Cytokinin Phytohormonal Effects on Crown Structure

A BRIEF DESCRIPTION OF CYTOKININS AND THEIR ROLES IN PLANT DEVELOPMENT

Cytokinins are one of the most important phytohormones in regulating development and growth in plants. Numerous endogenous cytokinins have been identified, and synthetic cytokinins are available on the market for exogenous application (Buban 2000; Wertheim 2000; NeSmith 2004; Carey 2008). Cytokinins play an essential role in plant development throughout the life cycle, ranging from the germination of seeds to fruit set (Sakakibara et al. 2006; Kyozuka 2007). Various cytokinins are synthesized in the plastids of plant tissues (including stems, leaf primordia, meristems, seeds, and especially roots), regulate development in neighboring tissues, and, after transport in xylem and phloem, regulate whole-plant developmental patterns. Root-produced cytokinins coordinate shoot:root resource allocation and are important for communicating plant nitrogen status (Pons et al. 2001). Naturally occurring cytokinins are nitrogen-rich molecules that contain either aliphatic isoprenoid or aromatic side chains (Sakakibara 2006). When cytokinins trigger receptors in the cell membranes of target cells, they activate kinases cascades (molecular chains of phosphate transferring molecules) that regulate gene transcription in nuclei. Cytokinin-activated kinase cascades may interact with signals from other phytohormones or photoreceptors, such as phytochrome (Boonman et al. 2007; Taiz and Zeiger 2010). Cytokinins may be present in both active forms and as potential regulatory molecules in inactive conjugates with sugars, sugar phosphates, and other molecules (Sakakibara 2006).

At tissue and organ levels, cytokinin concentrations change throughout the year due to phenology and maturation state of plant tissues (Andres et al. 2002; Juvany et al. 2013). Activity is generally associated with regulating cell division. In reproduction and embryo development, they regulate sex determination and pollination, seed dormancy, and germination (Schmulling 2002; Bishopp et al. 2006). After germination, they enhance cell division in root/shoot meristems, regulate the formation of vascular elements, and regulate leaf development (Moore 1998; Carey 2008). In mature tissues, they regulate genes associated with chlorophyll biosynthesis and nutrient partitioning, they open stomata, and they delay senescence. Important to plant crown formation, branch and meristem development are controlled by cytokinins (Roitsch and Ehneß 2000; Rasmussen et al. 2009).

Cytokinin and other phytohormones are controlled by a system of checks and balances within the plant. Cytokinin synthesis is autonomously down-regulated as total concentrations of cytokinin increase in plant organs, and as inactivation by enzymes are stimulated by other phytohormones, particularly auxin. Developing plant shoot organs compete with one another for water and nutrients by producing negative-effect hormones, which either down-regulate production of phytohormones required by competing organs or inactivate available phytohormones. For example, the influence of apical bud dominance over lateral buds led to the discovery of auxin as the first identified phytohormone (Thimann and Skoog 1933), where the removal of apical bud and a primary leaf of bean plants (Phaseolus vulgaris) induced rapid expansion of secondary leaves by interruption of auxin produced in the shoot apex, leading to increased gibberellin content (Humphries and Wheeler 1963). Unbeknownst to Thimann and Skoog, auxin and gibberellins phytohormonal checks and balances were at play.

Basic research into plant physiological processes is usually conducted on “model plants,” such as Arabidopsis and Nicotiana, where the accumulation of vast data bases permit connecting the dots between genomic and biochemical activity and whole-plant physiology. As most physiological processes are highly conserved, models derived from basic research can be applied to other species with the caveat that physiognomy and environment may require their modification. While definitive understanding of the action of phytohormones in particular situations may require species-specific studies, the diverse body of literature on phytohormones permits valuable generalization of phytohormone effects and activity.

CYTOKININ IN BRANCHING AND CROWN DEVELOPMENT

Crown architecture is due to a complex interaction of phytohormones, most commonly auxin, gibberellins, and ethylene, working in combination with cytokinins. Phytohormones are responsible for regulating branch angle, the amount of branching, shoot growth, and the activation of lateral buds (Cline and Dong 2002; Oates et al. 2004; Sansberro et al. 2006; Müller and Leyser 2011).

