The effect of mercury on the establishment of Pinus rigida seedlings and the development of their ectomycorrhizal communities

Metals are toxic to both plants and fungi, and elevated soil metal concentrations have been documented to change the structure of ectomycorrhizal communities. Mercury (Hg) is a highly toxic metal and inhibits the growth of ectomycorrhizal fungi (ECMF) in axenic culture. However, the effects of Hg on the growth of tree seedlings and the development of their ECMF communities have not been explored. In the current study, Pinus rigida seedlings were planted in soil amended with 0–366 μg g^?1 Hg and incubated for 5 months. Survival and growth of P. rigida seedlings was determined, and their ECMF communities were characterized by morphotype analysis. Seedling survival declined with increasing Hg additions, although no reduction in growth was detected among surviving seedlings. The addition of 88 μg g^?1 Hg to soil more than halved the total ectomycorrhizal colonization of root tips and reduced both richness and diversity of root tip morphotypes, while lower Hg additions did not significantly affect ECMF community composition relative to the no Hg control. This suggests that changes in the community of ECMF may occur in contaminated soils before any aboveground effects on surviving seedlings are noticeable, potentially altering the contribution of ECMF to the fitness of established host plants.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Two Cyclic Dipeptides from Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode and Their Toxicities to Japanese Black Pine Suspension Cells and Seedlings in vitro

Pseudomonas fluorescens GcM5-1A, isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, was cultured in Luria Broth medium (LB). The clarified culture was extracted with ethyl acetate, and two dipeptides were purified from the extract. The chemical structures of 1 and 2 were identified as cyclo(-Pro-Val-)and cyclo(-Pro-Tyr-), respectively, by MS, ¹H NMR, ¹³C NMR,¹H-¹H COSY, 1H -¹³C COSY spectra. Bioassay results showed that the two compounds were toxic to both suspension cells and seedlings of Pinus thunbergii, which may offer some clues to research the mechanism of pine wilt disease caused by PWN.     

Effects of arsenate on tobacco hairy root and seedling growth,and its removal

Arsenic (As) is a highly toxic environmental contaminant to which most living organisms are exposed. Plants have evolved several mechanisms to cope with this toxic metalloid; however, these mechanisms are only partially understood. The response of plants to As phytotoxicity is highly complex, with considerable variation among species. In this study, arsenate (As⁺⁵) effects on germination and early root development of tobacco (Nicotiana tabacum) seedlings were investigated. Also, As⁺⁵ tolerance and removal efficiency of tobacco hairy roots (HRs) and seedlings were assessed and compared. Total seed germination capacity was not affected by 10 to 200 μM As⁺⁵, while primary root length and root branching were reduced by As⁺⁵ concentrations that were at or above 100 μM. Both systems were able to tolerate As⁺⁵ concentrations of 10 μM since no growth inhibition was detected. For higher As⁺⁵ concentrations, phytotoxicity increased, but it was mitigated by higher phosphate (Pi) availability. Under the studied conditions, As⁺⁵ removal efficiency of HRs greatly exceeded that of seedlings. Further, tobacco HRs were able to accumulate As in their tissues. These results justify further investigations on As tolerance and detoxification mechanisms in tobacco, an easy-to-transform crop species with high biomass, which could allow evaluation of the possible application of wild type or alternatively transgenic tobacco plants for As phytoextraction.     

Effect of mineral composition and medium pH on Scots pine tolerance to toxic effect of zinc ions

Effect of mineral composition and pH of nutrient medium on the growth of Scots pine (Pinus sylvestris L.) seedlings and their tolerance to toxic effect of zinc ions was investigated by modifying the standard Knop-Hoagland medium (pH 6.6) (M1). The improved medium (M2) was more acid (pH 4.5) and notable for 2.7-fold lower osmolarity, with equimolar ratio between ammonia and nitrate nitrogen, assimilable form of iron (9 μM Fe²⁺), and the presence of 0.02 μM Co²⁺ and 2 mM Cl^?. Owing to balanced mineral composition and low pH, M2 enhanced seedling growth under normal conditions and reduced the toxic effect of 100 μM ZnSO₄. Rapid development of the root system (main root, the number and total length of lateral roots) on M2 was maintained even under stress conditions (100 μM Zn²⁺); as a result, the ratio between the biomass of aboveground organs and the biomass of the root system remained on the level of 2.5, whereas on M1, it increased to 3.9. At the same time, on M2 we observed accelerated production of the needles with elevated content of photosynthetic pigments therein. Investigation of the operation of individual components of antioxidant system (superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidases) and peculiarities of proline accumulation showed that Scots pine seedlings grown on M2 experienced less oxidative stress than the seedlings grown on M1. The probable physiological foundation of the observed phenomena is discussed.     

Effect of Chelate-Assisted Hexavalent Chromium on Physiological Changes,Biochemical Alterations,and Chromium Bioavailability in Crop Plants—An In Vitro Phytoremediation Approach

This study revealed heavy metal–induced physiological and biochemical alterations in crop seedlings by supplementing chelating agents in the nutrient solution. Hexavalent chromium (Cr⁺⁶) induces several toxic effects in hydroponically grown rice, wheat, and green gram seedlings. A noticeable decrease was observed in root length, shoot length, biomass content, and chlorophyll biosynthesis of the seedlings grown in the nutrient solutions supplemented with Cr⁺⁶ at 100 μM. The seedling growth was stimulated with supplement of chelating agents such as EDTA, DTPA, and EDDHA. An increase in proline content was noticed with the application of Cr⁺⁶ (100 μM) in nutrient solutions. Stimulated activities of antioxidant enzymes such as catalase and peroxidase were noticed with increasing concentrations of chromium. Cr bioaccumulation was significantly high in roots of seedlings treated with Cr⁺⁶ at 100 μM in nutrient solution. Shoot translocation of Cr as depicted by transportation index (Ti) values for different crops were enhanced with the application of chelating agents. The total accumulation rate (TAR) for Cr was enhanced with the supplementation of DTPA in rice and wheat, whereas the application of EDDHA was found effective for increasing the accumulation rate of Cr in green gram seedlings. This study demonstates the role of chelating agents in lessening the toxic effects of Cr⁺⁶. The chelating agents supplemented with Cr⁺⁶ in the culture medium enhanced the Cr bioavailability in plants.     

4-Hydroxyisoleucine from seed of Trigonella foenum-graecum

The principal free amino acid present in seed of Trigonella foenum-graecum has been isolated and identified as (2S, 3R, 4R)-4-hydroxyisoleucine. This compound has not been reported previously as a constituent of higher plants, but it is a component of the toxic peptide, γ-amanitin, produced by Amanita phalloides. The (2S, 3R, 4R)-isomer lactonizes readily under acidic conditions, whilst strong acid causes partial epimerization. The (2R, 3R, 4R)-isomer forms a minor component of Trigonella seed. The 4-hydroxyisoleucine content of fenugreek increases during the growth of seedlings and plants, and ¹⁴C-isoIeucine was used effectively as a biosynthetic precursor.     

Effect of Previous Nitrate Deprivation on 15N-nitrate Absorption and Assimilation by Wheat Seedlings

Nitrate uptake and assimilation were examined in intact 18 days old wheat (Triticum aestivum, cv Capitole) seedlings either permanently grown on nitrate (high-N seedlings) or N-stressed by transfer to an 0 N-solution for the final 7 days (low-N seedlings). The N-stressed seedlings were characterized by a lower organic N content (2.5 mg instead of 4.9 mg per seedling) and an increased root dry weight.The seedlings received ¹⁵NO₃K for 7 h in the light. Nitrate uptake was 2.8 times higher in low-N than in high-N seedlings. The assimilation rate was 35 and 16 μmol NO₃^?·^h?1· g^?1 dry weight respectively. Partitioning of NO₃^? to reduction and assimilation was the very same in both kinds of seedlings. The results support the view that 50 % of the nitrate reduction in Triticum aestivum, cv Capitole could be achieved in the roots.The present observations are interpreted as evidence that factors closely associated with the seedling N-status may have a major role in regulating NO₃^? uptake and assimilation. In low-N seedlings, the high amount of carbohydrates in roots may add its stimulus to the specific inducing effect of nitrate whereas in high-N seedlings, excess of nitrate or amino-acids may set the pace by negative feedback control.     

