Cellular changes induced by exogenous and endogenous gibberellins in shoot tips of the long-day plant Silene armeria

Stem elongation and flowering are two processes induced by long-day (LD) treatment in Silene armeria L. Whereas photoperiodic control of stem growth is mediated by gibberellins (GAs), the flowering response cannot be obtained by GA applications. Microscopic observations on early cellular changes in the shoot meristem following LD induction or GA treatment in short days (SD) were combined with GA analyses of stem sections at various distances below the shoot apex. The earliest effects of both LD and GA induction on the subapical meristem were an increase in the number of cells per cell file and a reduction of cell length in the meristematic tissue approx. 1.0–3.0 mm below the shoot apex. Within 8 d after the beginning of LD induction or after GA application, the cells in the subapical meristem were oriented in long files. In induced tips, cellulose deposition occurred mostly in longitudinal walls, indicating that many transverse cell divisions had taken place which, in turn, increased the length of the stem. In contrast to LD induction, GA treatments did not promote the transition from the vegetative to the floral stage. Endogenous GAs were analyzed by selected ion monitoring (SIM), using labeled internal standards, in extracts from transverse sections of the tip at various distances below the apical meristem. In control plants, the levels of the six 13-hydroxy GAs studied (GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈) decreased as the distance from the apical meristem increased. Except for GA₅₃, GA levels were higher in tips of LD-induced plants, particularly in the meristematic zone approx. 0.5–1.5 mm below the apical meristem. In comparison with SD, the highest increase observed was for GA₁, the content of which increased 30-fold in the zone 0.5–3.5 mm below the shoot apex. These data indicate a spatial correlation between the accumulation of GA₁ and its precursors, and the enhanced mitotic activity which occurs in the subapical meristem of elongating Silene apices.Abbreviations GA_n gibberellin A_n - LD long day(s) - SD short day(s) We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]- gibberellins, Dr. B.O. Phinney, University of California, Los Angeles, USA, for [¹³C]GA₈, Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility, for advice with mass spectrometry, and Mr. M. Chassagne, I.N.R.A. C.R. Bordeaux, for the photography. This work was supported, in part, by a fellowship from the Spanish Ministry of Agriculture (Instituto Nacional de Investigaciones Agrarias) to M.T., by the U.S. Department of Energy under contract DE-ACO2-76ERO-1338, and by the U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Predicting spring frost sensitivity by bud development and temperature sum in Norway spruce seedlings

Nurseries would benefit greatly if frost hardiness (FH) of seedlings could be predicted by some environmental variable or by bud development in spring. We investigated the FH of 1-year-old Norway spruce (Picea abies (L.) Karst.) seedlings of local origin. The seedlings were stored frozen until incubated in the growth chamber at six different temperature sums (TSs) (0, 55, 88, 142, 185 and 240 d.d., >5°C) from mid-February to mid-March. FH of the buds, stems and previous year needles was assessed on three occasions. When the TS was 88 d.d. or less, buds exhibited only microscopic signs of development, even when seedlings tolerated temperatures below −10°C. As TS increased, primordial needles and primordial stems of buds grew while FH weakened, especially in previous year needles. When the TS was at least 142 d.d., all plant parts were frost hardy to approximately −6°C. Monitoring TS and bud development can help predict FH of Norway spruce seedlings in spring. However, more studies with seedlings of different ages and from multiple locations are necessary to appreciate the generality of our results.     

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.     

Histochemical detection and image analysis of non-specific esterase activity and the amount of polyphenols during annual bud development in Norway spruce

