The electrical response ofAvena coleoptile cortex to auxins

Solute mobilities of 28 compounds in isolated cuticular membranes (CM) from Capsicum annuum L. fruit, Citrus aurantium L. and Pyrus communis L. leaves were studied using unilateral desorption from the outer surface. First-order rate constants of desorption (k^*), which are directly proportional to the diffusion coefficient in the waxy outer limiting skins of cuticles were measured. When log k^* was plotted vs. molar volumes of test compounds linear graphs were obtained. The y-intercepts of these graphs (k^*) represent the mobility of a hypothetical molecule having zero molar volume and the slopes of the graphs () represent the size selectivity of the barrier and are related to the free volume available for diffusion. Thus, solute mobilities in cuticles are composed of two independent terms which are subtractive. If k^* and are known, k^* can be estimated for any solute from its molar volume (V_x) using the equation log k^*=log k^* –V_x. These parameters were used to analyse the effects of plant species, extraction of cuticular waxes and molecular structure of solutes on solute mobilities in plant cuticles. For aliphatic solutes, k^* was a factor of 10 smaller than for cyclic compounds, while was 0.011 and 0.012, respectively. The k^*-values for CM of the three species were very similar, but was higher for bitter-orange CM (0.012) than for those of pepper fruits and pear leaves (0.009). This has the consequence that differences in solute mobilities (k^*) among cuticles from different plan species increase with increasing molar volumes of solutes. Our data and our analysis provide evidence that constituents of cuticular waxes are mobile, at least in the solid amorphous wax fraction, but mobility decreases rapidly with increasing molar volume. For instance, if amounts to 0.01, mobilities of wax monomers decrease by a factor of 10 for every increase in molar volume of 100 cm³ · mol^–1. Thus, hexadecanoic acid is quite mobile in the amorphous wax fraction of Citrus (k^*=1.5×10^–6·s^–1), but for dotriacontane having twice the molar volume, k^* was only 2.5×10^–9·s^–1, which is almost three orders of magnitude smaller. Wax esters have even higher molar volumes and their mobilities will be even smaller (about 4×10^–12·s^–1 for a C₄₈-ester). Since low chain mobilities are a prerequisite for low mobilities and permeabilities, the selective advantage of high-molecular-weight wax monomers in plant cuticular waxes becomes obvious. Extracting cuticular waxes from pear leaf CM increased solute mobilities by a factor of 182, but it had no effect on size selectivity. We interpret this result as evidence to the effect that cuticular waxes reduce mobility by increasing tortuosity of the diffusion path, rather than by decreasing the mean free path of diffusional jumps and jump frequencies of diffusants.Abbreviations CM cuticular membrane(s) - 2,4-D 2,4-dichloro-phenoxyacetic acid - LAB lactic acid buffer - MX polymer matrix membranes - UDOS unilateral desorption from the outer surface     

Transport properties of cuticular waxes of Fagus sylvatica L. and Picea abies (L.) Karst.: Estimation of size selectivity and tortuosity from diffusion coefficients of aliphatic molecules

Diffusion coefficients for thirteen lipophilic organic compounds in reconstituted waxes of Fagus sylvatica L. and Picea abies (L.) Karst. were measured to characterise the transport properties of the cuticular waxes that form the transport-limiting barrier of plant cuticles. Desorption kinetics (relative amounts desorbed versus time) were asymptotic, but could be linearized up to 50% desorption by plotting relative amounts desorbed versus the square root of time. Diffusion coefficients calculated from the slopes of the linear regressions ranged from 10^–22 to 10^–17 m²·s^–1 and decreased with increasing molecular size. This size dependency of diffusion coefficients was analysed, assuming an exponential dependence of the diffusion coefficients on molar volumes, which allowed cuticular transport properties to be related to the physical structure of the wax. Furthermore, the fact that the barrier properties of Fagus wax are less pronounced than those of Picea is interpreted as an ecological adaptation of the respective tree species to their habitats at the level of their cuticular transport barriers.Abbreviation D diffusion coefficient The authors gratefully acknowledge financial support by the Schwerpunkt Programm Baumphysiologie of the Deutsche Forschungsgemeinschaft.     

