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Article

Changes in the Differentiation Program of Birch Cambial Derivatives following Trunk Girdling

by
Aleksandra A. Serkova
*,
Tatiana V. Tarelkina
,
Natalia A. Galibina
,
Kseniya M. Nikerova
,
Yulia L. Moshchenskaya
,
Irina N. Sofronova
,
Nadezhda N. Nikolaeva
,
Diana S. Ivanova
,
Ludmila I. Semenova
and
Ludmila L. Novitskaya
†
Forest Research Institute of the Karelian Research Centre of the Russian Academy of Sciences, 11 Pushkinskaya St., 185910 Petrozavodsk, Russia
*
Author to whom correspondence should be addressed.
†
Deceased.
Forests 2022, 13(8), 1171; https://doi.org/10.3390/f13081171
Submission received: 28 June 2022 / Revised: 19 July 2022 / Accepted: 20 July 2022 / Published: 23 July 2022
(This article belongs to the Special Issue Intrinsic Regulation of Diameter Growth in Woody Plants)
Browse Figures
Review Reports Versions Notes

Abstract

:
The mechanisms regulating the tree trunk radial growth can be studied in original experiments. One technique for studying cambium activity (the meristem involved in radial growth) under conditions of an increased photoassimilate level is trunk girdling. We girdled the trunks of 17- to 22-year-old silver birch plants (Betula pendula Roth var. pendula) during the active growth period and collected xylem and phloem samples at two height levels (1 cm and 35 cm) above girdle, 10, 20, and 30 days after girdling. We investigated the changes that occurred at the anatomical level, as well as the activities of sucrose-metabolizing enzymes and antioxidant-system enzymes and the expression of genes that encode proteins involved in sucrose and auxin transport and metabolism. A moderate increase in photoassimilates (35 cm above the girdle) resulted in a change in the ratio of phloem to xylem increments and an increase in the proportion of parenchyma in the conducting tissues. The increase of photoassimilates above the level at which they can be used in the processes of normal tissue growth and development (1 cm above the girdle) led to xylogenesis suppression and the stimulation of phloem formation, a significant increase in the parenchyma proportion in the conducting tissues, and formation of large sclereid complexes. The differentiation of parenchyma and sclereid cells coincided with biochemical and molecular markers of abnormal conducting tissue formation in Karelian birch, which are also characterized by high proportions of parenchyma and sclereid near the cambium. The results obtained are important in understanding the cambium responses to the photoassimilate distribution changes and estimating tree productivity and survival under changing environmental conditions.

1. Introduction

Diameter growth of woody plants in temperate climatic zones occurs mainly because of cambium formation of secondary conducting trunk tissues—xylem (wood) and phloem. A constant sugar supply is necessary to maintain cell divisions in the cambial zone [1,2,3,4,5]. The strong dependence of cell growth processes, division, and differentiation on the availability of photoassimilate provides a basis for considering the transport of sugars as the main physiological process in the cambial zone [2]. The supply of sugar from leaves (source) to acceptor organs (sinks) occurs through the sieve elements of conducting phloem, and the priority of one or another organ in the carbohydrate allocation changes in both tree ontogenesis and the vegetation period [6,7]. During the growth period, the active acceptors of photoassimilates, apart from the cambium, are the growing leaves, the shoots, and the root system. In mature trees, the trunk cambium as an assimilate acceptor is considered to have a lower priority, as compared with roots under stress [8,9,10,11].
Understanding cambium responses to changes in source–sink relations is important for calculating the productivity and planning of tree death risks under changing environmental conditions [12,13,14]. One of the widespread methods for manipulating source–sink relations and photosynthate distribution is trunk girdling (see [13,15,16,17] and the references therein). During this procedure, the conducting phloem is physically removed, thereby blocking the downward transport of assimilates to the roots, removing the resource limitations for the cells of the cambial zone, and differentiating the conducting tissues above the girdle. Because the creation of genetically transformed trees is challenging, girdling is one of the easiest and most accessible methods for studying the responses of intact tree cambium to increases in photoassimilate [13,18,19,20].
The authors studied the effect of the phloem transport blockade on radial growth in different species of coniferous and angiosperm trees [13,15,16]. However, no definite reaction of cambium to the increased level of photoassimilates was observed. Some species (Acer rubrum, A. saccharum, Populus grandidentata [21], Liriodendron tulipifera [22], Prunus persica [23], Quercus robur [24], Juglans nigra × J. regia [25], Pinus contorta, P. rigida, Pseudotsuga menziesii, Tsuga canadensis [26], P. canariensis [27], and P. taeda [28]) were characterized by more active radial growth above the girdle, compared with the control. In other species (Q. petraea [29], P. deltoides × nigra [30], Q. rubra [21] Fraxinus pennsylvanica [31], Juniperus virginiana [26]) or no difference from the control conditions (Fagus grandifolia [21], Celtis occidentalis [31], and Tilia cordata [31]), a decrease in diameter growth was observed. Different radial growth reactions were observed even within the same species in different cultivars of Malus domestica [32]. When considering cambial responses to girdling, previous researchers paid the greatest attention to the xylogenesis process, while reporting much less often on the changes that occurred during phloem formation [33,34,35]. Very few works have examined the responses of enzyme systems and gene expression in the trunk tissues [27,36,37,38]. Thus, our understanding of how the cambial zone responds to increasing levels of photoassimilates at the anatomical, biochemical, and molecular levels is still very tenuous.
Our research was carried out in the territory of the Republic of Karelia, northwestern Russia. The study of cambium responses to changes in available assimilate levels is relevant to this region, because the territory’s variable climatic conditions during the growing season may lead to phloem transport restrictions on the ongoing photosynthesis background of the trees [39]. We conducted an experiment on the trunk girdling of Betula pendula Roth (silver birch), one of the economically and ecologically important tree species in northern Europe. Our further reasons for selecting this species were that the methods for determining enzyme activity have been previously adapted for silver birch [40,41,42,43] and the silver birch genome was sequenced in 2017 [44]. We studied the changes that occurred in the xylem and phloem structure and the activities of sucrose-degrading enzymes and antioxidant-system (AOS) enzymes, as well as the expression of genes involved in metabolism and the transport of sugars and auxin.
It is known that the zone immediately above the girdle is where sugars are most accumulated [24]. A prolonged disturbance of phloem transport leads to a further increase of sugar levels above the trunks and in the crowns of girdled trees [36,45]. Therefore, samples were taken at two heights above the girdle. A comparative analysis of tissues at different distances from the girdle, at different time points after girdling, allowed us to study the mechanisms of excess sugar utilization in the birch cambial zone at different levels of photoassimilate excess. Based on previous investigations on Karelian birch (a form of silver birch with an abnormal structure of conductive tissues) [46,47], we formulated the following working hypothesis: that an increase in available sugar levels in the cambial zone of silver birch with non-figured wood will lead to an intensive entry of hexoses into the cells, which in turn stimulates the differentiation of cambial derivatives into parenchyma cells.

