Drought until death do us part: a case study of the desiccation-tolerance of a tropical moist forest seedling-tree, Licania platypus (Hemsl.) Fritsch.

Studies of the desiccation tolerance of 15-month-old Licania platypus (Hemsl.) Fritsch seedlings were performed on potted plants. Pots were watered to field capacity and then dehydrated for 23-46 d to reach various visible wilting stages from slightly-wilted to dead. Root hydraulic conductance, k(r), was measured with a high-pressure flow meter and whole-stem hydraulic conductance, k(ws), was measured by a vacuum chamber method. Leaf punches were harvested for measurement of leaf water potential by a thermocouple psychrometer and for measurement of fresh- and dry-weight. L. platypus was surprisingly desiccation-tolerant, suggesting that most species of central Panama may be well adapted to the seasonality of rainfall in the region. The slightly-wilted stage corresponded to leaf water potentials and relative water contents of -2.7 MPa and 0.85, respectively, but plants did not die until these values fell to -7.5 MPa and 0.14, respectively. As desiccation proceeded k(r) and k(ws) declined relative to irrigated controls, but k(ws) was more sensitive to desiccation than k(r). Values of k(ws) declined by 70-85% in slightly-wilted to dead plants, respectively. By comparison, k(r) showed no significant change in slightly-wilted plants and fell by about 50% in plants having severely-wilted to dead shoots.

Translocation of amino acids in the xylem of apple (Malus domestica Borkh.) trees in spring as a consequence of both N remobilization and root uptake.

Nitrogen is remobilized from storage for the growth of Malus domestica leaves each spring. Seasonal patterns of N translocation in the xylem sap as a consequence of remobilization were determined in 2-year-old 'Golden delicious' trees grafted on M9 rootstocks. The trees were grown in sand culture and (15)NH(4)(15)NO(3) at 10.4 atom% abundance supplied during August-September. The following year no further N was supplied and destructive harvests were taken during bud burst and leaf growth to determine the patterns of N remobilization together with the isolation of xylem sap for an analysis of their amino acid profiles and (15)N enrichments by GC-MS. The concentration of amino acids in the xylem sap rose following bud burst, peaked at full bloom and then fell again during petal fall and fruit set. The peak in amino acid concentration corresponded with the period when the rate of N remobilization was the fastest. The majority of labelled N was recovered in Asn, Gln + Glu and Asp demonstrating that they were being translocated as a consequence of remobilization. In a second experiment, 8-year-old trees growing in an orchard were fertilized with N either in the autumn or spring. Xylem sap samples were collected in the spring and early summer and, by comparison with the amino acid profiles recovered in trees from both treatments, Asn was identified as the main compound translocated as a consequence of both remobilization and root uptake of N, although there was evidence that root uptake of N occurred later. The data are discussed in relation to quantifying the internal cycling of N in trees.

Pesticidal properties of parthenin (from Parthenium hysterophorus) and related compounds.

Eleven sesquiterpene lactone derivatives of parthenin (1), obtained from wild feverfew, Parthenium hysterophorus, were prepared by chemical and photochemical transformations. The compounds tested were a pyrazoline adduct (2) of parthenin, its cyclopropyl (3) and propenyl (4) derivatives, anhydroparthenin (5), a dihydro-deoxygenated product (6), a formate (7) and its corresponding alcohol (8) and acetate (9), a rearranged product (10), lactone (11) and hemiacetal (12). All these derivatives, along with parthenin, were tried for their antifeedant action against sixth-instar larvae of Spodoptera litura, for insecticidal activity against the adults of store grain pest Callosobruchus maculatus, for phytotoxic activity against Cassia tora, and for nematicidal activity against the juvenile stage-II (J2) of the root knot nematode Meloidogyne incognita. Antifeedent bioassay revealed that parthenin is moderately antifeedant. Among the derivatives, the saturated lactone (11) was found to be about 2.25 times more active than parthenin. The pyrazoline adduct (2) was found to be the most effective as an insecticide, with LC50 values after 24, 48 and 72 h of 96, 43 and 32 mg litre-1, respectively, which are comparable with neem extract. Compound 4 was found to be the most effective inhibitor of germination and seedling growth of C tora, with ID50 values for germination, plumule length and radicle length of 136, 326 and 172 compared with 364, 738 and 427 mg litre-1, respectively, for parthenin. Compound 10 was found to be the most effective in terms of nematicidal activity. The LC50 values for this compound were 273 and 104 mg litre-1, respectively, after 48 and 72 h compared with 862 and 512 mg litre-1 observed for parthenin after 48 and 72 h.

Influence of glyphosate on amino acid composition of Egyptian broomrape.

The parasitic plant broomrape is entirely dependent on its host for reduced carbon and nitrogen and is also susceptible to inhibition by glyphosate that is translocated to the parasite through a host. Studies were conducted to examine the effect of broomrape parasitism on amino acid concentrations of two hosts: common vetch that is tolerant of low levels of glyphosate and oilseed rape that has been genetically engineered for glyphosate resistance. The influence of glyphosate on the amino acid content of broomrape and the two hosts was also examined. Amino acid concentrations in leaves and roots of parasitized common vetch plants were generally similar to those of the corresponding tissues of nonparasitized plants. Amino acid concentrations in broomrape were lower than those of the parasitized common vetch root. For common vetch, glyphosate applied at rates that selectively inhibited broomrape growth did not alter individual amino acid concentrations in the leaves, but generally increased amino acid levels at 0.18 kg ha-1. Glyphosate application also increased the amino acid concentrations, with the exception of arginine, of broomrape growing on common vetch and did not generally influence concentrations in leaves or roots of common vetch. In oilseed rape, parasitization by broomrape generally led to higher amino acid concentrations in leaves but lower concentrations in roots of parasitized plants. Broomrape had higher amino acid concentrations than roots of the parasitized oilseed rape. Glyphosate applied at 0.25 and 0.5 kg ha-1 generally increased the amino acid concentrations in oilseed rape leaves, but the 0.75 kg ha-1 application caused the amino acid concentrations to decrease compared to those of untreated plants. In oilseed rape root the general trend was an increase in the concentration of amino acids at the two highest rates of glyphosate. Individual amino acid concentrations in broomrape attachments growing on oilseed rape were generally increased following glyphosate application of 0.25 kg ha-1. These results indicate that low rates of glyphosate alter amino acid profiles in both host and broomrape and raise questions about the regulation of amino acid metabolism in the parasite.

Inhibitors of ethylene synthesis inhibit auxin-induced stomatal opening in epidermis detached from leaves of Vicia faba L.

Using leaf epidermis from Vicia faba, we tested whether auxin-induced stomatal opening was initiated by auxin-induced ethylene synthesis. Epidermis was dark-incubated in buffered KNO3 containing 0.1 mM alpha-napthalene acetic acid or 1 mM indole-3-acetic acid. Maximum net opening was ca. 4 micron after 6 h. Opening was reversed by 20 microM ABA, 0.1 mM CaCl2. 1-Aminocyclopropane carboxylic acid (ACC) synthase catalyzes synthesis of ACC, the immediate precursor to ethylene. Auxin-induced stomatal opening was fully inhibited by 10 microM 1-aminoethoxyvinylglycine (AVG), an ACC synthase inhibitor. In solutions containing AVG, auxin-induced opening was restored in a concentration-dependent manner by exogenous ACC, but not in control solutions lacking an auxin. ACC-mediated reversal of AVG-inhibition of stomatal opening was inhibited by alpha-aminoisobutyric acid (AIB), an inhibitor of ACC oxidase, the last enzyme in the ethylene biosynthetic pathway, by 10 microM silver thiosulfate (STS), an inhibitor of ethylene action, and by 20 microM ABA, 0.1 mM CaCl2. CoCl2, an inhibitor of ethylene synthesis, also inhibited auxin-induced opening. Both STS and CoCl2 inhibited opening induced by light or by fusicoccin, but neither light- nor fusicoccin-induced opening was inhibited by AVG. These results support the hypothesis that auxin-induced stomatal opening is mediated through auxin-induced ethylene production by guard cells.

Extrafloral nectar production of the ant-associated plant, Macaranga tanarius, is an induced, indirect, defensive response elicited by jasmonic acid.

Plant species in at least 66 families produce extrafloral nectar (EFN) on their leaves or shoots and therewith attract predators and parasitoids, such as ants and wasps, which in turn defend them against herbivores. We investigated whether EFN secretion is induced by herbivory and/or artificial damage, and thus can be regarded as an induced defensive response. In addition, we studied the underlying signaling pathway. EFN secretion by field-grown Macaranga tanarius increased after herbivory, artificial leaf damage, and exogenous jasmonic acid (JA) application. Artificial damage strongly enhanced endogenous JA concentrations. The response in EFN production to artificial damage was much less pronounced in those leaves that were treated with phenidone to inhibit endogenous JA synthesis. Quantitative dose-response relations were found between the increase in nectar production and both the intensity of leaf damage and the amounts of exogenously applied JA. The amount of endogenously produced JA was positively correlated with the intensity of leaf damage. Increased numbers of defending insects and decreased numbers of herbivores were observed on leaves after inducing EFN production by exogenous JA treatment. Over 6 weeks, repeatedly applied JA or artificial damage resulted in a ten-fold reduction in herbivory. These results demonstrate that EFN production represents an alternative mechanism for induced, indirect defensive plant responses that are mediated via the octadecanoid signal transduction cascade.

