Artificial cellulosic leaf with adjustable enzymatic CO2 sequestration capability.

Developing artificial leaves to address the environmental burden of CO2 is pivotal for advancing our Net Zero Future. In this study, we introduce EcoLeaf, an artificial leaf that closely mimics the characteristics of natural leaves. It harnesses visible light as its sole energy source and orchestrates the controlled expansion and contraction of stomata and the exchange of petiole materials to govern the rate of CO2 sequestration from the atmosphere. Furthermore, EcoLeaf has a cellulose composition and mechanical strength similar to those of natural leaves, allowing it to seamlessly integrate into the ecosystem during use and participate in natural degradation and nutrient cycling processes at the end of its life. We propose that the carbon sequestration pathway within EcoLeaf is adaptable and can serve as a versatile biomimetic platform for diverse biogenic carbon sequestration pathways in the future.

A network-based modeling framework reveals the core signal transduction network underlying high carbon dioxide-induced stomatal closure in guard cells.

Stomata are pores on plant aerial surfaces, each bordered by a pair of guard cells. They control gas exchange vital for plant survival. Understanding how guard cells respond to environmental signals such as atmospheric carbon dioxide (CO2) levels is not only insightful to fundamental biology but also relevant to real-world issues of crop productivity under global climate change. In the past decade, multiple important signaling elements for stomatal closure induced by elevated CO2 have been identified. Yet, there is no comprehensive understanding of high CO2-induced stomatal closure. In this work, we assemble a cellular signaling network underlying high CO2-induced stomatal closure by integrating evidence from a comprehensive literature analysis. We further construct a Boolean dynamic model of the network, which allows in silico simulation of the stomatal closure response to high CO2 in wild-type Arabidopsis thaliana plants and in cases of pharmacological or genetic manipulation of network nodes. Our model has a 91% accuracy in capturing known experimental observations. We perform network-based logical analysis and reveal a feedback core of the network, which dictates cellular decisions in closure response to high CO2. Based on these analyses, we predict and experimentally confirm that applying nitric oxide (NO) induces stomatal closure in ambient CO2 and causes hypersensitivity to elevated CO2. Moreover, we predict a negative regulatory relationship between NO and the protein phosphatase ABI2 and find experimentally that NO inhibits ABI2 phosphatase activity. The experimental validation of these model predictions demonstrates the effectiveness of network-based modeling and highlights the decision-making role of the feedback core of the network in signal transduction. We further explore the model's potential in predicting targets of signaling elements not yet connected to the CO2 network. Our combination of network science, in silico model simulation, and experimental assays demonstrates an effective interdisciplinary approach to understanding system-level biology.

Effects of different light conditions on morphological, anatomical, photosynthetic and biochemical parameters of Cypripedium macranthos Sw.

In this study, the morphological (plant height, leaf length and width, stem diameter and leaf number), anatomical (epidermal cell density and thickness, Stomatal length and width), photosynthetic (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, relative humidity, leaf temperature and chlorophyll fluorescence parameters) and biochemical parameters (the content of soluble sugar, soluble protein, proline, malondialdehyde and electrical conductivity) of Cypripedium macranthos Sw. in Changbai Mountain were determined under different light conditions (L10, L30, L50, L100). The results showed that morphological values including plant height, leaf area, stem diameter and leaf number of C. macranthos were smaller under the condition of full light at L100. The epidermal cell density and epidermal thickness of C. macranthos were the highest under L30 and L50 treatments, respectively. It had the highest net photosynthetic rate (Pn) and chlorophyll content under L50 treatment. Meanwhile, correlation analysis indicated that photosynthetically active radiation (PAR) and water use efficiency (WUE) were the main factors influencing Pn. C. macranthos accumulated more soluble sugars and soluble proteins under L100 treatment, while the degree of membrane peroxidation was the highest and the plant was severely damaged. In summary, the adaptability of C. macranthos to light conditions is ranked as follows L50 > L30 > L10 > L100. Appropriate light conditions for C. macranthos are 30%-50% of full light, which should be taken into account in protection and cultivation.

Plasticity and implications of water-use traits in contrasting tropical tree species under climate change.

