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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, H 2 O 2 , 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., Competing Interests: Declaration of competing interest The authors declare that they have not used any AI tools for designing the experiment or for preparing the manuscript., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Stomatal movement is vital for plants to exchange gases and adaption to terrestrial habitats, which is regulated by environmental and phytohormonal signals. Here, we demonstrate that hydrogen peroxide (H 2 O 2 ) is required for light-induced stomatal opening. H 2 O 2 accumulates specifically in guard cells even when plants are under unstressed conditions. Reducing H 2 O 2 content through chemical treatments or genetic manipulations results in impaired stomatal opening in response to light. This phenomenon is observed across different plant species, including lycopodium, fern, and monocotyledonous wheat. Additionally, we show that H 2 O 2 induces the nuclear localization of KIN10 protein, the catalytic subunit of plant energy sensor SnRK1. The nuclear-localized KIN10 interacts with and phosphorylates the bZIP transcription factor bZIP30, leading to the formation of a heterodimer between bZIP30 and BRASSINAZOLE-RESISTANT1 (BZR1), the master regulator of brassinosteroid signaling. This heterodimer complex activates the expression of amylase, which enables guard cell starch degradation and promotes stomatal opening. Overall, these findings suggest that H 2 O 2 plays a critical role in light-induced stomatal opening across different plant species., (© 2024. The Author(s).)

Sperm nuclear basic proteins (SNBPs) were isolated from extracted antheridia-rich male gametophytes raised from spores of the swordfern, Polystichum munitum . Electrophoretic (acetic acid-urea PAGE and SDS-PAGE) and chromatographic (rp-HPLC) characterization of the nuclear proteins exhibited the characteristics of the histone (H-type). In both types of gel electrophoresis, histones H1, H2A, and H2B showed an altered electrophoretic mobility corresponding to that which is routinely observed for the histones in other plants. Histones present during spermatogenesis of the fern P. munitum were compared with the few current SNBPs known to be present in higher and lower evolutionary plant clades. A transition from an early protamine (P-type) SNBPs in charophytes and bryophytes to the (H-type) SNBP observed here is reminiscent of similar reversions observed in the animal kingdom., Competing Interests: The authors have no conflicting interests or competing interests, financial or otherwise.

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 H 2 O 2 ), 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., (© 2024. The Author(s).)

To combat the global loss of wetlands and their essential functions, the restoration and creation of wetlands is imperative. However, wetland development is challenging when soils have been in prolonged agricultural use, often resulting in a substantial nutrient legacy, especially of phosphorous (P). Inundating these soils typically leads to P mobilization, resulting in poor water quality and low biodiversity recovery. As a potential novel means to overcome this challenge, we tested whether cultivation of the floating fern Azolla filiculoides could simultaneously extract and recycle P, and provide a commercial product. Azolla has high growth rates due to the nitrogen fixing capacity of its microbiome and is capable of luxury consumption of P. Azolla cultivation may also accelerate soil P mobilization and subsequent extraction by causing surface water anoxia and the release of iron-bound P. To test this approach, we cultivated Azolla on 15 P-rich former agricultural soils in an indoor mesocosm experiment. Soils were inundated and either left unvegetated or inoculated with A. filiculoides during two 8-week cultivation periods. Biomass was harvested at different intervals (weekly/monthly/bimonthly) to investigate the effect of harvesting frequency on oxygen (O 2 ) and nutrient dynamics. We found that Azolla attained high growth rates only on soils with high mobilization of labile P, as plant cover did not reduce surface water O 2 concentrations in the first phase after inundation. This concurred with low porewater iron to P ratios (<10) and high porewater P concentrations. A. filiculoides cultivation substantially reduced surface water nutrient concentrations and extracted P at rates up to 122 kg ha -1 yr -1 . We conclude that rapid P extraction by A. filiculoides cultivation is possible on soils rich in labile P, offering new perspectives for wetland rehabilitation. Additional field trials are recommended to investigate long-term feasibility, seasonal variations, and the influence of potential grazers and pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Rare earth elements (REEs) are increasingly crucial for modern technologies. Plants could be used as a biogeochemical pathfinder and a tool to extract REEs from deposits. However, a paucity of information on suitable plants for these tasks exists., Methods: We aimed to discover new REE-(hyper)accumulating plant species by performing an X-ray fluorescence (XRF) survey at the Herbarium of the Muséum national d'Histoire naturelle (MNHN, Paris, France). We selected specific families based on the likelihood of containing REE-hyperaccumulating species, using known taxa that accumulate REEs. A total of 4425 specimens, taken in the two main evolutionary lineages of extant vascular plants, were analysed, including the two fern families Blechnaceae (n = 561) and Gleicheniaceae (n = 1310), and the two flowering plant families Phytolaccaceae (n = 1137) and Juglandaceae (n = 1417)., Key Results: Yttrium (Y) was used as a proxy for REEs for methodological reasons, and a total of 268 specimens belonging to the genera Blechnopsis (n = 149), Dicranopteris (n = 75), Gleichenella (n = 32), Phytolacca (n = 6), Carya (n = 4), Juglans (n = 1) and Sticherus (n = 1) were identified with Y concentrations ranging from the limit of detection (LOD) >49 µg g-1 up to 1424 µg g-1. Subsequently, analysis of fragments of selected specimens by inductively coupled plasma atomic emission spectroscopy (ICP-AES) revealed that this translated to up to 6423 µg total REEs g-1 in Dicranopteris linearis and up to 4278 µg total REEs g-1 in Blechnopsis orientalis which are among the highest values ever recorded for REE hyperaccumulation in plants. It also proved the validity of Y as an indicator for REEs in XRF analysis of herbarium specimens. The presence of manganese (Mn) and zinc (Zn) was also studied by XRF in the selected specimens. Mn was detected in 1440 specimens ranging from the detection limit at 116 µg g-1 up to 3807 µg g-1 whilst Zn was detected in 345 specimens ranging from the detection limit at 77 µg g-1 up to 938 µg g-1., Conclusions and Implications: This study led to the discovery of REE accumulation in a range of plant species, substantially higher concentrations in species known to be REE hyperaccumulators, and records of REE hyperaccumulators outside of the well-studied populations in China., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

