An original modi®cation of an analytical procedure has been developed to obtain puri®ed extracts for sugar measurement in different parts of ligneous plants by high-performance liquid chromatography. Extraction was performed at 4°C in the presence of water, methanol and chloroform. Only a representative part of the alcohol extract (methanol/water) was puri®ed for analysis. An internal marker such as maltotriose, added during extraction, makes it possible to estimate possible sugar loss and to correct concentrations. Compared with a more standard method based on total soluble sugar extraction, this new method is more precise. The use of internal calibration standards shows good linear progression but does not guarantee good accuracy in the absence of a reference method or plant powder standard. It is a robust procedure which can tolerate experimental variations without affecting the results. The high recovery rate (90%) of maltotriose proves the quality of the procedure and makes it possible to correct sugar concentrations to acceptable limits.
An enzymatic assay method in 96-well microplates (MP method) is proposed for non-structural carbohydrates (NSC) in ligneous plants. Soluble sugars were extracted from 50 mg of finely ground plant powder, in the presence of water, methanol and chloroform. The glucose, fructose and sucrose contents were successively determined in each well, although sorbitol could not be assayed under these conditions. Whatever the sugar, the precision (reproducibility), linearity (addition of specific amounts of sugars) and accuracy (comparison with method using high-performance liquid chromatography (HPLC)) were excellent. This method was more specific than with HPLC, so that the recovery rate of sugars was improved. In the absence of a significant matrix effect, purification of the extract was unnecessary, thus simplifying the procedure and contributing to its robustness. A micro-method is thus proposed which can be applied to 5 mg of plant powder. This miniaturization affects neither the precision of the MP method nor sugar concentrations. The results of starch assays further demonstrated that this micro-method was appropriate to the analysis of NSC in small samples of woody plant tissues. Reliable, rapid and simple to perform, this micro-method is less expensive than HPLC or other classic enzymatic methods.
In nature, plants are often exposed to a multitude of environmental constraints that severely limit crop productivity. Water deficit is one of the factors that most affects agricultural production. The aim of this work is to evaluate the effect of water deficit on morphology, development, nutritional behavior, as well as chlorophyll fluorescence and certain important metabolic parameters (soluble sugars, organic acids, starch, carotenoid, and vitamin C) of the cultivated tomato (Solanum lycopersicum cv Plovdiv). In this study, the water supply was reduced by 60% compared to control conditions. The conditions of water deficit showed that the size of the different organs (leaves, fruits) was reduced. A reduction in the number, width, and length of the leaves, respectively, 9%, 36%, and 37%, then the leaf surface was also observed. Reduction of fluorescence (Fo, Fm, and Fv) and total index performance were among the other symptoms of plants with water deficiency. For fruit, we observed a significant decrease in diameter, fresh weight, and moisture content during the cell division period, the cell expansion period, and the fruit ripening period. In contrast, the composition of the Plovdiv fruit changed only during cell division and expansion phase. On the other hand, the water deficit induces an increase in the total carotenoid and vitamin C content of the fruits.. Besides, water deficit induced a reduction of fruit size, moisture content, and production dry matter during different phases of development. Decrease levels of soluble sugars and organic acid but increase in vitamin C and carotenoid content.
Ligneous plants offer a wide variety of matrices that interfere with the assay of starch, which is present at low levels which vary considerably depending on the tissues considered and the phenological stage. Because no reference technique is available, a critical study was developed to validate a method for the enzymatic assay of starch. Analyses were performed on 50 mg samples of plant powder obtained from different parts of peach trees. After the total elimination of soluble components (including glucose) in a water/methanol/chloroform mixture, the starch was extracted from the ligneous residue and solubilised either by autoclaving in an aqueous medium or using 4 M potassium hydroxide (KOH) solution (dispersion stage). After hydrolysis with amyloglucosidase, the starch content was determined indirectly by the enzymatic assay of released sugar. The precision (repeatability and reproducibility), linearity (addition of specified amounts of starch) and accuracy (high rate of starch recovery, 95%) of the method were excellent, whichever dispersion method was employed. The highly significant analogy of the results obtained using such diverse dispersion techniques (autoclaving and dissolution in KOH) was indicative of their specificity and efficacy. The reliability of the enzymatic stages was generally excellent. However, some plant matrices may, during dispersion, release soluble compounds which interfere with amyloglucosidase activity, and this may explain the incomplete recovery of starch. This moderate and unpredictable interference was, in principle, the only critical point concerning the method for the enzymatic assay of starch in ligneous plants with very low starch content, the reliability of which was clearly demonstrated.
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