The raz1 mutant of Arabidopsis thaliana lacks the activity of a high-affinity amino acid transporter

The raz1 mutant of Arabidopsis thaliana (L.) Heynh. has been selected as resistant to the toxic proline analogue, azetidine-2-carboxylic acid (2AZ). Seedlings of the mutant tolerated fivefold higher concentrations of 2AZ (ED₅₀ = 0.25 mM) than the wild-type seedlings (ED⁵⁰ = 0.05 mM). The mutant gene was found to be semi-dominant and the corresponding RAZ1 locus was mapped on chromosome 5 at 69.6±1.8 cM. The resistance to 2AZ could be fully and exclusively accounted for by the lower uptake rate of the proline analogue in the mutant. The influx of L-proline in roots of wild-type seedlings could be dissected into two components: (i) a component with a high affinity and a low capacity for l-proline (K _m≈20 gmM, V _max≈60 nmol·(g FW)^-1·h^-1) and also a high affinity for L-2AZ (K _i≈40 μM) and (ii) a low-affinity, high-capacity component (K _m≈5 mM: V _max = 1300 nmol·(g FW)^-1·h^-1). Clearly, the raz1 mutation affects the activity of a high-affinity transporter, because the high-affinity uptake of proline in the mutant was at least fivefold lower than in the wild-type, whereas the low-affinity uptake was unchanged.     

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd²⁺ treatment. NO contributed caspase-3-like protease activation in Cd²⁺ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd²⁺-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd²⁺-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd²⁺-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd²⁺-induced plant PCD.     

Photosynthetic acclimation in shade-developed leaves of Euterpe edulis Mart (Arecaceae) after long-term exposure to high light

To analyze acclimation of Euterpe edulis seedlings to changes in light availability, we transferred three-year-old seedlings cultivated for six months under natural shade understory [≈ 1.3 mol(photon) m^?2 d^?1] to a forest gap [≈ 25.0 mol(photon) m^?2 d^?1]. After the transfer, changes in chlorophyll fluorescence and leaf gas-exchange parameters, as well as in the light-response curves of photosynthesis and photosynthetic induction parameters, were analyzed during the following 110 days. Simultaneously measured photosynthetic characteristics in the shaded seedlings grown in understory served as the control. Despite the fact that the understory seedlings were under suboptimal conditions to achieve their light-saturated net photosynthetic rate (P _Nmax), light-response curves and photosynthetic induction parameters indicated that the species had the low respiration rate and a fast opening of stomata in response to the intermittent occurrence of sunflecks, which exerted a feed-forward stimulation on P _Nmax. Sudden exposure to high light induced photoinhibition during the first week after the transfer of seedlings to gap, as it was shown by the abrupt decline of the maximal quantum yield of PSII photochemistry (F_v/F_m). The photoinhibition showed the time-dependent dynamics, as the F_v/F_m of the seedlings transferred to the forest gap recovered completely after 110 days. Furthermore, the net photosynthetic rate increased 3.5-fold in relation to priorexposure values. In summary, these data indicated that more than 21 days was required for the shade-acclimated seedlings to recover from photoinhibition and to relax induction photosynthetic limitations following the sudden exposure to high light. Moreover, the species responded very quickly to light availability; it highlights the importance of sunflecks to understory seedlings.     

