ZEITLUPE Promotes ABA-Induced Stomatal Closure in Arabidopsis and Populus.

Plants balance water availability with gas exchange and photosynthesis by controlling stomatal aperture. This control is regulated in part by the circadian clock, but it remains unclear how signalling pathways of daily rhythms are integrated into stress responses. The serine/threonine protein kinase OPEN STOMATA 1 (OST1) contributes to the regulation of stomatal closure via activation of S-type anion channels. OST1 also mediates gene regulation in response to ABA/drought stress. We show that ZEITLUPE (ZTL), a blue light photoreceptor and clock component, also regulates ABA-induced stomatal closure in Arabidopsis thaliana, establishing a link between clock and ABA-signalling pathways. ZTL sustains expression of OST1 and ABA-signalling genes. Stomatal closure in response to ABA is reduced in ztl mutants, which maintain wider stomatal apertures and show higher rates of gas exchange and water loss than wild-type plants. Detached rosette leaf assays revealed a stronger water loss phenotype in ztl-3, ost1-3 double mutants, indicating that ZTL and OST1 contributed synergistically to the control of stomatal aperture. Experimental studies of Populus sp., revealed that ZTL regulated the circadian clock and stomata, indicating ZTL function was similar in these trees and Arabidopsis. PSEUDO-RESPONSE REGULATOR 5 (PRR5), a known target of ZTL, affects ABA-induced responses, including stomatal regulation. Like ZTL, PRR5 interacted physically with OST1 and contributed to the integration of ABA responses with circadian clock signalling. This suggests a novel mechanism whereby the PRR proteins-which are expressed from dawn to dusk-interact with OST1 to mediate ABA-dependent plant responses to reduce water loss in time of stress.

Monitoring Seasonal Bud Set, Bud Burst, and Cold Hardiness in Populus.

Using a perennial model plant allows the study of reoccurring seasonal events in a way that is not possible using a fast-growing annual such as A. thaliana (Arabidopsis). In this study, we present a hybrid aspen (Populus tremula × P. tremuloides) as our perennial model plant. These plants can be grown in growth chambers to shorten growth periods and manipulate day length and temperature in ways that would be impossible under natural conditions. In addition, the use of growth chambers allows easy monitoring of height and diameter expansion, accelerating the collection of data from new strategies that allow evaluation of promoters or inhibitors of growth. Here, we describe how to study and quantify responses to seasonal changes (mainly using P. tremula × P. tremuloides) by measuring growth rate and key events under different photoperiodic cycles.

The Perennial Clock Is an Essential Timer for Seasonal Growth Events and Cold Hardiness.

Over the last several decades, changes in global temperatures have led to changes in local environments affecting the growth conditions for many species. This is a trend that makes it even more important to understand how plants respond to local variations and seasonal changes in climate.To detect daily and seasonal changes as well as acute stress factors such as cold and drought, plants rely on a circadian clock. This chapter introduces the current knowledge and literature about the setup and function of the circadian clock in various tree and perennial species, with a focus on the Populus genus.

Growing in time: exploring the molecular mechanisms of tree growth.

Trees cover vast areas of the Earth's landmasses. They mitigate erosion, capture carbon dioxide, produce oxygen and support biodiversity, and also are a source of food, raw materials and energy for human populations. Understanding the growth cycles of trees is fundamental for many areas of research. Trees, like most other organisms, have evolved a circadian clock to synchronize their growth and development with the daily and seasonal cycles of the environment. These regular changes in light, daylength and temperature are perceived via a range of dedicated receptors and cause resetting of the circadian clock to local time. This allows anticipation of daily and seasonal fluctuations and enables trees to co-ordinate their metabolism and physiology to ensure vital processes occur at the optimal times. In this review, we explore the current state of knowledge concerning the regulation of growth and seasonal dormancy in trees, using information drawn from model systems such as Populus spp.

GIGANTEA-like genes control seasonal growth cessation in Populus.

