Autotrophy in maize husk leaves : evaluation using natural abundance of stable isotopes

The natural abundance of carbon and hydrogen isotopic composition, expressed as a δ¹³C value of plant dry matter and cellulose in the hypsophylls (husk leaves) of maize (Zea mays L.) was measured and compared with that of leaves and cobs. The δ¹³C values of outer hypsophylls were usually 2 to 3%‰ more negative than leaves or other tissues, and became more negative with increasing chlorophyll content, indicating significant local C₃ pathway fixation of CO₂ in the outer hypsophylls. The δD values indicated a significant part of hypsophyll cellulose was derived from heterotrophic sources (sucrose from C₄ photosynthesis in other tissues). Isotopic mass balance calculations allowed quantitative estimation of these carbon sources and, in the samples examined, about 16% of hypsophyll cellulose was derived from local C₃ photosynthesis, about 62% from local C₄ photosynthesis, and about 22% from sucrose imported from other leaves.     

Carbon stable isotopic composition of soluble sugars in Tillandsia epiphytes varies in response to shifts in habitat

We studied C stable isotopic composition (δ¹³C) of bulk leaf tissue and extracted sugars of four epiphytic Tillandsia species to investigate flexibility in the use of crassulacean acid metabolism (CAM) and C₃ photosynthetic pathways. Plants growing in two seasonally dry tropical forest reserves in Mexico that differ in annual precipitation were measured during wet and dry seasons, and among secondary, mature, and wetland forest types within each site. Dry season sugars were more enriched in ¹³C than wet season sugars, but there was no seasonal difference in bulk tissues. Bulk tissue δ¹³C differed by species and by forest type, with values from open-canopied wetlands more enriched in ¹³C than mature or secondary forest types. The shifts within forest habitat were related to temporal and spatial changes in vapor pressure deficits (VPD). Modeling results estimate a possible 4% increase in the proportional contribution of the C₃ pathway during the wet season, emphasizing that any seasonal or habitat-mediated variation in photosynthetic pathway appears to be quite moderate and within the range of isotopic effects caused by variation in stomatal conductance during assimilation through the C₃ pathway and environmental variation in VPD. C isotopic analysis of sugars together with bulk leaf tissue offers a useful approach for incorporating short- and long-term measurements of C isotope discrimination during photosynthesis.     

Relevance of using whole-ring stable isotopes of black spruce trees in the perspective of climate reconstruction

Studies in dendroisotope chemistry suggested that latewood cellulose contains better climatic records than whole-ring cellulose. However, this approach has never been tested on northeastern Canadian spruce trees. This study compares dendroisotopic series of cellulose from late and whole ring, and analyses their statistical relationships with hydro-climatic variables with the aim of selecting the best suited protocol for future hydro-climatic reconstruction in the downstream sector of Churchill River basin of Labrador, Canada. To this end, δ¹³C and δ¹⁸O series from latewood (LW) and whole ring (WR) α-cellulose of black spruce trees (Picea mariana [Mill.] B.S.P.) were produced for the 1940–2010 period. The results show strong correlations between LW and WR isotopic series suggesting that there are no important variation in the isotopic ratios during the growing year and that black spruce trees use photosynthates of the current growing season to form their earlywood. Moreover, LW and WR δ¹³C and δ¹⁸O show similar relationships with both maximum temperature (T_max) and Churchill River discharge. Correlations are higher when combining δ¹³C and δ¹⁸O for LW and WR. Overall, those correlations support the indirect relationship between tree-ring isotopic series and river discharge, as they are integrators of several climatic variables and derived parameters (T_max, relative humidity, evapotranspiration, etc.). The LW and WR isotopic series give similar statistical relationships with hydro-climatic variables, and the WR treatment is faster (separation easier compared to LW). Thus, for black spruce the use of combined isotopic series in WR can be favored over LW for hydro-climatic reconstruction in the study region.     

Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in ¹³C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO₂. β-Carotene, an isoprenoid plant constituent, was depleted in ¹³C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in ¹³C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less ¹³C) than C₃ plant material but when plants are stressed the isotopic composition could vary.     

