Plant growth and development are regulated by phytohormones or growth regulators, low-molecular weight compounds present in cells in relatively low concentrations, acting in tissues remote from the place of their synthesis. Plant hormones may be divided into five basic groups: auxins, gibberellins, cytokinins, abscisic acid, and ethylene. Recently, polyamines and brassino-steroids have also been recognized as growth regulators. Of the many studies of plant hormones, studies of forest trees and particularly the conifers, constitute only a small fraction of the published works. With regard to papers dealing with coniferous trees, most of the studies on hormonal regulation of growth and development have been conducted on Scots pine (Pinus sylvestris). This review examines the role of plant hormones in regulating growth and development of Norway spruce [Picea abies (L.) Karst.] and evaluates the current state of research in this field.
7.1.1. The occurrence of phytohormones and their role in growth and development
Auxins belong to the best-known group of growth regulators. Auxins are natural and synthetic substances that stimulate the elongation of shoots and col-eoptiles. Indole-3-acetic acid (IAA) is the most important naturally occurring auxin in higher plants. In coniferous trees, IAA is responsible for many developmental processes, including shoot elongation, cambial activity, xylogenesis, and response to photoperiod. It is synthesized in the needles and buds and then transported to other organs. Auxin movement proceeds on the principle of a slow polar transport and fast, nonpolar transport through the phloem. There are no data in the literature on auxin transport in Norway spruce.
WODZICKI and WODZICKI (1981) suggest that the wave theory of basipetal auxin transport from the top to the base occurs in spruce as it does in pine. Indole-3-acetic acid occurs in plant tissues in both free and bound states. STEEN (1972) observed IAA in Picea abies buds and seedlings using bioassays. In the last few years, improved techniques, such as high-performance liquid chromatography (HPLC), mass spectrometry, and immunological tests have been used to determine the IAA content in plant tissues.
Bioassays were also used by IVONIS et al. (1983), who studied the annual dynamics of the growth regulator in needles of one- and two-year-old trees of Norway spruce. In that study, high levels of auxins were present during the period of bud dormancy. FACKLER et al. (1986b), using immunological assays to determine IAA distribution in the needles of Norway spruce, found that IAA levels depended on position in the tree crown, age, and season. These authors reported that free auxin concentration in the needles was higher in a shaded crown position than in full sun. The highest auxin content in the young needles of the crown apical zone was recorded in May and decreased to a minimum in July before increasing again in October. Seasonal differences in IAA content in the needles of the lower whorls were much less pronounced. Free IAA constituted 93% of the total IAA content in the buds, whereas bound IAA was predominant in the needles. In summary, the authors found that on the basis of the absolute contents of free and bound auxins in spruce needles, no conclusions can be drawn in relation to the potential growth abilities of tree organs. PSOTA et al. (1992) examined IAA content in the needles of 10- and 240-year-old trees of Picea abies throughout the year. They found that auxin content declined from October to November, after which it increased and achieved its maximum in July. IAA content in the needles of an old tree was significantly lower than that of a 10-year-old tree, whereas changes in its level in the annual cycle were significantly smaller.
DUNBERG (1976) found a marked increase in auxin content in spruce shoots from May to July, indicating a role of IAA in shoot elongation. Sandberg and Ernsten (1987) examined free and bound IAA contents in the seeds of P. abies before and during germination, using combined methods of HPLC and gas chromatography coupled with mass spectrometry. The content of bound auxin in seeds was several times higher than that of free auxins. During seed germination, bound auxin gradually decreased to a stable minimum, whereas the content of free auxin continually increased until the fifth day of germination, after which it also decreased to the initial level. The authors assumed that the increase in free IAA content resulted from the hydrolysis of conjugates of IAA, and not from de novo synthesis. A preliminary treatment of spruce seed with auxin had no influence on germination (Sandberg 1988).
