Light-emitting diode combined with humic acid improve the nutritional quality and enzyme activities of nitrate assimilation in rocket (Eruca sativa (Mill.) Thell.).

Belonging to the Brassicaceae family, rocket (Eruca sativa (Mill.) Thell.), is considered to be a nitrate-accumulating leafy vegetable. Many studies show that light-emitting diode (LED) lights can be a suitable tool to decrease anti-nutritional compounds (e.g., nitrate (and enhance antioxidant and nutritional quality for phytochemical-rich vegetable production. The positive influence of humic acid on health-promoting compounds in different crops is also well documented. This study aimed to investigate the effects of supplemental LED lights of various spectral compositions, namely 25-100% red, 25-100% blue, and 100% white, as well as their combination with humic acid on the physiological and biochemical responses of rocket plants. ANOVA results showed that almost all the measured traits were significantly affected by LED and humic acid treatments. Generally, LED combined with humic acid improved the accumulation of nutritional compounds (e.g., polyphenols, flavonoid, ascorbic acid, carbohydrate, tannin), increased the activity of key enzymes involved in nitrogen metabolism (e.g., nitrate reductase, nitrite reductase, and glutamine synthetase), and lowered nitrate and ammonium concentrations. The results of principal component analysis indicated that the combination of LED lights, regardless of the spectra, with humic acid was the most effective treatment to enhance the nutritional value and activity of enzymes involved in nitrate assimilation. In sum, these findings may be used as a reference in rocket production for supplemental LED light optimization and its combination with humic acid.

Structure and activity of a novel robust peroxidase from Alkanna frigida cell culture.

Demands for peroxidases (POX)s with diverse physicochemical properties have steadily grown as more applications of POXs are demonstrated. Plants are among the best sources of versatile POXs, and plant biotechnology, as an agricultural hassle-free technology, promises to circumvent the limitations of natural resource exploitation and to address the demands. Following this trend, it was shown that POX production steadily increased during the 31-day subculture of Alkanna frigida (from Boraginaceae) callus on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The purified cationic enzyme (POXalf) maintained its optimal activity over pH 4-7 for 2 years. It was resistant to H2O2 high concentrations (IC50 = 543.7 mM) and showed high specific activity in the reaction with phenol (4320.5 AU mg-1 > 20-fold of HRP AU). Furthermore, the specificity constant ratio of guaiacol to phenol indicated a 100 times faster reaction of POXalf with guaiacol. However, in contrast to HRP, it had little effect on diazo derivatives of aniline and meta-diaminobenzene. Based on the resulting primary structure from the tandem mass analysis, the POXalf 3D structure was constructed via homology modelling. Despite the high topological similarity between the HRP and POXalf structures, there were important differences between the active site pockets that could explain the observed differences in the corresponding substrate spectra and the specific activities. Considering the dynamics of POXalf production, its inactivity towards IAA and its high affinity for guaiacol, POXalf may have associated roles with A. frigida cell wall construction and monolignol metabolism.

Nitrate and ammonium differ in their impact on δ13C of plant metabolites and respired CO2 from tobacco leaves.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of N O 3 - vs. N H 4 + supply on δ13C of leaf metabolites and respired CO2 is largely unknown. We supplied tobacco plants with a gradient of N O 3 - to N H 4 + concentration ratios and determined gas exchange variables, concentrations and δ13C of tricarboxylic acid (TCA) cycle intermediates, δ13C of dark-respired CO2, and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble sugars and amino acids increased. As N O 3 - decreased, activities of all measured enzymes decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under N O 3 - supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13C enrichment in organic acids and respired CO2 under N O 3 - supply. Thus, the effect of N O 3 - vs. N H 4 + on δ13C is highly relevant, particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.

Effects of Potassium Fertilization on Oil Palm Fruit Metabolism and Mesocarp Lipid Accumulation.

