Hormonal Infl uence on Flowering

Acknowledgements

T. L. Davenport

5.6 Hormonal Infl uence on Flowering

T.L. Davenport 116

Photoperiod

Flowering in most trees does not appear to be under photoperiodic control (Kozlowski et al., 1991). Mango cultivation is concentrated between 27° N and 27° S where the shortest annual photoperiod is c.10.5 h and the longest photoperiod is c.13.5 h. Cultivars in the upper-latitude tropics and subtropics fl ower during the winter when photoperiods are short; however, trees in the low-latitude tropics, where a 12-h photoperiod is nearly constant, can fl ower at any time of the year. Furthermore, fl owering on spring-initiated shoots in the subtropics occurs during summer (Schaffer et al., 1994). Studies have failed to demonstrate a correlation between 8-h photoperiods and fl owering (Maiti, 1971; Maiti and Sen, 1978; Maiti et al., 1978). Núñez-Elisea and Daven- port (1995) studied the effects of 11-, 12-, 13- and 24-h photoperiods at 18°C day/10°C night, or 11- and 13-h photoperiods at 30°C day/25°C night on fl owering of container-grown trees. Photoperiod had no effect on the fate of buds, and the promotive effect of cool temperatures on fl owering was inde- pendent of photoperiod. Photoperiods of 11-, 12- or 13-h with 18°C day/10°C night, caused fl owering in trees within 40 days. The 24-h photoperiod with 12-h thermoperiods of 18°C and 10°C caused fl owering of trees within 35 days. Photoperiods of 11- or 13-h at 30°C day/25°C night resulted in vegeta- tive growth only. With warm temperatures, vegetative shoots were produced in 17 days. These results confi rm that fl oral induction is caused by cool tem- peratures and not by short photoperiods and that warm temperature, not a long photoperiod, caused vegetative induction.

Reproductive Physiology 117

of mangoes under specifi c conditions in the low-latitude tropics (Davenport and Núñez-Elisea, 1997).

The involvement of endogenous ethylene in fl owering is supported by observations that indirectly link it to symptoms of ethylene production.

Extrusion of latex from terminal buds occurs at the time of infl orescence ini- tiation, and epinasty of mature leaves near the apex during expansion of the panicle has been observed (Davenport and Núñez-Elisea, 1990, 1991). Both are symptoms of plants exposed to high ethylene levels (Abeles, 1973). Indi- rect support also comes from reports that KNO₃-stimulated fl owering of mango is mediated by increased levels of endogenous ethylene (Thuck-Thye, 1978; Lopez et al., 1984). Mosqueda-Vázquez and Avila-Resendiz (1985) reported that the effi cacy of KNO₃ was negated by cobalt chloride (CoCl₂) and silver nitrate (AgNO₃), which inhibit the synthesis and action of ethyl- ene, respectively, when sprayed 1–4 h after KNO₃. Saidha et al. (1983) reported a gradual increase in endogenous leaf ethylene production as the season of fl oral initiation approached. Ethylene production by stems producing repro- ductive shoots was up to fi vefold that of resting stems.

Inconsistent (Pandey et al., 1973; Sen et al., 1973; Winston and Wright, 1986) or non-responsive results with ethephon (Pandey and Narwadkar, 1984; Ou and Yen, 1985; Pandey, 1989) or smudging (Sen and Roy, 1935), especially during warm, non-inductive conditions, have been reported. Dav- enport and Núñez-Elisea (1990, 1991) reported elevated ethylene production in mango stems in response to ethephon sprays without an accompanying fl oral response. Experiments were conducted during fl oral-inductive and non-inductive periods. Unlike Saidha et al. (1983), they observed no increase in ethylene production rates prior to or during panicle development.

The effect of ethylene on fl owering is unresolved. It is likely that ethylene stimulates shoot initiation by inhibiting auxin transport from leaves to buds and stems (Morgan and Gausman, 1966; Beyer and Morgan, 1971; Riov and Goren, 1979, 1980; Ramina et al., 1986). This may increase the ratio of cytoki- nin to auxin in buds and stimulate shoot initiation (Davenport, 2000). Other factors (i.e. cool temperatures or aged leaves) may be responsible for fl oral induction (Ona and de Guzman, 1982; Davenport, 1993).

