Genome Evolution in Plants: Complex Thalloid Liverworts (Marchantiopsida).

Why do some genomes stay small and simple, while others become huge, and why are some genomes more stable? In contrast to angiosperms and gymnosperms, liverworts are characterized by small genomes with low variation in size and conserved chromosome numbers. We quantified genome evolution among five Marchantiophyta (liverworts), measuring gene characteristics, transposable element (TE) landscape, collinearity, and sex chromosome evolution that might explain the small size and limited variability of liverwort genomes. No genome duplications were identified among examined liverworts and levels of duplicated genes are low. Among the liverwort species, Lunularia cruciata stands out with a genome size almost twice that of the other liverwort species investigated here, and most of this increased size is due to bursts of Ty3/Gypsy retrotransposons. Intrachromosomal rearrangements between examined liverworts are abundant but occur at a slower rate compared with angiosperms. Most genes on L. cruciata scaffolds have their orthologs on homologous Marchantia polymorpha chromosomes, indicating a low degree of rearrangements between chromosomes. Still, translocation of a fragment of the female U chromosome to an autosome was predicted from our data, which might explain the uniquely small U chromosome in L. cruciata. Low levels of gene duplication, TE activity, and chromosomal rearrangements might contribute to the apparent slow rate of morphological evolution in liverworts.

PIF-independent regulation of growth by an evening complex in the liverwort Marchantia polymorpha.

Previous studies in the liverwort Marchantia polymorpha have shown that the putative evening complex (EC) genes LUX ARRHYTHMO (LUX) and ELF4-LIKE (EFL) have a function in the liverwort circadian clock. Here, we studied the growth phenotypes of MpLUX and MpEFL loss-of-function mutants, to establish if PHYTOCHROME-INTERACTING FACTOR (PIF) and auxin act downstream of the M. polymorpha EC in a growth-related pathway similar to the one described for the flowering plant Arabidopsis. We examined growth rates and cell properties of loss-of-function mutants, analyzed protein-protein interactions and performed gene expression studies using reporter genes. Obtained data indicate that an EC can form in M. polymorpha and that this EC regulates growth of the thallus. Altered auxin levels in Mplux mutants could explain some of the phenotypes related to an increased thallus surface area. However, because MpPIF is not regulated by the EC, and because Mppif mutants do not show reduced growth, the growth phenotype of EC-mutants is likely not mediated via MpPIF. In Arabidopsis, the circadian clock regulates elongation growth via PIF and auxin, but this is likely not an evolutionarily conserved growth mechanism in land plants. Previous inventories of orthologs to Arabidopsis clock genes in various plant lineages showed that there is high levels of structural differences between clocks of different plant lineages. Here, we conclude that there is also variation in the output pathways used by the different plant clocks to control growth and development.

The Physcomitrium patens egg cell expresses several distinct epigenetic components and utilizes homologues of BONOBO genes for cell specification.

Although land plant germ cells have received much attention, knowledge about their specification is still limited. We thus identified transcripts enriched in egg cells of the bryophyte model species Physcomitrium patens, compared the results with angiosperm egg cells, and selected important candidate genes for functional analysis. We used laser-assisted microdissection to perform a cell-type-specific transcriptome analysis on egg cells for comparison with available expression profiles of vegetative tissues and male reproductive organs. We made reporter lines and knockout mutants of the two BONOBO (PbBNB) genes and studied their role in reproduction. We observed an overlap in gene activity between bryophyte and angiosperm egg cells, but also clear differences. Strikingly, several processes that are male-germline specific in Arabidopsis are active in the P. patens egg cell. Among those were the moss PbBNB genes, which control proliferation and identity of both female and male germlines. Pathways shared between male and female germlines were most likely present in the common ancestors of land plants, besides sex-specifying factors. A set of genes may also be involved in the switches between the diploid and haploid moss generations. Nonangiosperm gene networks also contribute to the specification of the P. patens egg cell.

Rates and patterns of molecular evolution in bryophyte genomes, with focus on complex thalloid liverworts, Marchantiopsida.

Plants commonly referred to as "bryophytes" belong to three major lineages of non-vascular plants: the liverworts, the hornworts and the mosses. They are unique among land plants in having a dominant haploid generation and a short-lived diploid sporophytic generation. The dynamics of selection acting on a haploid genome differs from those acting on a diploid genome: new mutations are directly exposed to selection. The general aim of this paper is to investigate the diversification rateof bryophytes - measured as silent site substitution rate representing neutral evolution (mutation rate) and the nonsynonymous to synonymous substitution rate ratio (dN/dS) representing selective evolution - and compare it with earlier studies on vascular plants. Results show that the silent site substitution rate is lower for liverworts as compared to angiosperms, but not as low as for gymnosperms. The selection pressure, measured as dN/dS, isnot remarkably lower for bryophytes as compared to other diploid dominant plants as would be expected by the masking hypothesis, indicating that other factors are more important than ploidy.