Use of an Acylcyclohexanedione Growth Retardant, LAB 198 999, to Determine Whether Gibberellin A(20) Has Biological Activity per se in Dark-Grown Dwarf (le) Seedlings of Pisum sativum

Dwarf (le⁵⁸³⁹) seedlings of Pisum sativum respond to gibberellin A₂₀ (GA₂₀) in the dark, although the same dosage of GA₂₀ applied to light-grown le⁵⁸³⁹ seedlings elicits no growth response. The acylcyclohexanedione growth retardant, LAB 198 999, which is known to inhibit gibberellin oxidation and in particular 3β-hydroxylation such as the conversion of GA₂₀ to GA₁, also inhibits the growth response of dark-grown dwarf (le⁵⁸³⁹) seedlings to GA₂₀. Thus, the biological activity of GA₂₀ in the dark appears to be a consequence of its conversion to GA₁, even though it is known from studies with light-grown seedlings that the le mutation reduces the conversion of GA₂₀ to GA₁.     

De novo synthesis of d-alanine in germinating Pisum sativum seedlings

The amounts of d-alanine derivatives, γ-l-glutamyl-d-alanine and N-malonyl-d-alanine, increase rapidly during the early growth of pea seeds. Pyruvate-[1?¹⁴C], l-alanine-[U?¹⁴C], d-alanine-[U?¹⁴C], l-alanine-[¹⁵N] and ¹⁵NH₄Cl were therefore fed to the seedlings and the incorporation investigated. Labelling results revealed that pea seedlings can utilize these erogenous compounds to form d-alanine and that labelled l-alanine is effectively converted to the d-enantiomer with retention of ¹⁴C and, largely, ¹⁵N label. Enzyme analyses in vitro provided additional evidence that the extract of pea seedlings catalyzes the direct conversion of l-alanine to d-alanine. The data suggest that the de novo synthesis of d-alanine in pea seedlings occurs by a racemase reaction.     

Paclobutrazol mitigates salt stress in <Emphasis Type="Italic">indica</Emphasis> rice seedlings by enhancing glutathione metabolism and glyoxalase system

Stress-induced methylglyoxal (MG) functions as a toxic molecule, inhibiting plant physiological processes such as photosynthesis and antioxidant defense systems. In the present study, an attempt was made to investigate the MG detoxification through glutathione metabolism in indica rice [Oryza sativa L. ssp. indica cv. Pathumthani 1] under salt stress by exogenous foliar application of paclobutrazol (PBZ). Fourteen-day-old rice seedlings were pretreated with 15 mg L^?1 PBZ foliar spray. After 7 days, rice seedlings were subsequently exposed to 0 (control) or 150 mM NaCl (salt stress) for 12 days. Prolonged salt stress enhanced the production of MG molecules and the oxidation of proteins, leading to decreased activity of glyoxalase enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II). Consequently, the decreased glyoxalase activities were also associated with a decline in reduced glutathione (GSH) content and glutathione reductase (GR) activity. PBZ pretreatment of rice seedlings under salt stress significantly lowered MG production and protein oxidation, and increased the activities of both Gly I and Gly II. PBZ also increased GSH content and GR activity along with the up-regulation of glyoxalase enzymes, under salt stress. In summary, salinity induced a high level of MG and the associated oxidative damage, while PBZ application reduced the MG toxicity by up-regulating glyoxalase and glutathione defense system in rice seedlings.     

Some Effects of Triazines on Growth, Photosynthesis and Translocation of Photosjuthate in Pinus Species

The effects of triazines on growth, ¹⁴CO₂-fixation and translocation of ¹⁴C-assimilates by young Pinus seedlings were investigated. Post-emergenceroot application of moderate and high concentrations of simazine and atrazine resulted in severe toxicity to the seedlings of Pinus nigra and P. coniorta as depicted by reduction in dry weight, chlorosis, wilting and mortality. Atrazine was more toxic than simazine and P. nigra was more susceptible than P. contorta to both the triazines. Simazine treatment to the roots of P. nigra seedlings, not only caused inhibition of ¹⁴CO₂-fixation but also reduced the short-term downward transloca-tion of ^l4 C-assimilates. Various possible mechanisms whereby simazine reduced the downward movement of ^I4C-assimilates in Pinus seedlings are discussed.     