2. Materials and Methods

2.1. Plant Selection and Sampling

Thirty-two trees of Betula pendula Roth were selected for the experiment, each growing under the same soil and climatic conditions at the experimental plot of the Forest Institute of the Karelian Research Centre of the Russian Academy of Science, which is 2 km south of Petrozavodsk. Trees of similar age (19 to 22 years), height (10 ± 0.5 m), and trunk diameter at 1.3 m (10 ± 0.2 cm) were selected. All trees had a well-developed crown and no visible signs of damage. On 19 June 2017, we performed girdling on the trunks of 20 of the birch trees. At heights of 1.3 m, 5 cm-wide girdles of bark tissues, up to the zone of forming xylem, were removed with a sharp knife. We marked 12 trees as control, and made shallow punctures of bark tissues on their trunks to mark the beginning of the experiment.
Trunk tissues were sampled at two levels, 1 cm and 35 cm above the girdles (levels AG1 and AG35, respectively). In the control trees, we took samples at heights that corresponded to the levels of AG1 and AG35 in the girdled trees.
Ten, 20, and 30 days after girdling (on 29 June 2017, 10 July 2017, and 20 July 2017, here in after referred to as 10DAG, 20DAG, and 30DAG), and after the growing season in October, we took samples for microscopic analysis and relative water content of tissues. On each date, samples were taken from five girdled and three control trees. For each sample, we selected eight closely growing trees so that they were in similar conditions. At the same time, we decided to include more trees within the experimental group to account for individual reactions. On each date, samples were taken from eight other trees, because during the sampling process the trees were subjected to serious injuries. Bark and wood samples were taken from deep notches 1 cm high and 1 cm wide. After the vegetation period, samples for wood maceration, were taken from the parts of the wood that were closer to the barrier zones.
Samples for biochemical and molecular genetic analysis were taken at 10DAG and 20DAG. A 5 cm-high bark girdle was cut out around the circumference of the tree at corresponding levels. A thin tissue layer containing the cambial zone, conducting phloem, and a thin layer of non-conducting phloem were scraped off the side of the bark with a razor. Because phloem cells dominated this fraction, it is referred to as phloem. On the wood side, the tissues containing the zone of xylem cell expansion and differentiation were similarly co-sculpted. We took samples under microscopic control. Because the volume of the tissues for biochemical and molecular genetic studies was too small, materials from three control trees and from five girdled trees were combined into two samples, control and girdled. The tissues were frozen in liquid nitrogen immediately after sampling.

2.2. Microscopic Analysis

Bark and wood samples were fixed in glutaric aldehyde, dehydrated in a series of alcohols of increasing concentrations, and embedded in an Araldite-Epon-812 mixture according to a well-known technique [48]. Panoramic transverse sections with areas of 7 mm2 to 10 mm2 and thicknesses of 2 μm were formed using Ultrotome IV (LKB, Bromma, Sweden) and stained with a 1% aqueous safranin solution. Permanent slides were made using the synthetic mounting medium Vitrogel (BioVitrum, St. Petersburg, Russia).
For maceration, we immersed small pieces of fresh birch wood from the AG35 level in a solution of one part 30% hydrogen peroxide, four parts distilled water, and five parts glacial acetic acid, and placed the solution in an oven at 60 °C for about 24 h. The samples were then taken out and washed until the smell of acid was gone. The cells were stained with safranin and alcian blue 1% aqueous solutions. Temporary slides were made using glycerol as a mounting medium.
Studies of permanent and temporary slides were performed using an AxioImager A1 light microscope (Carl Zeiss, Jena, Germany) with an ADF Pro camera (ADF Optics, Wuhan, China) and ImageJ software v.1.53e (NIH, Bethesda, MD, USA). The recommendations guided the measurements [49,50,51].
The studied parameters and the peculiarities of their determination are stated below:
(1)
Phloem and xylem increments that formed after the girdling were measured on transverse sections in 6-fold replications for each sample. The late phloem in birch is clearly distinguishable from the early phloem, because the sieve tubes have a smaller diameter therein [52].
(2)
The ratio of different cell types in the phloem and xylem increments was determined using the grid method [53]. A grid of points spaced 50 µm apart in the horizontal and vertical directions was superimposed on the image of the transverse section. A minimum of 300 points for each sample were analyzed. The percentage ratio of each cell type was estimated as a number of points of a particular type divided by the total number of points analyzed. The ratio of the cell types in the xylem was determined only in the AG35 level. We calculated the proportion of elements in the part closest to the barrier zone in order to compare the anatomical changes that occurred in the xylem with the level of gene expression, the samples for determination of which were taken at 10DAG and 20DAG.
(3)
The length and width of the xylem vessels were measured on macerated material in 30-fold replications for each sample.

2.3. Relative Water Content Determination

Bark and wood pieces were weighed immediately after sampling, then dried at 105 °C for at least 3 days and weighed again. The relative water content was calculated by applying the following formula:
RWC = (FW − DW)/DW × 100%,
where RWC is the relative water content, FW is the fresh weight of the sample, and DW is the dry weight of the sample.

