Evidence that cytokinin controls bud size and branch form in Norway spruce

Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current-year whorl shoots of 15- to 20-year-old trees of Norway spruce [ Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds.
We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.     

Relations between cytokinin level, bud development and apical control in Norway spruce, Picea abies

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA), and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.     

Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings

Cuttings were taken from 4-week-old seedlings of Norway spruce ( Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL).
Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.     

Ectomycorrhizal community structure varies among Norway spruce (Picea abies) clones

In northern boreal forests, the diversity of ectomycorrhizal (ECM) species is much greater than that of their host trees. This field study investigated the role of individual trees in shaping the ECM community. We compared ECM communities of eight Norway spruce (Picea abies) clones planted in a clear-cut area in 1994 with a randomized block design. In 2003, the ECM fungi were identified from randomly sampled root tips using denaturing gradient gel electrophoresis (DGGE) and rDNA internal transcribed spacer (ITS) sequence similarity. ECM diversity varied among clone groups, showing twofold growth differences. Moreover, according to detrended correspondence analysis (DCA), ECM community structure varied not only among but also within slow-growing or fast-growing clones. Results suggest that ECM diversity and community structure are related to the growth rate or size of the host. A direct or indirect influence of host genotype was also observed, and we therefore suggest that individual trees are partly responsible for the high diversity and patchy distribution of ECM communities in boreal forests.     

Winter hardening in first-year black spruce (Picea mariana) seedlings

Winter hardening of first-year black spruce [ Picea mariana (Mill.) B.S.P.] seedlings was studied by assessing a number of morphological and physiological changes under three hardening regimes: 1) early removal (ER), in which seedlings were exposed to natural daylengths and low ambient temperatures outside. 2) extended greenhouse culture (EG), in which seedlings were exposed to natural daylengths and warm temperatures, and 3) short day (SD), in which seedlings were exposed to short daylengths and low ambient temperatures outside. Measurements included needle primordia initiation, embryonic shoot volume, terminal bud mitotic index, embryonic shoot average cell volume, and shoot tip frost hardiness. EG seedlings formed buds containing 4 times as many needle primordia as ER stock. Embryonic shoot volume increased with number of needle primordia initiated, until late in the hardening period, when significant reductions in meristem volumes of SD and EG stock were observed. Frost hardiness increased sooner in seedlings which set bud in response to short days, but SD treatment did not result in significantly greater frost hardiness at the end of the trial. Frost hardiness was correlated with mitotic index of the embryonic shoot. Cell size in the embryonic shoot declined in seedlings of all treatments during hardening, however, EG seedlings had significantly lower cell volumes by the end of the trial in comparison to ER and SD seedlings.     

Abscisic acid content at defined levels of bud dormancy and frost tolerance in two contrasting populations of Picea abies grown in a phytotron

Seedlings of a southern (Romanian) and a northern (Swedish) population of Picea abies were cultivated under continuous light and 20°C for 10 weeks. To arrest growth, induce terminal bud dormancy and promote frost tolerance the seedlings were then exposed to 16 h nights for 12 weeks, with gradually lower temperature during the last 6 weeks. Samples for estimating the abscisic acid content of the needles were taken just before the onset of the night treatment, at day 3 of the treatment, and then with one, and later 2 week, intervals. From the second week onwards (third week for frost tolerance) bud dormancy and frost tolerance were assessed at the same time as abscisic acid (ABA) determinations. Phosphate-buffered saline extracts were purified on mini-columns (in some cases immunoaffinity colums) and quantified by HPLC. The degree of dormancy was estimated by transferring the seedlings to growth conditions and determining the number of days until growth was resumed. The frost tolerance of the needles exposed to –10°C and –20°C was classified in 6 classes. The frost tolerance of the terminal buds was estimated as the number of seedlings that showed some growth after 6 weeks in growth conditions. The night treatment rapidly induced terminal bud dormancy in both populations, but the release of dormancy occurred earlier in the northern population. The needles and the terminal buds became highly frost tolerant more rapidly in the northern than in the southern population and before the temperature decrease. The degree of dormancy began to decline before full frost tolerance was obtained in the southern population and this decline continued in both populations, while frost tolerance remained at a high level. The southern population showed a transient peak in ABA content at day 3. Although the ABA content of the northern population was lower than in the southern before the 16-h night treatment, it increased in the northern population during the treatment period, in particular after the temperature decrease.     

Growth of coniferous trees exposed to SO2 and O3 using an open-air fumigation system

Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) Karst.) and Sitka spruce (Picea sitchensis Bong. Carr.) were planted as 2-year-old seedlings in an open-air fumigation facility at Liphook in southern England in March 1985. The soil was a humoferric podzol of pH 4. SO₂ fumigation began in May 1987 and continued until December 1990. Long-term mean SO₂ concentrations were 4,13 and 22 nmol mo^?1. Three plots, one at each SO₂ level, were also exposed to O₃ at an average of 1–3.times the ambient level. O₃ fumigation ran from March to December 1988, May to December 1989 and February to December 1990. Each species reacted differently to treatment. Scots pine showed no growth response to either pollutant, although other work on the site demonstrated a number of deleterious effects of SO₂ on this species, including increased leaf loss and foliar injury. Stem basal diameter growth of Norway spruce was depressed in SO₂-treated plots. In contrast, extension growth of shoots of Sitka spruce increased in SO₂-treated plots, in apparent response to codeposition of NH₃-N. However, diameter growth of Sitka spruce main stems did not increase. No effects of O₃ on growth were recorded for any species.