Morphological,anatomical, and ultrastructural changes (visualized through scanning electron microscopy) inducedin Triticum aestivum by Pb2+ treatment

Lead (Pb) causes severe damage to crops, ecosystems, and humans, and alters the physiology and biochemistry of various plant species. It is hypothesized that Pb-induced metabolic alterations could manifest as structural variations in the roots of plants. In light of this, the morphological, anatomical, and ultrastructural variations (through scanning electron microscopy, SEM) were studied in 4-day-old seedlings of Triticum aestivum grown under Pb stress (0, 8, 16, 40, and 80 mg Pb²⁺ l^?1; mild to highly toxic). The toxic effect was more pronounced in radicle growth than on the plumule growth. The SEM of the root of T. aestivum depicted morphological alterations and surface ultrastructural changes. Compared to intact and uniform surface cells in the control roots, cells were irregular and desiccated in Pb²⁺-treated roots. In Pb²⁺-treated roots, the number of root hairs increased manifold, showing dense growth, and these were apparently longer. Apart from the deformity in surface morphology and anatomy of the roots in response to Pb²⁺ toxicity, considerable anatomical alterations were also observed. Pb²⁺-treated root exhibited signs of injury in the form of cell distortion, particularly in the cortical cells. The endodermis and pericycle region showed loss of uniformity post Pb²⁺ exposure (at 80 mg l^?1 Pb²⁺). The cells appeared to be squeezed with greater depositions observed all over the tissue. The study concludes that Pb²⁺ treatment caused structural anomalies and induced anatomical and surface ultrastructural changes in T. aestivum.     

Biosyntheses of the uracilylalanines willardiine and isowillardiine in higher plants

The syntheses of willardiine and isowillardiine were studied in vivo in pea seedlings by feeding uracil-[2-¹⁴C] and in vitro with enzyme extracts from Pisum, Lathyrus, Albizia, Leucaena and Fagus seedlings by using uracil and O-acetyl-l-serine as substrates. The fate of isowillardiine in the intact seedlings has also been studied by feeding isowillardiine-[¹⁴C] through the roots and determining its distribution. Some properties of willardiine and isowillardiine synthase(s) are described. Willardiine was also obtained by a B₆-catalysed chemical reaction under mild conditions. The question whether two enzymes are involved in the formation of the two isomeric uracilylalanines is discussed.     

The K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter <Emphasis Type="Italic">AhNHX1</Emphasis> improved tobacco tolerance to NaCl stress by enhancing K<Superscript>+</Superscript> retention

High salinity is the one of important factors limiting plant growth and crop production. Many NHX-type antiporters have been reported to catalyze K⁺/H⁺ exchange to mediate salt stress. This study shows that an NHX gene from Arachis hypogaea L. has an important role in K⁺ uptake and transport, which affects K⁺ accumulation and plant salt tolerance. When overexpressing AhNHX1, the growth of tobacco seedlings is improved with longer roots and a higher fresh weight than the wild type (WT) under NaCl treatment. Meanwhile, when exposed to NaCl stress, the transgenic seedlings had higher K⁺/H⁺ antiporter activity and their roots got more K⁺ uptake. NaCl stress could induce higher K⁺ accumulation in the roots, stems, and leaves of transgenic tobacco seedlings but not Na⁺ accumulation, thus, leading to a higher K⁺/Na⁺ ratio in the transgenic seedlings. Additionally, the AKT1, HAK1, SKOR, and KEA genes, which are involved in K⁺ uptake or transport, were induced by NaCl stress and kept higher expression levels in transgenic seedlings than in WT seedlings. The H⁺-ATPase and H⁺-PPase activities were also higher in transgenic seedlings than in the WT seedlings under NaCl stress. Simultaneously, overexpression of AhNHX1 increased the relative distribution of K⁺ in the aerial parts of the seedlings under NaCl stress. These results showed that AhNHX1 catalyzed the K⁺/H⁺ antiporter and enhanced tobacco tolerance to salt stress by increasing K⁺ uptake and transport.