There are many available reports of secondary metabolites as bioactive molecules from culturable endophytes, nevertheless, there are scarce research pertaining to the levels of metabolites in plants with respect to the incidence and colonisation of fungal endophytes in the same foliar tissues. Therefore, the study was focussed to examine whether fungal endophyte colonisation and the accumulation of secondary metabolites, such as flavonoids and phenols, in the plants are related in any way. For this reason, the study aims to analyse phenols and flavonoids from the fronds of eleven pteridophytes along with the culture-dependent isolation of fungal endophytes from the host plants subsequently assigning them to morphological category and their quantitative analysis and further resolving its identities through molecular affiliation. The results revealed that nine morpho-categories of fungal endophytes were allotted based on culture attributes, hyphal patterns and reproductive structural characters. Highest numbers of species were isolated from Adiantum capillus-veneris and least was recorded from Pteris vittata and Dicranopteris linearis. Maximum phenol content was analysed from the fronds of P. vittata and lowest was recorded in A. capillus-veneris. Highest flavonoid content was measured in D. linearis and lowest was detected in Christella dentata. Significant negative correlation was observed between phenol content of ferns and species richness of fungi. Moreover, significant positive correlation was observed with the relative abundance of Chaetomium globosum and flavonoid content of ferns and negative significant relation was found between relative abundance of Pseudopestalotiopsis chinensis and phenol content of pteridophytes. The occurrence and the quantitative aspects of endophytes in ferns and their secondary metabolites are discussed., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Human skin is exposed daily to oxidative stress factors such as UV light, chemical pollutants, and invading organisms. Reactive oxygen species (ROS) are intermediate molecules that cause cellular oxidative stress. In order to survive in an oxygen-rich environment, all aerobic organisms, including mammals, have evolved enzymatic and non-enzymatic defence systems. The interruptins from an edible fern Cyclosorus terminans possess antioxidative properties and can scavenge intracellular ROS in adipose-derived stem cells.

Objectives: This study aimed to evaluate the antioxidative efficacy of interruptins A, B, and C in cultured human dermal fibroblasts (HDFs) and epidermal keratinocytes (HEKs). Moreover, the anti-photooxidative activity of interruptins in ultraviolet (UV)-exposed skin cells was investigated.

Methods: The intracellular ROS scavenging capacity of interruptins in skin cells was measured by flow cytometry. Their induction effects on gene expression of the endogenous antioxidant enzymes was monitored using real-time polymerase chain reaction.

Results: Interruptins A and B, but not interruptin C, were highly effective in ROS scavenging, particularly in HDFs. Interruptins A and B upregulated gene expression of superoxide dismutase (SOD)1, SOD2, catalase (CAT), and glutathione peroxidase (GPx) in HEKs, but they only induced SOD1, SOD2, and GPx gene expression in HDFs. Additionally, interruptins A and B efficiently suppressed UVA- and UVB-induced ROS generation in both HEKs and HDFs.

Conclusion: The results suggest that these naturally occurring interruptins A and B are potent natural antioxidants and therefore may have the potential in the future of inclusion in antiaging cosmeceutical products.

This study attempted to explore the metabolome profile of Azolla filiculoides subjected to two different concentrations of BPA (1 and 30 mg L -1 ) in congruence with two different durations (3 and 9 days) of treatment. Bisphenol A (BPA) is a ubiquitously occurring environmental pollutant that imparts acute toxicity in aquatic plants. Therefore, studying the variations in the fern metabolome profile and identifying stress-responsive metabolites can help develop criteria for assessing the aquatic ecosystem. In recent times, metabolomics has drawn attention for its ability to detect biochemical processes and help link plant responses with environmental stresses. However, the studies concerning the metabolome profile of A. filiculoides exposed to environmental contaminants are limited. In the present study, the untargeted metabolomics study allowed the detection of a large array of metabolites, with 767 shared metabolites representing 41 crucial pathways. Exposure to 30 mg L -1 BPA seemingly disrupted the primary metabolism of the fern and induced a shift toward defense-related pathways. Additionally, BPA stress triggered the expression of metabolites like 3,4-dihydroxyphenylglycol, perillic acid, and perillaldehyde in BPA&#95;L3 (1 mg L -1 for 3 days) and BPA&#95;L9 (1 mg L -1 for 9 days) samples indicating protective mechanism of the plants. Conversely, the BPA&#95;H3 (30 mg L -1 for 3 days) and BPA&#95;H9 (30 mg L -1 for 9 days) samples expressed a distinct set of markers like luteolin, 3-hydroxyanthranilic acid, cinnamaldehyde, and l-DOPA indicating the onset of senescence and apoptosis related pathways can help in the health assessment of freshwater ecosystems and also appraisal of ecotoxicological risks imposed by BPA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ageing in dry chlorophyllous propagules is leaded by photooxidation through the photosynthetic machinery, but why species differ in longevity and the ageing mechanisms of when light and oxygen are absent are unknown. We hypothesize that the cellular antioxidant capacity is key for the inter- and intra-specific differences in the ageing process. We have tested this hypothesis in chlorophyllous spores of two ferns. They were subjected to four different storage regimes resulting from light/dark and normoxia/hypoxia combinations. Lipophilic and hydrophilic antioxidants, reactive oxygen species (ROS), and photosynthetic pigments were analysed in parallel to germination and the recovery of Fv/Fm over a storage period of up to 22-months. We show that light and oxygen accelerate the ageing process, but their mechanisms (ROS, increase, antioxidant capacity decrease, loss of efficiency of the photosystem II, pigment degradation) appear the same under all conditions tested. The end of the asymptomatic phase of longevity, when a sudden drop of germination occurs, seems to be determined by a threshold in the depletion of antioxidants. Our results support the hypothesis that ageing kinetics in dry plant propagules is determined by the antioxidant system, but also suggests an active role of the photosynthetic machinery during ageing, even in darkness and hypoxia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Land plant sexual reproduction involves the transition of cells from somatic to reproductive identity during post-embryonic development. In Arabidopsis, the leucine-rich repeat receptor-like kinase EXCESS MICROSPOROCYTES1 (EXS/EMS1) restricts the number of sporogenous cells during the transition from diploid tissue to haploid spore production by promoting the formation of the tapetum cell layer within the anther. Although all land plants studied contain EMS1 genes, its function is unknown beyond a few angiosperms. In the model fern Ceratopteris (Ceratopteris richardii), we discovered an EMS1 homolog (CrEMS1) that functions to suppress formation of reproductive structures on vegetative leaves of the fern sporophyte, a role not found in angiosperms. Suppression of CrEMS1 by RNAi did not affect sporogenesis on reproductive leaves but did affect antheridium production of the fern gametophyte. Expression patterns of CrEMS1 across developmental stages suggest threshold levels of CrEMS1 control the specification of reproductive organs during both generations of the fern. Additional EMS1 homologs present in the fern genome suggest a dynamic role of EMS1 receptors in the evolution of reproductive development in vascular plants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Despite its small size, the water fern Azolla is a giant among plant symbioses. Within each of its leaflets, a specialized leaf cavity is home to a population of nitrogen-fixing cyanobacteria (cyanobionts). Although a number of plant-cyanobiont symbioses exist, Azolla is unique in that its symbiosis is perpetual: the cyanobionts are inherited during sexual and vegetative propagation. What underpins the communication between the two partners? In angiosperms, the phytohormone salicylic acid (SA) is a well-known regulator of plant-microbe interactions. Using high-performance liquid chromatography-tandem mass spectrometry, we pinpoint the presence of SA in the fern. Comparative genomics and phylogenetics on SA biosynthesis genes across Chloroplastida reveal that the entire Phenylalanine ammonia-lyase-dependent pathway likely existed in the last common ancestor of land plants. Indeed, Azolla filiculoides secondarily lost its isochorismate synthase but has the genetic competence to derive SA from benzoic acid; the presence of SA in artificially cyanobiont-free Azolla supports the existence of this route. Global gene expression data and SA levels from cyanobiont-containing and -free A. filiculoides link SA synthesis with the symbioses: SA appears to induce cyanobacterial proliferation, whereas removal of the symbiont results in reduced SA levels in a nitrogen-dependent manner., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Progressive climate changes cause disturbance of water relations in tropical rainforests, where epiphytic ferns are an important element of biodiversity. In these plants, the efficiency of photosynthesis is closely related to the efficiency of water transport. In addition, due to the lack of contact with the soil, epiphytes are extremely susceptible to water-deficit stress. The aim of this experiment was to determine the response of the photosynthetic apparatus of Platycerium bifurcatum to a 6-week water deficit. The hydration and pigment composition of leaves were determined using reflectance spectroscopy and epifluorescence microscopy. Chlorophyll a fluorescence kinetics parameters, fluorescence induction curves (OJIP), low-temperature fluorescence curves at 77 K and proline concentration were analyzed at seven time points. After a decrease in leaf hydration by 10-15%, there were disturbances in the oxidation-reduction balance, especially in the initial photochemical reactions, a rapid decrease in plant vitality (PI) and significant fluctuations in chlorophyll a fluorescence parameters. The relative size of PSI antenna structures compared to PSII decreased in the following weeks of water deficit. Changes in photochemical reactions were accompanied by a decrease in gross photosynthesis and an increase in proline concentration. Changes in the functioning of photosynthesis light phase and the pigment composition of leaves are related to the resistance of elkhorn fern to long-term water deficit.