2.4. Enzyme Activity Analysis

To determine the activity of the enzymes, plant tissues were ground to powder in liquid nitrogen and homogenized at 4 °C in the following buffer: 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 20 min of extraction, the homogenate was centrifuged at 10,000× g for 20 min (MPW-351R centrifuge, Warsaw, Poland). The residue was rinsed three times with a washing buffer. The residue and the combined supernatant were dialyzed at 4 °C for 18 to 20 h against a 10× diluted homogenizing buffer. The residue was assayed for cell wall invertase (CWInv), and the supernatant for vacuolar (VacInv), cytosolic (CytInv) invertases, sucrose synthase (SuSy), and antioxidant-system (AOS) enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO)). Protein content was determined using a Bradford assay. Enzymatic activity was determined spectrophotometrically (Spectrophotometer SF-2000, OKBSpectr, St. Petersburg, Russia).
The activity of acid (CWInv and VacInv) invertases (Inv, EC 3.2.1.26) was determined in the incubation medium containing 50 mM acetate buffer (pH 4.7) and 25 mM sucrose. The glucose amount formed during incubation was determined by the glucose oxidation technique (Glukoza-Agat kit, Moscow, Russia). The CWInv activity was determined after incubation at 30 °C for 30 min and expressed in μmol of degraded sucrose per g fresh weight (μmol sucrose/g FW). The activities of CytInv and SuSy (EC 2.4.1.13) were determined by the amount of fructose formed, as described in [54]. The incubation medium CytInv activity determination contained 50 mM Hepes (pH 7.5) and 100 mM sucrose. The incubation medium for SuSy activity determination contained 50 mM Hepes (pH 7.5), 100 mM sucrose, and 1 mM uridine diphosphate. Incubation was at 30 °C for 30 min. Fructose that formed in the process of incubation was determined in the reaction with 2,3,5-triphenyltetrazolium chloride (TTC) by increasing the optical density at λ = 495 nm; its quantity was calculated according to the preconstructed calibration. SuSy and Inv activities were expressed in μmol of degraded sucrose per mg of protein (μmol sucrose/mg protein).
The activity of superoxide dismutase (SOD, EC 1.15.1.1) was determined by the photoreduction inhibition of nitro blue tetrazolium (NBT). The incubation medium for determining the activity of SOD contained 50 mm K, Na-phosphate buffer (pH 7.8), 172 µm NBT, 210 µm methionine, 24 µm riboflavin, and 0.1% Triton X-100. To determine the activity of SOD, the decrease in the optical density at 560 nm after 30 min of incubation under the light of fluorescent lamps was measured. The SOD activity was expressed in conventional units per mg of protein per 30 min (U/mg protein) [42,55].
The enzymatic decomposition of hydrogen peroxide determined the catalase activity (CAT, EC 1.11.1.6). The incubation medium contained 50 mm K, Na-phosphate buffer (pH 7.8), and 14.7 mm hydrogen peroxide. The incubation time was 4 min. To determine the activity of CAT, the decrease in the optical density at 240 nm was measured; the H2O2 content was calculated according to a preconstructed calibration. The CAT activity was expressed in µmol of reduced hydrogen peroxide per mg of protein per 4 min (µmol H2O2/mg protein) [43,55].
To determine the peroxidase activity (POD, EC 1.11.1.7), guaiacol was used as the hydrogen donor. Hydrogen peroxide was used as the substrate. The incubation medium for determining the activity of POD contained 50 mm K, Na-phosphate buffer (pH 5; 7.8), 2.6 mm hydrogen peroxide, and 21.5 mm guaiacol. The incubation time was 30 min. The POD activity was determined by the formation rate of the tetraguaiacol (TG) reaction product. To determine the content of the formed TG, the increase in the optical density at 470 nm was measured and the amount of TG was calculated, taking into account the extinction coefficient (ε470nm = 0.0266 µM−1 cm−1). The POD activity was expressed in µmol TG formed per mg of protein per 30 min (µmol TG/mg protein) [56,57].
The activity of polyphenoloxidase (PPO, 1.10.3.1) was determined using pyrocatechol as the substrate. The incubation medium for determining the PPO activity contained 50 mm K, Na-phosphate buffer (pH 5; 7.8), and 16.4 mm pyrocatechol. To determine the activity of PPO, the increase in the optical density was measured at a wavelength of 420 nm, where pyrocatechol oxidation products were absorbed. The reaction monitoring time was 20 min. The activity of PPO was expressed in units per mg of protein per 1 min (U/mg protein) [42].

2.5. Choice of Primers for Determination of Gene Expression Level

Specific primers for amplifying fragments of the target genes and the reference gene (Table 1) (Evrogen, Moscow, Russia) were constructed using the Primer Express Software v.3.0 (Applied Biosystems, Waltham, MA, USA), which relied on the gene sequences identified for Betula pendula Roth [44]. A real-time polymerase chain reaction (PCR) analyzed the accumulation of gene transcripts.
We used actin-encoding gene (ACTIN1) as the stable reference gene for normalization of the expression level of target genes [58]. The PCR efficiencies of primer pairs were determined according to the standard curve calculated from a dilution series of cDNA samples [59].

2.6. Total RNA Isolation and Complementary DNA Synthesis

Total RNA was extracted by ExtractRNA reagent (Evrogen, Moscow, Russia). The reaction of cDNA synthesis (reverse transcription, RT) was carried out using the MMLV RT kit (Evrogen, Moscow, Russia). The isolation of total RNA and the reverse transcription reaction were performed following the user manuals (https://evrogen.com (accessed on 19 June 2017)). The quality control of isolated RNA was performed using 1% agarose gel electrophoresis. To control the contamination of total RNA with genomic DNA, RT-PCR was performed on a total of RNA samples with primers to all genes. The RT-PCR with total RNA showed no reaction product for all pairs of primers.

2.7. Real-Time PCR

The RT-PCR was performed in an iCycler with an iQ5 optical system (Bio-Rad, Hercules, CA, USA). The qPCRmix-HS SYBR (Evrogen, Moscow, Russia) was used for amplification of fragments of the target and reference genes. Amplification was carried out according to the manufacturer’s recommendations (https://evrogen.com (accessed on 19 June 2017)) at 48 to 60 °C primer pairs’ annealing temperature. The reaction specificity was estimated using melting curves and 8% acrylamide gel electrophoresis. The reaction products of expected sizes were obtained.
The relative quantification (RQ) of gene transcription followed the following formula:
RQ = 2−ΔCt,
where ΔCt = Ct (reference gene) − Ct (target gene) and 2 is the PCR efficiency. The transcription levels of the specific genes were expressed in relative units (r.u.).
The RT-PCR data were analyzed by Relative Expression Software Tool 2009 v.2.0.13 (REST 2009, https://www.gene-quantification.de/rest-2009.html (accessed on 19 July 2022)).
All assays were performed at the core facility of the Karelian Research Centre of the Russian Academy of Science.

2.8. Statistical Analysis

The results were statistically processed with Statistica v.10 software (StatSoft, Moscow, Russia). The significance of differences between variants was estimated by Wilcoxon’s and Student’s tests.