Survival of trees growing in temperate zones requires cycling between active growth and dormancy. This involves growth cessation in the autumn triggered by a photoperiod shorter than the critical day length. Variations in GIGANTEA (GI)-like genes have been associated with phenology in a range of different tree species, but characterization of the functions of these genes in the process is still lacking. We describe the identification of the Populus orthologs of GI and their critical role in short-day-induced growth cessation. Using ectopic expression and silencing, gene expression analysis, protein interaction and chromatin immunoprecipitation experiments, we show that PttGIs are likely to act in a complex with PttFKF1s (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) and PttCDFs (CYCLING DOF FACTOR) to control the expression of PttFT2, the key gene regulating short-day-induced growth cessation in Populus. In contrast to Arabidopsis, in which the GI-CONSTANS (CO)-FLOWERING LOCUS T (FT) regulon is a crucial day-length sensor for flowering time, our study suggests that, in Populus, PttCO-independent regulation of PttFT2 by PttGI is more important in the photoperiodic control of growth cessation and bud set.

Circadian clock components control daily growth activities by modulating cytokinin levels and cell division-associated gene expression in Populus trees.

Trees are carbon dioxide sinks and major producers of terrestrial biomass with distinct seasonal growth patterns. Circadian clocks enable the coordination of physiological and biochemical temporal activities, optimally regulating multiple traits including growth. To dissect the clock's role in growth, we analysed Populus tremula × P. tremuloides trees with impaired clock function due to down-regulation of central clock components. late elongated hypocotyl (lhy-10) trees, in which expression of LHY1 and LHY2 is reduced by RNAi, have a short free-running period and show disrupted temporal regulation of gene expression and reduced growth, producing 30-40% less biomass than wild-type trees. Genes important in growth regulation were expressed with an earlier phase in lhy-10, and CYCLIN D3 expression was misaligned and arrhythmic. Levels of cytokinins were lower in lhy-10 trees, which also showed a change in the time of peak expression of genes associated with cell division and growth. However, auxin levels were not altered in lhy-10 trees, and the size of the lignification zone in the stem showed a relative increase. The reduced growth rate and anatomical features of lhy-10 trees were mainly caused by misregulation of cell division, which may have resulted from impaired clock function.

Autumn senescence in aspen is not triggered by day length.

Autumn senescence in mature aspens, grown under natural conditions, is initiated at almost the same date every year. The mechanism of such precise timing is not understood but we have previously shown that the signal must be derived from light. We studied variation in bud set and autumn senescence in a collection of 116 natural Eurasian aspen (Populus tremula) genotypes, from 12 populations in Sweden and planted in one northern and one southern common garden, to test the hypothesis that onset of autumn senescence is triggered by day length. We confirmed that, although bud set seemed to be triggered by a critical photoperiod/day length, other factors may influence it. The data on initiation of autumn senescence, on the other hand, were incompatible with the trigger being the day length per se, hence the trigger must be some other light-dependent factor.

Circadian and Plastid Signaling Pathways Are Integrated to Ensure Correct Expression of the CBF and COR Genes during Photoperiodic Growth.

The circadian clock synchronizes a wide range of biological processes with the day/night cycle, and correct circadian regulation is essential for photosynthetic activity and plant growth. We describe here a mechanism where a plastid signal converges with the circadian clock to fine-tune the regulation of nuclear gene expression in Arabidopsis (Arabidopsis thaliana). Diurnal oscillations of tetrapyrrole levels in the chloroplasts contribute to the regulation of the nucleus-encoded transcription factors C-REPEAT BINDING FACTORS (CBFs). The plastid signal triggered by tetrapyrrole accumulation inhibits the activity of cytosolic HEAT SHOCK PROTEIN90 and, as a consequence, the maturation and stability of the clock component ZEITLUPE (ZTL). ZTL negatively regulates the transcription factor LONG HYPOCOTYL5 (HY5) and PSEUDO-RESPONSE REGULATOR5 (PRR5). Thus, low levels of ZTL result in a HY5- and PRR5-mediated repression of CBF3 and PRR5-mediated repression of CBF1 and CBF2 expression. The plastid signal thereby contributes to the rhythm of CBF expression and the downstream COLD RESPONSIVE expression during day/night cycles. These findings provide insight into how plastid signals converge with, and impact upon, the activity of well-defined clock components involved in circadian regulation.