Exposure to high light intensity (HL) and cold treatment (CT) induces reddish pigmentation in Azolla filiculoides , an aquatic fern. Nevertheless, how these conditions, alone or in combination, influence Azolla growth and pigment synthesis remains to be fully elucidated. Likewise, the regulatory network underpinning the accumulation of flavonoids in ferns is still unclear. Here, we grew A. filiculoides under HL and/or CT conditions for 20 days and evaluated the biomass doubling time, relative growth rate, photosynthetic and non-photosynthetic pigment contents, and photosynthetic efficiency by chlorophyll fluorescence measurements. Furthermore, from the A. filiculoides genome, we mined the homologs of MYB , bHLH , and WDR genes, which form the MBW flavonoid regulatory complex in higher plants, to investigate their expression by qRT-PCR. We report that A. filiculoides optimizes photosynthesis at lower light intensities, regardless of the temperature. In addition, we show that CT does not severely hamper Azolla growth, although it causes the onset of photoinhibition. Coupling CT with HL stimulates the accumulation of flavonoids, which likely prevents irreversible photoinhibition-induced damage. Although our data do not support the formation of MBW complexes, we identified candidate MYB and bHLH regulators of flavonoids. Overall, the present findings are of fundamental and pragmatic relevance to Azolla 's biology.

Rare earth elements (REEs) are critical for numerous modern technologies, and demand is increasing globally; however, production steps are resource-intensive and environmentally damaging. Some plant species are able to hyperaccumulate REEs, and understanding the biology behind this phenomenon could play a pivotal role in developing more environmentally friendly REE recovery technologies. Here, we identified a REE transporter NRAMP REE Transporter 1 (NREET1) from the REE hyperaccumulator fern Dicranopteris linearis . Although NREET1 belongs to the natural resistance-associated macrophage protein (NRAMP) family, it shares a low similarity with other NRAMP members. When expressed in yeast, NREET1 exhibited REE transport capacity, but it could not transport divalent metals, such as zinc, nickel, manganese, or iron. NREET1 is mainly expressed in D. linearis roots and predominantly localized in the plasma membrane. Expression studies in Arabidopsis thaliana revealed that NREET1 functions as a transporter mediating REE uptake and transfer from root cell walls into the cytoplasm. Moreover, NREET1 has a higher affinity for transporting light REEs compared to heavy REEs, which is consistent to the preferential enrichment of light REEs in field-grown D. linearis . We therefore conclude that NREET1 may play an important role in the uptake and consequently hyperaccumulation of REEs in D. linearis . These findings lay the foundation for the use of synthetic biology techniques to design and produce sustainable, plant-based REE recovery systems.