The study focused on Norway spruce trees during their annual development and, specifically, on the histochemical analysis of the activity of the non-specific esterase and the amount of polyphenols in the vegetative buds. Azo-coupling reaction with naphthol-AS-acetate as a substrate and Fast Blue B Salt as a coupling reagent was applied to the longitudinal median cryo-sections of the formalin-fixed buds. A method of image analysis was developed to evaluate the intensity of the histochemical reaction quantitatively. High activity of non-specific esterase was detected in the cells of the collenchymatic plate, the youngest bud scales, the leaf primordia, in the zone of differentiation of vascular tissues and procambium of the dormant bud. The activity of non-specific esterase decreased in all tissues during bud break. During the growth of a new shoot enzyme activity was detected only in the vascular bundles of the shoot. Enzyme activity re-occurred at the beginning of the autumn in the cells of the collenchymatic plate of the newly establishing buds. Three different types of polyphenols at different intracellular localizations (as verified by scanning electron microscopy) were observed: granular, vacuolar and drop-like polyphenols. The amount and localization of those polyphenols changed during the bud growing cycle. The positive reaction of all three types of polyphenols are tannin in nature. These findings are discussed from the viewpoint of frost tolerance and tissue differentiation.     

The apical control of lateral bud development in excised shoot tips of Cuscuta reflexa cultured in vitro

Excised shoot tips of Cuscuta reflexa Roxb. (dodder), a rootless and leafless angiospermic plant parasite, were cultured in vitro for the study of the control of lateral bud development by the apex. In a chemically defined medium lacking hormones, the basal bud alone developed into a shoot. The addition of coconut milk to the growth medium induced the activation of multiple lateral buds, but only a single bud developed further into a shoot. The decapitation of this shoot induced the development of another shoot and the process could be repeated. This showed the controlling effect of the apex in correlative control of bud development. Application of indole-3-acetic acid to the shoot tip explant delayed the development of the lateral bud. Gibberellic acid A₃ induced a marked elongation growth of the explant and reinforced apical dominance. The direct application of cytokinin to an inhibited bud relieved it from apical dominance. A basipetally decreasing concentration gradient of auxin may prevail at the nodes. Bud outgrowth is probably stimulated by cytokinin produced locally in the bud.     

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.     

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.     

Stem elongation and changes in the levels of gibberellins in shoot tips induced by differential photoperiodic treatments in the long-day plant Silene armeria

The effects of differential photoperiodic treatments applied to shoot tips and mature leaves of the long-day (LD) plant Silene armeria L. on growth and flowering responses, and on the levels of endogenous gibberellins (GAs), were investigated. Gibberellins were analyzed by gaschromatography-mass spectrometry and the use of internal standards. Exposure of mature leaves to LD, regardless of the photoperiodic conditions of the shoot tips, short days (SD), LD, or darkness, promoted elongation of the stems and of the immature leaves. Long-day treatment of the mature leaves modified the levels of endogenous GAs in shoot tips kept under LD, SD, or darkness. In shoot tips kept in LD or darkness the levels of GA₅₃ were reduced, whereas the levels of GA₁₉ and GA₂₀ were increased. The contents of GA₁ were increased in all three types of shoots: SD twofold, LD fivefold, and darkness eightfold. Dark treatment of the shoot tips on plants of which the mature leaves were grown in SD promoted elongation of the immature etiolated leaves and increased the GA₁ content of the shoot tips threefold. However, this treatment did not cause stem elongation. The different photoperiodic treatments applied to the shoot tips did not change the levels of GAs in mature leaves. These results indicate that both LD and dark treatments result in an increase in GA₁ in shoot tips. In addition, it is proposed that LD treatment induces the formation of a signal that is transmitted from mature leaves to shoot tips where it enhances the effect of GA on stem elongation.Abbreviations GA_n gibberellin A_n - LD long day(s) - SD short day(s) We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]-gibberellins and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility, East Lansing, for advice with mass spectrometry. This work was supported, in part, by a fellowship from the Spanish Ministry of Agriculture (Instituto Nacional de Investigaciones Agrarias) to M.T., by the U.S. Department of Energy grant No. DE-FG02-91ER20021, and by the U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Conserved expression profiles of circadian clock-related genes in two Lemna species showing long-day and short-day photoperiodic flowering responses

