Microclimate influence on mineral and metabolic profiles of grape berries.

The grape berry microclimate is known to influence berry quality. The effects of the light exposure of grape berry clusters on the composition of berry tissues were studied on the "Merlot" variety grown in a vineyard in Bordeaux, France. The light exposure of the fruiting zone was modified using different intensities of leaf removal, cluster position relative to azimuth, and berry position in the cluster. Light exposures were identified and classified by in situ measurements of berry temperatures. Berries were sampled at maturity (>19 Brix) for determination of skin and/or pulp chemical and metabolic profiles based on (1) chemical and physicochemical measurement of minerals (N, P, K, Ca, Mg), (2) untargeted 1H NMR metabolic fingerprints, and HPLC targeted analyses of (3) amino acids and (4) phenolics. Each profile defined by partial least-square discriminant analysis allowed us to discriminate berries from different light exposure. Discriminant compounds between shaded and light-exposed berries were quercetin-3-glucoside, kaempferol-3-glucoside, myricetin-3-glucoside, and isorhamnetin-3-glucoside for the phenolics, histidine, valine, GABA, alanine, and arginine for the amino acids, and malate for the organic acids. Capacities of the different profiling techniques to discriminate berries were compared. Although the proportion of explained variance from the 1H NMR fingerprint was lower compared to that of chemical measurements, NMR spectroscopy allowed us to identify lit and shaded berries. Light exposure of berries increased the skin and pulp flavonols, histidine and valine contents, and reduced the organic acids, GABA, and alanine contents. All the targeted and nontargeted analytical data sets used made it possible to discriminate sun-exposed and shaded berries. The skin phenolics pattern was the most discriminating and allowed us to sort sun from shade berries. These metabolite classes can be used to qualify berries collected in an undetermined environment. The physiological significance of light and temperature effects on berry composition is discussed.

Allometric relationships to estimate seasonal above-ground vegetative and reproductive biomass of Vitis vinifera L.

A procedure is described for obtaining allometric regression equations to estimate non-destructively and in a cost-effective manner the current year's above-ground vegetative and reproductive biomass of Vitis vinifera L. Merlot' throughout the growing season. Significant relationships were obtained over a 3-year period (1998-2000) between the dimensions of an individual shoot per vine (i.e. diameter and length) and dry weights of its primary stem, primary leaves and lateral growth. The dry mass of a grape was best estimated from measurements of the basal diameter of the bunch peduncle. Introducing cumulative degree-days as an additional explanatory variable in the equations allowed them to be used irrespective of year and growth stage. Multi-year regressions were used to quantify in detail the seasonal evolution of mature grapevine biomass under the climatic conditions of the Bordeaux area, France, and for differing levels of soil nitrogen.

Generalized Münch coupling between sugar and water fluxes for modelling carbon allocation as affected by water status.

A model of within-plant carbon allocation is proposed which makes a generalized use of the Münch mechanism to integrate carbon and water functions and their involvement in growth limitations. The plant is envisioned as a branched network of resistive pathways (phloem and xylem) with nodal organs acting as sources and sinks for sucrose. Four elementary organs (leaf, stem, fruit, root) are described with their particular sink functions and hydraulic attributes. Given the rates of photosynthesis and transpiration and the hydraulic properties of the network as inputs, the model calculates the internal fluxes of water and sucrose. Xylem water potential (Psi), phloem sucrose concentration (C) and turgor pressure (P) are calculated everywhere in the network accounting for osmotic equilibrium between apoplasm and symplasm and coupled functioning of xylem and phloem. The fluxes of phloem and xylem saps are driven by the gradients of P and Psi, respectively. The fruit growth rate is assumed as turgor pressure dependent. To demonstrate its ability to address within-plant competition, the model is run with a simple-branched structure gathering three leaves, eight stem segments, three competing growing fruits and one root. The model was programmed with P-Spice, a software specifically designed for simulating electrical circuits but easily adaptable to physiology. Simulations of internal water fluxes, sucrose concentrations and fruit growth rates are given for different conditions of soil water availability and hydraulic resistances (sensitivity analysis). The discussion focuses on the potential interest of this approach in functional--structural plant models to address water stress-induced effects.