Plants face a trade-off between hydraulic safety and growth, leading to a range of water-use strategies in different species. However, little is known about such strategies in tropical trees and whether different water-use traits can acclimate to warming. We studied five water-use traits in 20 tropical tree species grown at three different altitudes in Rwanda (RwandaTREE): stomatal conductance (gs), leaf minimum conductance (gmin), plant hydraulic conductance (Kplant), leaf osmotic potential (ψo) and net defoliation during drought. We also explored the links between these traits and growth and mortality data. Late successional (LS) species had low Kplant, gs and gmin and, thus, low water loss, while low ψo helped improve leaf water status during drought. Early successional (ES) species, on the contrary, used more water during both moist and dry conditions and exhibited pronounced drought defoliation. The ES strategy was associated with lower mortality and more pronounced growth enhancement at the warmer sites compared to LS species. While Kplant and gmin showed downward acclimation in warmer climates, ψo did not acclimate and gs measured at prevailing temperature did not change. Due to distinctly different water use strategies between successional groups, ES species may be better equipped for a warmer climate as long as defoliation can bridge drought periods.

PIF transcriptional regulators are required for rhythmic stomatal movements.

Stomata govern the gaseous exchange between the leaf and the external atmosphere, and their function is essential for photosynthesis and the global carbon and oxygen cycles. Rhythmic stomata movements in daily dark/light cycles prevent water loss at night and allow CO2 uptake during the day. How the actors involved are transcriptionally regulated and how this might contribute to rhythmicity is largely unknown. Here, we show that morning stomata opening depends on the previous night period. The transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) accumulate at the end of the night and directly induce the guard cell-specific K+ channel KAT1. Remarkably, PIFs and KAT1 are required for blue light-induced stomata opening. Together, our data establish a molecular framework for daily rhythmic stomatal movements under well-watered conditions, whereby PIFs are required for accumulation of KAT1 at night, which upon activation by blue light in the morning leads to the K+ intake driving stomata opening.

Evaluation of potential increase in photosynthetic efficiency of cassava (Manihot esculenta Crantz) plants exposed to elevated carbon dioxide.

Cassava (Manihot esculenta Crantz), an important tropical crop, is affected by extreme climatic events, including rising CO2 levels. We evaluated the short-term effect of elevated CO2 concentration (ECO2 ) (600, 800 and 1000ppm) on the photosynthetic efficiency of 14 cassava genotypes. ECO2 significantly altered gaseous exchange parameters (net photosynthetic rate (P n ), stomatal conductance (g s ), intercellular CO2 (C i ) and transpiration (E )) in cassava leaves. There were significant but varying interactive effects between ECO2 and varieties on these physiological characteristics. ECO2 at 600 and 800ppm increased the P n rate in the range of 13-24% in comparison to 400ppm (ambient CO2 ), followed by acclimation at the highest concentration of 1000ppm. A similar trend was observed in g s and E . Conversely, C i increased significantly and linearly across increasing CO2 concentration. Along with C i , a steady increase in water use efficiency [WUEintrinsic (P n /g s ) and WUEinstantaneous (P n /E )] across various CO2 concentrations corresponded with the central role of restricted stomatal activity, a common response under ECO2 . Furthermore, P n had a significant quadratic relationship with the ECO2 (R 2 =0.489) and a significant and linear relationship with C i (R 2 =0.227). Relative humidity and vapour pressure deficit during the time of measurements remained at 70-85% and ~0.9-1.31kPa, respectively, at 26±2°C leaf temperature. Notably, not a single variety exhibited constant performance for any of the parameters across CO2 concentrations. Our results indicate that the potential photosynthesis can be increased up to 800ppm cassava varieties with high sink capacity can be cultivated under protected cultivation to attain higher productivity.

How and why do species break a developmental trade-off? Elucidating the association of trichomes and stomata across species.

PREMISE: Previous studies have suggested a trade-off between trichome density (Dt) and stomatal density (Ds) due to shared cell precursors. We clarified how, when, and why this developmental trade-off may be overcome across species. METHODS: We derived equations to determine the developmental basis for Dt and Ds in trichome and stomatal indices (it and is) and the sizes of epidermal pavement cells (e), trichome bases (t), and stomata (s) and quantified the importance of these determinants of Dt and Ds for 78 California species. We compiled 17 previous studies of Dt-Ds relationships to determine the commonness of Dt-Ds associations. We modeled the consequences of different Dt-Ds associations for plant carbon balance. RESULTS: Our analyses showed that higher Dt was determined by higher it and lower e, and higher Ds by higher is and lower e. Across California species, positive Dt-Ds coordination arose due to it-is coordination and impacts of the variation in e. A Dt-Ds trade-off was found in only 30% of studies. Heuristic modeling showed that species sets would have the highest carbon balance with a positive or negative relationship or decoupling of Dt and Ds, depending on environmental conditions. CONCLUSIONS: Shared precursor cells of trichomes and stomata do not limit higher numbers of both cell types or drive a general Dt-Ds trade-off across species. This developmental flexibility across diverse species enables different Dt-Ds associations according to environmental pressures. Developmental trait analysis can clarify how contrasting trait associations would arise within and across species.