Exogenously applied auxins promote rooting. POPIVSHCHII and SHAPKIN (1986) found that soaking spruce seedlings in IAA and NAA (naphthale-
neacetic acid) enhanced growth, increasing height, diameter, and biomass of needles, shoots, and roots for two years following treatment. Mauer and Palatkova (1989) as well as SEABY and SELBY (1990) found increased growth of lateral roots in Picea abies seedlings following treatment with natural auxins and NAA.
Changes in auxin levels may occur in plants in response to environmental stress. For example, WESSLER and WILD (1993) tracked the IAA content in needles of Picea abies in declining stands on industrial sites in Germany. The auxin level in the needles of visibly injured trees was significantly lower than that of healthy trees.
Gibberellins (GA) constitute a large class of compounds (about 80 have been described to date) with a characteristic, basic terpenoid structure. Not all of them are biologically active and not all of them occur in higher plants. Kato et al. (1962) first detected gibberellins in conifers in the cones of Juniperus chinensis. DUNBERG (1973) first investigated gibberellin-like substances in the shoots of Picea abies, finding that these substances as well as auxins both influence shoot elongation (DUNBERG 1974). Gibberellins are synthesized in leaves. They occur in both free and bound states in cells. Their prime role consists of regulating plant height growth, shoot elongation, and the transition of the plant from the vegetative to the reproductive stage and associated processes, including the induction of flowering and regulation of seed germination. In the case of Norway spruce, the majority of studies have concerned the role of gibberellins in flowering.
Cytokinins occur in young, actively dividing meristemic cells of shoots and roots. They are synthesized in roots and are transported to other plant organs through the xylem along with water and solutes. The best-known cytokinins are zeatin and 2-isopentenyladenine, which are known to also occur in coniferous trees. Cytokinins participate in the regulation of many plant processes, such as cell division, shoot and root morphogenesis as well as organ senescence. ROGOZINSKA (1967) first noted the presence of cytokinins in Scots pine. SEBANEK et al. (1991) examined cytokinin content in the needles of 10-and 240-year-old individuals of Norway spruce. They found no differences in cytokinin content between young and old trees, but noted two characteristic annual minima in November and March. BOLLMARK et al. (1995) found that zeatin riboside (ZR) was the most abundant cytokinin in Norway spruce buds and that also isopentenyladenosine (iPA) was present in all samples. The level of zeatin-type cytokinins was correlated with bud size. CHEN et al. (1996) followed the levels of endogenous ZR and iPA in the terminal buds, whorl buds, and lower lateral buds in shoots of the uppermost whorl of 15- to 20-year-old trees of Norway spruce from June to September. Bud growth was greatest during August and September and was correlated with ZR levels, indicating the importance of cytokinin for bud development. Differences in ZR levels among bud types also suggested that this hormone may control the form of individual branches in the tree.
Schwartzenberg and Hahn (1991) found a high content of free and bound cytokinins in the needles of declining Picea abies trees growing on a site contaminated with industrial pollutants. Cytokinin content was correlated with the degree of the needle injury. KRAIGHER and HANKE (1996) determined the level of isopentenyladenine- types of cytokinins in needles of Norway spruce seedlings growing on soil substrates from polluted and unpolluted forest research plots. They found higher levels of cytokinins in seedlings grown on polluted soils.
In some conifers, e.g. Douglas fir (Pseudotsuga menziesii), cytokinin is involved in the induction of flowering (IMBAULT et al. 1988). Cytokinins can be used exogenously to rejuvenate plant organs as has been shown in Norway spruce (BOURIQUET et al. 1985; MATSCHKE et al. 1991).
Abscisic acid (ABA) belongs to a group of growth inhibitors commonly occurring in plants. It is synthesized in all plant cells having plastids. Its content in tissues can be successfully determined by gas chromatography, liquid chromatography, and immunological assays. ABA plays a major role in regulating bud and seed dormancy, water relations, as well as in the processes of plant acclimation and response to stress factors.
