A Norway spruce FLOWERING LOCUS T homolog is implicated in control of growth rhythm in conifers

Growth in perennial plants possesses an annual cycle of active growth and dormancy that is controlled by environmental factors, mainly photoperiod and temperature. In conifers and other nonangiosperm species, the molecular mechanisms behind these responses are currently unknown. In Norway spruce (Picea abies L. Karst.) seedlings, growth cessation and bud set are induced by short days and plants from southern latitudes require at least 7 to 10 h of darkness, whereas plants from northern latitudes need only 2 to 3 h of darkness. Bud burst, on the other hand, is almost exclusively controlled by temperature. To test the possible role of Norway spruce FLOWERING LOCUS T (FT)-like genes in growth rhythm, we have studied expression patterns of four Norway spruce FT family genes in two populations with a divergent bud set response under various photoperiodic conditions. Our data show a significant and tight correlation between growth rhythm (both bud set and bud burst), and expression pattern of one of the four Norway spruce phosphatidylethanolamine-binding protein gene family members (PaFT4) over a variety of experimental conditions. This study strongly suggests that one Norway spruce homolog to the FT gene, which controls flowering in angiosperms, is also a key integrator of photoperiodic and thermal signals in the control of growth rhythms in gymnosperms. The data also indicate that the divergent adaptive bud set responses of northern and southern Norway spruce populations, both to photoperiod and light quality, are mediated through PaFT4. These results provide a major advance in our understanding of the molecular control of a major adaptive trait in conifers and a tool for further molecular studies of adaptive variation in plants.     

Alteration of PHYA expression change circadian rhythms and timing of bud set in Populus

In many temperate woody species, dormancy is induced by short photoperiods. Earlier studies have shown that the photoreceptor phytochrome A (phyA) promotes growth. Specifically, Populus plants that over-express the oat PHYA gene (oatPHYAox) show daylength-independent growth and do not become dormant. However, we show that oatPHYAox plants could be induced to set bud and become cold hardy by exposure to a shorter, non-24 h diurnal cycle that significantly alters the relative position between endogenous rhythms and perceived light/dark cycles. Furthermore, we describe studies in which the expression of endogenous Populus tremula × P. tremuloides PHYTOCHROME A (PttPHYA) was reduced in Populus trees by antisense inhibition. The antisense plants showed altered photoperiodic requirements, resulting in earlier growth cessation and bud formation in response to daylength shortening, an effect that was explained by an altered innate period that leads to phase changes of clock-associated genes such as PttCO2. Moreover, gene expression studies following far-red light pulses show a phyA-mediated repression of PttLHY1 and an induction of PttFKF1 and PttFT. We conclude that the level of PttPHYA expression strongly influences seasonally regulated growth in Populus and is central to co-ordination between internal clock-regulated rhythms and external light/dark cycles through its dual effect on the pace of clock rhythms and in light signaling.     

Growth and Dormancy in Norway Spruce Ecotypes (Picea abies) I. Interaction of Photoperiod and Temperature

Growth and dormancy as affected by photoperiod and temperature have been studied in Norway spruce ecotypes of different latitudinal and altitudinal origin. First-year seedlings were used. In all ecotypes apical growth cessation and terminal bud formation occurred within 2 weeks after exposure to SD at temperatures of 18 to 24°C. At lower temperatures or at near-critical photoperiods the response was delayed. The critical photoperiod for apical growth cessation varied from 21 hours in ecotype Steinkjer, Norway (64°N) to about 15 hours in ecotype Lankowitz, Austria (47°04′N). High-elevation ecotypes also had longer critical pholoperiods than low-elevation ecotypes from the same latitude. A detectable growth depression resulted from as little as 1 or 2 SDs of 10 hours, and with 4 or more SDs apical growth cessation took place. In contrast to the situation in the shoot, root growth was not affected by photoperiod. Accordingly, the top:root ratio is drastically affected by photoperiod. The critical photoperiod for cambial growth was shorter than that for apical growth in all ecotypes and cambial growth cessation was delayed for several weeks compared with cessation of apical growth. A transition to formation of late-wood tracheids with thick walls and narrow lumens took place upon exposure to SD. The photoperiodic effects were significantly modified by temperature, but the critical photoperiods were only slightly changed by temperature in the range of 12 to 24°C. However, a 10-hour “night” at 4°C caused growth cessation in continuous light in four ecotypes tested. Temperature optimum for apical growth under non-limiting photoperiods (24 hours) was 21°C in all ecotypes, but with little difference among 18,21 and 24°C. The Q₁₀ for apical growth was 3.5 in the temperature range 12 to 18°C. The growth potential as determined in 24-hour photoperiods was not significantly different among the various ecotypes except for one northern eco-type which was clearly inferior to the others. However, the growth of ecotype Steinkjer (64°N) was greatly suppressed even by the long midsummer days at 59°40′N, thus demonstrating the misleading impression one gets of the growth potential of northern ecotypes when they are moved southwards.     