The transduction of the signal for grape bud dormancy breaking induced by hydrogen cyanamide may involve the SNF-like protein kinase GDBRPK.

Alterations in gene expression during early stages of dormancy release in grapevine buds were analyzed to facilitate the identification of gene products that may mediate the signal transduction of a dormancy-release signal, or derepression of meristematic activity. In the present report we describe the identification of GDBRPK, a transcript for an SNF-like protein kinase that is up-regulated upon chemical induction of dormancy release by hydrogen cyanamide (HC). Since SNF and SNF-like protein kinases are known as sensors of stress signals, we hypothesize that GDBRPK may be involved in the perception of a stress signal induced by HC. We also describe a simultaneous and remarkable induction of both PDC and ADH transcripts that was observed shortly after HC application, and was of a transient nature. These data may imply that HC application leads to a transient respiratory stress, which likely results in a temporary increase in the AMP/ATP ratio. Since AMP is known as a stress signal that is sensed by SNF-like kinases, we suggest that the SNF-like GDBRPK could serve as the sensor of this signal.

Antioxidant activity and radioprotective effects against chromosomal damage induced in vivo by X-rays of flavan-3-ols (Procyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic and organic compounds.

The quantitative distribution of several flavan-3-ols was determined using HPLC in a grape (Vitis vinifera) seed extract (GSE) of four cultivars grown in the region of Murcia. Polymer >/= C(4) units made up the largest group of procyanidins in the GSE (90.92%, expressed as HPLC % area). The antioxidant activity of GSE and other reference compounds was investigated by measuring their ability to scavenge the ABTS(*)(+) radical cation (TEAC). The most effective compounds were, in order: GSE > rutin > (+)-catechin > diosmin >/= ascorbic acid. The radioprotective effects of GSE and other reference compounds were determined by using the micronucleus test for anticlastogenic activity, any reduction of the frequency of micronucleated polychromatic erythrocytes (MnPCEs) being evaluated in the bone marrow of mouse exposed to X-rays. The most effective compounds were, in order: GSE > rutin > dimethyl sulfoxide (DMSO) > ascorbic acid > 6-n-propyl-2-thiouracil-6c (PTU) > diosmin. The higher ABTS(*)(+) scavenging capacity and anticlastogenic activity of GSE can be explained, structurally, by the high number of conjugated structures between the catechol groups in the B-rings and the 3-OH free groups of the polymeric polyphenolic skeleton and, in addition, by the stability of the aroxyl flavonoid radical generated in the above processes.

Salicylates of intact Salix myrsinifolia plantlets do not undergo rapid metabolic turnover.

Salicylates, the main phenolic glucosides of northern willow (Salix spp.), play an important role in plant-herbivore interactions. Salicylates are labile metabolites that are thought to undergo metabolic turnover. Salicylates are synthesized from phenylalanine (Phe) via the shikimate pathway. 2-Aminoindan-2-phosphonic acid (AIP), a strong inhibitor of Phe ammonia-lyase (EC 4.3.1.5), was used to block the biosynthesis of salicylates. The aim of this study was to investigate long-term turnover of salicylates in intact micropropagated plantlets of Salix myrsinifolia Salisb. The biosynthesis of salicylates was inhibited efficiently but not completely by 30 microM 2-aminoindan-2-phosphonic acid. Inhibitor treatment, aside from leading to a high accumulation of Phe, also led to an increase in tyrosine and tryptophan, indicating that 2-aminoindan-2-phosphonic acid may also inhibit enzymes other than Phe ammonia-lyase. Salicylates were shown to be unexpectedly stable metabolites that did not undergo marked metabolic turnover in intact plants; in leaves no significant turnover occurred, and in the stems the five salicylates studied were turned over slowly, with half-lives of 11 to 25 d. The total amount of salicylate in mature shoots decreased only 0.6% per day.

Binding affinity of the methyl ester of AK-toxin I to membrane fractions from Japanese pear leaves.