Composition of suberin-associated waxes from the subterranean storage organs of seven plants

The waxes associated with the suberin in the periderm of the underground storage organs of parsnip (Pastinaca sativa L.), carrot (Daucus carota L.), rutabaga (Brassica napobrassica Mill.), turnip (Brassica rapa L.), red beet (Beta vulgaris L.), sweet potato (Ipomoea batatas L.) and potato (Solanum tuberosum L.) were isolated, fractionated into hydrocarbon, wax ester, free fatty alcohol and free fatty acid fractions, and analyzed by combined gas chromatography and mass spectrometry. The amount of wax extracted from the periderm of the storage organs ranged from 2 to 32 μg/cm². The hydrocarbons from the suberin layer have a broader chain-length distribution, a predominance of shorter carbon chains, and a higher proportion of even-numbered carbon chains than the leaf alkanes from the same plants. The major components of the free and esterified fatty alcohols and fatty acids have an even number of carbon atoms, and are similar in chain-length distribution to their counterparts found covalently attached to the suberin polymers; however, these suberin components are shorter in chain length than their cuticular analogues from the leaves. Also extracted from the storage organs were polar components which included fatty alcohols and fatty acids in a conjugated form, and ω-hydroxy acids and dicarboxylic acids. Evidence is presented that removal of the wax from the periderm of whole storage organs results in a decrease in diffusion resistance to moisture. Scientific Paper No. 5516, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

Wax Layers on Cosmos bipinnatus Petals Contribute Unequally to Total Petal Water Resistance