Ecotype-dependent control of growth,dormancy and freezing tolerance under seasonal changes in <Emphasis Type="Italic">Betula pendula</Emphasis> Roth

Woody plants in the temperate and boreal zone undergo annual cycle of growth and dormancy under seasonal changes. Growth cessation and dormancy induction in autumn are prerequisites for the development of substantial cold hardiness in winter. During evolution, woody plants have developed different ecotypes that are closely adapted to the local climatic conditions. In this study, we employed distinct photoperiodic ecotypes of silver birch (Betula pendula Roth) to elucidate differences in these adaptive responses under seasonal changes. In all ecotypes, short day photoperiod (SD) initiated growth cessation and dormancy development, and induced cold acclimation. Subsequent low temperature (LT) exposure significantly enhanced freezing tolerance but removed bud dormancy. Our results suggested that dormancy and freezing tolerance might partially overlap under SD, but these two processes were regulated by different mechanisms and pathways under LT. Endogenous abscisic acid (ABA) levels were also altered under seasonal changes; the ABA level was low during the growing season, then increased in autumn, and decreased in winter. Compared with the southern ecotype, the northern ecotype was more responsive to seasonal changes, resulting in earlier growth cessation, cold acclimation and dormancy development in autumn, higher freezing tolerance and faster dormancy release in winter, and earlier bud flush and growth initiation in spring. In addition, although there was no significant ecotypic difference in ABA level during growing season, the rates and degrees of ABA alterations were different between the ecotypes in autumn and winter, and could be related to ecotypic differences in dormancy and freezing tolerance.     

Identification of genes associated with growth cessation and bud dormancy entrance using a dormancy-incapable tree mutant

Background  

In many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points.     

Proteomic analysis of shoot tissue during photoperiod induced growth cessation in <Emphasis Type="Italic">V. riparia</Emphasis> Michx. grapevines

Background  

Growth cessation, cold acclimation and dormancy induction in grapevines and other woody perennial plants native to temperate continental climates is frequently triggered by short photoperiods. The early induction of these processes by photoperiod promotes winter survival of grapevines in cold temperate zones. Examining the molecular processes, in particular the proteomic changes in the shoot, will provide greater insight into the signaling cascade that initiates growth cessation and dormancy induction. To begin understanding transduction of the photoperiod signal, Vitis riparia Michx. grapevines that had grown for 35 days in long photoperiod (long day, LD, 15 h) were subjected to either a continued LD or a short photoperiod (short day, SD, 13 h) treatment. Shoot tips (4-node shoot terminals) were collected from each treatment at 7 and 28 days of LD and SD for proteomic analysis via two-dimensional (2D) gel electrophoresis.     

Transitions in the functioning of the shoot apical meristem in birch (Betula pendula) involve ethylene

In many trees, a short photoperiod (SD) triggers substantial physiological adjustments necessary for over-wintering. We have used transgenic ethylene-insensitive birches (Betula pendula), which express the Arabidopsis ethylene receptor gene ETR1 carrying the dominant mutation etr1-1, to investigate the role of ethylene in SD-induced responses in the shoot apical meristem (SAM). Under SD, the ethylene-insensitive trees ceased elongation growth comparably to the wild-type. In contrast, the formation of terminal buds, which in trees is typically induced by SD, was abolished. However, although delayed, endo-dormancy did eventually develop in the ethylene-insensitive trees. This, together with the rapid resumption of growth in the ethylene-insensitive trees after transfer from non-permissive to permissive conditions suggests that ethylene facilitates the SD-induced terminal bud formation, as well as growth arrest. In addition, apical buds of the ethylene-insensitive birch did not accumulate abscisic acid (ABA) under SD, suggesting interaction between ethylene and ABA signalling in the bud. Alterations in SAM functioning were further exemplified by reduced apical dominance and early flowering in ethylene-insensitive birches. Gene expression analysis of shoot apices revealed that the ethylene-insensitive birch lacked the marked increase in expression of a beta-xylosidase gene typical to the SD-exposed wild-type. The ethylene-dependent beta-xylosidase gene expression is hypothesized to relate to modification of cell walls in terminal buds during SD-induced growth cessation. Our results suggest that ethylene is involved in terminal bud formation and in the timely suppression of SAM activity, not only in the shoot apex, but also in axillary and reproductive meristems.     