In the shoot apical meristem, cytokinin is a major regulator of meristem characteristics and branch initiation. Arabidopsis mutants that lacked cytokinin showed a severe reduction in shoot apical meristem size, indicating the importance of cytokinins in shoot development (Kyozuka 2007). Cytokinin controls the outcome of undifferentiated cells in shoot apical meristems (Kurakawa et al. 2007). In a developing shoot, there is a cell elongation zone of approximately 10 to 15 cm in length subtending the meristem. The elongation zone is an area of undifferentiated cells that begin to form into determined organs, as phytohormone balances shift in nearby tissues. In the apical meristems of tobacco plants (Nicotiana tabacum L.), cytokinins induce the biosynthesis of auxin, which regulates the formation of bud primordia in the expanding shoot (Nakagawa et al. 2005; Cortleven and Valcke 2012). Bud distance from an active growing meristem determines the amount of cytokinin, which is correlated with triggering lateral bud burst and further branch development. Therefore, the development of a new shoot meristem depends on whether the activated bud (and its associated undifferentiated cells) is associated with the internode that has not begun to elongate or one that has already partially completed the elongation process (Elfving and Visser 2006). The two principal regulatory pathways are apical dominance (when the terminal bud of a shoot or branch prevents activity of dormant lateral buds) and apical control (when the terminal bud inhibits the growth of branches from activated lateral buds).

It has been long known that the apical meristems on dominant shoots inhibit bursting of lateral buds. The power of control of lateral bud bursting by shoot apices is the primary mechanism by which woody plants assume their typical crown form. Strong apical dominance leads to monopodial crowns, typical of many conifers, while weaker dominance results in highly branched sympodial-shaped crowns, typical of angiosperm trees and shrubs. The process of apical dominance is maintained by down-regulating cytokinin supply to lateral buds by the phytohormones auxin and abscisic acid (ABA) (Ward and Leyser 2004) from shoot apical meristems and developing foliage, and by strigolactones, a form of steroid produced in roots (Hayward et al. 2009). Auxin produced in the shoot apex travels basipetally through xylem parenchyma and stimulates the production of cytokinin dehydrogenase, which inactivates cytokinins diffusing toward lateral buds. ABA appears to have a role in suppressing lateral bud development in woody plants that acts independently of auxin (Frewen et al. 2000). The mechanism associated with strigolactones, is less well understood, but their presence also down-regulates cytokinin activity. Both auxin and ABA may also down-regulate cytokinin synthesis in root plastids (Frewen et al. 2000; Tanaka et al. 2006). These phytohormonal mechanisms maintain dominant shoot status by sustaining its larger sink for water, nutrients, and carbohydrates.

Branch buds are initiated in the shoot apex (apical meristem and developing foliage), and their flushing activity requires activation by cytokinin. The effect of the shoot apex on lateral bud activity diminishes as distance between apical and lateral buds lengthen. As the influence of negative-effect phytohormones decreases and cytokinin and resource availability to lateral buds increase with distance from the apical meristem, lateral buds are released from suppression (Rasmussen et al. 2009). When lateral buds are released from suppression, their endogenous cytokinin levels increase, high nutrient levels are associated with high rates of cell division, and new branches form (Humphries and Wheeler 1963).

Auxin synthesized in the shoot apex has also been identified as the principle regulator of apical control (Cline 1991; Wilson 2000). Both apical dominance and apical control are regulated by phytohormones through their effects on vascular tissue development and by altering sink strength for carbohydrates and nutrients. In apical dominance, connections between the vascular system and dormant buds are prevented. In apical control, cambial stimulation by phytohormones from the shoot apex results in selective enhancement of vascular tissue supplying the shoot apex (Cline and Harrington 2006). What both developmental mechanisms have in common is the reduction or elimination of cytokinin availability to lateral buds and branches. In apical dominance, cytokinins are actively deactivated. While in apical control, the transpiration streams (with its root-produced cytokinins) are diverted to the shoot apex. Selective loading mechanisms for cytokinins are believed to be active in both xylem (i.e., transpiration streams) and phloem, regulating transport to sinks for cytokinin in other plant tissues (Sakakibara et al. 2006). In turn, cytokinins affect growth metabolism, enzyme activity, and the biosynthesis of other phytohormones through regulation of gene activity (Sakakibara et al. 2006). Cytokinin effects are largely limited to aboveground via manipulation of sink strength.

Branch density and angle are affected by both exogenous and endogenous cytokinin and its interaction with auxin. When the terminal bud is clipped (auxin supply is interrupted), many of these dormant buds develop, and the branch density increases as apical control is lost, resulting in a ‘full’ tree. Once the apical meristem is excised, auxin levels in the stem decrease, repression of cytokinin biosynthesis is released, and cytokinins levels increase to promote lateral bud growth. After a lateral bud is activated, it assumes control as the apical meristem of its subtending shoot. An endogenous supply of auxin is synthesized in the new apical meristem and represses cytokinin availability to lateral buds on its branch (Tanaka et al. 2006).