Auxin

Although auxin may have a critical role in fl oral induction of mango (Chadha and Pal, 1986; Hegele et al., 2006), there is little supporting evidence. The application (L.B. Singh, 1961; Singh and Singh, 1963; Bakr et al., 1981; Pandey and Narwadkar, 1984) and analysis of auxin in leaves (Paulas and Shanmu- gavelu, 1989; Sivagami et al., 1989), stems (Chen, 1987) and shoots (Chacko et al., 1972b) have been reported in relation to mango fl owering. These studies are inconclusive due to inconsistencies in purifi cation and analytical methodolo- gies (Davenport and Núñez-Elisea, 1997).

Auxin may indirectly stimulate root-produced cytokinins through initia- tion of new root growth. Auxin is transported basipetally from growing

T.L. Davenport 118

shoots and leaves to roots (Goldsmith, 1968; Cane and Wilkins, 1970; Wilkins and Cane, 1970; Goldsmith and Ray, 1973; Lomax et al., 1995) and stimulates root initiation (Hassig, 1974; Wightman et al., 1980). The effi cacy of various auxins for stimulating adventitious rooting of mango marcots and cuttings was reviewed by Davenport and Núñez-Elisea (1997).

Auxin inhibits shoot initiation (Davies, 1995) and confers apical domi- nance by preventing axillary bud break. Leaf-produced auxin and petiolar auxin transport capacity declines as leaves age (Veen, 1969; Veen and Jacobs, 1969; Davenport et al., 1980). The interaction of decreasing auxin and accu- mulating cytokinins in resting buds may explain the cyclic nature of shoot initiation. The ratio of cytokinin to auxin levels in buds regulates shoot initiation (Skoog and Miller, 1957; Bangerth, 1994; Cline et al., 1997; Beveridge et al., 2003).

Cytokinins

Relationships between mango fl owering and the endogenous levels of cyto- kinins in leaves (Paulas and Shanmugavelu, 1989; Kurian et al., 1992), stem tips (Agrawal et al., 1980) and xylem sap (Chen, 1987) and the effect of cyto- kinin applications on bud break and shoot development have been reported.

Chen (1985) described precocious fl owering of mango shoots in response to early October application of 6-benzylaminopurine (BA). Flowering was observed 1 month following application and 3 months later on non-treated trees. Núñez-Elisea et al. (1990) reported numerous reproductive shoots per stem in response to the synthetic cytokinin, thidiazuron, during cool, fl oral inductive conditions; however, numerous vegetative shoots per stem were initiated when thidiazuron was applied during warm, vegetatively induc- tive conditions. Early bud break was not achieved following foliar applica- tion of Promalin (commercial formulation of BA and gibberellins A₄+A₇) (Oosthuyse, 1991b), BA (A.K. Singh and Rajput, 1990) or kinetin (Singh and Singh, 1974).

Chen (1987) reported the lowest levels of putative trans zeatin and its riboside were translocated from roots during the vegetative shoot growth and resting stages, whereas the highest levels occurred during early fl ower- ing and full bloom. Paulas and Shanmugavelu (1989) observed no signifi cant difference in cytokinin levels of the fourth and fi fth leaves during resting bud and fl owering. Cytokinin levels in mango stem buds increased during expo- sure to cool, fl oral inductive temperatures (Bangerth et al., 2004). Agrawal et al. (1980) described 11 cytokinin-like substances isolated from stem tips of an alternate-bearing cultivar in ‘on’ and ‘off’ years. Kurian et al. (1992) reported a link between PBZ applications and reduction in cytokinins in mango leaves with treatments, perhaps caused by reduction in feeder root development and formation of thick, blunt roots (Bausher and Yelenosky, 1987; Peng et al., 1991; Burrows et al., 1992; Yelenosky et al., 1993). Concurrent with this response was suppression of bud initiation and reduced internode lengths for c.2 years.

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The role of cytokinins in fl owering is unresolved due to sampling of dif- ferent organs at non-comparable times or conditions. The elevated cytokinin levels found prior to and during fl owering and the fl owering response to applied BA led to the conclusion that cytokinins are involved in fl owering of mango (Chen, 1985, 1987; Bangerth, 2006); however, such responses can be explained if cytokinins are involved in stimulation of bud break (i.e. shoot initiation) during fl oral inductive conditions.

