Wolfgang Höll

Food reserves of Scots pine (Pinus sylvestris L.)

SummaryThe amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.

Soluble carbohydrates of Pinus sylvestris L. sapwood and heartwood

SummaryThe amounts of glucose, fructose, sucrose, arabinose/galactose, raffinose/stachyose and starch were investigated in the outer sapwood, innermost sapwood, transition zone and heartwood of four stems of Pinus sylvestris L. The samples were taken in October and the determination of the compounds was done enzymatically. It was not possible to distinguish arabinose from galactose and raffinose from stachyose. The amounts of glucose, fructose and sucrose were greatest in the outer sapwood and decreased gradually towards the innermost sapwood and the heartwood. In the outermost heartwood glucose, fructose and sucrose were only present in trace amounts. Raffinose/stachyose showed highest concentrations in the outer sapwood and decreased towards the heartwood. In contrast, the concentrations of arabinose/galactose increased towards the heartwood and the greatest amount was found in the inner heartwood. When identified by thin-layer chromatography (TLC), arabinose was found to be present in greater amounts than galactose. The amount of starch decreased markedly towards heartwood. However, the amounts of sugars in all the studied stems was very variable. The changes in the amounts of carbohydrates in the different zones of the stems and the possible relationships of these phenomena with heartwood formation are discussed.

Oxidative pentose phosphate pathway and pyridine nucleotides in relation to heartwood formation in Robinia pseudoacacia L.

Most tree species show in the inner parts of their woody axes often a dark colored zone, the heartwood. Its formation is a genetically determined, programmed cell death which is characterized by the activation of metabolic pathways which lead to the formation of phenolic heartwood extractives. In the present paper we report on the key position of the oxidative pentose phosphate pathway (OPP) for this process. The OPP plays a crucial role in anabolic processes and is involved in the interconversion and rearrangements of sugar-phosphates with the net production of NADPH. In tissues of Robinia pseudoacacia L. which are transferred to heartwood, enhanced activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are present. A consequence of these increased enzyme activities is a shift in the pyridine nucleotide pool towards NADP+NADPH at the expense of NAD+NADH. These alterations in the metabolism and the redox status probably provide precursors and reduction equivalents being required for the synthesis of heartwood phenolics. The non heartwood forming species Acer pseudoplatanus L. shows neither a radial gradient nor seasonal changes in the amounts of pyridine nucleotides across the trunkwood. The results are discussed in connection with programmed cell death, mitochondrial activity, and heartwood formation.

Respiration in the sapwood and heartwood of Robinia pseudoacacia

Abstract The respiratory activity of distinct sapwood and heartwood annual rings of the stem of Robinia pseudoacacia L. has been investigated. The oxygen uptake and the carbon dioxide release in the inner parts of the sapwood is enhanced in comparison with that in the outer parts. The heartwood rings have no measureable gas exchange.

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial profile of lipase and phospholipase activities was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and alterations in the enzymatic activities were investigated across the sapwood and heartwood. Methods employed include gaschromatographic, colorimetric and enzymatic assays. On a dry weight basis, the hydrolysis of the artificial substrate pnitrophenylpalmitate shows a maximum activity in growth ring 4; however, the assay has proved not to be specific for lipase. In contrast, lipase analyses (triacylglycerol acylhydrolase; E.C. 3.1.1.3) with an authentic substrate show activity peaks in growth rings 1 and 4. With protein as a reference the highest activity is found in growth ring 5. A similar tendency is observed for phospholipase A1 (E.C. 3.1.1.32) and phospholipase A2 (E.C. 3.1.1.4). Phospholipase C (E.C. 3.1.4.3) activity decreases towards the sapwood-heartwood boundary; negligible traces of activity are detected in the heartwood, whereas, based on the protein content, growth ring 4 yields maximal activity. Phospholipase D (3.1.4.4) exhibits the same radial pattern with regard to protein content as a reference. On a dry weight basis there is a significant increase within the sapwood area, while in the heartwood the activity drastically decreases. The enzyme activities are discussed in relation to degradative processes within the plasma membranes and the hydrolysis of reserve lipids during heartwood formation.

Free Sterols and Steryl Esters in the Trunkwood of Picea abies (L.) Karst.

Summary Free sterols and steryl esters were analyzed in discs from mature xylem in four trunks of Picea abies (L.) Karst. (77–86 years old). The wood of the individual discs was separated into a) outer sapwood, b) rest of the sapwood, c) sapwood-heartwood transition zone, d) outer heart-wood and e) innermost heartwood. After extraction of the wood with acetone, free sterols and steryl esters were separated by column chromatography and quantitated by color reaction. In all trunks analyzed the innermost heartwood contains the highest amounts of free sterols and steryl esters. Lipid-phosphorus, on the other hand, decreases from the younger to the older parts of the trunks. The heartwood of all trunks contains only trace amounts of lipid-phosphorus. The free sterols are made up of cholesterol, desmosterol, campesterol and sitosterol. Desmosterol was absent in the steryl ester fraction. Sitosterol was dominant in both free sterols and steryl esters. The percentage of cholesterol and desmosterol to the total free sterols decreased from the younger to the older trunkwood while campesterol and sitosterol showed no gradient in the trunks. In the steryl ester fraction, the percentage of cholesterol decreased, campesterol seemed to increase and sitosterol showed no gradient towards the center of the trunks. Calculated on dry weight basis, only cholesterol had the tendency to decrease from the younger to the older trunkwood zones.

