Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

Phytoconstituents of Selaginella effusa Alston and Their α-Glucosidase as well as Urease Inhibitory Activities.

Three new compounds (1-2, 14), as well as 22 known compounds (3-13, 15-25), were extracted for the first time from the Selaginella effusa Alston (S. effusa). For the unknown compounds, the planar configurations were determined via NMR and by high-resolution mass spectrometry, while their absolute configurations were determined by calculated electronic circular dichroism (ECD), and the configuration of the stereogenic center of biflavones 4-5 were established for the first time. The pure compounds (1-25) were tested in vitro to determine the inhibitory activity of the enzyme-catalyzed reactions. Compounds 1-9 inhibited α-glucosidase with IC50 values ranging from 0.30±0.02 to 4.65±0.04 µM and kinetic analysis of enzyme inhibition indicated that biflavones 1-3 were mixed-type α-glucosidase inhibitors. Compounds 12-13 showed excellent inhibitory activity against urease, with compound 12 (IC50 =4.38±0.31 µM) showing better inhibitory activity than the positive control drug AHA (IC50 13.52±0.61 µM). In addition, molecular docking techniques were used to simulate inhibitor-enzyme binding and to estimate the binding posture of the α-glucosidase and urease catalytic sites.

Impacts of methyl jasmonate on Selaginella martensii: volatiles, transcriptomics, phytohormones, and gas exchange.

Methyl jasmonate (MeJA) induces various defence responses in seed plants, but for early plant lineages, information on the potential of jasmonates to elicit stress signalling and trigger physiological modifications is limited. The spikemoss Selaginella martensii was exposed to a range of MeJA concentrations (0, 10, 25, and 50 mM), and biogenic volatile organic compound (BVOC) emissions, photosynthetic rate (A), and stomatal conductance (gs) were continuously measured. In addition, changes in phytohormone concentrations and gene expression were studied. Enhancement of methanol, lipoxygenase pathway volatiles and linalool emissions, and reductions in A and gs, were MeJA dose-dependent. Before MeJA treatment, the concentration of 12-oxo-phytodienoic acid (OPDA) was 7-fold higher than jasmonic acid (JA). MeJA treatment rapidly increased OPDA and JA concentrations (within 30 min), with the latter more responsive. Some genes involved in BVOC biosynthesis and OPDA-specific response were up-regulated at 30 min after MeJA spraying, whereas those in the JA signalling pathway were not affected. Although JA was synthesized in S. martensii, OPDA was prioritized as a signalling molecule upon MeJA application. MeJA inhibited primary and enhanced secondary metabolism; we propose that fast-emitted linalool could serve as a marker of elicitation of stress-induced metabolism in lycophytes.

Selaginella tamariscina Inhibits Glutamate-Induced Autophagic Cell Death by Activating the PI3K/AKT/mTOR Signaling Pathways.

Glutamate-induced neural toxicity in autophagic neuron death is partially mediated by increased oxidative stress. Therefore, reducing oxidative stress in the brain is critical for treating or preventing neurodegenerative diseases. Selaginella tamariscina is a traditional medicinal plant for treating gastrointestinal bleeding, hematuria, leucorrhea, inflammation, chronic hepatitis, gout, and hyperuricemia. We investigate the inhibitory effects of Selaginella tamariscina ethanol extract (STE) on neurotoxicity and autophagic cell death in glutamate-exposed HT22 mouse hippocampal cells. STE significantly increased cell viability and mitochondrial membrane potential and decreased the expression of reactive oxygen species, lactate dehydrogenase release, and cell apoptosis in glutamate-exposed HT22 cells. In addition, while glutamate induced the excessive activation of mitophagy, STE attenuated glutamate-induced light chain (LC) 3 II and Beclin-1 expression and increased p62 expression. Furthermore, STE strongly enhanced the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) phosphorylation activation. STE strongly inhibited glutamate-induced autophagy by activating the PI3K/Akt/mTOR signaling pathway. In contrast, the addition of LY294002, a PI3K/Akt inhibitor, remarkably suppressed cell viability and p-Akt and p62 expression, while markedly increasing the expression of LC3 II and Beclin-1. Our findings indicate that autophagy inhibition by activating PI3K/Akt/mTOR phosphorylation levels could be responsible for the neuroprotective effects of STE on glutamate neuronal damage.

Rapid screening for acetylcholinesterase inhibitors in Selaginella doederleinii Hieron by using functionalized magnetic Fe3O4 nanoparticles.