Relations of sugar composition and δ13C in phloem sap to growth and physiological performance of Eucalyptus globulus (Labill)

We characterized differences in carbon isotopic content (δ¹³C) and sugar concentrations in phloem exudates from Eucalyptus globulus (Labill) plantations across a rainfall gradient in south‐western Australia. Phloem sap δ¹³C and sugar concentrations varied with season and annual rainfall. Annual bole growth was negatively related to phloem sap δ¹³C during summer, suggesting a water limitation, yet was positively related in winter. We conclude that when water is abundant, variations in carboxylation rates become significant to overall growth. Concentrations of sucrose in phloem sap varied across sites by up to 600 mm, and raffinose by 300 mm . These compounds play significant roles in maintaining osmotic balance and facilitating carbon movement into the phloem, and their relative abundances contribute strongly to overall δ¹³C of phloem sap. Taken together, the δ¹³C and concentrations of specific sugars in phloem sap provide significant insights to functions supporting growth at the tree, site and landscape scale.     

Metabolism of ent-Kaurene to Gibberellin A(12)-Aldehyde in Young Shoots of Normal Maize

Young shoots of normal maize (Zea mays L.) were used to determine both the stepwise metabolism of ent-kaurene to gibberellin A₁₂-aldehyde and the endogenous presence of the members in this series. Each of the five steps in the sequence was established by feeds of 17-¹³C, ³H-labeled kauranoids to cubes from the cortex of elongating internodes, to homogenates from the cortex of elongating internodes, and/or to homogenates from dark-grown seedlings. The ¹³C-metabolites were identified by Kovats retention indices (KRI) and full-scan capillary gas chromatography-mass spectrometry (GC-MS). Five substrates and the final product in this sequence were shown to be native by the isotopic dilution of 17-¹³C, ³H-labeled substrates added as internal standards to extracts obtained from elongating internodes. Evidence for the isotopic dilution was obtained by KRI and full-scan capillary GC-MS. Thus, we document the presence in young maize shoots of the metabolic steps, ent-kaurene → ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-7 α-hydroxykaurenoic acid → gibberellin A₁₂-aldehyde.     

Tree-ring isotopes from Araucaria araucana as useful proxies for climate reconstructions

Tree-ring width (TRW) chronologies have been widely and long-time used to reconstruct past climate variations in the Andes in South America. The use of tree-ring isotopic chronologies is still not widespread in this region although they have proved to be very efficient climate proxies. Araucaria araucana (Molina) K. Koch is a conifer tree species with some multi-century-old individuals that offers an excellent opportunity to measure stable carbon (δ¹³C) and oxygen (δ¹⁸O) isotopes in cellulose from long tree-ring records. Here, we explore whether current or stored carbohydrates are used for A. araucana radial growth and we assess the potential of a tree-ring isotopic record of to study past climate variability. Eleven A. araucana cores from a dry and high-elevation forest at the northern border of Patagonia, Argentina (38°55’S, 70°44’W) were selected for stable isotopes analyses. The strong correlation between the isotopic composition of the first and second parts of the same ring, but also the strong relationships between δ¹³C and δ¹⁸O records with climate parameters of the current growing season such as temperature, show that tree-rings are built mostly with carbohydrates produced during the current growing season with little or no supply from storage or reserves. This finding leads to reconsidering the interpretation of the legacy effect (i.e. ecological memory effects) based on the previously described strong negative correlation between A. araucana TRW chronologies and previous growing season temperature and suggests a dependence of radial tree growth on the level of development of organs. Regarding climate sensitivity, the A. araucana tree-ring δ¹³C chronology is strongly related to current summer temperature (r = 0.82, p < 0.001), vapour pressure deficit (VPD; r = 0.79, p < 0.001), precipitation (r = −0.53, p < 0.001) and SPEI2 (r = −0.73, p < 0.001). These strong relationships support the use of δ¹³C of A. araucana tree-ring cellulose to reconstruct past temperature variations at regional scale in relation with large-atmospheric drivers of climate variability such as the Southern Annular Mode. The A. araucana tree-ring δ¹⁸O chronology is also correlated with temperature (r = 0.42, p < 0.01) and VPD (r = 0.45, p < 0.01) of the winter preceding the growing season. This suggests that trees are using water from precipitation infiltrated in the soil during the previous recharge period (autumn-winter). The weak correlations of δ¹⁸O with current summer atmospheric conditions and the decoupling between δ¹⁸O and δ¹³C, may be due to a high rate of oxygen exchange between sugars and xylem water (P_ex) during cellulose synthesis, which dampens evaporative isotopic fractionation.     

Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch

We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, δ¹⁸O cellulose = 0.48 δ¹⁸O water + 24.1%‰. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of ¹⁸O in the form of O₂ bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O₂ of normal ¹⁸O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water.     

Source water, phenology and growth of two tropical dry forest tree species growing on shallow karst soils

Seasonally dry tropical forests are dominated by deciduous and evergreen tree species with a wide range of leaf phenology. We hypothesized that Piscidia piscipula is able to extend leaf senescence until later in the dry season due to deeper and more reliable water sources than Gymnopodium floribundum, which loses leaves earlier in the dry season. Physiological performance was assessed as timing of leaf production and loss, growth, leaf water potential, depth of water uptake determined by stable isotopes, and leaf stable isotopic composition of carbon (δ¹³C) and oxygen (δ¹⁸O). P. piscipula took water primarily from shallow sources, whereas G. floribundum took water from shallow and deep sources. The greatest variation in water sources occurred during the onset of the dry season, when G. floribundum was shedding old leaves and growing new leaves, but P. piscipula maintained its leaves from the previous wet season. P. piscipula showed greater relative growth rate, greater leaf expansion rates, and more negative predawn and midday water potentials than G. floribundum. P. piscipula also exhibited greater leaf organic δ¹³C and lower δ¹⁸O values, indicating that the decrease in photosynthetic carbon isotope discrimination was associated with greater stomatal conductance and greater photosynthesis. Our results indicate that the contrasting early and late dry season leaf loss phenology of these two species is not simply determined by rooting depth, but rather a more complicated suite of characteristics based on opportunistic use of dynamic water sources, maximizing carbon gain, and maintenance of water potential during the dry season.     

Translocation from leaves to fruits of a legume,studied by a phloem bleeding technique: Diurnal changes and effects of continuous darkness

Summary Diurnal changes in the carbohydrates of leaf laminae and fruits and in the bleeding of sugar and amino acids from fruit phloem were followed by successive sampling from a population of Lupinus albus L. plants. Phloem sap was collected for a standard 5 min period from cut distal tips of attached fruits. Daily fluctuations in leaf dry matter resulted largely from changes in starch and sugar. Leaf sugar rose to a maximum in the afternoon, starch to a maximum at, or shortly after, dusk. Leaves lost sugar and starch from dusk to dawn. Phloem bleeding rate varied little over a daily cycle but sucrose levels fluctuated from a noon maximum of 12–13% (w/v) to a dawn minimum of 9–10%. The rhythm of phloem sugar levels matched closely those of fruit and leaf. Phloem amino acid levels fluctuated in phase with that of sucrose: the relative composition of the amino fraction did not vary significantly over the daily cycle. Pulse feeding of source leaves with ¹⁴CO₂ at different times in the photoperiod allowed study of the pattern of release of labelled photosynthate to the fruit phloem and the build up and depletion of ¹⁴C starch in leaves. Plants transferred to continuous darkness showed a rapid decline in output and concentration of phloem sap solutes, and translocated nitrogen to their fruits at only one quarter of the rate of control plants retained in natural daylight. The combined data from the experiments showed that the rate of output of sugar from cut phloem of a fruit was directly related to the current level of sugar in leaves. When leaf sugar levels were low (5–10 mg ml tissue water^-1) sugar in phloem was 10–11 times more concentrated than in source leaves, but at high leaf sugar levels (25–30 mg ml^-1) this concentration difference was only 3–4 fold.     

Spatial and temporal variations in phloem sap composition of plantation-grown Eucalyptus globulus

