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Article

Differences in Environmental and Hormonal Regulation of Growth Responses in Two Highly Productive Hybrid Populus Genotypes

1
Institute of Forestry, Lithuanian Research Centre for Agriculture and Forestry, Liepų 1, 53101 Girionys, Lithuania
2
Faculty of Natural Sciences, Vytautas Magnus University, Universiteto 10-314, 53361 Akademija, Lithuania
3
Department of Silviculture and Forest Tree Genetics, Forest Research Institute, Braci Leśnej 3, 05-090 Sękocin Stary, Poland
*
Authors to whom correspondence should be addressed.
Academic Editors: Jorge Canhoto, Paloma Moncaleán and Sandra Correia
Forests 2022, 13(2), 183; https://doi.org/10.3390/f13020183
Received: 7 December 2021 / Revised: 11 January 2022 / Accepted: 21 January 2022 / Published: 26 January 2022
(This article belongs to the Special Issue Application of Biotechnology Techniques on Tree Species)

Abstract

Phenotypic plasticity, in response to adverse conditions, determines plant productivity and survival. The aim of this study was to test if two highly productive Populus genotypes, characterised by different in vitro etiolation patterns, differ also in their responses to hormones gibberellin (GA) and abscisic acid (ABA), and to a GA biosynthesis inhibitor paclobutrazol (PBZ). The experiments on shoot cultures of ‘Hybrida 275′ (abbr. H275; Populus maximowiczii × P. trichocarpa) and IBL 91/78 (Populus tremula × P. alba) were conducted by either modulating the physical in vitro environment or by adding specific chemicals to the nutrient medium. Our results revealed two main sets of differences between the studied genotypes in environmental and hormonal regulation of growth responses. First, the genotype H275 responded to darkness with PBZ-inhibitable shoot elongation; in contrast, the elongation of IBL 91/78 shoots was not affected either by darkness or PBZ treatment. Secondly, the explants of H275 were unable to recover their growth if it was inhibited with ABA; in contrast, those of IBL 91/78 recovered so well after the temporal inhibition by ABA that, when rooted subsequently, they developed longer shoots and roots than without a previous ABA treatment. Our results indicate that GA catabolism and repressive signalling provide an important pathway to control growth and physiological adaptation in response to immediate or impending adverse conditions. These observations can help breeders define robust criteria for identifying genotypes with high resistance and productivity and highlight where genotypes exhibit susceptibility to stress.
Keywords: dark treatment; hybrid poplar; plant hormone; rooting; shoot culture dark treatment; hybrid poplar; plant hormone; rooting; shoot culture