Cytokinins generally induce branching, but the concentrations and effects vary widely among species and by interactions with other phytohormones (Carey 2008). In addition to interactions with auxin, gibberellins and cytokinins are mutually antagonistic. Cytokinins inhibit gibberellin formation, and gibberellin inhibits cytokinins responses (Weiss and Ori 2007). Abscisic acid inhibits seed germination, stimulates ethylene production, and causes stomata to close (Kong et al. 2009). Cytokinin inhibits the activity of ABA on all of these processes (Carey 2008; Kong et al. 2009), maintaining more rapid growth and, in many cases, preventing phase change of vegetative meristems to floral meristems and prolonging juvenile maturation states.

Additional factors that affect cytokinin activity in branching are the level of the particular cytokinin (Carey 2008), the sensitivity/responsiveness of the tissue to cytokinin, and for exogenous applications, the location of cytokinin application (Bangerth et al. 2000). High levels of cytokinins in branch shoots stimulate bud growth and lateral stem formation (Bangerth et al. 2000; Carey 2008). A particular tissue may or may not be competent to recognize the presence of cytokinins (Carey 2008). The intensities of these factors may vary due to tissue age, stress, environment, life-stage of plant development or other conditions. For example, younger Picea rubens trees show a higher reaction to exogenous cytokinin application with the increase of needles within a given area on the branch, increase in stem mass, and an increase in stem length (Figure 1). The concentration of cytokinin had the same effect across the three variables. In apples, certain cultivars, such as ‘Gala’ and ‘Delicious,’ show greater response to exogenous cytokinin application than others.

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Figure 1.

Differential effects on foliage and shoot growth of cytokinin application related to tree age in red spruce (Picea rubens Sarg.). The x-axis gives tree age as (S) sapling mean 25 years and old-growth (O) mean 160 years and 0 to 500 mg L⁻¹ exogenous B-6-P application. Both species exhibit maximum treatment effects at 50–100 mg L⁻¹ (Autio 2013), and age differentials consistent with those previously reported for red spruce (Greenwood et al. 2008). Bars indicate standard errors.

Exogenous auxin application inhibits growth of lateral branches (Cline 2002), but the inhibition can be canceled by endogenous cytokinin (Durbak et al. 2012). Exogenously applied cytokinin stimulates the cell of elongation zone in plants (Cortleven and Valcke 2012), and can delay maturation and competency to flower (Taiz and Zeiger 2010). Rasmussen et al. (2009) found exogenous cytokinin application increased leader length, bud width, bud count, and needle number when applied to the leading shoot of a branch in Abies Nordmanniana. While exogenous application of cytokinins can produce dramatic effects in the short-term, cytokinins do not affect apical meristems in the long-term. Once suppressed lateral buds begin to grow, plants return to prior balances of auxin and cytokinins (Carey 2008).

ENVIRONMENTAL EFFECTS

Little is known about the interactions of cytokinin activity with environmental stress factors (i.e., moisture, temperature stress, and pathogen infections). Many environmental variables have yet to be tested and reported. However, scientists do know that genes regulating endogenous cytokinin production are directly coordinated with macronutrient content, particularly nitrate content. Cytokinin has a negative effect on root growth and on stem growth. As cytokinins are widely reported to prevent or delay reproductive maturation and seasonal dormancy, potential interactions and/or feedbacks may occur between environmental factors, such as photoperiod, temperature, and drought as exogenous signals for reproduction or dormancy.

Both cytokinin synthesis and water movement through roots are up-regulated by soil nitrate availability. Cytokinin is translocated through the transpiration stream in xylem conduits, and up-regulates nitrate responsive genes in the phloem of roots and shoots. The rate of nitrogen uptake increases cytokinin synthesis, the rate of water transport, and the rate of cytokinin transport (Sakakibara et al. 2006).

CYTOKININS AS TOOLS IN ARBORICULTURE AND HORTICULTURE

Industries that could benefit from hormonal technology include the tree care industry, woody plant horticulture, and fruit care industries. Synthetic plant growth regulators (PGRs) are usually structural analogues of hormones (Moore 1998). Cytokinins are based on nitrogen-carbon rings and naturally occur in both isoprenoid and aromatic forms. Forms vary in their side chains, which impart receptor specificity in target cells (Sakakibara 2006). Analogous manufactured chemicals that mimic the structures and functions of natural phytohormones can also have significant effects on plant growth and development.