The enzyme flavone synthase Is (FNS Is) converts flavanones to flavones, whereas flavanone 3β-hydroxylases (F3Hs) catalyze the formation of dihydroflavonols, a precursor of flavonols and anthocyanins. Canonical F3Hs have been characterized in seed plants, which are evolutionarily related to liverwort FNS Is. However, as important evolutionary lineages between liverworts and seed plants, ferns FNS Is and F3Hs have not been identified. In the present study, we characterized a bifunctional enzyme PnFNS I/F3H from the fern Psilotum nudum. We found that PnFNS I/F3H catalyzed the conversion of naringenin to apigenin and dihydrokaempferol. In addition, it catalyzed five different flavanones to generate the corresponding flavones. Site-directed mutagenesis results indicated that the P228-Y228 mutant protein displayed the FNS I/F2H activity (catalyzing naringenin to generate apigenin and 2-hydroxynaringenin), thus having similar functions as liverwort FNS I/F2H. Moreover, the overexpression of PnFNS I/F3H in Arabidopsis tt6 and dmr6 mutants increased the content of flavones and flavonols in plants, further indicating that PnFNS I/F3H showed FNS I and F3H activities in planta. This is the first study to characterize a bifunctional enzyme FNS I/F3H in ferns. The functional transition from FNS I/F3H to FNS I/F2H will be helpful in further elucidating the relationship between angiosperm F3Hs and liverwort FNS Is., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this study, the potential of Azolla filiculoides , a freshwater fern species, on phenanthrene phytoremediation and biodegradation was investigated. Furthermore, the effect of phenanthrene on growth performance, photosynthetic activity and biosynthesis, and accumulation of secondary metabolites of A. filiculodes were evaluated. Plants were grown in a nitrogen-free Hoagland and exposed to different phenanthrene concentrations (0, 1, 5, and 10 mg/L). Exposure to 10 mg/L phenanthrene caused a significant reduction (42%) in Azolla filiculoides growth compared to control on day 14. The photosynthetic pigment content of A. filiculoides treated with 1 and 5 mg/L was almost the same as the control, while 10 mg/L phenanthrene was significantly reduced. In comparison to unplanted controls, the biodegradation percentages obtained from the planted growth medium were found to be 88, 69, and 60%, respectively, for the application of 1, 5, and 10 mg/L phenanthrene. Data on plant growth, photosynthetic pigments, secondary metabolite contents, and biodegradation percentages indicated the tolerance level and the effective phytoremediation potential of A. filiculoides for phenanthrene was <10 mg/L. The results indicated that A. filiculoides is highly effective in phytoremediation of low concentrations of phenanthrene pollution in a short time.

During fern spore germination, lipid hydrolysis primarily provides the energy to activate their metabolism. In this research, fatty acids (linoleic, oleic, palmitic and stearic) were quantified in the spores exposed or not to priming (hydration-dehydration treatments). Five fern species were investigated, two from xerophilous shrubland and three from a cloud forest. We hypothesised that during the priming hydration phase, the fatty acids profile would change in concentration, depending on the spore type (non-chlorophyllous and crypto-chlorophyllous). The fatty acid concentration was determined by gas chromatograph-mass spectrometer. Chlorophyll in spores was vizualised by epifluorescence microscopy and quantified by high-resolution liquid chromatography with a DAD-UV/Vis detector. Considering all five species and all the treatments, the oleic acid was the most catabolised. After priming, we identified two patterns in the fatty acid metabolism: (1) in non-chlorophyllous species, oleic, palmitic, and linoleic acids were catabolised during imbibition and (2) in crypto-chlorophyllous species, these fatty acids increased in concentration. These patterns suggest that crypto-chlorophyllous spores with homoiochlorophylly (chlorophyll retained after drying) might not require the assembly of new photosynthetic apparatus during dark imbibition. Thus, these spores might require less energy from pre-existing lipids and less fatty acids as 'building blocks' for cell membranes than non-chlorophyllous spores, which require de novo synthesis and structuring of the photosynthetic apparatus., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

The interactions between ferns and the environment have been frequently researched. However, detailed data on how ferns respond to specific stresses and a combination of stress factors during cultivation are lacking. This study assessed the effects of salinity and full sunlight and the combination of both stresses on the growth and selected metabolic parameters of two hardy ferns ( Athyrium nipponicum cv. Red Beauty and Dryopteris erythrosora ) under production conditions. Hardy ferns are highly interesting ornamental plants that can serve as a potential source of antioxidants for the pharmaceutical, cosmetic, and food industries. The results showed that in both ferns, salinity and salinity combined with full sunlight lowered the dry weight of the aerial part and potassium/sodium and calcium/potassium ratio compared with control plants. Salinity, full sunlight, and multi-stress did not affect the total polyphenol content in both ferns but increased the total free amino acids and flavonoids in D. erythrosora . In A. nipponicum cv. Red Beauty, all stressors decreased the total free amino acids content and the antioxidant activities determined by ABTS, DPPH, FRAP, and reducing power assays. By contrast, plants of D. erythrosora grown under full sunlight are characterized by higher antioxidant activities determined by DPPH, FRAP, and reducing power assays. Overall, a greater adaptive potential to abiotic stresses was found in D. erythrosora than in A. nipponicum cv. Red Beauty. Our findings shed some light on the physiological mechanisms responsible for sensitivity/tolerance to salinity, full sunlight, and combined stresses in hardy ferns.

Background: This study determined the effect of processing conditions on protein extractability from Azolla pinnata fern, and their influence on the physicochemical, structural, techno-functional properties and protein quality., Results: The protein extraction from A. pinnata fern was optimized through response surface methodology obtaining a maximum yield of 18.93% with a recovery rate of 73.66%. The A. pinnata fern protein concentrate (AFPC) had five protein bands with a molecular weight ranging from 17 to 56 kDa. AFPC contained high β-sheet structure (36.61%), favouring its good thermal properties with three endothermic peaks at 54.28, 86.52 and 166.25 °C. The AFPC scored ≥ 1 for all essential amino acids, except for lysine and histidine. The AFPC exhibited exceptionally high techno-functional properties, particularly for water holding (5.46 g g -1 ) and fat absorption capacity (10.08 g g -1 ), and gelling properties (5% gelation concentration). The AFPC had high in vitro digestibility of 73%, signifying its high availability for human consumption., Conclusion: The underexploited A. pinnata fern is a potential source of edible protein, thus a promising nutraceutical or ingredient of functional and health-promoting foods. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Cadmium (Cd) or nickel (Ni) were applied as a foliar spray (1 µM solution over one month) to mimic air pollution and to monitor metabolic responses and oxidative stress in the pteridophyte species. Exogenous metals did not affect the metal content of the soil and had relatively little effect on the essential elements in leaves or rhizomes. The amounts of Cd and Ni were similar in treated leaves (7.2 µg Cd or 5.3 µg Ni/g DW in mature leaves compared with 0.4 µg Cd or 1.2 µg Ni/g DW in the respective control leaves), but Ni was more abundant in rhizomes (56.6 µg Ni or 3.4 µg Cd/g DW), resulting in a higher Cd translocation and bioaccumulation factor. The theoretical calculation revealed that ca. 4% of Cd and 5.5% of Ni from the applied solution per plant/pot was absorbed. Excess Cd induced stronger ROS production followed by changes in SOD and CAT activities, whereas nitric oxide (NO) stimulation was less intense, as detected by confocal microscopy. The hadrocentric vascular bundles in the petioles also showed higher ROS and NO signals under metal excess. This may be a sign of increased ROS formation, and high correlations were observed. Proteins and amino acids were stimulated by Cd or Ni application in individual organs, whereas phenols and flavonols were almost unaffected. The data suggest that even low levels of exogenous metals induce an oxidative imbalance, although no visible damage is observed, and that the responses of ferns to metals are similar to those of seed plants or algae.

