Shedding light on an extremophile lifestyle through transcriptomics.

The tropical intertidal ecosystem is defined by trees - mangroves - which are adapted to an extreme and extremely variable environment. The genetic basis underlying these adaptations is, however, virtually unknown. Based on advances in pyrosequencing, we present here the first transcriptome analysis for plants for which no prior genomic information was available. We selected the mangroves Rhizophora mangle (Rhizophoraceae) and Heritiera littoralis (Malvaceae) as ecologically important extremophiles employing markedly different physiological and life-history strategies for survival and dominance in this extreme environment. For maximal representation of conditional transcripts, mRNA was obtained from a variety of developmental stages, tissues types, and habitats. For each species, a normalized cDNA library of pooled mRNAs was analysed using GSFLX pyrosequencing. A total of 537,635 sequences were assembled de novo and annotated as > 13,000 distinct gene models for each species. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology annotations highlighted remarkable similarities in the mangrove transcriptome profiles, which differed substantially from the model plants Arabidopsis and Populus. Similarities in the two species suggest a unique mangrove lifestyle overarching the effects of transcriptome size, habitat, tissue type, developmental stage, and biogeographic and phylogenetic differences between them.

Carbon and phosphorus partitioning in Pinus serotina seedlings growing under hypoxic and low-phosphorus conditions.

Ten-week-old pond pine (Pinus serotina Michx.) seedlings were grown in solution culture at 5 or 100 microM P and under aerobic or hypoxic solution conditions. After 6 and 10 weeks in the treatments, changes in relative growth rate (RGR), P acquisition and allocation, and carbohydrate partitioning were determined by analyzing tissue for total P, soluble sugars and starch. Six weeks of low-P growth conditions decreased seedling dry weight and the ratio of shoot dry weight to root dry weight (S/R) by 39 and 51%, respectively, in comparison to seedlings from the aerobic, high-P (control) treatment. Mean RGRs of shoots in the low-P treatment were reduced by 33%, whereas root growth was unaffected. After 10 weeks of low-P growth conditions, however, both shoot and root RGRs were significantly reduced, and plants had lower S/R ratios than in any other treatment. Slowed shoot growth was accompanied by starch and nonstructural carbohydrate accumulation in needles, indicating that needle growth was not limited by carbohydrate supply. Six weeks of low-P growth conditions decreased total seedling P by 75%, reflecting a 97% reduction in the net uptake rate (NUR). Shoot NUR as a fraction of seedling NUR was also greatly reduced in the low-P treatment, indicating that low-P growth conditions affected P translocation to the shoot more than P accumulation by roots. In contrast, 6 weeks of hypoxic growth conditions decreased total dry weight of seedlings in the high-P treatment by 41% relative to their aerobic counterparts. Root growth was affected more than shoot growth, however, and S/R ratios increased. After 10 weeks, S/R ratios doubled, primarily because of the reduction in root RGR. Nevertheless, roots of hypoxic seedlings contained a higher percentage of total seedling P than their aerobic counterparts. Net P acquisition per seedling decreased by more than 50% under hypoxic growth conditions, as a result of reductions in both root RGR and seedling NUR. Starch accumulation in shoots of hypoxic seedlings reflected reductions both in root growth and in transport of carbohydrates to nonwoody roots. Carbohydrate availability did not appear to be limiting growth of hypoxic woody roots, which are well-aerated internally, but it may have limited metabolic processes in nonwoody roots of seedlings from the high-P treatment.

The analysis of photosynthetic performance in leaves under field conditions: A case study using Bruguiera mangroves.