Sink feedback regulation of photosynthesis in vines: measurements and a model.

An experimental and modelling study of source-sink interactions in Vitis vinifera L., cv. Cabernet Sauvignon, rooted cuttings under non-limiting environmental conditions with a 12 h photoperiod is presented here. After 4 h, measured photosynthesis, stomatal conductance and leaf carbohydrate content reached maximum values. Over the remainder of the photoperiod, photosynthesis and stomatal conductance decreased continuously, whereas leaf carbohydrate content remained relatively constant. Because the experiment took place in a non-limiting environment, the results suggest that stomatal regulation of photosynthesis was mediated by an internal factor, possibly related to sink activity. A simple 1-source, 2-sink model was developed to examine the extent to which the data could be explained by a hypothetical sink-to-source feedback mechanism mediated by carbohydrate levels in either the mesophyll, the source phloem or the phloem of one of the two sinks. Model simulations reproduced the data well under the hypothesis of a phloem-based feedback signal, although the data were insufficient to elucidate the detailed nature of such a signal. In a sensitivity analysis, the steady-state response of photosynthesis to sink activity was explored and predictions made for the partitioning of photosynthate between the two sinks. The analysis highlights the effectiveness of a phloem-based feedback signal in regulating the balance between source and sink activities. However, other mechanisms for the observed decline in photosynthesis, such as photoinhibition, endogenous circadian rhythms or hydraulic signals in the leaf cannot be excluded. Nevertheless, it is concluded that the phloem-based feedback model developed here may provide a useful working hypothesis for incorporation into plant growth models and for further development and testing.

Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings.

During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555-1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities).

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.

Modeling Carbon Export Out of Mature Peach Leaves.

The characteristics of sorbitol and sucrose export out of mature leaves in seedlings of peach (Prunus persica L. Batsch cv GF 305) were investigated by simulating carbon fluxes through the leaf. Three treatments were employed: a control treatment and two treatments modifying leaf export, the latter using either shading or girdling. Photosynthesis and 14C partitioning into sorbitol and sucrose were measured during carbohydrate pool buildup at the beginning of the photoperiod, and the export rate of sorbitol and sucrose was modeled using a PSPICE (Simulation Program with Integrated Circuit Emphasis) simulator. The simulation allowed prediction of the resulting sorbitol and sucrose contents, which were compared to experimental carbohydrate contents. The apparent Km for sorbitol and sucrose phloem loading, estimated by carbon flux modeling, was 6.6 and 4 mol m-3, respectively. The predicted export capacity of the leaf, characterized by the estimated Vmax values for phloem loading of sorbitol and sucrose, was similar to the photosynthetic carbon flux measured under the leaf growth conditions. This export capacity was enhanced in plants in which all leaves except those studied were shaded. The mature leaf had a higher storage capacity for sorbitol than for sucrose in control plants, especially in the girdled treatment. Sucrose content appears to be tightly regulated.

Physiological Aspects of Sugar Exchange between the Gametophyte and the Sporophyte of Polytrichum formosum.

The sporophyte of bryophytes is dependent on the gametophyte for its carbon nutrition. This is especially true of the sporophytes of Polytrichum species, and it was generally thought that sucrose was the main form of sugar for long distance transport in the leptom. In Polytrichum formosum, sucrose was the main soluble sugar of the sporophyte and gametophyte tissues, and the highest concentration (about 230 mm) was found in the haustorium. In contrast, sugars collected from the vaginula apoplast were mainly hexoses, with traces of sucrose and trehalose. p-Chloromercuribenzene sulfonate, a nonpermeant inhibitor of the cell wall invertase, strongly reduced the hexose to sucrose ratio. The highest cell wall invertase activity (pH 4.5) was located in the vaginula, whereas the highest activity of a soluble invertase (pH 7.0) was found in both the vaginula and the haustorium. Glucose uptake was carrier-mediated but only weakly dependent on the external pH and the transmembrane electrical gradient, in contrast to amino acid uptake (S. Renault, C. Despeghel-Caussin, J.L. Bonnemain, S. Delrot [1989] Plant Physiol 90: 913-920). Furthermore, addition of 5 or 50 mm glucose to the incubation medium induced a marginal depolarization of the transmembrane potential difference of the transfer cells and had no effect on the pH of this medium. Glucose was converted to sucrose after its absorption into the haustorium. These results demonstrate the noncontinuity of sucrose at the gametophyte/sporophyte interface. They suggest that its conversion to glucose and fructose at this interface, and the subsequent reconversion to sucrose after hexose absorption by haustorium cells, mainly governs sugar accumulation in this latter organ.

Carbon fluxes in mature peach leaves.

The turnover and transport of sugars are described in peach (Prunus persica L. Batsch), a species exporting both sucrose and sorbitol. Apparent export rate was slower in peach leaves than in leaves of herbaceous species. Sorbitol was the major soluble end product of photosynthesis and the major soluble carbohydrate in the leaf (higher than sucrose). Carbon fluxes were described using (14)C labeling, radioactivity loss curves, and compartmental analysis during the second half of the photoperiod when chemical steady state was reached for soluble carbohydrates. The measured specific radioactivity of sucrose was typical of a primary product. The delayed decrease in specific radioactivity of sorbitol indicated that part of it was secondarily synthesized. Sucrose is proposed to be the carbon source for the delayed synthesis of sorbitol in the light. The sorbitol to sucrose ratio was higher in the petiole than in the leaf tissues. In phloem sap, obtained using stylectomy of aphids and collected from the main stem between source leaves and apex, this ratio was lower than in the petiole, suggesting a preferential sorbitol demand by sinks.

Carbon and nitrogen partitioning in peach/plum grafts.

Modifications in root-shoot relationships induced by graft incompatibility were studied in peach/plum graft combinations by means of carbohydrate and nitrogen analyses and isotope labeling. Mobilization of stored carbon, phloem transport of carbon, and mobilization, assimilation and distribution of nitrogen were studied in one compatible peach/plum graft (Prunus persica L. Batsch cv. springtime grafted on Prunus cerasifera L. Ehrh cv. myrobalan P 2032) and one incompatible graft (Prunus persica L. Batsch cv. Springtime grafted on Prunus cerasifera L. Ehrh cv. myrobalan P 18) for 89 days after grafting. Carbon and nitrogen reserves were mobilized in the rootstock in both graft combinations during the first 78 days following grafting. After that, sorbitol concentration was lower in the roots of the incompatible graft than in the roots of the compatible graft, whereas soluble sugars and starch accumulated in the peach scion of the incompatible graft. In both graft types, carbon was allocated mainly to the scion. Labeling with (13)CO(2) from 78 to 81 days after grafting showed that carbon partitioning among the different plant organs was only slightly affected by graft incompatibility. Carbohydrate concentrations provided indirect evidence that carbon transfer to the roots was hindered in the incompatible graft. Labeling with (15)NO(3) showed that nitrogen distribution and the rate of nitrogen assimilation were similar in the two graft combinations from 57 to 78 days after grafting. Nitrogen assimilation in the incompatible graft ceased 78 days after grafting, whereas it continued in the compatible graft.

Fructan chemical structure and sensitivity to an exohydrolase.

A fructan exohydrolase selective for (2----1)-linked terminal fructosyl linkages, isolated from barley (Hordeum vulgare L. cv. Morex) stems and leaf sheaths, was used to elucidate the chemical structures of several oligomeric fructans extracted from liliaceous and graminaceous species. Products released by enzymic and mild acid hydrolysis were separated by reversed-phase high-performance liquid chromatography. Gas-liquid chromatography-mass spectrometry of partially methylated alditol acetates permitted unequivocal deduction of many linkage sequences, first of the hydrolysis products and then of the original oligomers. We found that bifurcose, a tetrasaccharide formed by addition of a fructosyl unit to O-6 of the central fructose residue of 1-kestose, was a central molecule in the generation of the branched, oligomeric fructans of wheat (Triticum aestivum L. cv. Fidel). These arise by the extension of both (2----1)- and (2----6)-linked chains from the bifurcose branch-point residue. Some of the (2----6)-linked units that slowly accumulate in oligomers may arise in vivo from selective hydrolysis, by fructan exohydrolases, of (2----1)-linked terminal units at branch point residues rather than by the action of (2----6)-specific synthases. Limited hydrolysis by specific exohydrolases in vitro coupled with separation of the oligomeric products constitutes an effective approach to the sequence analysis of complex oligosaccharides.

Rate of accumulation of fructan oligomers in wheat seedlings (Triticum aestivum L.) during the early stages of chilling treatment.

The accumulation of soluble carbohydrates was studied during chilling of 2-week-old wheat (Triticum aestivum L.) seedlings. The extracts were analysed using a C18 HPLC method separating hexoses, sucrose and a wide range of oligofructan. Isomers were recognized and their content determined. The amounts of hexoses, sucrose and the trisaccharides 1-kestose and 6-kestose increased at the beginning of the treatment. The accumulation rate of 1-kestose, the trisaccharide generally considered to be the precursor of fructan, increased ten fold with some delay. Heavier fructan accumulated even later. In leaf blades, 1-3 molecular species with a degree of polymerization from 4 to 8 were detected. Five of these accounted for 90% of this pool at day 5. In leaf bases the amount of carbohydrates was lower but more species of fructan were detected. The sequence of accumulation was the same as in the blades. These results are discussed in relation to current models ot fructan synthesis.

Effects of periodic fluctuations of photon flux density on anatomical and photosynthetic characteristics of soybean leaves.

The development of soybean leaves grown at fluctuating photon flux density between 100 and 1500µM m(-2)s(-1) with a period of 160 sec were compared to leaves developed under continuous light with the same mean photon flux density. Number of epidermal cells and stomata, leaf area and specific leaf weight were not affected by the periodic fluctuation of photon flux density. Chloroplastic pigment concentration and chlorophyll fluorescence reveal some photoinhibitory effects of the high photon flux density phase. Stomatal and internal CO2 conductance and the quantum yield were not affected by the light regime. In contrast ribulose 1.5 bisphosphate carboxylase/oxygenase activity before in vitro activation by CO2 and Mg(++) was stimulated by the periodic illumination whereas the total amount of the enzyme and the internal leaf CO2 conductance remained steady. In conclusion, there was no major difference between leaves of plant grown either under a steady or under a periodic fluctuation of the photon flux density except some photoinhibitory symptoms under fluctuating illumination, and a higher in vivo level of activation of the Rubisco.

Postillumination CO2 fixation by wheat leaves.

Postillumination CO2 fixation by wheat leaves was studied following light-limited photosynthetic conditions. Dark CO2 fixation showed two phases differing by their rates of CO2 uptake and carbon metabolism. These two phases are related to preillumination light flux density. During the first 30s of darkness, assimilated CO2 was found in PGA, alanine, malate and aspartate. After 5 min of darkness, it was additionally found in phosphorylated sugars.The lack of labelling of glycolate pathway intermediates shows that the Calvin cycle cannot run in the dark.The synthesized compounds indicate that reducing power but not ATP is available after turning the light off. This observation suggests that during pre-illumination, when light strictly limits photosynthesis, ATP supply would be the first limiting factor.