seedlings of p35S::SPCH-YFP, and p35S::YFP were grown on ½ MS solid medium supplemented with 1% Sucrose for 12 days under a long-day photoperiod, harvested, and cross-linked in 1% formaldehyde for 30 min under vacuum. Immunoprecipitation was performed as previously described⁵³, using GFP-Trap agarose beads (Chromotek). ChIP products were analyzed by qPCR, and the fold enrichment was calculated as the ratio between p35S::SPCH-YFP, and p35S::YFP, and then normalized by PP2A (At1g13320) gene, which was used as an internal control. The ChIP experiments were performed with three biological replicates, from which the means and standard deviations were calculated.

RT-qPCR analysis

Seedlings for wild type and indicated plants were grown on ½ MS solid medium supplemented with 1% sucrose for 6 days under a long-day photoperiod, harvested to extract total RNA (TransGen Biotech). First-strand cDNAs were synthesized using RevertAid reverse transcriptase (Thermo Fisher Scientific) and used as qPCR templates. Quantitative PCR analysis was performed on a CFX connect real-time PCR detection system (Bio-Rad) using synergy brands (SYBR) green reagent (Roche) with gene-specific primers (Supplementary Data 1).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

All data in this study are available in the manuscript or the Supplementary materials. Source data are provided with this paper.

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Acknowledgements

We thank Prof. Dominique C. Bergmann and Prof. Jie Le for providing the seeds of pSPCH::nucGFP, pSPCH::SPCH-GFP, Prof. Changle Ma for providing the seeds of cat1 cat2 cat3, and Prof. Zhong Zhao for providing the seeds of ndufs4. We thank Haiyan Yu and Xiaomin Zhao from the Analysis and Testing Center of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for assistance with the laser scanning confocal microscopy. This work was funded by the National Natural Science Foundation of China (grant nos. 31870262, 31970306, and 32070210) and by the Shandong Province Natural Science Foundation (grant nos. ZR2019ZD16 and 2019LZGC-015).

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These authors contributed equally: Wen Shi, Lingyan Wang.

Authors and Affiliations

The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, 266237, China
Wen Shi, Lingyan Wang, Lianmei Yao, Wei Hao, Chao Han, Min Fan & Ming-Yi Bai
College of Horticulture, College of Life Sciences, Hai xia Institute of Science and Technology, Fujian Agriculture and Forestry University, 350002, Fuzhou, China
Wenfei Wang

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Contributions

W.S. and M.B. together designed the experiments. W.S. and L.W. performed the ROS staining, microscopy analysis, stomatal index analysis, western blot, subcellular location analysis, pull down, CoIP assay. W.S., L.Y., W.H., C.H., and M.F. generated pCAT2::GFP, pAPX1::GFP, pSPCH::CAT2-RFP, kinβ1 kinβ2, p35S::KINβ2-YFP. C.H. and W.W provided the critical discussion on the work. W.S. performed all other experiments. W.S. and M.B. wrote the manuscript.

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Correspondence to Ming-Yi Bai.

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Shi, W., Wang, L., Yao, L. et al. Spatially patterned hydrogen peroxide orchestrates stomatal development in Arabidopsis. Nat Commun 13, 5040 (2022). https://doi.org/10.1038/s41467-022-32770-7

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Received: 12 October 2021
Accepted: 16 August 2022
Published: 26 August 2022
DOI: https://doi.org/10.1038/s41467-022-32770-7

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