Insufficient acetylcholine (ACh) can cause cognitive and memory dysfunction, clinically known as, Alzheimer's disease (AD). Acetylcholinesterase (AChE) can hydrolyze ACh into acetic acid and inactivate choline. Therefore, inhibiting the activity of AChE would help to improve the effectiveness of AD treatment. Currently, the methods for rapid screening of AChE inhibitors are limited. This study reports the application of AChE-immobilized magnetic nanoparticles as a drug screening tool to screen AChE inhibitors for natural products. First, AChE was immobilized on a surface of amino-modified magnetic nanoparticles using covalent binding and the AChE concentration, and the pH as well as time was optimized to obtain the maximum enzyme immobilization yield (61.4 µg/mg), and the kinetic model indicated that AChE-immobilized magnetic nanoparticles and the substrate had the high affinity and specificity. Then, a ligand fishing experiment was carried out using a mixed model of tacrine (an inhibitor of AChE) and caffeic acid (a non-inhibitor of AChE) to verify the specificity of the immobilized AChE, and the conditions for ligand fishing were further optimized. Finally, the optimized immobilized AChE was combined with UPLC-MS to screen for AChE inhibitors in Selaginella doederleinii Hieron extracts. Four compounds were confirmed to be potent AChE inhibitors. Among the four compounds, amentoflavone had a stronger AChE inhibitory effect than tacrine (positive control) with an IC50 of 0.73 ± 0.009 µmol/L. The results showed that AChE-functionalized magnetic nanoparticles can be used in the discovery of target drugs from complex matrices.

Anatomical and Biochemical Traits Related to Blue Leaf Coloration of Selaginella uncinata.

Selaginella uncinata shows particularly rare blue leaves. Previous research has shown that structural interference by the cell wall of adaxial epidermal cells imparts blue coloration in leaves of S. uncinata; the objective of this study was to see whether anthocyanins might additionally contribute to this color, as changes in pH, and conjugation with metals and other flavonoids is also known to result in blue coloration in plants. We compared anatomical and biochemical traits of shade-grown (blue) S. uncinata leaves to high light (red) leaves of the same species and also to a non-blue (green) leaves of a congeneric S. kraussiana. By examining the anatomical structure, we found that the shape of adaxial epidermis of S. uncinata leaves was convex or lens-shaped on the lateral view and irregular circles with smooth embossment on the top view. These features were different from those of the abaxial and adaxial epidermis of S. kraussiana. We suspect that these structures increase the proportion of incident light entering the cell, deepening the leaf color, and therefore may be related to blue leaf color in S. uncinata. By examining biochemical traits, we found little difference in leaf pH value among the leaf types; all leaves contained several metal ions such as Mg, Fe, Mn, and copigments such as flavones. However, because there was no anthocyanin in blue S. uncinata leaves, we concluded that blue coloration in S. uncinata leaves is not caused by the three hypotheses of blue coloration: alkalization of the vacuole pH, metal chelation, or copigmentation with anthocyanins, but it may be related to the shape of the leaf adaxial epidermis.

Backbone and sidechain 1H, 15N and 13C resonance assignments of the free and RNA-bound tandem zinc finger domain of the tristetraprolin family member from Selaginella moellendorffii.

Members of the tristetraprolin (TTP) family of RNA binding proteins (RBPs) regulate the metabolism of a variety of mRNA targets. In mammals, these proteins modulate many physiological processes, including immune cell activation, hematopoiesis, and embryonic development. Regulation of mRNA stability by these proteins requires that the tandem zinc finger (TZF) domain binds initially and directly to target mRNAs, ultimately leading to their deadenylation and decay. Proteins of this type throughout eukarya possess a highly conserved TZF domain, suggesting that they are all capable of high-affinity RNA binding. However, the mechanism of TTP-mediated mRNA decay is largely undefined. Given the vital role that these TTP family proteins play in maintaining RNA homeostasis throughout eukaryotes, we focused here on the first, key step in this process: recognition and binding of the TZF domain to target RNA. For these studies, we chose a primitive plant, the spikemoss Selaginella moellendorffii, which last shared a common ancestor with humans more than a billion years ago. Here we report the near complete backbone and side chain resonance assignments of the spikemoss TZF domain, including: (1) the assignment of the RNA-TZF domain complex, representing one of only two data sets currently available for the entire TTP family of proteins; and (2) the first NMR resonance assignments of the entire TZF domain, in the RNA-free form. This work will serve as the basis for further NMR structural investigations aimed at gaining insights into the process of RNA recognition and the mechanisms of TTP-mediated mRNA decay.