Spontaneous bleeding of sugar-rich sap from cambial-deep incisions in the bark of trunks was demonstrated for Eucalyptus globulus and other eucalypts across a range of localities and seasonal conditions in south-west Australia. High levels of sucrose and raffinose (up to 31% w/v total sugars) were present in the exudates, and upward and downward gradients in exudate sugar concentrations were recorded between samples obtained at different heights up trunks of E. globulus. The data indicated a phloem origin for the exudates, with source:sink pressure gradients driving translocation. Concentration ratios of sugars to amino acids were consistently lower in exudate from upper (distal) than basal regions of trunks, suggesting preferential partitioning of nitrogen upwards towards the trunk apex. A comparison of phloem and xylem sap composition from one plantation over a season showed nitrate in xylem but not phloem and substantial amounts of sodium, and high concentrations of chloride and sulphate relative to phosphate in xylem and phloem. Phloem sap sampled across a range of 29 contrasting plantations of E. globulus at peak stress (autumn) showed great inter-site variability in concentrations of amino acids, sulphur, sodium and certain trace elements and in C:N and Na:K ratios of sap. Carbon isotope ratios (δ¹³C) were strongly correlated with sugar concentrations of the sap samples from these and other plantations. Use of sap compositional attributes of phloem and δ¹³C values of translocated carbon is suggested for assessing the current nutritional condition and water status of E. globulus plantings. Received: 9 April 1998 / Accepted: 20 August 1998     

Water relations link carbon and oxygen isotope discrimination to phloem sap sugar concentration in Eucalyptus globulus

A strong correlation was previously observed between carbon isotope discrimination (Delta(13)C) of phloem sap sugars and phloem sap sugar concentration in the phloem-bleeding tree Eucalyptus globulus Labill. (J. Pate, E. Shedley, D. Arthur, M. Adams [1998] Oecologia 117: 312-322). We hypothesized that correspondence between these two parameters results from covarying responses to plant water potential. We expected Delta(13)C to decrease with decreasing plant water potential and phloem sap sugar concentration to increase, thereby maintaining turgor within sieve tubes. The hypothesis was tested with analyses of E. globulus trees growing on opposite ends of a rainfall gradient in southwestern Australia. The Delta(13)C of phloem sap sugars was closely related to phloem sap sugar concentration (r = -0.90, P < 0.0001, n = 40). As predicted, daytime shoot water potential was positively related to Delta(13)C (r = 0.70, P < 0.0001, n = 40) and negatively related to phloem sap sugar concentration (r = -0.86, P < 0.0001, n = 40). Additional measurements showed a strong correspondence between predawn shoot water potential and phloem sap sugar concentration measured at midday (r = -0.87, P < 0.0001, n = 30). The Delta(13)C of phloem sap sugars collected from the stem agreed well with that predicted from instantaneous measurements of the ratio of intercellular to ambient carbon dioxide concentrations on subtending donor leaves. In accordance, instantaneous ratio of intercellular to ambient carbon dioxide concentrations correlated negatively with phloem sap sugar concentration (r = -0.91, P < 0.0001, n = 27). Oxygen isotope enrichment (Delta(18)O) in phloem sap sugars also varied with phloem sap sugar concentration (r = 0.91, P < 0.0001, n = 39), consistent with predictions from a theoretical model of Delta(18)O. We conclude that drought induces correlated variation in the concentration of phloem sap sugars and their isotopic composition in E. globulus.     

Tree-ring widths and isotopes of artificially defoliated balsam firs: A simulation of spruce budworm outbreaks in Eastern Canada

Defoliation by insects is a major disturbance influencing the forest dynamics in many ecosystems and can affect forest productivity worldwide. The main objective of this research was to further investigate the potential use of tree-ring widths and isotopic compositions to identify different degrees of past spruce budworm defoliation episodes. A secondary objective was to understand the responses of trees to defoliation episodes using carbon isotopes as a proxy to provide insights into subsequent physiological changes. Tree-ring widths, carbon and oxygen isotopic compositions in wood cellulose and gas exchange measurements were compared among 288 balsam fir (Abies balsamea Mill.) seedlings grown in a controlled experiment that involved different intensities of defoliation. Observations were performed over four growing periods. Moderate to heavy-defoliated seedlings showed reduced radial growth and enriched their cellulose carbon isotopic composition probably as a result of mobilized stored carbohydrates enriched in ¹³C. Less severely defoliated seedlings did not show significant reductions in growth and ¹³C enrichments. The gas exchange observations and wood cellulose oxygen isotope compositions do not suggest photosynthetic compensation in the remaining needles although a positive trend in the response of both assimilation rate (A) and stomatal conductance (g_s) to defoliation was observed in the first growing period. Thus it remains open as to which mechanisms were employed to compensate for the reduced carbon source in the mildly defoliated seedlings. While further investigations are advised, the results of this study still help promote the utilization of tree-ring widths in combination with carbon isotopic compositions for reconstructing severe past defoliation events.     