The best-known hormonal cytokinin found in plants is zeatin, and the most common forms of synthetic cytokinin are its analogs based on benzyladenine, or BA (Moore 1998). One cytokinin product registered for use on ornamental plants is Configure®; a benzyladenine-based PGR. It can be applied as a foliar spray or a substrate drench at concentrations from 1 to 3000 mg·L⁻¹ (Carey 2008; Table 1; Table 2). A few others, such as Topolin® (Buban 2000), 9-Benzyladenine (Canal et al. 2000), Triacanthine, 6-Chloroprine, and 4PU (Zhang and Hasenstein 1999) have resulted in larger fruit sizes and increased return bloom the following year, a movement from inactive to active forms of BA, an increase in lateral budbreak, and a decrease in terminal budbreak as seen in experimental studies (Table 1; Table 2). In addition, many other chemicals are known to have partial cytokinin activity (Carey 2008).

Many attempts have been made over the years to experimentally integrate cytokinin products into horticultural practices focused on lateral branch stimulation in young nursery trees (Elfving and Visser 2005). Benzyladenine+GA4,7 was first registered in the late 1970s for agricultural crops, and pyranylbenzyladenine PBA was registered in the late 1970s for use on ornamentals. From the 1970s until the 1990s, a product called Pro-shear® (2% BA) was used on white pine trees (Pinus strobus L. in Christmas tree plantations) to increase branch development by increasing lateral bud set in the year of application. However, it is no longer produced (Baker 2001). Some products are not labeled for use in every state or country due to variations in state regulation laws and often by marketing decisions by manufacturers. In many cases, cost–benefit analysis may discourage efforts at registration compliance in multiple jurisdictions. Cytokinins-based PGRs are commonly used in a 1:1 ratio with Gibberellin 4,7 in a product called Promalin® (Carey 2008).

Exogenous cytokinins are usually applied to plants via foliar sprays or drenches and are absorbed through the cuticle (Canal et al. 2000). Plants can absorb cytokinins through leaf or root epidermis and transport them to growing points. It is likely that younger tissues with thinner cuticle and epidermal layers absorb more cytokinins than older tissues (Carey 2008). Activity level depends on the form of synthetic cytokinin (Hartmann et al. 2001; Davies 2004b) and the amount applied to a particular site (Carey 2008).

Currently, arborists use a mixture of chemicals and synthetic hormones to manipulate tree growth, shape, and increase branching, while avoiding unwanted damage to other plants, animals, and humans. Extrinsic regulation of development and form can be accomplished at any growth stage. Tree shape alteration can begin in embryos. Both Lyyra et al. (2006) and San-Jose (2001) found that a liquid BAP cytokinin pulse induced adventitious shoot formation in cotyledons of black willow (Salix nigra Marsh.) and sweet chestnut (Castanea sativa Mill.). At the seedling stage, spray treatments of cytokinin have been used to alter tree shape by hindering stem elongation and promoting adventitious bud formation in Douglas-fir seedlings (Lisheng, et al. 2009). Cline et al. (2006) found that whole-tree (but not terminal leader) foliar BA applications promoted both bud formation and second flushing in six-year-old Douglas-fir seedlings. During release from quiescence in mature trees, cytokinin levels increase, and branching is controlled by relative levels of cytokinin and auxin as the crown develops (Mazri 2013).

Generally, cytokinin in stem tissue is required for activation of dormant lateral buds, and auxin transported basipetally from shoot apices interferes with this process through apical dominance. Branching agents work by interrupting apical dominance, which triggers lateral bud growth, which ‘fills in’ the plant. Apical dominance can be interrupted in several ways, including by pruning of apical meristems, to reduce auxin, or by applying exogenous cytokinins (Carey 2008). Christmas tree growers have also experimented with applying artificial cytokinins and increasing cytokinin to auxin ratios.

The complexity of the branching pattern depends on the temporal and spatial development of branches. These characteristics, although they are plastic in their response to environmental cues and human intervention, are in large part genetically determined. Therefore, regulation of crown structure is determined by an interaction between the developmental program that specifies branching patterns in different plant species generating species-specific plant forms and natural and/or artificial influences.

Terminal bud dominance and control can be manually manipulated by pruning. Growers and horticulturists commonly use pruning to shape the crown of commercial and urban trees, interrupting the basipetal flow of auxin. Arborists use a multitude of pruning techniques to achieve their goals of aesthetics, safety, and tree health. However, pruning is time-consuming and requires substantial skill and training to avoid unintended consequences.