A well-documented role for cytokinins in higher plants, especially evi- dent in vitro, is bud organogenesis (Skoog and Miller, 1957; Miller, 1963;

Takahashi, 1986; Salisbury and Ross, 1992; Davies, 1995; Haberer and Kieber, 2002). The primary cytokinins in higher plants are trans zeatin, dihydrozeatin, isopentenyl adenine and their ribosides. They are translocated from roots and accumulate in resting buds (Hendry et al., 1982a, b) or can possibly be synthe- sized in nearby tissues as regulated by auxin (Nordstrom et al., 2004; Tanaka et al., 2006). Their rate of accumulation may relate to periodic root fl ushes that alternate with shoot fl ushes (Krishnamurthi et al., 1960; Bevington and Castle, 1986; Cull, 1987, 1991; Parisot, 1988; Williamson and Coston, 1989).

Gibberellins

Gibberellins are tetracyclic diterpenoid compounds that vary in biological activity according to the type and location of substituted side groups on a basic ent-gibberellane skeleton. The number of known gibberellins is > 100 (Pearce et al., 1994). Reproductive shoot initiation is suppressed in many woody angiosperms by gibberellic acid (GA₃) (Pharis and King, 1985). GA₃ inhibits mango fl owering (older literature reviewed in Davenport and Núñez-Elisea, 1997; Núñez-Elisea and Davenport, 1998).

GA₃ inhibition of mango fl owering is correlated with the applied con- centration (Kachru et al., 1971, 1972) and may cause buds to develop vegeta- tively under fl oral-inductive conditions. Núñez-Elisea and Davenport (1991a, 1998) reported a delay in initiation of axillary shoots when GA₃ was foliar applied to deblossomed stems during cool, fl oral inductive temperatures.

Higher concentrations caused longer delays in shoot initiation. GA₃ did not inhibit fl oral induction, so long as cool, inductive temperatures were present during axillary shoot initiation. Late initiating buds, which grew during warm, spring temperatures, however, formed vegetative shoots. Similar delays in reproductive shoot initiation in response to GA₃ application was reported by Shawky et al. (1978) and Turnbull et al. (1996). Multiple applica- tions, even at lower rates, are more effective than a single application (Tomer, 1984; Turnbull et al., 1996; Davenport and Smith, 1997). GA₃ treatment has been recommended in the Canary Islands to delay fl owering until the danger of frost has passed (Galán-Saúco, 1990). In the subtropics of Australia, it is used to prevent fl owering in newly planted trees during the spring so that the full growing period can be utilized for vegetative growth, thereby hastening orchard establishment (A.W. Whiley, personal communication, Queensland, 1996).

T.L. Davenport 120

Response to GA₃ varies among cultivars, growing conditions and timing of application (Tomer, 1984; Oosthuyse, 1995a; Turnbull et al., 1996; Sánchez- Sánchez et al., 2004). GA₃ can delay shoot initiation beyond the fl oral induc- tive window, resulting in a vegetative fl ush when shoots develop in warm weather (Kachru et al., 1971, 1972; Núñez-Elisea and Davenport, 1991a, 1998;

S. Gazit, personal communication, Israel, 1993). The variable response to GA₃ may be related to levels of active gibberellin in buds at the time of applica- tion, inconsistent uptake or differential sensitivity of buds, depending on their position (apical versus axillary) or age (Núñez-Elisea and Davenport, 1991a, 1998). Effi cacy is related to the timing of application; immediately prior to normal shoot initiation appears to be most effective (Davenport and Smith, 1997).

Reports of endogenous gibberellins in mango tissues, especially in buds, are diffi cult to interpret with respect to a regulatory role in bud break or fl owering. Problems include sampling of tissues other than apical buds, i.e.

whole stems (Tongumpai et al., 1991b), leaves (Paulus and Shanmugavelu, 1989; Sivagami et al., 1989) and xylem sap (Chen, 1987), or at times when developing shoots may contribute to the overall result (Chen, 1987). Pal and Ram (1978) tentatively identifi ed the presence of gibberellins A₁, A₃, A₄, A₅, A₆, A₇ and A₉. Chen (1987) identifi ed gibberellins A_1/3, A_4/7, A₅, A₁₇, A₂₀ and A₂₉. The estimated levels of gibberellins in apical buds for 6 months prior to the fl owering season were reported to be higher in the ‘off’ year than in the