Accurate photosynthetic parameter estimation at low stomatal conductance: effects of cuticular conductance and instrumental noise.

Accurate estimation of photosynthetic parameters is essential for understanding plant physiological limitations and responses to environmental factors from the leaf to the global scale. Gas exchange is a useful tool to measure responses of net CO2 assimilation (A) to internal CO2 concentration (Ci), a necessary step in estimating photosynthetic parameters including the maximum rate of carboxylation (Vcmax) and the electron transport rate (Jmax). However, species and environmental conditions of low stomatal conductance (gsw) reduce the signal-to-noise ratio of gas exchange, challenging estimations of Ci. Previous works showed that not considering cuticular conductance to water (gcw) can lead to significant errors in estimating Ci, because it has a different effect on total conductance to CO2 (gtc) than does gsw. Here we present a systematic assessment of the need for incorporating gcw into Ci estimates. In this study we modeled the effect of gcw and of instrumental noise and quantified these effects on photosynthetic parameters in the cases of four species with varying gsw and gcw, measured using steady-state and constant ramping techniques, like the rapid A/Ci response method. We show that not accounting for gcw quantitatively influences Ci and the resulting Vcmax and Jmax, particularly when gcw exceeds 7% of the total conductance to water. The influence of gcw was not limited to low gsw species, highlighting the importance of species-specific knowledge before assessing A/Ci curves. Furthermore, at low gsw instrumental noise can affect Ci estimation, but the effect of instrumental noise can be minimized using constant-ramping rather than steady-state techniques. By incorporating these considerations, more precise measurements and interpretations of photosynthetic parameters can be obtained in a broader range of species and environmental conditions.

Heat Waves Coupled with Nanoparticles Induce Yield and Nutritional Losses in Rice by Regulating Stomatal Closure.

The frequency, duration, and intensity of heat waves (HWs) within terrestrial ecosystems are increasing, posing potential risks to agricultural production. Cerium dioxide nanoparticles (CeO2 NPs) are garnering increasing attention in the field of agriculture because of their potential to enhance photosynthesis and improve stress tolerance. In the present study, CeO2 NPs decreased the grain yield, grain protein content, and amino acid content by 16.2, 23.9, and 10.4%, respectively, under HW conditions. Individually, neither the CeO2 NPs nor HWs alone negatively affected rice production or triggered stomatal closure. However, under HW conditions, CeO2 NPs decreased the stomatal conductance and net photosynthetic rate by 67.6 and 33.5%, respectively. Moreover, stomatal closure in the presence of HWs and CeO2 NPs triggered reactive oxygen species (ROS) accumulation (increased by 32.3-57.1%), resulting in chloroplast distortion and reduced photosystem II activity (decreased by 9.4-36.4%). Metabolic, transcriptomic, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that, under HW conditions, CeO2 NPs activated a stomatal closure pathway mediated by abscisic acid (ABA) and ROS by regulating gene expression (PP2C, NCED4, HPCA1, and RBOHD were upregulated, while CYP707A and ALMT9 were downregulated) and metabolite levels (the content of γ-aminobutyric acid (GABA) increased while that of gallic acid decreased). These findings elucidate the mechanism underlying the yield and nutritional losses induced by stomatal closure in the presence of CeO2 NPs and HWs and thus highlight the potential threat posed by CeO2 NPs to rice production during HWs.

Transcription factor ZmDof22 enhances drought tolerance by regulating stomatal movement and antioxidant enzymes activities in maize (Zea mays L.).