Pruning lateral branches will change stem taper and therefore affect wood quality and strength. Pruning and lopping of trees in ways that remove large amounts of foliage disrupts both auxins and cytokinins levels. As a consequence of poor pruning practice, large numbers of dormant buds, epicormic buds may develop rapidly. They are often poorly attached but grow quickly under proper environmental conditions. Because of their weak attachment, the branches are easily shed and present a significant hazard in urban settings (Moore 1998). Furthermore, overpruning of a tree will result in lower photosynthetic and hormonal transport levels, which may directly decrease fruiting (Carey 2008).

Plant growth regulators have outstanding potential for arboricultural manipulation of urban trees and avoiding some of the negative aspects of manual pruning. They are currently used in some contexts but, like pruning, require competent and professional workers to avoid situations where direct application of hormones and PGRs result in unacceptable environmental risks. Cytokinins have impressive effects on plant growth and development, but there is an inherent danger in their use (Moore 1998). Many PGRs can be harmful or toxic, and should be used with extreme care (Harrison et al. 2014). Documented and established protocols are available for best PGR practices, and as in any chemical released into the environment users must be thoroughly trained in best practices (as outlined in Table 2).

Although cytokinins contribute to many plant processes, they are not yet widely used in horticulture outside of a few specialized areas, such as tissue culture, apple/cherry production, and Christmas tree farms. In the apple industry, when applied to flowering trees, they promote bloom thinning, which results in higher fruit production in future years (Buban 2000; Baker 2001). When sprayed onto young fruit, cytokinins improve the shape of fruit and increase size. They are used on young apple trees to promote lateral bud break and increase radial growth on branches (Wilson 2000). They are also used on pear trees and nut trees for similar reasons (Andres et al 2002). Cytokinin PGRs are also used on table grapes to increase the size of fruits (Cruz-Castillo et al. 1999).

The bioregulators Cyclanilide® (CYC) and Promalin (PR) show species specific differences in their effects of induced branch height in apple and sweet cherry trees in nurseries (Elfving and Visser 2005; Elfving and Visser 2006). Application of the cytokinin 6-benzyladenine (BA) with or without gibberellic acid isomers GA4 and GA7 (GA4 and GA7) also improves branch formation in apple and sweet cherry trees (Elfving and Visser 2006). Cytokinins generally increase branch size and the number of branches of apple trees, which in turn increases fruit yield. Cytokinins are promising as fruit thinners because they are not harmful to beneficial insects (as carbaryl thinners are) and also promote branching and flowering the following year (Elfving and Visser 2006; Mickelbart 2011).

Both CYC and PR act by altering auxin to cytokinin ratios. CYC is an auxin transport and action inhibitor (Pederson et al. 1997), and PR contains the cytokinin growth promoter 6-benzyladenine (Sachs and Thimann 1967). When Elfving and Visser (2006) applied both to separate tree plots of nursery sweet cherry trees, they found that although the mode of action of CYC and PR products are different, their effects on branching and tree structure seem to be dictated more by tree species than product. CYC induced new lateral shoots starting above the bud union, and lower than spontaneously formed branches that developed on untreated trees. PR did not affect the final height of the lowest induced branch. CYC produced branches starting at a progressively lower height above the bud union on the central leader as the product concentration increased. In contrast, PR did not affect the final height of the lowest induced branch.

In another study, a cytokinin treatment of 0.5 mM BA was applied five to six weeks following two weeks of cytokinin foliar spray treatments of the terminal spring-flushing leader shoots, which resulted in a small increase in the total number of lateral buds and a large increase in current bud outgrowth (Cline et al. 2006). A second terminal bud flushing occurred in only 28.4% of the control seedlings in the five experiments compared with 81.5% of the BA-treated seedlings, indicating that BA substantially enhanced second flushing of the terminal buds. After testing BA applications in different concentrations, both as drops and sprays, 0.5mM BA foliar spray was found to be optimal under greenhouse conditions.

Cytokinins are currently registered for use on apples (Accel®, Promalin) and sweet cherries (Promalin) as fruit thinners, to improve ‘typiness’, and to induce lateral branching and budbreak (Buban 2000; Western Plant Growth Regulator Society 2000; Baker 2001; Yu et al. 2008).

THE FUTURE OF CYTOKININS IN ARBORICULTURE AND HORTICULTURE

Potential uses for cytokinin phytohormones include: to decelerate senescence and to promote growth and bud formation, to improve grafting results, to be used as branching agents, and to improve branching angles (Mok 1994).