Lipids and lipolytic enzymes in the trunkwood of Robinia pseudoacacia L. during heartwood formation. I. Radial distribution of lipid classes

The radial distribution of membrane and storage lipids was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and fluctuations in the amount and composition of phospholipids, free sterols, steryl esters, diand triacylglycerols, and free fatty acids were investigated across the sapwood-heartwood boundary. The individual compounds were identified and quantified by thin layer chromatography, enzymatic and colorimetric assays, and by capillary gas chromatography. Phospholipids show a significant decrease towards the boundary area, and in the heartwood only trace amounts can be detected. The same pattern is observed for free sterols in the sapwood; in the heartwood, however, they reach maximum values with increasing depth of the trunk. Steryl esters exhibit a complementary behaviour by accumulating at the periphery of the heartwood. No concentration changes are found in the total amounts of diacylglycerols and free fatty acids. In contrast, the triacylglycerol concentration declines steadily across the trunk. With regard to qualitative composition, free fatty acids and the fatty acid moieties of the esterified constituents vary in their chain length from 14 to 24 carbon atoms and have up to three double bonds. A radial gradient in the ratio saturated/unsaturated fatty acids can be observed: except for the phospholipid fraction the relative amounts of unsaturated fatty acids increase in centripetal direction. Seven phospholipids were identified: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and phosphatidic acid, which constitutes the major proportion. In the sterol group, sitosterol is the most abundant component. The composition of the esterified sterols remains constant across the trunk cross-section, whereas the relative frequencies of individual free sterols change markedly.

Lipids in the Sap- and Heartwood of Picea abies (L.) Karst.

Summary Lipids were analyzed across the cross section of a trunk of Picea abies (L.) Karst . The amount of petroleum soluble lipids decreases from 1.1 % (outer sapwood) to 0.7 % (inner heartwood), while chloroform-methanol soluble lipids increase from 2.1 % to 4.6 %. No lipid phosphorus is present in the heartwood. Neutral glycerides represent 26 % of the lipids (petroleum soluble) in the outer sapwood and 8 % in the heartwood. 67 % of the glycerides from the sapwood are triglycerides, those from the heartwood contain 50 %. The ratio of triglycerides to di- and monoglycerides is 2 : 1 in the sapwood and 1 : 1 in the heartwood. The major free fatty acids in sapwood lipids are 16 : 0, 18 : 1 and 18 : 2. Heartwood lipids contain higher amounts of 18 : 2 and 20 :0 in the free form. The proportion of fatty acids with a chain length of 12 to 18 carbon atoms is higher in the total ester fraction extracted from the heartwood than that from the sapwood. The amount of total sterols increases from outer sapwood (10 % of petroleum solubles) to the inner heartwood (22 %). The free sterols account for most of the sterol increase. With the exception of palmitoleic acid there is no pronounced difference in the proportion of fatty acids in sterol esters across the trunkwood. The results indicate, that the dead heartwood of Picea abies (L.) Karst. contains neutral lipids, and these might be part of a defense mechanism against wood destroying organisms.

Alteration of physiological parameters in needles of cloned spruce trees (Picea abies (L.) Karst.) by ozone and acid mist.

Three-year-old clonal spruce trees, kept in growth chambers, were treated with ozone and acid mist during a period of 14 months. One half of the trees were grown on an acidic sandy soil, the other half on a calcareous soil rich in carbonate. At the end of the fumigation period, carbohydrates (glucose, fructose, sucrose, raffinose, starch, glucose-1-phosphate and fructose-6-phosphate) and parameters of the energy status (ATP-, AdN-(ATP + ADP + AMP)- levels, ATP/ADP-ratios and adenylate-energy-charge-(AEC)-values) were determined in the current-years needles. The results indicate that the metabolic status of a plant tissue is not only influenced by the nature of the air pollutants. Soil factors play an important role in metabolic changes within the plant and are thus of relevance in the manifestation of damage symptoms.

Lipoprotein Lipase as a New Tool in Steryl Ester Analysis

A new method for the quantitative hydrolysis of steryl esters is described. Using lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34), steryl esters are cleaved to yield free sterols and fatty acids. In comparison to alkaline hydrolysis, the method is more gentle, more accurate and less laborious. Quantification of the hydrolysis products was done with capillary gas chromatography. The practicability of the method is demonstrated for authentic steryl esters and steryl esters as they occur in needles of Scots pine (Pinus sylvestris L.).