High Aluminum Resistance in Buckwheat : II. Oxalic Acid Detoxifies Aluminum Internally

Buckwheat (Fagopyrum esculentum Moench. cv Jianxi), which shows high Al resistance, accumulates Al in the leaves. The internal detoxification mechanism was studied by purifying and identifying Al complexes in the leaves and roots. About 90% of Al accumulated in the leaves was found in the cell sap, in which the dominant organic acid was oxalic acid. Purification of the Al complex in the cell sap of leaves by molecular-sieve chromatography resulted in a complex with a ratio of Al to oxalic acid of 1:3. A ¹³C-nuclear magnetic resonance study of the purified cell sap revealed only one signal at a chemical shift 164.4 ppm, which was assigned to the Al-chelated carboxylic group of oxalic acid. A ²⁷Al-nuclear magnetic resonance analysis revealed one major signal at the chemical shift of 16.0 to 17.0 ppm, with a minor signal at the chemical shift of 11.0 to 12 ppm in both the intact roots and their cell sap, which is consistent with the Al-oxalate complexes at 1:3 and 1:2 ratios, respectively. The purified cell sap was not phytotoxic to root elongation in corn (Zea mays). All of these results indicate that Al tolerance in the roots and leaves of buckwheat is achieved by the formation of a nonphytotoxic Al-oxalate (1:3) complex.     

Beyond CO2-fixation by Rubisco – an interpretation of 13C/12C variations in tree rings from novel intra-seasonal studies on broad-leaf trees

Evidence is presented for a very specific, seasonally recurring tri‐phase carbon isotope pattern in tree rings of broad‐leaf deciduous tree species. It is derived from highly resolved intra‐annual measurements of ¹³C/¹²C ratios of wood and cellulose from tree rings of Fagus sylvatica, Populus nigra, Quercus petraea and Morus alba. Investigations on δ¹³C from buds and leaves of Fagus sylvatica revealed a similar tri‐phase δ¹³C pattern. At the very beginning of a growing season, the δ¹³C trend of tree rings and foliage shows a marked increase of up to 5‰. The maximum δ¹³C‐value of each vegetation period always occurs in young heterotrophic leaves shortly after bud burst and persistently in the early wood of each tree ring, when growth depends on carbon reserves. Thereafter, δ¹³C profiles represent the autotrophic stage of the leaves, which show different patterns of variation, by and large characterized by a decline. The minimum δ¹³C‐value always shows up in the late wood of each tree ring. At the very end of each tree ring δ¹³C‐values start rising again. This increase in δ¹³C marks the gradual switch‐over to storage‐dependent growth and can also be observed in senescent leaves. Seasonal changes of more than 4‰ were measured, whereas contiguous δ¹³C values rarely differed from each other by more than 0.3‰. This tri‐phase pattern cannot be explained by the common model of carbon isotope fractionation during photosynthesis. It appears to be primarily an indication of seasonal changes in down‐stream processes of the carbohydrate metabolism. Environmental influences on the carbon isotope fractionation during photosynthesis are presumably of secondary importance and expressed by certain peculiarities showing up during the autotrophic phase, i.e. the mid‐section of the seasonal δ¹³C pattern.     

Variation in the carbon isotope composition of a plant with crassulacean Acid metabolism

The content of ¹³C varies in plants with Crassulacean acid metabolism. Differences up to 3.5‰ in the ¹³C/¹²C ratios were observed between leaves of different age in the same plant of Bryophyllum daigremontianum. Soluble and insoluble carbon in the same leaf differed up to 8‰, the largest difference occurring in the leaves with the highest Crassulacean acid metabolism activity. Models to account for the isotope discrimination by C₃, C₄, and Crassulacean acid metabolism plants are proposed.     

Physiological and isotopic aspects of photosynthesis in peperomia

Physiological and isotopic aspects of several Peperomia species were investigated. All but one species had C₃-like stomatal behavior, in that stomata were open during the day and closed during the night. In these species, most atmospheric CO₂ uptake occurred during the day. Concurrent with this stomatal behavior, there were Crassulacean acid metabolism-like acid fluctuations in most species. Carbon and hydrogen isotope ratios of cellulose nitrate from Peperomia reflect their physiological behavior. The δ¹³C values of cellulose nitrate from Peperomia species were similar to values observed in C₃ plants and consistent with the daytime uptake of exogeneous CO₂ via the C₃ photosynthetic pathway. The δD values of cellulose nitrate from Peperomia species approach those of Crassulacean acid metabolism plants. These elevated δD values are caused by fractionations occurring during biochemical reactions and not as a consequence of water relations.     