1. Introduction

Forest trees have tremendous economic and ecological value and possess unique biological properties that are of fundamental scientific interest [1]. However, experiments with very large, long-lived organisms have been fraught with difficulties. With the great technological advances in molecular tools for genetic engineering and genomics [2,3], poplar (Populus L., Salicaceae Mirb.) has achieved the status of a ‘model biological material’. Populus is the most widely distributed genus in the Northern Hemisphere and indeed has several advantages as a model system, including rapid growth, prolific sexual reproduction, ease of cloning, a small genome, facile transgenesis, and a tight coupling between physiological traits and biomass productivity [1].
Due to their fast growth and favourable wood properties (fibre parameters) and chemical composition (high cellulose content), poplar genetic resources are widely used as a fibre source for the pulp and paper industry and, more recently, for biomass and biofuel production, carbon sequestration, and phytoremediation [4,5,6,7]. For these reasons, breeding programs have been developed for poplars in many European and North American countries, including controlled crossing strategies and selection of the best hybrid genotypes [4,5,6,7]. Interspecific poplar hybrids are commonly used in commercial biomass plantations and planted as clonal stands [8]. The selection of appropriate genotypes in tree plantations should therefore be directed to many traits of economic and adaptive importance.
Phenotypic plasticity in response to adverse conditions determines plant productivity and survival [9]. Plants reduce their growth under unfavourable conditions to avoid potentially lethal stress [10]. In addition, plants can use environmental cues to detect and anticipate impending unfavourable conditions to adjust their growth accordingly [11]. Environmental signals, such as photoperiod, may, for example, induce dormancy in deciduous plants in anticipation of the onset of freezing conditions in winter [11,12].
The phenomenon of different plant development patterns in the dark and light has previously been investigated by different research groups. These studies have largely focused on seedling development in the model plant Arabidopsis [13,14] and in some domesticated plant species, e.g., pea [15,16] and tomato [17]. Typically, seedling development in the dark (skotomorphogenesis) is characterised by intense elongation of the hypocotyl [13]. However, such a response is not universal among plants. For instance, dark-grown seedlings of conifer species, such as Pinus sylvestris and Picea abies, do not become more elongated than light-grown seedlings [18]. The roles of various hormones have also previously been investigated, with respect to dark-induced growth. Gibberellin (GA) has unanimously been found to be a positive regulator of dark-induced hypocotyl elongation [13,15]. Meanwhile, the influence of abscisic acid (ABA), a hormone known for its antagonistic action with respect to GA, remains more controversial. A relatively high amount of exogenous ABA has been found to have an inhibitory effect on dark-induced hypocotyl elongation in Arabidopsis [14]. However, endogenous ABA was also found to be necessary for dark-induced hypocotyl elongation in tomato [17]. Most previous studies on the relation of the action of ABA to GA have indicated an antagonistic interaction between them. Lorrai et al. [14] reported that the ABA-treatment, which led to decreased hypocotyl length in Arabidopsis had a negative effect on the expression of the genes responsible for GA biosynthesis. Similarly, in an earlier study, Toh et al. [19] found that high temperature-induced ABA in Arabidopsis seeds suppressed germination, through the inhibition of GA biosynthesis. Hence, in the model plant, the ABA-induced responses were found to be closely related to GA action. In turn, GA action in plants is dependent on other internal factors, such as DELLA proteins that interfere with gene transcription, until GA directs them towards proteasomal degradation [20]. A variety of DELLA proteins, with different expression patterns in different plant parts, have been reported in the most widely studied plant species [21]. Furthermore, some data have shown that GA action might also occur independently from DELLA proteins [22]. Thus, the response of a specific plant genotype, either to an environmental factor known to induce GA activity or to a GA synthesis inhibitor, might be quite different from the responses of other genotypes. This may lead to genotype-specific responses to other plant hormones whose action, as reported e.g., in the case of ABA [14], is potentially related to GA action.
Besides seedlings, another popular plant system for the investigation of etiolation (growing in the dark) effects is stem cuttings. Historically, the etiolation of stem cuttings was used mostly to induce or investigate adventitious root (AR) formation, as in the study by Nanda and Jain [23] on Populus nigra and, consequently, the main focus of interest in such studies was the AR-inducing hormone class of auxins, rather than ABA or GA. However, the importance of the latter hormones in the etiolation of cuttings was recently demonstrated by Lu et al. [24], who studied the hormone level and gene expression differences between the etiolated and non-etiolated black locust (Robinia pseudoacacia) cuttings. The authors [24] found that the juvenile branches sprouted from the etiolated cuttings, if compared to those from the non-etiolated cuttings, did not only have higher levels of auxin indole acetic acid (IAA), but also higher levels of gibberellin GA3 and lower levels of ABA compared to those of the non-etiolated cuttings.
In the genus Populus, which is considered a model for the study of tree biology [25], the influence of light and dark conditions on some other developmental processes have been investigated. For example, Stiles and Van Volkenburgh [26] studied the light-dependent patterns of leaf expansion in two Populus species, P. trichocarpa and P. deltoides. The authors [26] found that light stimulates the growth rate and acidification of cell walls in the former but not the latter species, which, instead, maintains leaf growth in the dark. Hence, a huge genetic and phenotypic variety is found within the different species, hybrids, and individual selected genotypes from the genus Populus [27], providing a potentially rich resource for research into different responses to environmental signals and chemical growth regulators. This may result in beneficial scientific findings and practical applications for the propagation of selected Populus genotypes, including micropropagation via in vitro cultures. Although, with respect to the use of plant hormones for Populus micropropagation, the application of different concentrations of cytokinins and auxins remains the most popular approach [28,29,30], positive developmental changes in shoot cultures were reported after GA- and ABA-related chemical regulations. For instance, in the case of aspen (P. tremula), the application of paclobutrazol (PBZ), a well-reviewed GA biosynthesis inhibitor [31], resulted in a higher number of ARs per explant, while ABA had a positive effect on AR elongation [32]. Hence, new insights about specific developmental patterns induced by GA or ABA signalling seems to be particularly important.
The aim of this study was to assess the relation between in vitro etiolation pattern and responses to specific growth regulators in the shoot cultures of distinct Populus genotypes. Specifically, the following three factors were tested depending on the poplar genotype: response to darkness, response to PBZ and ABA. We used two hybrid Populus genotypes: ‘Hybrida 275′ syn. NE-42, OP-42 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba). Both ‘Hybrida 275′ (abbreviation: H275) and IBL 91/78 were previously involved in different comparative studies on Populus growth and wood properties, and showed high potential in biomass productivity for commercial plantations in central European and northern countries [33,34,35]. The intent of our study was to address knowledge gaps regarding the main differences between the genotypes in their responses to the analysed factors, possible biological interactions, and the prospects for practical application in Populus breeding.

2. Materials and Methods

2.1. Plant Material and Standard Culture Conditions

In vitro shoot cultures of the hybrid Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba) were established at the Forest Research Institute, Poland, from the vegetative buds of 6–7-year-old cloned trees. Before starting the experiments, shoot cultures of these genotypes were maintained in vitro for approximately 1.5 years, through bimonthly subcultures on a solid Murashige and Skoog (MS) nutrient medium [36], which contained 20 g L−1 sucrose and 4 g L−1 Gelrite (all the components were purchased from Duchefa Biochemie, Haarlem, The Netherlands). The medium used for shoot multiplication before the start of the experiments was supplemented additionally with 0.6 mg L−1 6-benzylaminopurine and 0.1 mg L−1 1-naphthylacetic acid (Duchefa Biochemie). The above-mentioned hormones, however, were not included in the medium used during the experiments. In both the pre-experimental and the experimental treatments, the pH value of the medium for the Populus shoot cultures was set at 5.8 before autoclaving for 30 min at 121 °C.
For all the experiments described below, 10-mm-long apical segments of in vitro-developed shoots, with leaves removed, were used as explants. The temperature in the growth chambers for the shoot cultures was kept at 20 °C under the different light conditions. The standard illumination (indicated as “culturing in the light”, in contrast to culturing under continuous darkness) indicates a 16-h white-light (irradiance 30 μmol m−2 s−2) photoperiod.

2.2. Experimental Design

The study was conducted using four separate experiments. The MS nutrient medium used for the experiments was free of any hormones or hormone-related plant growth regulators (PGRs), except for the cases where one of the following PGRs was added for experimental purposes: PBZ, the gibberellin mixture GA4 + 7, or ABA. GA4 + 7 (mixture of GA4 and GA7 at the rate 2:1; Duchefa Biochemie) and ABA (Duchefa Biochemie) were first dissolved in a drop of ethanol or NaOH, respectively, and then diluted with distilled water to an appropriate volume for the stock solution. PBZ (Sigma–Aldrich Chemie GmbH, Taufkirchen, Germany) was directly dissolved in distilled water. The pH value of the stock solutions of these chemicals was adjusted to 5.8 (the same as the medium). The basal solutions of these chemicals were filtered through syringe-driven membrane filters (pore size 0.1 μm) prior to adding them, at the appropriate volume, to the autoclaved nutrient medium. The exact concentrations of the PGRs in the medium are given in the following descriptions of separate experiments.
One of the experiments was designed to test the effects of darkness and to estimate how Populus responses to darkness are changed by PBZ. This experiment was conducted in 55 mm× 12 mm polystyrene Petri dishes (volume—28.5 mL). Each Petri dish contained 11 mL of medium and five Populus explants placed horizontally on the medium. The possibility to produce good-quality Populus shoots by forced horizontal elongation in Petri dishes was confirmed by a previous experience [37]. For the control variant of this experiment, the H275 and IBL 91/78 explants were cultured on PGR-free medium under the 16-h white-light photoperiod. Besides control, two experimental variants were included. In one of these, the explants were cultured on the identical PGR-free medium but under continuous darkness; in the other, the explants were also cultured in darkness but on the medium with PBZ (1 μmol L−1). This experiment covered a time span of four weeks.
In another experiment, the explants were cultured in 62 × 70 mm glass jars (approximate volume—210 mL). Each jar contained 30 mL of medium and four vertically inserted explants. This experiment was conducted under the 16-h white-light photoperiod. The H275 and IBL 91/78 explants were cultured on either PGR-free or PBZ-supplemented media. As in the previously described experiment, PBZ was added to the medium at a concentration of 1 μmol L−1, and the experiment’s duration was four weeks.
In another experiment, designed to test the effects of exogenous GA, the H275 and IBL 91/78 explants were cultured for three weeks in 62 mm × 70 mm jars under the 16-h white-light photoperiod. Each jar contained 30 mL of medium and four vertically inserted explants. The medium variants included PGR-free (control) and gibberellin-supplemented (GA4 + 7 1 µmol L−1) media. In this experiment, the same calculated equivalent of ethanol which was added to the medium with the GA4 + 7 stock solution (0.005% of the final medium volume) was added also to the control medium.
To examine the short-term and long-term Populus responses to exogenous ABA, the H275 and IBL 91/78 explants were first cultured for three weeks in 55 mm × 12 mm polystyrene Petri dishes under continuous darkness either on the PGR-free or ABA-supplemented medium. In the latter variant, ABA was added to the medium at a concentration of 3 μmol L−1. Each Petri dish contained 11 mL of medium and five explants placed horizontally on the medium. After the initial stage of the experiment (three weeks), visually viable shoots from both the control and the ABA-supplemented medium variants were used for the preparation of new 10-mm-long apical explants. These were then transferred onto fresh PGR-free medium in 62 mm × 70 mm jars and cultured in the light for an additional five weeks. In this second stage of the ABA testing, each jar contained 30 mL of medium and four vertically inserted explants.
In all experiments, each experimental variant (genotype × treatment) consisted of three replicates, 20–25 explants per replicate, and they were all organised in completely randomised designs.

2.3. Processing of Results

After an appointed period of culturing (three to five weeks, depending on the specified conditions of each experiment, as described above), the morphometric parameters for the development of the explants were evaluated. For each explant, the number of adventitious roots (ARs) was counted visually and the general rate of rooting for a given explant group evaluated. The lengths of the main shoot (increase from the primary 10 mm) and all ARs (if present) were recorded using a ruler after the explants were taken out of their culture vessels. Due to the generally low rooting rate in the dark, the data on root length are given only for the experiments or experimental stages conducted in the light. Furthermore, in the experiment with ABA, the rate of explants with visible shoot apex necrosis (browning) was scored.
For the comparison of the obtained means, a two-tailed Student’s t-test was performed to calculate the probability that the means of the two different treatments were equal. The differences between the two treatments were considered significant at the calculated probability p < 0.05.

3. Results

3.1. Populus Shoot Culture Responses to Darkness and the Response Alterations by PBZ

The results of the experiment, testing the responses of the two Populus genotypes to darkness and how these responses can be changed by the GA biosynthesis inhibitor PBZ, are shown in Figure 1.
Prominent differences between the two genotypes were observed in the pattern of shoot growth regulation by darkness and by PBZ (Figure 1a). The shoots of H275, grown in the dark, were 3.7 times longer than in light. This dark-induced shoot elongation in H275 was suppressed by the PBZ back to the control (light) level. A quite different situation was observed with the IBL 91/78 explants. In contrast to H275, the average shoot length of IBL 91/78 was not increased by the dark treatment and, correspondingly, not affected by the PBZ applied in the dark.
With respect to the rooting rate (Figure 1b), H275, in comparison to IBL 91/78, was generally characterised by a much poorer performance. For instance, in the control variant, the rooting rate of H275 (17.1%) was 3.6 times lower than that of IBL 91/78. Both genotypes had lower rooting rates in the dark than in light; however, this difference was statistically significant only in IBL 91/78, where the rooting rate dropped from 62% to 20% in response to darkness. Furthermore, the combination of darkness and PBZ resulted in zero rooting in both genotypes.

3.2. Alterations in Populus Shoot and Root Development Induced by PBZ in Light

A further comparison of the responses of the two Populus genotypes to PBZ was done by culturing explants in the jars under the 16-h photoperiod. In H275, both the average shoot length (Figure 2a) and rooting rate (Figure 2b) were significantly decreased on the medium with PBZ, in comparison to the control. In contrast, the IBL 91/78 explants did not decrease significantly either their average shoot length or rooting rate in response to PBZ.
A further comparison of the two genotypes’ responses to PBZ in the jars, was done also with respect to the root system parameters of rooted explants. The number of adventitious roots per explant is shown in Figure 2c, and both largest root length and total root length are shown in Figure 2d. Although PBZ was found to decrease the general rooting rate among the H275 explants (Figure 2b), those explants that were still able to root with the PBZ treatment had a 2.5-times higher average root number than their counterparts on the control medium (Figure 2c). In contrast, the IBL 91/78 culture remained unaffected by the PBZ in this respect. However, the PBZ effects on root elongation were similar in both genotypes, causing a 3.5-fold and 2.9-fold decrease in the average largest root length of the H275 and IBL 91/78 explants, respectively (Figure 2d). The PBZ-induced adventitious root proliferation in the H275 culture did not fully compensate for the loss in root length, and the average total root length of the rooted H275 explants in the PBZ-treated variant was still 1.9 times lower than in the control. This difference, in relative terms, was very similar to the 2.1-fold decrease that PBZ also caused in the total root length of the IBL 91/78 explants (Figure 2d).

3.3. Populus Shoot Culture Responses to Exogenous GA

The effects of exogenous GA on the H275 and IBL 91/78 explants are shown in Figure 3. Most explants responded to the presence of GA4 + 7 in the nutrient medium by abnormal shoot elongation; the average shoot length was doubled, in comparison to the control, in both genotypes (Figure 3a). Other typical features of GA-treated explants included lack of adventitious roots, relatively narrow leaves (Figure 3b,c) and, particularly among the IBL 91/78 explants, shoot outgrowth from axillary buds (Figure 3c).

3.4. Short-Term and Long-Term Responses to Exogenous ABA in Populus Shoot Cultures

The short-term and long-term responses to the exogenous ABA in the two studied Populus genotypes are shown in Figure 4.
With respect to the average shoot length (Figure 4a), both H275 and IBL 91/78 genotypes were affected negatively by the ABA in the first stage of the experiment, during which the explants were cultured either on the hormone-free or ABA-supplemented medium in the dark. In H275, the effect of the ABA proved to be long-lasting and led to even stronger shoot growth inhibition in the second stage, when the newly excised explants from the ABA-treated shoots, as well as from their control counterparts, were transferred to the hormone-free medium in the jars and cultured under a 16-h photoperiod for an additional five weeks. In contrast to H275, the shoot growth of the IBL 91/78 explants in the second stage was not impaired at all by the previous ABA treatment, if compared to the untreated control (Figure 4a).
In the culture of H275, ABA-induced shoot growth inhibition was often accompanied by shoot apex necrosis (Figure 4b). The apex necrosis rate was approximately two times higher with the ABA medium than with the ABA-free medium. Interestingly, the corresponding difference of almost the same size also remained after the transfer of both ABA-treated and control H275 explants onto the fresh hormone-free medium for the second culture stage, considering that these new subcultures were prepared only from the shoots without visible signs of apex necrosis. In contrast to H275, the IBL 91/78 did not suffer increased shoot apex necrosis rate under or after the ABA treatment (Figure 4b).
With respect to rooting (Figure 4c), no effect of the ABA could be demonstrated for the H275 explants during the first culture stage, because of the almost total absence of rooted explants in both control and ABA-treated groups. During the second stage, a small portion (21.3%) of the H275 explants from the control treatment formed roots; however, the explants from the ABA-treatment remained largely incapable, not only of shoot elongation but also of root formation, with a significant difference from the control (Figure 4c). For the IBL 91/78 explants, the ABA effect on the rooting rate was negative during the first culture stage, with an eight-fold drop from the control level. However, this negative effect was lost during the second culture stage on the hormone-free medium; here, the IBL 91/78 explants from the control group showed 50% rooting rate and the ABA-treated group followed closely behind, with an insignificant difference (Figure 4c).
The above-described rooting pattern of the IBL 91/78 explants during the second culture stage, allowed for a further comparison between the control and ABA-treated variants, because, inside either variant, two almost equally sized groups, those of unrooted and rooted explants, were obtained. Accordingly, Figure 5a shows the effect of the previous ABA treatment on the shoot growth in the groups of unrooted and rooted IBL 91/78 explants separately, and Figure 5b shows the corresponding effect on the root growth of the rooted explants. It was found that the unrooted explants were able to develop only very short shoots, with the average length of approximately 3 mm, and no difference was observed between the control and the ABA treatments (Figure 5a). In contrast, the rooted explants from the control variant developed shoots with the average length of 12.4 ± 1.8 mm and, furthermore, the rooted explants obtained after the previous ABA treatment had, on the average, even 75% longer shoots than their control counterparts (Figure 5a). Moreover, the analysis of root growth (Figure 5b) showed that the explants prepared from the ABA-treated shoots surpassed the control by the average length of an explant’s largest root, which was about 60% higher in the former variant than in the latter. Still, the two variants did not differ from each other in regard to the total root length (Figure 5a).

4. Discussion

4.1. Analysis of Populus Genotype Responses to the Darkness and GA-Related Growth Regulators

The obtained results indicate that there are essential differences between the studied Populus genotypes, H275 and IBL 91/78, in regard to the environmental and hormonal regulation of growth responses. Increased shoot elongation of the H275 explants in the dark and its inhibition by the GA biosynthesis inhibitor PBZ (Figure 1a) suggests that the in vitro shoot development mechanism of this genotype may be similar to that reported in Arabidopsis seedlings, where dark-induced hypocotyl elongation was found to be dependent on GA [13,38]. Even in the light, the H275 explants were seemingly more dependent on GA for their shoot growth than the IBL 91/78 explants, because when the culturing was conducted in the jars under a 16-h photoperiod, the PBZ treatment suppressed shoot elongation for the H275, but not the IBL 91/78 (Figure 2a). Interestingly, while PBZ did not affect the shoot growth of IBL 91/78, it significantly suppressed its adventitious root (AR) elongation (Figure 2d). Although the bulk of GA-related studies have historically focused on the action of this hormone in plant shoots, the importance of GA for root elongation has also been reported; particularly, in the context of root-accumulated DELLA proteins [39]. DELLAs function as negative transcription regulators and are degraded by the GA signal [40]; hence, the importance of GA for plant growth in a specific situation may be dependent on the absence or abundance of these regulatory proteins. The analysis of the DELLA genes by Liu et al. [41] in one selected genotype of hybrid poplar Populus deltoides × P. euramericana resulted in the isolation and characterisation of four DELLA genes which had different expression levels in different plant parts, such as ARs, stems, and leaves. Three of the four DELLA genes had their strongest expression levels in the 2-3-week-old ARs rather than in the stems or leaves [41]. In this context, the present investigation tested if the inability of PBZ to suppress IBL 91/78 shoot elongation, in contrast to the AR elongation, is associated with a decreased sensitivity of the IBL 91/78 shoot segments to GA, which, according to King et al. [42], might indicate the shortage of DELLA proteins. However, the obtained results did not support this possibility, as the IBL 91/78 shoot segments were no less sensitive to the exogenously applied GA than those of H275 (Figure 3).
To suggest another explanation for the resistance of IBL 91/78 shoot growth to the PBZ, it can be speculated that the relatively intense growth of this genotype in the light, surpassing that of H275, may indicate a more important role of other hormonal factors here, instead or alongside GA. Root formation differences, observed between the H275 and IBL 91/78 explants, suggest a stronger action of the endogenous auxin in the latter genotype. On the hormone-free medium, IBL 91/78 clearly surpassed H275 in both rooting rate (Figure 1b and Figure 2b) and the number of roots per explant (Figure 2c). Auxin is the most widely reported hormonal inducer of adventitious root formation both in Populus [43] and in other plant species [44,45]. Furthermore, the positive role of auxin in the promotion of shoot elongation is well reported in Arabidopsis [46], and confirmed in Populus [47]. Moreover, in Arabidopsis seedlings, it was found that auxin was required particularly for shoot elongation in light conditions [48], and the idea of the specific importance of auxin for plant growth in the light was further developed in the review by Halliday et al. [49]. One of the reported mechanisms for how auxin promotes shoot [50,51,52] and root [53] elongation, is through enhanced GA biosynthesis, as auxin increases the level of GA oxidases, which convert the immediate GA precursors to active gibberellins. However, the ability of auxin to promote shoot growth independently of GA has also been established in a previous investigation [54]. Hence, the two Populus genotypes from this study, which were found to contrast in their responses to the darkness and to an inhibitor of GA synthesis, could provide a good experimental material for further research on auxin-GA interactions.

4.2. Analysis of Populus Genotype Responses to the ABA Treatment

The differences between the H275 and IBL 91/78 development patterns were also extended to their responses to exogenous ABA. The exogenous ABA was found to have persistent inhibitory effects on the development of H275, but not IBL 91/78, shoot cultures.
ABA is a well-reported antagonist of GA and contradicts the action of the latter hormone through several mechanisms [55,56]. The repression of the dark-induced shoot elongation by ABA in the H275 explants was essentially similar to the effect which was found for ABA in respect of hypocotyl growth in Arabidopsis seedlings by Lorrai et al. [14]. The latter authors reported that the ABA action resulted in decreased expression of GA biosynthetic genes. It is of note that in the present study, the effect of ABA on H275 explants corresponded well with that of the GA biosynthesis inhibitor PBZ, as both of these growth regulators similarly decreased H275 shoot elongation in the dark (Figure 1a and Figure 4a). However, a GA-independent path for ABA-induced inhibition cannot be excluded as well. In the process of seedling development, as it was for instance reported in Arabidopsis [57], ABA can act as an inhibitor of cotyledon greening and prevents seedlings from establishing their full photosynthetic capacity. In the present case, almost 44% of the ABA-treated H275 explants, instead of developing green leaves after being transferred from the continuous darkness to a 16-h photoperiod, got necrotic shoot tips (Figure 4b), and remained incapable of any further development. Whether the ABA-induced greening inhibition processes both in Arabidopsis seedlings and the H275 shoot explants share the same signalling pathways, requires further investigation.
In contrast to H275, the IBL 91/78 explants responded to ABA treatment in a more complex manner. Direct culturing of IBL 91/78 explants on the medium with ABA, resulted in a relatively slight, although statistically significant, decrease in shoot length (Figure 4a) and nearly complete inhibition of adventitious rooting (Figure 4c). Both effects could have resulted from ABA antagonism with auxin, rather than with GA because, as discussed above, neither the IBL 91/78 explants showed dark-induced shoot elongation, nor their shoot growth was inhibited by PBZ. In turn, various modes of antagonism between ABA and auxin are well-reported in model plant systems. For instance, ABA was found to prevent the outgrowth of Arabidopsis axillary buds by suppressing auxin accumulation in these buds through the down-regulation of certain genes responsible for auxin biosynthesis and transport [58]. Moreover, the inhibition of Arabidopsis hypocotyl elongation by ABA, as reported by Lorrai et al. [14], was achieved not only through the regulation of GA metabolic genes, but also through the repression of auxin biosynthetic genes. Moreover, a study conducted on grey poplars (Populus × canescens)—the hybrids whose parent species, P. tremula and P. alba, are the same as for the genotype IBL 91/78—showed that the poplars transformed with the mutant Arabidopsis abi1 gene for ABA-insensitivity, did not differ from their wild-type counterparts, with respect to their GA levels [59].
The most intriguing aspect of the differences between the H275 and IBL 91/78 responses to the exogenous ABA, however, became evident after the explants from the medium with ABA were transferred to hormone-free medium. In contrast to the H275 explants whose development remained suppressed by the previous ABA treatment, the ABA-affected IBL 91/78 explants recovered their growth particularly well, surpassing their counterparts from the control variant both in shoot length (Figure 5a) and, to a lesser extent, root length (Figure 5b). Thus, instead of continuing to inhibit shoot growth, the ABA from the previous subculture had a growth-promoting effect long term. Considering the reported data about the auto-regulation of ABA biosynthesis, it seems unlikely that the increased growth in IBL 91/78 cultures after the ABA treatment could have resulted from a restrictive effect of ABA on its own biosynthesis, because the genes involved in ABA biosynthesis are up-regulated rather than suppressed by ABA [60]. In turn, the potential role of ABA in the regulation of auxin conjugation should be considered. Auxin homeostasis in plants is regulated not only through their biosynthesis but also through their conjugation with certain organic molecules into inactive compounds. For instance, GH3 proteins that are widespread in plants may control the level of active auxins by binding excess auxin indole-3-acetic acid to amino acids [61]. A study of the GH3-encoding gene isolated from hybrid larch (gene LaGH3) revealed that the promoter region of LaGH3 had ABA-inducible elements, and the expression of this gene was strongly down-regulated by exogenous ABA [62]. Hence, ABA seems not to only be able to decrease auxin biosynthesis [14], but, on the other side, also induce the maintenance of auxin in an active state. If, under certain circumstances, the latter effect of ABA overcomes the former, auxin-inducible growth responses, such as increased shoot growth, may be obtained as a result of the ABA treatment. Hence, the actual involvement of auxin in the increase of IBL 91/78 shoot growth, after the ABA treatment, should be investigated further.
Although ABA has a long history of being considered as plant growth inhibitor, various applications of this hormone for practical plant breeding purposes have also been suggested. Several reports indicated that exogenous ABA may be used to increase adventitious root (AR) formation on plant cuttings. Such effects were reported early in pea [63] and, also, in Populus [64]. A study on Vigna radiata stem cuttings [65] suggested that the increase in AR formation that followed the ABA treatment may have been related to ABA-induced changes in the levels of active and conjugated auxin. In hydroponic rice cultures [66] and in in vitro silver birch cultures [32], ABA was found to promote lateral root formation. Another use of ABA in vitro is known from the studies on tobacco cultures [67,68], with respect to the transition of in vitro-grown plants to ex vitro conditions. ABA was reported both as an internal regulator, which accumulated in response to ex vitro conditions [67] and it was found that the addition of ABA to the last in vitro subculture of tobacco plants, increased their water use efficiency ex vitro and prevented them from wilting [68]. In the present study of Populus, the morphometric parameters of ABA-affected IBL 91/78 shoots, such as being, on average, longer than 2 cm and having approximately 7 cm long adventitious roots, corresponded well to the suggestions by García-Angulo et al. [30] about the qualifications of Populus in vitro plants for ex vitro transfer. However, the current results also indicated that certain limits for the application of ABA should be considered. The genotype H275 (P. maximowiczii × P. trichocarpa), whose shoot growth, as discussed above, seemed to be largely GA-dependent, suffered ABA-induced long-term inhibition with respect to both shoot growth and AR formation. Thus, out of the two studied Populus genotypes, only IBL 91/78 (P. tremula × P. alba), whose shoot elongation was neither induced by darkness nor inhibited by PBZ, showed a positive long-term response to the ABA treatment. ABA is known as one of the most important chemical signals that enables poplars to cope with various abiotic stresses, including environmental toxicity [69,70] and drought [71]. It was shown that, to be able to survive water shortage, poplars must be responsive to ABA [59] but, also, that stress-induced hyperaccumulation of ABA might be a negative factor for drought resistance in poplars [72]. Hence, the ability of genotype IBL 91/78 both to respond to ABA directly and to recover and even increase its growth during the subsequent culture stage, after an ABA treatment, makes this genotype an interesting object for future studies, aimed at the assessment of stress responses in Populus.

5. Conclusions

Our results clearly showed that there are significant differences between the studied genotypes of Populus, H275 and IBL 91/78 in terms of environmental and hormonal regulation of growth responses. The genotype H275 (P. maximowiczii × P. trichocarpa) responded to darkness with PBZ-inhibitable shoot elongation; in contrast, the elongation of IBL 91/78 (P. tremula × P. alba) shoots was not affected either by darkness or PBZ treatment. Moreover, the explants of H275 were unable to recover their growth if it was inhibited with ABA; in contrast, those of IBL 91/78 recovered so well after the temporal inhibition by ABA that, when rooted subsequently, they developed longer shoots and roots than without a previous ABA treatment. Our results indicate that GA catabolism and repressive signalling provide an important pathway to control growth and physiological adaptation in response to immediate or impending adverse conditions. These observations can help breeders to define robust criteria for identifying genotypes with high resistance and productivity and highlight where genotypes exhibit susceptibility to stress. The need to combine the selection of genotypes resistant to abiotic stress with productivity in poplar breeding programmes is particularly important in the face of rapid climate change.

Author Contributions

Conceptualization, J.Ž., I.S.-B. and M.N.; methodology, J.Ž., I.S.-B. and S.K.; validation, J.Ž. and M.N.; formal analysis, J.Ž. and G.S.; investigation, J.Ž., G.S., and I.S.-B.; resources, I.S.-B., S.K. and M.N.; data curation, J.Ž.; writing—original draft preparation, J.Ž. and G.S.; writing—review and editing, J.Ž. and M.N.; visualization, J.Ž. and G.S.; supervision, J.Ž. and M.N.; project administration, M.N.; funding acquisition, M.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was carried out under a scholarship grant to Jonas Ziauka within a scholarship fund of the Forest Research Institute, Poland, pursuant to the decision of the Head of the Institute dated 23 April 2018 (based on agreement no. 1/2018 concluded on 2 May 2018) and funding provided by the Directorate General of the State Forests (Poland) (project nos. 500 425, 500 468) for Marzena Niemczyk and Iwona Szyp-Borowska.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the first (and corresponding) author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Explant development parameters of Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba) after four weeks of culturing in Petri dishes (55 mm × 12 mm): (a) shoot length (increase from the original 10 mm explant length); (b) rate of explants with adventitious roots. Different culture conditions/treatments are denoted: “C”—control variant, cultured on PGR-free medium under the 16-h white-light photoperiod; “D”—differs from the control variant in that the explants were cultured in the dark; “D + PBZ”—the explants were cultured in the dark on the nutrient medium with paclobutrazol (1 µmol L−1). Different lower-case letters indicate that means are significantly different from each other at p < 0.05, with comparisons made for each genotype separately.
Figure 1. Explant development parameters of Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba) after four weeks of culturing in Petri dishes (55 mm × 12 mm): (a) shoot length (increase from the original 10 mm explant length); (b) rate of explants with adventitious roots. Different culture conditions/treatments are denoted: “C”—control variant, cultured on PGR-free medium under the 16-h white-light photoperiod; “D”—differs from the control variant in that the explants were cultured in the dark; “D + PBZ”—the explants were cultured in the dark on the nutrient medium with paclobutrazol (1 µmol L−1). Different lower-case letters indicate that means are significantly different from each other at p < 0.05, with comparisons made for each genotype separately.
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Figure 2. Shoot and root development parameters of Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba) after four weeks of culturing in glass jars (62 mm × 70 mm) under a 16-h white-light photoperiod. (a) Shoot length (increase from the original 10 mm explant length); (b) Rate of explants with adventitious roots; (c) Root number per explant (with only rooted explants included); (d) Largest and total root length for an individual rooted explant. Nutrient medium differences are denoted: “P−”—control, without plant growth regulators; “P+”—with 1 µmol L−1 of paclobutrazol (PBZ). Significant differences between the control and the PBZ treatment are indicated: * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2. Shoot and root development parameters of Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba) after four weeks of culturing in glass jars (62 mm × 70 mm) under a 16-h white-light photoperiod. (a) Shoot length (increase from the original 10 mm explant length); (b) Rate of explants with adventitious roots; (c) Root number per explant (with only rooted explants included); (d) Largest and total root length for an individual rooted explant. Nutrient medium differences are denoted: “P−”—control, without plant growth regulators; “P+”—with 1 µmol L−1 of paclobutrazol (PBZ). Significant differences between the control and the PBZ treatment are indicated: * p < 0.05; ** p < 0.01; *** p < 0.001.
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Figure 3. Gibberellin (GA) effects on the development of Populus explants from genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (Populus tremula × P. alba) after three weeks of culturing in glass jars (62 mm × 70 mm) under a 16-h white-light photoperiod. Different variants of nutrient medium are denoted: “C”—control, without plant growth regulators; “GA”—with 1 µmol L−1 of GA4 + 7. (a) Comparison of the average shoot length (increase from the original 10 mm explant length); * indicates significant differences between the control and the GA4 + 7 treatments at p < 0.05. (b) Morphology of H275 explants; (c) morphology of IBL 91/78 explants.
Figure 3. Gibberellin (GA) effects on the development of Populus explants from genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (Populus tremula × P. alba) after three weeks of culturing in glass jars (62 mm × 70 mm) under a 16-h white-light photoperiod. Different variants of nutrient medium are denoted: “C”—control, without plant growth regulators; “GA”—with 1 µmol L−1 of GA4 + 7. (a) Comparison of the average shoot length (increase from the original 10 mm explant length); * indicates significant differences between the control and the GA4 + 7 treatments at p < 0.05. (b) Morphology of H275 explants; (c) morphology of IBL 91/78 explants.
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Figure 4. Explant development parameters of Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba), as affected by abscisic acid (ABA) treatment. (a) Shoot length (increase from the original 10 mm explant length); (b) rate of explants with visible shoot apex necrosis; (c) rate of explants with adventitious roots. Nutrient medium differences are denoted: “Control”—hormone-free medium; “ABA”—medium with 3 µmol L−1 of ABA. In the first stage, explants were cultured for three weeks in the dark, while being placed on different nutrient media in Petri dishes (55 mm × 12 mm). In the second stage, explants from the two different first stage media were transferred onto fresh hormone-free medium in glass jars (62 mm × 70 mm) and cultured for five weeks under a 16-h white-light photoperiod. Significant differences between the control and the ABA treatment are indicated: ** p < 0.01; *** p < 0.001.
Figure 4. Explant development parameters of Populus genotypes H275 (P. maximowiczii × P. trichocarpa) and IBL 91/78 (P. tremula × P. alba), as affected by abscisic acid (ABA) treatment. (a) Shoot length (increase from the original 10 mm explant length); (b) rate of explants with visible shoot apex necrosis; (c) rate of explants with adventitious roots. Nutrient medium differences are denoted: “Control”—hormone-free medium; “ABA”—medium with 3 µmol L−1 of ABA. In the first stage, explants were cultured for three weeks in the dark, while being placed on different nutrient media in Petri dishes (55 mm × 12 mm). In the second stage, explants from the two different first stage media were transferred onto fresh hormone-free medium in glass jars (62 mm × 70 mm) and cultured for five weeks under a 16-h white-light photoperiod. Significant differences between the control and the ABA treatment are indicated: ** p < 0.01; *** p < 0.001.
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Figure 5. Growth parameters of the genotype IBL 91/78 (Populus tremula × P. alba) in the second experimental stage, as affected by abscisic acid (ABA) treatment in the first stage. (a) Shoot length (increase from the original 10 mm explant length), shown separately for unrooted and rooted explants; (b) the largest and total root length for an individual rooted explant. In the first stage, explants were cultured for three weeks in the dark, while being placed on different nutrient media in Petri dishes (55 mm × 12 mm). In the second stage, explants from the two different first stage media were transferred onto fresh hormone-free medium in glass jars (62 mm × 70 mm) and cultured for five weeks under a 16-h white-light photoperiod. The first stage nutrient medium differences are denoted: “Control”—hormone-free medium; “ABA”—medium with 3 µmol L−1 of ABA. Significant differences between the control and the ABA treatment are indicated: * p < 0.05.
Figure 5. Growth parameters of the genotype IBL 91/78 (Populus tremula × P. alba) in the second experimental stage, as affected by abscisic acid (ABA) treatment in the first stage. (a) Shoot length (increase from the original 10 mm explant length), shown separately for unrooted and rooted explants; (b) the largest and total root length for an individual rooted explant. In the first stage, explants were cultured for three weeks in the dark, while being placed on different nutrient media in Petri dishes (55 mm × 12 mm). In the second stage, explants from the two different first stage media were transferred onto fresh hormone-free medium in glass jars (62 mm × 70 mm) and cultured for five weeks under a 16-h white-light photoperiod. The first stage nutrient medium differences are denoted: “Control”—hormone-free medium; “ABA”—medium with 3 µmol L−1 of ABA. Significant differences between the control and the ABA treatment are indicated: * p < 0.05.
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