Cuticular waxes coat all primary aboveground plant organs as a crucial adaptation to life on land. Accordingly, the properties of waxes have been studied in much detail, albeit with a strong focus on leaf and fruit waxes. Flowers have life histories and functions largely different from those of other organs, and it remains to be seen whether flower waxes have compositions and physiological properties differing from those on other organs. This work provides a detailed characterization of the petal waxes, using Cosmos bipinnatus as a model, and compares them with leaf and stem waxes. The abaxial petal surface is relatively flat, whereas the adaxial side consists of conical epidermis cells, rendering it approximately 3.8 times larger than the projected petal area. The petal wax was found to contain unusually high concentrations of C₂₂ and C₂₄ fatty acids and primary alcohols, much shorter than those in leaf and stem waxes. Detailed analyses revealed distinct differences between waxes on the adaxial and abaxial petal sides and between epicuticular and intracuticular waxes. Transpiration resistances equaled 3 × 10⁴ and 1.5 × 10⁴ s m⁻¹ for the adaxial and abaxial surfaces, respectively. Petal surfaces of C. bipinnatus thus impose relatively weak water transport barriers compared with typical leaf cuticles. Approximately two-thirds of the abaxial surface water barrier was found to reside in the epicuticular wax layer of the petal and only one-third in the intracuticular wax. Altogether, the flower waxes of this species had properties greatly differing from those on vegetative organs.The flowers of many plants are especially adapted to ensure reproductive success by attracting, orienting, and assisting pollinators. Petals must also resist unfavorable environmental conditions such as a desiccating atmosphere. Some characteristics that increase reproductive success, including their high surface areas and surface permeability to small scent molecules, may also make petals more vulnerable to drying out (Goodwin et al., 2003; Bergougnoux et al., 2007). Thus, despite their ephemeral nature, petals may need to compromise between competing physiological and ecological functions. This raises questions: How effective are petal skins at blocking water? Do petal skin compositions differ from those on other plant parts in order to balance multiple functions?To answer these questions, both the chemical composition and the transpiration barrier properties of petal skins must be determined. It is well established that petals are covered by cuticles comparable to those on vegetative organs (Whitney et al., 2011). The waxes coating all primary parts of shoots consist of very-long-chain compounds, including alkanes, aldehydes, primary and secondary alcohols, fatty acids, esters, and ketones ranging in chain length from 20 to 70 carbons (Jetter et al., 2007). The ratio between these derivatives varies temporally and spatially between organs and layers within the cuticle (Jenks et al., 1995, 1996; Jetter and Schäffer, 2001). As well, wax may contain cyclic compounds such as pentacyclic triterpenoids (Buschhaus and Jetter, 2011). Even though it has long been known that the waxes, rather than the accompanying cutin polymer, are essential for the cuticular transpiration barrier (Schönherr, 1976), it is currently not clear how individual wax components contribute to this physiological function.In contrast to other organs, relatively few studies so far have addressed the chemical composition of petal waxes. Noteworthy exceptions are detailed analyses of petal waxes for Crataegus monogyna and three cultivars of Rubus idaeus (Griffiths et al., 2000), Antirrhinum majus (Goodwin et al., 2003), Vicia faba (Griffiths et al., 1999), Cistus albidus (Hennig et al., 1988), Petunia hybrida (King et al., 2007), Arabidopsis (Arabidopsis thaliana; Shi et al., 2011), and Rosa damascena (Stoianova-Ivanova et al., 1971). Selected compound classes have been investigated for some more species, including selected Ericaceae (Salasoo, 1989), Rosaceae (Wollrab, 1969a, 1969b), and Asteraceae (Akihisa et al., 1998) species. Some major plant families, such as the Asteraceae, have not been investigated in much detail.Along with chemical analyses, the physiological properties of waxes on fruits and leaves of diverse plant species also have been investigated in the past. The effectiveness of a water barrier may be characterized by quantifying the permeance for water (P; m s⁻¹) or, inversely, the transpiration resistance (s m⁻¹; Riederer and Schreiber, 1995). These characteristics may, in turn, be determined by measuring the water flux (J; kg m⁻² s⁻¹) across the cuticle under controlled conditions according to the equation P = J/Δc (where Δc is the water concentration gradient driving the diffusion across the barrier). Because both permeance and resistance are physiological characteristics independent of water concentration, their values enable comparisons between water barriers of different plant species and organs. Water permeance values and the corresponding barrier effectiveness vary widely between plant species and organs, with a range of 0.36 to 200 × 10⁻⁶ m s⁻¹ (Kerstiens, 1996; Schreiber and Riederer, 1996). The mean and median leaf permeances (1.42 × 10⁻⁵ and 0.58 × 10⁻⁵ m s⁻¹, respectively) were lower than those of fruit (9.93 × 10⁻⁵ and 9.46 × 10⁻⁵ m s⁻¹), leading to the conclusion that leaves typically produce a better barrier against water movement than does fruit (Kerstiens, 1996). This difference in the physiological performance of waxes on different organs raises the question of how effective the transpiration barrier of cuticular waxes on petals may be. However, to date, water permeance values for petals have not been published and thus cannot be compared with those for other organs.To fill important gaps in our understanding of cuticle function and composition, we initiated a detailed analysis of petal waxes using Cosmos bipinnatus as a first model. We recently reported the identification of novel compounds from the C. bipinnatus petal waxes (Buschhaus et al., 2013) but not the overall wax composition of the petal waxes. Therefore, the ray flowers of this species were examined here to determine (1) the wax composition on the adaxial and abaxial petal surfaces in comparison with the stem and leaf wax and (2) the corresponding petal water permeances.     

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

CYP86A33-Targeted Gene Silencing in Potato Tuber Alters Suberin Composition,Distorts Suberin Lamellae,and Impairs the Periderm's Water Barrier Function

