WUSCHEL (WUS)-related homeobox (WOX) protein family members play important roles in the maintenance and proliferation of the stem cells in the cambium, the lateral meristem that forms all the wood structural elements. Most studies have examined the function of these genes in angiosperms, and very little was known about coniferous trees. Pine is one of the most critical forest-forming conifers globally, and in this research, we studied the distribution of WOX4, WOX13, and WOXG genes expression in Pinus sylvestris L. trunk tissues. Further, we considered the role of TDIF(CLE41/44)/TDR(PXY) signaling in regulating Scots pine cambial activity. The distribution of CLE41/44-PXY-WOXs gene expression in Scots pine trunk tissues was studied: (1) depending on the stage of ontogenesis (the first group of objects); and (2) depending on the stage of cambial growth (the second group of objects). The first group of objects is lingonberry pine forests of different ages (30-, 80-, and 180-year-old stands) in the middle taiga subzone. At the time of selection, all the trees of the studied groups were at the same seasonal stage of development: the formation of late phloem and early xylem was occurring in the trunk. The second group of objects is 40-year-old pine trees that were selected growing in the forest seed orchard. We took the trunk tissue samples on 27 May 2022, 21 June 2022, and 21 July 2022. We have indicated the spatial separation expressed of PsCLE41/44 and PsPXY in pine trunk tissues. PsCLE41/44 was differentially expressed in Fraction 1, including phloem cells and cambial zone. Maximum expression of the PsPXY gene occurred in Fraction 2, including differentiating xylem cells. The maximum expression of the PsCLE41/44 gene occurred on 27 May, when the number of cells in the cambial zone was the highest, and then it decreased to almost zero. The PsPXY gene transcript level increased from May to the end of July. We found that the highest transcript level of the PsWOX4 gene was during the period of active cell proliferation in the cambial zone, and also in the trees with the cambial age 63 years, which were characterized by the largest number of cell layers in the cambial zone. In this study, we have examined the expression profiles of genes belonging to the ancient clade (PsWOXG and PsWOX13) in stem tissues in Scots pine for the first time. We found that, in contrast to PsWOX4 (high expression that was observed during the period of active formation of early tracheids), the expression of genes of the ancient clade of the WOX genes was observed during the period of decreased cambial activity in the second half of the growing season. We found that PsWOX13 expression was shifted to Fraction 1 in most cases and increased from the phloem side, while PsWOXG expression was not clearly bound to a certain fraction. Based on the data, the role of the CLE41/44-PXY-WOX signaling module in regulating P. sylvestris cambial growth is discussed.

The processes of cambial activity and secondary xylem and phloem differentiation are completely dependent on the influx of photoassimates. Trunk girdling is a frequently used method for studying cambial growth under conditions of different assimilate supply. We girdled 20-year-old birch trees (Betula pendula Roth) and took samples 1 cm (AG1) and 35 cm above the girdle (AG35). Tissues of ungirdled trees served as a control. A sharp increase in carbohydrates level (AG1) inhibited xylogenesis and stimulated phloemogenesis. A moderate increase (AG35) also stimulated phloemogenesis; however, xylogenesis continued. The activity of the APL gene encoding a phloem-specific transcription factor correlates with the active phloemogenesis, as it was 2.18 (AG1) and 3 (AG35) times higher than in the control. The SUC gene encoding the transmembrane sucrose transporter was up-regulated in the AG1 and AG35 zones by 2.24 and 2.51 times, respectively, compared with the control, which indicates an active sucrose loads into the cells and correlates with the preferential differentiation of parenchyma. The activity of the PIN1 gene encoding the auxin transporter was highest in zone AG35 (2.1 times higher than in the control). In zone AG1, the PIN1 activity was 1.7 times lower than that of AG35, which corresponds to the impaired differentiation of the phloem sieve tubes. The data obtained can be useful for a better understanding of physiological processes and predicting changes in the forest’s productivity under conditions of a changing climate.

Summary: Girdling is often used as an experimental method to study source/sink controls of cambial growth. However, the phloem responses to girdling have not been well investigated. The aim of this research was to characterize the anatomical changes in the phloem and xylem of different species following trunk girdling. Different species of gymnosperm (Abies sibirica, Pinus sylvestris) and angiosperm (Alnus incana, Populus tremula) woody plants were selected. We girdled trunks during active growth and sampled tissues at two levels (1 cm and 35 cm) above the girdle at the end of the growing season. General responses to girdling were recorded for the studied species, such as increased phloem increments, parenchymatization of conducting tissues, reduction in the size of conducting elements, and an increase in the size of axial parenchyma cells in the phloem and xylem. We observed the suppression of xylogenesis in 3 out of 4 species. Differences in the structure of conducting tissues were found, which are due to species differences in the initial tissue structure. In gymnosperms, noticeable differences in the number of resin ducts in the xylem were observed between control and girdled trees. In angiosperms, we found the formation of cells with thickened cell walls in girdled trees (i.e., the formation of phloem and xylem fibers with thickened cell walls in aspen and sclereids in the phloem of alder). Based on the literature data, the observed responses may be due to both the wounding effect and the influence of the high sugar content above the girdle.

The balance between cell proliferation and differentiation into other cell types is crucial for meristem indeterminacy, and both growth aspects are under genetic control. The peptide-receptor signaling module regulates the activity of the cambial stem cells and the differentiation of their derivatives, along with cytokinins and auxin. We identified the genes encoding the signaling module CLE41-PXY and the regulator of vascular cambium division WOX4 and studied their expression during the period of cambial growth in the radial row: the conducting phloem/cambial zone and the differentiating xylem in two forms of Betula pendula, silver birch and Karelian birch. We have shown that the expression maximum of the BpCLE41/44a gene precedes the expression maximum of the BpPXY gene. Non-figured Karelian birch plants with straight-grained wood are characterized by a more intensive growth and the high expression of CLE41/44-PXY-WOX4. Figured Karelian birch plants, where the disturbed ratio and spatial orientation of structural elements characterizes the wood, have high levels of BpWOX4 expression and a decrease in xylem growth as well as the formation of xylem with a lower vessel density. The mutual influences of CLE41-PXY signaling and auxin signaling on WOX4 gene activity and the proliferation of cambium stem cells are discussed.

Silver birch (Betula pendula Roth) is an economically important species in Northern Europe. The current research focused on the molecular background of different xylogenesis scenarios in the birch trunks. The study objects were two forms of silver birch, silver birch trees, and Karelian birch trees; the latter form is characterized by the formation of two types of wood, non-figured (straight-grained) and figured, respectively, while it is currently not clear which factors cause this difference. We identified VND/NST/SND genes that regulate secondary cell wall biosynthesis in the birch genome and revealed differences in their expression in association with the formation of xylem with different ratios of structural elements. High expression levels of BpVND7 accompanied differentiation of the type of xylem which is characteristic of the species. At the same time, the appearance of figured wood was accompanied by the low expression levels of the VND genes and increased levels of expression of NST and SND genes. We identified BpARF5 as a crucial regulator of auxin-dependent vascular patterning and its direct target—BpHB8. A decrease in the BpARF5 level expression in differentiating xylem was a specific characteristic of both Karelian birch with figured and non-figured wood. Decreased BpARF5 level expression in non-figured trees accompanied by decreased BpHB8 and VND/NST/SND expression levels compared to figured Karelian birch trees. According to the results obtained, we suggested silver birch forms differing in wood anatomy as valuable objects in studying the regulation of xylogenesis.