The timing of the onset and release of dormancy impacts the survival, productivity and spatial distribution of temperate horticultural and forestry perennials and is mediated by at least three main regulatory programs involving signal perception and processing by phytochromes (PHYs) and PHY-interacting transcription factors (PIFs). PIF4 functions as a key regulator of plant growth in response to both external and internal signals. In poplar, the expression of PIF4 and PIF3-LIKE1 is upregulated in response to short days, while PHYA and PHYB are not regulated at the transcriptional level. Integration of light and environmental signals is achieved by gating the expression and transcriptional activity of PIF4. During this annual cycle, auxin promotes the degradation of Aux/IAA transcriptional repressors through the SKP–Cullin-F–boxTIR1 complex, relieving the repression of auxin-responsive genes by allowing auxin response factors (ARFs) to activate the transcription of auxin-responsive genes involved in growth responses. Analyses of transcriptome changes during dormancy transitions have identified MADS-box transcription factors associated with endodormancy induction. Previous studies show that poplar dormancy-associated MADS-box (DAM) genes PtMADS7 and PtMADS21 are differentially regulated during the growth-dormancy cycle. Endodormancy may be regulated by internal factors, which are specifically localized in buds. PtMADS7/PtMADS21 may function as an internal regulator in poplar. The control of flowering time shares certain regulatory hierarchies with control of the dormancy/growth cycle. However, the particularities of different stages of the dormancy/growth cycle warrant comprehensive approaches to identify the causative genes for the entire cycle. A growing body of knowledge also indicates epigenetic regulation plays a role in these processes in perennial horticultural and forestry plants. The increased knowledge contributes to better understanding of the dormancy process and consequently to precise manipulation of dormancy-related horticultural traits, such as flowering time.
Because of their multiyear life spans, temperate perennial horticultural and forestry plants have an adaptive mechanism that alters active growth and vegetative dormancy in accord with seasonal climate changes. These plants use cyclically changing environmental signals, such as day-length and temperature, to coordinate their growth and development with seasonal changes in climate (Figure 1).1,
The overarching question of the molecular mechanisms that govern the dormancy and growth changes in perennial plants in response to seasonal climatic variation remains largely unanswered. While temporal coincidence between the seasonal changes and the internal biological clock is thought to be prerequisite for these cyclical processes, the underlying genetic control must be more complex than simply a response to shifts in these two environmental stimuli given the highly complex and physiologically different responses to these stimuli in spring and fall. Indeed, in many localities, comparable temperatures and day lengths are present during spring and fall, yet in one instance, dormancy is induced and in the other the initiation of annual growth occurs. Fulfilling a chilling requirement prior to breaking dormancy has been suggested as the reason for this differential response,5 but the genes or regulation of gene expression associated with such a mechanism have not been identified. By what mechanisms then do perennial plants, such as fruit trees, use the roughly equivalent photoperiods and temperatures of spring and fall, as well as the intervening seasons of warmth and cold, to regulate these two dramatically different physiological responses?
Better understanding of the molecular mechanisms controlling the annual growth and dormancy cycle has the potential to help mitigate the impact of climate change on plant productivity and survival by providing vital information about how temperate perennials utilize the environmental cues to trigger adaptive mechanisms. Increases in winter temperature would be expected to cause delayed and erratic flower development in tree species with large chilling requirements and both early spring bud break and increased risk of frost damage in species with low chilling requirements. Dormancy also determines winter survival of both native and invasive perennials and thus affects agricultural productivity and biodiversity in the ecosystem. Identification of expression-based molecular markers for dormancy regulation may facilitate cultivar selection and breeding for development of regionally suited crops in accord with changes in the global climate.
Recent efforts to elucidate the transcriptional regulatory mechanisms by which woody plants use environmental stimuli to coordinate their periods of growth with the annual cycle of environmental conditions have contributed much to our understanding of these processes. The growth-dormancy cycle in temperate perennials appears to be tightly controlled by complex genetic regulatory networks in response to endogenous and environmental signals.3,8,
Perception of short-day photoperiods induces growth cessation and transition to dormancy
Due to their lack of variation from year to year, seasonal changes in photoperiod provide a consistent source of information for the timing of developmental events in perennial plants. Plants monitor photoperiod changes by means of light-sensitive pigments, including PHY, which play central roles in modulating key physiological and developmental processes.19,20 PHYs, red and far-red light photoreceptors, are synthesized in the cytosol and translocated to the nucleus as the active Pfr form in response to red light.21,
PIF4 functions as a key regulator of plant growth in response to external signals, such as temperature and light, as well as internal signals, including gibberellin and the circadian clock. Light-activated photoreceptor PHYs (Pfr) directly interact with the basic helix–loop–helix transcription factors named phytochrome interacting factors, PIFs.28,30,
Both light and temperature signaling serve as critical environmental cues for growth cessation and dormancy onset. Perception of SD photoperiods induces growth cessation and transition to dormancy.38 Poplar responds to SDs with a PHYA-mediated cessation of elongation growth, terminal bud set and dormancy.7,15,
Cellular response to plant growth regulators is a part of seasonal dormancy regulation
The plant hormone auxin regulates virtually every aspect of plant growth and development. Therefore, how the auxin signal is perceived and interpreted by plant cells is also a central question in the biology of dormancy. In poplar, SD-induced growth cessation and dormancy onset involves dormancy cycle stage-specific alterations in auxin responsiveness of the transcriptome in the stem tissues.8 The cellular response to auxin is quite rapid, with transcription regulation of a large and diverse set of genes detectable within minutes of exposure.47 Proteins involved in auxin distribution and signaling are induced by auxin, including auxin efflux machinery components (e.g., PIN proteins), influx (e.g., LAX proteins) carriers and Aux/IAA proteins that are unstable transcriptional repressors of auxin responses.48 The expression of Aux/IAAs, primary auxin response genes, is rapidly upregulated by auxin. The Aux/IAA proteins interact with auxin response factors (ARFs), a family of transcription factors that bind auxin-responsive promoters and regulate transcription.49 This interaction negatively regulates auxin-responsive gene expression by keeping ARFs from forming functional dimers.50 Auxin promotes the degradation of Aux/IAA transcriptional repressors through the ubiquitin protein ligase SKP–Cullin-F–boxTIR1 complex (SCFTIR1),51,52 thereby relieving the active repression of the auxin-responsive genes by allowing ARFs to activate the transcription of auxin-responsive genes.53
The F-box protein subunit of the SCF complex (TIR1 in the case of Aux/IAA) confers target specificity. Direct binding of auxin to TIR1 allows the TIR1-Aux/IAA binding. Crystal structures show that auxin binds to a single surface pocket of TIR1 and the Aux/IAA substrate peptides, docked on top of auxin, occupy the rest of the TIR1 pocket.54,55 TIR1 is required for auxin response in Arabidopsis.56 Quadruple mutants of TIR1 and its three homologs AFB (tir1 afb1 afb2 afb3) are auxin-insensitive,57 further supporting the hypothesis that auxin stimulates binding of SCFTIR1 to the AUX/IAA repressor, and its degradation, resulting in the activation of auxin-responsive genes. The expression of PttTIR1, a poplar homolog of TIR1, was reduced by 50% after 42 SDs preceding endodormancy, suggesting its role in SD-induced growth cessation and dormancy onset in poplar.8 In order to gain insight into how auxin signaling induces growth cessation and dormancy onset, it is imperative to identify the molecular regulators that mediate SD modulation of auxin responsiveness in perennial plants.
In Japanese apricot (Prunus persica), various hormone response genes, such as INDOLE-3-ACETIC ACID-INDUCIBLE 33 (IAA33), the gibberellin-regulated gene family, ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5 (ERF5), the auxin-responsive gene family and a member of the F-box/RNI-LIKE gene family, were differentially regulated at four different seasonal dormancy stages, suggesting their involvement in dormancy regulation.58 Exogenous application of GA4 resulted in earlier budbreak in Japanese apricot.59 The proteomic and transcriptomic analysis after the GA4 treatment showed that proteins and genes associated with energy metabolism appear to be involved in dormancy release.
Dam genes may function as a positive regulator of endodormancy induction
Analyses of transcriptome changes during dormancy transitions in several perennial plant species, including poplars, have identified several MADS-box transcription factors associated with endodormancy induction. The first definitive evidence that DAM genes directly impact endodormancy was obtained from the analysis of evergrowing (evg) mutant of peach [Prunus persica (L.) Batsch], which fails to cease growth and enter dormancy under dormancy-inducing conditions.60,61 The DAM genes are expressed in terminal buds of the wild type, whereas their expression is lost in the evg mutant.62 Expression of DAM3, DAM5 and DAM6 during winter indicated involvement of the transcription factors in endodormancy were expressed during winter, with expression of DAM5 and DAM6 higher in high chill cultivars and at a minimum coincident to bud break, indicating a potential role in repressing bud break.63 Deletion of six tandem-arrayed DAM genes (PpMADS1-6) in the EVG locus resulted in loss of endodormancy induction.64,65 DAM homologs in leafy spurge (Euphorbia esula), EeDAM1 and EeDAM2, were associated with endodormancy induction.13
DAM genes have a high sequence similarity to two different transcription factors, SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE24 (AGL24), which were previously associated with flowering regulation in the winter annual Arabidopsis. Yamane et al.66 identified a MADS-box gene with endodormancy-associated expression in Japanese apricot (Prunus mume). This gene has a high sequence similarity with SVP and AGL24 and is upregulated during endodormancy induction and downregulated during endodormancy release.63 SVP inhibits flowering through negative regulation of a floral integrator FLOWERING LOCUS T (FT),67,68 while AGL24 acts as a promoter of flowering.69 SVP is upregulated under short day conditions in Arabidopsis.13 In poplar, overexpression of FT inhibits SD-induced growth cessation.70,71 Transgenic poplars overexpressing PHYA have elevated expression levels of FT and FT-like genes, such as CENL1.69 Expression of FT or CENL1 is downregulated in poplar18,72 and spruce during induction of endodormancy.73 FT expression in leafy spurge buds was coordinately down regulated as DAM gene expression was induced,13 indicating coordinated regulation of genes associated with flowering and with dormancy onset and release in accord with cyclical changes in the growth environment.
The analysis of the phylogenetic relationship between Arabidopsis, peach and poplar MIKCC-type MADS-box genes shows that the peach DAM genes belong to a clade with two Arabidopsis (AGL22/SVP and AGL24) and eight poplar (PtMADS7, PtMADS21, PtMADS26, PtMADS27, PtMADS28, PtMADS29, PtMADS47 and PtMADS48) MADS-box genes.74 In the clade, two poplar homologs, PtMADS7 and PtMADS21, formed a monophyletic group with the six peach homologs. Transgenic poplar plants constitutively overexpressing an apricot DAM (PmDAM6) showed growth cessation and terminal bud set under environmental conditions that are conducive to continued growth.75 Our preliminary data show that the two poplar DAM genes (PtMADS7 and PtMADS21) are differentially regulated during the growth-dormancy cycle. Based on the findings reported in the literature and our preliminary study, it is possible that endodormancy is regulated by internal factors (e.g., internal growth inhibitors), which are specifically localized in buds during endodormancy and prevent the buds from resuming growth. Thus, it is prudent to consider that PtMADS7/PtMADS21 may function as such an internal regulator in poplar.
Transcriptome changes occurring during the growth-dormancy cycle
Studies on phenological traits in Populus suggest that the timing of bud flush may be controlled by a modest number of major genes with low environmental variation.76,77 However, no such genes have yet been identified. The changes in gene expression during both the onset and breaking of dormancy in poplar have been described.6,7,18 Schrader et al.78,79 and Druart et al.10 reported a comprehensive analysis of cambial dormancy utilizing cryosection-isolated cambial cells from P. tremula during active growth and dormancy. These studies used cDNA microarray experiments (POP1)80 to investigate transcriptional changes underlying cambial dormancy. However, the POP1 cDNA microarray consists of only 13 490 clones with high redundancy, which is not enough to cover the entire transcriptome of poplar in which currently at least 45 778 protein-coding loci have been identified (Phytozome, http://www.phytozome.net/poplar.php). Nonetheless, very significant insights into dormancy/growth cycle regulation were gained when Bhalerao and his colleagues carried out global transcript profiling (POP2 cDNA Arrays) during the activity-dormancy transition in the cambial meristem in a hybrid aspen.8 Their findings suggested that dormancy stage-specific changes of auxin responsiveness of the transcriptome might be involved in SD-induced growth cessation. Recently, Ko et al.14 obtained whole genome transcriptional profiles (Affymetrix Poplar GeneChip Arrays) covering the winter and summer stems of field-grown P. trichocarpa trees. The study showed high levels of transcriptional activity and differential regulation of a broad array of genes beyond those associated with environmental stress during the dormancy period. In grape (Vitis vinifera), global transcriptome analysis during bud development showed that a MADS-box gene (VvFLC2) and other transcripts with similar expression patterns appear to be involved in the regulation of endodormancy induction.81 In kiwifruit, upregulation of the glutathione S-transferase class of genes, which are associated with stress responses, was observed in response to application of hydrogen cyanamide used to synchronize budbreak.82 In raspberry, microarray analysis of gene expression during dormancy transition enabled identification of key dormancy-related genes, with a high percentage of stress-response genes present in the expression profiles.83 Hedley et al.84 identified 842 differentially expressed ESTs (expressed sequence tags) during dormancy release in blackcurrant (Ribes nigra) with high representation of genes encoding transcription factors such as zinc finger family members (B-box, CCCH, C2H2 and C3HC4/RING types), which are known to play important roles in signal transduction pathways. The EST analysis also showed that expression of a Short Vegetative Phase (SVP)-type MADS box transcription factor gene peaked in early winter and gradually declined into spring-time. Homologues of SVP in apricot also showed similar expression pattern to that of blackcurrant under dormancy release inducing condition.66,83
Recent discoveries have shown that the control of flowering time shares certain regulatory hierarchies with control of the seasonal dormancy/growth cycle.85,86 Overexpression of a poplar homolog of FLOWERING LOCUS T2 (FT2) suppressed SD-induced growth cessation and dormancy onset and its downregulation by RNAi led to less vegetative growth.71 In addition, overexpression of a poplar FLC-like MADS box gene delayed SD-induced growth cessation and leaf senescence in poplar.87 In leafy spurge, analysis of 1000 genes differentially expressed through dormancy transitions led to the identification of a MADS-box transcription factor related to the DAM genes from peach. The authors hypothesize that it may play a direct role in dormancy induction and maintenance through regulation of FLOWERING LOCUS T.13 Rinne et al.88 investigated the molecular and cellular-level changes occurring during the dormancy cycle at the shoot apical meristem (SAM) of poplar. That study showed that chilling-induced expression of 1,3-β-glucanases at the SAM reopens signal conduits (i.e., plasmodesmata) and subsequently enables the symplastic movement of FT to its target, resulting in dormancy release.89 While these commonalities between flowering and dormancy regulation offer new avenues to critically assess the transcriptional network for seasonal regulation of the dormancy/growth cycle, the particularities of different stages of the dormancy/growth cycle, especially in terms of gene expression in the meristems, warrant various comprehensive approaches to identify the causative genes involved in the entire dormancy cycle.
Epigenetic regulation of dormancy
There is growing evidence that genome-wide epigenetic regulation of gene expression is involved in dormancy regulation (for reviews) (Table 1).90,91 Whole-genome DNA methylation and acetylated histone 4 (H4) analyses in chestnut (Castanea sativa) showed higher DNA methylation ratios and lower H4 acetylation levels in dormant buds compared to actively growing tissues,92,93 suggesting gene silencing concomitant with bud dormancy. In hybrid aspen, putative histone deacetylases (HDA14 and HDA08), histone lysine methyltranferase (SUVR3) and histone ubiquitination involved gene (HUB2) were upregulated during growth-dormancy transition, while several Trithorax family genes counteracting the repressive effect of Polycomb complex and putative DEMETER-like DNA glycosylases are downregulated.89 These results suggest that some unknown active growth regulating genes might be repressed during dormancy induction through chromatin compaction mechanisms, such as histone deacetylation and methylation, histone ubiquitination, DNA methylation and Polycomb activity. Ko et al.14 also reported that many genes involved in chromatin remodeling/modification in poplar (e.g., including BRAT1, SWI2/SNF2, NFC2 and PAF1) showed upregulation in winter/dormancy stems, suggesting the dynamic nature of winter/dormancy-responsive gene regulation at chromatin level (Figure 2).
Two genes similar to FERTILIZATION INDEPENDENT ENDOSPERM (FIE) and PICKLE (PKL), which are involved in epigenetic regulation, were strongly up-regulated in poplar immediately after transfer to SD conditions.18 FIE is known as a component of the Polycomb repressive complex 2 involved in the trimethylation of H3 lysine 27 (H3K27me3), while PKL is an ATP-dependent chromatin remodeler that regulates embryo identity traits.94,95 In fact, transgenic hybrid aspen lines with RNAi-mediated suppression of FIE were not able to establish dormancy, even though growth cessation and bud formation processes did not seem to be affected.96
A time period of quantitative and cumulative chilling is required for both dormancy release and vernalization for flowering in Arabidopsis. In this process, transcriptional regulation of MADS box genes, FLOWERING LOCUS C (FLC) and DAM, appears to include common epigenetic mechanisms. Horvath et al.86 reported that the chromatin in the promoter region of leafy spurge DAM1 had a decrease in the level of trimethylation of H3 lysine 4 (H3K4me3) (i.e., active histone modification) and an increase of H3K27me3 (i.e., repressive histone modification) after a prolonged cold exposure. Likewise, the two histone modifications were also found in the vernalization-dependent repression of FLC in Arabidopsis.97,98 In addition, downregulation of PdDAM6 after dormancy release was associated with a decrease of H3 acetylation in the chromatin around the ATG region.99
Taken together, epigenetic mechanisms appear to be involved in the seasonal growth-dormancy regulation of perennial plants. However, the details of these mechanisms are so far unknown. Further research, such as annual growth-dormancy stage-specific genome-wide DNA methylation and histone acetylation analysis in perennial trees, is required to identify the main components.
Many avenues of research remain to be explored before the genetic regulation of the complex physiological processes associated with the onset and breaking of dormancy and the integration of these processes with the regulation of flowering are understood. It is apparent that this involves a broad array of genes beyond those just associated with environmental stress. That auxin plays a major role has long been evident, but changes in the auxin responsiveness of the transcriptome need to be more fully addressed as PIF involvement is better understood. Continued identification and classification of DAM genes from additional species is needed to identify the common functional features of clades of these sequence-specific proteins. As microarray technologies continue to evolve and be widely used in gene expression studies, next-generation sequencing technologies such as Illumina, 454 Life Sciences (Roche) and Applied Biosystems (ABI) offer unparalleled depth, specificity and sensitivity for confident discovery of novel and rare transcripts. The growing availability of high quality annotation in many horticultural species now provides the opportunity for large-scale RNA-Seq-based Digital Gene Expression to examine the critical changes in transcriptome during growth-dormancy cycle. Such future work should yield deeper insights into the transcriptional regulation of the annual growth cycle. Clearly also, genome-wide epigenetic regulation of gene expression is involved in dormancy regulation with possible regulatory gene repression through chromatin compaction. Future research to elucidate the role of gene silencing in dormancy onset is needed.
Economically, dormancy regulation represents a very significant physiological problem that is critical to fruit and other horticultural sectors, and also to attaining increased, sustainably produced energy and fuel from intensive, short rotation plantation production of biomass. In agriculture, inappropriate dormancy responses in fruit tree crops will result in reduced productivity due to winter damage in flower buds. In fruit trees with a low chilling requirement for spring dormancy release, increased or erratic winter temperature will result in early spring bud-break and an increased risk of frost damage. On the other hand, species with a greater chilling requirement may not undergo proper flower development with higher winter temperatures. Increased understanding of the genetic regulation of the timing of dormancy induction and release, as well as the depth of endodormancy and thus cold hardiness, could profoundly impact horticultural production systems. In many regions, local climate is a key factor in selecting sites for the production of cherries, blueberries, peaches, apricots, wine grapes and other fruits and vegetables. For these crops, the lower limit of the sustained cold temperatures during winter and other microclimatic factors involving erratic spring temperatures or early frosts are often as critical as soil type and water availability in defining, and thus limiting, production zones. In these areas, producers are challenged annually by seasonal climate variation. For instance, a warm spring followed by a cold snap resulted in about 90% crop loss in tart cherries and apples in Michigan in 2012. In many fruits including apples and cherries, developing cultivars with even a one-week delay in the breaking of dormancy would greatly reduce the annual risk of crop loss to early frost and thus increase farm profitability in regions where early frosts regularly cause severe damage. It would also enable the development of new cultivars with growing seasons and hardiness levels tailored to specific planting zones and sites where these crops cannot currently be grown due to erratic frosts or cold temperatures, thereby increasing the geographic range over which some fruit crops could be produced and expand the production of locally grown fruits and vegetables.
More efficient woody biomass production for biofuels could also be accomplished by tailoring the growth and dormancy cycle to the environmental conditions where the plants are grown and could lead to increased primary productivity of temperate perennials grown in short rotation biomass plantations. For instance, carefully shortening the dormancy period, with the understanding that excessive prolonging of the growth period carries a tradeoff between winter survival and biomass production, may result in significant increase in growing period. This would be particularly effective for increasing biomass accumulation by indeterminant growth habit species like Populus, where current growth rates for biofuels are often marginally profitable. Tailoring the onset of dormancy to maximize utilization of the full growing season would result in increased late season growth and thus, accumulation of additional feedstock annually. Although this would not be of as great of economic significance as potentially expanding fruit and vegetable production regions, it could well lift these short rotation biofuel systems above the cutoff for profitability and enable successful deployment of short rotation woody biofuel crops in areas where the climate and marginal profitability currently limit production of these crops on retired farm lands.