Suberin is a cell wall lipid polyester found in the cork cells of the periderm offering protection against dehydration and pathogens. Its biosynthesis and assembly, as well as its contribution to the sealing properties of the periderm, are still poorly understood. Here, we report on the isolation of the coding sequence CYP86A33 and the molecular and physiological function of this gene in potato (Solanum tuberosum) tuber periderm. CYP86A33 was down-regulated in potato plants by RNA interference-mediated silencing. Periderm from CYP86A33-silenced plants revealed a 60% decrease in its aliphatic suberin load and greatly reduced levels of C18:1 ω-hydroxyacid (approximately 70%) and α,ω-diacid (approximately 90%) monomers in comparison with wild type. Moreover, the glycerol esterified to suberin was reduced by 60% in the silenced plants. The typical regular ultrastructure of suberin, consisting of dark and light lamellae, disappeared and the thickness of the suberin layer was clearly reduced. In addition, the water permeability of the periderm isolated from CYP86A33-silenced lines was 3.5 times higher than that of the wild type. Thus, our data provide convincing evidence for the involvement of ω-functional fatty acids in establishing suberin structure and function.Periderm, the boundary tissue that replaces the epidermis in the secondary organs of plants, provides efficient protection against dehydration, UV radiation, and pathogens (Esau, 1965). Periderm is regularly found in the bark of woody plants, but herbaceous plants may also form a well-developed periderm in roots, tubers, and the oldest portions of stem. The periderm has been widely studied in potato (Solanum tuberosum) tubers because of the latter''s great agronomic significance (Schmidt and Schönherr, 1982; Vogt et al., 1983; Lulai and Freeman, 2001; Sabba and Lulai, 2002). Shrinkage and flaccidity occur in tubers if the protection afforded by the periderm against water loss is compromised (Lulai et al., 2006). Suberin and waxes embedded into the suberin matrix are considered essential for periderm imperviousness (Franke and Schreiber, 2007). Chemically, suberin is a complex lipid polymer consisting of a fatty acid-derived domain (aliphatic suberin) cross-linked by ester bonds to a polyaromatic lignin-like domain (aromatic suberin; Kolattukudy, 2001; Bernards, 2002; Franke and Schreiber, 2007). Aliphatic suberin has been widely analyzed in potato periderm (Kolattukudy and Agrawal, 1974; Graça and Pereira, 2000; Schreiber et al., 2005). The main monomers released from potato aliphatic suberin are a mixture of ω-hydroxyacids and α,ω-diacids with chain lengths ranging from C16 to C28 (mainly C18), together with glycerol. Small amounts of monofunctional fatty acids, alcohols, and ferulic acid are also released. Waxes are complex mixtures of lipids extractable with chloroform that in potato periderm consist mostly of linear very-long-chain aliphatics up to C32 (Schreiber et al., 2005). Suberin is deposited in the cell wall as a continuous deposit or secondary cell wall that overlays the polysaccharide primary cell wall from the inside (Esau, 1965). These suberin deposits appear under the transmission electron microscope (TEM) as regularly alternating opaque and translucent lamellae (Schmidt and Schönherr, 1982). Although several molecular models for suberin have been proposed (Kolattukudy, 1980; Bernards, 2002; Graça and Santos, 2007), how the suberin and wax components are organized in the lamellated suberin secondary cell wall is a matter of debate (Graça and Santos, 2007). Moreover, to what extent suberin and wax deposition and composition determine sealing properties of periderm still remains unclear (Schreiber et al., 2005). Several studies confirm the importance of waxes for the diffusion barrier (Soliday et al., 1979; Vogt et al., 1983; Schreiber et al., 2005), but the significance of aliphatic suberin has hardly been investigated at all. Interestingly, an Arabidopsis (Arabidopsis thaliana) knockout mutant for the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE5 gene (GPAT5) with altered suberin showed higher tetrazolium salt permeability in the seed coat (Beisson et al., 2007).ω-Hydroxylation of fatty acids, a reaction carried out in plants by cytochrome P450 monooxygenases, is a crucial step in the biosynthesis of plant biopolymers (Kolattukudy, 1980; Nawrath, 2002). The Arabidopsis mutant lacerata, which shows phenotype defects compatible with a cutin deficiency, is defective in CYP86A8 encoding a fatty acid ω-hydroxylase (Wellesen et al., 2001). The aberrant induction of type three genes1 (att1) mutant, showing an altered cuticle ultrastructure and a higher transpiration rate than wild type, is defective in CYP86A2 and contains reduced amounts of ω-functionalized cutin monomers (Xiao et al., 2004). Moreover, a genome-wide study of cork oak (Quercus suber) bark highlighted a member of the cytochrome P450 of the CYP86A subfamily as a strong candidate gene for aliphatic suberin biosynthesis (Soler et al., 2007); and a role for CYP86A1 in the biosynthesis of suberin has recently been confirmed in the primary root of Arabidopsis knockout mutants (Li et al., 2007; Hofer et al., 2008). However, how the lack of fatty acid ω-hydroxylase activity may affect the structural features and sealing properties of suberin in periderm cell walls has not been documented.To provide experimental evidence of the role of CYP86A genes in periderm fine structure and water transpiration properties, especially quantitative permeability studies so far unexplored in Arabidopsis, we followed a strategy based on the potato (cv Desirée). The potato is a model of choice for such studies because transgenic plants can be produced in reasonable time and sufficient amounts of periderm can easily be obtained from tubers for chemical and physiological studies (Vogt et al., 1983; Schreiber et al., 2005). Based on the CYP86A gene that was highlighted in cork oak periderm as a strong suberin candidate for aliphatic suberin biosynthesis, we isolated the putative ortholog in potato and used a reverse genetic approach to analyze the effects of down-regulation on the chemical and ultrastructural features of suberin and on the physiological properties of the tuber periderm. Our findings indicate that down-regulation of CYP86A33, as this gene was designated, results in a strong decrease of the ω-functionalized monomers in aliphatic suberin, which are necessary for the suberin typical lamellar organization and for the periderm resistance to water loss.     

