Elisabeth Magel

Formation of heartwood substances in the stemwood of Robinia pseudoacacia L. II. Distribution of nonstructural carbohydrates and wood extractives across the trunk

SummaryThe distributions of reserve carbohydrates and of three dominant heartwood extractives were determined in the trunkwood of Robinia pseudoacacia L. The trees were cut at different times of the year (September, November, January, and April). With the exception of the tree felled in January, all trunks exhibited highest contents of nonstructural storage carbohydrates (glucose, fructose, sucrose, and starch) in the youngest, outermost sapwood zone. With increasing depth of the trunk, the levels of carbohydrates decreased. At the sapwood-heartwood transition zone, only trace amounts of nonstructural carbohydrates were present. The heartwood itself contained no storage material. The wood zones of different ages of the trees cut in September, November, and January exhibited glucose/fructose ratios of approximately 1. In April, however, there was a shift to glucose. In the youngest sapwood the amounts of soluble sugars were higher in the earlythan in the latewood. Older zones of the sapwood and the sap-wood-heartwood transition zone showed the opposite behaviour. Three main wood extractives of Robinia were characterized and quantified: the flavanonol dihydrorobinetin (DHR), the flavonol robinetin (ROB) and a hydroxycinnamic acid derivative (HCA). Only DHR was present — in very low amounts — in the younger sapwood of all trunks investigated. Higher amounts (>1 μmol/g dry weight) of this compound and the HCA were present in the sapwood-heartwood transition zone. DHR augmented within the heartwood up to a more or less constant level. HCA increased towards the heartwood and decreased again in the inner heartwood parts. ROB appeared in the innermost parts of the sapwood-heartwood transition zone and reached maximum values in older parts of the heart-wood. The results indicate that starch is hydrolyzed at the sapwood-heartwood boundary and thus represents a primary major source of hydroxycinnamic acid and flavonoid synthesis.

Extractable activities and protein content of sucrose-phosphate synthase, sucrose synthase and neutral invertase in trunk tissues of Robinia pseudoacacia L. are related to cambial wood production and heartwood formation

Abstract. The presence of sucrose synthesizing and degrading enzymes and the correlation of their enzyme activity with cambial growth and heartwood formation are demonstrated in trunks of Robinia pseudoacacia L., black locust. Sucrose is formed by sucrose-phosphate synthase (SPS; EC 2.4.1.14), predominantly in the storage part of the sapwood. In the cambial differentiation zone and the sapwood-heartwood transition zone, both of which constitute carbohydrate sinks, sucrose is primarily cleaved by sucrose synthase (SuSy; EC 2.4.1.13) and a neutral invertase (NI; EC 3.2.1.26). In spring, enhanced activities of SuSy and NI were found in the differentiating xylem tissues. This coincided with elevated SPS rates at the sites of starch mobilization. Heartwood formation in autumn, a period of intense accumulation of phenolics in the innermost living wood tissues, was accompanied by high activities of SuSy and NI. Increased SPS and NI activities in all tissues of winter samples could be correlated with cold acclimation. Probing of SPS and SuSy protein from black locust with heterologous antibodies revealed a subunit size of 130u2009kDa for SPS and of 89u2009kDa for SuSy. Both SPS and SuSy exhibited a linear correlation between catalytic activity and amount of enzyme protein with respect to the radial profile from bark to inner core and with respect to the seasonal course. The highest amounts of SuSy-specific mRNA were detected in differentiating xylem in summer and the sapwood-heartwood transition zone in autumn. These data are taken as evidence for a pivotal role of SuSy in supplying carbon skeletons for the biosynthesis of secondary substances in woody axes.

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.

Carbohydrates in trees

Nonstructural carbohydrates are important back bones of life strategies of long-living trees. In conifers and broadleaf trees, sucrose, glucose, and fructose constitute the dominating soluble, starch the pivotal non-soluble storage carbohydrates. Sucrose is the preferred transport sugar. In addition, species-specific sugar alcohols can be found. Like in herbaceous plants, photoassimilates are allocated from green (source) tissues to nongreen (sink) areas. In addition, leaves of evergreen trees do not only deliver carbohydrates but can also serve as storage organs. Woody axes (branches and the trunk) are not only involved in long distance transport (bark) of carbon. The living wood tissues (sapwood) and the bark are also the major storage compartments of carbon. During heartwood formation, carbohydrates sustain the formation of phenolic extractives, components which ascribe for the natural durability of wood. As roots of most trees are part of plant microbe interactions (mycorrhiza), their carbohydrate status and the role of sugars in this interaction is also of importance. Allocation and partitioning of carbohydrates between and within the individual organs of a tree depend on ontogenetic (eg stage of organ maturation) and environmental (eg mineral nutrition, toxic gases, climate, pathogenic and symbiotic interactions) impacts.

Differences in genomic DNA extracted from bark and from wood of different zones in Robinia trees using RAPD-PCR

Abstractu2002Determination of genetic differences and levels of gene expression in mature and old tissues (e.g. wood) is often difficult based on morphological and anatomical characteristics, levels of metabolites or enzymatic activity. The use of molecular markers allows assessment of polymorphic (genetic) variation amongst individuals and between closely related species directly at the DNA level, but such techniques have not been generally applied to the bark and wood of mature trees. In this study we have applied the technique of random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR) to analyse the relationship between the bark and variously aged wood zones of Robiniapseudoacacia. The use of micro polyacrylamide gel electrophoresis coupled to silver staining for DNA provided a quick, reliable and sensitive method of detecting polymorphisms. It was necessary to test a small number of ten-base synthetic oligonucleotide primers before arriving at a set of five which clearly identified post-transcriptional differences between bark, sapwood, transition zone and heartwood even in the one individual tree. The variability of the technique, and in particular the origin and quality of the DNA extracted was analysed. We demonstrated that the procedures and protocols developed are applicable to all tissue types tested from bark to the inner heartwood zones. Our results show that RAPD-PCR technology is a versatile and sensitive method of detecting genomic changes in trees.

Pyridine nucleotide levels and activities of dehydrogenases in cambial derivatives ofRobinia pseudoacacia L.

Despite the importance of the vascular cambial differentiation, little is known about its regulation. In order to address this problem we attempted to biochemically characterize differentiating xylem and phloem elements during the early stages of development. By applying techniques of quantitative histochemistry we show that the total pool size of pyridine nucleotides is similar in the phloem (PD) and xylem (XD) oriented derivatives of the cambial zone of trees ofRobinia pseudoacacia L. Within the PD zone, the amount of NAD + NADH exceeded that of NADP + NADPH [around 600 versus 200 pmol (mg dry weight)-1], possibly indicative of a preponderance of catabolic pathways (ratio of NADH∶NAD about 1). In contrast, the NADP(H) system dominated in the XD zone. This coincided with a high activity of NAD kinase. In addition, the extractable activities of the key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were greatly increased. At a ratio of NADPH∶NADP of approximately 1, this could be indicative of increased rates of reductive biosyntheses, and could thus well be involved in early steps of the formation of phenols and lignin monomers. Taken together, this first approach clearly shows that phloem-oriented and xylem-oriented cambial descendents exhibit distinct differences in their biochemical patterns even in early stages of differentiation.