There are several reports concerning a regulatory role of ABA in Norway spruce. HEIDE (1986) injected ABA to the apical buds of Picea abies seedlings and caused a transient inhibition of shoot elongation. She did not confirm a decisive role of ABA in inducing bud dormancy. QAMARUDIN etal. (1993) determined the ABA content in seedlings of two Picea abies populations from Sweden and Romania in a study of bud dormancy and frost tolerance. They found no correlation between ABA content and bud dormancy, or frost hardiness of these two populations of spruce. FACKLER et al. (1986a) found an increased ABA content in the needles of five-year-old spruce trees exposed to ozone. Yang et al. (1993) revealed the influence of ABA on the synthesis of the ethylene precursor, amino-cyclopropane-carboxylate (ACC), in needles of Norway spruce injured by industrial pollution. In that study, ABA restricted ethylene synthesis and thereby assisted in needle response to environmental stress.
There are no separate reports in the literature on the role of abscisic acid in regulating water relations and drought resistance of Norway spruce. However, a number of studies have involved Picea mariana, P. glauca, P. sitchensis, and other spruce species. The role of ABA in plant drought resistance consists of the regulation of stomatal conductance and in maintaining a suitable osmotic potential as well as inducing gene expression and synthesis of specific proteins that protect cells from dehydration injury. Generally, plants exposed to drought stress accumulate ABA in their cells. However, a higher concentration of abscisic acid does not always ensure a higher drought tolerance. ROBERTS and DUMBROFF (1986) found that ABA content in seedlings of P mariana and P. glauca exposed to a drought stress initially increased, but declined with a large increase in water stress. Increasing ABA content was correlated with declining transpiration rates. After re-hydration of the seedlings, ABA levels returned to their initial values and transpiration increased. SILIM et al. (1993) using the retardant-mefluidide, which caused an increase in the dehydration tolerance of P. glauca seedlings, found that it induces an increase in ABA content in the needles, a decrease of stomatal conductance, and the resultant maintenance of a high osmotic potential in shoots. Tan and BLAKE (1993) also revealed ABA accumulation in P. mariana seedlings exposed to drought stress, but they found no correlation between the amount of ABA and the degree of resistance to dehydration among progenies. Tan and BLAKE (1993) observed that ABA accumulation in the needles of P. mariana exposed to drought was concomitant with an increase in electrolyte leakage from the cells, which according to them, was a result of the interaction between abscisic acid and membrane lipids, leading to changes in membrane permeability. ABA alone applied exogenously had a similar effect. The same phenomenon occurred in the needles of Pinus banksiana and in the leaves of Eucalyptus grandis. JACKSON et al. (1995) compared the dehydration stress tolerance of 3-year-old individuals of Picea sitchensis and Pinus sylvestris. In both species the concentration of ABA in cells increased 11-fold as the water deficit increased. However, Pinus sylvestris displayed a higher tolerance to drought than spruce. In spruce, the response of stomatal cells to an increased level of abscisic acid was delayed, and consequently, the osmotic potential declined during dehydration.
Ethylene also belongs to the group of growth inhibitors. It is the only hormone in a gaseous form. It is synthesized in the majority of plant organs. It is detected and measured using gas chromatography. Ethylene regulates fruit ripening, leaf and flower senescence, leaf and fruit drop, seedling growth, seed and bud dormancy, and participates in root and flower induction. The mechanism of its action is not fully understood. Environmental conditions and other hormones, such as auxin, promote ethylene biosynthesis.
EKLUND (1993) suggested that the maintenance of a low ethylene content in shoots is important for the process of wood formation. Later studies (EKLUND and TILTU 1999; EKLUND et al. 2003) indicated a positive relation ship between spiral grain angle in the last growth ring and ethylene concentration in stems of Picea abies. The authors confirmed that ethylene affects wood formation quantitatively, as measured by increased compression wood and an increase in left-handed spiral grain angle. INGEMARSSON (1995) found that ethylene stimulates lignification and cell wall formation in P. abies seedlings. BOLLMARK and ELIASSON (1990a) examined the participation of ethylene in root induction in excised 4-week-old spruce seedlings. They found a stimulating effect of ethylene on root formation along with a simultaneous degradation of cytokinins.
EKLUND et al. (1992) investigated the influence of drought stress on ethyl-ene production in spruce and found no correlation between ethylene production and drought stress. In contrast, DRIESSCHE and LANGEBARTELS (1994) observed an in increase in ethylene synthesis and its precursor, ACC (1-amino-cyclopropane-carboxylate), in 4-year-old spruce trees subjected to drought and ozone stresses separately. However, in the combined ozone and drought treatment the synthesis of ethylene and ACC was reduced and was associated with reduced injury. The authors suggest that in young spruce trees exposed to ozone, drought stress has less of an effect on tissue injury, and consequently ethylene production is also decreased. An increase in ethylene synthesis and ACC was also observed in spruce needles following treatment with acid mist (CHEN and WELLBURN 1989). A simultaneous application of other growth regulators, such as IAA, ABA, kinetin, and gibberellic acid, had no effect on ethylene production. WILKSCH et al. (1998) studied the emission of ethylene and its ACC and MACC (malonyl-amino-cyclopropane-carboxylate) precursors in Norway spruce growing on contaminated and uncontaminated sites in Germany. The emission of ethylene and its precursors was considerably higher in injured trees.
In the recent years, a considerable number of studies have examined the role of polyamines in plant growth and development (SLOCUM et al. 1984; BAGNI and BIONDI1987; GALSTON and KAUR-SAWHNEY1987). KONIGSHOFER (1989, 1991) found seasonal changes in polyamine content in juvenile and mature trees of Norway spruce that were dependent upon age. In contrast to juvenile organs, shoots of mature spruce individuals grown from seed, grafted trees, as well as buds prior to sprouting, are characterized by a pronounced increase of putrescine content at the time of intensive shoot sprouting. A transient increase in polyamine content in juvenile shoots was observed prior to shoot sprouting. Polyamine content varied annually from peak concentrations in the newly formed needles to lowest concentrations during the period of shoot sprouting (May-July). Similar changes were observed in juvenile as well as in mature spruce individuals, indicative of polyamine involvement in the regulation of shoot elongation. The occurrence of abundant levels of putrescine in the shoots in the cambial region during its intensive activity suggests that polyamines in Norway spruce may also be involved in diameter growth.
Polyamines occur also in seeds of Norway spruce. Santanen and SIMOLA (1999) observed that putrescine levels were approximately ten times higher in the embryo than in megagametophyte tissues, whereas spermidine and spermine levels were nearly identical in both tissues. The authors suggest that polyamines may play a role in the accumulation of seed storage proteins and in the maturation of P. abies seeds.
Tenter and WILD (1991) showed that the putrescine content in needles of injured and declining spruce trees was several-times higher in comparison to healthy trees. In that study, the marked differences in the content of putrescine and the ratio of putrescine to spermidine may serve as indicators of the general condition of trees growing in areas subjected to industrial pollutants. SANDERMAN etal. (1989) andDOHMEN etal. (1990) observed putrescine accumulation in spruce needles in response to ozone stress, suggesting a role of polyamines as factors stabilizing membranes under oxidative stress conditions. LANCHERT and WILD (1995) found a negative correlation between the potassium ion content and putrescine content in needles of Norway spruce. Stress-induced membrane injury is often manifested by increased electrolyte leakage, followed by a decline in potassium content in cells. A strong negative correlation of putrescine with K ions in Norway spruce trees was also observed by Kaunisto and Sarjala (1997) and suggested that putrescine concentration can be used for describing symptoms of potassium demand in spruce trees of various sizes and varying nutritional status.
7.1.2. Hormone participation in the regulation of flowering
Flowering is one indication of the transition of a plant from its juvenile stage to physiological maturity. PHARIS et al. (after JACKSON and SWEET 1972) suggested that the juvenile period in coniferous trees encompasses the time period when the appropriate hormones have not achieved a sufficient concentration to induce flowering. Further investigations demonstrated a key role of gibberellins in flowering in terms of both absolute level and composition. The influence of gibberellins on the induction of flowering depends on their molecular structure, specifically on the number and location of the double bonds and hydroxyl groups. Gibberellins differ in terms of their effects on plant proteins, such as enzymes, transporter proteins, or receptors.