A feruloyl transferase involved in the biosynthesis of suberin and suberin‐associated wax is required for maturation and sealing properties of potato periderm

Suberin and waxes embedded in the suberin polymer are key compounds in the control of transpiration in the tuber periderm of potato (Solanum tuberosum). Suberin is a cell‐wall biopolymer with aliphatic and aromatic domains. The aliphatic suberin consists of a fatty acid polyester with esterified ferulic acid, which is thought to play an important role in cross‐linking to the aromatic domain. In potato, ferulic acid esters are also the main components of periderm wax. How these ferulate esters contribute to the periderm water barrier remains unknown. Here we report on a potato gene encoding a fatty ω‐hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT), and study its molecular and physiological relevance in the tuber periderm by means of a reverse genetic approach. In FHT RNAi periderm, the suberin and its associated wax contained much smaller amounts of ferulate esters, in agreement with the in vitro ability of the FHT enzyme to conjugate ferulic acid with ω‐hydroxyacid and fatty alcohols. FHT down‐regulation did not affect the typical suberin lamellar ultrastructure but had significant effects on the anatomy, sealing properties and maturation of the periderm. The tuber skin became thicker and russeted, water loss was greatly increased, and maturation was prevented. FHT deficiency also induced accumulation of the hydroxycinnamic acid amides feruloyl and caffeoyl putrescine in the periderm. We discuss these results in relation to the role attributed to ferulates in suberin molecular architecture and periderm impermeability.     

An extracellular sulfated fucose-rich polysaccharide produced by a tropical strain of Cryptomonas obovata (Cryptophyceae)

A tropical strain of Cryptomonas obovata Skuja, isolated from a shallow oxbow lake,releaseda sulfated fucose-rich polysaccharide. The polysaccharide is composed mainly offucose (42%), N-acetyl-galactosamine (26%) and rhamnose (15%), with smallquantities of glucuronic acid, mannose, galactose, xylose and glucose. Sulfateaccounted for 1.7% total polysaccharide. Quantitative release was studied withcells exposed to optimal culture conditions contrasted with high irradiance andnitrate depletion. This latter set of conditions could simulate stresssituations usually found in the place from which this strain was isolated. Themonosaccharide composition of the polysaccharide was evaluated using PAD-HPLCand gas chromatography. The two irradiances tested (165 molm^–2 s^–1 and 2000 molm^–2 s^–1) had no significant effect onamounts of polysaccharide released by the cells. Differences were observed whenthe nitrate availability was varied. In the nitrate-depleted situation,extracellular polysaccharide production was 2.5 times higher than replete cellsafter 6 h at 165 mol m^–2s^–1, and 2.25 times higher at 2000 molm^–2 s^–1.     

