GENETIC EVIDENCE FOR DIPLOIDY IN EQUISETUM

Sporophytes and gametophytes of Equisetum arvense, E. laevigatum, and E. telmateia were analyzed using enzyme electrophoresis to estimate isozyme number. Despite their uniformly high chromosome numbers (2n = 216), these three species exhibited isozyme numbers typical of diploid seed plants for the enzymes AAT, ADH, ALD, GDH, [NADP]IDH, LAP, MDH, [NADP]ME, PGI, PGM, SkDH, and 6PGDH. All three species exhibited an additional isozyme for TPI. There is, therefore, no genetic evidence for low base numbers such as x = 9 and x = 12 suggested for Equisetum. Intact chloroplasts were isolated from E. arvense and the chloroplast extract compared electrophoretically to whole plant extracts. The single enzymes observed for LAP, GDH, [NADP]IDH, and [NADP]ME were absent from the chloroplast extract. Isozymes AAT-1, ALD-2, MDH-3, PGI-1, PGM-2, SkDH-2, 6PGDH-2, TPI-2, and TPI-3 were active in the chloroplast fraction; 6PGDH-1, PGI-2, PGM-1, and TPI-1 were lacking from the chloroplast fraction and were considered cytosolic. Isozymes AAT-2, MDH-1, MDH-2, MDH-4, and SkDH-1 were also lacking from the chloroplast fraction but because AAT, MDH, and SkDH have been reported from several subcellular compartments, their localization is unknown. These findings indicate that isozymes in Equisetum species are subcellularly compartmentalized as has also been demonstrated for homosporous ferns, gymnosperms, and angiosperms.     

Allozyme differentiation in four species of the Crocus cartwrightianus group and in cultivated saffron (Crocus sativus)

The genetic variability and divergence of four species of the genus Crocus L., related to Crocus cartwrightianus group, namely Crocus thomasii Ten., Crocus hadriaticus Herbert, Crocus oreocreticus B.L. Burtt, C. cartwrightianus Herbert, has been studied by means of starch gel isozyme electrophoresis. For each population the following enzymatic loci were analyzed: PGI-1, PGI-2, G6PDH-1, G6PDH-2, IDH-1, IDH-2, 6PGDH-1, 6PGDH-2, SKDH-1, SKDH-2, AK-1 and AK-2. The genetic variability was estimated through the parameters A (mean number of alleles per locus), P (percent of polymorphic loci), H_o (mean observed heterozygosity), and H_e (mean expected heterozygosity). The genetic differentiation has been assayed by Wrigth's F-statistics, and the genetic divergence by Nei's index. Our data confirmed that the taxa are distinct species, in spite of their similar morphology and karyology. C. thomasii is more genetically similar to C. cartwrightianus and C. oreocreticus than to C. hadriaticus. We hypothesized an autopolyploid origin of saffron, probably from C. cartwrightianus, considering the genotypic classes of Crocus sativus and the other related species of the C. cartwrightianus group studied here.     

Changes in energy status of leaf cells as a consequence of mitochondrial genome rearrangement

The MSC16 cucumber (Cucumis sativus L.) mutant with lower activity of mitochondrial Complex I was used to study the influence of mitochondrial metabolism on whole cell energy and redox state. Mutant plants had lower content of adenylates and NADP(H) whereas the NAD(H) pool was similar as in wild type. Subcellular compartmentation of adenylates and pyridine nucleotides were studied using the method of rapid fractionation of protoplasts. The data obtained demonstrate that dysfunction of mitochondrial respiratory chain decreased the chloroplastic ATP pool. No differences in NAD(H) pools in subcellular fractions of mutated plants were observed; however, the cytosolic fraction was highly reduced whereas the mitochondrial fraction was more oxidized in MSC16, as compared to WTc. The NADP(H) pool in MSC16 protoplasts was greatly decreased and the chloroplastic NADP(H) pool was more reduced, whereas the extrachloroplastic pool was much more oxidized, than in WTc protoplast. Changes in nucleotides distribution in cucumber MSC16 mutant were compared to changes found in tobacco (Nicotiana sylvestris) CMS II mitochondrial mutant. In contrast to MSC16 cucumber, the content of adenylates in tobacco mutant was much higher than in tobacco wild type. The differences were more pronounced in leaf tissue collected after darkness than in the middle of the photoperiod. Results obtained after tobacco protoplast fractionating showed that the increase in CMS II adenylate content was mainly due to a higher level in extrachloroplast fraction. Both mutations have a negative effect on plant growth through perturbation of chloroplast/mitochondrial interactions.     