In this report, we analyze the photosynthetic capacity and performance of leaves under field conditions with a case study based on the mangroves Bruguiera parviflora and B. gymnorrhiza. Using a tower through a closed canopy at a field sight in North Queensland and portable infra-red gas analyzers, a large data set was collected over a period of 11 days early in the growing season. The set was used to analyze the relationship between net photosynthesis (Pnet) and light, leaf temperature, stomatal conductance and intracellular CO2 (Ci).There are three objectives of this report: (1) to determine photosynthetic potential as indicated by the in situ responses of Pnet to light and stomatal conductance, (2) to determine the extent to which photosynthetic performance may be reduced from that potential, and (3) to explore the basis for and physiological significance of the reduction.The results indicate that even under harsh tropical conditions, the mangrove photosynthetic machinery is capable of operating efficiently at low light and with maximal rates of more than 15 µmol CO2 m(-2) s(-1). Though stomata were more often limiting than light, in any single measurement the average reduction of Pnet from the maximum value predicted by light or conductance responses was 35%. Analysis of single leaf light and CO2 responses indicated that photosynthetic performance was under direct photosynthetic, or non-stomatal, control at all light and conductance levels. Capacity was adjustable rapidly from a maximum value to essentially nil such that Ci varied inversely with Pnet from ca. 150 µL L(-1) at the highest rates of CO2 exchange to ambient at the lowest.

Sodium and Potassium Compartmentation and Transport across the Roots of Intact Spergularia marina.

The Na(+) and K(+) transport characteristics of Spergularia marina (L.) Griseb. were considered in order to compare the systems by which these two physiologically different cations are managed during initial acquisition and subsequent partitioning in midvegetative plants. Uptake of (22)Na(+) and (42)K(+) and redistribution of labels in pulse-chase studies were compared under steady state growth conditions or with the concentration of one of the ions elevated. At high external concentrations, the initial (42)K(+) accumulation and transport to the shoot was associated with a small, rapidly exchanging, cellular compartment similar to that previously indicated for Na(+) (D Lazof, JM Cheeseman 1986 Plant Physiol 81: 742-747). At 1 mol m(-3), K(+) was conducted to the shoot through a root compartment, the specific activity of which rose much more slowly than the rapidly exchanging compartment. After a lag of approximately 5 minutes, (42)K(+) translocation approached a constant rate with a half-time of 14 minutes compared to 5 minutes for (22)Na(+) or for (42)K(+) at higher external levels. At all external levels, prolonged translocation of (42)K(+) was measured when a 10 minute pulse was followed by an unlabeled chase, again suggesting a conducting compartment distinct from that for Na(+). It is suggested that the K(+) conducting compartment, possibly the ;bulk cytoplasm,' is associated with the active K(+) transport system generally found in higher plants.

Sodium and potassium compartmentation and transport in the roots of intact lettuce plants.

In this report, we consider the accumulation in roots, and transport to the shoot, of Na(+) and K(+) in intact lettuce plants (Lactuca sativa cv Black-seeded Simpson). Plants were grown in modified Hoagland medium supplemented with 10 moles NaCl per cubic meter. At this salinity, significant levels of Na(+) were accumulated in roots and shoots, but there was no reduction in plant growth. Transport characteristics for both Na(+) and K(+) were qualitatively similar to those previously reported, for Spergularia marina, indicating that the results obtained with these experimental protocols are not limited to one unconventional experimental plant. The most pronounced difference in transport of the two ions was evident when transport was followed in a chase period after a 10 minute uptake pulse. For Na(+), there was an initially rapid, but small, loss of label to the medium, and very little movement to the shoot. For K(+), little label was lost from the plants, but translocation to the shoot proceeded for at least 60 minutes. The transport systems were further distinguished by treating the roots during labeling with 20 micrograms per milliliter cycloheximide. For K(+), both uptake and translocation were reduced by about 50%. For Na(+), root accumulation was stimulated more than five-fold, while transport to the shoot was reduced about 20%. Cycloheximide also modified the Na(+) transport characteristics such that continued translocation occurred during the chase period of pulse-chase studies.

Mechanisms of salinity tolerance in plants.

The mechanisms of salt stress response and tolerance have eluded definition despite reasonable success in defining their physiological manifestations. In this review, we consider the integrated salt metabolism of plants, essentially as a problem in meganutrient physiology. Two critical aspects of cellular and organismal metabolism are given particular attention-those involved in the control and integration of Na(+) acquisition and allocation in plants and those involved in readjustment of other aspects of metabolism, especially those involving carbon as a resource.

Sodium Transport and Compartmentation in Spergularia marina: Partial Characterization of a Functional Symplasm.