Background: Flavonoid C-glycosides have many beneficial effects and are widely used in food and medicine. However, plants contain a limited number of flavonoid C-glycosides, and it is challenging to create these substances chemically., Results: To screen more robust C-glycosyltransferases (CGTs) for the biosynthesis of flavonoid C-glycosides, one CGT enzyme from Stenoloma chusanum (ScCGT1) was characterized. Biochemical analyses revealed that ScCGT1 showed the C-glycosylation activity for phloretin, 2-hydroxynaringenin, and 2-hydroxyeriodictyol. Structure modeling and mutagenesis experiments indicated that the glycosylation of ScCGT1 may be initiated by the synergistic action of conserved residue His26 and Asp14. The P164T mutation increased C-glycosylation activity by forming a hydrogen bond with the sugar donor. Furthermore, when using phloretin as a substrate, the extracellular nothofagin production obtained from the Escherichia coli strain ScCGT1-P164T reached 38 mg/L, which was 2.3-fold higher than that of the wild-type strain. Finally, it is proved that the coupling catalysis of CjFNS I/F2H and ScCGT1-P164T could convert naringenin into vitexin and isovitexin., Conclusion: This is the first time that C-glycosyltransferase has been characterized from fern species and provides a candidate gene and strategy for the efficient production of bioactive C-glycosides using enzyme catalysis and metabolic engineering., (© 2022. The Author(s).)

Copper (Cu) and zinc (Zn) have a high demand in the industry. However, these ions, at high concentrations, can cause severe damage to both fauna and flora. Phytoremediation has gained international importance because its relatively low cost and it is environmentally friendly. The aim of the present study was to evaluate the capacity of Salvinia minima of accumulating Cu and Zn from aqueous solutions of various external concentrations (20, 40 and 80 µmol L -1 of CuSO 4 and ZnSO 4 , separately). In addition, to estimate the effect of exposure of S. minima plants to those metals, on various physiological parameters (growth potential, maximum quantum efficiency of PSII, electrolyte leakage: as a cell membrane integrity index). S. minima was able of accumulating more Zn than Cu in its tissues, reaching values of 6.96 mg Cu g -1 dry weight (DW) and 19.6 mg Zn g -1 DW when exposed to 80 μM of each metal during 96 h, that were stored mainly at roots. Despite accumulating less Cu in its tissues, Cu had more severe reductions in various physiological parameters than Zn (in maximum quantum efficiency, integrity of cell membranes, and growth). We conclude that this species can be useful in the phytoremediation for copper and zinc in relatively short time, as maximum accumulation occurred within the first 24 h. However, in the long term, the accumulation of such metals is accompanied by a negative impact in the appearance, physiology, and growth of this plant species, which was more severe for copper exposure than for zinc., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In photosynthetic organisms except angiosperms, an alternative electron sink that is mediated by flavodiiron proteins (FLVs) plays the major role in preventing PSI photoinhibition while cyclic electron flow (CEF) is also essential for normal growth under fluctuating light. However, the dynamic changes of FLVs and CEF has not yet been well clarified. In this study, we measured the P700 signal, chlorophyll fluorescence, and electrochromic shift spectra in the fern Cyrtomium fortune and the gymnosperm Nageia nagi . We found that both species could not build up a sufficient proton gradient (∆pH) within the first 30 s after light abruptly increased. During this period, FLVs-dependent alternative electron flow was functional to avoid PSI over-reduction. This functional time of FLVs was much longer than previously thought. By comparison, CEF was highly activated within the first 10 s after transition from low to high light, which favored energy balancing rather than the regulation of a PSI redox state. When FLVs were inactivated during steady-state photosynthesis, CEF was re-activated to favor photoprotection and to sustain photosynthesis. These results provide new insight into how FLVs and CEF interact to regulate photosynthesis in non-angiosperms.

Studies investigating the mechanisms underlying the variation of photosynthesis along plant phylogeny and especially during domestication are of great importance, and may provide new insights to further improve crop photosynthesis. In the present study, we compiled a database including 542 sets of data of leaf gas exchange parameters and leaf structural and chemical traits in ferns and fern allies, gymnosperms, non-crop angiosperms, and crops. We found that photosynthesis was dramatically improved from ferns and fern allies to non-crop angiosperms, and further increased in crops. The improvement of photosynthesis during phylogeny and domestication was related to increases in carbon dioxide diffusional capacities and, to a lesser extent, biochemical capacity. Cell wall thickness rather than chloroplast surface area facing intercellular airspaces drives the variation of mesophyll conductance. The variation of the maximum carboxylation rate was not related to leaf nitrogen content. The slope of the relationship between mass-based photosynthesis and nitrogen was lower in crops than in non-crop angiosperms. These findings suggest that the manipulation of cell wall thickness is the most promising approach to further improve crop photosynthesis, and that an increase of leaf nitrogen will be less efficient in improving photosynthesis in crops than in non-crop angiosperms., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (βααβ) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc) n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc) n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc) n -binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc) n . Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Phytoremediation potential of Azolla in removal of nitrogen from wastewater has been promising. However, little is known about the response of Azolla to high concentrations of nitrogen. In this study, the responses of four Azolla species to different concentrations of total nitrogen ranging from 0 to 180 mg L -1 were examined. The responses varied among different species, and the high nitrogen-tolerant species A. caroliniana and A. microphylla could remove nitrogen from aqueous solutions with higher efficiencies. We further performed transcriptome analysis to explore the molecular mechanism underlying the response to high nitrogen stress in Azolla. RNA-seq analysis revealed a synergistic regulatory network of differentially expressed genes (DEGs) involved in nitrogen transport and metabolism in A. microphylla, mainly in the roots. Under high nitrogen treatment, the DEGs encoding nitrate transporters or nitrate transporter 1/peptide transporters (NRTs/NPFs), ammonium transporters (AMTs), nitrate reductase (NIA), nitrite reductase (NIR) and glutamine synthetases/glutamate synthases (GSs/GOGATs) were down-regulated, and the DEGs encoding glutamate dehydrogenases (GDHs) were up-regulated, suggesting that A. microphylla possessed high tolerance against excess nitrogen through down-regulation of nitrate and ammonium uptake and fine regulation of nitrogen assimilation in the roots. Our results provided a theoretical foundation for better utilization of Azolla for wastewater treatment., (Copyright © 2022. Published by Elsevier B.V.)

Phytohormones play an important role in the adaptive evolution of terrestrial plants. Brassinosteroids (BRs) are essential hormones that regulate multiple aspects of plant growth and development in angiosperms, but the presence of BR signaling in non-seed plants such as ferns remains unknown. Here, we found that BR promotes the growth of Ceratopteris richardii , while the synthetic inhibitor PCZ inhibits the growth. Using full-length transcriptome sequencing, we identified four BRI1-like receptors. By constructing chimeric receptors, we found that the kinase domains of these four receptors could trigger BR downstream signaling. Further, the extracellular domains of two receptors were functionally interchangeable with that of BRI1. In addition, we identified a co-receptor, CtSERK1, that could phosphorylate with CtBRL2s in vitro. Together, these proved the presence of a receptor complex in Ceratopteris richardii that might perceive BR and activate downstream hormone signaling. Our results shed light on the biological and molecular mechanisms of BR signaling in ferns and the role of BR hormone signaling in the adaptive evolution of terrestrial plants.

The aim of our research was to describe the structure and growth potential of a cell suspension of the tree fern Cyathea smithii . Experiments were performed on an established cell suspension with ½ MS medium supplemented with 9.05 µM 2,4-D + 0.88 µM BAP. In the experiments, attention was paid to the microscopic description of cell suspension, evaluation of cell growth dependent on the initial mass of cells and organic carbon source in the medium, the length of the passage, the content of one selected flavonoid in the post-culture medium, nuclear DNA content, ethylene production, and the antimicrobial value of the extract. For a better understanding of the cell changes that occurred during the culture of the suspension, the following structures of the cell were observed: nucleus, lipid bodies, tannin deposits, starch grains, cell walls, primary lamina, and the filaments of metabolites released into the medium. The nuclear DNA content (acriflavine-Feulgen staining) of cell aggregates distinctly indicated a lack of changes in the sporophytic origin of the cultured cell suspension. The physiological activity of the suspension was found to be high because of kinetics, intensive production of ethylene, and quercetin production. The microbiological studies suggested that the cell suspension possessed a bactericidal character against microaerobic Gram-positive bacteria. A sample of the cell suspension showed bacteriostatic activity against aerobic bacteria.

Pteris vittata is an arsenic (As) hyperaccumulator that can accumulate several thousand mg As kg -1 DW in aboveground biomass. A key factor for its hyperaccumulation ability is its highly efficient As long-distance translocation system. However, the underlying molecular mechanisms remain unknown. We isolated PvAsE1 through the full-length cDNA over-expression library of P. vittata and characterized it through a yeast system, RNAi gametophytes and sporophytes, subcellular-location and in situ hybridization. Phylogenomic analysis was conducted to estimate the appearance time of PvAsE1. PvAsE1 was a plasma membrane-oriented arsenite (AsIII) effluxer. The silencing of PvAsE1 reduced AsIII long-distance translocation in P. vittata sporophytes. PvAsE1 was structurally similar to solute carrier (SLC)13 proteins. Its transcripts could be observed in parenchyma cells surrounding the xylem of roots. The appearance time was estimated at c. 52.7 Ma. PvAsE1 was a previously uncharacterized SLC13-like AsIII effluxer, which may contribute to AsIII long-distance translocation via xylem loading. PvAsE1 appeared late in fern evolution and might be an adaptive subject to the selection pressure at the Cretaceaou-Paleogene boundary. The identification of PvAsE1 provides clues for revealing the special As hyperaccumulation characteristics of P. vittata., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Recent results suggest that metabolism-mediated stomatal closure mechanisms are important to regulate differentially the stomatal speediness between ferns and angiosperms. However, evidence directly linking mesophyll metabolism and the slower stomatal conductance (g s ) in ferns is missing. Here, we investigated the effect of exogenous application of abscisic acid (ABA), sucrose and mannitol on stomatal kinetics and carried out a metabolic fingerprinting analysis of ferns and angiosperms leaves harvested throughout a diel course. Fern stomata did not respond to ABA in the time period analysed. No differences in the relative decrease in g s was observed between ferns and the angiosperm following provision of sucrose or mannitol. However, ferns have slower g s responses to these compounds than angiosperms. Metabolomics analysis highlights that ferns have a higher accumulation of secondary rather than primary metabolites throughout the diel course, with the opposite being observed in angiosperms. Our results indicate that metabolism-mediated stomatal closure mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms, in which the slower stomatal closure in ferns is associated with the lack of ABA-responsiveness, to a reduced capacity to respond to mesophyll-derived sucrose and to a higher carbon allocation toward secondary metabolism, which likely modulates both photosynthesis-g s and growth-stress tolerance trade-offs., (© 2021 John Wiley & Sons Ltd.)

The tree ferns are an important component of tropical forests. In view of this, the enhancement of in vitro production of these plants is needed. Thus, the effect of different light-emitting diodes (LEDs) as well as control fluorescent lamps (Fl) and a 3-week-long period of darkness at the beginning of in vitro culture on micropropagation of the tree fern Cyathea delgadii Sternb. was analysed. Moreover, the photosynthetic pigment content and secondary metabolite profiles were estimated. The period of darkness contributed to a high production of somatic embryo-derived sporophytes and a low production of gametophytes. The formation of new sporophytes was stimulated by RBY (35% red, 15% blue, and 50% yellow) and B (100% blue) lights when the stipe explants or whole young sporophytes were used in the culture, respectively. The elongation of the roots and leaves was stimulated by RBfR light (35% red, 15% blue, and 50% far red), while root production increased under RBY light. The RB (70% red and 30% blue) and B lights stimulated the accumulation of chlorophyll better than Fl light. The most abundant metabolite found in the plant extracts was trans -5- O -caffeoylquinic acid (1.013 µg/mg of dry weight). The extract obtained from plants growing in a greenhouse had the best antioxidant activity.