3. Results

3.1. Structure of Conducting Tissues

In all trees at the beginning of the experiment, the early phloem (part of the current year’s increment of the conducting phloem, which is formed at the beginning of the growing season) was fully formed, and xylem was actively forming. During the first 30 days after the beginning of the experiment, control trees in levels AG1 and AG35 had active processes of both phloem formation and xylogenesis. Tissue formation was typical of the species (data not shown). In girdled trees in the AG35 level, we observed cambial activity toward both phloem and xylem in the first 30 days after the start of the experiment (Figure 1).
In the AG1 level of the girdled trees, xylogenesis ceased immediately after girdling. Deposition of new xylem elements by the cambium was observed, for the first time, only at 30DAG. Active phloem formation, comprising small sieve tubes and parenchyma cells, was observed immediately after girdling. At 30DAG, we observed groups of sclereids that were absent in the 20DAG samples. The sclereids were larger than the surrounding parenchyma cells and had thickened lignified cell walls (Figure 2).
In the 10DAG samples, a barrier zone was present in the xylem in both zones of the girdled trees (Figure 1d and Figure 2d). It formed in response to injury and looked like a layer of radially flattened cells with thick secondary walls [60]. In the xylem of the control trees, we observed a narrow barrier zone (1 to 3 cell layers) that was formed in response to the puncturing of bark tissue that occurred at the beginning of the experiment. Thus, the barrier zone in the xylem of the control and girdled trees served as a marker of the beginning of the experiment. In samples taken at the end of the growing season, we measured the width of the phloem and xylem increments formed after girdling. In phloem, this indicator was measured from the cambium to the border of the late phloem, and in xylem it was measured from the border of the barrier zone to the cambium.
In the control trees, the widths of the phloem increment in levels AG1 and AG35 did not differ significantly. There were small but statistically significant differences in the xylem increments at the AG1 and AG35 levels. The girdled trees had wide phloem increments by the end of the growing season, while the xylem increments were narrower compared with similar levels in the control trees (Figure 3 and Figure 4).
The structure of late phloem in both levels of the control trees was typical for the species. Sieve tubes dominated, parenchyma cells represented the remaining part, and sclereids were absent (Figure 5a; Supplementary Materials, Figure S2). In the girdled trees, late phloem in the AG1 level was almost entirely represented by parenchyma cells and large groups of sclereids, small sieve tubes were found singularly. In the AG35 level, parenchyma cells dominated in late phloem in the same trees, but the proportion of sieve tubes was higher compared with the proportion in the AG1 level; sclereids were found singularly.
We compared the structure of xylem formed immediately after the beginning of the experiment in the control and girdled trees in the AG35 level. In the control trees, the xylem structure was typical for the species: fibrous tracheids dominated, while vessels and parenchyma cells were less common. In the girdled trees, the proportion of fibrous tracheids was lower, with parenchyma cells occupying up to 30.2% of the xylem area. The proportion of vessels was virtually unchanged compared with the control trees (Figure 5b; Supplementary Materials, Figure S2). Visually, there were more vessels, but they were smaller. The length and width of the vessel elements in the AG35 level were greater in the control trees than in girdled trees (Figure 5c,d).

3.2. Enzyme Activity

3.2.1. Sucrose-Metabolizing Enzymes

Girdling considerably affected the activity of the sucrose-metabolizing enzymes in the trunk tissues. In the control trees, high CWInv activity was observed in phloem, while the activity of the intracellular enzymes was relatively low. In xylem, sucrose metabolism during normal growth and development occurred to a greater extent with SuSy participation (Figure 6).
Girdled trees at 10DAG and 20DAG showed very high CWInv activity in the AG1 level on both the phloem and xylem sides. The cytoplasmic enzyme activity (CytInv, SuSy) at 10DAG was lower in phloem and higher in xylem in the AG1 level, compared to both control trees and tissues in the AG35 level of the girdled trees. At 20DAG, the activity of sucrose-degrading enzymes localized in cytoplasm did not differ from the control trees in both levels, except for SuSy in the xylem of the AG1 level. The VacInv activity in both phloem and xylem was low at both sampling dates. However, in the xylem at both levels, the VacInv activity was higher in the girdled trees compared to with the xylem of the control trees (Figure 6).

3.2.2. AOS Enzymes

At 10DAG, SOD activity was not detected in the control trees but was quite high in both levels in the phloem of the girdled trees. The enzyme activity in the xylem of the girdled trees was considerably lower than in the phloem. In both tissues, the SOD activity was higher in the AG1 level compared with the AG35 level. At 20DAG, SOD activity was detected in both the phloem and xylem of both groups of trees. However, it was higher in the AG1 level in both tissues in the girdled trees, while in the AG35 level in the girdled trees, the SOD activity was lower compared with the control trees (Figure 7a,b).
The CAT activity on the first date of sampling in both tissues did not differ in the control and girdled trees. At 20DAG, the CAT activity was higher in both tissues in the girdled trees in the AG1 level and below (phloem), or did not differ (xylem) from the control trees in the AG35 level (Figure 7c,d).
The most noticeable differences between the trunk tissues of the control and girdled trees were recorded for POD and PPO activities. At both sampling dates, both in the phloem and in the xylem, the activities of these enzymes in the AG1 level of the girdled trees considerably exceeded the values in both the AG35 level of the girdled trees and in the trunk tissues of the control trees (Figure 8).

3.3. Gene Expression

3.3.1. Genes Involved in Sugar Metabolism and Transport

On both sampling dates, the SUS expression was higher on the xylem side than on the phloem side. There was no noticeable difference in SUS gene expression in control and girdled tree tissues, except for SUS1 expression in the AG1 level at 10DAG (Figure 9a,d; Supplementary Materials, Figure S3). In contrast, the expression of genes encoding CWInv was higher on the phloem side than on the xylem side in all selection terms. In both tissues, the CWINV expression was slightly higher in the girdled trees than in the control trees at both sampling dates (Figure 9b,e; Supplementary Materials, Figure S3). Considerable differences between the control and girdled trees were detected in the expression levels of the genes encoding the protein inhibitor of CWInv activity (CIF). In the trunk tissues of the girdled trees, its expression was 2 times lower at both sampling dates (Figure 9c,f).
Genes encoding sucrose (SUC) and hexose transporters (HEX1 and HEX2) showed higher activity on the phloem side than on the xylem side. At both sampling dates, the expression of all the genes we studied was higher in the trunk tissues of the girdled trees than in the control trees (Figure 10; Supplementary Materials, Figure S4).

3.3.2. Genes Encoding Proteins Involved in Auxin Transport and Metabolism

The activity of PIN genes activity was higher on the phloem side than on the xylem side, with PIN1 and PIN3 gene expression levels 1.5 to 2 times higher in the phloem of the girdled trees than in the phloem of the control trees (Figure 11; Supplementary Materials, Figure S5).
The UGT84B1 gene was more active on the xylem side. At 10DAG, the expression level did not differ between the control and girdled trees, while at 20DAG, the expression level in the xylem of the girdled trees was 1.4 to 1.6 times higher than the expression level of the control trees. On the phloem side, the expression level in the girdled trees in both levels was 2 to 3.4-fold higher than the expression level in the control trees at both sampling dates (Figure 12).

3.3.3. Genes Involved in Phloem and Xylem Formation

Expression of phloem- and xylem-specific transcription factors was observed in the respective tissues. At both sampling dates, the level of APL expression in both the AG1 and AG35 levels was 2 to 3 times higher in the phloem of girdled trees than in the phloem of the control trees. The expression level of VND7 did not differ in tissues of the control and girdled trees at 10DAG. At 20DAG, the VND7 expression was higher in both levels in the girdled trees than in the control trees (Figure 13; Supplementary Materials, Figure S6).
The expression of genes encoding cellulose synthases was detected only in the xylem part of the samples. The expression level of both genes was considerably higher in the xylem of the girdled trees than in the controlled trees. However, an increase in both genes was noticeable at 10DAG (CESA3 4.4–4.9 and CESA7 were 1.7 to 2 times higher than in the control genes, respectively). At 20DAG, distinct differences in the expression of the girdled and control trees were maintained only the CESA3 gene in the AG1 level (Figure 13; Supplementary Materials, Figure S6).

4. Discussion

4.1. Experiment Design

Interruption of phloem transport by girdling is associated with extensive injury to trunk tissues. In woody plants, tissue injury can cause changes in enzyme activity and in the expression of wound-inducible genes [61,62,63]. The most pronounced reaction of enzymes and gene activity is observed in the first 5 days post-wounding [64,65,66,67,68]. For several enzymes involved in the biosynthesis of high-molecular-weight compounds, such as monoterpene cyclases, hydroxycinnamyl alcohol dehydrogenase, and polyphenol oxidase, increased activity can be observed 1 to 2 weeks post-wounding [65,69,70]. In our experiment, we sampled tissues to analyze anatomical and biochemical changes at 10DAG, 20DAG, and 30DAG to minimize the effects of injury. However, the observed differences between the control trees and the girdled trees at the first sampling date (10DAG) may partly have resulted from the effects of wounding, especially in the AG1 level located 1 cm from the girdle border.
In intact trunks, both of the conducting tissues interact closely, so phloem removal could affect the water transport through the xylem [24,71,72,73], which could ultimately affect cambium activity. Several studies have shown that girdling has no significant effect on water transport and tissue moisture if the xylem is not damaged by girdling [74,75,76]. In our experiment, we observed no significant differences in the relative water content of bark and wood in the control and girdled trees (Supplementary Materials, Table S1), a result that agrees with the data of other authors indicating that tissue moisture correlates closely with sugar concentrations [77,78]. Therefore, the results are discussed further in terms of the cambium response to increased sugars.
We considered that changes in the anatomical structure of tissues occur more slowly than gene and enzyme responses. Therefore, we compared the observed anatomical patterns with enzyme activity and gene expression data from the previous selection. We did not divide the phloem and cambium while sampling from the bark side (see Section 2.1). However, these tissues may differ in enzyme activity and gene expression. It should be noted that in our study, both cambium and phloem contributed to the data but their relative contributions were not resolved.

4.2. Increased Sucrose Concentration in the Cambial Zone Causes Activation of Phloem Formation

The anatomical pattern observed in the first weeks after girdling results from the cambium response to the changed conditions, i.e., the accumulation of sucrose and auxin in the tissues above the girdle [24,25,36,79]. The increase in sucrose concentration appears to be a signal to the cambium that the sieve tubes do not ensure normal phloem conduction [80,81]. As a result, in both levels of the girdled trees, the cambium actively produced phloem cells. At the same time, xylogenesis was stopped in the AG1 level, where the sucrose concentration was highest, a result that agrees with the data on the higher sensitivity of xylem cambium derivatives to increased sugar levels [19,80,82,83,84,85]. In the AG35 level, xylogenesis does not cease, but the proportion of vessels in the xylem decreases, and the vessels are smaller than those in the control trees. Previously, impaired vessel differentiation in conditions of increased sugar content in the cambial zone was shown during the formation of birch and oak wood [19,46,47,86,87].

4.3. High Levels of Sugars Stimulate Differentiation of Cambial Derivatives into Parenchyma Cells

In trees, the transport of assimilates through the phloem occurs as a pressure-driven bulk flow from assimilated sources to sinks [88,89]. During the day and during the vegetation period, the source and sink activities change. The leakage-retrieval mechanism controls the maintenance of the concentration gradients, in which the phloem apoplast plays the role of a carbohydrate buffer [90,91]. The concentration of sugars in phloem apoplast is supposed to be maintained at a constant level [92]; therefore, when the level of sugars in apoplast is significantly increased or decreased, their translocation to or from the symplast of living parenchyma cells occurs [91].
In our study, the level of sugars at 10DAG in the phloem apoplast in the AG1 level was expected to be very high, consistent with the data on a considerable increase in CWInv and a decrease in the activity of cytoplasmic enzymes. The gene expression data show that regulation of CWInv activity occurs at the post-translational level [93]. Sucrose and hexoses formed during sucrose cleavage were actively loaded into the symplast of the phloem cells with the participation of transmembrane transporters. We believe that the active sugar loading from the apoplast was a signal for a change in the program of cambial derivative development toward active differentiation of parenchyma cells, many of which were found in the late phloem at 20DAG. The data obtained agreed with the previously expressed hypothesis that hexoses coming from the apoplast can stimulate differentiation of parenchyma cells in the conducting tissues of trees [46,94].
The mechanism involved in switching the differentiation program of cambial derivatives may be a decrease in the free (physiologically active) auxin concentration because of its metabolism or because of transport to other tissues (e.g., involving UGT84B1 and PIN3 genes, which are upregulated after sucrose or glucose treatments) [46,47]. A similar conducting tissue structure has been described in the cambial zone in the trunks of figured Karelian birch trees, in which low levels of free auxin and high levels of its conjugates have been recorded [94,95,96]. In addition, high values of POD activity show ROS accumulation, which can also reduce the level of physiologically active auxin and/or auxin signaling [97,98,99].
At 10DAG, the increased activity of enzymes involved in sucrose metabolism on the xylem side in the AG1 level seems to be explained by their participation in cellulose synthesis and by the formation of thick cell walls of cells forming a barrier zone in the girdled trees [100,101,102]. This suggestion is supported by the high activity of the CESA genes.
In the AG35 level of the girdled trees at 10DAG, the sugar level in the phloem were expected to be slightly higher than the level in the control trees, stimulating phloem cell differentiation. The increased expression level of genes encoding transporters shows the activation of sugar transport from the apoplast into the cells. However, in contrast to the AG1 level, sucrose was used predominantly in the cell cytoplasm. As a result, conducting tissues were formed, in which the ratio of conducting and mechanical elements to parenchyma was more similar to that typical of the species.