In this paper, a combination of tracer uptake, efflux, and pulse-chase techniques is applied to the problem of compartmentation of Na(+) ((24)Na(+)) in the roots of intact, midvegetative Spergularia marina (L.) Griseb. plants. An approach is presented for conducting useful compartmental analysis when it is known that the assumptions required for straightforward interpretations of influx and efflux studies are invalid. Linear rates of (24)Na(+) accumulation in both roots and shoots were attained within at most a few minutes following the start of labeling. Shoot (24)Na(+) contents equaled root contents within about 20 minutes. Analysis of root accumulation rates, and compartmental and pulse-chase efflux studies indicated that the unidirectional flux rates involved were at least an order of magnitude greater than linear rates of root and shoot accumulation. These rapid fluxes involved only a small portion of the total root Na(+) (about 1%). The results suggest the existence of a small symplastic compartment, distinct from the ;bulk cytoplasm,' rapidly exchanging with the medium, and responsible for delivery of Na(+) to the xylem. The physical identity of this compartment and its physiological significance are discussed with respect to precedents in the literature.

Compartmental efflux analysis: an evaluation of the technique and its limitations.

Efflux analysis is an established tool for characterizing the exchange properties of multicomponent systems. In this report, we have simulated several three- and four-compartment systems with error-free and imperfect data, the errors being designed to mimic actual, nonbiological variability in isotope efflux studies. The data sets were analyzed using computerized nonlinear regression techniques to identify the important aspects of actual experimental design (uptake times, efflux collection schedules, and total efflux times), and to consider the possibility that a properly designed and executed experiment might fail to resolve compartmentation correctly. The results showed that for any of the systems simulated, including those with error-free four-component data, a reasonable three-component fit was obtainable. Resolution of the additional compartment was not always possible. In correctly resolved systems, failure to estimate the correct decay constants was common, especially when the half-times were separated by less than an order of magnitude. We conclude that efflux analysis, by itself, lacks the power to provide reliable information about multicompartment systems.

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L.

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-(3)H]+/-ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

Uptake and distribution of sodium and potassium by corn seedlings : I. Role of the mesocotyl in ;sodium exclusion'.

The distribution of sodium and potassium throughout corn (Zea mays L. [A632 x Crows 3640] x Oh 43) plants is not simply a matter of uptake by cortical cells and irreversible delivery to the xylem for upward transport. We show that sodium, but not potassium, accumulates in the mesocotyl of corn seedlings grown on NaCl medium. Upon transfer to NaCl-free medium, total sodium is reduced by export through the roots but remains at high levels within the mesocotyl. We report experiments which consider uptake from the xylem.Shoots excised at the seed were allowed to transpire solutions containing (22)Na and (42)K. Potassium uptake within the mesocotyl was very sensitive to concentration, increasing 27-fold between 1 and 10 millimolar. Sodium uptake was dependent upon the square root of the concentration suggesting active accumulation. At sodium concentrations below 1 millimolar, more than 80% of the sodium in the plant was retained in the mesocotyl. Both the uptake by and retention within the mesocotyl were dependent upon transpiration rate as well as concentration. We discuss the limitations of measuring uptake from a finite, depletable medium. The mesocotyl is a modified root with a cuticularized epidermis. We discuss the feasibility of using this ;plastic-coated root' as a model for root transport studies.

Uptake and Distribution of Sodium and Potassium by Corn Seedlings : II. Ion Transport within the Mesocotyl.

In this paper, uptake and distribution of sodium and potassium within the mesocotyl are considered in excised, 8-day-old corn (Zea mays L.) seedlings supplied with label via the transpiration stream. The stele and cortex were dissected following uptake and analyzed separately. At equal concentrations, sodium uptake by the stele was much more rapid than potassium uptake, and sodium was preferentially retained within the stele. Transport of sodium to the cortex halted when the supply of ions in the transpiration stream was interrupted. Potassium would not substitute for sodium in restoring this transport but neither did it compete with sodium for transport to the cortex. In the presence of continued sodium supply, transport was temperature sensitive.By labeling first with (22)Na for 2 hours and subsequently with (24)Na for up to 21 hours, three sodium pools were identified within the stele. The first was rapidly transportable to the cortex. The second equilibrated rapidly with the first but was not itself directly available for transport. We postulate that these represent the stelar symplasm and apoplasm, respectively. A third pool was not transported and probably represents sequestration within the vacuoles of some cell type. Transport of label acquired during the initial 2 hours proceeded with a half-time of approximately 10 hours with 10 millimolar sodium present during the redistribution period, and with a half-time of approximately 30 hours at 1 millimolar sodium.A working model is presented which explains these characteristics and supplies approachable questions for subsequent study.