KEY MESSAGE: The Dof22 gene encoding a deoxyribonucleic acid binding with one finger in maize, which is associated with its drought tolerance. The identification of drought stress regulatory genes is essential for the genetic improvement of maize yield. Deoxyribonucleic acid binding with one finger (Dof), a plant-specific transcription factor family, is involved in signal transduction, morphogenesis, and environmental stress responses. In present study, by weighted correlation network analysis (WGCNA) and gene co-expression network analysis, 15 putative Dof genes were identified from maize that respond to drought and rewatering. A real-time fluorescence quantitative PCR showed that these 15 genes were strongly induced by drought and ABA treatment, and among them ZmDof22 was highly induced by drought and ABA treatment. Its expression level increased by nearly 200 times after drought stress and more than 50 times after ABA treatment. After the normal conditions were restored, the expression levels were nearly 100 times and 40 times of those before treatment, respectively. The Gal4-LexA/UAS system and transcriptional activation analysis indicate that ZmDof22 is a transcriptional activator regulating drought tolerance and recovery ability in maize. Further, overexpressed transgenic and mutant plants of ZmDof22 by CRISPR/Cas9, indicates that the ZmDof22, improves maize drought tolerance by promoting stomatal closure, reduces water loss, and enhances antioxidant enzyme activity by participating in the ABA pathways. Taken together, our findings laid a foundation for further functional studies of the ZmDof gene family and provided insights into the role of the ZmDof22 regulatory network in controlling drought tolerance and recovery ability of maize.

Reponses of morphological and biochemical traits of bamboo trees under elevated atmospheric O3 enrichment.

Dwarf bamboo (Indocalamus decorus) is an O3-tolerant plant species. To identify the possible mechanism and response of leaf morphological, antioxidant, and anatomical characteristics to elevated atmospheric O3 (EO3) concentrations, we exposed three-year-old I. decorus seedlings to three O3 levels (low O3-LO: ambient air; medium O3-MO: Ambient air+70 ppb high O3-HO: Ambient air+140 ppb O3) over a growing season using open-top chambers. Leaf shape and stomatal characteristics, and leaf microscopic structure of I. decorus were examined. The results indicated that 1) the stomata O3 flux (Fst) of HO decreased more rapidly under EO3 as the exposure time increased. The foliar O3 injury of HO and MO occurred when AOT40 was 26.62 ppm h and 33.20 ppm h, respectively, 2) under EO3, leaf number, leaf mass per area, leaf area, and stomata length/width all decreased, while leaf thickness, stomatal density, width, and area increased compared to the control, 3) MDA and total soluble protein contents all showed significantly increase under HO (36.57% and 32.77%) and MO(31.91% and 19.52%) while proline contents only increased under HO(33.27%). 4) MO and HO increased bulliform cells numbers in the leaves by 6.28% and 23.01%, respectively. HO reduced the transverse area of bulliform cells by 13.73%, while MO treatments had no effect, and 5) the number of fusoid cells interspace, the transverse area of fusoid cells interspace, and mesophyll thickness of HO significantly increased by 11.16%, 28.58%, and 13.42%, respectively. In conclusion, I. decorus exhibits strong O3 tolerance characteristics, which stem from adaptions in the leaf's morphological, structural, antioxidant, and anatomical features. One critical attribute was the enlargement of the bulliform cell transverse area and the transverse area of fusoid cells interspace that drove this resistance to O3. Local bamboo species with high resistance to O3 pollution thus need to be promoted for sustained productivity and ecosystem services in areas with high O3 pollution.

Effects of high-temperature stress on gene expression related to photosynthesis in two jujube (Ziziphus jujuba Mill.) varieties.

Elevated temperatures critically impact crop growth, development, and yield, with photosynthesis being the most temperature-sensitive physiological process in plants. This study focused on assessing the photosynthetic response and genetic adaptation of two different heat-resistant jujube varieties 'Junzao' (J) and 'Fucuimi' (F), to high-temperature stress (42°C Day/30°C Night). Comparative analyses of leaf photosynthetic indices, microstructural changes, and transcriptome sequencing were conducted. Results indicated superior high-temperature adaptability in F, evidenced by alterations in leaf stomatal behavior - particularly in J, where defense cells exhibited significant water loss, shrinkage, and reduced stomatal opening, alongside a marked increase in stomatal density. Through transcriptome sequencing 13,884 differentially expressed genes (DEGs) were identified, significantly enriched in pathways related to plant-pathogen interactions, amino acid biosynthesis, starch and sucrose metabolism, and carbohydrate metabolism. Key findings include the identification of photosynthetic pathway related DEGs and HSFA1s as central regulators of thermal morphogenesis and heat stress response. Revealing their upregulation in F and downregulation in J. The results indicate that these genes play a crucial role in improving heat tolerance in F. This study unveils critical photosynthetic genes involved in heat stress, providing a theoretical foundation for comprehending the molecular mechanisms underlying jujube heat tolerance.

Potassium deficiency enhances imbalances in rice water relations under water deficit by decreasing leaf hydraulic conductance.