4.4. Sclerification of Parenchyma Cells in the Phloem May Be Involved in the Reduction of Sugars in the Cambial Zone

The renewal of cambial activity toward deposition of new xylem elements in the AG1 level coincided with the differentiation of some parenchyma cells in the phloem into sclereids. The data agree with the hypothesis that the formation of sclerenchyma cells in the phloem of trees may be one mechanism for the utilization of excess sugars and the maintenance of their concentration at the level that does not cause xylogenesis disruption [20,80,103]. Sclereids have thick lignified walls, the formation of which requires significant carbohydrate resources for the synthesis of cellulose and lignin components [104,105]. While parenchyma cells differentiate from cambial cells, sclerification represents a stage in the development of pre-existing phloem parenchyma cells.
During the normal growth of most trees in the temperate zones, the sclerification of parenchyma cells occurs in the nonconducting phloem [51,106]. One of the activity peaks of genes that are involved in the biosynthesis of lignin components is observed in this tissue [107]. The localization of sclereids in proximity to cambium is encountered less frequently, for example, in wound healing [62], in extreme growing conditions [108], and during the formation of Karelian birch and “birdseye maple” figured wood [39,109]. In addition, the sclerification of parenchyma in the bark of the girdled trees in the AG1 level at 20DAG is preceded by high activity of CWInv, SuSy, and AOS enzymes, which are biochemical indicators of abnormal conducting tissue formation in Karelian birch [93,110,111]. By the second sampling date, an increase in SOD and CAT was observed in the girdled trees, followed by a large increase in POD and PPO activities. It is known that coordinated AOS functioning is determined by local and cell-wide cascade interactions [112]. Here, the interaction was supported by the AOS and phenolic substrates [111]. Hydrogen peroxide could become a signaling molecule that links both invertase and PPO functioning via a gene cascade [113,114,115]. We suggest that the subsequent high activity of POD and PPO at various pH values probably shows the involvement of many isoforms in the running reactions and the high general metabolic activity of living phloem and xylem cells in the girdled trees, directed toward neutralization of the formed ROS and toward lignification reactions [111,116,117].
In the AG35 level, where the increase in sugars was not as considerable as in the AG1 level, the proportion of sclereids in the phloem composition was significantly lower. The excess of the photoassimilates was apparently consumed in the processes of forming secondary cell walls of the xylem elements [4,5].

5. Conclusions

The cambium gives rise to all cell types of conducting tissues and can dynamically respond to changing environmental conditions, translated through changes in the endogenous regulation system (the signaling of hormones, sugars, and other regulators of cambial activity). Reactions of cambium can be expressed through the reorientation of cambial divisions toward phloem or xylem, and through the changing of a program of differentiation of cambial derivatives. The final cellular composition of phloem and xylem reflects the conditions under which tissue formation takes place. The main results of our experiment can be summarized as follows:
(1)
the compromise between xylogenesis and phloem formation, as described in the literature, can be controlled by the level of incoming photoassimilates;
(2)
an increase in the level of photoassimilates causes an increase in the proportion of parenchyma in the composition of the conducting tissues. The potential mechanism involved in switching the cell differentiation program can be the active supply of apoplast sugars and the inactivation of auxin;
(3)
the formation of thick sclereid cell walls may be one of the mechanisms involved in regulating sugar level in the cambial zone.
The findings are important in understanding source–sink relationships of organs and tissues in trees, assessing tree productivity and mortality risks under changing climate conditions, and planning biotechnological and breeding programs that are aimed at obtaining wood and bark with desirable properties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f13081171/s1, Figure S1: Width of phloem increment (a) and xylem increment (b), formed after the girdling in control (1C–3C) and girdled (1G–5G) trees 1 cm and 35 cm above girdle (AG1 and AG35, respectively); Figure S2: Anatomical characteristics of conducting tissues. (a) the proportion of different cell types in late phloem; (b) the proportion of different cell types in xylem of AG35 level. 1C–3C: control trees; 1G–5G: girdled trees; Figure S3: Relative gene expression of SUS2, CWINV1, and CWINV3 genes in the phloem (a–c) and xylem (d–f) of control and girdled trees, 10 and 20 days after girdling (10DAG, 20DAG, respectively); Figure S4: Relative gene expression of SUC (a), HEX1 (b), and HEX2 (c) genes in the xylem of control and girdled trees; Figure S5: Relative gene expression of PIN1 (a), PIN2 (b), and PIN3 (c) genes in the xylem of control and girdled trees; Figure S6: Relative gene expression of APL (a), VND7 (b), CESA3 (c), and CESA7 (d) genes in the xylem (a) and xylem (b–d) of control and girdled trees; Table S1: The relative water content of bark and wood in control and girdled trees, 10, 20, and 30 days after girdling (10DAG, 20DAG, 30DAG, respectively) and at the end of the growing season (autumn), 1 cm and 35 cm above girdle (AG1 and AG35, respectively).