Transgenic hybrid aspen trees with increased gibberellin (GA) concentrations suggest that GA acts in parallel with FLOWERING LOCUS T2 to control shoot elongation.

Bioactive gibberellins (GAs) have been implicated in short day (SD)-induced growth cessation in Populus, because exogenous applications of bioactive GAs to hybrid aspens (Populus tremula × tremuloides) under SD conditions delay growth cessation. However, this effect diminishes with time, suggesting that plants may cease growth following exposure to SDs due to a reduction in sensitivity to GAs. In order to validate and further explore the role of GAs in growth cessation, we perturbed GA biosynthesis or signalling in hybrid aspen plants by overexpressing AtGA20ox1, AtGA2ox2 and PttGID1.3 (encoding GA biosynthesis enzymes and a GA receptor). We found trees with elevated concentrations of bioactive GA, due to overexpression of AtGA20ox1, continued to grow in SD conditions and were insensitive to the level of FLOWERING LOCUS T2 (FT2) expression. As transgenic plants overexpressing the PttGID1.3 GA receptor responded in a wild-type (WT) manner to SD conditions, this insensitivity did not result from limited receptor availability. As high concentrations of bioactive GA during SD conditions were sufficient to sustain shoot elongation growth in hybrid aspen trees, independent of FT2 expression levels, we conclude elongation growth in trees is regulated by both GA- and long day-responsive pathways, similar to the regulation of flowering in Arabidopsis thaliana.

The perennial clock is an essential timer for seasonal growth events and cold hardiness.

Over the last several decades, changes in global temperatures have led to changes in local environments affecting the growth conditions for many species. This is a trend that makes it even more important to understand how plants respond to local variations and seasonal changes in climate. To detect daily and seasonal changes as well as acute stress factors such as cold and drought, plants rely on a circadian clock. This chapter introduces the current knowledge and literature about the setup and function of the circadian clock in various tree and perennial species, with a focus on the Populus genus.

Monitoring seasonal bud set, bud burst, and cold hardiness in Populus.

Using a perennial model plant allows the study of reoccurring seasonal events in a way that is not possible using a fast-growing annual such as Arabidopsis thaliana (Arabidopsis). In this study, we present a hybrid aspen (Populus tremula × P. tremuloides) as our perennial model plant. These plants can be grown in growth chambers to shorten growth periods and manipulate day length and temperature in ways that would be impossible under natural conditions. In addition, the use of growth chambers allows easy monitoring of height and diameter expansion, accelerating the collection of data from new strategies that allow evaluation of promoters or inhibitors of growth. Here, we describe how to study and quantify responses to seasonal changes (mainly using P. tremula × P. tremuloides) by measuring growth rate and key events under different photoperiodic cycles.

A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides).

BACKGROUND: The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches. RESULTS: We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter. CONCLUSIONS: The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.

The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms.

In tree species native to temperate and boreal regions, the activity-dormancy cycle is an important adaptive trait both for survival and growth. We discuss recent research on mechanisms controlling the overlapping developmental processes that define the activity-dormancy cycle, including cessation of apical growth, bud development, induction, maintenance and release of dormancy, and bud burst. The cycle involves an extensive reconfiguration of metabolism. Environmental control of the activity-dormancy cycle is based on perception of photoperiodic and temperature signals, reflecting adaptation to prevailing climatic conditions. Several molecular actors for control of growth cessation have been identified, with the CO/FT regulatory network and circadian clock having important coordinating roles in control of growth and dormancy. Other candidate regulators of bud set, dormancy and bud burst have been identified, such as dormancy-associated MADS-box factors, but their exact roles remain to be discovered. Epigenetic mechanisms also appear to factor in control of the activity-dormancy cycle. Despite evidence for gibberellins as negative regulators in growth cessation, and ABA and ethylene in bud formation, understanding of the roles that plant growth regulators play in controlling the activity-dormancy cycle is still very fragmentary. Finally, some of the challenges for further research in bud dormancy are discussed.

Plant cell responses to cold are all about timing.

Changes in temperature present the cells of plants with particular challenges. Fortunately, many changes in temperature can be anticipated due to the rhythms of day/night and the seasons. To anticipate changes in the environment most organisms have a circadian clock to optimize daily and seasonal timing of gene expression, metabolism, physiology and cell biology. Circadian clocks comprised positive and negative feedback loops which ensure an internal period of approximately 24 hours. We describe the role of the circadian clock in modulating cellular cold signalling networks to prepare the cell for the onset of winter.

Full genome re-sequencing reveals a novel circadian clock mutation in Arabidopsis.

Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process, specifically if the phenotype is subtle and scoring labour intensive. Here, we have re-sequenced the 120-Mb genome of a novel Arabidopsis clock mutant early bird (ebi-1) in Wassilewskija (Ws-2). We demonstrate the utility of sequencing a backcrossed line in limiting the number of SNPs considered. We identify a SNP in the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype.

Partners in time: EARLY BIRD associates with ZEITLUPE and regulates the speed of the Arabidopsis clock.

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.

Circadian clock components regulate entry and affect exit of seasonal dormancy as well as winter hardiness in Populus trees.

This study addresses the role of the circadian clock in the seasonal growth cycle of trees: growth cessation, bud set, freezing tolerance, and bud burst. Populus tremula x Populus tremuloides (Ptt) LATE ELONGATED HYPOCOTYL1 (PttLHY1), PttLHY2, and TIMING OF CAB EXPRESSION1 constitute regulatory clock components because down-regulation by RNA interference of these genes leads to altered phase and period of clock-controlled gene expression as compared to the wild type. Also, both RNA interference lines show about 1-h-shorter critical daylength for growth cessation as compared to the wild type, extending their period of growth. During winter dormancy, when the diurnal variation in clock gene expression stops altogether, down-regulation of PttLHY1 and PttLHY2 expression compromises freezing tolerance and the expression of C-REPEAT BINDING FACTOR1, suggesting a role of these genes in cold hardiness. Moreover, down-regulation of PttLHY1 and PttLHY2 causes a delay in bud burst. This evidence shows that in addition to a role in daylength-controlled processes, PttLHY plays a role in the temperature-dependent processes of dormancy in Populus such as cold hardiness and bud burst.

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 x 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.

Changes in diurnal patterns within the Populus transcriptome and metabolome in response to photoperiod variation.

Changes in seasonal photoperiod provides an important environmental signal that affects the timing of winter dormancy in perennial, deciduous, temperate tree species, such as hybrid aspen (Populus tremula x Populus tremuloides). In this species, growth cessation, cold acclimation and dormancy are induced in the autumn by the detection of day-length shortening that occurs at a given critical day length. Important components in the detection of such day-length changes are photoreceptors and the circadian clock, and many plant responses at both the gene regulation and metabolite levels are expected to be diurnal. To directly examine this expectation and study components in these events, here we report transcriptomic and metabolomic responses to a change in photoperiod from long to short days in hybrid aspen. We found about 16% of genes represented on the arrays to be diurnally regulated, as assessed by our pre-defined criteria. Furthermore, several of these genes were involved in circadian-associated processes, including photosynthesis and primary and secondary metabolism. Metabolites affected by the change in photoperiod were mostly involved in carbon metabolism. Taken together, we have thus established a molecular catalog of events that precede a response to winter.

Forward genetic analysis of the circadian clock separates the multiple functions of ZEITLUPE.

The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.