Potassium fertilization is commonly practiced in oil palm (Elaeis guineensis) plantations to increase yield. However, its effects on fruit oil content and composition are not well documented. Here, we conducted bunch, metabolomics, and oil composition analyses in two contrasting crosses (Deli × La Mé and Deli × Yangambi) grown under different K fertilization conditions. K availability impacted bunch oil content, resulting in lower water content and higher oil proportion in fruit mesocarp, in Deli × La Mé only, thus showing differential responses of crosses to K. Oil composition at maturity did not significantly change under low K conditions despite clear alterations in fruit metabolism associated with lipid production during maturation, demonstrating the resilience of oil biosynthetic metabolism. However, the analysis of variance in oil content (across K treatments and crosses) demonstrates that sugar availability, lipid synthesis rates, and metabolic recycling are all important in determining the oil content.

Stomatal and non-stomatal limitations are responsible in down-regulation of photosynthesis in melon plants grown under the saline condition: Application of carbon isotope discrimination as a reliable proxy.

Salinity is one of the most severe environmental stresses limiting agricultural crop production worldwide. Photosynthesis is one of the main biochemical processes getting affected by such stress conditions. Here we investigated the stomatal and non-stomatal factors during photosynthesis in two Iranian melon genotypes "Ghobadlu" and "Suski-e-Sabz", as well as the "Galia" F1 cultivar, with an insight into better understanding the physiological mechanisms involved in the response of melon plants to increasing salinity. After plants were established in the greenhouse, they were supplied with nutrient solutions containing three salinity levels (0, 50, or 100 mM NaCl) for 15 and 30 days. With increasing salinity, almost all of the measured traits (e.g. stomatal conductance, transpiration rate, internal to ambient CO2 concentration ratio (Ci/Ca), Rubisco and nitrate reductase activity, carbon isotope discrimination (Δ13C), chlorophyll content, relative water content (RWC), etc.) significantly decreased after 15 and 30 days of treatments. In contrast, the overall mean of water use efficiency (intrinsic and instantaneous WUE), leaf abscisic acid (ABA) and flavonol contents, as well as osmotic potential (ΨS), all increased remarkably with increasing stress, across all genotypes. In addition, notable correlations were found between Δ13C and leaf gas exchange parameters as well as most of the measured traits (e.g. leaf area, biomass, RWC, ΨS, etc.), encouraging the possibility of using Δ13C as an important proxy for indirect selection of melon genotypes with higher photosynthetic capacity and higher salinity tolerance. The overall results suggest that both stomatal and non-stomatal limitations play an important role in reduced photosynthesis rate in melon genotypes studied under NaCl stress. This conclusion is supported by the concurrently increased resistance to CO2 diffusion, and lower Rubisco activity under NaCl treatments at the two sampling dates, and this was revealed by the appearance of lower Ci/Ca ratios and lower Δ13C in the leaves of salt-treated plants.

Pyrrolizidine Alkaloids-Free Extract from the Cell Culture of Lithospermum officinale with High Antioxidant Capacity.

The benefits of Lithospermum officinale has encouraged people to continue using its extract (CAS 90063-58-4) in both medicinal and cosmetic industries despite the fact that chemical analysis confirms the presence of pyrrolizidine alkaloids (PAs) in the extract. While the cultivation of L. officinale takes, at least, 2 years to produce usable crops, its callus culture proliferated 8.3 times with 4.9-fold biomass in less than 30 days under the applied conditions in this study. Under the applied conditions, the cell extract contained no toxic PAs while phenylpropanoid pathway was active toward phenolic acids formation not toward naphthoquinone derivatives. Rosmarinic acid was produced as the main constituent. Total phenolic content and antioxidant capacity of the proliferated cell extracts were similar to those of the extracts of the natural plant tissues, in particular from the root. These results support the idea that the extract of L. officinale cells can be a reliable substitute for the extract of the natural plant tissues.

The validity of carbon isotope discrimination as a screening criterion for grain yield in two barley landraces under deficit irrigation with saline water in southern Tunisia.