The Lemna genus is a group of monocotyledonous plants with tiny, floating bodies. Lemna gibba G3 and L. paucicostata 6746 were once intensively analyzed for physiological timing systems of photoperiodic flowering and circadian rhythms since they showed obligatory and sensitive photoperiodic responses of a long-day and a short-day plant, respectively. We attempted to approach the divergence of biological timing systems at the molecular level using these plants. We first employed molecular techniques to study their circadian clock systems. We developed a convenient bioluminescent reporter system to monitor the circadian rhythms of Lemna plants. As in Arabidopsis, the Arabidopsis CCA1 promoter produced circadian expression in Lemna plants, though the phases and the sustainability of bioluminescence rhythms were somewhat diverged between them. Lemna homologs of the Arabidopsis clock-related genes LHY/CCA1, GI, ELF3 and PRRs were then isolated as candidates for clock-related genes in these plants. These genes showed rhythmic expression profiles that were basically similar to those of Arabidopsis under light-dark conditions. Results from co-transfection assays using the bioluminescence reporter and overexpression effectors suggested that the LHY and GI homologs of Lemna can function in the circadian clock system like the counterparts of Arabidopsis. All these results suggested that the frame of the circadian clock appeared to be conserved not only between the two Lemna plants but also between monocotyledons and dicotyledons. However, divergence of gene numbers and expression profiles for LHY/CCA1 homologs were found between Lemna, rice and Arabidopsis, suggesting that some modification of clock-related components occurred through their evolution.     

Activation of latent origins of DNA replication in florally determined shoot meristems of long-day and short-day plants: Silene coeli-rosa and Pharbitis nil

Replicon spacing was measured during the S-phase of the cell cycle in shoot meristems of Silene coeli-rosa L., a long-day (LD) plant, and Pharbitis nil Chois, a short-day (SD) plant to examine the hypothesis that activation of latent origins of DNA replication is a feature of floral determination. Silene coeli-rosa was germinated and grown in SD for 28 d and then exposed to either a florally inductive combination of 7 LD + 2 SD, the last day of which coincides with determination of the sepal and stamen whorls, or was germinated and grown in 37 non-inductive SD. Pharbitis nil was germinated and grown in continuous light (CL) for 5 d and then given either 48 h of inductive darkness followed by 1 d of CL, the last day of which coincides with determination of the sepal, petal and stamen whorls, or given one of two independent non-inductive treatments: 48 h dark interrupted by red light (R) + 1 d of CL, or 8 d of CL. Following these treatments, each batch of plants was exposed to tritiated [methyl-³H]thymidine for 30, 60, 90 or 120 min. Apical domes were dissected, nuclei lysed and prepared as fibre autoradiographs from which replicon size was recorded. In S. coeli-rosa, replicon size was in the range 10–15 μm in SD (non-inductive) and 0–5 μm in LD (inductive) while in P. nil it was 10–15 μm in the 48 h dark interrupted by R, 5–10 μm in CL (both non-inductive) but was reduced to 0–5 μm in the 48 h dark treatment (inductive). Therefore, the recruitment of additional initiation points for DNA replication occurred in both a LD and a SD plant immediately before the appearance of floral organs. The data are consistent in showing that a shortening of S-phase, which is a characteristic feature of florally determined shoot meristems for both species, is brought about by the activation of latent origins of DNA replication. Received: 14 May 1998 / Accepted: 20 August 1998     

Changes in endogenous hormones in apple during bud burst induced by defoliation

Under the tropical conditions of East Java, terminal buds of apple burst at any time of the year in response to removal of the subtending leaves. Following two such defoliations, two weeks apart on separate trees, there was a decrease in abscisic acid (ABA), a three-fold increase in gibberellin-like substances (GAs) and only a slight increase in cytokinin-like substances (CKs) in the apex tissue of closed buds. These changes preceded bud opening and the associated increases in fresh and dry weight, and may be causally related to bud burst. In open buds (i.e. young expanding leaves) the concentration of CKs was greater, and the concentrations of ABA and GAs less, than the concentrations in closed buds. As the leaves expanded, ABA increased and GAs and CKs decreased in concentration. The decrease in concentration of GAs and CKs, however, was due to the rise in dry weight of the expanding tissue; the amounts of all three hormones (per apex) increased. During bud burst there was a concurrent decrease in the CKs of subtending stems, suggesting a transfer into the expanding bud tissues. Removal of the old leaves by defoliation may remove the source of ABA and allow the amount of GAs in the apex to rise, bud burst following. Stem CKs may be utilized in the expansion of the new leaves in the bursting buds.     

