Azolla pinnata as a phytoremediator: improves germination, growth and yield of maize irrigated with Ni-polluted water.

The removal of pollutants from the environment has become a global demand. The current study aimed to relieve the Ni toxicity effect on the germination, growth, and grain yield of maize by using Azolla pinnata as a phytoremediator. Azolla-treated and untreated nickel solutions [0 (control), 24, 70, 140 and 190 ppm] were applied for germination and pot experiments. Electron microscope examination cleared the Ni accumulation in Azolla's cell vacuole and its adsorption on the cell wall. The inhibition of the hydrolytic enzyme activity reduces maize germination; maximal inhibition was 57.1% at 190 ppm of Ni compared to the control (100%). During vegetative growth, Ni stimulated the generation of H2O2 (0.387 mM g-1 F Wt at 190 ppm of Ni), which induced maximal lipid peroxidation (3.913 µMDA g-1 F Wt) and ion leakage (74.456%) compared to control. Chlorophyll content and carbon fixation also showed significant reductions at all Ni concentrations; at 190 ppm, they showed maximum reductions of 56.2 and 63%, respectively. However, detoxification enzymes' activity such as catalase and antioxidant substances (phenolics) increased. The highest concentration of Ni (190 ppm) had the most effect on constraining yield, reaching zero for the weight of 100 grains at 190 ppm of Ni. Azolla-treated Ni solutions amended all determinant parameters, indicating a high percentage of changes in hydrolytic enzyme activity (125.2%) during germination, chlorophyll content (77.6%) and photosynthetic rate (120.1%). Growth measurements, carbon fixation, and yield components showed a positive association. Thus, we recommended using Azolla as a cost-effective and eco-friendly strategy to recover Ni-polluted water.

Athyrium yokoscense, a cadmium-hypertolerant fern, exhibits two cadmium stress mitigation strategies in its roots and aerial parts.

Athyrium yokoscense is hypertolerant to cadmium (Cd) and can grow normally under a high Cd concentration despite Cd being a highly toxic heavy metal. To mitigate Cd stress in general plant species, Cd is promptly chelated with a thiol compound and is isolated into vacuoles. Generated active oxygen species (ROS) in the cytoplasm are removed by reduced glutathione. However, we found many differences in the countermeasures in A. yokoscense. Thiol compounds accumulated in the stele of the roots, although a long-term Cd exposure induced Cd accumulation in the aerial parts. Synchrotron radiation-based X-ray fluorescence (SR-XRF) analysis indicated that a large amount of Cd was localized in the cell walls of the roots. Overexpression of AyNramp5a, encoding a representative Fe and Mn transporter of A. yokoscense, increased both Cd uptake and Fe and Mn uptake in rice calli under the Cd exposure conditions. Organic acids are known to play a key role in reducing Cd availability to the plants by forming chelation and preventing its entry in free form into the roots. In A. yokoscense roots, Organic acids were abundantly detected. Investigating the chemical forms of the Cd molecules by X-ray absorption fine structure (XAFS) analysis detected many compounds with Cd-oxygen (Cd-O) binding in A. yokoscense roots, whereas in the aerial parts, the ratio of the compounds with Cd-sulfur (Cd-S) binding was increased. Together, our results imply that the strong Cd tolerance of A. yokoscense is an attribute of the following two mechanisms: Cd-O compound formation in the cell wall is a barrier to reduce Cd uptake into aerial parts. Thiol compounds in the region of root stele are involved in detoxication of Cd by formation of Cd-S compounds.

Phytoremediation Potential of Azolla filiculoides: Uptake and Toxicity of Seven Per- and Polyfluoroalkyl Substances (PFAS) at Environmentally Relevant Water Concentrations.