Measurement of apoplasmic and cell-to-cell components of root hydraulic conductance by a pressure-clamp technique

A pressure-clamp technique was devised for the direct measurement of cell-to-cell and apoplasmic components of root hydraulic conductance; the experimental results were analyzed in terms of a theoretical model of water and solute flow, based on a composite membrane model of the root. When water is forced under a constant pressure into a cut root system, an exponential decay of flow is observed, until a constant value is attained; when pressure is released, a reverse water flow out of the root system is observed which shows a similar exponential behavour. The model assumes that the transient flow occurs through a cell-to-cell pathway and the observed decrease is the result of accumulation of solutes in front of the root semi-permeable membrane, whilst the steady-state component results from the movement of water through the parallel apoplasmic pathway. Root conductance components are estimated by fitting the model to experimental data. The technique was applied to the root systems of potted cherry (Prunus avium L.) seedlings; average apoplasmic conductance was 15.5 × 10^–9m³· s^–1· MPa^–1, with values ranging from 12.0 × 10^–9 to 18.5 × 10^–9m³· s^–1· MPa^–1; average cell-to-cell conductance was 11.7 × 10⁹ m³· s^–1· MPa^–1, with values ranging from 8.5 × 10^–9 to 15.3 × 10^–9 m³ · s^–1·MPa^–1. Cell-to-cell conductance amounted on average to 43% of total root conductance, with values between 41 and 45%. Leaf specific conductance (conductance per unit of leaf area supported) of the root systems ranged from 2.7 × 10^–8 to 5.6 × 10^–8 m· s^–1·MPa^–1, with an average of 3.7 × 10^–8 m · s^–1·MPa^–1. The newly developed technique allows the interaction of mass flow of water and of solutes to be explored in the roots of soil-grown plants.Abbreviations and Symbols A L_p root hydraulic conductance - A^aL _p ^a root apoplasmic conductance - A^ccL _p ^cc root cell-to-cell conductance - C_s(t) concentration of solutes in apical root compartment at time t - J_v flow of water through the root - J _v ^a apoplasmic flow of water - J_v/cc cell-to-cell flow of water - LSC leaf specific conductance of the root system - P root hydrostatic pressure - P_appl applied pressure - _s(t) root osmotic pressure at time t - _m osmotic pressure of rooting medium - reflection coefficient of root membrane - time constant of cell-to-cell flow decay This research was funded within the EC Project Long-term effects of CO₂-increase and climate change on European forests (LTEEF) (EV5V-CT94-0468); F.M. was supported by a Ministero dell' Universitá e della Ricerca Scientifica e Tecnologica — British Council agreement (Project The ecological significance of cavitation in woody plants); M.C. was supported by a Consiglio Nazionale delle Ricerche — British Council agreement. We gratefully thank Prof. P.G. Jarvis (University of Edinburgh, UK) for revising an earlier version of this paper and Prof. E. Steudle (University of Bayreuth, Germany) for helpful comments.     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization

This study reports the effects of light availability during the acclimatization phase on photosynthetic characteristics of micropropagated plantlets of grapevine (Vitis vinifera L.) and of a chestnut hybrid (Castanea sativa × C. crenata). The plantlets were acclimatized for 4 weeks (grapevine) or 6 weeks (chestnut), under two irradiance treatments, 150 and 300 mol m^–2 s^–1 after in vitro phases at 50 mol m^–2 s^–1. For both treatments and both species, leaves formed during acclimatization (so-called `new leaves') showed higher photosynthetic capacity than the leaves formed in vitro either under heterotrophic or during acclimatization (so-called `persistent leaves'), although lower than leaves of young potted plants (so-called `greenhouse leaves'). In grapevine, unlike chestnut, net photosynthesis and biomass production increased significantly with increased light availability. Several parameters associated with chlorophyll a fluorescence indicated photoinhibition symptoms in chestnut leaves growing at 300 mol m^–2 s^–1. The results taken as a whole suggest that 300 mol m^–2 s^–1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.     