Non-structural carbohydrate status in Norway spruce buds in the context of annual bud structural development as affected by acidic pollution

The present study focused on changes in the annual dynamics of the contents of non-structural saccharides (NSS) of Norway spruce vegetative buds related to their structural development under the effect of acidic pollution during the year 1995. Two types of material were analysed: (1) 4-year-old trees treated for 2 years by simulated acid rain (SAR; pH 2.9 and 3.9), and (2) 40-60-year-old trees growing in natural mountain stands exhibiting different degrees of macroscopic damage. Our study revealed that the dynamics of the NSS content reflected the major morphogenetic and developmental changes occurring during the annual bud developmental cycle. No systematic changes in the annual dynamics of NSS content were observed in buds from both mountain sites, or as a consequence of the SAR. The total sugar content of bud tissues was composed of a combination of five main sugar components: sucrose, glucose, fructose, raffinose family oligosaccharides (RFO; combination of raffinose and stachyose), and a pinitol fraction (PF) probably of cyclitols with pinitol as a main member. The dynamics of individual sugar components also reflected possible carbohydrate mediated bud frost protection. Interesting results were obtained from buds in dormant state. In dormant buds of the SAR experiment the higher value of the ratio PF:RFO of the pinitol fraction and raffinose family oligosaccharides followed the higher dose of SAR treatment. When evaluating the ratio from both types of material we assumed that changes in PF:RFO ratio corresponded to early stages of damage or acute metabolic reaction. Thus, we suggest the ratio PF:RFO as a possible non-specific metabolic marker of early bud stress reaction which is, among other stress factors, sensitive to increasing load of acidic pollutants.     

Seasonal profiles of sulphur, phosphorus, and potassium in Norway spruce wood

Seasonal profiles of sulphur, phosphorus, and potassium content in the wood of trees have been established for the first time. This became possible by using a novel laser ablation system coupled to HR-ICP-MS for measuring these elements in Norway spruce drill cores. This technique combines excellent spatial resolution with superior detection power, and makes it possible to measure low element concentrations even in relatively narrow annual rings. Despite its low quantity in wood, sulphur is an important macronutrient for plants and seems to display seasonal variations of its concentration, which correspond to actual theories of sulphur metabolism in plants. A similar seasonal pattern was also found for phosphorus, another crucial element in tree nutrition. This was unexpected, because it was previously assumed that the distribution of phosphorus remains constant throughout the year. Potassium, the third element measured, seems to be especially accumulated in the latewood. The profiles presented in this article suggest a seasonal variation, revealing some new aspects of Norway spruce (PICEA ABIES) metabolism.     

Heat induced changes in protein expression profiles of Norway spruce (Picea abies) ecotypes from different elevations

Although tree species typically exhibit low genetic differentiation between populations, ecotypes adapted to different environmental conditions can vary in their capacity to withstand and recover from environmental stresses like heat stress. Two month old seedlings of a Picea abies ecotype adapted to high elevation showed lower level of thermotolerance and higher level of tolerance to oxidative stress relative to a low elevation ecotype. Protein expression patterns following exposure to severe heat stress of the two ecotypes were compared by means of 2-DE. Several proteins exhibiting ecotype and tissue specific expression were identified by MS/MS. Among them, small heat shock proteins of the HSP 20 family and proteins involved in protection from oxidative stress displayed qualitative and quantitative differences in expression between the ecotypes correlated with the observed phenotypic differences. On the basis of these results, it can be speculated that the observed interpopulation polymorphism of protein regulation in response to heat stress could underlie their different capacities to withstand and recover from heat stress. These local adaptations are potentially relevant for the species adaptation to the conditions predicted by the current models for climate change.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.