In Norway spruce, ODEN et al. (1982,1987) identified the following gibberellins: GA1, GA3, GA4, and GA9. DUNBERG (1974) isolated six gibberellins from spruce shoots and found that a high content of endogenous gibbe-rellin-like compounds during the initiation of flower buds ensures abundant flowering. IVONIS et al. (1981) and IVONIS (1988) also found a correlation between gibberellin content in spruce needles and the initiation of flower buds.
CHALUPKA et al. (1982) found that covering shoot apices of spruce with polyethylene resulted in an increased production of male flowers and a simultaneous increase in content of a less-polar gibberellin in shoots. ODEN et al. (1994) determined the content of the endogenous gibberellins GA1, GA3, GA4, and GA9 in elongating shoots of Norway spruce grafts during the period of flower bud differentiation in response to two treatments: cool and wet vs. hot and dry. The cool and wet treatment exhibited decreased numbers of flower buds, whereas the hot and dry treatment stimulated flowering. They also found that gibberellin GA9 was the dominant GA and that the content of all gibberellins increased during the period of shoot elongation and declined when shoot elongation ceased. The ratio of GA9 to GA1 was 12.5 in the cool and wet treatment and 36.6 in the hot and dry treatment, suggesting that this ratio may serve as an index of reproductive buds.
In many studies, exogenously applied gibberellins have been used to induce flowering with the aim of understanding of the role of gibberellins in the flowering process. BLEYMULLER (1976,1978) sprayed spruce shoots with GA3 solution, which stimulated the differentiation of female flowers. DUNBERG (1980) applied several gibberellins as well as NAA to induce the production of seed and pollen cones. He obtained the best results with a direct shoot injection with a GA4/7 mixture, followed by an additional application of GA9. The same mixture of the less-polar GA4/7 gibberellins appeared to be the most effective in inducing flowering in spruce in the experiment of SCHACHLER and Matschke (1991). In contrast, JOHNSEN etal. (1994b) concluded that the regulation of flowering and the sex determination process is also possible under strictly controlled external temperature conditions. A single application of gibberellin GA4/7 to a Norway spruce stem cross section during the late stage of shoot elongation in the experiment of Fogal etal. (1996) resulted in a noticeable increase in the number of female strobili and a reduction in the number of vegetative buds.
Exogenously applied gibberellin GA4/7 is also known to stimulate flowering in other spruce species, such as Picea mariana (HO 1991), Picea glauca (Marquard and HANOVER 1984; ROSS 1988), and Picea engelmanni (ROSS 1990). However, the effect of GA4/7 on the sex of the set flowers differs among studies. HO (1991) and MARQUARD and HANOVER (1984) obtained an increase in male flowers, whereas CECICH (1985) and ROSS (1988, 1990) found increases in the abundance of both male and female flowers. Degree-day accumulation during the period of shoot elongation in Picea glauca modified the production of pollen cones (ROSS 1991). ROSS (1990) was able to stimulate male and female flower set in Picea mariana using a GA4/7 mixture and NAA. Smith and Greenwood (1995) also succeeded in stimulating seed and pollen cone production in P. mariana as a result of shoot injection with a GA4/7 mixture and a simultaneous root pruning. The effect of gibberellins and root pruning decreased after the application of cytokinin. The authors concluded that a decrease in cytokinin content in developing buds as a result of root pruning may enhance flowering.
ODEN et al. (1995) investigated gibberellin transport and metabolism during bud differentiation in Picea abies, using deuterium- and tritium-labeled gibberellin GA4 injected into the xylem below an elongating shoot and needles. This study revealed that gibberellins were transported first to the needles and then back to the stem and lateral buds. In other experiments, a mixture of deuterium-labeled GA9 and tritium-labeled GA4 were injected into elongating shoots of one abundantly flowering family and one sparsely flowering family and grown either under hot and dry conditions and under cool and wet conditions conducive for flowering. In all treatments, gibberellin GA9 was transformed into GA51, GA4, GA34, and GA1; whereas gibberellin GA4 was transformed into GA34, GA1, and GA8. In cold-treated clones the main metabolite of GA9 was GA51. In clones treated with heat, more GA9 was transformed into GA4. The main metabolite of GA4 was GA34. The authors suggest that active forms of gibberellins involved in bud differentiation are regulated in specific shoot regions and that their metabolism is influenced by different environmental factors, such as root activity, water potential, and temperature. These findings concur with those of earlier experiments of MORITZ and ODEN (1990) on the metabolism of deuterium- and tritium-labeled gibberellin GA9 in the shoots of Norway spruce during bud differentiation.
7.1.3. Application of growth regulators in somatic embryogenesis
In recent years, there has been an increased interest in the relatively new method of plant propagation through somatic embryogenesis. This method is particularly useful in coniferous trees, where other methods of vegetative propagation are somewhat less successful. Norway spruce is easily cultured in vitro and there are comparatively many reports on obtaining somatic embryos of different spruce species. Somatic embryos of spruce are obtained through multiplication of cotyledon, seed, and entire zygotic embryo tissues on appropriate media. Growth regulators are of primary importance in generating somatic embryos. In the first stage, the applied medium contains auxins and cytokinins. The most frequently used auxins are: NAA (ARNOLD and Hakman 1988; JAIN et al. 1988; AFELE et al. 1992; CHALUPA 1987), 2,4 D (2,4-dichlorophenoxyacetic acid) (BELLAROSA et al. 1992; SUSS et al. 1990) as well as IBA (indole-3-butyric acid) (ROBERTS et al. 1990; CHALUPA 1987). Benzyladenine (BA) is the most frequently used cytokinin, though SUSS et al. (1990) also used kinetin. BELLAROSA et al. (1992) obtained a high yield of somatic embryos of Norway spruce on a medium with both 2,4 D and BA.
Abscisic acid plays an important role in the process of somatic embryo production at the final stage of culture to obtain fully viable embryos (ATTREE et al. 1990). BOZHKOV et al. (1992) found that ABA applied simultaneously with
BA improves the quality of somatic embryos of Norway spruce. Abscisic acid promotes the maturation of somatic embryos, which consists of the synthesis of certain storage proteins (HAKMAN etal. 1990; ROBERTS etal. 1990). Only after an adequate period of growth in the presence of ABA are somatic embryos capable of generating an entire plant on a medium lacking the hormone. Abscisic acid is a factor governing the expression of genes responsible for the synthesis of storage proteins (ROBERTS et al. 1990; HAKMAN 1993; FLINN et al. 1993). In addition, an appropriately selected ABA concentration and an osmotic agents, such as mannitol or polyethylene glycol (PEG), together increase the resistance of the embryo to dehydration (ROBERTS 1991; ATTREE et al. 1991,1994; STASOLLAetal. 2002). Then, the so-called "artificial seeds" can be prepared for germination. According to Leal et al. (1995), somatic embryos of coniferous trees may be useful models in studies of gene expression.
VAGNER et al. (1998) studied the content of endogenous growth regulators during the development and maturation of somatic embryos of Norway spruce. They found that the level of endogenous ABA depends to a large extent on its prior concentration in a given medium. After removal of ABA from the medium, ABA levels in embryos are maintained for some time. The IAA content decreases during the development of somatic embryos and increases again in the period of late maturation. Cytokinins (isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside) behave similarly. Embryogenic tissue produces 40 times less ethylene than non-embryogenic tissue. FIND (1997) also showed that endogenous ABA levels in somatic spruce embryos were dependent upon on the exogenous concentration of ABA and on the osmotic potential of the medium. A decrease in osmotic potential of the medium caused a decrease in ABA concentration and an increase in embryo germination.
Stanislawa Pukacka, Polish Academy of Sciences, Institute of Dendrology, Kornik.
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