Photosynthesis and growth rates of Laurencia brongniartii J. Agardh (Rhodophyta, Ceramiales) in preparation for cultivation

Laurencia brongniartii is usually found at depths below 4 m, but can be found in shallow subtidal areas in crevices and on the walls of a coral reef in Amami Oshima Island, Kagoshima Prefecture, Japan, where irradiances were significantly lower than those at similar depths in open water. In preparation for the possible cultivation of this species for its antibiotic compounds, the effects of temperature and irradiance on photosynthesis and growth were measured. Photosynthesis and growth rates of L. brongniartii explants were highest at 26 and 28 °C, which closely corresponded to temperatures found during August to late December when it was most abundant. The estimated maximum photosynthesis rate (P _max) was 4.41 mol photon m^–2 s^–1 at 26 °C and 4.07 mol photon m^–2 s^–1 at 28 °C. Saturating irradiance occurred at 95 mol photon m^–2 s^–1 at 26 °C and 65 mol photon m^–2 s^–1 at 28 °C. In contrast, growth experiments at 41.7 mol photon m^–2 s^–1 caused bleaching of explants and the maximum growth rate observed during the study was 3.02 ± 0.75% day^–1 at 28 °C and 25 mol photon m^–2 s^–1. The difference in the saturating irradiance for photosynthesis and the irradiance that caused bleaching in growth experiments suggests that long-term exposure to high irradiance was detrimental and should be addressed before the initiation of large scale cultivation.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Effects of α-bungarotoxin on acetylcholine-induced response at the Helix neuronal membrane

The effects of -bungarotoxin (-BT) on two patterns of acetylcholine (ACh)-induced response differing in desensitization rate were investigated in isolated mollusk neurons using intracellular dialysis and concentration clamping techniques. It was found that -BT depressed both types of ACh-induced response — a reversible action in the majority of experiments performed. It also exerted a blocking effect on ACh-induced currents dependent on the presence of albumin, although albumin itself produced no noticeable change in ACh-induced response. Concentration dependence of -BT-induced blockade on both types of currents evoked by 1 and 10 µM ACh was investigated. The -BT concentrations producing a 50% suppression of the current evoked by 1 µM ACh were calculated by approximating concentration plots as (13.85±1.25)×10^–9 and (5.56±1.0)×10^–8 g/ml for type A and B cells respectively.Institute of Experimental Biology. Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 729–735, November–December, 1989.     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Transport number effects in the transverse tubular system and their implications for low frequency impedance measurement of capacitance of skeletal muscle fibers

Summary It has been shown in an earlier paper that the slow transient decrease in conductance, somtimes referred to as creep, obtained with small-to-medium hyperpolarizing current or voltage pulses is due to K⁺ transport number differences across the walls of the transverse tubular system. Using the same basic numerical analysis and the parameters already obtained experimentally in the previous paper for frog skeletal muscle in a sulphate Ringer's solution, this paper predicts the equivalent membrane capacitance and dynamic resistance due to transport number effects for very low amplitude and low frequency sinusoidal currents from the phase lag of the voltage response behind the current. Such sinusoidal currentper se give rise to an equivalent capacitance which increased from less than 1F·cm^–2 at 10 Hz to about 16F·cm^–2 at 0.01 Hz and to an equivalent dynamic membrane resistance which increases from its instantaneous slope resistance value of 11.7kcm² at 10 Hz to about 16kcm² at 0.01 Hz. Similar small sinusoidal components of current superimposed on depolarizing and hyperpolarizing pulses (25–45 mV) give rise to even greater capacitances at low frequencies (e.g., 24–28F·cm^–2 at 0.01 Hz). The response due to large sinusoidal currents was also investigated. These transport number effects help to explain the small discrepancies obtained by some workers between experimental and predicted values of skeletal muscle fiber impedances measured in the 1–10 Hz range and would seem to be critical for the interpretation of any skeletal muscle fiber impedance studies done at frequencies less than 1 Hz.     