Author Contributions

Conceptualization, L.L.N. and T.V.T.; formal analysis, A.A.S., T.V.T., N.A.G., K.M.N., Y.L.M., I.N.S., N.N.N., D.S.I. and L.I.S.; writing—original draft preparation, A.A.S., T.V.T., N.A.G., K.M.N. and Y.L.M.; writing—review and editing, A.A.S., T.V.T., N.A.G., K.M.N. and Y.L.M.; visualization, A.A.S. and L.I.S.; funding acquisition, N.A.G. and L.L.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Foundation for Basic Research, grants 16-04-01191-a, 16-44-100639-p_a, 19-04-00622_a. The anatomical studies were carried out under state orders that were made to the Karelian Research Centre of the Russian Academy of Sciences (Forest Research Institute KarRC RAS).

Acknowledgments

We warmly thank Vitaly V. Vorobiev and the specialists from the Analytic Laboratory of the Forest Research Institute of the Karelian Research Centre of the Russian Academy of Science for their technical assistance in sampling and carrying out anatomical and chemical analyses for the study.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Transverse sections of phloem (a–c) and xylem (d–f) 35 cm above girdle (AG35) of girdled trees. (a,d) 10 days after girdling (10DAG); (b,e) 20 days after girdling (20DAG); (c,f) 30 days after girdling (30DAG). Colors show the following tissue layers. Green: xylem formed before the experiment; blue: xylem formed after the experiment; black: cambial zone; yellow: late phloem; orange: early phloem. The letters in the figures are vs: vessels; ft: fibrous tracheids; rp: ray parenchyma; ap: axial parenchyma; st: sieve tubes. Scale bar is 100 µm.
Figure 1. Transverse sections of phloem (a–c) and xylem (d–f) 35 cm above girdle (AG35) of girdled trees. (a,d) 10 days after girdling (10DAG); (b,e) 20 days after girdling (20DAG); (c,f) 30 days after girdling (30DAG). Colors show the following tissue layers. Green: xylem formed before the experiment; blue: xylem formed after the experiment; black: cambial zone; yellow: late phloem; orange: early phloem. The letters in the figures are vs: vessels; ft: fibrous tracheids; rp: ray parenchyma; ap: axial parenchyma; st: sieve tubes. Scale bar is 100 µm.
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Figure 2. Transverse sections of phloem (a–c) and xylem (d–f) 1 cm above girdle (AG1) of girdled trees. (a,d) 10DAG; (b,e) 20DAG; (c,f) 30DAG. Colors show the following tissue layers. Green: xylem formed before the experiment; blue: xylem formed after the experiment; black: cambial zone; yellow: late phloem; orange: early phloem. The cambium is not defined in figures (c,f), because all the trees had tissue discontinuity along the cambium during sampling. The letters in the figures are vs: vessels; ft: fibrous tracheids; rp: ray parenchyma; ap: axial parenchyma; st: sieve tubes; sc: sclereids. Scale bar is 100 µm.
Figure 2. Transverse sections of phloem (a–c) and xylem (d–f) 1 cm above girdle (AG1) of girdled trees. (a,d) 10DAG; (b,e) 20DAG; (c,f) 30DAG. Colors show the following tissue layers. Green: xylem formed before the experiment; blue: xylem formed after the experiment; black: cambial zone; yellow: late phloem; orange: early phloem. The cambium is not defined in figures (c,f), because all the trees had tissue discontinuity along the cambium during sampling. The letters in the figures are vs: vessels; ft: fibrous tracheids; rp: ray parenchyma; ap: axial parenchyma; st: sieve tubes; sc: sclereids. Scale bar is 100 µm.
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Figure 3. Width of phloem increment (a) and xylem increment (b), formed after the girdling. Representative values are given for one control (tree 2C) and one girdled tree (tree 3G). Characteristics for all trees can be found in the Supplementary Materials, Figure S1. The data in the diagrams are presented as M ± SD. Asterisks mark significant differences between groups (* p < 0.05), n.s.—no differences.
Figure 3. Width of phloem increment (a) and xylem increment (b), formed after the girdling. Representative values are given for one control (tree 2C) and one girdled tree (tree 3G). Characteristics for all trees can be found in the Supplementary Materials, Figure S1. The data in the diagrams are presented as M ± SD. Asterisks mark significant differences between groups (* p < 0.05), n.s.—no differences.
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Figure 4. Structure of trunk conducting tissues at the end of the growing season: (a) control tree; (b) girdled tree, AG35 level; (c) girdled tree, AG1 level. Colors show the following tissue layers: Green: xylem formed before the experiment; blue: xylem formed after the experiment; black: cambial zone: yellow: late phloem; orange: early phloem. Scale bar is 100 µm.
Figure 4. Structure of trunk conducting tissues at the end of the growing season: (a) control tree; (b) girdled tree, AG35 level; (c) girdled tree, AG1 level. Colors show the following tissue layers: Green: xylem formed before the experiment; blue: xylem formed after the experiment; black: cambial zone: yellow: late phloem; orange: early phloem. Scale bar is 100 µm.
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Figure 5. Anatomical characteristics of conducting tissues: (a) the proportion of different cell types in late phloem; (b) the proportion of different cell types in xylem in AG35 level; (c) length of vessel elements in AG35 level; (d) width of vessel elements in AG35 level. For (a,b), representative values are given for one control (tree 2C) and one girdled tree (tree 3G). Characteristics of all trees are provided in the Supplementary Materials, Figure S2. The data in the diagrams are presented as M ± SD. Asterisks mark significant differences between groups (*** p < 0.001), n.s.—no differences.