Potassium (K+) is an essential macronutrient for appropriate plant development and physiology. However, little is known about the mechanisms involved in the regulation of leaf water relations by K under water deficit. A pot experiment with two K supplies of 0.45 and 0 g K2O per pot (3 kg soil per pot) and two watering conditions (well-watered and water-deficit) was conducted to explore the effects of K deficiency on canopy transpiration characteristics, leaf water status, photosynthesis, and hydraulic traits in two rice genotypes with contrasting resistance to drought. The results showed that K deficiency reduced canopy transpiration rate by decreasing stomatal conductance, which led to higher canopy temperatures, resulting in limited water deficit tolerance in rice. In addition, K deficiency led to further substantial reductions in leaf relative water content and water potential under water deficit, which increased the imbalance in leaf water relations under water deficit. Notably, K deficiency limited leaf gas exchange by reducing leaf hydraulic conductance, but decreased the intrinsic water use efficiency under water deficit, especially for the drought-resistant cultivar. Further analysis of the underlying process of leaf hydraulic resistance revealed that the key limiting factor of leaf hydraulic conductance under K deficiency was the outside-xylem hydraulic conductance rather than the xylem hydraulic conductance. Overall, our results provide a comprehensive perspective for assessing leaf water relations under K deficiency, water deficit, and their combined stresses, which will be useful for optimal rice fertilization strategies.

H2S is involved in drought-mediated stomatal closure through PLDα1 in Arabidopsis.

MAIN CONCLUSION: PLDα1 promoted H2S production by positively regulating the expression of LCD. Stomatal closure promoted by PLDα1 required the accumulation of H2S under drought stress. Phospholipase Dα1 (PLDα1) acting as one of the signal enzymes can respond to drought stress. It is well known that hydrogen sulfide (H2S) plays an important role in plant responding to biotic or abiotic stress. In this study, the functions and relationship between PLDα1 and H2S in drought stress resistance in Arabidopsis were explored. Our results indicated that drought stress promotes PLDα1 and H2S production by inducing the expression of PLDα1 and LCD genes. PLDα1 and LCD enhanced plant tolerance to drought by regulating membrane lipid peroxidation, proline accumulation, H2O2 content and stomatal closure. Under drought stress, the H2O2 content of PLDα1-deficient mutant (pldα1), L-cysteine desulfhydrase (LCD)-deficient mutant (lcd) was higher than that of ecotype (WT), the stomatal aperture of pldα1 and lcd was larger than that of WT. The transcriptional and translational levels of LCD were lower in pldα1 than that in WT. Exogenous application of the H2S donor NaHS or GYY reduced the stomatal aperture of WT, pldα1, PLDα1-CO, and PLDα1-OE lines, while exogenous application of the H2S scavenger hypotaurine (HT) increased the stomatal aperture. qRT-PCR analysis of stomatal movement-related genes showed that the expression of CAX1, ABCG5, SCAB1, and SLAC1 genes in pldα1 and lcd were down-regulated, while ACA1 and OST1 gene expression was significantly up-regulated. Thus, PLDα1 and LCD are required for stomatal closure to improve drought stress tolerance.

Simultaneous and Independent Abaxial and Adaxial Gas Exchange Measurements.

Stomata can be distributed exclusively on the abaxial or adaxial leaf surface, but they are most commonly found on both leaf surfaces. Variations in stomatal arrangement, patterning, and the impact on photosynthesis can be measured using an infrared gas exchange system. However, when using standard gas exchange techniques, both surfaces are measured together and averaged to provide leaf-level values. Employing an innovative gas exchange apparatus with two infrared gas analyzers, separate gaseous flux from both leaf surfaces can be quantified simultaneously and independently. Here, we provide examples of typical measurements that can be performed using a "split chamber" gas exchange system.

Using Infrared Thermography for High-Throughput Plant Phenotyping.

Infrared thermography offers a rapid, noninvasive method for measuring plant temperature, which provides a proxy for stomatal conductance and plant water status and can therefore be used as an index for plant stress. Thermal imaging can provide an efficient method for high-throughput screening of large numbers of plants. This chapter provides guidelines for using thermal imaging equipment and illustrative methodologies, coupled with essential considerations, to access plant physiological processes.

Assessing the High Temperature Effects on Stomatal Production.

The production of stomata, the epidermal pores of plants, is influenced by diverse environmental signals including high temperature. To assess its impact on stomatal formation, researchers need to grow plants in a carefully designed regime under controlled conditions and capture clear, microscopic views of the epidermis. Here, we describe a procedure to study the effect of high temperature on stomatal formation. This method can generate high-quality epidermal images of cotyledons, leaves, and hypocotyl of young Arabidopsis seedlings, which allow the determination of the pattern, density, and index of stomata on these tissues. Besides temperature, the protocol can serve as a general approach to examine stomatal phenotype and the effect of other external signals on stomatal formation.

Plant responses to co-occurring heat and water deficit stress: A comparative study of tolerance mechanisms in old and modern wheat genotypes.

Global climate change increases the likelihood of co-occurrence of hot and dry spells with increased intensity, frequency, and duration. Studying the impact of the two stresses provide a better understanding of tolerance mechanisms in wheat, and our study was focused on revealing plant stress responses to different severities of combined stress at two phenophases in old and modern wheat genotypes. During the stem elongation and anthesis stages, plants were exposed to four treatments: control, deficit irrigation, combined heat, and deficit irrigation at 31 °C (HD31) and 37 °C (HD37). The modern genotypes were less affected by deficit irrigation at stem elongation as they maintained higher photosynthesis, stomatal conductance, and leaf cooling than old genotypes. When the HD37 stress was imposed during anthesis, the modern genotypes exhibited superior performance compared to the old, which was due to their higher photosynthetic rates resulting from improved biochemical regulation and a higher chlorophyll content. The plant responses varied during two phenophases under the combined stress exposure. Genotypes subjected to HD37 stress during stem elongation, photosynthesis was mainly controlled by stomatal regulation, whereas at anthesis it was predominated by biochemical regulation. These findings contribute to a deeper comprehension of plant tolerance mechanisms in response to different intensities of co-occurring hot and dry weather conditions.

Guard cell and subsidiary cell sizes are key determinants for stomatal kinetics and drought adaptation in cereal crops.

Climate change-induced drought is a major threat to agriculture. C4 crops have a higher water use efficiency (WUE) and better adaptability to drought than C3 crops due to their smaller stomatal morphology and faster response. However, our understanding of stomatal behaviours in both C3 and C4 Poaceae crops is limited by knowledge gaps in physical traits of guard cell (GC) and subsidiary cell (SC). We employed infrared gas exchange analysis and a stomatal assay to explore the relationship between GC/SC sizes and stomatal kinetics across diverse drought conditions in two C3 (wheat and barley) and three C4 (maize, sorghum and foxtail millet) upland Poaceae crops. Through statistical analyses, we proposed a GCSC-τ model to demonstrate how morphological differences affect stomatal kinetics in C4 Poaceae crops. Our findings reveal that morphological variations specifically correlate with stomatal kinetics in C4 Poaceae crops, but not in C3 ones. Subsequent modelling and experimental validation provide further evidence that GC/SC sizes significantly impact stomatal kinetics, which affects stomatal responses to different drought conditions and thereby WUE in C4 Poaceae crops. These findings emphasize the crucial advantage of GC/SC morphological characteristics and stomatal kinetics for the drought adaptability of C4 Poaceae crops, highlighting their potential as future climate-resilient crops.

Birch WRKY transcription factor, BpWRKY32, confers salt tolerance by mediating stomatal closing, proline accumulation, and reactive oxygen species scavenging.

Plant WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, how WRKY facilitate physiological changes to confer salt tolerance still needs to be studied. Here, we identified a WRKY TF from birch (Betula platyphylla Suk), BpWRKY32, which is significantly (P < 0.05) induced by salt stress. BpWRKY32 binds to W-box motif and is located in the nucleus. Under salt stress conditions, fresh weights (FW) of OE lines (BpWRKY32 overexpression lines) are increased by 66.36% than that of WT, while FW of knockout of BpWRKY32 (bpwrky32) lines are reduced by 39.49% compared with WT. BpWRKY32 regulates the expression of BpRHC1, BpNRT1, and BpMYB61 to reduce stomatal, and width-length ratio of the stomatal aperture in OE lines are reduced by 46.23% and 64.72% compared with in WT and bpwrky32 lines. BpWRKY32 induces P5CS expression, but inhibits P5CDH expression, leading to the proline content in OE lines are increased by 33.41% and 97.58% compared with WT and bpwrky32 lines. Additionally, BpWRKY32 regulates genes encoding SOD and POD family members, which correspondingly increases the activities of SOD and POD. These results suggested that BpWRKY32 regulates target genes to reduce the water loss rate, enhance the osmotic potential, and reduce the ROS accumulation, leading to improved salt tolerance.