Isoforms of glucose 6-phosphate dehydrogenase in Deinococcus radiophilus

Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in Deinococcus radiophilus, an extraordinarily UV-resistant bacterium, was investigated to gain insight into its resistance as it was shown to be involved in a scavenging system of superoxide (O2-1) and peroxide (O2-2) generated by UV and oxidative stresses. D. radiophilus possesses two G6PDH isoforms: G6PDH-1 and G6PDH-2, both showing dual coenzyme specificity for NAD and NADP. Both enzymes were detected throughout the growth phase; however, the substantial increase in G6PDH-1 observed at stationary phase or as the results of external oxidative stress indicates that this enzyme is inducible under stressful environmental conditions. The G6PDH-1 and G6PDH-2 were purified 122- and 44-fold (using NADP as cofactor), respectively. The purified G6PDH-1 and G6PDH-2 had the specific activity of 2,890 and 1,033 U/mg protein (using NADP as cofactor) and 3,078 and 1,076 U/mg protein (using NAD as cofactor), respectively. The isoforms also evidenced distinct structures; G6PDH-1 was a tetramer of 35 kDa subunits, whereas G6PDH-2 was a dimer of 60 kDa subunits. The pIs of G6PDH-1 and G6PDH-2 were 6.4 and 5.7, respectively. Both G6PDH-1 and G6PDH-2 were inhibited by both ATP and oleic acid, but G6PDH-1 was found to be more susceptible to oleic acid than G6PDH-2. The profound inhibition of both enzymes by beta-naphthoquinone-4-sulfonic acid suggests the involvement of lysine at their active sites. Cu2+ was a potent inhibitor to G6PDH-2, but a lesser degree to G6PDH-1. Both G6PDH-1 and G6PDH-2 showed an optimum activity at pH 8.0 and 30 degrees .     

Evidence for intergeneric incompatibility in ferns

The archegonial mucilage ofAthyrium filix-femina andA. distentifolium paralyses spermatozoids ofDryopteris filix-mas (and in one caseD. inaequalis) before they penetrate the archegonial venter. The archegonial mucilage ofDryopteris filix-mas has a weak positive chemotactic influence on the spermatozoids of the twoAthyrium species. The spermatozoids ofDryopteris were never observed in the archegonia ofAthyrium. Incompatibility was not observed within and between the twoAthyrium species, withinDryopteris filix-mas or betweenAthyrium filix-femina and twoAsplenium species.Contribution No. 327.     

Redox Transfer across the Inner Chloroplast Envelope Membrane

In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO₂ the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10⁻³ is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved. From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane.     

Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme

An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction.     

Ameliorative effect of dandelion (Taraxacum officinale) peptides on benzo(a)pyrene-induced oxidative stress and inflammation in human umbilical vein endothelial cells

Dandelion (Taraxacum officinale) is widely consumed as a health food and a traditional medicine. However, the protective effect of dandelion bio-active peptides (DPs) against polycyclic aromatic hydrocarbon-induced blood vessel inflammation and oxidative damage is not well documented. In the current study, four novel DPs were isolated using an activity tracking method. The protective activity of the DPs against benzo(a)pyrene (Bap)-induced human umbilical vein endothelial cell (HUVEC) damage was explored. The results indicated that DP-2 [cycle-(Thr-His-Ala-Trp)] effectively inhibited Bap-induced reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction and reinforced antioxidant enzyme activity while inhibiting the production of inflammatory factors in HUVECs. Moreover, DP-2 increased NAD(P)H:quinone oxidoreductase 1, heme oxygenase-1, and nuclear factor E2-releated factor 2 expression levels by activating the PI3K/Akt signaling pathway. In addition, DP-2 attenuated Bap-induced HUVEC apoptosis via the Bcl-2/Bax/cytochrome c apoptotic pathway. These results suggest that DP-2 is a promising compound for protecting HUVECs from Bap-induced inflammatory and oxidative damage.     

Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter‐glacial CO2

C₄ photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C₄ plants were not conducted at the low atmospheric CO₂ prevailing during their evolution. Here, 24 C₄ grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD‐ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP‐ME)] and six major evolutionary lineages were grown under ambient (400 μL L^?1) and inter‐glacial (280 μL L^?1) CO₂. We hypothesized that nitrogen‐related and water‐related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO₂ treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD‐ME group, while NADP‐ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO₂ and by high conductance at inter‐glacial CO₂. In conclusion, the evolutionary and biochemical diversity among C₄ grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.     

NADK2, an <Emphasis Type="Italic">Arabidopsis</Emphasis> Chloroplastic NAD Kinase,Plays a Vital Role in Both Chlorophyll Synthesis and Chloroplast Protection

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H⁺ to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.     

Long‐term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity

Ammonium nutrition has been suggested to be associated with alterations in the oxidation‐reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)⁺ ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over‐reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)⁺ ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.     

Electrophoretic study and genetic affinity in theCampanula elatines andC. fragilis (Campanulaceae) rock-plants group from Italy and W. Jugoslavia

Variability and genetic divergence of 11 field populations of seven species of theCampanula elatines andC. fragilis rock-plants group from the Mediterranean and pre-Alpine areas have been studied by starch-gel electrophoretic techniques.Campanula isophylla, C. elatines, C. elatinoides, C. fragilis subsp.fragilis and subs.cavolinii, C. garganica were collected in Italy, whilstC. fenestrellata subsp.istriaca andC. portenschlagiana came from W. Jugoslavia. Twelve enzymatic loci for each population were genetically analysed: PGI-1 and 2, PGM-2, IDH-1 and -2, SKDH, ME, ADH, GOT-2, MPI-1 and -2, SOD-1. The genetic distances among the above mentioned entities have been calculated by Nei's index and depicted in a dendrogram.     

Cytosolic ratios of free [NADPH]/[NADP+] and [NADH]/[NAD+] in mouse pancreatic islets, and nutrient-induced insulin secretion.

When the extracellular concentration of glucose was raised from 3 mM to 7 mM (the concentration interval in which beta-cell depolarization and the major decrease in K+ permeability occur), the cytosolic free [NADPH]/[NADP+] ratio in mouse pancreatic islets increased by 29.5%. When glucose was increased to 20 mM, a 117% increase was observed. Glucose had no effect on the cytosolic free [NADH]/[NAD+] ratio. Neither the cytosolic free [NADPH]/[NADP+] ratio nor the corresponding [NADH]/[NAD+] ratio was affected when the islets were incubated with 20 mM-fructose or with 3 mM-glucose + 20 mM-fructose, although the last-mentioned condition stimulated insulin release. The insulin secretagogue leucine (10 mM) stimulated insulin secretion, but lowered the cytosolic free [NADPH]/[NADP+] ratio; 10 mM-leucine + 10 mM-glutamine stimulated insulin release and significantly enhanced both the [NADPH]/[NADP+] ratio and the [NADH]/[NAD+] ratio. It is concluded that the cytosolic free [NADPH]/[NADP+] ratio may be involved in coupling beta-cell glucose metabolism to beta-cell depolarization and ensuing insulin secretion, but it may not be the sole or major coupling factor in nutrient-induced stimulation of insulin secretion.     