Figure 5. Anatomical characteristics of conducting tissues: (a) the proportion of different cell types in late phloem; (b) the proportion of different cell types in xylem in AG35 level; (c) length of vessel elements in AG35 level; (d) width of vessel elements in AG35 level. For (a,b), representative values are given for one control (tree 2C) and one girdled tree (tree 3G). Characteristics of all trees are provided in the Supplementary Materials, Figure S2. The data in the diagrams are presented as M ± SD. Asterisks mark significant differences between groups (*** p < 0.001), n.s.—no differences.
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Figure 6. Activity of sucrose-metabolizing enzymes in the phloem (a–d) and xylem (e–h) in control and girdled trees. CWInv: cell wall invertase; VacInv: vacuolar invertase; CytInv: cytosolic invertase; SuSy: sucrose synthase.
Figure 6. Activity of sucrose-metabolizing enzymes in the phloem (a–d) and xylem (e–h) in control and girdled trees. CWInv: cell wall invertase; VacInv: vacuolar invertase; CytInv: cytosolic invertase; SuSy: sucrose synthase.
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Figure 7. The activity of SOD (a,b) and CAT (c,d) in phloem (a,c) and xylem (b,d) of control and girdled trees.
Figure 7. The activity of SOD (a,b) and CAT (c,d) in phloem (a,c) and xylem (b,d) of control and girdled trees.
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Figure 8. The activity of POD (a,b,e,f) and PPO (c,d,g,h) in the phloem (a–d) and xylem (e–h) of control and girdled trees.
Figure 8. The activity of POD (a,b,e,f) and PPO (c,d,g,h) in the phloem (a–d) and xylem (e–h) of control and girdled trees.
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Figure 9. Relative gene expression of SUS1 (a,d), CWINV2 (b,e), and CIF (c,f) genes in the phloem (a–c) and xylem (d–f) of control and girdled trees.
Figure 9. Relative gene expression of SUS1 (a,d), CWINV2 (b,e), and CIF (c,f) genes in the phloem (a–c) and xylem (d–f) of control and girdled trees.
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Figure 10. Relative gene expression of SUC (a), HEX1 (b), and HEX2 (c) genes in the phloem of control and girdled trees.
Figure 10. Relative gene expression of SUC (a), HEX1 (b), and HEX2 (c) genes in the phloem of control and girdled trees.
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Figure 11. Relative gene expression of PIN1 (a), PIN2 (b), and PIN3 (c) genes in the phloem of control and girdled trees.
Figure 11. Relative gene expression of PIN1 (a), PIN2 (b), and PIN3 (c) genes in the phloem of control and girdled trees.
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Figure 12. Relative gene expression of UGT84B1 gene in the phloem (a) and xylem (b) of control and girdled trees.
Figure 12. Relative gene expression of UGT84B1 gene in the phloem (a) and xylem (b) of control and girdled trees.
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Figure 13. Relative gene expression of APL (a), VND7 (b), CESA3 (c), and CESA7 (d) genes in the phloem (a) and xylem (b–d) of control and girdled trees.
Figure 13. Relative gene expression of APL (a), VND7 (b), CESA3 (c), and CESA7 (d) genes in the phloem (a) and xylem (b–d) of control and girdled trees.
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Table
Table 1. Nucleotide sequence of primers for real-time PCR and brief characterization of target and reference genes.
Table 1. Nucleotide sequence of primers for real-time PCR and brief characterization of target and reference genes.
Gene NameForward Primer (5′→3′)Reverse Primer (5′→3′)Locus [44]Product Function
ACTIN1GGTGGTGAATGAGTAGCCTTCTTTCCCTTTATGCCBpev01.c0427.g0027Component of the cytoskeleton
CWINV3TATCAGACTCAAGCACCCAGATTACACGCCCAGAACAGACBpev01.c0237.g0050CWInv isoforms
CWINV1AGTGCCCCGATTTCTTCCCTGGTCCACCTGCCCCTTGTCCGBpev01.c0333.g0031
CWINV2GCTCTACCACAATCCTCCCAGCACTCGCATTCATCCCCTCBpev01.c0516.g0006
SUS1TAGCATCAACCCCTGTCCCTGTTCAGTTCCTCAACCGTCABpev01.c0294.g0013SuSy isoforms
SUS2CTGCTAACCGCAACGAAATACCGCCAAGGCAACCCACBpev01.c0051.g0185
CIFGCAAGCAGACACCCTTTTATGTTTAGTTTTGGGCTACCGTBpev01.c0932.g0004Protein inhibitor of CWInv
CESA3TGTCTGCTGCATCACCTGAAAGAGTCATCCACAAGCACATBpev01.c0777.g0012Cellulose synthase isoforms
CESA7GTAATAGCCGGTGGTAGATCCTGCTCGAAGCAATCGGTABpev01.c0603.g0003
VND7CCACTGCTGCTGGATTCTACCATTGGGTGCTCGTBpev01.c0411.g0006Xylem-specific transcription factor
APLGAAGCTCAAGCTGGTCACGGAGAAAGCCTGTCAAACBpev01.c0189.g0073Phloem-specific transcription factor
HEX1GGGGTGGTTGATTCCTATCCAGCAGAGCATTGTGBpev01.c0329.g0008Hexose transporters
HEX2GGATTTGCTTGGTCATGGGGTCCATTTATACCCTTGGTCTCTGGBpev01.c0151.g0005
SUCCTTCATCTGGCTCTGCGGGTTTTCGTCGTCTTGTCBpev01.c0594.g0012Sucrose transporter
UGT84B1CAGCATCGTAGGCTCAAGTCTGTTCTCACGGTCAAAGTCCBpev01.c0157.g0047Auxin conjugation with UGT-glucose
PIN1CACTCCCAGACCCTCAAATTTCCTCCCACCAGCCATCABpev01.c0147.g0002Polar auxin transport
PIN2CCATTGTGCCTTTATACGTTGCCGAAATTTCCGTACATGGCCTTCAGBpev01.c0606.g0011
PIN3CCCAACCCAGAGTTCTCGTCCACACCGAATCCCTCACTTTCBpev01.c1162.g0001
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Serkova, A.A.; Tarelkina, T.V.; Galibina, N.A.; Nikerova, K.M.; Moshchenskaya, Y.L.; Sofronova, I.N.; Nikolaeva, N.N.; Ivanova, D.S.; Semenova, L.I.; Novitskaya, L.L. Changes in the Differentiation Program of Birch Cambial Derivatives following Trunk Girdling. Forests 2022, 13, 1171. https://doi.org/10.3390/f13081171

AMA Style

Serkova AA, Tarelkina TV, Galibina NA, Nikerova KM, Moshchenskaya YL, Sofronova IN, Nikolaeva NN, Ivanova DS, Semenova LI, Novitskaya LL. Changes in the Differentiation Program of Birch Cambial Derivatives following Trunk Girdling. Forests. 2022; 13(8):1171. https://doi.org/10.3390/f13081171

Chicago/Turabian Style

Serkova, Aleksandra A., Tatiana V. Tarelkina, Natalia A. Galibina, Kseniya M. Nikerova, Yulia L. Moshchenskaya, Irina N. Sofronova, Nadezhda N. Nikolaeva, Diana S. Ivanova, Ludmila I. Semenova, and Ludmila L. Novitskaya. 2022. "Changes in the Differentiation Program of Birch Cambial Derivatives following Trunk Girdling" Forests 13, no. 8: 1171. https://doi.org/10.3390/f13081171

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