As a result of the project's first year, a network of permanent sample plots was established in a latitudinal gradient (middle taiga subzone - northern taiga subzone - northern border of the northern taiga subzone). It consists of plantations of different ontogenesis stages (15-30 years, 70-80 years, 180 years ), growing in a wide range of forest growing conditions (blueberry pine forest - cowberry pine forest - lichen pine forest). A sample plots database has been prepared, including complete biogeocenosis characteristics (soil, living ground cover, forest stand). The result obtained (network of permanent sample plots) is important for future long-term monitoring studies (not only within this project).
We have studied the intensity of the heartwood formation process depending on the tree age and the cambium age within the same tree in North-West Russia (middle taiga subzone) in Scots pine in different forest conditions for the first time. We found that the lifespan of xylem parenchyma cells ranged from 10-15 years in 20-year-old plants and up to 70 years in 180-year-old trees in Scots pine in a lingonberry pine forest. Heartwood formation rate (HW) also depended on the tree age. It averaged 0.3 rings per year for 20-30-year-old trees, 70-80-year-olds - 0.4-0.5 rings per year, and 180-year-old plants - about 0.7 rings per year. Based on the data for lingonberry pine forest, we constructed models of the dependence of the number of annual rings in HW on the cambium age (SA) (√ (HW) = a * √ (CA) + b; Lg (HW) = a * Lg (CA ) + b). Validation of the models developed on randomly selected pine trees has proven successful in predicting the number of annual rings in HW. The HW formation intensity in different forest growing conditions was studied in two contrasting forest types: blueberry pine forest (a highly productive type of forest that forms on soils with a sufficiently high fertility level and a sufficient water supply) and lichen pine forest (a low-productivity type of forest on poor and dry soils). The rate of HW formation was, on average, 0.54 annual rings per year in 80-year-old trees from the blueberry pine forest. Its maximum values were observed at a trunk height of 1.5 to 4.5 m (1 annual ring per year), minimum - from 4.5 to 10.5 m (0.32 annual rings per year, on average). We found that heartwood was detected up to a height of 4.5 m in a 70-year-old lichen pine forest; heartwood in the trunks was not found in this type of forest at 7.5 m. The HW formation rate in model trees averaged 0.56 annual rings per year in a lichen pine forest. At the same time, its values were also the highest (0.68 annual rings per year) at the height of 1.5–4.5 m. Verification of the models obtained for the lingonberry pine forest showed their success in predicting the number of annual rings in HW in blueberry pine forest and lichen pine forest. Thus, we obtained that the cambial age and the number of growth rings in HW are related (for different trunk heights within the same tree, for trees of different ages, for different forest growing conditions). A critical biological conclusion follows from the data obtained that heartwood formation is, to a greater extent, determined by the number of annual rings in the radial row of the xylem, so it is inextricably linked with the cambial cell's ageing.
The HW proportion in the trunk of mature trees is an essential indicator for characterizing the wood quality and assessing the role of pine forests in carbon sequestration. Old-growth forests are critical to biodiversity conservation and global carbon sinks. Carbon absorbed from the atmosphere is deposited for a long time in living tissues of woody plants, in the litter, etc. In most studies, when calculating carbon stocks in forest ecosystems, the carbon content in wood is taken as 50%. We have shown a decrease in the proportion of cellulose and an increase in the proportion of phenolic polymers (lignin, extractives) in the radial row "sapwood - transition zone - heartwood". At the same time, plants growing in different climatic conditions did not differ among themselves in the content of the studied polymers. As a result of the accumulation of phenolic polymers in heartwood, compared to sapwood, (1) the carbon content is higher (~ 59%), (2) resistance to biodegradation (heartwood decomposition when the tree dies is a longer process). According to our data, HW reaches 50%, in 70-80-year-old plants - 30% for a lingonberry pine forest in the trunk (up to 10.5 m) in 180-year-old plants, calculations for a blueberry pine forest show that HW already reaches 45 -50% in 80-year-old plants. We believe that all of the above suggests a unique (as opposed to sapwood) participation of heartwood in long-term carbon storage, which has not been fully assessed to date. Further research in this area may allow redefining the role of old-growth boreal forests in the global carbon balance.
We studied the anatomical features of the transition zone between sapwood and heartwood in pine, depending on the tree's age in different forest growing conditions. For this, marker events detected by histochemical tests were noted on the radial sections of the transition zone, namely: (1) a change in the colour of the torus of bordered pits of the tracheids (in sapwood, tori of the bordered pits were stained with alcian blue, which indicates the presence of cellulose and pectins in their composition; in heartwood, tori were stained with safranin in a dirty pink colour due to the deposition of extractives and lignin on the pit membrane); (2) the disappearance of starch grains from the cells of the radial and axial parenchyma; (3) the disappearance of nuclei from the cells of the radial and axial parenchyma. In all studied trees, changes in the chemical composition of pit membranes (associated with the water transport cessation) and parenchyma cells death occurred within 1–2 neighbouring annual rings. In this case, the chemical properties of the pit membranes of the tracheids changed in the ring (or part of the growth ring) located closer to the trunk periphery. We observed the disappearance of nuclei closer to the centre of the trunk either in the layer of earlywood of the same growth ring or in the previous growth ring. We showed for the first time that the transition zone width in all studied trees, regardless of (1) the tree age, (2) forest growing conditions, ranged from 1 to 2 annual rings.
As a result of the project's first year, we studied the expression of PXY, WOX (WOX13, WOX4, WOXA and WOXG) genes, which are patterns of cell division in the cambial zone, as well as genes encoding endonucleases (BFN, BFN1, BFN2, BFN3) and peptidase (cysteine endopeptidase (CEP) and metacaspase (MC5)) in the radial row "conducting phloem, cambial zone - differentiating xylem" during the active cambial growth period using the example of pine trees of different ages: 30-, 70-80-, and 180-years old. We revealed a high level of relative expression of WOX family genes during the period of active cambial growth. We showed that WOX4, WOXA, and WOXG were expressed predominantly in the xylem, indicating active cell proliferation in this zone. In contrast, WOX13 showed greater expression in the phloem than in the xylem. In the differentiating xylem of the studied plants of all ages, we revealed the expression of genes involved in the PCD of cells - MC5 and CEP. The BFN genes expression encoding endonucleases, both with RNase and DNase activity, was lower than that of genes encoding peptidases. We showed that the level of BFN1 and BFN2 transcripts prevailed on the phloem side, while BFN and BFN3 expression was higher in the differentiating xylem. Obtaining new data will allow us to compare the molecular genetic mechanisms of PCD regulation during the formation of conductive and mechanical elements of the xylem and PCD of parenchyma cells during the heartwood formation in Scots pine.