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases (NAD-ICDH, EC 1.1.1.41, and NADP-ICDH, EC 1.1.1.42) by the level of reduced and oxidized pyridine nucleotides has been investigated in pea (Pisum sativum L.) leaves. The affinities of mitochondrial and cytosolic ICDH enzymes to substrates and inhibitors were determined on partially purified preparations in forward and reverse directions. From the kinetic data, it follows that NADP(+)- and NAD(+)-dependent isocitrate dehydrogenases in mitochondria represent a system strongly responding to the intramitochondrial NADPH and NADH levels. The NADPH, NADP(+), NADH and NAD(+) concentrations were determined by subcellular fractionation of pea leaf protoplasts using membrane filtration in mitochondria and cytosol in darkness and in the light under saturating and limiting CO(2) conditions. The cytosolic NADPH/NADP ratio was about 1 and almost constant both in darkness and in the light. In mitochondria, the NADPH/NADP ratio was low in darkness (0.2) and increased in the light, reaching 3 in limiting CO(2) conditions compared to 1 in saturating CO(2). At high reduction levels of NADP and NAD observed at limiting CO(2) in the light, i.e. when photorespiratory glycine is the main mitochondrial substrate, isocitrate oxidation in mitochondria will be suppressed and citrate will be transported to the cytosol ('citrate valve'), where the cytosolic NADP-ICDH supplies 2-oxoglutarate for the photorespiratory ammonia refixation.     

Proline-Dehydrogenase from Pumpkin (Cucurbita moschata) Cotyledons

A NAD specific proline-dehydrogenase was found in pumpkin (Cucurbita moschata Poir. cv. Dickinson Field) which oxidized proline to Δ¹-pyrroline-5-carboxylate. NADP did not substitute for NAD and L-proline-methyl-ester and thiazolidine-4-carboxylate were substrates in the reaction, at a rate of 107% and 33% respectively, of the rate with L-proline. Pumpkin cotyledons contained the bulk of the enzyme activity with 90% of the activity being in the soluble fraction. Proline-dehydrogenase, which was not treated at high temperature, was stable at –10°C for 4 months in the presence of high ammonium sulfate concentration. The Michaelis constant for NAD was 2.2 mM and for L-proline was 2.5 mM. At 5 mM NADP, a 40% non-competitive inhibition of proline-dehydrogenase was obtained, while 50 μM NADP was sufficient to induce 20% inhibition.     

Form of inorganic carbon involved as a product and as an inhibitor of c(4) Acid decarboxylases operating in c(4) photosynthesis

These studies demonstrated that CO₂ rather than HCO₃⁻ is the inorganic carbon metabolite produced by the C₄ acid decarboxylases involved in C₄ photosynthesis (chloroplast located NADP malic enzyme, mitochondrial NAD malic enzyme, and cytosolic phosphoenolpyruvate [PEP] carboxykinase). The effect of varying CO₂ or HCO₃⁻ as a substrate for the carboxylation reaction catalyzed by these enzymes or as inhibitors of the decarboxylation reaction was also determined. The K_mCO₂ was 1.1 millimolar for NADP malic enzyme and 2.5 millimolar for PEP carboxykinase. For these two enzymes the velocity in the carboxylating direction was substantially less than for the decarboxylating direction even with CO₂ concentrations at the upper end of the range of expected cellular levels. Activity of NAD malic enzyme in the carboxylating direction was undetectable. The decarboxylation reaction of all three enzymes was inhibited by added HCO₃⁻. For NADP malic enzyme CO₂ was shown to be the inhibitory species but PEP carboxykinase and NAD malic enzyme were apparently inhibited about equally by CO₂ and HCO₃⁻.     

