Cambial Age Influences PCD Gene Expression during Xylem Development and Heartwood Formation.

Heartwood formation is an important ontogenetic stage in Scots pine (Pinus sylvestris L.). The amount of heartwood determines the proportion of functionally active sapwood in the total trunk biomass as well as the quality of wood. The key criterion for heartwood formation is the death of xylem ray parenchyma cells. Previously, models that described the patterns of heartwood formation, depending on the cambial age, were derived from Scots pine trees of different ages. The cambial age is the number of annual xylem layers at the core sampling site at a certain trunk height. We studied the features of the occurrence of programmed cell death (PCD) processes during the xylem differentiation and heartwood formation of 80-year-old Scots pine trees, depending on the cambial age, under the lingonberry pine forest conditions in the transition area of the northern taiga subzone and tundra. We have shown that the distance from the cambial zone to the heartwood boundary does not change significantly with stem height. As the cambial age increases, the lifespan of the formed xylem ray parenchyma cells increases and the activity of PCD genes decreases during the formation of both (1) xylem (in the outer layers of sapwood) and (2) heartwood (in the inner layers of sapwood and transition zone). We hypothesized that the decisive factor in the PCD initiation during heartwood formation is the distance of the xylem ray parenchyma cells from the cambial zone. The younger cambium forms wider annual increments, and therefore the xylem ray parenchyma cells in these parts of the trunk reach the distance from the cambial zone earlier, which is necessary for PCD initiation.

Identification and Expression Profile of CLE41/44-PXY-WOX Genes in Adult Trees Pinus sylvestris L. Trunk Tissues during Cambial Activity.

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.

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.

Changes in the Activity of the CLE41/PXY/WOX Signaling Pathway in the Birch Cambial Zone under Different Xylogenesis Patterns.

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.

Molecular Genetic Characteristics of Different Scenarios of Xylogenesis on the Example of Two Forms of Silver Birch Differing in the Ratio of Structural Elements in the Xylem.

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.

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.