Photosystem II photoinhibition and photoprotection in a lycophyte, Selaginella martensii.

The Lycophyte Selaginella martensii efficiently acclimates to diverse light environments, from deep shade to full sunlight. The plant does not modulate the abundance of the Light Harvesting Complex II, mostly found as a free trimer, and does not alter the maximum capacity of thermal dissipation (NPQ). Nevertheless, the photoprotection is expected to be modulatable upon long-term light acclimation to preserve the photosystems (PSII, PSI). The effects of long-term light acclimation on PSII photoprotection were investigated using the chlorophyll fluorometric method known as "photochemical quenching measured in the dark" (qPd ). Singularly high-qPd values at relatively low irradiance suggest a heterogeneous antenna system (PSII antenna uncoupling). The extent of antenna uncoupling largely depends on the light regime, reaching the highest value in sun-acclimated plants. In parallel, the photoprotective NPQ (pNPQ) increased from deep-shade to high-light grown plants. It is proposed that the differences in the long-term modulation in the photoprotective capacity are proportional to the amount of uncoupled LHCII. In deep-shade plants, the inconsistency between invariable maximum NPQ and lower pNPQ is attributed to the thermal dissipation occurring in the PSII core.

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1).

Nonclassical P450s of the CYP74 family catalyse the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. The model organism, spikemoss Selaginella moellendorffii Hieron, possesses at least ten CYP74 genes of novel J, K, L, and M subfamilies. The cloning of three CYP74L genes and catalytic properties of recombinant proteins are described in the present work. The CYP74L1 possessed mainly hydroperoxide lyase (HPL) activity towards the 13(S)-hydroperoxide of α-linolenic acids (13-HPOT) and nearly equal HPL and allene oxide synthase (AOS) activities towards the 13(S)-hydroperoxide of linoleic acids (13-HPOD). The 9-hydroperoxides were poor substrates for CYP74L1 and led to the production of mainly the α-ketols (AOS products) and minorities of HPL and epoxyalcohol synthase (EAS) products. The CYP74L2 possessed the AOS activity towards all tested hydroperoxides. CYP74L3 possessed low HPL/EAS activity. Besides, the aerial parts of S. moellendorffii plants possessed complex oxylipins patterns including divinyl ethers, epoxyalcohols, and 12-oxo-phytodienoic acid. Characterization of the CYP74L enzymes and oxylipin pattern updates the knowledge on the complex oxylipin biosynthetic machinery in the surviving oldest taxa of vascular plants.

Cytokinin Perception in Ancient Plants beyond Angiospermae.

Cytokinins (CKs) control many plant developmental processes and responses to environmental cues. Although the CK signaling is well understood, we are only beginning to decipher its evolution. Here, we investigated the CK perception apparatus in early-divergent plant species such as bryophyte Physcomitrium patens, lycophyte Selaginella moellendorffii, and gymnosperm Picea abies. Of the eight CHASE-domain containing histidine kinases (CHKs) examined, two CHKs, PpCHK3 and PpCHK4, did not bind CKs. All other CHK receptors showed high-affinity CK binding (KD of nM range), with a strong preference for isopentenyladenine over other CK nucleobases in the moss and for trans-zeatin over cis-zeatin in the gymnosperm. The pH dependences of CK binding for these six CHKs showed a wide range, which may indicate different subcellular localization of these receptors at either the plasma- or endoplasmic reticulum membrane. Thus, the properties of the whole CK perception apparatuses in early-divergent lineages were demonstrated. Data show that during land plant evolution there was a diversification of the ligand specificity of various CHKs, in particular, the rise in preference for trans-zeatin over cis-zeatin, which indicates a steadily increasing specialization of receptors to various CKs. Finally, this distinct preference of individual receptors to different CK versions culminated in vascular plants, especially angiosperms.

Identification of resurrection genes from the transcriptome of dehydrated and rehydrated Selaginella tamariscina.