Arid and semiarid regions with rain shortage and scarce good quality water must make use of low-quality water for irrigation. Consequently, improved plant cultivars for use in these areas should show adaptation capacities to confer drought and salt resistance and allow the cultivation under limited water availabiltiy. The present study was conducted to determine the effect of deficit irrigation with saline water on two local barley landraces, "Karkeni" and "Bengardeni". Plants were saline-irrigated with three watering regimes during tillering, heading, and grain filling stages. Biochemical traits, carbon isotope discrimination (Δ13C), mineral composition, grain yield (GY) and water use efficiency based on grain yield (WUEgy) were evaluated as performance indicators. Almost all of the studied traits (e.g. soluble carbohydrates, proline, ∆13C, Na concentration, and GY) were significantly affected by deficient saline-irrigation regimes at different growth stages. The hierarchical clustering analysis clearly showed that Δ13C placed very close to GY averaging two barley landraces, which was in accordance with the scatter plot result. Multiple linear regression performed between GY as the dependent variable and other traits studied as the independent variables indicated that WUEgy, Δ13C, and soluble carbohydrates significantly explained the variability in GY (R 2=95.64%). A significant positive correlation that observed between ∆13C and GY at three growth stages, indicated that ∆13C may be an important proxy component for indirect selection of yield potential in barley under deficient irrigation regimes with saline water. According to our result, "Karkeni" seems to be more efficient in terms of higher GY, WUEgy, proline and carbohydrate contents, K, Mg and Zn concentrations, as well as lower Δ13C and lipid peroxidation as compared with "Bengardeni", under low osmotic potential imposed by deficient irrigation treatments with saline water, "Karkeni" can thus be selected and used as a parent in order to obtain more tolerant plants against such stresses in future breeding programs.

Genotypic differences in physiological and biochemical responses to salinity stress in melon (Cucumis melo L.) plants: Prospects for selection of salt tolerant landraces.

Melon (Cucumis melo L.) is one of the most important horticultural crops in Iran often cultivated in arid and semiarid regions of the country with salinity problems. The objective of this work was to better understand the mechanisms of physiological and biochemical responses to salinity stress of five Iranian melon landraces "Samsuri", "Kashan", "Khatouni", "Suski-e-Sabz", and "Ghobadlu" from different geographical origins, and "Galia" F1 cultivar. Plants were grown under greenhouse conditions and irrigated with half-strength Hoagland solution containing 0, 30, 60, or 90 mM NaCl for 60 days. Increase in the external salt concentration was accompanied by an obvious depression in leaf relative water content, membrane stability index, chlorophyll a and b and carotenoid contents, stomata and trichome density, leaf area, specific leaf area, biomass, leaf and stem K+ concentrations as well as leaf and stem K+/Na+ ratios in all landraces studied. In contrast, hydrogen peroxide, lipid peroxidation, proline and soluble carbohydrate contents, activity of antioxidant enzymes as well as leaf and stem Na+ and Cl- concentrations, all increased significantly with increasing stress over all plants. Moreover, carbon isotope discrimination (Δ13C), determined on leaf organic matter, was found to be associated with evaluated traits. For example, a highly positive correlation between Δ13C and both biomass production and salt tolerance index was notable when all saline treatments were averaged (r = 0.998 and 0.998, respectively). Also, scatter plot and clustering analysis showed that "Suski-e-Sabz" and "Ghobadlu" were placed close to "Galia" F1, a salt tolerant cultivar, indicating that their similar behavior under salinity. Overall, the present results indicated a significant genetic variability for most of the traits studied, suggesting that "Suski-e-Sabz" and "Ghobadlu" could be introduced as the superior landraces and the most promising tolerant parents in the future melon breeding programs due to their suitable performance, in terms of responses to salt stress as compared with other landraces. Also, Δ13C can be used as a powerful criterion in melon breeding programs aimed at selection of salt tolerant landraces.

Intraspecific variability of carbon isotope discrimination and its correlation with grain yield in safflower: prospects for selection in a Mediterranean climate.

The goals of the present study were to obtain a first estimate of intraspecific variability of carbon isotope discrimination (Δ) in safflower, a thistle-like herbaceous plant, and to determine the statistical relationship between Δ and grain yield as well as its components in a collection of 45 accessions of different origins. Grain yield and aboveground biomass, harvest index, average grain weight, and Δ (measured on the bulk leaf organic matter) were investigated in experimental field conditions. A large variability was noted for all traits but a principal component analysis (PCA) allowed to identify several homogeneous groups of accessions. Average grain yield per plant varied between 1 and 39 g. Δ varied between 21.3 and 25.2 ‰, i.e. a large variation of 3.9 ‰. In our experiment, the variation of Δ was not significantly related to that of grain yield in the whole accession sample. However, we found contrasting trends for this relation within accession groups. These initial results motivate further experiments to assess more in depth correlation between Δ and yield in safflower and are encouraging regarding the possibility of using Δ as an effective selection index in safflower to obtain genotypes that efficiently consume water. This study also highlighted one accession that combines the two characters required in the Mediterranean regions, i.e. high yield performance and high water-use efficiency.

Changes and their possible causes in δ13C of dark-respired CO2 and its putative bulk and soluble sources during maize ontogeny.

The issues of whether, where, and to what extent carbon isotopic fractionations occur during respiration affect interpretations of plant functions that are important to many disciplines across the natural sciences. Studies of carbon isotopic fractionation during dark respiration in C3 plants have repeatedly shown respired CO2 to be (13)C enriched relative to its bulk leaf sources and (13)C depleted relative to its bulk root sources. Furthermore, two studies showed respired CO2 to become progressively (13)C enriched during leaf ontogeny and (13)C depleted during root ontogeny in C3 legumes. As such data on C4 plants are scarce and contradictory, we investigated apparent respiratory fractionations of carbon and their possible causes in different organs of maize plants during early ontogeny. As in the C3 plants, leaf-respired CO2 was (13)C enriched whereas root-respired CO2 was (13)C depleted relative to their putative sources. In contrast to the findings for C3 plants, however, not only root- but also leaf-respired CO2 became more (13)C depleted during ontogeny. Leaf-respired CO2 was highly (13)C enriched just after light-dark transition but the enrichment rapidly decreased over time in darkness. We conclude that (i) although carbon isotopic fractionations in C4 maize and leguminous C3 crop roots are similar, increasing phosphoenolpyruvate-carboxylase activity during maize ontogeny could have produced the contrast between the progressive (13)C depletion of maize leaf-respired CO2 and (13)C enrichment of C3 leaf-respired CO2 over time, and (ii) in both maize and C3 leaves, highly (13)C enriched leaf-respired CO2 at light-to-dark transition and its rapid decrease during darkness, together with the observed decrease in leaf malate content, may be the result of a transient effect of light-enhanced dark respiration.

Natural (13) C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern.

Oil palm has now become one of the most important crops, palm oil representing nearly 25% of global plant oil consumption. Many studies have thus addressed oil palm ecophysiology and photosynthesis-based models of carbon allocation have been used. However, there is a lack of experimental data on carbon fixation and redistribution within palm trees, and important C-sinks have not been fully characterized yet. Here, we carried out extensive measurement of natural (13) C-abundance (δ(13) C) in oil palm tissues, including fruits at different maturation stages. We find a (13) C-enrichment in heterotrophic organs compared to mature leaves, with roots being the most (13) C-enriched. The δ(13) C in fruits decreased during maturation, reflecting the accumulation in (13) C-depleted lipids. We further used observed δ(13) C values to compute plausible carbon fluxes using a steady-state model of (13) C-distribution including metabolic isotope effects ((12) v/(13) v). The results suggest that fruits represent a major respiratory loss (≈39% of total tree respiration) and that sink organs such as fruits are fed by sucrose from leaves. That is, glucose appears to be a quantitatively important compound in palm tissues, but computations indicate that it is involved in dynamic starch metabolism rather that C-exchange between organs.

Changes in δ(13)C of dark respired CO2 and organic matter of different organs during early ontogeny in peanut plants.

Carbon isotope composition in respired CO2 and organic matter of individual organs were measured on peanut seedlings during early ontogeny in order to compare fractionation during heterotrophic growth and transition to autotrophy in a species with lipid seed reserves with earlier results obtained on beans. Despite a high lipid content in peanut seeds (48%) compared with bean seeds (1.5%), the isotope composition of leaf- and root-respired CO2 as well as its changes during ontogeny were similar to already published data on bean seedlings: leaf-respired CO2 became (13)C-enriched reaching -21.5‰, while root-respired CO2 became (13)C-depleted reaching around -31‰ at the four-leaf stage. The opposite respiratory fractionation in leaves vs. roots already reported for C3 herbs was thus confirmed for peanuts. However, contrarily to beans, the peanut cotyledon-respired CO2 was markedly (13)C-enriched, and its (13)C-depletion was noted from the two-leaf stage onwards only. Carbohydrate amounts being very low in peanut seeds, this cannot be attributed solely to their use as respiratory substrate. The potential role of isotope fractionation during glyoxylate cycle and/or gluconeogenesis on the (13)C-enriched cotyledon-respired CO2 is discussed.

Opposite carbon isotope discrimination during dark respiration in leaves versus roots - a review.

In general, leaves are (13) C-depleted compared with all other organs (e.g. roots, stem/trunk and fruits). Different hypotheses are formulated in the literature to explain this difference. One of these states that CO2 respired by leaves in the dark is (13) C-enriched compared with leaf organic matter, while it is (13) C-depleted in the case of root respiration. The opposite respiratory fractionation between leaves and roots was invoked as an explanation for the widespread between-organ isotopic differences. After summarizing the basics of photosynthetic and post-photosynthetic discrimination, we mainly review the recent findings on the isotopic composition of CO2 respired by leaves (autotrophic organs) and roots (heterotrophic organs) compared with respective plant material (i.e. apparent respiratory fractionation) as well as its metabolic origin. The potential impact of such fractionation on the isotopic signal of organic matter (OM) is discussed. Some perspectives for future studies are also proposed .

On the 13C/12C isotopic signal of day and night respiration at the mesocosm level.

While there is currently intense effort to examine the (13)C signal of CO(2) evolved in the dark, less is known on the isotope composition of day-respired CO(2). This lack of knowledge stems from technical difficulties to measure the pure respiratory isotopic signal: day respiration is mixed up with photorespiration, and there is no obvious way to separate photosynthetic fractionation (pure c(i)/c(a) effect) from respiratory effect (production of CO(2) with a different delta(13)C value from that of net-fixed CO(2)) at the ecosystem level. Here, we took advantage of new simple equations, and applied them to sunflower canopies grown under low and high [CO(2)]. We show that whole mesocosm-respired CO(2) is slightly (13)C depleted in the light at the mesocosm level (by 0.2-0.8 per thousand), while it is slightly (13)C enriched in darkness (by 1.5-3.2 per thousand). The turnover of the respiratory carbon pool after labelling appears similar in the light and in the dark, and accordingly, a hierarchical clustering analysis shows a close correlation between the (13)C abundance in day- and night-evolved CO(2). We conclude that the carbon source for respiration is similar in the dark and in the light, but the metabolic pathways associated with CO(2) production may change, thereby explaining the different (12)C/(13)C respiratory fractionations in the light and in the dark.

On the resilience of nitrogen assimilation by intact roots under starvation, as revealed by isotopic and metabolomic techniques.

The response of root metabolism to variations in carbon source availability is critical for whole-plant nitrogen (N) assimilation and growth. However, the effect of changes in the carbohydrate input to intact roots is currently not well understood and, for example, both smaller and larger values of root:shoot ratios or root N uptake have been observed so far under elevated CO(2). In addition, previous studies on sugar starvation mainly focused on senescent or excised organs while an increasing body of data suggests that intact roots may behave differently with, for example, little protein remobilization. Here, we investigated the carbon and nitrogen primary metabolism in intact roots of French bean (Phaseolus vulgaris L.) plants maintained under continuous darkness for 4 days. We combined natural isotopic (15)N/(14)N measurements, metabolomic and (13)C-labeling data and show that intact roots continued nitrate assimilation to glutamate for at least 3 days while the respiration rate decreased. The activity of the tricarboxylic acid cycle diminished so that glutamate synthesis was sustained by the anaplerotic phosphoenolpyruvate carboxylase fixation. Presumably, the pentose phosphate pathway contributed to provide reducing power for nitrate reduction. All the biosynthetic metabolic fluxes were nevertheless down-regulated and, consequently, the concentration of all amino acids decreased. This is the case of asparagine, strongly suggesting that, as opposed to excised root tips, protein remobilization in intact roots remained very low for 3 days in spite of the restriction of respiratory substrates.

Is there any 12C/13C fractionation during starch remobilisation and sucrose export in potato tubers?

The delta(13)C (carbon isotope composition) variations in respired CO(2), total organic matter, proteins, sucrose and starch have been measured during tuber sprouting of potato (Solanum tuberosum) in darkness. Measurements were carried out both on tubers and on their growing sprouts for 23 days after the start of sprout development. Sucrose was slightly (13)C-depleted compared with starch in tubers, suggesting that starch breakdown was associated with a small isotope fractionation. In sprouts, all biochemical fractions including sucrose were (13)C-enriched compared with source tuber-sucrose, suggesting that sucrose translocation from tuber to sprouts fractionated against (12)C. However, both apparent fractionations were explained by the consumption of (13)C-depleted carbon for respiration or growth that enriched in the (13)C sucrose molecules left behind. In addition, whole tuber sucrose is constantly composed of recent sucrose from starch breakdown and old sucrose associated with an inherited, slightly (13)C-depleted pool. We therefore conclude that any fractionation at either the starch breakdown or the sucrose translocation level is unlikely under our conditions.

Carbon stable isotope ratio of phloem sugars in mature pine trees throughout the growing season: comparison of two extraction methods.

The study presents a comparison of two phloem sugar extraction methods. The amount of phloem sugar extracted and the carbon isotope composition (delta(13)C) of the total extracts and of the main phloem compounds separated by high-performance liquid chromatography (sucrose, glucose, fructose and pinitol) are compared. These two phloem sap extraction methods are exudation in distilled water and a new method using centrifugation, which avoids the addition of any solvent. We applied both extraction methods on phloem discs sampled from 38-year-old Pinus pinaster trees in south-western France throughout the period from June 2007 to December 2008 on different time-scales: hourly, daily and monthly. We found that the centrifugation method systematically extracted ca. 50% less compounds from the phloem discs than the exudation method. In addition, the two extraction methods provided similar delta(13)C values of the total extracts, but the values obtained by the exudation method were 0.6 per thousand more negative than those calculated from the mass balance using the individual constituents. Over the growing season, both extraction methods exhibited lower total sugar content and more (13)C-enriched phloem sap in summer compared with winter values. These findings suggest that both extraction methods can be applied to study the carbon isotope composition of phloem sap, and the centrifugation method has the advantage that no solvent has to be added. The exudation method, however, is more appropriate for the quantification of the amounts of phloem sugars.

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates.

The natural (13)C/(12)C isotope composition (delta(13)C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the (12)C/(13)C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific delta(13)C of individual carbohydrates. Here, we examined two complementary methods for measuring the delta(13)C value of sucrose, glucose and fructose, that is, off-line high-performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA-IRMS) analysis, and gas chromatography-combustion (GC-C)-IRMS. We also used these methods to determine the in vitro (12)C/(13)C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC approximately EA-IRMS, and being sensitive to derivatization conditions, the GC-C-IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the (12)C/(13)C isotope effect is rather small and it is not affected by the use of heavy water (D(2)O).

Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

Consistent patterns in leaf lamina and leaf vein carbon isotope composition across ten herbs and tree species.

Wide-spread post-photosynthetic fractionation processes deplete metabolites and plant compartments in (13)C relative to assimilates to varying degrees. Fragmentation fractionation and exchange of metabolites with distinct isotopic signatures across organ boundaries further modify the patterns of plant isotopic composition. Heterotrophic organs tend to become isotopically heavier than the putative source material as a result of respiratory metabolism. In addition fractionation may occur during metabolite transport across organ and tissue boundaries. Leaf laminae, veins and petioles are leaf compartments that are arranged along a gradient of increasing weight of heterotrophic processes and along a transport chain. Thus, we expect to find consistent patterns of isotopic signatures associated with this gradient. Earlier studies on leaves of Fagus sylvatica, Glycine max, and Saccharum officinarum showed that the organic mass and cellulose of major veins or petioles were consistently more positive than the respective fraction in leaf laminae. The objective of the current study was to assess whether this pattern can be detected in a greater set of plant species. Leaves from ten species were collected in the summer of 2006 outdoors and in glasshouses. Leaf laminae including small veins were separated from the major veins and the isotopic signatures of the organic mass, and the soluble and non-soluble fractions were measured for laminae and veins separately. The organic mass, and the soluble and non-soluble fractions of leaf laminae, were depleted in (13)C relative to the veins in all cases. A general trend for the signature of organic mass being more depleted in (13)C than the soluble fraction is in accordance with well-known patterns of fractionation between metabolites.