Environmental contamination of aquatic systems by per- and polyfluoroalkyl substances (PFAS) has generated significant health concerns. Remediation of contaminated sites such as the fire-fighting emergency training grounds that use aqueous film-forming foams is a high priority. Phytoremediation may help play a part in removing PFAS from such contaminated waters. We investigated the potential of the water fern Azolla filiculoides, which is used for phytoremediation of a wide range of contaminants, to uptake seven common PFAS (perfluorobutanoic acid [PFBA], perfluorobutane sulfonic acid [PFBS], perfluoroheptanoic acid [PFHpA], perfluorohexanoic acid [PFHxA], perfluorohexane sulfonic acid [PFHxS], perfluorooctanoic acid [PFOA], and perfluoropentanoic acid [PFPeA]), during a 12-day exposure to environmentally relevant concentrations delivered as equimolar mixtures: low (∑PFAS = 0.0123 ± 1.89 µmol L-1), medium (∑PFAS = 0.123 ± 2.88 µmol L-1), and high (∑PFAS = 1.39 µmol L-1) treatments, equivalent to approximately 5, 50, and 500 µg L-1 total PFAS, respectively. The possible phytotoxic effects of PFAS were measured at 3-day intervals using chlorophyll a content, photosystem II efficiency (Fv/Fm), performance index, and specific growth rate. The PFAS concentrations in plant tissue and water were also measured every 3 days using ultra-high-performance liquid chromatography-tandem mass spectrometry. Treatments with PFAS did not lead to any detectable phytotoxic effects. All seven PFAS were detected in plant tissue, with the greatest uptake occurring during the first 6 days of exposure. After 12 days of exposure, a maximum bioconcentration factor was recorded for PFBA of 1.30 and a minimum of 0.192 for PFBS. Consequently, the application of Azolla spp. as a stand-alone system for phytoremediation of PFAS in aquatic environments is not sufficient to substantially reduce PFAS concentrations. Environ Toxicol Chem 2024;43:2157-2168. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The impact of polystyrene nanoplastics on plants in the scenario of increasing temperatures: The case of Azolla filiculoides Lam.

There are great concerns for the accumulation in the environment of small dimension plastics, such as micro- and nanoplastics. Due to their small size, which facilitates their uptake by organisms, nanoplastics are of particular concern. The toxic effects of nanoplastics on plants are already reported in the literature, however nothing is known, to date, about the possible effects of climate change, in particular of increasing temperatures, on their toxicity for plants. To address this issue, plants of the water fern Azolla filiculoides were grown at optimal (25 °C) or high (35 °C) temperature, with or without polystyrene nanoplastics, and the effects of these stressors were assessed using a multidisciplinary approach. Green fluorescent polystyrene nanoplastics were used to track their possible uptake by A. filiculoides. The development and physiology of our model plant was adversely affected by both nanoplastics and high temperatures. Overall, histological, morphological, and photosynthetic parameters worsened under co-treatment, in accordance with the increased uptake of nanoplastics under higher temperature, as observed by fluorescence images. Based on our findings, the concern regarding the potential for increased toxicity of pollutants, specifically nanoplastics, at high temperatures is well-founded and warrants attention as a potential negative consequence of climate change. Additionally, there is cause for concern regarding the increase in nanoplastic uptake at high temperatures, particularly if this phenomenon extends to food and feed crops, which could lead to greater entry into the food chain.

Evaluation of arsenic phytoremediation potential in Azolla filiculoides Lam. plants under low pH stress conditions.

The Azolla filiculoides plants were challenged with different arsenic (As) concentration under low pH stress conditions. The growth rate and doubling time of the plants were severely affected by higher As treatments at pH 5.00 when compared with stress pH 4.75 treatments. Hence, pH 5.00 was considered for further studies. In 10-30 µM As treated cultures, after 6 days, the relative growth rate (RGR) of Azolla plants was significantly reduced and in higher concentration of As, the RGR was negatively regulated. The root trait parameters were also significantly affected by increasing concentrations of As. Further, photosynthetic performance indicators also show significant decline with increasing As stress. Overall, the plants treated with 40 and 50 µM of As displayed stress phenotypes like negative RGR, reduced doubling time and root growth, browning of leaves and root withering. The total proline, H2O2, POD and Catalase activities were significantly affected by As treatments. Meantime, 30 µM of As treated cultures displayed 15 µg/g/Fw As accumulation and moderate growth rate. Thus, the Azolla plants are suitable for the phytoremediation of As (up to 30 µM concentration) in the aquatic environment under low pH conditions (5.00). Furthermore, the transcriptome studies on revealed that the importance of positively regulated transporters like ACR3, AceTr family, ABC transporter super family in As (10 µM) stress tolerance, uptake and accumulation. The transporters like CPA1, sugar transporters, PiT were highly down-regulated. Further, expression analysis showed that the MATE1, CIP31, HAC1 and ACR3 were highly altered during the As stress conditions.

Azolla filiculoides extract improved salt tolerance in wheat (Triticum aestivum L.) is associated with prompting osmostasis, antioxidant potential and stress-interrelated genes.

The growth and productivity of crop plants are negatively affected by salinity-induced ionic and oxidative stresses. This study aimed to provide insight into the interaction of NaCl-induced salinity with Azolla aqueous extract (AAE) regarding growth, antioxidant balance, and stress-responsive genes expression in wheat seedlings. In a pot experiment, wheat kernels were primed for 21 h with either deionized water or 0.1% AAE. Water-primed seedlings received either tap water, 250 mM NaCl, AAE spray, or AAE spray + NaCl. The AAE-primed seedlings received either tap water or 250 mM NaCl. Salinity lowered growth rate, chlorophyll level, and protein and amino acids pool. However, carotenoids, stress indicators (EL, MDA, and H2O2), osmomodulators (sugars, and proline), antioxidant enzymes (CAT, POD, APX, and PPO), and the expression of some stress-responsive genes (POD, PPO and PAL, PCS, and TLP) were significantly increased. However, administering AAE contributed to increased growth, balanced leaf pigments and assimilation efficacy, diminished stress indicators, rebalanced osmomodulators and antioxidant enzymes, and down-regulation of stress-induced genes in NaCl-stressed plants, with priming surpassing spray in most cases. In conclusion, AAE can be used as a green approach for sustaining regular growth and metabolism and remodelling the physio-chemical status of wheat seedlings thriving in salt-affected soils.

Cryopreservation of Fern Spores and Pollen.

Fern spores and pollen are haploid plant germplasm of microscopic nature that can be used to regenerate full plants through germination (fern spores) or to fertilize seed-bearing plants through breeding programs (pollen). Due to their short life span in conventional storage (i.e., dry at -20 °C), the use of cryopreservation has been indicated for long-term ex situ conservation. While fern spores of most species and pollen from many seeded plants tolerate desiccation and can be stored dry at liquid nitrogen temperatures, some pollen is desiccation sensitive, and cryopreservation protocols require controlled drying and cooling and some level of cryoprotection. In this chapter we describe the cryopreservation process for fern spores used in the Millennium Seed Bank of Royal Botanic Gardens, Kew, including some details of the fern spores harvest and cleaning methods. In addition, two protocols for pollen cryopreservation are described, one generic for desiccation-tolerant pollen that can be used for multiple species and one specific for a desiccation sensitive pollen (Zea mays).

Fabrication and Characterization of a Novel Herbicide Delivery System with Magnetic Collectability and Its Phytotoxic Effect on Photosystem II of Aquatic Macrophyte.

The use of nano- and microparticles as a release system for agrochemicals has been increasing in agricultural sector. However, the production of eco-friendly and smart carriers that can be easily handled in the environment is still a challenge for this technology. In this context, we have developed a biodegradable release system for the herbicide atrazine with magnetic properties. Herein, we investigated the (a) physicochemical properties of the atrazine-loaded magnetic poly(ε-caprolactone) microparticles (MPs:ATZ), (b) in vitro release kinetic profile of the herbicide, and (c) phytotoxicity toward photosynthesis in the aquatic fern Azolla caroliniana. The encapsulation efficiency of the herbicide in the MPs:ATZ was ca. 69%, yielding spherical microparticles with a diameter of ca. 100 µm, a sustained-release profile, and easily manipulated with an external magnetic field. Also, phytotoxicity issues showed that the MPs:ATZ maintained their herbicidal activity via inhibition of PSII, showing lower toxicity compared with the nonencapsulated ATZ at 0.01 and 0.02 µmol·L-1. Therefore, this technology may conveniently promote a novel magnetic controlled release of the herbicide ATZ (with the potential to be collected from a watercourse) and act as a nutrient boost to the nontarget plant, with good herbicidal activity and reduced risk to the environment.

De novo transcriptome analysis reveals an unperturbed transcriptome under high cadmium conditions in the Cd-hypertolerant fern Athyrium yokoscense.

Athyrium yokoscense shows strong tolerance to cadmium exposure, even at levels that are many times greater than the toxic levels in ordinary plants. To determine the mechanism of Cd tolerance in A. yokoscense, we grew these plants under high Cd conditions and observed the tissue-specific accumulation of Cd and generation of reactive oxygen species, which is one of the major physiological responses to Cd stress. Fuchsin staining indicated the existence of a casparian strip in A. yokoscense roots, which may participate in Cd hypertolerance in A. yokoscense. Moreover, we performed RNA-seq of RNA samples from A. yokoscense plants treated with or without Cd exposure and obtained comprehensive RNA sequences as well as the Cd-responsive expression patterns of individual genes. Through de novo transcriptome assembly and gene expression analysis, we found that A. yokoscense showed normal features with no significant change in the expression levels of any transporter genes, even under high Cd exposure conditions. Our results demonstrate that A. yokoscense has an unusual mechanism that allows the invading Cd to partition into the distal roots, thus avoiding translocation of Cd into the xylem.

Pteridophytes in phytoremediation.

Soil contamination by heavy metals and metalloids is a serious problem which needs to be addressed. There are several methods for removal of contaminants, but they are costly, while the method of phytoremediation is eco-friendly and cost-effective. Pteridophytes have been found to remediate heavy metal-contaminated soil. Pteridophytes are non-flowering plant that reproduces by spores. Pteris vittata has been reported as the first fern plant to hyperaccumulate arsenic. The Pteris species belongs to the order Pteridales. Other ferns that are known phytoremediators are, for example, Nephrolepis cordifolia and Hypolepis muelleri (identified as phytostabilisers of Cu, Pb, Zn and Ni); similarly Pteris umbrosa and Pteris cretica accumulate arsenic in leaves. So, pteridophytes have a number of species that accumulate contaminants. Many of them have been identified, while various other are being explored. The present review article describes the phytoremediation potential of pteridophytes plants and suggests as a potential asset for phytoremediation programs.

TiO2 nanoparticles may alleviate cadmium toxicity in co-treatment experiments on the model hydrophyte Azolla filiculoides.

The hydrophyte Azolla filiculoides can be a useful model to assess if TiO2 NPs may in some way alleviate the Cd injuries and improve the ability of the plant to cope with this metal. With this mechanistic hypothesis, after a pre-treatment with TiO2 NPs, A. filiculoides plants were transferred to cadmium-contaminated water with or without TiO2 nanoparticles. After 5 days of treatment, cadmium uptake, morpho-anatomical, and physiological aspects were studied in plants. The continuous presence of TiO2 nanoparticles, though not increasing the uptake of cadmium in comparison with a priming treatment, induced a higher translocation of this heavy metal to the aerial portion. Despite the translocation factor was always well below 1, cadmium contents in the fronds, generally greater than 100 ppm, ranked A. filiculoides as a good cadmium accumulator. Higher cadmium contents in leaves did not induce damages to the photosynthetic machinery, probably thanks to a compartmentalization strategy aimed at confining most of this pollutant to less metabolically active peripheral cells. The permanence of NPs in growth medium ensured a better efficiency of the antioxidant apparatus (proline and glutathione peroxidase and catalase activities) and induced a decrease in H2O2 content, but did not suppress TBARS level.

How does aquatic macrophyte Salvinia auriculata respond to nanoceria upon an increased CO2 source? A Fourier transform-infrared photoacoustic spectroscopy and chlorophyll a fluorescence study.

With the continued increase of technological uses of cerium oxide nanoparticles (CeO2 NPs or nanoceria) and their unregulated disposal, the accumulation of nanoceria in the environment is inevitable. Concomitantly, atmospheric carbon dioxide (CO2) levels continue to rise, increasing the concentrations of bicarbonate ions in aquatic ecosystems. This study investigates the influence of CeO2 NPs (from 0 to 100 µgL-1) in the presence and absence of an elevated bicarbonate (HCO3-) ion concentration (1 mM), on vibrational biochemical parameters and photosystem II (PSII) activity in leaf discs of Salvinia auriculata. Fourier transform-infrared photoacoustic spectroscopy (FTIR-PAS) was capable of diagnostic use to understand biochemical and metabolic changes in leaves submitted to the CeO2 NPs and also detected interactive responses between CeO2 NPs and HCO3- exposure at the tissue level. The results showed that the higher CeO2 NPs levels in the presence of HCO3- increased the non-photochemical quenching (NPQ) and coefficient of photochemical quenching in dark (qPd) compared to the absence of HCO3. Moreover, the presence of HCO3- significantly decreased the NPQ at all levels of CeO2 NPs demonstrating that HCO3- exposure may change the non-radiative process involved in the operation of the photosynthetic apparatus. Overall, the results of this study are useful for providing baseline information on the interactive effects of CeO2 NPs and elevated HCO3- ion concentration on photosynthetic systems.

Fluctuations in cell cycle, morphology and metabolism of Anemia phyllitidis gametophytes are the most important hallmarks of GA3-induced antheridiogenesis.

The research object concerns partially explained mechanisms of plant hormone participation in male sex determination in plants, among them in A. phyllitidis gametophytes during GA3-induced antheridiogenesis. To provide an explanation of the mechanisms of fluorescence and white-light microscopy, cytophotometric, autoradiographic and spectrophotometic methods were used to study cell cycle, the number of nucleoli, the amount of DiOC6-stained IMN/ER, in which endoplasmic reticulum (ER) constitutes the main part, and its distribution as well as the amounts of proteins and chlorophylls and activities of acidic (Ac) and basic (Ba) phosphatases (Phases). It was revealed that antheridiogenesis was accompanied by cell cycle arrest at S-phase, changes of the number of nucleoli with simultaneous changes of the amount of IMN/ER and its distribution as well as fluctuations of protein amounts and of activities of acidic (Ac) and basic (Ba) phosphatases (Phases). The results indicated that initiation of antheridiogenesis in A. phyllitidis gametophytes by GA3 was related to the elevation of GAs/ANs in the culture media, during its induction phase, and the elevation of IMN/ER and GAs/ANs amounts, during expression phase of this process.

Misleading conclusions from exogenous ABA application: a cautionary tale about the evolution of stomatal responses to changes in leaf water status.

Stomatal responses to changes in leaf water status are critical for minimizing excessive water loss during soil drought. A major debate has surrounded the evolution of stomatal responses to water status and this debate has particularly focused on the evolution of the regulatory role of the drought hormone abscisic acid (ABA). Studies relying on the application of high levels of exogenous ABA have occasionally concluded that all stomata respond to ABA and that stomatal regulation in response to this hormone has not evolved over the past 450 million years. In contrast, studies which have investigated stomatal function in intact plants, as well as the role of endogenous ABA in regulating stomatal aperture, have found major evolutionary transitions in the functional regulation of stomata across land plant lineages. We show that endogenous ABA plays no role in closing the stomata of the fern Nephrolepis exaltata during natural soil drought, in contrast to a recent finding using isolated epidermis and exceptionally high levels of exogenous ABA. We conclude that stomatal behavior in intact plants has evolved over time, and may have shaped the evolutionary and ecological success of successive land plant lineages.

Effect of daily exposure to Pb-contaminated water on Salvinia biloba physiology and phytoremediation performance.

Lead (Pb) removal from water column was evaluated in batch experiments using naturally occurring Salvinia biloba Raddi (S. biloba) specimens collected from Middle Paraná River and exposed every 24 h to a fresh discharge of water contaminated with 2.65 ± 0.07, 12.62 ± 0.02 or 30.57 ± 0.01 mg L-1 Pb, during 10 consecutive days. S. biloba demonstrated a great ability for metal concentration-dependent Pb removal under these stressful conditions. Additionally, Pb toxicity in plants was assessed by the quantification of physiological parameters in root-like modified fronds (named "roots"), and its aerial leaf-like fronds (named "leaves") of submerged S. biloba. Photosynthetic (carotenoids, chlorophyll a, b, and total) and antioxidant pigments (anthocyanins and flavonoids), soluble carbohydrate content, and membrane stability index of both roots and leaves were affected as the metal concentration increased. In general, root deterioration was more pronounced than that in leaves, suggesting a greater implication of the former organs in Pb removal by S. biloba. All of these deleterious effects were well correlated with qualitative changes observed at plant phenotype during the assay. In conclusion, S. biloba may be considered as a water fern useful in phytoremediation strategies towards management of residual water bodies contaminated with Pb. In addition, these macrophytes could also be valuable for water biomonitoring contributing to improve risk assessments related to metal presence in wastewaters.

Differential responses of stomatal kinetics and steady-state conductance to abscisic acid in a fern: comparison with a gymnosperm and an angiosperm.

Origins of abscisic acid (ABA)-mediated metabolic control of stomatal conductance have been suggested to be recent, based on a gradualistic model of stomatal evolution. In ferns, steady-state stomatal conductance (gs ) was unresponsive to ABA in some studies, supporting this model. Stomatal kinetic responses to ABA have not been considered. We used dynamic gas exchange methods to characterise half times of stomatal opening and closing in response to step changes in light, across a range of ABA exposures in three diverse taxa. All taxa had asymmetric kinetics, with closure slower than opening in fern and cedar, but faster than opening in soybean. Closing was fastest in soybean but opening was slowest. Stomatal kinetics, particularly for closure, responded to ABA in all three taxa. Steady-state gs did not respond significantly to ABA in fern or cedar but responded strongly in soybean. Stomatal kinetics were responsive to ABA in fern. This finding supports a contrasting, single origin model, with ABA-mediated regulation of stomata arising early, in conjunction with stomata themselves. Stomatal kinetics are underutilised. Differential responses of opening and closing rates to environmental and hormonal stimuli may provide insights into phylogeny and stomatal regulatory strategies with potential application to selection for crop improvement.

Are the green synthesized nanoparticles safe for environment? A case study of aquatic plant Azolla filiculoides as an indicator exposed to magnetite nanoparticles fabricated using microwave hydrothermal treatment and plant extract.

It has been claimed that the green synthesized NPs possess no toxicity in comparison to the NPs fabricated via conventional protocols like reduction by sodium borohydride. Therefore, it is necessary to test the toxic effects of NPs on environment. In the current study, we report the binding of Fe3O4 NPs to galate ions containing biomaterial namely "galate bio-capping agent". The bio-capping agent is simply mixed with the Fe3+ cations at pH 8 to produce negatively charged bio-capped Fe3O4 NPs. Finally, the toxic effects of the Fe3O4 NPs were investigated on some growth and developmental indices of the aquatic plant species Azolla filiculoides. The relative frond number and relative growth rate were calculated after treatment of plants with different concentrations of bio-capped Fe3O4 NPs. In addition, the content of phenolics as well as antioxidant enzymes' activity including superoxide dismutase and peroxidase were assessed. The Fe3O4 NPs led to growth reduction and significant changes in total phenol and flavonoid content as well as in antioxidant enzymes' activity. All these findings confirm reactive oxygen species formation due to the nanoparticle toxicity. In consequence, the enzymatic and non-enzymatic antioxidant defense systems of plant were stimulated against oxidative stress.

Separate and combined effects of glyphosate and copper on growth and antioxidative enzymes in Salvinia natans (L.) All.

The coexistence of glyphosate and copper is widely found in bodies of water and terrestrial ecosystems due to widespread application of herbicides and heavy metal. However, their joint ecotoxicological risks in aquatic environments remain unknown. The experiment investigated the individual and combined effects of glyphosate and copper on the growth and physiological response in Salvinia natans (L.) All. The results showed that their joint toxicity is related to concentration. Antagonistic effects were induced when plants were exposed to low concentrations of glyphosate and copper (≤1 + 0.2 mg l-1). Synergistic effects were elicited at higher doses (≥5 + 1 mg l-1). In addition, increased hydrogen peroxide levels indicated the occurrence of oxidative stress at individual or combined exposures. To cope with oxidative stress, S. natans can activate the antioxidant defense systems, including increased superoxide dismutase and changes in peroxidase, ascorbate peroxidase and catalase. High concentrations of combined pollution exceed the oxidative defense capabilities of plants, and therefore, malondialdehyde content increased significantly. Our results indicated that the ecotoxicity of glyphosate or copper may be exacerbated in aquatic environments and caused obvious damage to S. natans.

Hydraulics Regulate Stomatal Responses to Changes in Leaf Water Status in the Fern Athyrium filix-femina.

Stomatal responses to changes in leaf water status are important for the diurnal regulation of gas exchange and the survival of plants during drought. These stomatal responses in angiosperm species are well characterized, yet in species of nonseed plants, an ongoing debate surrounds the role of metabolism, particularly the role of the hormone abscisic acid (ABA), in functionally regulating stomatal responses to changes in leaf water status. Here, we measured the stomatal response to changes in vapor pressure difference (VPD) in two natural forms of the fern species Athyrium filix-femina, recently suggested to have stomata that are regulated by ABA. The two forms measured had considerable differences in key hydraulic traits, including leaf hydraulic conductance and capacitance, as well as the kinetics of stomatal response to changes in VPD. In both forms, the stomatal responses to VPD could be accurately predicted by a dynamic, mechanistic model that assumes guard cell turgor changes in concert with leaf turgor in the light, and not via metabolic processes including the level of ABA. During drought, endogenous ABA did not play a role in stomatal closure, and exogenous ABA applied to live, intact leaves did not induce stomatal closure. Our results indicate that functional stomatal responses to changes in leaf water status in ferns are regulated by leaf hydraulics and not metabolism. With ferns being sister to seed plants, this result has implications for the evolutionary reconstruction of functional stomatal responses across vascular land plant lineages.

Differential expression of proteins in the leaves and roots of cadmium-stressed Microsorum pteropus, a novel potential aquatic cadmium hyperaccumulator.

Microsorum pteropus is a fully or partially submerged Polypodiaceae fern that has been proven to be a potential Cd aquatic hyperaccumulator. Proteomic analysis was used in this study to investigate the resistance mechanisms of M. pteropus root and leaf tissues under Cd stress. M. pteropus plants were exposed to up to 500 µM Cd in hydroponics for 7 days. The plant can accumulate >4,000 mg/kg Cd in both root and leaf dry mass. Meanwhile, the proteins in roots and leaves in the 500 µM Cd treatment were separated and analyzed by proteomics. Eight proteins with altered expression in roots and twenty proteins with altered expression in leaves were identified using MALDI-TOF/TOF-MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) in this study. The proteins were involved in energy metabolism, antioxidant activity, cellular metabolism and protein metabolism. However, just three proteins were significantly differentially expressed in both tissues, and they were all involved in basal metabolism, indicating different resistance mechanisms between roots and leaves. Root tissues of M. pteropus mainly resist Cd damage by antioxidants and the enhancement of energy metabolism, while leaf tissues of M. pteropus mainly protect themselves by maintaining photosynthetic functions and the regulation of cellular metabolism.