Selaginella tamariscina is a lycophyta species that survives under extremely dry conditions via the mechanism of resurrection. This phenomenon involves the regulation of numerous genes that play vital roles in desiccation tolerance and subsequent rehydration. To identify resurrection-related genes, we analyzed the transcriptome between dehydration conditions and rehydration conditions of S. tamariscina. The de novo assembly generated 124,417 transcripts with an average size of 1,000 bp and 87,754 unigenes. Among these genes, 1,267 genes and 634 genes were up and down regulated by rehydration compared to dehydration. To understand gene function, we annotated Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The unigenes encoding early light-inducible protein (ELIP) were down-regulated, whereas pentatricopeptide repeat-containing protein (PPR), late embryogenesis abundant proteins (LEA), sucrose nonfermenting protein (SNF), trehalose phosphate phosphatase (TPP), trehalose phosphate synthase (TPS), and ABC transporter G family (ABCG) were significantly up-regulated in response to rehydration conditions by differentially expressed genes (DEGs) analysis. Several studies provide evidence that these genes play a role in stress environment. The ELIP and PPR genes are involved in chloroplast protection during dehydration and rehydration. LEA, SNF, and trehalose genes are known to be oxidant scavengers that protect the cell structure from the deleterious effect of drought. TPP and TPS genes were found in the starch and sucrose metabolism pathways, which are essential sugar-signaling metabolites regulating plant metabolism and other biological processes. ABC-G gene interacts with abscisic acid (ABA) phytohormone in the stomata opening during stress conditions. Our findings provide valuable information and candidate resurrection genes for future functional analysis aimed at improving the drought tolerance of crop plants.

Biochemical Characterization and Function of Eight Microbial Type Terpene Synthases from Lycophyte Selaginella moellendorffii.

Selaginella moellendorffii is a lycophyte, a member of an ancient vascular plant lineage. Two distinct types of terpene synthase (TPS) genes were identified from this species, including S. moellendorffii TPS genes (SmTPSs) and S. moellendorffii microbial TPS-like genes (SmMTPSLs). The goal of this study was to investigate the biochemical functions of SmMTPSLs. Here, eight full-length SmMTPSL genes (SmMTPSL5, -15, -19, -23, -33, -37, -46, and -47) were functionally characterized from S. moellendorffii. Escherichia coli-expressed recombinant SmMTPSLs were tested for monoterpenes synthase and sesquiterpenes synthase activities. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by typical plant TPSs when provided with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as the substrates. Meanwhile, SmMTPSL23, -33, and -37 were up-regulated when induced by alamethicin (ALA) and methyl jasmonate (MeJA), suggesting a role for these genes in plants response to abiotic stresses. Furthermore, this study pointed out that the terpenoids products of SmMTPSL23, -33, and -37 have an antibacterial effect on Pseudomonas syringae pv. tomato DC3000 and Staphylococcus aureus. Taken together, these results provide more information about the catalytic and biochemical function of SmMTPSLs in S. moellendorffii plants.

In an ancient vascular plant the intermediate relaxing component of NPQ depends on a reduced stroma: Evidence from dithiothreitol treatment.

In plants, the non-photochemical quenching of chlorophyll fluorescence (NPQ) induced by high light reveals the occurrence of a multiplicity of regulatory processes of photosynthesis, primarily devoted to photoprotection of photosystem I and II (PSI and PSII). The study of NPQ relaxation in darkness allows the separation of three kinetically distinct phases: the fast relaxing high-energy quenching qE, the intermediate relaxing phase and the nearly non-relaxatable photoinhibitory quenching. Several processes can underlie the intermediate phase. In the ancient vascular plant Selaginella martensii (Lycopodiophyta) this component, here termed qX, was previously proposed to reflect mainly a photoprotective energy-spillover from PSII to PSI. It is hypothesized that qX is induced by an over-reduced photosynthetic electron transport chain from PSII to final acceptors. To test this hypothesis the leaves were treated with the reductant dithiothreitol (DTT) and the chlorophyll fluorescence changes were analysed during the induction with high irradiance and the subsequent relaxation in darkness. DTT treatment caused the well-known decrease in NPQ induction and expectedly resulted in a disturbed photosynthetic electron flow. The relaxation curves of Y(NPQ), formally representing the quantum yield of the regulatory thermal dissipation, revealed a DTT dose-dependent decrease in amplitude not only of qE, but also of qX, up to the complete disappearance of the latter. Modelling of the relaxation curves under alternative scenarios led to the conclusion that DTT is permissive with respect to qX induction but suppresses its dark relaxation. The strong dependence of qX on the chloroplast redox state is discussed with respect to its proposed energy-spillover photoprotective significance in a lycophyte.

An Evolutionarily Primitive and Distinct Auxin Metabolism in the Lycophyte Selaginella moellendorffii.

Auxin is a key regulator of plant growth and development. Indole-3-acetic acid (IAA), a plant auxin, is mainly produced from tryptophan via indole-3-pyruvate (IPA) in both bryophytes and angiosperms. Angiosperms have multiple, well-documented IAA inactivation pathways, involving conjugation to IAA-aspartate (IAA-Asp)/glutamate by the GH3 auxin-amido synthetases, and oxidation to 2-oxindole-3-acetic acid (oxIAA) by the DAO proteins. However, IAA biosynthesis and inactivation processes remain elusive in lycophytes, an early lineage of spore-producing vascular plants. In this article, we studied IAA biosynthesis and inactivation in the lycophyte Selaginella moellendorffii. We demonstrate that S. moellendorffii mainly produces IAA from the IPA pathway for the regulation of root growth and response to high temperature, similar to the angiosperm Arabidopsis. However, S. moellendorffii exhibits a unique IAA metabolite profile with high IAA-Asp and low oxIAA levels, distinct from Arabidopsis and the bryophyte Marchantia polymorpha, suggesting that the GH3 family is integral for IAA homeostasis in the lycophytes. The DAO homologs in S. moellendorffii share only limited similarity to the well-characterized rice and Arabidopsis DAO proteins. We therefore suggest that these enzymes may have a limited role in IAA homeostasis in S. moellendorffii compared to angiosperms. We provide new insights into the functional diversification of auxin metabolic genes in the evolution of land plants.

Two New Abietane Diterpenoids from Selaginella moellendorffii Hieron.

Two new abietane diterpenoids, (3S,5R,10S)-3-hydroxy-12-O-demethyl-11-deoxy-19(4→3)-abeo-cryptojaponol, 12,19-dihydroxyabieta-8,11,13-trien-7-one, were isolated from Selaginella moellendorffii Hieron., together with one known abietane diterpenoid and four known tetracyclic triterpenoids. Their structures were characterized by their 1D- and 2D-NMR, ECD and mass spectral studies. All compounds were tested for their inhibitory effects on proliferation of three human cancer cells (human non-small-cell lung carcinoma cell lines A549 and human breast adenocarcinoma cell lines MDA-MB-231 and MCF-7) in vitro. Among them, three compounds displayed modest cytotoxic activities against the above three human cancer cell lines with IC50 values ranging from 16.28 to 40.67 µM.

Selaginella convolute extract mediated synthesis of ZnO NPs for pain management in emerging nursing care.

Zinc oxide (ZnO), an inorganic metal oxide established in the form of nanoparticles, has considerable biological properties. The current research uses Selaginella convolute (S. convolute) leaf extract to establish ZnO NPs and to assess their use in pain management. S. Convolute leaf extract mediated ZnO NPs were characterized by modern techniques and instruments such as Fourier transforms infrared spectroscopy (FTIR), electron microscopy, X-ray diffraction (XRD), and Ultraviolet-vis-spectroscopy (UV-vis), energy dispersive X-ray spectroscopy (EDX), indicating the emergence of spherical NPs of which is around 40 nm. The FTIR spectrum also signified that S. convolute plant extract polyphenols acted as a capping ligand for the fabricated ZnO NPs. Possessed ZnO NPs have shown important characteristics of muscle relaxing and antinociceptive. A concentration dependent acetic acid induced writhing effect was noted for both S. convolute extract and ZnO NPs. S. convolute plant extract and ZnO NPs are found to exhibit highest muscle relaxation effect in both traction and chimney tests and no sedative effect was shown by both ZnO NPs and plant extract. The present results showed that the S. convolute leaves extract is a very effective green reducing agent for the preparation of ZnO NPs and the prepared NPs can be used in pain management in emerging nursing care in future.

Functional study of the brassinosteroid biosynthetic genes from Selagnella moellendorfii in Arabidopsis.

Brassinosteroids (BRs) are essential hormones for plant growth and development. Enzymes DET2 and CYP90 family are responsible for BR biosynthesis in seed plants. Yet, their roles in non-seed plants are unknown. Here, we report the first functional study of DET2 and all 4 CYP90 genes isolated from Selaginella moellendorfii. Sm89026 (SmCPD) belonged to a clade with CYP90A1 (CPD) and CYP90B1 (DWF4) while Sm182839, Sm233379 and Sm157387 formed a distinct clade with CYP90C1 (ROT3) and CYP90D1. SmDET2, SmCPD and Sm157387 were highly expressed in both leaves and strobili while Sm233379 was only highly expressed in the leaves but not strobili, implying their differential functions in a tissue-specific manner in S. moellendorfii. We showed that only SmDET2 and SmCPD completely rescued Arabidopsis det2 and cpd mutant phenotypes, respectively, suggestive of their conserved BR biosynthetic functions. However, neither SmCPD nor other CYP90 genes rescued any other cyp90 mutants. Yet overexpression of Sm233379 altered plant fertility and BR response, which means that Sm233379 is not an ortholog of any CYP90 genes in Arabidopsis but appears to have a BR function in the S. moellendorfii leaves. This function is likely turned off during the development of the strobili. Our results suggest a dramatic functional divergence of CYP90 family in the non-seed plants. While some of them are functionally similar to that of seed plants, the others may be functionally distinct from that of seed plants, shedding light for future exploration.

Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense.

Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long-distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate-limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4-coumaroyl-shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4-coumaroyl-esters and -amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4-coumaroyl-shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4-coumaroyl-shikimate poorly in vitro, but were able to use alternative substrates, in particular 4-coumaroyl-anthranilate. Thus, caffeoyl-shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non-seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4-coumaroyl-anthranilate, while 4-coumaroyl-shikimate was converted to lower extents. Despite having in vitro activity with 4-coumaroyl-shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss-of-function phenotype, suggesting distinct properties also in vivo.

The effects of environmental light on the reorganization of chloroplasts in the resurrection of Selaginella tamariscina.

Chloroplast repair and reorganization are crucial for the rehydration of resurrected plants. As one of the most important organelles in plant, photosynthesis takes place in chloroplasts. Meanwhile, light is important to the biosynthesis and activity regulation of chloroplasts. Here, we investigate the recovery of the chloroplasts and photosynthetic system in plant: Selaginella tamariscina under dark condition and environmental light (dark-light transition) condition. This study used the S. tamariscina grown in a culturing room, dehydrated S. tamariscina and S. tamariscina rehydrated in environmental light and dark conditions for 72 h as experimental material to measure and observed the chlorophyll content, chloroplast ultrastructure, photosynthesis, chlorophyll a fluorescence parameters. Specific leaf area and relative water content recovered in dark-rehydration conditions and were higher than those of light-rehydration, while dark-rehydration did not fully recover the chlorophyll content, net photosynthetic rate, water-use efficiency, nor the Fv/Fm. Dehydration did not destroy the chloroplast envelop, but increased the number of plastoglobules and disturbed the granum structure. As a homeochlorophyllous resurrection plant, reorganization, not the rebuilding of chloroplasts, occurs during the dehydration and rehydration processes in S. tamariscina. Environmental light signals play an important role in the recovery of photosynthetic systems.

Detection of the first higher plant epoxyalcohol synthase: Molecular cloning and characterisation of the CYP74M2 enzyme of spikemoss Selaginella moellendorffii.

The CYP74M2 gene of a model plant, the spikemoss Selaginella moellendorffii Hieron, was cloned and the catalytic properties of corresponding recombinant protein were studied. The recombinant CYP74M2 protein was active towards 13-hydroperoxides of linoleic and a-linolenic acids (13-HPOD and 13-HPOT, respectively). In contrast to previously studied CYP74M1 and CYP74M3, which possessed the divinyl ether synthase activity, CYP74M2 behaved as a dedicated epoxyalcohol synthase (EAS). For instance, the 13-HPOD was converted to three epimeric oxiranyl carbinols 1-3 (formed at a ratio ca. 4:2:1), namely the (11R,12S,13S), (11R,12R, 13S), and (11S,12S,13S) epimers of (9Z)-11-hydroxy-12,13-epoxy-9-octadecenoic acid. Besides these products, a minority of oxiranyl vinyl carbinols like (10E)-11-hydroxy-12,13-epoxy-9-octadecenoic acid was formed. The 13-HPOT conversion by CYP74M2 afforded two stereoisomers of 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the 1H-NMR, 2D-COSY, HSQC, and HMBC. Thus, the CYP74M2 is the dedicated epoxyalcohol synthase. To our knowledge, no enzymes of this type have been detected in higher plants yet.