The binding site of AK-toxin, a host-specific toxin against Japanese pear, was searched for in the membrane fractions of the pear leaves, using 3H-labeled AK-toxin I methyl ester. Binding activity, which was displaceable by the unlabeled ligand, was observed for microsomal fraction from a toxin-susceptible cultivar, Nijisseiki. However, the binding was also observed for those from toxin-resistant cultivars, Kosui and Hosui. Detection of the specific binding failed for the plasma membrane fraction which was prepared from microsomal fraction of the toxin-susceptible cultivar by aqueous two-phase separation, and the hitherto presumed model of the AK-toxin receptor in the plasma membrane could not be verified.

In vitro multiplication of Peganum harmala--an important medicinal plant.

The frequency of shoot regeneration and multiplication of P. harmala was influenced by the type of explant and kind and concentration of hormones. Of the various seedling explants, cotyledonary node exhibited maximum shoot regeneration frequency from axillary region on MS medium supplemented with 5 microM BAP. Addition of 0.1 microM NAA enhanced the efficacy of BAP for multiple shoot regeneration as well as improved the growth of shoots. BAP (5 microM) in combination with NAA (0.1 microM) was found to be the optimal for inducing an average of 4-5 shoots per explant in 75% of the cultures within 5 weeks. Replacement of BAP with other cytokinins at equimolar concentration of BAP i.e. 5 microM was not effective in inducing multiple shoots. Regenerated shoots were rooted on MS medium containing IBA (8 microM) with 80% efficiency. The plantlets were successfully established in soil where 80% of them developed into morphological normal plants.

Stimulation of anthraquinone production in suspension cultures of Cassia acutifolia by salt stress.

Suspension cultures of Cassia acutifolia were established by transferring callus tissues derived from root, hypocotyl and cotyledon explants onto liquid MS-medium supplemented with 1.0 mg/l 2.4-D and 0.1 mg/l kinetin and containing increasing levels of NaCl. The stress induced by salt NaCl raised anthraquinone content and reduced growth of cultures. The levels of anthraquinones and their glycosides as sennosides showed distinct changes in cells and media as well as in the different cultures initiated from various explants. Furthermore, the salt stress tended to affect more drastically the productivity of anthraquinones in hypocotyl and cotyledon cell cultures than in root cultures.

Effects of copper exposure in tissue cultured Vitis vinifera.

The present study determined the effects of copper treatment on some biochemical parameters in a closed system. Sauvignon grapevines were cultured in agar and exposed to copper levels ranging from 0.07 to 10 microg Cu/g medium. Chlorophylls, carotenoids, lipids, sucrose, soluble sugars, starch, cellulose, and minerals in root, leaves, and sap were determined. Copper levels over 5 microg Cu/g inhibit root and plantlet development. Copper-exposed plants have higher levels of chlorophylls and carotenoids as well as total lipids. Soluble sugars decrease without changes in starch or sucrose. Copper concentration increases dramatically in roots and leaves. Iron concentrations are lower in leaves, although they increase in roots, with respect to control plants. Sap flow and translocation of essential elements are reduced. Reduction of K translocation can be related to limited use of water by the plant and, thus, reduction in growth and physiological activity.

Comparison among 18 hexyl esters of 1-naphthylacetic acid used on grapevine.

Eighteen esters derived from 1-naphthylacetic acid (NAA) and from hexyl isomer alcohols were synthesized and sprayed on grapevine suckers at 1, 2, 4, and 8% w/w concentrations to test their sucker control efficiency 2, 10, and 30 days and 1 year after their application. All compounds gave a prompt control of suckers. After 1 year, sucker growth was inhibited by the derivatives from primary and secondary alcohols, whereas the number of suckers was reduced only by application of naphthyl acetates of tertiary alcohols.

The generation of active oxygen species differs in tobacco and grapevine mesophyll protoplasts.

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H(2)O(2) can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109-114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O(2)(*-)) and H(2)O(2), and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L. ) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O(2)(*-) and H(2)O(2) accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O(2)(*-) production, and a second NAD(P)H activity that was sensitive to KCN and NaN(3), contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.

Does ozone exposure protect from photoinhibition?

Tropospheric ozone and high light intensities are two stress factors that often occur simultaneously under natural conditions. Ozone is well known to form oxygen radicals in the apoplastic water and long lasting photoinhibition can cause photooxidative damage also by formation of several species of oxygen radicals. We were interested whether moderate levels of ozone would be able to modulate the response of leaves to photoinhibitory conditions naturally occurring around noon on a bright day. Cuttings of Populus sp. were cultivated in two separate greenhouse-compartments adapted as fumigation chambers. In the two compartments plants were grown in ambient air containing about 20 nmol mol(-1) ozone and in elevated ozone concentrations supplied for 8 h per day. During the midday of bright days Fv/Fm decreased by the same amount in all leaves, indicating photoinhibition. At the same time Fo increased in control leaves more than in ozone-exposed leaves indicating a higher amount of heat-deactivating PSII centres in the latter. This was confirmed by a higher epoxidation state in ozone-exposed leaves during midday of a bright day. The contents of chlorophyll a and chlorophyll b were significantly decreased in ozone-exposed leaves. In older leaves the ratios chlorophyll a : b, and xanthophylls: chlorophyll b were increased indicating an adaptation to higher light stress. From this we conclude that by increasing the amount of heat-deactivating centres ozone seems to protect PSII from photoinhibition.

Calcium chloride sprays decrease physiological disorders following long-term cold storage of apple.

Field experiments were carried out at the Horticultural Research Institute, and in five commercial apple orchards, located in southwest Finland including the Aland Islands during 1994 and 1995. The experimental cultivars were Melba, Raike, Red Atlas, Akerö, Aroma, and Lobo. The treatments were untreated control and preharvest calcium chloride (CaCl2) sprays at Ca 2.0 g/l. Fruit samples were stored for two to six months at 2-4 degrees C and 85-95% RH. The percentage of the incidence of physiological disorders of stored apples was scored. Fruit macronutrients, firmness, diameter and juice titratable acidity and percentage of soluble solids were determined. Only a few nutrient effects in the flesh of the apple cultivars were a result of CaCl2 sprayings. Preharvest CaCl2 sprays increased fruit firmness and the titratable acidity but decreased soluble solids, soluble solids/titratable acidity ratio, and the incidence of physiological storage disorders of some cultivars. When looking at the mean of all cultivars, CaCl2 sprayings increased titratable acidity and decreased soluble solids/titratable acidity ratio after four months of storage during 1995 and reduced the incidence of physiological disorders after three months of storage during 1994 and after four months of storage in 1995.

Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions.

Very short exposures of embryogenic calli of Vitis vinifera cv. Superior Seedless grape plants to diluted cultures of Agrobacterium resulted in plant tissue necrosis and subsequent cell death. Antibiotics used for Agrobacterium elimination or as plant selectable markers were not responsible for this necrotic response. Rather, cell death seemed to be oxygen-dependent and correlated with elevated levels of peroxides. Therefore, we studied the effects on necrosis of various combinations of antioxidants during and after grape-Agrobacterium cocultivation. The combination of polyvinylpolypyrrolidone and dithiothreitol was found to improve plant viability. Tissue necrosis was completely inhibited by these antioxidants while Agrobacterium virulence was not effected. These treatments enabled the recovery of stable transgenic grape plants resistant to hygromycin.

Identification of naturally occurring calmodulin inhibitors in plants and their effects on calcium- and calmodulin-promoted protein phosphorylation.

While studying the calmodulin activity in post-climacteric apples, a heat stable, dialyzable component that inhibited calmodulin-promoted phosphodiesterase activity was detected. The compound(s) that inhibited calmodulin activity did not bind to Dowex-50, H+ or Dowex-2, Cl- and was exclusively present in the neutral fraction. The inhibitors irreversibly bound to polyvinylpolypyrrolidone indicating their phenolic nature. Fractionation of the neutral fraction on a C18-microbondapak column and analysis for the inhibition of calmodulin-promoted phosphodiesterase activity showed significant inhibitory activity associated with fractions eluted 5 min, 15 min and 18 min after injection. Perdeuteriomethylation and combined gas chromatographic-mass spectrometric analysis of the inhibitors showed them to be flavonoids. (+)-Catechin was identified in the fraction eluted 5 min after injection that also showed maximum inhibition. Other flavonoids such as epicatechin, quercetin and naringenin also inhibited calmodulin-promoted phosphodiesterase activity. Among the phenolic compounds commonly encountered in plant tissue only caffeic acid inhibited calmodulin-promoted phosphodiesterase activity. Inhibition by catechin and caffeic acid could be reversed by increasing the calmodulin concentration in the assay mixture. Both catechin and caffeic acid inhibited Ca- and calmodulin-promoted phosphorylation of soluble proteins from corn coleoptiles. The physiological properties of flavonoids are discussed in light of this evidence.