‘on’ year of an alternate-bearing cultivar (Pal and Ram, 1978). Chen (1987) reported the highest levels of gibberellins in xylem sap during leaf differen- tiation and lower concentrations during rest, panicle emergence and full fl owering. Tongumpai et al. (1991b) observed increasing levels of gibberellins in whole stems over the 16 weeks prior to vegetative shoot emergence and decreasing levels over the same period prior to panicle development. Gib- berellins A₁, epi-A₁, A₃, A₁₉, A₂₀ and an unidentifi ed gibberellin in buds and leaves from shoot and stem tips of different ages have been quantifi ed (Dav- enport et al., 2001b). The detected gibberellins are members of the early 13-hydroxylation pathway of gibberellin synthesis (Takahashi, 1986; Pearce et al., 1994). Gibberellins A₃ and A₁₉ were the most abundant gibberellins in apical stem buds. The concentration of GA₃ increased within buds with increasing age of stems, although concentrations of other GAs were variable.

The concentration of GA₃ did not change signifi cantly with age in leaves, whereas that of most of the other GAs declined. Davenport et al. (2001b) concluded that elevated GA₃ levels in buds may enhance or maintain the synthesis or activity of endogenous auxin to maintain low cytokinin/auxin ratios and enhance inhibition of shoot initiation (Jacobs and Case 1965; Scott et al., 1967; Pharis et al., 1972; Ross et al., 1983; Law 1987; Law and Hamilton, 1989).

The roles of gibberellins and other phytohormones in shoot initiation and induction is unclear. Endogenous levels in buds and leaves must be cor- related with physiological events in individual stems. Experimental approaches should include examination of resting buds up to both vegetative and repro- ductive shoot initiation to avoid misinterpretation of results. Experiments

Reproductive Physiology 121

should utilize plants grown under defi ned conditions with specifi c environ- mental controls for evaluation of cause and effect. Finally, extraction and purifi cation protocols should include quantifi able internal standards and use of sensitive unambiguous analytical techniques.

Plant growth retardants

Plant growth retardants have been evaluated to stimulate early or more intense fl owering, especially in the ‘off’ year of alternate-bearing cultivars (Davenport and Núñez-Elisea, 1997). They are in three main classes: (i) the gibberellin transport inhibitor, daminozide (N-dimethylamino-succinamic acid), known as alar or B-Nine; (ii) the onium type, chloremquat chloride (2-chloroethyl trimethylammonium chloride), known as cycocel and CCC;

and (iii) the steroid-synthesis-inhibiting triazoles, for example PBZ (PP-333), known as Cultar^®, and uniconazole, known as XE-1019 or Sumagic (Rademacher, 1991, 2000a). The latter two classes of compounds inhibit ent-kaurene syn- thetase, an enzyme in the gibberellin synthesis pathway (Nickell, 1983;

Dalziel and Lawrence, 1984; Rademacher, 1991, 2000a). Applying daminoz- ide results in increased gibberellin levels, perhaps due to the inability to dis- tribute it properly (Rademacher, 1991). Plant responses may depend upon whether target tissues are near the site of gibberellin synthesis or suffi ciently removed from it to be affected by the inhibited translocation.

Daminozide and cycocel

The effi cacy of daminozide and cycocel for increasing fl owering in the ‘off’ sea- son of alternate-bearing cultivars has been studied (Maiti et al., 1972; Mukhopad- hyaya, 1978; Rath and Das, 1979; Suryanarayana, 1980; Rath et al., 1982; Ou and Yen, 1985), together with their ability to stimulate early fl owering (Suryanarayana and Rao, 1977; Chen, 1985; Kurian and Iyer, 1993a, b). Enhanced, inconsistent fl owering occurs in response to these compounds, especially cycocel.

Triazoles

PBZ is being used (except in the USA where it has not been cleared for use) to stimulate enhanced or early fl owering. It is best applied to the soil due to its low solubility, long residual activity and lack of effi cient foliar uptake (Rademacher, 2000b). PBZ applied as a soil drench (1–20 g active ingredient (ai)/tree) reduces internode lengths and causes earlier and enhanced fl ower- ing in mango trees (Hasdiseve and Tongumpai, 1986; Haw, 1986; Hongsb- hanich, 1986). Depending on climate, residual activity lasts for c.2 years (Kulkarni, 1988a). These results have been confi rmed in different locations in the tropics (Davenport and Núñez-Elisea, 1997; Yeshitela et al., 2004a, b).