Temperature-driven plasticity in growth cessation and dormancy development in deciduous woody plants: a working hypothesis suggesting how molecular and cellular function is affected by temperature during dormancy induction

The role of temperature during dormancy development is being reconsidered as more research emerges demonstrating that temperature can significantly influence growth cessation and dormancy development in woody plants. However, there are seemingly contradictory responses to warm and low temperature in the literature. This research/review paper aims to address this contradiction. The impact of temperature was examined in four poplar clones and two dogwood ecotypes with contrasting dormancy induction patterns. Under short day (SD) conditions, warm night temperature (WT) strongly accelerated timing of growth cessation leading to greater dormancy development and cold hardiness in poplar hybrids. In contrast, under long day (LD) conditions, low night temperature (LT) can completely bypass the short photoperiod requirement in northern but not southern dogwood ecotypes. These findings are in fact consistent with the literature in which both coniferous and deciduous woody plant species’ growth cessation, bud set or dormancy induction are accelerated by temperature. The contradictions are addressed when photoperiod and ecotypes are taken into account in which the combination of either SD/WT (northern and southern ecotypes) or LD/LT (northern ecotypes only) are separated. Photoperiod insensitive types are driven to growth cessation by LT. Also consistent is the importance of night temperature in regulating these warm and cool temperature responses. However, the physiological basis for these temperature effects remain unclear. Changes in water content, binding and mobility are factors known to be associated with dormancy induction in woody plants. These were measured using non-destructive magnetic resonance micro-imaging (MRMI) in specific regions within lateral buds of poplar under SD/WT dormancing inducing conditions. Under SD/WT, dormancy was associated with restrictions in inter- or intracellular water movement between plant cells that reduces water mobility during dormancy development. Northern ecotypes of dogwood may be more tolerant to photoinhibition under the dormancy inducing LD/LT conditions compared to southern ecotypes. In this paper, we propose the existence of two separate, but temporally connected processes that contribute to dormancy development in some deciduous woody plant: one driven by photoperiod and influenced by moderate temperatures; the other driven by abiotic stresses, such as low temperature in combination with long photoperiods. The molecular changes corresponding to these two related but distinct responses to temperature during dormancy development in woody plants remains an investigative challenge.     

Florigen is involved in axillary bud development at multiple stages in Arabidopsis

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.     

Apical Growth Cessation and Shoot Tip Abscission in Salix

Time course of apical shoot growth and shoot tip abortion in northern ecotypes (lat. 69°39′N, long. 18°37′E) of Salix pentandra and S. caprea have been investigated. In trees more than 15 years old growing under natural climatic conditions apical growth cessation and shoot tip abortion normally occurred in June-July when the day length still was 24 h. Application of GA₃, in spring to the apex effectively delayed growth cessation and shoot tip abortion. Application of kinetin was without effect. First-year seedlings of both species grew continuously at temperatue of 9 to 24°C in 24 h photoperiod. Short days induced apical growth cessation, but two to four (S. pentandra) or three to five (S. caprea) weeks of 12 h photoperiod were required to stop the elongation growth. The results indicated that the critical photoperiod for apical growth cessation in the used ecotype of S. pentandra was 16 to 18 h at 18°C. Short days had a minor effect only on the formation of apical leaf primordia in small seedlings. Development of axillary buds and radial growth were stimulated by short days when compared with long days. Small seedlings of both species (3 to 8 cm high at the start) formed terminal buds in short days, but in large seedlings (more than about 15 cm high) apical growth cessation was accompanied by shoot tip abortion. Abscisic acid applied to the apex or through a leaf did not induce growth cessation in S. pentandra seedlings grown in continuous light. The growth retardants CCC, B-9 and Phosphon D reduced growth rate under continuous light and induced shoot tip abortion in some plants. The effect of CCC was counteracted by GA₃. Apical growth cessation in short days was significantly delayed by a single GA₁ application.     

Growth and Dormancy in Norway Spruce Ecotypes

Height and radial growth of spruce in years n+ 1, n+ 2, and n+ 3 as affected by photoperiod and temperature in year n have been studied in controlled environments and in the field. In agreement with common opinion apical shoot growth in year n+ 1 was strongly dependent on the temperature conditions prevailing in the period following budset in year n. This was found mainly to be a direct effect upon the differentiation of the shoot and needle primordia for next year's growth. A similar, although less pronounced effect, was found also on radial growth, possibly an indirect effect elicited through the effect on apical growth. A rather wide temperature optimum of 18 to 24°C was found in three Norwegian ecotypes and a somewhat lower optimum (15 to 18 C) in an Austrian high altitude ecotype. The shorter the bud differentiation period, the higher was the temperature optimum in all ecotypes (heat sum effect). Photoperiod which is the main factor controlling the time of budset, thus had a great after-effect. The after-effect was strongly modified by photoperiod and to a lesser extent also by temperature in the current growing season. It is concluded that in second-year or older spruce plants the important effect of photoperiod in the current growing season is to control quantitatively and qualitatively the amount of secondary (lammas) shoot formation, and to modify shoot extension in the main growth flush. Longer photoperiods were needed for continuation or resumption of growth in second-year plants than for maintenance of uninterrupted growth in first-year seedlings. Delayed flushing was observed in plants maturing at high temperatures, indicating that these plants had entered a deeper state of dormancy than those maturing at lower temperatures. Also in years n+ 2 and n+ 3 apical and radical growth was significantly related to photoperiod and temperature conditions in year n. This effect gradually became an indirect one through the effects on general plant size (leaf and root area). The results are discussed in the light of previous work in the field.     

Gibberellins and Subapical Cell Divisions in Relation to Bud Set and Bud Break in Salix pentandra

In young plants of Salix pentandra, a temperate zone deciduous woody species, elongation growth ceases and a terminal bud is formed at day lengths shorter than a critical length. This is the first step in dormancy development, making survival under harsh winter conditions possible. Early studies strongly indicate that gibberellin is involved in the photoperiodic control of bud set and bud break. GA₁ action was studied by application under short days to plants where cessation of shoot elongation had occurred, followed by subsequent anatomic investigations of shoot tips. Under short days the frequency of cell division decreased rapidly along with the earlier observed decrease in GA₁ levels. Application of GA₁ to short-day–induced terminal buds rapidly stimulated cell division in apices several days before visible shoot elongation in response to this treatment was observed. One day after GA₁ application a fourfold increase in cell division frequency in apices was observed, increasing to a maximum of sevenfold 2 days after application. Long-day treatment leading to induction of bud break after about 4–6 days was followed by slowly increasing frequency of cell divisions. In earlier studies of this species, short days and gibberellins had no effect on cell elongation. These data show that increased GA₁ content, by application or long-day treatment, results in increased frequency of mitosis. This strongly indicates that GA₁ affects stem elongation in connection with bud set and bud break primarily by affecting cell divisions in subapical tissues. Received February 26, 1999; accepted October 8, 1999     

Alterations in ultrastructure and subcellular localization of Ca2+ in poplar apical bud cells during the induction of dormancy

In poplar (Populus deltoides Bartr. ex Marsh), bud dormancyand freezing tolerance were concomitantly induced by short-day(SD) photoperiods. Ultrastructural changes and the alterationin subcellular localization of calcium in apical bud cells associatedwith dormancy development were investigated. During the developmentof dormancy, the thickness of cell walls increased significantly,the number of starch granules increased, and there was a significantaccumulation of storage proteins in the vacuoles of the apicalbud cells. The most striking change was the constriction andblockage of the plasmodesmata. It was demonstrated that antimonate precipitation is a reliabletechnique for studying subcellular localization of calcium inpoplar apical bud cells. Under the long day (LD) photoperiod,electron-dense calcium antimonate precipitates were mainly localizedin vacuoles, intercellular spaces and plastids. Some antimonateprecipitates were also found in the cell walls and at the entranceof the plasmodesmata. However, there were few Ca²⁺ depositsfound in the cytosol and nucleus. After 20 d of SD exposure,when development of bud dormancy was initiated, calcium depositsin intercellular spaces were decreased, whereas some depositswere found in the cytosol and nuclei. From 28–49 d ofSD exposure, while dormancy was developing, a large number ofCa²⁺ precipitates were found in the cytosol and nuclei. Whendeep dormancy was reached after 77 d of SD exposure, Ca²⁺ depositsbecame fewer in both cytosol and nuclei, whereas numerous depositswere again observed in the cell walls and in the intercellularspaces. These results suggest that under the influence of SDphotoperiods, there are alterations in subcellular Ca²⁺ localization,and changes in ultrastructure of apical bud cells during thedevelopment of dormancy. The constriction and blockage of plasmodesmatamay cause the cessation of symplastic transport, limit cellularcommunication and signal transduction between adjacent cells,which in turn may lead to events associated with growth cessationand dormancy development in buds. Key words: Poplar, apical bud cells, Ca²⁺ subcellular localization, dormancy