Purification and Some Catalytic Properties of Glucose-6-Phosphate Dehydrogenase Isoforms from Barley Leaves

The cytosolic and chloroplastic isoforms of glucose-6-phosphate dehydrogenase (G₆PDH) were separated and purified from barley leaves (Hordeum vulgare L.). In etiolated leaves, only the cytosolic isoform was expressed. The molecular mass of the cytosolic enzyme, G₆PDH₁, was 112±8 kDa and that of the chloroplast enzyme, G₆PDH₂, was 136±7 kDa. The K_m values for glucose-6-phosphate and NADP were 0.133 and 0.041 mM for G₆PDH₁, and 0.275 and 0.062 mM for G₆PDH₂, respectively. The pH optimum was 8.2 for G₆PDH₁ and 7.8 for G₆PDH₂. The enzyme is absolutely specific for NADP. NADPH is a competitive inhibitor of the G₆PDH₁ in respect to glucose-6-phosphate (G₆P) and NADP (K_i = 0.050 and 0.025 mM, respectively). NADPH is a competitive inhibitor of the G₆PDH₂ in respect to NADP (K_i = 0.010 mM), but a non-competitive inhibitor in respect to the G₆P. ADP, AMP, UTP, NAD, and NADH had no effect on the activity of G₆PDH. ATP inhibited the G₆PDH₂ activity.     

90. K. Jeremias und Elisabeth Frank: Das jahresperiodische Verhalten der Zucker und der Stärke in Wedeln und Rhizomen; von Athyrium filix-femina und Dryopteris dilatata

1. In Wedeln und Rhizomen von Athyrium filix-femina und von Dryopteris dilatata wird der Jahresgang der Zucker und der Stärke bestimmt. 2. Die sommergrünen Wedel von Athyrium besitzen im Juni und im November je ein Zuckermaximum, das von Glucose, Fructose und Saccharose gebildet wird. Ein Zuckerminimum, das mit dem Stärkemaximum zusammenfällt, liegt im Mai. 3. Die Wedel von Dryopteris und die Rhizome beider Arten zeigen ein ausgeprägtes winterliches Zuckermaximum. In den Rhizomen wird während der kalten Jahreszeit Raffinose angehäuft. Der Stärkegehalt ist relativ hoch, nur während der Hauptwachstumsperiode geht er deutlich zurück. 4. Das jahresperiodische Verhalten der Zucker und der Stärke zeigt keine grundsätzlichen Unterschiede zu dem aus den Spermatophyten bekannt gewordenen.     

The effect of NADP on the subunit structure and activity of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase

Spinach chloroplast glyceraldehyde phosphate dehydrogenase (d-glyceraldehyde-3-phosphate: NADP oxidoreductase, phosphorylating; EC 1.2.1.13) is an equilibrium mixture of aggregates of a basic protomer (M_r about 145,000) and is active with both NADP and NAD. The enzyme is primarily “tetrameric” (M_r about 600,000), although minor amounts of smaller and larger oligomers are also found. Gel chromatography in buffer containing 30 μm NADP results in depolymerization of the enzyme, mainly to protomers. NAD does not dissociate and counteracts this effect of NADP.The apparent K_m values of the protomers are 7 μm (NADP) and 8 μm (NAD). The aggregates with a M_r > 10⁶ have properties similar to the protomers. The tetramer as first isolated has higher M_m values for NADP (380 μm) and NAD (48 μm), but its apparent affinity for NADP is further decreased by repeated gel filtrations in buffer or by a single one in buffer containing NAD. Such preparations display nonlinear kinetics when NADP is the varied substrate and have a K_m (NADP) of about 1.5–3.3 μm. All these effects are reversible.V values are apparently the same in all enzyme forms and the $\text{V (}\text{NADP}\text{)}\text{V (}\text{NAD}\text{)}$ ratio always approaches 2. Since, however, the enzyme is presumably dissociated by the NADP concentrations required for a “saturating” assay, the significance of V (NADP) seems questionable.     

Biosystematic investigations on the lady fern(Athyrium filix-femina)

In EuropeAthyrium filix-femina has a constant chromosome number (2n = 80) and is sexual. The normal type of reproduction is intergametophytic crossing. In the gametophyte phase there is a hormone system which induces dark germination of spores and antheridium formation. Sporophytes originating from single prothalli show that a genetic load is present in all population examined. It appears not to be a simple allelic load but a complicated balanced system. Morphological variability can be interpreted as the expression of the genetic heterogeneity of populations. There are no crossing barriers, not even between insular populations hundreds of kilometers apart.