Nartvaranant et al. (2000) recommended soil application of PBZ at 1–1.5 g ai/m of canopy diameter to achieve fl owering in 90–120 days if the trees are stimulated to fl ush. Davenport (2003) observed that such treatments allowed a reduction of c.1 month in the time required for stem rest before stimulating them to initiate reproductive shoots using KNO₃. PBZ also reduces alternate

T.L. Davenport 122

bearing of some cultivars (Hillier and Rudge, 1991; Burondkar and Gunjate, 1993; Rao, 1997; Rao et al., 1997; Rao and Srihari, 1998; Vijayalakshmi and Srinivasan, 1999). Cultivars that tend to fl ower with minimal inductive impe- tus are more responsive and can be induced to fl ower out-of-season using PBZ (Tongumpai et al., 1989). Núñez-Elisea et al. (1993) demonstrated that application of PBZ and uniconazole advanced bud break of containerized trees in controlled environment chambers, but cool temperatures were neces- sary to induce fl owering. Initiated shoots were induced to be vegetative in warm temperatures. The greater proportion of purely reproductive panicles in treated plants (compared with controls) suggests that triazoles impact the level of a putative VP, probably a gibberellin. Whiley (1993) suggested a sec- ondary mechanism for the fl oral promotive action of PBZ on mangoes, not- ing inconsistent responses in the literature between cultivars, environments and application times.

Application of PBZ reduces the number of panicles, despite increased fruit set (Goguey, 1990). Davenport (1987, 1994) observed neither growth inhibition nor enhanced or early fl owering in response to root drenches or bark banding with uniconazole (1–5 g ai/tree) in trees growing in alkaline, calcareous soil. He reported that new shoot growth was stunted with extremely short internodes when trees were severely pruned soon after or as long as 3 years after treatment. Yield was severely reduced due to the lack of normal growth fl ushes. The growth stunting effect continued for 7 years after pruning. Davenport (1994) warned that use of triazole plant growth retar- dants for control of tree growth, fl owering or yield must be done with con- siderable caution, especially if severe pruning of the trees is anticipated.

Residual uniconazole or PBZ applied as a soil drench or bark band is appar- ently retained in high concentrations in main scaffolding branches. In Cen- tral and South America, growers utilize PBZ annually to stimulate early fl owering. A test tree should be severely pruned to determine if the trees are affected by PBZ to anticipate the orchard response to later severe pruning.

Certain gibberellins (i.e. GA₁) are necessary for shoot elongation. Inhibi- tion of bud break and shoot elongation in response to application of the growth retardants cycocel (Maiti et al., 1972) and triazoles (Kulkarni, 1988a;

Burondkar and Gunjate, 1991, 1993; Tongumpai et al., 1991a; Kurian et al., 1992; Winston, 1992; Kurian and Iyer, 1993a, b; Núñez-Elisea et al., 1993; Wer- ner, 1993) have been reported. Elongation of panicles is inhibited, especially by high levels of triazoles (Kulkarni, 1988b; Winston, 1992; Davenport, 1994;

Salomon and Reuveni, 1994). Infl orescences in treated trees may become compact, improving opportunities for disease and insect attack (Winston, 1992). Kurian et al. (1992) associated reduced cytokinin levels in leaves with inhibition of shoot initiation in plants treated with soil drenches of PBZ. Ele- vated, concentration-dependent levels of phenolic compounds were also found in resting apical buds of PBZ-treated trees (Kurian et al., 1994). They suggested that low cytokinin activity and high phenolic levels in buds con- tributed to inhibition of shoot initiation.

The combined impact of the gibberellin synthesis-inhibiting triazoles on shoot initiation, induction, and elongation implies that several different

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gibberellins regulate specifi c activities in mango plants. This is supported by the inhibitory effect of GA₃ on shoot initiation in contrast with early initia- tion of fl owering in triazole-treated trees. Compression of reproductive and vegetative shoot internodes may involve inhibition of GA₁ synthesis. Stimu- lation of fl owering instead of vegetative growth during early initiation in triazole-treated plants in marginal or non-fl oral inductive conditions, sug- gests that the putative VP, a gibberellin other than GA₃ or GA₁, is reduced when gibberellin synthesis is inhibited.