Response of maize and rice to 9-β-L(+) adenosine applied under different environmental conditions

The increase in growth, determined by dry weight gain, of rice (Oryza sativa L.) and maize (Zea mays L.) caused by foliar applications of 9--L(+) adenosine, a putative second messenger elicited by triacontanol, was studied under different environmental conditions. Maize seedlings cultured in the greenhouse under approximately 100 mol m^–2s^–1 of light prior to treatment with L(+) adenosine did not respond unless they received supplemental light (250–300 mol m^–2s^–1) after treatment. Exposure of rice seedlings growing for 16 h at 150 mol m^–2s^–1 to short periods of 450 mol m^–2s^–1 (< than 20 min) had no effect on the positive response of rice to L(+) adenosine; however, exposure for 60 min or more increased the growth of rice and obviated the effect of L(+) adenosine. Rice seedlings treated with L(+) adenosine at different times during the day responded only when treated 9 to 12h after initiation of the photoperiod. Normal growing temperatures under different light intensities had little or no direct effect on the response of plants to L(+) adenosine.     

Nitrate and phosphate removal by<Emphasis Type="Italic"> Spirulina platensis</Emphasis>

The cyanobacterium Spirulina platensis was used to verify the possibility of employing microalgal biomass to reduce the contents of nitrate and phosphate in wastewaters. Batch tests were carried out in 0.5 dm³ Erlenmeyer flasks under conditions of light limitation (40 mol quanta m^–2 s^–1) at a starting biomass level of 0.50 g/dm³ and varying temperature in the range 23–40°C. In this way, the best temperature for the growth of this microalga (30°C) was determined and the related thermodynamic parameters were estimated. All removed nitrate was used for biomass growth (biotic removal), whereas phosphate appeared to be removed mainly by chemical precipitation (abiotic removal). The best results in terms of specific and volumetric growth rates ( =0.044 day^–1, Q _x =33.2 mg dm^–3 day^–1) as well as volumetric rate and final yield of nitrogen removal ( =3.26 mg dm^–3 day^–1, =0.739) were obtained at 30°C, whereas phosphorus was more effectively removed at a lower temperature. In order to simulate full-scale studies, batch tests of nitrate and phosphate removal were also performed in 5.0 dm³ vessels (mini-ponds) at the optimum temperature (30°C) but increasing the photon fluence rate to 80 mol quanta m^–2 s^–1 and varying the initial biomass concentration from 0.25 to 0.86 g/dm³. These additional tests demonstrated that an increase in the inoculum level up to 0.75 g/dm³ enhanced both NO₃ ^– and PO₄ ^3– removal, confirming a strict dependence of these processes on biomass activity. In addition, the larger surface area of the ponds and the higher light intensity improved removal yields and kinetics compared to the flasks, particularly concerning phosphorus removal ( =0.032–0.050 day^–1, Q _x =34.7–42.4 mg dm^–3 day^–1, =3.24–4.06 mg dm^–3 day^–1, =0.750–0.879, =0.312–0.623 mg dm^–3 day^–1, and =0.224–0.440).     

Seasonal CO<Subscript>2</Subscript>-exchange variations of temperate semi-desert grassland in Hungary

CO₂ exchange components of a temperate semi-desert sand grassland ecosystem in Hungary were measured 21 times in 2000–2001 using a closed IRGA system. Stand CO₂ uptake and release, soil respiration rate (R _s), and micrometeorological values were determined with two types of closed system chambers to investigate the daily courses of gas exchange. The maximum CO₂ uptake and release were –3.240 and 1.903 mol m^–2 s^–1, respectively, indicating a relatively low carbon sequestration potential. The maximum and the minimum R _s were 1.470 and 0.226 mol(CO₂) m^–2 s^–1, respectively. Water shortage was probably more effective in decreasing photosynthetic rates than R _s, indicating water supply as the primary driving variable for the sink-source relations in this ecosystem type.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence