In our previous work [13] we studied the impact of temperature on growth cessation, dormancy development, and cold acclimation of four poplar cultivars. These temperature regimes changed the kinetics of dormancy development patterns with the 18/3 °C treatment inducing the widest separation of dormancy depth. Therefore, to elucidate if there is a relationship between the expression levels of CBF genes and dormancy in poplar, two cultivars differing in their dormancy acquirement based on night temperature responses were tested under short-day conditions in our current research.
Dormancy development
Growth cessation
Growth cessation is the first indication of dormancy induction [57] and was induced in both genotypes (Fig. 1). Consistent with Kalcsits et al. (2009), a significantly earlier and steeper drop could be observed in the ‘Okanese’ compared to ‘Walker’ cultivar, between the 3rd and the 4th week of dormancy inducing conditions. After the 5th week, no further growth was recorded. ‘Walker’ showed higher growth rate at every time-point, with the exception of the 1st week. By the end of the experiment, no significant difference was found between these two cultivars (0.36 and 0.51 cm * week-1 in Okanese and Walker, respectively).
Dormancy Induction
While growth cessation was a more sensitive indicator, significant differences in the number of days to bud break were found between the two cultivars from day 40 (Fig. 2). Okanese buds took 10 days to break bud at Day 0, while at the end of the experiment (i.e. on the 50th and 60th day) this value was increased and levelled off at 13.5 and 14.1 days (respectively). Conversely, the duration of bud break was hardly changed in ‘Walker’ over the whole 60 days treatment period, just a slight fluctuation was recorded (Fig. 2). No difference was detected between the first and the last days (days to bud break: 8.4 and 8.2 days, respectively).
The depth of dormancy was reflected by the parameter ΔDBB (Differences between the first and last Days to Bud Break) and dormancy was not induced at all in ‘Walker’ (ΔDBB=0.2). Conversely in ‘Okanese’, the dormant state started to be induced after 30 days. At the end of the experiment, Okanese had a ΔDBB of 4.1 (Fig. 2). The data on growth cessation rate and the bud break analysis indicates that ‘Okanese’ reached a deeper dormant state than ‘Walker’. Our results are consistent with the outcome of Kalcsits et al. (2009) [13], who reported characteristic differences between these two cultivars – ‘Okanese’ was shown to be more capable of endodormancy development under the 18/3oC day/night temperature treatment under 12 h and 10 h daylengths although a larger difference was found between the two cultivars (ΔDBB: 13.9) in that study.
Expression pattern of CBF genes
The expression patterns of six CBF genes were recorded over the whole experiment. Samples were collected from leaf and bud tissues every ten days, taking into account the circadian rhythms of many CBFs, in the same period of the day, i.e. 4 – 6 hours after the start of the light period. The expression of each gene in a given time-point was normalized to the level measured at the beginning (i.e. on the 0 day) of the given treatment.
Differences in the kinetics and spatial localization of the overall CBF expression were observed between the two cultivars. The highest levels of CBF expression across the entire experiment were recorded in the bud tissue, isolated from ‘Okanese’ on the 10th day (Fig. 3A) and on the first day in ‘Walker’ leaf samples (Fig. 3D). The expression levels in ‘Okanese’ poplar buds peaked at the 10th day and were at least an order of magnitude (10-20 fold) higher than in ‘Walker’ buds, and at any other time during the experiment for all the CBFs (with the exception of PtCBF5). There was differential expression of bud PtCBFs between the two cultivars in that PtCBF1 and PtCBF5 showed the highest and lowest expression in ‘Okanese’ buds, respectively, while the reverse was observed in ‘Walker’. In ‘Walker’ leaves on the 1st day, PtCBF1 and PtCBF2 expression levels were roughly equivalent to ‘Okanese’, however, ‘Walker’ leaf expression of PtCBF3, PtCBF4, PtCBF5 and PtCBF6 spiked on the 1st day and were 150 – 200 times greater than ‘Okanese’ (Fig. 3C, D).
The expression patterns of the unique CBF genes are described in detail in the Supplemented Fig 1. In bud tissue, PtCBF2, PtCBF3 and PtCBF5 were induced only in the beginning of the experiment, on the 10th day, while PtCBF1 and PtCBF6 were induced not only at the beginning but also at the end of the treatment, on the 50th and 60th (PtCBF1) or on the 40th day (PtCBF6). The induction level was always an order of magnitude higher in the ‘Okanese’ buds compared to ‘Walker’ for each CBF. The repression of CBF genes was more pronounced in the ‘Walker’ buds. A repressed period was recorded in the middle of the experiment for PtCBF1, PtCBF5 and PtCBF6 genes in ‘Walker’ buds, while only one repressed stage was found in ‘Okanese’ in the mid period of PtCBF3 expression (Supp. Fig. 1A, D and E).
By contrast in leaf tissue, two induction waves could be observed in the leaf samples for all CBFs in ‘Walker’: the first was at the beginning (on the 1st and 20th day), while the second was at the end (50th and 60th day). Induction waves were also found in ‘Okanese’ leaves, but in the opposite direction, since repression of all CBFs was detected in the period 1st-10th and 30th-40th and finally on the 60th day. It is interesting to note the differential responses between the cultivars in leaf tissues in that CBF induction was found in ‘Walker’ leaves, while repression was found almost in every case in ‘Okanese’ leaves (Supp. Fig. 1F-K). Thus, these two cultivars had similar but opposite PtCBF expression under dormancy inducing conditions based on buds or leaves.
Differences in the CBF expression kinetics and levels measured in the meristematic (bud, stem) and leaf tissues were studied in several cases in woody plants, among them poplar. Benedict et al. (2006) described different CBF induction patterns in P. balsamifera ssp. trichocarpa showing that all four PtCBFs are cold-inducible in leaves, while only two (PtCBF1 and PtCBF3) were cold induced in the stem [35]. Under a short-expression period (24 hours), they concluded, ‘the perennial driven evolution of winter dormancy led to the development of specific roles for abiotic stress response regulators, such as the CBFs, in annual and perennial tissues’. CBF expression was followed in leaf and leaf bud tissues in Prunus mume during one year by Zhao et al. (2018) [33]. They also found a differential gene expression pattern for all six CBFs studied, with specific induction kinetics. In that study, all six CBFs were induced in vegetative buds, in the cold period (November – January); PmCBF4, PmCBF5 and PmCBF6 being the most intensively expressed. These three CBFs were also the most induced in the leaf tissues. But interestingly, in leaves, the highest expression for all 6 CBFs was recorded during the warmest period, from June to July. This finding is in accordance with our results, i.e. that the CBF expression was much more intense in leaves of the non-dormant cultivar, may indicate that their role in the development in dormancy is organ-specific. Six PmCBFs in 7 different organs were determined in P. mume [33]. The induction levels were high in stems, moderate in flower buds, leaf buds, and leaves, poor in flowers, fruits, and seeds.
Gene duplication and multiplication produced a large number of CBFs in many species. This redundancy makes possible the divergence of functionality, and the possibility for fine-tuning of adequate response for any environmental stimuli, such as stress. As mentioned above, 6 CBFs encoded in the P. mume genome exhibited different expression kinetics during the year: PmCBF1, PmCBF2, and PmCBF3 were up-regulated in the stem tissues not only in the cold period but also in late spring [34]. Additionally, low temperature up-regulated 8 CBFs in Prunus mume which subsequently induced all six DAM genes resulting in dormancy development [36]. Under natural dormancy induction conditions, 3 out of 4 CBFs showed similar expression trends in Pyrus pyrifolia bud tissues, while PpCBF1 showed a different induction kinetic [37]. During an artificial chilling test, PpCBF1 was the only CBF highly expressed, while PpCBF2 was repressed intensively, and the levels of PpCBF3 and PpCBF4 were undetectable.
These results show that although CBF expression kinetics may be similar, differences in the individual expression patterns can be distinguished. Shortening the light period by 2 hours/day to account for the variance in nature (at the same temperature regime) may have caused a moderate functional polymorphism in our experiment. PtCBF4 was detectable only in ‘Walker’ leaves, while PtCBF1 and PtCBF6 were the most intensively expressed genes in ‘Okanese’ buds. Whether they have different functions, as was suggested for PpCBF4 [37] in pear, is still unclear. It is also remarkable that PtCBF5 was the only gene which was not induced during the CBF-burst on the 10th day in ‘Okanese’ buds but was the most intensively up-regulated in ‘Walker’ buds on the 1st day. Therefore, we assume PtCBF5 is not related to dormancy development.
Leaf samples of Populus balsamifera ssp. trichocarpa genotypes originating from northern and southern populations were examined [58]. A growth chamber study showed all PtCBF genes were induced by cold, indicating functional redundancy. On the other hand, under field conditions, a more diverse gene expression pattern was described. The expression of PtCBFs increased as the growing season progressed, but among the six genes, only PtCBF3 was marginally differentially expressed across latitudes. In our experiment, leaf samples also showed a certain level of functional polymorphism, but the most common outcome of the two systems is that in leaves, no dormancy dependent expression pattern was found, such a relation was present only in the bud tissues.
PtDAM1 identification and its expression kinetics
DAMs (Dormancy-Associated MADS-Box) are well-characterized genes in perennial plants, associated with various components of the dormancy cycle but particularly dormancy induction. DAM sequences had already been published in woody plants, all containing K-box and SRF-TF motifs [33, 59, 60]. The P. trichocarpa genome has been sequenced [61], however, it is still poorly annotated. We have found 151 candidates for the DAM genes. From these, we suggested the XP_024452024.1 protein entry as a putative PtDAM1 product (Fig. 4, Fig. 5). Howe et al. (2015) studied transcriptome changes during endodormancy induction by microarray in P. trichocarpa and found several DAM-like SVP genes were differentially expressed but were downregulated during endodormancy [53]. Since sampling was conducted on a once per month basis, it is not clear if upregulated peaks were missed.
Having identified a PtDAM1 gene in Populus, we decided to evaluate its potential role in dormancy development, using cultivars known to be differentially responsive to night temperatures. Therefore, primers were developed to study the encoding PtDAM1 gene expression. Compared to the first sampling day, mild up-regulation of the identified putative XP_024452024.1 (is corresponds to older versions as MADS7, Potri.002G105600) sequence was recorded in ‘Okanese’ leaves through the experiment, while lower induction was found in ‘Walker’. PtDAM1 was repressed from the middle of the experiment (Fig. 6) and the expression of PtDAM1 was almost unchanged throughout the 60 days in leaf tissues. The bud tissues showed much more pronounced induction than the leaf tissues. In more dormant ‘Okanese’, the maximum expression (2.8-fold) was recorded on the 10th day then the induction gradually declined. Repression was recorded in both cultivars at the end of the treatment. PtDAM1 induction in buds was weaker in the first half of the treatment in ‘Walker’ (1.1 – 1.6-fold induction) which did not enter endodormancy.
Similar expression trend for PmDAM1 gene was described in Japanese apricot (Prunus mume) bud tissue, but differently in the leaf samples [47]. In the vegetative buds, expression of PmDAM1 (as well as PmDAM2 and PmDAM3) was upregulated from June to July, i.e. long before the start of growth cessation, then expression started to decrease. We also showed an initial PtDAM1 induction in our system, well before the start of growth cessation, or dormancy development. We found no characteristic changes in leaf tissues, however, in Prunus mume, different kinetic patterns were described in this organ [47]. Two seasonal expression trends were shown for P. mume DAMs, PmDAM1 (together with PmDAM2 and PmDAM3) was rapidly up-regulated in spring, being gradually down-regulated in autumn. This difference in the expression in leaf tissue might be explained by the two different experimental systems. In other studies in peach (Prunus persica), differential DAM gene expression appeared to be related to dormancy induction or fulfillment of the chilling requirement phases. Based on the ever-growing peach mutant system, Li et al. (2009) reported DAM1, DAM2 and DAM4 were the most likely candidates associated with growth cessation and dormancy induction [45]. Using the same system, Yamane et al. (2011) showed under both field and controlled environment conditions and in leaves and stems, DAM5 and DAM6 gene expression levels were up-regulated during endodormancy induction and downregulated during endodormancy release which appeared to be tied to chilling requirement satisfaction [62]. Furthermore, DAM5 and DAM6 gene expression levels were higher in high chill cultivars and reduced with chilling requirement satisfaction [63]. DAM5 and DAM6 genes were negative regulators of bud break.
Dynamics of hormone changes during dormancy development
Phytohormones have been long known to be involved in the dormancy cycle [23, 25, 64–72]. Recently, mechanistic relationships between phytohormones and dormancy are being revealed [36, 73, 74].
In our study, due to the very small size of poplar buds and only limited capacity of growth chambers, hormone analysis was conducted only in leaf samples. Overall, phytohormonal response in ‘Okanese’ was different than in the ‘Walker’ poplar hybrid cultivar with most significant distinction for Ox-IAA, phaseic Acid, DAM1, cis-zeatin riboside-O-glucoside (cZROG) (Fig. 7, 8). Exposure of poplar plants to short photoperiod and low night temperatures was associated with down-regulation of ABA content in leaves of both genotypes (Fig. 8). However, an early (on the 1st day) transient elevation of the ABA metabolite, phaseic acid, indicated enhanced ABA degradation in the ‘Okanese’ cultivar, suggesting a preceding short-term up-regulation of ABA content early after temperature drop. This assumption is supported by the report on transient up-regulation of ABA in cold-stressed wheat leaves [75]. The ethylene precursor ACC was elevated in both clones. Ruttink et al. (2007) showed ethylene rise preceded ABA during dormancy induction [64]. Jasmonate has been known to be involved in several stress responses [76]. Inactivation of the repressors of JA signaling pathway - jasmonate ZIM-domain (JAZ) proteins, which physically interact with ICE1 and ICE2 transcription factors, results in up-regulation of CBFs [77]. CBF genes promote gibberellin deactivation and thus growth inhibition [78]. In our study in leaf tissue, JA levels were suppressed in both genotypes during the entire experimental period, and more in ‘Okanese’. However, JA level in leaves need not correlate with its content in buds. Moreover, JAZ inactivation may be achieved by their interaction with DELLA proteins [79, 80], which accumulate at low temperature and are stabilized by gibberellin down-regulation. In contrast to JA, SA levels were increased at the beginning of the experiment, one week longer in ‘Okanese’. This agrees with the positive effect of SA on plant cold tolerance [81]. After the 3rd week, the SA content was unchanged in both cultivars, however, the concentration was lower in the less cold-hardy ‘Walker’. Benzoic acid, the precursor of SA and other phenolic compounds, was elevated during the experiment; in ‘Okanese’ until dormancy initiation, in ‘Walker’ during the whole experiment. These changes demonstrate differences in hormonal dynamics between the clones during leaf senescence (Fig. 8).
The auxin, indole-3-acetic acid (IAA), had varying levels across the 60-day treatment in both cultivars. However, the main IAA catabolite, Ox-IAA, had a more consistent response, being up-regulated in ‘Okanese’ and down-regulated in ‘Walker’, which indicates stronger IAA deactivation in ‘Okanese’ leaves. Dormancy initiation, associated with substantial suppression of growth rate, was accompanied by IAA down-regulation, which was not observed in the non-dormant clone. Baldwin et al. (2000) showed that while the auxin naphthaleneacetic acid was not required for bud scale development, its absence was critical [82].
The whole cytokinin pathway was downregulated in Okanese compared to Walker: the precursors iPRMP and tZR increased only in Walker, the active form (iP) decreased only in Okanese, and the deactivated form iP7G was accumulating in Okanese and decreasing in Walker. Other compounds did not show any major changes between both trees.
Cytokinin analysis clearly showed that promotion of dormancy in ‘Okanese’ was associated with a general decrease of cytokinin biosynthesis and profound elevation of their deactivation products in leaves (Fig. 7, Fig. 9, Supple. Fig. 2). Collectively, these results provide new evidence that the degradation of growth-promoting phytohormones such as IAA and cytokinins may be an important mechanism of endodormancy induction.
The relation between PtCBFs and PtDAM1 expression, hormone level and the development of dormancy
A CBF-burst occurred on the 10th day of the short photoperiod and low night temperature treatment in ‘Okanese’ bud tissues, while in ‘Walker’ CBF levels were an order of magnitude lower (Fig. 3). In ‘Okanese’ which was able to enter endodormancy (Fig. 2), CBF1 had the highest relative expression at the initiation of dormancy. PtDAM1 expression peaked in ‘Okanese’ exactly on the same 10th sampling day (Fig. 6). By contrast, ‘Walker’ which did not attain endodormancy (Fig. 2) had a lower CBF expression on the 1st day (Fig. 3), while PtDAM1 expression was also low and unchanged during the experiment (Fig. 6). Growth rate started to decline in both cultivars by the 3rd week, but at a much faster rate in ‘Okanese’ (Fig. 1). These findings support the possible relationship between PtCBF1, PtDAM1 induction and endodormancy development.
The dormancy-associated phytohormone, ABA, was surprisingly down-regulated in leaves of ‘Walker’ and even more downregulated in ‘Okanese’. However, the concentration of the ABA degradation intermediate, phaseic acid, increased in ‘Okanese’ while it was reduced in ‘Walker’ and therefore, an ABA induction peak in ‘Okanese’ leaves may have been missed (Fig. 8). Recent evidence indicates a role of DAM1 in activating NCED3 through binding to its promoter and upregulating ABA biosynthesis in Japanese pear [83]. The same study found high concentrations of ABA can also reduce DAM1 in a feedback regulatory loop. DAM proteins are similar to SVP (Short Vegetative Phase), one of the flowering time regulators in Arabidopsis. In kiwifruit, Wu et al. (2017) performed a transcriptomic analysis and found AcSVP2 may mimic ABA action [85]. They further indicated that SVP2 was mediated by ABA to decrease meristem activity and prevent premature bud break. DAMs also appear to play a regulatory role in the ABA signaling pathway [85]. Thus, there is increasing evidence that CBF and DAM gene actions are linked with phytohormonal concentration and action in dormancy. The reverse has also been demonstrated in that Knight et al. (2004) earlier showed ABA to upregulate CBF expression [86]. Singh et al. (2019) reported that SVL is the ortholog of SVP in aspen (Populus tremula x tremuloides), which mediates photoperiodic dormancy induction via callose synthase, operating downstream of ABA [74]. Singh et al. (2018) also showed ABA induced the expression of the DAM/SVL gene in hybrid aspen [87]. For an excellent recent review, see Liu and Sherif (2019) [25].
In a recent study, analysis of a transformant hybrid aspen (Populus tremula x tremuloides) showed that expression of SVL, a negative regulator of bud break, was down-regulated in hybrid aspen buds after low temperature treatment. It was noted that nonetheless, SVL is similar to DAM genes, clustering closer to SVP in Arabidopsis and apple than to hybrid aspen or peach DAM genes [74, 87]. Interestingly, SVL induced the expression of callose synthase and negatively regulated the gibberellin pathway. Moreover, CBF14 and 15 upregulated the GA2ox5 gene which deactivates gibberellins in barley [88].
Dormancy is known to be induced primarily by temperature in some fruit species, such as apple and pear [89]. Increasing evidence highlights the role of temperature, especially in the case of northern woody cultivars. While the main regulator of growth cessation and dormancy induction in woody species is short photoperiod, it may be moderated by, and interact with temperature [17]. The increasing confirmation of direct regulation by cold-induced CBFs on DAM gene expression [34, 37, 56], Niu et al. (2016) provided evidence and proposed a model in which CBF induces DAM and DAM downregulates FT which then suppresses growth and stimulates the development of dormancy [38]. Liu and Sherif (2019) further outlined a model integrating multiple phytohormonal networks regulated by DAM [25]. Key among them was the direct suppression by DAM of cytokinins, gibberellins and direct activation by DAM of ABA and callose deposition. Our study provides additional evidence that cytokinin and IAA degradation may be an important regulatory mechanism to endodormancy induction.