Non-statistical isotope fractionation as a novel “retro-biosynthetic” approach to understanding alkaloid metabolic pathways

During the biosynthesis of natural products, isotope fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs. Thus, the position-specific ¹³C/¹²C ratios in these compounds can be related to their known precursors and to the known isotope effects of enzymes involved in their biosynthesis, or similar reaction mechanisms. This can be accessed by isotope ratio monitoring NMR spectrometry. In this short review, how isotope fractionation occurs and when it is manifest is described. Then, the way that ¹³C NMR spectrometry has been applied to study certain aspects of the biosynthesis of the solanaceous alkaloids S-(−)-nicotine and tropine is outlined. Notably, it is shown how similar isotope fractionation is found in the steps of the pathway to the common intermediate, N-methyl-Δ¹-pyrrolinium, but that in the moieties derived from different origins no such similarity is found, the isotopic composition of these atoms reflecting their specific metabolic ancestry. In a second example, tramadol, it is shown how this technique can be used in retro-biosynthesis to give direction as to what precursors and pathway intermediates are probable. It is shown how the observed fractionation in the site-specific ¹³C/¹²C ratios can be effectively explained by known metabolism and the properties of enzymes proposed for the pathway. Furthermore, it can give indications of possible mechanisms of those enzymes that are as yet to be described for a number of key steps.     

Spatial and Temporal Variations in Stable Carbon (δ13C) and Nitrogen (δ15N) Isotopic Composition of Symbiotic Scleractinian Corals

Tropical scleractinian corals are considered autotrophic as they rely mainly on photosynthesis-derived nutrients transferred from their photosymbionts. Corals are also able to capture and ingest suspended particulate organic matter, so heterotrophy can be an important supplementary trophic pathway to optimize coral fitness. The aim of this in situ study was to elucidate the trophic status of 10 coral species under contrasted environmental conditions in a French Polynesian lagoon. Carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopic compositions of coral host tissues and photosymbionts were determined at 3 different fringing reefs during wet and dry seasons. Our results highlighted spatial variability in stable isotopic compositions of both coral host tissues and photosymbionts. Samples from the site with higher level of suspended particulate matter were ¹³C-depleted and ¹⁵N-enriched relative to corals and photosymbionts from less turbid sites. However, differences in both δ¹³C and δ¹⁵N between coral host tissues and their photosymbionts (Δ^{host-photosymbionts 13}C and Δ^{host-photosymbionts 15}N) were small (0.27 ± 0.76‰ and 1.40 ± 0.90‰, respectively) and similar at all sites, thus indicating no general increases in the heterotrophic pathway. Depleted δ¹³C and enriched δ¹⁵N values of coral host tissues measured at the most turbid site were explained by changes in isotopic composition of the inorganic nutrients taken up by photosymbionts and also by changes in rate of isotopic fractionation with environmental conditions. Our results also highlighted a lack of significant temporal variations in δ¹³C and δ¹⁵N values of coral host and photosymbiont tissues and in Δ^{host-photosymbionts 13}C and Δ^{host-photosymbionts 15}N values. This temporal stability indicated that corals remained principally autotrophic even during the wet season when photosymbiont densities were lower and the concentrations of phytoplankton were higher. Increased coral heterotrophy with higher food availability thus appears to be species-specific.     

Gramine Accumulation in Leaves of Barley Grown under High-Temperature Stress

The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as `Arimar' and CI 12020 accumulate gramine (N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as `Proctor' do not. When grown at optimal temperatures (21°C/16°C, day/night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two- to three-leaf stage from 21°C/16°C to supra-optimal temperatures (≥30°C/25°C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight).

Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan →→ AMI → MAMI → gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [¹⁴C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [¹⁴C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-¹⁴C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [¹⁴C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [¹⁴C]formate label into gramine and MAMI when supplied with AMI; the ratio [¹⁴C]gramine/[¹⁴C]MAMI fell with leaf age, suggesting that the two N-methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific.

In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures.