TY - JOUR
AU - Cregg, Bert M
AB - Abstract Forest tree improvement programs provide the basis for most of our knowledge of cone induction in conifers. Since World War II, forest geneticists and tree breeders have largely selected for traits that improve productivity, and improved seed is now commonly produced in seed orchards. However, seed production in many conifers varies widely from year to year, regulated in part by environmental conditions. Therefore, much of the research in conifer reproduction has focused on enhancement of cone development during summers when weather conditions are unfavorable to reproductive bud initiation. In this review, we synthesize current knowledge about the manipulation of cone production in temperate conifers. We provide an overview of conifer reproductive biology, describe the progression from reproductive incompetence to reproductive maturity, and discuss both endogenous (e.g., genetic, epigenetic, hormonal) and exogenous (e.g., temperature, moisture, tree size) regulation of reproductive development. Finally, we summarize the most common approaches to cone enhancement in seed orchards, provide direction for future research, and suggest possible mechanisms that might govern reproductive development, such as the GA-DELLA and miR156/SPL modules. forest tree improvement, conifer reproduction, seed orchard, tree stress, seed cone Much of what we know about strobilus induction in conifers stems from tree improvement programs that service the timber and pulpwood industries. Since World War II, forest geneticists and tree breeders have systematically selected for traits that improve wood quality and productivity (i.e., growth rate). Improved seed is commonly produced in seed orchards (Miller and DeBell 2013). However, many conifers are mast-seeding species, producing copious amounts of seed across broad geographic areas under favorable conditions, only to be followed by several years of low seed production (Kelly 1994). Thus, considerable research has centered on enhancement of cone development under unfavorable conditions. This review attempts to synthesize what we have learned about the biology, regulation, and manipulation of cone production in temperate conifers. Although our focus is on reproductive maturation and development in conifers, we turn frequently to model systems (e.g., Arabidopsis, apple, pea) because maturation mechanisms are at least partially conserved in conifers (Carlsbecker et al. 2013). We are hopeful this will prove useful to researchers interested in exploring basic reproductive biology in conifers and to seed orchard managers looking to refine their technique. Biology of Reproductive Development in Conifers Stages of Strobilus Development Strobilus development in conifers is a complex, multistage process. Three distinct stages—induction, initiation, and differentiation—are of particular importance in understanding cone development. During strobilus induction, the meristem in the lateral bud is reprogrammed to transition from vegetative to reproductive growth. This stage is identifiable by the upregulation of certain proteins and nucleic acids required for further development. The second stage, strobilus initiation, is characterized by the first detectable morphological changes (shape or size) within the meristem, which reorganizes to prepare for the production of reproductive structures. The third stage in early strobilus development is differentiation, which is marked by the organogenesis of reproductive structures and represents an irreversible change that persists even if the tree is released from the conditions that originally promoted strobilus induction (Sedgley and Griffin 1989). We will follow the lead of Owens and Blake (1985) in using initiation to refer to the first two stages (induction and initiation) combined. This is to avoid confusion because induction is commonly used in the literature to refer to promotion of cone development by chemical or cultural means, such as in a seed orchard (e.g., Owens and Blake 1985). In addition, the precise timing of induction has not been determined for most species because it precedes any morphological change; therefore, it is more difficult to detect than subsequent stages. Timing of Strobilus Initiation and Differentiation The timing of strobilus initiation and differentiation is governed by thermal time (growing degree-days) and varies by genus and species. For example, in Abies, axillary bud primordia develop on newly expanding shoots approximately 1 week before vegetative bud-break (Powell 1974, Owens and Blake 1985). Bud-scale initiation commences, and continues for several weeks, coinciding with shoot elongation. Strobilus initiation occurs in early summer, when lateral shoot elongation begins to slow, toward the end of bud-scale initiation. Biochemical and anatomical changes occur over the next several weeks, with differentiation complete near the end of lateral shoot elongation. The timing of strobilus initiation and differentiation is essentially the same for Pseudotsuga and Picea, with differentiation complete by the end of bud-scale initiation and lateral shoot elongation (Owens and Blake 1985). Strobilus development for many conifers is summarized in Figure 1. Figure 1. View largeDownload slide Times and methods of cone initiation. From Forest tree seed production. 1985. Owens, J.N; Blake, M.D. Agriculture Canada, Can. For. Serv., Petawawa National Forestry Institute, Chalk River, Ont. Information Report PI-X-53. 161 p. Reproduced with permission, 2016. Figure 1. View largeDownload slide Times and methods of cone initiation. From Forest tree seed production. 1985. Owens, J.N; Blake, M.D. Agriculture Canada, Can. For. Serv., Petawawa National Forestry Institute, Chalk River, Ont. Information Report PI-X-53. 161 p. Reproduced with permission, 2016. Phases of Reproductive Development Conifers change in their sensitivity to environmental signaling as they progress through the three phases (incompetent, competent, and mature) of postembryonic reproductive development (Poethig 1990, Greenwood 1995). In the reproductively incompetent phase, only vegetative growth is possible and the conifer cannot form reproductive structures, even under strongly inductive conditions that would otherwise result in reproductive development. In the reproductively competent phase, a conifer may form reproductive structures, but only in the presence of strong external cues, such as drought stress or exogenous application of gibberellin (GA). In the reproductively mature phase, reproductive structures are produced regularly, regardless of environmental conditions (Bond 2000, Williams 2009). To avoid ambiguity, we will restrict the terms juvenile and adult to refer to phases of vegetative maturation. Advancing through developmental phases is a long, complex process in conifers that enables them to respond to changes in environment, size, and complexity as their life cycle progresses (Day and Greenwood 2011). Phase change is not governed by a single, regulatory event but is facilitated by multiple, overlapping processes that affect both vegetative and reproductive development (Hackett and Murray 1993). Meristem maturation involves complex changes that alter the meristem’s response to internal and external signals, engendering reproductive competence and morphological changes, while reducing regenerative potential and vigor (Trewavas 1983, Hackett 1987, Hutchison et al. 1990, Greenwood 1995, Poethig 2003). Gene expression, operating at the level of the meristem, interacts with internal and external factors to regulate the morphological and physiological changes that offer strategic advantages specific to the current life stage and position in the tree (Poethig 1990, Day and Greenwood 2011, Wendling et al. 2014a). Management and Policy Implications Cone production in conifers is controlled by internal and environmental factors, often resulting in highly variable cone crops. Applications of GAs can often help to stabilize cone production. Developing knowledge of genetic control of coning may further increase the ability of seed orchard managers to produce reliable cone crops. Masting Many conifers are mast-seeding species, meaning that seed production is highly variable across years but synchronized across a population (Janzen 1976, Kelly 1994). Depending on the species, the synchrony may be detectable across sites for hundreds or even thousands of kilometers (Koenig and Knops 1998), although plasticity in response to local site conditions is evident (Crone et al. 2011, Roland et al. 2014). Mast seeding comes at the expense of vegetative growth, and is thought to be an evolutionary strategy that involves mass-scale reproductive output to increase fitness by, for example, satiating seed predators (Janzen 1976) and improving wind pollination (Kelly 1994, Koenig and Knops 1998). Weather and the availability of resources, such as nutrients, water, and light, likely serve as environmental signals that synchronize mast seeding. Long-term accumulation of resources, such as nutrients and photosynthates, may create an additional, internal trigger (Koenig and Knops 2000). Sensitivity to these factors varies by species and decreases with age. In general, trees accumulate resources during periods conducive to vegetative growth, when rainfall and temperatures are in the optimal range. In reproductively competent trees, the availability of sufficient reserves within the tree may then create a permissive state for reproductive development. Subsequent hot, dry conditions during bud development encourage strobilus initiation and differentiation. A mast year with heavy cone production follows 1–2 years later, depending on the length of the reproductive cycle (Owens and Blake 1985, Roland et al. 2014). Growing and developing strobili are strong sinks for photosynthates and other resources, such as nitrogen and phosphorus. Reserves must be replenished to repeat the masting cycle (Powell 1977, Sala et al. 2012). In biennially bearing species, such as balsam fir (Abies balsamea [L.] Miller), cone production generally occurs every other year, indicating that the temporal pattern of cone production is under endogenous control. In contrast, the number of strobili produced in a mast year is highly regulated by weather and resource availability. The biennial pattern is most simply explained by source–sink relations: Initiation of new strobili is likely limited by competition for local resources among existing, growing and developing cones, which are a strong sink for photosynthates (Powell 1977). Markers of Developmental Phase Change Much of the information on developmental phase change in plants is provided by studies in model systems, such as Arabidopsis, in which flowering may occur just weeks after germination. In long-lived conifers, phase-change transitions may occur over months or years, and it may be decades before reproductive maturity is attained. The ability to gauge maturation state is critical in clonal forestry and tree improvement programs, in which mature material must often be rejuvenated to propagate trees with desirable traits. Phase-change indicators in forest trees have been comprehensively reviewed by Wendling et al. (2014a) and include decreased regeneration potential; changes in foliar and stem properties; changes in vegetative vigor and habit; and various hormonal, physiological, and biochemical changes. These gauges give an approximate idea of the developmental state, particularly when strong correlations have been noted between the marker and developmental phase in a particular species. However, such indicators are often imprecise and may confound vegetative and reproductive phase change, which often overlap but appear to be genetically and physiologically distinct (Wiltshire et al. 1994, 1998, Hasan and Reid 1995, Abedon et al. 1996, Jordan 1999). Plastic responses to environmental signals may also mimic ontogenetic morphological or physiological changes. Identification of a highly conserved biochemical marker would greatly improve the rate and precision of scientific discovery in the area of reproductive phase change in woody plants (Wendling et al. 2014a). Reproductive Structures At present, the most reliable indicator of reproductive competence is the formation of reproductive structures. However, because formation of reproductive structures is dependent on environmental signaling and other pathways, lack of reproductive structures does not rule out the possibility that reproductive competence has already been physiologically attained. Vegetative Traits Because reproductive structures form in vegetatively mature regions of the tree, such as on branch tips and in the upper crown (Grace 1939, Hackett 1987), markers of vegetative maturation may be useful as a proxy for markers of reproductive maturation in the absence of observable reproductive structures. Morphological Traits.—Various morphological and physiological changes may occur as a tree progresses from the juvenile to adult vegetative phase (Poethig 1990). Before onset of reproductive structures, these traits offer the best gauge of the degree of reproductive maturation (Wendling et al. 2014a). Needle and stem morphology, such as increasing needle width with age, are useful indicators of vegetative phase change in temperate conifers. However, in reciprocal grafting studies, red spruce (Picea rubens Sarg.) needle characteristics were influenced by both scion and rootstock age, suggesting interactions between multiple control pathways (Day and Greenwood 2011). This limits their usefulness in rejuvenation efforts, in which needle morphology may not accurately predict other age-related traits, such as rooting potential. In addition, it is important to differentiate ontogenetic traits, which develop in a predictable pattern independent of environmental conditions (Poethig 2013), from plastic traits, such as morphological changes during organogenesis in response to environmental cues. For example, it has long been known that leaf morphology of heteroblastic plants changes as the plants mature (Goebel 1900). For these plants, leaf morphology may be used as an ontogenetic marker. However, leaves on plants grown in shade may exhibit juvenile morphological traits. This has been interpreted as evidence that shade slows whole plant development, increasing the duration of juvenility (Goebel 1908, Njoku 1956). Jones (1995) was the first to demonstrate that developmental plasticity at the level of the individual leaf could account for the morphological changes attributed to juvenility. Thus, the shape of the leaf was a function of light interception during development, not delayed vegetative maturation, although the leaf morphologically resembled a juvenile leaf. It is not easy to separate out which factors are actually influencing the rate of maturation and not simply triggering a local plastic response to environmental conditions. Vegetative Regeneration Potential.—Woody plants, especially conifers, generally exhibit declining vegetative regeneration potential as the tree matures (Greenwood 1995). In many species, roots or shoots may be induced adventitiously from the vascular parenchyma or cambial cells in juvenile cuttings (Díaz-Sala et al. 1996, Ballester 1999, de Klerk et al. 1999). This competence is lost with age, and adult cuttings may not readily form roots even in response to exogenous application of auxins (Geneve and Kester 1991, Ballester 1999, Díaz-Sala 2014). Rejuvenation techniques (e.g., tissue culture, serial grafting, and hedging) are used in tree improvement programs, in which propagation from adult cuttings is often necessary (Hackett 1985, Greenwood 1987, 1995). Tissue culture involves culturing adult explants in vitro, generally in the presence of a cytokinin (CK), such as 6-benzylaminopurine (BA). Newly formed plantlets retain adult traits, and serial subculture (repeatedly culturing cuttings of new plantlets) is required before juvenile traits are restored. Similarly, serial grafting results in the gradual restoration of juvenile traits by recurrent grafting of scions onto juvenile rootstock. Juvenile traits may be maintained in some species, such as Norway spruce (Picea abies L. Karst.) and Monterey pine (Pinus radiata D. Don), by annual hedging, which stimulates production of shoots with high rooting capacity (Greenwood 1987). It is unclear to what degree rejuvenation attempts result in true rejuvenation (reduced maturation) rather than just reinvigoration (increased vigor). Frequently, regenerative potential is temporarily restored whereas other adult traits remain (Greenwood 1995, Wendling et al. 2014b). Thus, regenerative potential should not be used as the sole marker for vegetative maturation state without consideration of additional traits (Hackett 1985, Greenwood 1987). For a thorough review of juvenility maintenance and methods of rejuvenation, see Wendling et al. (2014b). Vigor.—As they age, temperate conifers decrease in shoot vigor (Greenwood 1995), which inversely correlates with increasing reproductive capacity (Day and Greenwood 2011). Thus, vigor is often useful as an indicator of reproductive maturation state. However, in grafting studies on red spruce (Greenwood et al. 2010), juvenile scions produced more vegetative growth than did older scions only when grafted to juvenile rootstock, and total vegetative growth was similar for scions of all ages when grafted onto middle-aged or old-growth rootstock. However, growth habit was different: Middle-aged rootstock promoted terminal shoot elongation, and old-growth rootstock promoted branch density. This suggests that juvenile meristems have greater growth potential that may be limited by distance to the roots as the tree grows taller, but that growth potential does not continue to decrease as the tree further matures from middle aged to old growth. This is consistent with work on several tree species, including Scots pine (Pinus sylvestris L.) (Mencuccini et al. 2005, Vanderklein et al. 2007) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco; Bond et al. 2007), which established that decreases in shoot growth in later life stages are not due to decreased vigor in more mature meristems but are a function of tree size—a factor extrinsic to the meristem. This evidence suggests that vigor may be useful as an indicator of vegetative phase change early in the life history of a tree, but growth habit may become more important in later years. In addition, the growth rate of more mature grafts matches that of juvenile grafts after the first growing season, suggesting that the growth advantage of juvenility decreases with tree size and that adult scions may be reinvigorated (Greenwood et al. 1989, Bond et al. 2007, Mencuccini et al. 2007). In Douglas-fir, all scions of all ages from juvenile to old growth exhibit the vegetative growth traits of the rootstock within 2 years of grafting (Bond et al. 2007). This suggests that vegetative vigor, although still useful as a proxy, is regulated quite differently than reproductive maturation, which is related to changes in the mature meristem and is generally irreversible in grafting studies (Bond et al. 2007, Greenwood et al. 2010, Day and Greenwood 2011). Biochemical Markers Maturation (vegetative and reproductive) in woody plants is a complex process involving changes in levels of numerous metabolic compounds and gene products, many of which have been evaluated as potential markers of phase change (Haffner et al. 1991). However, efforts to identify simple and reliable biochemical phase-change markers in forest tree species have not been successful (Wendling et al. 2014a). Many potential markers, such as differentially expressed gene products, appear to be species specific (von Aderkas and Bonga 2000). Others are involved in intricate crosstalk between multiple pathways, requiring complex analysis of multiple parameters that are difficult to interpret (Haffner et al. 1991). A reliable marker would greatly simplify many forest tree improvement tasks, such as monitoring rejuvenation efforts or selecting appropriate material for seed orchards. The marker would ideally not only identify the current developmental phase of a forest tree but would gradually change in gradient according to developmental progression from one phase to the next (von Aderkas and Bonga 2000, Valdés et al. 2003a, 2003b). Hormones are important regulators of phase change in plants (Trewavas 1983, Haffner et al. 1991, Valdés et al. 2002, Davies 2010, Turnbull 2011). Unfortunately, individual hormone levels can fluctuate dramatically because of crosstalk between signaling pathways and simultaneous involvement in multiple processes. Although individual hormones may be unsuitable as markers, certain endogenous hormone ratios may correlate with maturation, but this varies by species. For example, in Monterey pine (Valdés et al. 2002), but not Stone pine (Pinus pinea L.; Valdés et al. 2004), the ratio of isopentenyladenine-type to zeatin-type CKs decreases as vegetative maturation advances and increases during reinvigoration by grafting (Valdés et al. 2003b). Polyamines (PAs) are small, broadly conserved polycations that function in diverse cellular processes, including growth, development, and stress response (Kusano et al. 2008, Takahashi and Kakehi 2010). Because PAs are upregulated in actively growing and dividing cells, they are differentially expressed in juvenile and mature tissues (Kumar et al. 1997), making them potential indicators of phase change in woody plants (Rey et al. 1994). In peach (Prunus persica [L.] Batsch) and Monterey pine, free putrescine (an important PA) and the ratio of free PAs to conjugates decrease as the tree matures from reproductively incompetent to reproductively mature (Fraga et al. 2004). However, PA metabolism varies greatly among species (Fraga et al. 2004, Wendling et al. 2014a) and inresponse to seasonal growth patterns and stress (Königshofer 1989), which could complicate its use as an ontogenetic gauge. The most promising candidate for a molecular marker is microRNA156 (miR156). miRNAs are small, nonencoding RNAs that regulate numerous important processes in plants, including vegetative phase change (Wang et al. 2011), flowering time (Aukerman and Sakai 2003, Chen 2004), and biotic (Katiyar-Agarwal and Jin 2010) and abiotic (Sunkar et al. 2007) stress responses. miR156 has emerged as a general regulator of vegetative phase change in herbaceous (Wu and Poethig 2006, Xie et al. 2006, 2012, Chuck et al. 2007, Fu et al. 2012, Salinas et al. 2012, Shikata et al. 2012) and woody plants (Wang et al. 2011). The expression level of miR156 is highest when the plant is young and decreases as the plant ages (Wu and Poethig 2006, Xie et al. 2006, Chuck et al. 2007, Wu et al. 2009, Jung et al. 2012). Therefore, it is possible to use miR156 as a molecular marker to distinguish between juvenile and adult plants (Poethig 2013), but it remains unknown how well miR156 levels correlate with subtle changes in morphology and physiology associated with maturation. In Cole’s wattle (Acacia colei Maslin & L.A.J.Thomson), high levels of miR156 correlated with juvenile leaf morphology, intermediate levels correlated with transition leaf morphology, and low levels correlated with adult leaves (Wang et al. 2011). Thus, it may be possible to gauge incremental changes in a tree’s maturation by monitoring changes in miR156 expression, long before the development of reproductive structures, or even the competence to form such structures. miR156 is conserved in gymnosperms (Qiu et al. 2009, Huijser and Schmid 2011), but to what degree ontogenetic pathways are conserved is unknown. Endogenous Control of Reproduction Genetic Control of Reproduction Fecundity and the timing of reproductive onset in plants are mediated through intricately controlled gene expression (Hackett 1985, Greenwood and Hutchison 1993, Greenwood 1995, Cardon et al. 1999, Greenwood et al. 2010, Wendling et al. 2014a). Genotype establishes the general parameters for reproductive development at the whole-tree level and at the level of the individual meristem (Diggle 1993, Day and Greenwood 2011). Various intrinsic and extrinsic factors regulate the expression of genes that mediate phase change, influencing the fate of cells produced by the meristem (Day et al. 2002). Fecundity Evidence for genetic control of fecundity comes from forestry tree improvement research. In red spruce seed orchards, strobilus production among clones may differ by orders of magnitude, which cannot be accounted for by variation in environmental variables (Greenwood et al. 2010, Day and Greenwood 2011). In loblolly pine (Pinus taeda L.), more than 50% of female and 40% of male reproductive output may be attributable to genetic effects (Schmidtling 1983). Reproductive Onset Several genetic studies have demonstrated that time to reproductive onset is an inheritable trait in woody plants (Johnsson 1949, Stern 1961, Visser 1976), including conifers (Heimburger and Fowler 1969, Johnson and Critchfield 1978). Evidence for genetic control of reproductive maturation in conifers is provided by studies following a common-rootstock approach, in which scions from juvenile or mature trees are grafted onto rootstock of equal age. This approach controls for the age and size of the tree and for environmental conditions. Production of strobili increased with increasing age of the scion in red spruce (Greenwood et al. 2010), Monterey pine (Sweet 1973), and loblolly pine (Greenwood 1984). However, more strobili were produced on younger scions in eastern larch, which the authors attributed to the smaller size threshold for reproductive onset in larch (Larix laricina [Du Roi] C. Koch; Greenwood et al. 1989). Possible Mechanisms In Arabidopsis, flowering time is regulated by photoperiod, temperature, and endogenous signaling primarily through five independent, but partially overlapping, genetic pathways: photoperiodic, vernalization, autonomous, GA, and vegetative phase-change path-ways (Koornneef et al. 1991, Simpson and Dean 2002, Mencuccini et al. 2005, Bäurle and Dean 2006, Amasino and Michaels 2010, Lee and Lee 2010, Srikanth and Schmid 2011). Although independent, these pathways interact to regulate flowering. For example, the vernalization and vegetative phase-change pathways do not directly induce flowering but create the permissive state required for induction of flowering through the photoperiodic pathway (Poethig 2013). In gymnosperms, little is known about the pathways that govern reproduction, but at least some of the genetic mechanisms found in Arabidopsis are conserved in woody plants, including conifers (Castillo et al. 2013, Uddenberg et al. 2013, Wendling et al. 2014a). Some of the more than 50 genes known to regulate flowering time in Arabidopsis (see Srikanth and Schmid 2011) are known to exist in conifers (Purugganan 1997, Sundstrom et al. 1999, Carlsbecker et al. 2004, 2013), suggesting possible mechanisms for genetic control of reproductive maturation in conifers. CONSTANS/FLOWERING LOCUS T Regulatory Module.—FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) are floral integrators, meaning that they integrate signals that converge from multiple flowering pathways (Amasino and Michaels 2010). A growing body of evidence suggests that the FT protein is a mobile, florigenic signal conserved among all flowering plants (Samach et al. 2000, Putterill et al. 2004, Lifschitz et al. 2006, Yang et al. 2007, Turck et al. 2008). In the Arabidopsis photoperiodic flowering pathway, CONSTANS (CO) activates FT, which upregulates SOC1, which in turn activates floral meristem identity genes that initiate flowering (Koornneef et al. 1991, Wigge et al. 2005, Yoo et al. 2005). The CO/FT regulatory module has been shown to regulate the timing of reproductive onset and photoperiodic bud-set in Populus spp. (Böhlenius et al. 2006). FT orthologs were recently found to regulate bud-set in Norway spruce (Gyllenstrand et al. 2007) and Scots pine (Avia et al. 2014), suggesting that the CO/FT regulatory module is conserved in conifers. This module may control reproductive development by upregulating florigenic FT protein as the tree matures (Day and Greenwood 2011). MADS Box Genes and Other Transcription Factors.— Flowering pathways converge on SOC1 and other integrators that promote floral transition of the meristem by activating transcription factors, notably LEAFY (LFY) and the MADS box genes FRUITFULL (FUL) and APETALA1 (AP1; Benlloch et al. 2007, Yamaguchi et al. 2009). Homologs of LFY have been found in gymnosperms and implicated in reproductive identity of the meristem, but expression patterns differ from those of angiosperms, and their activity and role are less well characterized (Benlloch et al. 2007). In Norway spruce, MADS box genes have been identified that are believed to regulate the duration of the reproductively incompetence phase and the reproductive or vegetative identity of a meristem (Carlsbecker et al. 2003, 2004). Expression of DEFICIENS AGAMOUS-LIKE1 (DAL1) is upregulated as the tree ages and distributed in a pattern that precisely predicts the changes in morphological and physiological traits that occur during reproductive phase change. In addition, the time to reproductive onset is shortened or nonexistent in transgenic Arabidopsis expressing DAL1 (Carlsbecker et al. 2004). A related gene, DAL10, appears to regulate reproductive identity in Norway spruce (Carlsbecker et al. 2002). Phylogenetic analysis indicates that DAL19 (in Norway spruce) and SOC1 (in Arabidopsis) are in orthologous clades, and DAL19 is upregulated in reproductive shoots (Uddenberg et al. 2013), which may suggest similar mechanisms in specification of reproductive identity. miR156/SPL Module.—SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode transcription factors that regulate gene expression to control many aspects of plant development, including floral transition (Cardon et al. 1997, 1999, Poethig 2013). This age-related floral transition pathway, also known as the vegetative phase-change pathway (Poethig 2013), is regulated by miR156, which inhibits flowering during the reproductively incompetent phase (Wang et al. 2009). The primary function of miR156 is to repress SPL gene expression (Poethig 2013). miR156 levels are high in reproductively incompetent plants and decrease over time, whereas SPL protein levels are low in reproductively incompetent plants and increase over time. Mutations that prevent miR156 from binding to SPL gene transcripts result in precocious flowering (Cardon et al. 1997, Wu and Poethig 2006, Gandikota et al. 2007, Usami et al. 2009, Wang et al. 2009, Yamaguchi et al. 2009). Despite the high functional redundancy within the gene family, loss-of-function mutations of certain SPL genes result in delayed flowering (Schwarz et al. 2008; Wang et al. 2008). Constitutive overexpression of miR156 by the 35S promoter likewise delays reproductive onset in the poplar hybrid Populus × canadensis by suppressing expression of two SPL genes (Wang et al. 2011). miR156 maintains the period of reproductive incompetence and governs the phase-change transition whereby a meristem becomes competent to flower, but miR156 does not induce flowering (Poethig 2013). The decline in miR156 as the plant ages releases SPL from miR156-mediated suppression, creating a permissive state for other pathways (vernalization, photoperiodic, and GA) to regulate SPL transcription and thus control floral initiation (Wang et al. 2009, Jung et al. 2011, 2012, Yu et al. 2012). In Arabidopsis, SPL functions in parallel with CO to upregulate FT (Yu et al. 2012) and directly activates MADS box genes in the meristem to initiate flowering (Wang et al. 2009). miR156 is conserved in gymnosperms (Huijser and Schmid 2011, Zhang et al. 2012, Wang and Wang 2015). Levels of many miRNAs involved in growth and development change in response to biotic and abiotic stress signals (Sunkar and Zhu 2004, Fujii et al. 2005, Navarro et al. 2008, Khraiwesh et al. 2012, Kruszka et al. 2012, Sunkar et al. 2012), suggesting integration of stress-response and developmental pathways (Sunkar et al. 2012). For example, miR156 is upregulated in response to heat (Lee et al. 2010, Kim et al. 2012, Stief et al. 2014), drought (Sun et al. 2012), nutrient deficiency (Hsieh et al. 2009), and salt stress (Ding et al. 2009, Sun et al. 2012), resulting in a corresponding decrease in SPL levels. These stress-mediated changes in miRNA expression levels may account for reduced growth and development in response to stress (Sunkar et al. 2012). Sugars.—In Arabidopsis, leaf removal results in delayed reproductive and vegetative phase change and increased miR156 expression. This suggests that a repressive signal from the leaves inhibits miR156 (Yang et al. 2011). Nutrients—especially sugars—have long been implicated in vegetative phase change because of their effects on morphology and physiology in heteroblastic plants (Goebel 1900, 1908, Allsopp 1952, 1953a, 1953b, Feldman and Cutter 1970, Njoku 1971). Sugars accumulate rapidly in the meristem before initiation of flowering in Arabidopsis and could serve as a mobile signal (Eriksson et al. 2006). Vegetative phase change is delayed in chlorophyll-deficient Arabidopsis mutants, chlorina1-4 (Yang et al. 2013, Yu et al. 2013). This delay results from decreased production of sugars and is mediated by HEXOKINASE1 (HXK1), a glucose signaling protein that helps maintain juvenility when sugar levels are low, such as under low-light conditions. HXK1 acts by repressing miR156 transcription in response to glucose signaling (Yang et al. 2013). The gradual accumulation of sugars from seed germination to reproductive maturation may serve as an endogenous aging signal that regulates developmental timing by downregulating miR156 as the plant ages (Yu et al. 2013). Epigenetic Regulation Epigenetic regulation results in phenotypic changes by persistently modifying gene expression without altering the underlying DNA (Yakovlev and Fossdal 2012). Many of these changes are not transmitted to progeny. Examples include changes in foliar morphology and branching habit with increasing age and size (Greenwood et al. 2010). In contrast, epigenetic inheritance is transgenerational, adjusting progeny performance for many years, based on the parental environmental conditions—especially temperature, photoperiod, and irradiance—at the time of seed initiation and development (Day and Bonduriansky 2011, Yakovlev et al. 2011, Yakovlev and Fossdal 2012). These aftereffects vary by trait and among species (Andersson 1994). For example, a colder maternal environment results in earlier bud-break and bud-set, which are important in cold hardiness (Johnsen et al. 2005). The timing of bud-set also regulates reproductive bud initiation and differentiation in conifers. A slight delay may expose the developing bud to warmer conditions, thus encouraging reproductive over vegetative development (Owens and Blake 1985, Day and Greenwood 2011). Plants are particularly sensitive to changes in environmental conditions when young and during sporogenesis and gametogenesis (Day et al. 2002, Bazhina 2014). Seed orchards must be carefully sited, or action must be taken to modify the microclimate around the trees during spore and seed development to prevent undesired epigenetic effects on progeny (Schmidtling 1987, Funda and El-Kassaby 2013). Meristem Maturation Day et al. (2002) describe intrinsic and extrinsic controls that regulate ontogeny. Intrinsic controls modify gene expression in the meristem through epigenetic changes, such as DNA methylation, histone modification, and telomere shortening. The signals prompting these changes may initiate in the meristem or follow transduction pathways from the external environment (Day and Greenwood 2011). Grafting studies on red spruce indicate that reproductive phase change is primarily determined by the maturation state of the meristem (Greenwood et al. 2010), which is largely the result of epigenetic changes to gene expression that are generally permanent and irreversible (Day and Greenwood 2011). Other epigenetically governed traits, such as branching habit, may be partially reversed by altering external conditions (Greenwood et al. 2010). It is difficult to separate size effects from age (maturation) effects, but progress has been made in recent years. Changes in shoot elongation are largely a function of tree size. Foliar morphology and branching habit are governed by size and meristem maturation. Reproductive development is primarily regulated by meristem maturation (Bond et al. 2007, Greenwood et al. 2010). This is important for seed orchard management because cone-inducing treatments differentially affect meristems based on degree of maturation. Reproductively incompetent meristems rarely respond to such treatments (Day and Greenwood 2011). Hormonal Regulation of Reproductive Development Plant development is regulated by hormone signaling pathways that integrate endogenous and exogenous cues through complex, overlapping regulatory networks. Hormone-mediated developmental and biotic and abiotic stress response pathways interact to precisely control the final gene expression, generating a robust, yet dynamic system that enables developmental plasticity in response to changing environmental conditions (Achard et al. 2007, Vanstraelen and Benková 2012). miRNAs have emerged as important mediators of this crosstalk between hormone signaling pathways (Curaba et al. 2014). The miR156/SPL module integrates environmental signals into the vegetative and reproductive phase-change pathways it regulates. miR156 levels are primarily determined by age and decrease over time (Yamaguchi and Abe 2012) but may be influenced by auxin (Marin et al. 2010) and ethylene (Zuo et al. 2012) in some instances and are upregulated in response to stress (Sunkar et al. 2012). miR156 maintains juvenility by repressing SPL transcription factors (Poethig 2013). GA, auxin, ethylene, and abscisic acid (ABA) all regulate the stability of DELLA proteins that target some of the same SPLs (Vanstraelen and Benková 2012). Thus, hormone signaling may serve to dynamically integrate environmental information into developmental programming through the miR156/SPL module (Wang and Wang 2015). Plant hormones are important regulators of maturation and reproductive development in temperate conifers (Haffner et al. 1991, Greenwood 1995, Valdés et al. 2002). Mitotic activity, controlled by auxin, CK, and GA, maintains the stability of the developmental state of the meristem (Haffner et al. 1991, Valdés et al. 2002, Vanstraelen and Benková 2012). Auxin, CK, and strigolactones regulate shoot branching, and GAs and brassinosteroids add additional control to tune the levels of auxin and CK in several developmental processes (Vanstraelen and Benková 2012). Hormonal changes during strobilus initiation and differentiation suggest a role in regulating gender determination (Chałupka 2008, Kong et al. 2012) and strobilus distribution within the crown, although responses vary by genotype (Chałupka 2008). GAs The role of GAs in reproductive development has been extensively studied in temperate conifers for tree improvement purposes (Owens 1995). In Cupressaceae and Taxodiaceae, GA3 promotes strobilus formation. In Pinaceae, the less-polar GAs, such as GA4 and GA7, are promotive. In addition, the ratio of polar to less-polar GAs decreases with age, suggesting a role for GA in regulating reproductive phase change (Pharis and Kuo 1977). These less-polar GAs are important in reproductive organogenesis and accumulate at the meristem from neighboring tissues immediately before strobilus initiation. Indeed, induction of strobili by exogenous application of GA4/7 is possible in many species. Similarly, certain GAs, especially less-polar GAs, may be upregulated in response to environmental stresses that promote cone production, such as drought and heat stress, whether naturally occurring or culturally induced in seed orchards (Pharis and Kuo 1977). The mechanism by which GAs regulate strobilus initiation or differentiation (e.g., Smith 1998) is not known, but similarities shared with angiosperms and other plants suggest that molecular pathways are at least partially conserved. In vascular plants, DELLA proteins play an important role in regulation of GA homeostasis and serve as integrators of hormonal and environmental signaling pathways that constrain growth under adverse conditions (Davière et al. 2008). In Arabidopsis and most conifers, the less-polar GA4 serves as a mobile, florigenic signal, rapidly accumulating at the meristem shortly before reproductive initiation (Odén et al. 1995, Eriksson et al. 2006, Fornara et al. 2010). In Arabidopsis, there is some overlap between the GA and age-related, vegetative phase-change pathways, both of which regulate reproductive development (Yu et al. 2012). In both pathways, SPL transcription factors are released from suppression and activate FT and MADS box genes—notably the SOC1 floral meristem identity gene—to trigger floral initiation (Moon et al. 2003, Achard et al. 2007, Yu et al. 2012). In the age-related pathway, aging reduces miR156 levels, reducing suppression of SPL. In the GA flowering pathway, GAs bind to GA INSENSITIVE DWARF1 (GID1) receptors, promoting degradation of DELLA proteins through the ubiquitin-proteasome pathway and thus reducing DELLA-mediated transcriptional suppression of SPLs (Sun 2010, Yu et al. 2012). GA also independently regulates changes in morphological traits associated with miR156-mediated vegetative phase transition (Chien and Sussex 1996, Poethig 2003), which provides further evidence for crosstalk between GA and age-related pathways. The GA-GID1-DELLA regulatory module of the GA signaling pathway is conserved in gymnosperms (Vandenbussche et al. 2007). In addition, the ability of the meristem to respond to GA increases with age (Eysteinsson and Greenwood 1993, Greenwood et al. 2010), suggesting an overlap between the GA and phase-change pathways, as in Arabidopsis (Poethig 2013). For example, reproductively incompetent conifers will not produce strobili even when high levels of endogenous GAs are present nor consistently in response to exogenous application of GA (Zimmerman et al. 1985, Eysteinsson and Greenwood 1993). Likewise, very few strobili form on reproductively incompetent scions grafted into mature crowns, despite importation of GAs from neighboring branches (Greenwood et al. 2010, Day and Greenwood 2011). Thus, GA regulation of strobilus formation is dependent on the state of the vegetative phase-change pathway that governs the maturation of the meristem, just as is flowering in Arabidopsis. Because FT and SOC1 orthologs have been identified in conifers, it is likely they are involved in GA-mediated reproductive development (Day and Greenwood 2011). However, application of exogenous GAs to mature English ivy (Hedera helix L.; Rogler and Dahmus 1974) and Australian blackwood (Acacia melanoxylon R. Br.; Borchert 1965) results in production of morphologically juvenile leaves, indicating that GAs suppress vegetative phase change in some plants (Poethig 2013) and that underlying mechanisms are only partially conserved among spermatophytes. CKs Bud differentiation in conifers is partially regulated by CKs (Morris et al. 1990, Bollmark et al. 1995, Kong et al. 2012). Changes in CK biosynthesis and metabolism occur during maturation and often revert during reinvigoration, allowing ratios of various CKs to serve as phase-change indicators in some species (Valdés et al. 2002, 2004, Wendling et al. 2014a). Similar changes occur during bud initiation and differentiation (Kong et al. 2009), and differences have been noted between CK ratios in male and female buds (Kong et al. 2012). When applied after strobilus initiation, but before differentiation, exogenous CKs enhance female cone formation in Japanese red (Pinus densiflora Sieb. et Zucc.) and black (Pinus thunbergii Parl.) pine through conversion of male cones to female cones, possibly by changing the balance between CK and auxin in the developing bud (Wakushima 2004). Although these results have not been consistent across species, they suggest multiple roles for CK in reproductive development. CKs appear to inhibit GA-mediated strobilus initiation, leading to the hypothesis that as a tree grows, the distance increases between the inducible meristem and the roots, where much of the CK is thought to originate (Smith and Greenwood 1995). This may produce a CK gradient that regulates both the timing of reproductive onset and the increase in reproductive potential as the tree ages (Greenwood et al. 2010). In support of this hypothesis, exogenous application of CKs before strobilus initiation reduces strobilus formation, whereas root pruning increases strobilus formation, possibly by limiting the availability of root-produced CK (Smith and Greenwood 1995). However, in Arabidopsis, flowering time is not affected in transgenic plants lacking roots, indicating that roots have no significant role in phase change (Yang et al. 2011). In common pea (Pisum sativum L.), removal of the apical bud results in local CK synthesis in the stem, suggesting that auxins downregulate CK biosynthesis in the stem (Tanaka et al. 2006). In Nordmann fir (Abies nordmanniana [Steven] Spach), large differences in CK profiles in adjacent buds suggest local CK synthesis, and CK profiles correlate with different bud fates, suggesting a role for CK in bud-fate determination (Rasmussen et al. 2009). In addition, CK levels in the bud and stem decrease after bud-break, reaching a minimum just as shoot expansion reaches a maximum, then increasing as shoot elongation slows. This might be due to auxin-mediated regulation of local CK biosynthesis in the bud and stem. Taken together, it appears that local CK biosynthesis may be more important than root-derived CKs in regulating reproduction. The mechanism by which root pruning enhances reproductive output remains unclear; it may be through changes in ratios of CKs, a reduction in total CKs, or through another pathway, possibly the miR156/SPL regulatory module implicated in stress response (Stief et al. 2014). Auxins Auxins are central to plant growth and development (Curaba et al. 2014); therefore, it would not be surprising to find a role for them in phase change and reproductive development in conifers. Auxins, together with CKs and GAs, regulate the mitotic activity that maintains the state of maturation of the meristem (Haffner et al. 1991, Valdés et al. 2002). Concentrations of the endogenous auxin indole-3-acetic acid (IAA) increase during the period of active, vertical growth during juvenility and are then maintained or decrease during the adult vegetative and reproductively competent and mature phases (Valdés et al. 2002), coincident with declining vigor and changes in branching habit (Greenwood et al. 2010). High concentrations of IAA during shoot elongation, which spans reproductive bud initiation and differentiation, correlate with heavy megastrobilus production in highly productive genotypes of Douglas-fir, but no trend is observed in less-productive genotypes, suggesting that high concentrations of IAA upregulate megastrobilus production (Kong et al. 2009). Similarly, IAA concentrations are higher in adult than juvenile buds and needles in several species (Andrés et al. 2002, Valdés et al. 2002, 2003a), but not all (Valdés et al. 2004). Auxins may also play a role in gender determination. Application of exogenous auxin increased microstrobilus and decreased megastrobilus formation in Picea, Pinus, and Larix spp., although results were inconsistent (Sheng and Wang 1990). During the period of microstrobilus formation in lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), concentrations of IAA are higher in proximal regions of shoots, where microstrobili form, than in distal regions, where megastrobili will subsequently develop (Kong et al. 2012). This may suggest local production of auxin (Zhao 2008) or that developing male cone buds are particularly strong sinks for apically produced auxin. ABA ABA levels increase from the reproductively incompetent to reproductively competent phases and decrease during reinvigoration, indicating that phase transition is partially regulated by ABA in conifers (Haffner et al. 1991, Valdés et al. 2002, 2003b, 2004, Materán et al. 2009). After reproductive onset, the role of ABA is less clear (Valdés et al. 2004), and ABA levels may plateau or even decline (Munné-Bosch 2007). In addition to the general upward trend during early maturation, ABA levels are fine-tuned in response to environmental stress signals, including extreme temperature, drought, and physical wounding (Tuteja 2007), thus serving to integrate environmental cues into developmental pathways, possibly through interaction with the GA-DELLA or miR156/SPL module. Ethylene Local ethylene biosynthesis is rapidly upregulated in response to stress (Davies 2010). For example, ethylene mediates the response of plants to mechanical perturbation, including wind stress and physical wounding, resulting in increased radial growth and decreased elongation growth (Biro and Jaffe 1984, Telewski and Jaffe 1986). In an apple (Malus domestica Borkh.) hybrid seedling (Jonathan × Golden Delicious), an ethylene response factor (37–416_J) is up-regulated during the reproductively incompetent phase, suggesting possible heighted sensitivity to environmental stimuli (Gao et al. 2013). Application of BA (a synthetic CK) + ethephon (an ethylene-releasing compound) induces reproductive onset in apple trees that would not ordinarily flower at that size but had reached reproductive competence (Zhang et al. 2007). Ethylene has also been implicated in regulation of photoperiodic flowering, but effects are species specific (Thomas and Vince-Prue 1997, Davies 2010). These results suggest that ethylene mediates developmental responsiveness to stress, probably through its interaction with the GADELLA module (Achard et al. 2007) or the miR156/SPL module. Exogenous Regulation Tree Size Although reproduction is more a function of meristem maturation than tree size (Greenwood et al. 2010), there is generally a positive relationship between tree size and reproductive output. This relationship is weakened by genetic variability and plasticity in response to environmental conditions, but remains significant (Haymes and Fox 2012, Santos-Del-Blanco et al. 2013). Reproductive onset is also related to size, with a genetically determined critical size required before reproduction may commence in many plants (Roff 2000), including apple and pear (Hackett 1985). This is supported by grafting studies in conifers: Few cones are produced by reproductively mature scions in the first few years (Greenwood et al. 1989, Day and Greenwood 2011), suggesting that the minimum size for reproduction has not been reached (Day and Greenwood 2011). There exists an allocational tradeoff between reproduction and vegetative growth: For many trees, reproductive output is reduced and reproduction is delayed under conditions favorable to vegetative growth (Day and Greenwood 2011, Santos-Del-Blanco et al. 2013). For example, in red spruce, irrigation increases vegetative growth while simultaneously reducing cone production (Day and Greenwood 2011). This is particularly true for trees that have reached reproductive competence but not reproductive maturity, which will not generally reproduce except in the presence of sufficiently strong environmental signals (Williams 2009). However, the time to reproductive onset may be shortened in environments that encourage rapid growth, such as under controlled greenhouse conditions, presumably because the size threshold is reached earlier. Likewise, time to reproductive onset may be extended by conditions that repress growth, such as low temperature or lack of resources (e.g., nutrients, water, light) (Hackett 1985, Poethig 1990, Bond et al. 2007, Amasino and Michaels 2010, Turnbull 2011). However, once the size threshold has been reached, those same stress conditions may encourage reproductive precocity and fecundity, resulting in early and heavy reproduction in smaller trees (Santos-Del-Blanco et al. 2013). Other factors, such as status in the canopy and competition, are also important, resulting in cone production that is highly variable and difficult to predict by size alone (Haymes and Fox 2012). Environmental Factors Temperature Hot, dry summers increase reproductive output for many forest trees (Owens and Blake 1985). Studies have found significant positive correlations between cone or seed production and temperature during the summer of strobilus initiation for many species, such as Norway spruce (Solberg 2004), western hemlock (Tsuga heterophylla [Raf.] Sarg.; Pollard and Portlock 1984), Scots pine (Ozolincius and Sujetoviene in Ozolinčius et al. 2009), balsam fir, and white spruce (Picea glauca [Moench] Voss; Messaoud et al. 2007). No correlation was found for black spruce (Picea mariana [Miller] B.S.P.), which yields heavier and more frequent cone crops independent of climate conditions (Messaoud et al. 2007). Increased precipitation and cooler temperatures during all other years correlates with higher seed production in white spruce. This suggests that resource accumulation and allocation is critical during the years before cone initiation and likewise during the year after as seeds develop and that hot, dry conditions are required to trigger strobilus initiation (Roland et al. 2014). Moisture The results of studies in Norway spruce suggest that high temperatures during cone initiation are a stronger determinant of cone production than is precipitation, yet precipitation remains a significant factor (Solberg 2004). Drought stress reduces soil water potential, making it more difficult for a plant to take up water and nutrients, which alters the physiology and reduces growth (Smith and Greenwood 1997, Sardans et al. 2008). Under moderate or even severe drought conditions, reproductive development may be enhanced, as an allocation tradeoff with vegetative growth (Owens and Blake 1985, Muller-Starck and Seifert 2008, Ozolinčius et al. 2009). However, under severe, prolonged drought, generative growth may be reduced. In Scots pine, reproductive bud initiation occurs two years before cone emergence. However, under multiyear, induced drought conditions, reproductive output dropped dramatically 2 years after the treatment was set in place—concurrent with 40% defoliation—and remained suppressed until the canopy had recovered 3 years after removal of the water-excluding, subcanopy roofs (Ozolinčius et al. 2009). It is possible that drought had enhanced cone bud initiation in the first year, but that bud survival declined under prolonged drought (cf. Ebell 1971). Cones initiated the first year of drought may have been aborted in the second year as limited resources were allocated to the growth of existing cones. Note that in the second year of drought, cone production was equal to that of control trees, despite significant defoliation, indicating a tradeoff in resource allocation. Light Although temperature is the more important factor driving cone production across larger scales, higher light interception locally increases cone production. Whereas temperate conifers on southern slopes (higher irradiance) tend to produce the most cones (Despland and Houle 1997), shaded trees may produce no cones (Chałupka and Giertych 1977). Likewise, for a given tree, the section of the crown receiving the most irradiance yields the most cones (Despland and Houle 1997). Light interception explains part of the variation in cone production based on canopy status. For example, a white spruce tree will produce cones in a plantation in full sun once it is 3 m high, but it must be 14 m high to produce cones as a subcanopy tree (Greene et al. 2002). Scots pine trees produce much heavier cone crops subsequent to release thinning, and most cones form further down the tree, in areas now exposed to light (Karlsson 2000). Experimentally, female strobili may be induced in Scots pine and Norway spruce by channeling natural light directly into the apical dome via optical fibers (Kosiński and Giertych 1982). Photoperiod In north temperate species, photoperiod regulates growth cessation, bud-set, and entry into dormancy (Ekberg et al. 1979). Photoperiod is an important regulator of reproductive development because the timing of strobilus initiation and differentiation is closely tied to shoot phenology (Owens and Blake 1985). In Picea, Pinus, Abies, and Larix, the maximum rate of growth corresponds to maximum day length, not temperature, indicating photoperiodic control of vegetative growth (Rossi et al. 2006). In Pinus spp., female strobili differentiate only 2–3 weeks before male strobili, but they seem to have different photoperiod requirements, suggesting photoperiod regulation of sex expression (Giertych 1967). Experimental manipulation of photoperiod demonstrates the role of photoperiod in gender determination in western hemlock: An 18-h photoperiod during cone bud initiation increases male strobili whereas a 13-h photoperiod increases female strobili (Pollard and Portlock 1984). Nutrition Cone production generally correlates positively with nutrition (Owens and Blake 1985, Rothstein and Cregg 2005, Owens et al. 2001). Phosphorus, and to a lesser extent nitrogen, was found to limit cone production in Fraser fir (Abies fraseri [Pursh] Poir.) when comparing populations with high and low reproductive output (Arnold et al. 1992). In ponderosa pine (Pinus ponderosa Dougl. ex Laws.), cone production increased linearly in response to increasing rates of urea ammonium phosphate (Heidmann 1984). The linear response suggests that fertilization may enhance cone production when nutrients are limiting. Managing Cone Induction Early efforts to enhance seed production in seed orchards relied on cultural treatments, such as fertilization, girdling, root pruning, and drought (Puritch 1972). Results were inconsistent and occasionally detrimental (Puritch 1979). This is due in part to interspecies variability and in part to the complex nature of regulation of maturation and reproductive development in conifers. Subsequent work with plant growth regulators (PGRs), particularly exogenous application of GAs, resulted in greatly enhanced cone production, especially when combined with cultural treatments, although results varied by species, application method and timing, and environmental conditions (Puritch 1979). In a few species, early reproductive onset could be induced by application of GAs, resulting in precocious cone formation (Chałupka 1991), but results were generally temporary (Longman 1987). Reproductive incompetence and environmental conditions during the reproductive phases remain the primary factors that constrain our ability to induce cone formation (Bonnet-Masimbert and Webber 1995). The most common treatments used to enhance cone production are briefly reviewed here, but they have been extensively reviewed elsewhere (see Owens and Blake 1985, Ross and Pharis 1985, Bonnet-Masimbert and Webber 1995). Because many cone enhancement techniques stress the tree and heavy cone yields are strong sinks for photosynthates (Dickmann and Kozlowski 1968, 1970), use of enhancing techniques on very young trees may have a detrimental effect on long-term reproductive output (Ebell 1971, Roff 2000, Obeso 2002). Rather, practices that promote rapid vegetative growth in young trees will help them to more quickly attain reproductive size (Hackett 1985), after which time cone enhancement techniques may be applied but must be balanced against effects on the long-term health of the tree. PGRs Application of GAs is the most broadly effective way to promote strobilus induction in temperate conifers. Efficacy is affected by many factors, including product quality; application rate and timing; and size, physiological age, and genotype of the tree. GA application is much more effective when combined with stress treatments, such as girdling, tenting, or water exclusion. Treatments must be applied during the period of strobilus initiation and differentiation to be effective (Bonnet-Masimbert and Zaerr 1987, Pharis et al. 1987, Owens 1995, Owens et al. 2001). In Cupressaceae and Taxodiaceae, GA3 is the most effective (Longman et al. 1982). In Pinaceae, the less-polar GAs (i.e., GA4 and GA7) are most effective (Owens and Blake 1985). GA is commonly applied by stem injection or foliar spray, beginning shortly after vegetative bud-break, and reapplied several times until strobilus initiation is complete (Owens and Blake 1985, Bonnet-Masimbert 1987, Funda and El-Kassaby 2013). Although exogenous GA application has been shown to promote early strobilus formation in a few conifers (Chałupka 1991), with carryover effects that might last a few years (Johnsen et al. 1994), exogenous application of GA does not promote phase change (Zimmerman et al. 1985). The effects of CKs on cone formation are species specific. Exogenous application of (BA) enhanced female cone formation in Japanese red and black pines (Wakushima 2004) but had the opposite effect in black spruce (Smith and Greenwood 1995) and Chinese pine (Pinus tabuliformis Carr.; Sheng and Wang 1990). GA + BA enhanced male and female strobili production in Douglas-fir (Ross and Pharis 1976) and Sitka spurce (Picea sitchensis [Bong.] Carr.; Tompsett 1977). Other PGRs may act synergistically with GA to enhance cone formation, but results are species specific and often variable (Bonnet-Masimbert 1987), perhaps because of differences in product quality or application rate and timing. In combination with GA, the synthetic auxin 1-naphthaleneacetic acid (NAA) may enhance female but not male strobilus production in some species, such as lodgepole pine (Wheeler et al. 1980). In a few species, such as Sitka spurce (Tompsett 1977) and Douglas-fir (Ross 1975), GA + NAA application promotes male strobilus formation at the expense of female development, although low rates of NAA may promote female strobilus formation in Douglas-fir (Pharis et al. 1980). Similarly, chlorocholine chloride (CCC), a GA biosynthesis inhibitor, promotes male strobilus formation at high rates and female strobilus production at lower rates in Chinese pine, but the female strobili do not persist to maturity (Zhao et al. 2011). Application of paclobutrazol, another GA biosynthesis inhibitor, results in precocious, profuse cone formation in Eucalyptus (Griffin et al. 1993, Williams et al. 2003) but reduces cone formation in Fraser fir (Crain and Cregg 2017). Ethephon (metabolizes to ethylene) enhances female strobilus formation in Norway spruce when applied alone (Remrod in Bonnet-Masimbert 1989). Ethephon enhances male and female strobilus production in Cupressaceae but only when applied in combination with GA3 (Bonnet-Masimbert 1971, Hashizume 1975). Cultural Treatments Cultural treatments, such as girdling, root pruning, fertilization, heat, and drought, are frequently used in seed orchards as an adjunct to GAs to synergistically enhance cone production, particularly in young conifers, or when weather conditions during bud initiation are not conducive to cone development. Cone enhancement may occasionally be obtained by application of a single cultural treatment, but most work better in conjunction with other cultural treatments, and often no treatment effects are observed unless GA is also applied (Owens and Blake 1985, Bonnet-Masimbert 1987, Pharis et al. 1987). A few of the more common cultural techniques are briefly reviewed here. Fertilization Nitrogen fertilizer is often applied to increase cone production in conifers, but results are highly variable (Owens and Blake 1985, Bonnet-Masimbert and Webber 1995, Miller and DeBell 2013), and addition of nitrogen alone may sometimes increase vegetative growth at the expense of generative growth (Krannitz and Duralia 2004). In general, cone production is positively correlated with nutrition (Owens and Blake 1985, Owens et al. 2001, Rothstein and Cregg 2005). In Pacific silver fir (Abies amabilis [Douglas ex Loudon] Douglas ex Forbes), the combination of fertilization, GA4/7, and girdling promotes strobilus formation, but fertilizer alone was not tested (Owens et al. 2001). In Eucalyptus nitens, nitrogen fertilizer promotes strobilus development, but phosphorus fertilizer has no effect. Nitrogen fertilization results in increased tree size, which partially accounts for the increase in reproductive output (Williams et al. 2003). In Douglas-fir, application of nitrate nitrogen increases cone production, but application of ammonium nitrogen does not (Stoate et al. 1961, Ebell 1966). There are no differences in foliar nitrogen content, bud density, or growth, indicating that the reproductive response is independent of any growth response resulting from increased nitrogen uptake. Nitrate nitrogen fertilization results in changes in nitrogen metabolism that increase arginine and other free amino acid levels, with a corresponding increase in lateral bud survival during shoot elongation. Thus, more buds are available to develop into strobili (Ebell 1972b). Whether nitrogen fertilization acts in other ways to promote strobilus initiation or development is not known (Bonnet-Masimbert and Webber 1995). Unfortunately, even in Douglas-fir results are not consistent across sites (Ebell 1972b, Miller and DeBell 2013), and a similar stimulatory effect of fertilization on reproductive output has not been reliably demonstrated in most conifers (Owens and Blake 1985). Girdling Of the various cultural practices tested, stem girdling is one of most effective at stimulating strobilus production in conifers (Wheeler and Masters 1985). Girdling refers to the removal of a thin strip of bark around the circumference of a stem and results in accumulation of photosynthates above the girdle because of interruption of phloem transport. A common technique is to remove a strip of bark (3–30 mm wide) from just over half of the circumference of the trunk and a second, partially overlapping strip from higher up (e.g., 70 cm) on the other side of the tree. This effectively disrupts the phloem while reducing long-term detrimental effects (Ebell 1971, Wheeler and Masters 1985). Even so, repeated girdling over many years may damage tree health and decrease reproductive output (Owens and Blake 1985). Strangulation (banding) is similar to girdling but is generally less effective at cone enhancement. It involves the tightening of a restrictive band around the phloem to disrupt translocation (Owens and Blake 1985). Girdling alone increases cone production in Douglas-fir (Ebell 1971, Wheeler and Masters 1985), but girdling is more effective in combination with GA (Owens and Blake 1985, Kolpak et al. 2014). In Douglas-fir trees of seedling origin that had not yet reached reproductive maturity, girdling alone had no effect, but girdling combined with low levels of applied GA4/7 did (Munoz-Gutierrez et al. 2010). Girdling is generally applied approximately 1 month before bud-break, although optimal timing varies based on site-specific factors (Miller and DeBell 2013). The inductive effects may persist for several years. Cone production may be reduced if girdling is applied after the first week subsequent to bud-break (Ebell 1971). Girdling does not generally promote cone formation in most species unless used in combination with GA (Ross and Greenwood 1979, Miller and DeBell 2013). In ponderosa pine, girdling alone increases cone production, with no carryover effect to subsequent years (Shearer and Schmidt 1970). In western larch (Larix occidentalis Nutt.), girdling alone increases cone development during a low cone production year, but not during a high cone production year (Chałupka 2008), suggesting an interaction between multiple signaling pathways. The mechanism by which girdling acts to enhance cone production is unclear. In Douglas-fir, girdling does not change the number of buds initially formed, but it does increase bud survival (Ebell 1971), identical to nitrate fertilization (Ebell 1972b). However, reproductive bud survival did not correlate with carbohydrate levels, suggesting that disruption of phloem translocation is not the driver of bud survival in girdled trees (Ebell 1971). Root Pruning and Transplanting Although root pruning alone is occasionally effective in cone enhancement (e.g., Marquard and Hanover 1984, Webber et al. 1985), it is more commonly used to synergistically increase the responsiveness of a tree to GA treatments and is most effective when applied near the time of vegetative bud-break (Smith and Greenwood 1995). Although root pruning reduces the surface area of the root that is available for water absorption, it seems unlikely that cone induction results from a drought-response mechanism because predawn water potential decreases in response to drought but may be unaffected by root-pruning treatments (Smith and Greenwood 1997). Root pruning may act by altering hormone ratios or reducing the concentration of an inhibitory substance produced by the roots. Because application of CK can reduce cone formation and offset the effects of exogenous GA, root pruning may act by altering the GA to CK ratio (Smith and Greenwood 1995, 1997, Greenwood et al. 2010). Transplanting has effects similar to root pruning, presumably by the same mechanism (Owens and Blake 1985). Heat Heat treatment alone, often implemented by placing the tree under polyethylene to trap solar heat, results in increases in strobilus production for some conifers, such as Sitka spruce (Tompsett 1977). Timing is critical. In Engelmann spruce (Picea engelmannii Parry ex Engelm.), heat represses cone production when applied during early, rapid shoot elongation, but it enhances cone production when applied toward the end of lateral shoot elongation, when growth has slowed and lateral bud determination occurs. The treatment window to promote female strobili is several weeks later than that for male strobili, which develop on lower, less vigorous branches that complete growth sooner (Ross 1985). This may explain why heat alone increased male strobilus production, but had no effect on female strobilus production, in Norway spruce (Chałupka and Giertych 1977, Johnsen et al. 1994). Note that when combined with GA, timing is less critical, and heat increases male and female cone production in many species, including Picea spp. (Philipson 1992, Johnsen et al. 1994), western hemlock (Pollard and Portlock 1981), and Pacific silver fir (Owens et al. 2001). However, in Scots pine, solar heating suppresses female cone production, alone and in combination with GA (Chałupka 1981). In Picea spp., the effects of heat and GA are only additive (Tompsett 1977, Chałupka 1981), suggesting a common mechanism for cone enhancement. Indeed, the levels of less-polar GAs believed to play a role in generative determination increase after 1 day of heat treatment in Norway spruce and remain elevated for 2–3 weeks (Chałupka et al. 1982). This may explain why heat treatment is only effective in enhancing female cone production when applied during late growth, precisely at the time of strobilus initiation. When applied early in the season, the elevated levels of endogenous GAs may return to normal levels before generative determination. Drought Drought alone is sufficient to enhance cone production in some conifers, such as Engelmann spruce (Ross 1985), Douglas-fir (Ebell 1967), and jack pine (Pinus banksiana Lamb.) (Riemenschneider 1985). Timing is critical and varies by species. Spring irrigation followed by summer drought increases female strobilus production in loblolly pine (Dewers and Moehring 1970). In Engelmann spruce, the timing of inductive drought is opposite to that of heat: Drought enhances cone production when applied during early, rapid shoot elongation, but it represses cone production when applied toward the end of lateral shoot elongation, when growth has slowed and lateral bud determination occurs (Ross 1985). Drought is more commonly applied together with GA and other cultural treatments, such as heat or girdling (Owens and Blake 1985, Smith and Greenwood 1997). In Sitka spruce, drought + heat + GA resulted in increased production of male and female cones during a cool, wet summer, when GA alone had no treatment effect (Philipson 1992). In Douglas-fir, inductive drought increases levels of arginine and other amino acids (Ebell and McMullan 1970), which might suggest a common mechanism with nitrate fertilization (Ebell 1972a). Possible Mechanisms When cultural practices are combined with exogenous GAs, the effect on cone enhancement is often synergistic, suggesting multiple reproductive pathways (Pharis et al. 1987). When cultural practices are used alone, effects are generally much less dramatic or nonexistent. This supports the hypothesis that there are multiple pathways that must each create a permissive state for reproduction to occur (Ross and Pharis 1985). However, environmental factors, such as photoperiod, temperature, and nitrogen availability, regulate GA biosynthesis and metabolism, suggesting a possible common mechanism for environmental control of reproductive development in conifers (Chałupka et al. 1982, Pollard and Portlock 1984, Odén et al. 1995). Indeed, many cultural treatments that enhance cone production, including drought, girdling, heat, root pruning, and nitrate fertilization, alter the levels of GAs, frequently increasing the levels of the less-polar, inductive GAs and decreasing the levels of the more-polar GAs. These changes in GA biosynthesis and metabolism may in part explain the promotive effect of such treatments (Pharis et al. 1987, Bonnet-Masimbert 1989). Other changes occur in the levels of ABA, CK, and ethylene in response to certain cultural treatments; therefore, crosstalk between multiple hormonal signaling pathways may be involved in regulating reproduction in response to such treatments (Bonnet-Masimbert 1989). Environmental and stress signaling also regulate the levels of many miRNAs, notably miR156 (Hsieh et al. 2009, Lee et al. 2010, Kim et al. 2012, Sun et al. 2012, Stief et al. 2014). Because miR156 regulates phase change, cultural treatments may indirectly act to prolong or shorten the period of reproductive incompetence by altering miR156 levels (Yamaguchi and Abe 2012). Controlling Ratio of Male to Female Cones Spatial separation of male and female cones into zones within the crown is common among conifers (Ross and Pharis 1987). In some conifers, such as Pinus and Tsuga spp., development of male and female cones is temporally (female cones differentiate a few weeks after male cones) and spatially (Owens and Blake 1985) separated. Thus, male strobili develop under and may require slightly different environmental conditions (longer photoperiod, less direct light, different temperature) than do female strobili (Giertych 1967). Because GA acts in male and female strobilus development, GA application can be timed to preferentially enhance one sex over the other in species that display temporal separation of male and female bud differentiation (Chałupka 1981). Manipulation of photoperiod can influence sex expression in some species. For example, in western hemlock, an 18-h photoperiod favors male strobili and a 13-h photoperiod favors female development (Pollard et al. 1984). Apical meristems exhibit considerable sexual plasticity during differentiation, as evidenced by the occasional formation of bisporangiate cones (containing micro- and megasporophylls) in nearly all conifer species (Steil 1918, Holmes 1932, Ross and Pharis 1987). Therefore, it is possible to manipulate sex expression in some species by altering the environmental conditions or hormone levels during differentiation to favor one sex over the other (Ross and Pharis 1987). For example, auxin may be involved in male strobilus development. IAA is maintained at higher concentrations in proximal areas, where male cones form, during the period of male cone bud differentiation in lodgepole pine (Kong et al. 2012). Auxin applied alone stimulates male and reduces female cone production in some species, such as Chinese pine (Sheng and Wang 1990). CKs also appear to have a role in sex determination in some conifers, and changes in CK biosynthesis and metabolism have been implicated in sex determination (Kong et al. 2012). When applied before strobilus initiation, CK inhibits GA-induced strobilus initiation. When applied after male strobilus initiation, but before differentiation, CKs enhance female cone formation in Japanese pines through conversion of male cones to female cones (Wakushima 2004). Thus, the ratio of CK to auxin may be important, with auxins favoring male development and CKs favoring female development. It is likely that other hormones, such as ABA (Kong et al. 2012) and ethylene (Ross and Pharis 1987), play some role in sex determination. There is considerable indirect evidence to support a role for vigor in sex expression, with female cone production associated with sites of high vigor, and male cone production associated with sites of lower vigor. These effects appear to be indirect, with sex determination mediated through changes in hormone signaling. Nevertheless, treatments that affect vigor may be useful in regulating sex determination (Ross and Pharis 1987). Seed Orchard Panmixia Most seed orchards are designed to encourage panmictic equilibrium, meaning that each clone or family is equally likely to mate with any other clone or family, ensuring equal contribution to the genetic diversity of the progeny (Funda and El-Kassaby 2013). However, there is considerable variation in fecundity and reproductive phenology among clones, families, and individuals, making such orchard harmonization difficult (El-Kassaby et al. 1989, El-Kassaby and Askew 1991). Half of the parentage in a seed orchard may be attributed to just 20% of the trees (Funda 2012), and less than half of the trees in a seed orchard may contribute meaningfully to genetic diversity (Chałupka 2008). Cone enhancement techniques may be used on individual trees to improve panmixia. In addition, because cone enhancement techniques often have a greater effect on less-productive trees, they may improve genetic contribution even when applied broadly across an orchard (Chałupka 2008). Techniques that preferentially promote the formation of cones of one sex over the other may be useful to correct problems with sexual asymmetry in species with unequal male and female fertility (Choi et al. 2004, Codesido and Fernández-López 2013). Mist cooling may be used to delay bud-break and compress reproductive phenology, reducing background pollen contamination and improving synchrony within an orchard (Silen and Keane 1969, Funda and El-Kassaby 2013). Summary Our understanding of reproductive maturation in temperate conifers has advanced steadily over the past decades. Vegetative and reproductive maturation are often confused in the literature, but they are now known to be distinct, but overlapping, genetic and physiological processes. This separation makes it difficult to use vegetative traits as markers for reproductive maturation, and no suitable biochemical marker has been found. Improved understanding of the mechanisms underlying reproductive maturation in conifers is overdue and could prove useful in probing for markers of reproductive maturation. The search for a genetic, biochemical, or biophysical marker is an important area for future research because identifying this marker could accelerate tree improvement efforts. Since the discovery that exogenous GA enhances cone production, application of GA has emerged as the most consistently reliable technique for cone induction. 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TI - Regulation and Management of Cone Induction in Temperate Conifers
JF - Forest Science
DO - 10.5849/fs-20l6-131
DA - 2018-02-01
UR - https://www.deepdyve.com/lp/springer-journals/regulation-and-management-of-cone-induction-in-temperate-conifers-8vMRHrJUKT
SP - 82
EP - 101
VL - 64
IS - 1
DP - DeepDyve
ER -