Some Minor Characteristics of Spectrophotometric Determination of Antioxidant System and Phenolic Metabolism Enzyme Activity in Wood Plant Tissues of Pinus sylvestris L.

INTRODUCTION: A comprehensive study of enzymes of the antioxidant system (AOS) and phenolic metabolism is an actual subject of biochemical research; changes in the activity of these enzymes can be used as a diagnostic sign. At the same time, practically little attention has been paid to describing the regularities of these enzymatic reactions. The article presents the chemical kinetics study of reactions catalyzed by superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues (Pinus sylvestris L.). The dependence of the enzyme reaction rate on the enzyme concentration and the substrate concentration is presented, and the pH-optimum for each reaction is established. BACKGROUND: Determination of AOS enzyme activity and PAL activity in woody plants has many difficulties. The chemical composition of pine trunk tissues affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics. OBJECTIVE: This study aimed at determining the AOS enzyme activity in 40-year-old Scots pine (Pinus sylvestris L.) plants growing in the Karelian (Russia) forest seed plantation. METHODS: Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4 °C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 15-min of extraction, the homogenate was centrifuged at 12000 g for 10 min (MPW-351R centrifuge, Poland). The supernatant was purified on 20 cm3 columns with Sephadex G-250. Aliquots with the highest protein amount were collected. In tissues, the protein concentration was 10-50 µg/ml. Proteins in the extracts were quantified by a Bradford assay. The enzyme activity was determined spectrophotometrically on a SpectroStar Nano plate spectrophotometer (BMG Labtech, Germany). RESULTS: Our study made it possible to modify the methods for determining the activity of superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues. The enzymatic reaction rate dependence on the enzyme concentration and the substrate concentration was determined, and pH-optimum was also noted. This methodological article also provides formulas for calculating the activities of the enzymes. CONCLUSION: We found that determining AOS enzyme activity and PAL activity in woody plants is challenging. The chemical composition of the xylem and phloem of pine affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics.

Plant-Programmed Cell Death-Associated Genes Participation in Pinus sylvestris L. Trunk Tissue Formation.

Molecular genetic markers of various PCD (programmed cell death) variants during xylo- and phloemogenesis have been identified for the first time in Scots pine under lingonberry pine forest conditions in Northwest Russia (middle taiga subzone). PCD is a genetically determined process. Gene profiles of serine and cysteine proteases (endopeptidases), endonucleases, and metacaspases families are often considered markers of the final xylogenesis stage. In the present study, we examined the gene expression profiles of the BFN (bifunctional endonuclease) family-BFN, BFN1, BFN2, BFN3, and peptidase (cysteine endopeptidase, CEP and metacaspase, MC5) in the radial row, in addition to the vascular phloem and cambium (F1), differentiating xylem (F2), sapwood (SW), and transition zone during the active cambial growth period of uneven-aged pine trees (25-, 63- and 164-cambial age (c.a.) years old). We have shown that the expression patterns of the PCD-related genes did not depend on the cambial age but were largely determined by plant tissue type. In the radial row F1-F2-SW, we studied the activities of enzymes, including sucrose in metabolism (sucrose synthase, three forms of invertase); antioxidant system (AOS) enzymes (superoxide dismutase, catalase); and peroxidase andpolyphenol oxidase, which belonged to AOS enzymes and were involved in the synthesis of phenolic components of cell walls. The activity of the enzymes indicated that the trunk tissues of pine trees had varying metabolic status. Molecular genetic PCD regulation mechanisms during xylem vascular and mechanical element formation and parenchyma cells' PCD during the formation of Scots pine heartwood were discussed.

Expression Analysis of Key Auxin Biosynthesis, Transport, and Metabolism Genes of Betula pendula with Special Emphasis on Figured Wood Formation in Karelian Birch.

Auxin status in woody plants is believed to be a critical factor for the quantity and quality of the wood formed. It has been previously demonstrated that figured wood formation in Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hämet-Ahti) is associated with a reduced auxin level and elevated sugar content in the differentiating xylem, but the molecular mechanisms of the abnormal xylogenesis remained largely unclear. We have identified genes involved in auxin biosynthesis (Yucca), polar auxin transport (PIN) and the conjugation of auxin with amino acids (GH3) and UDP-glucose (UGT84B1) in the B. pendula genome, and analysed their expression in trunk tissues of trees differing in wood structure. Almost all the investigated genes were overexpressed in Karelian birch trunks. Although Yucca genes were overexpressed, trunk tissues in areas developing figured grain had traits of an auxin-deficient phenotype. Overexpression of GH3s and UGT84B1 appears to have a greater effect on figured wood formation. Analysis of promoters of the differentially expressed genes revealed a large number of binding sites with various transcription factors associated with auxin and sugar signalling. These data agree with the hypothesis that anomalous figured wood formation in Karelian birch may be associated with the sugar induction of auxin conjugation.