Effect of ATPase inhibitors on cell potential and k influx in corn roots.

Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Psi) and K(+) ((86)Rb) influx of corn root tissue over a wide range of K(+) activity. N,N'Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K(+) influx and depolarized Psi at low, but not at high, K(+) activity (K degrees ). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K(+) influx over a wide range of K degrees , with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Psi(p)) above 0.2 millimolar K degrees . Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K(+) influx and Psi(p) at low K degrees can be attributed to inhibition of the plasmalemma ATPase.It is concluded that by DCCD treatment, the energy-linked electrogenic system at high K degrees is separated from the energy-linked K(+) influx system at low K degrees . The results are analyzed in terms of electrical analogue models of the membrane. The presence of two, algebraically additive electrogenic components is indicated; one is better modeled as a current source (system I) and one as a voltage source (system II). No K(+) stimulation of system II is required to produce the observed K degrees dependence of Psi(p).

Energy-linked Potassium Influx as Related to Cell Potential in Corn Roots.

Cell potentials and K(+) ((86)Rb) influx were determined for corn roots over a wide range of external K(+) activity (K degrees ) under control, anoxic, and uncoupled conditions. The data were analyzed using Goldman theory for the contribution of passive influx to total influx. For anoxic and uncoupled roots the K(+) influx shows the functional relationship with K degrees predicted with constant passive permeability, although K(+) permeability in uncoupled roots is about twice that of anoxic roots. In control roots the equation fails to describe K(+) influx at low K degrees , but does so at high K degrees , with a gradual transition over the region where the electrical potential becomes equal to the equilibrium potential for K(+) (psi = E(K)). In the low K degrees range, where net K(+) influx is energetically uphill, participation of an energy-linked K(+) carrier is indicated. In the high K degrees range, K(+) influx becomes passive down the electrical gradient established by the cell potential. Since the cell potential includes a substantial electrogenic component, anoxia or uncoupling reduces passive influx.

Comparison of the responses of corn root tissue to fusicoccin and washing.

A comparison has been made of the effects of fusicoccin with those of washing on segments of corn (Zea mays L.) root tissue. Both fusicoccin and washing caused increases in K(+)((86)Rb) influx, net H(+) efflux, and electrogenic cell membrane potential, but with no effect on respiration rate. The similarity was most evident with fresh tissue during the initial phases of washing, prior to the developmental changes which augment the anion and general solute transport rates of the tissue. After the development of enhanced transport capacity the proportional response to fusicoccin was much diminished. It is suggested that the fusicoccin-like response to washing may be a manifestation of recovery from injury.

Mathematical analysis of the dependence of cell potential on external potassium in corn roots.

The K(+) dependence of normal (psi) and diffusion (psi(D)) potentials in corn roots [Zea mays L., hybrid (A619 x Oh43) x A632] was determined experimentally and analyzed with respect to the parameter xi [defined as exp (F psi/RT)]. In the presence of 10 micromolar carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), psi behaved as expected of a diffusion potential. Based upon the assumptions (a) that FCCP did not change any term of the Goldman-Hodgkin-Katz equation, and (b) that total potential was functionally the algebraic sum of psi(D) and psi(P) (the deviation from psi(D) due to an electrogenic system), psi(P) was found to be a complex function of external potassium and to have a minimum value of 0.69 millimolar K ion activity outside the cell. Analysis of psi allowed us to develop an equation which predicts a complicated K(+) dependence of psi such as that found by Mertz and Higinbotham (Membrane Transport in Plants and Plant Organelles. Springer-Verlag 1974).