Plants can produce cellular metal nanoparticles (NPs) from the uptake of metal ions, but the mechanism remains unclear. This work reported the new insight into different fates of iron (Fe) and nickel (Ni) ions to transform into the metal NPs in Azolla pinnata roots. After exposing to ferric nitrate, nickel nitrate, and a combination of both for 12 h, the energy dispersive X-ray fluorescence analysis indicated the efficient uptakes of both metal ions in the roots and their transports into the shoots. Transmission electron microscope images revealed the accumulation of spherical FeNPs, but not NiNPs, near the cell wall and cell membrane, and inside vacuoles and multivesicular bodies in cortical and vascular cells at the root tips. The energy dispersive X-ray analysis suggested that the formation of metal NPs depended on the sufficient concentration of metal ions localized in the roots. FeNPs were identified to ɑ-Fe 2 O 3 and Fe 3 O 4 by selected area electron diffraction analysis. The formation of FeNPs might involve the increase of superoxide dismutase activity. This work is the first report about the cellular biogenesis of metal NPs in plant roots that likely depends on cellular metal content and involves the reducing activity of antioxidant enzymes.

Stomata evolved as plants transitioned from water to land, enabling carbon dioxide uptake and water loss to be controlled. In flowering plants, the most recently divergent land plant lineage, stomatal pores actively close in response to drought. In this response, the phytohormone abscisic acid (ABA) triggers signaling cascades that lead to ion and water loss in the guard cells of the stomatal complex, causing a reduction in turgor and pore closure. Whether this stimulus-response coupling pathway acts in other major land plant lineages is unclear, with some investigations reporting that stomatal closure involves ABA but others concluding that closure is passive. Here, we show that in the model fern Ceratopteris richardii active stomatal closure is conditional on sensitization by pre-exposure to either low humidity or exogenous ABA and is promoted by ABA. RNA-seq analysis and de novo transcriptome assembly reconstructed the protein-coding complement of the C. richardii genome, with coverage comparable to other plant models, enabling transcriptional signatures of stomatal sensitization and closure to be inferred. In both cases, changes in abundance of homologs of ABA, Ca 2+ , and ROS-related signaling components were observed, suggesting that the closure-response pathway is conserved in ferns and flowering plants. These signatures further suggested that sensitization is achieved by lowering the threshold required for a subsequent closure-inducing signal to trigger a response. We conclude that the canonical signaling network for active stomatal closure functioned in at least a rudimentary form in the stomata of the last common ancestor of ferns and flowering plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We sequenced the sporophyte transcriptome of Killarney fern ( Vandenboschia speciosa (Willd.) G. Kunkel). In addition to being a rare endangered Macaronesian-European endemism, this species has a huge genome (10.52 Gb) as well as particular biological features and extreme ecological requirements. These characteristics, together with the systematic position of ferns among vascular plants, make it of high interest for evolutionary, conservation and functional genomics studies. The transcriptome was constructed de novo and contained 36,430 transcripts, of which 17,706 had valid BLAST hits. A total of 19,539 transcripts showed at least one of the 7362 GO terms assigned to the transcriptome, whereas 6547 transcripts showed at least one of the 1359 KEGG assigned terms. A prospective analysis of functional annotation results provided relevant insights on genes involved in important functions such as growth and development as well as physiological adaptations. In this context, a catalogue of genes involved in the genetic control of plant development, during the vegetative to reproductive transition, in stress response as well as genes coding for transcription factors is given. Altogether, this study provides a first step towards understanding the gene expression of a significant fern species and the in silico functional and comparative analyses reported here provide important data and insights for further comparative evolutionary studies in ferns and land plants in general.

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

In a laboratory based study, Salvinia natans L. was pre-treated with reduced glutathione (GSH) following transfer under 2, 4-Dicholro phenoxy acetic acid (2,4-D), peroxide (H 2 O 2 ), dark and irradiation. Plants recorded 2, 4-D bio-accumulation and tolerance maximally under 500 µM following absorption kinetics modulated with GSH in changes of relative water content (20.98%), growth rate (3.04%) and net assimilation rate (1.3 fold) over control. GSH pre-treatment minimized the oxidative revelation with reactive oxygen species (ROS) by 5.55% decrease under 2, 4-D and 1.3, 1.2, 0.8 fold increase through the other stresses. Apoplastic NADPH-oxidase expression was moderated by GSH with 11.76% less over the control. Also the activity of alcohol dehydrogenase and glutathione-S-transferase had their altered values by 1.5 and 9.0 fold increases respectively and may serve as biomarkers. The oxidized:reduced glutathione was positively correlated with glutathione-peroxidase (r=+0.99) and negatively with glutathione reductase (r=-0.04). The induced activities sustained oxidized:reduced GSH pool by 1.09 fold and had varied polymorphic gene expression under 2, 4-D and allied stresses. This study may be relevant to consider Salvinia as a potent weed species remediating 2, 4-D toxicity in soil with its wider hyper-accumulating efficiency. The cellular responses in tolerance to oxidative stress and thereby, induced physiological attributes may opt for selection pressures in other weed flora for broader aspects of phytoremediation against xenobiotics like 2, 4-D., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Several plant species survive in the metal-contaminated environment by minimization of detrimental effects of metal exposure and cellular accumulation, but little is known about their capability to transform the uptake metal ions into nanoparticles, especially in nonspherical shapes. This work firstly reported the in vivo formation of spherical and rod-shaped lead nanoparticles (PbNPs) from the uptake lead ions in root cells of water velvet (Azolla pinnata). The energy-dispersive X-ray fluorescence analysis revealed the high level of lead (67.21 ± 0.70%) and the modulated levels of sulfur, potassium, and calcium in the treated roots. Fourier-transform infrared spectroscopy spectral analysis suggested the changes of biochemical constituents in Pb-treated roots, including carbohydrates, organic compounds, proteins, and nucleic acids. Transmission electron microscope (TEM) images revealed the formation of spherical, short rod, and long rod PbNPs dominantly in epidermal, cortical, and vascular cells in the plant roots, respectively. The analyses of energy-dispersive X-ray spectroscopy, high-resolution TEM, and selected area diffraction TEM indicated the body-centered tetragonal lattice of lead oxide nanoparticles (PbONPs) in the root cells., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

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.

Despite the implications leaves have for life, their origin and development remain debated. Analyses across ferns and seed plants are fundamental to address the conservation or independent origins of megaphyllous leaf developmental mechanisms. Class I KNOX expression studies have been used to understand leaf development and, in ferns, have only been conducted in species with divided leaves. We performed expression analyses of the C lass I KNOX and Histone H4 genes throughout the development of leaf primordia in two simple-leaved and one divided-leaved fern taxa. We found Class I KNOX are expressed (1) throughout young and early developing leaves of simple and divided-leaved ferns, (2) later into leaf development of divided-leaved species compared to simple-leaved species, and (3) at the leaf primordium apex and margins. H4 expression is similar in young leaf primordia of simple and divided leaves. Persistent Class I KNOX expression at the margins of divided leaf primordia compared with simple leaf primordia indicates that temporal and spatial patterns of Class I KNOX expression correlate with different fern leaf morphologies. However, our results also indicate that Class I KNOX expression alone is not sufficient to promote divided leaf development in ferns. Class I KNOX patterns of expression in fern leaves support the conservation of an independently recruited developmental mechanism for leaf dissection in megaphylls, the shoot-like nature of fern leaves compared with seed plant leaves, and the critical role marginal meristems play in fern leaf development., Competing Interests: The authors declare no conflict of interest.

Main Conclusion: A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.

The distribution of highly de-esterified homogalacturonans (HGs) in dividing protodermal cells of the monocotyledon Zea mays , the dicotyledon Vigna sinensis , and the fern Asplenium nidus was investigated in order to examine whether the cell wall region adjoining the preprophase band (PPB) is locally diversified. Application of immunofluorescence revealed that de-esterified HGs were accumulated selectively in the cell wall adjacent to the PPB in: (a) symmetrically dividing cells of stomatal rows of Z. mays , (b) the asymmetrically dividing protodermal cells of Z. mays , (c) the symmetrically dividing guard cell mother cells (GMCs) of Z. mays and V. sinensis , and (d) the symmetrically dividing protodermal cells of A. nidus . A common feature of the above cell types is that the cell division plane is defined by extrinsic cues. The presented data suggest that the PPB cortical zone-plasmalemma and the adjacent cell wall region function in a coordinated fashion in the determination/accomplishment of the cell division plane, behaving as a continuum. The de-esterified HGs, among other possible functions, might be involved in the perception and the transduction of the extrinsic cues determining cell division plane in the examined cells.

The fossil record and models of atmospheric concentrations of O 2 and CO 2 suggest that past shifts in plant ecological dominance often coincided with dramatic changes in Earth's atmospheric composition. This study tested the effects of past changes in atmospheric composition on the photosynthetic physiology of a limited range of early-diverging angiosperms (eight), gymnosperms (three), and ferns (two). We performed physiological measurements on all species and used the results to parameterize simulations of their photosynthetic paleophysiology using three independent modeling approaches. Unique physiological attributes were identified for the three evolutionary groups: angiosperm taxa displayed significantly higher mesophyll conductance ( g m ), yet their stomatal conductance ( g s ) was lower than that of ferns. Gymnosperm taxa displayed low g s and g m , but they partially offset their significant diffusional limitations on photosynthesis through their higher maximum Rubisco carboxylation rate. Despite their high total conductance to CO 2 , fern taxa lacked an optimized control of g s , which was reflected in their low intrinsic water use efficiency. Simulations of the photosynthetic physiology of ferns, angiosperms, and gymnosperms through Earth's history demonstrated that past fluctuations in O 2 and CO 2 concentrations may have resulted in significant shifts in the relative competitiveness of the three evolutionary groups. Although preliminary because of limited species sampling, these findings hint at a potential mechanistic basis for the observed broad temporal correlation between atmospheric change and shifts in plant evolutionary group-level richness observed in the fossil record and are presented as a framework to be tested with paleophotosynthetic proxies and through increased species sampling., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

The hydrophyte Azolla filiculoides can be a useful model to assess if TiO 2 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 TiO 2 NPs, A. filiculoides plants were transferred to cadmium-contaminated water with or without TiO 2 nanoparticles. After 5 days of treatment, cadmium uptake, morpho-anatomical, and physiological aspects were studied in plants. The continuous presence of TiO 2 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 H 2 O 2 content, but did not suppress TBARS level.

With the continued increase of technological uses of cerium oxide nanoparticles (CeO 2 NPs or nanoceria) and their unregulated disposal, the accumulation of nanoceria in the environment is inevitable. Concomitantly, atmospheric carbon dioxide (CO 2 ) levels continue to rise, increasing the concentrations of bicarbonate ions in aquatic ecosystems. This study investigates the influence of CeO 2 NPs (from 0 to 100 μgL -1 ) in the presence and absence of an elevated bicarbonate (HCO 3 - ) 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 CeO 2 NPs and also detected interactive responses between CeO 2 NPs and HCO 3 - exposure at the tissue level. The results showed that the higher CeO 2 NPs levels in the presence of HCO 3 - increased the non-photochemical quenching (NPQ) and coefficient of photochemical quenching in dark (qP d ) compared to the absence of HCO 3 . Moreover, the presence of HCO 3 - significantly decreased the NPQ at all levels of CeO 2 NPs demonstrating that HCO 3 - 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 CeO 2 NPs and elevated HCO 3 - ion concentration on photosynthetic systems., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Plants undergo a phase transition from vegetative to reproductive development that triggers floral induction. Genes containing an AAI (α-amylase inhibitor) domain form a large gene family, but there have been no comprehensive analyses of this gene family in any plant species. Here, we identified 336 AAI genes from nine plant species including122 AAI genes in cotton (Gossypium hirsutum). The AAI gene family has evolutionarily conserved amino acid residues throughout the plant kingdom. Phylogenetic analysis classified AAI genes into five major clades with significant polyploidization and showing effects of genome duplication. Our study identified 42 paralogous and 216 orthologous gene pairs resulting from segmental and whole-genome duplication, respectively, demonstrating significant contributions of gene duplication to expansion of the cotton AAI gene family. Further, GhAAI66 was preferentially expressed in flower tissue and as responses to phytohormone treatments. Ectopic expression of GhAAI66 in Arabidopsis and silencing in cotton revealed that GhAAI66 triggers a phase transition to induce early flowering. Further, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of RNA sequencing data and qRT-PCR (quantitative reverse transcription-PCR) analysis indicated that GhAAI66 integrates multiple flower signaling pathways including gibberellin, jasmonic acid, and floral integrators to trigger an early flowering cascade in Arabidopsis. Therefore, characterization of the AAI family provides invaluable insights for improving cotton breeding., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)