DE-ETIOLATED1 has a role in the circadian clock of the liverwort Marchantia polymorpha.

Previous studies of plant circadian clock evolution have often relied on clock models and genes defined in Arabidopsis. These studies identified homologues with seemingly conserved function, as well as frequent gene loss. In the present study, we aimed to identify candidate clock genes in the liverwort Marchantia polymorpha using a more unbiased approach. To identify genes with circadian rhythm we sequenced the transcriptomes of gemmalings in a time series in constant light conditions. Subsequently, we performed functional studies using loss-of-function mutants and gene expression reporters. Among the genes displaying circadian rhythm, a homologue to the transcriptional co-repressor Arabidopsis DE-ETIOLATED1 showed high amplitude and morning phase. Because AtDET1 is arrhythmic and associated with the morning gene function of AtCCA1/LHY, that lack a homologue in liverworts, we functionally studied DET1 in M. polymorpha. We found that the circadian rhythm of MpDET1 expression is disrupted in loss-of-function mutants of core clock genes and putative evening-complex genes. MpDET1 knock-down in turn results in altered circadian rhythm of nyctinastic thallus movement and clock gene expression. We could not detect any effect of MpDET1 knock-down on circadian response to light, suggesting that MpDET1 has a yet unknown function in the M. polymorpha circadian clock.

Evolutionary History of the Marchantia polymorpha Complex.

The potential role of introgression in evolution has gained increased interest in recent years. Although some fascinating examples have been reported, more information is needed to generalize the importance of hybridization and introgression for adaptive divergence. As limited data exist on haploid dominant species, we analyzed genomes of three subspecies of the liverwort Marchantia polymorpha. We used available genomic data for subsp. ruderalis and carried out whole-genome (PacBio) sequencing for one individual each of subsp. montivagans and subsp. polymorpha as well as Illumina resequencing of additional genomes for all three subspecies. The three subspecies were compared against M. paleacea as outgroup. Our analyses revealed separation of the three taxa, but all three possible topologies were richly represented across the genomes, and the underlying divergence order less obvious. This uncertainty could be the result of the divergence of the three subspecies close in time, or that introgression has been frequent since divergence. In particular, we found that pseudo-chromosome 2 in subsp. montivagans was much more diverged than other parts of the genomes. This could either be explained by specific capture of chromosome 2 from an unknown related species through hybridization or by conservation of chromosome 2 despite intermittent or ongoing introgression affecting more permeable parts of the genomes. A higher degree of chromosomal rearrangements on pseudo-chromosome 2 support the second hypothesis. Species tree analyses recovered an overall topology where subsp. montivagans diverged first and subsp. ruderalis and subsp. polymorpha appeared as sister lineages. Each subspecies was associated with its own chloroplast and mitochondrial haplotype group. Our data suggest introgression but refute a previous hypothesis that subsp. ruderalis is a new stabilized hybrid between the other two subspecies.

Nyctinastic thallus movement in the liverwort Marchantia polymorpha is regulated by a circadian clock.

The circadian clock coordinates an organism's growth, development and physiology with environmental factors. One illuminating example is the rhythmic growth of hypocotyls and cotyledons in Arabidopsis thaliana. Such daily oscillations in leaf position are often referred to as sleep movements or nyctinasty. Here, we report that plantlets of the liverwort Marchantia polymorpha show analogous rhythmic movements of thallus lobes, and that the circadian clock controls this rhythm, with auxin a likely output pathway affecting these movements. The mechanisms of this circadian clock are partly conserved as compared to angiosperms, with homologs to the core clock genes PRR, RVE and TOC1 forming a core transcriptional feedback loop also in M. polymorpha.

An ancestral signalling pathway is conserved in intracellular symbioses-forming plant lineages.

Plants are the foundation of terrestrial ecosystems, and their colonization of land was probably facilitated by mutualistic associations with arbuscular mycorrhizal fungi. Following this founding event, plant diversification has led to the emergence of a tremendous diversity of mutualistic symbioses with microorganisms, ranging from extracellular associations to the most intimate intracellular associations, where fungal or bacterial symbionts are hosted inside plant cells. Here, through analysis of 271 transcriptomes and 116 plant genomes spanning the entire land-plant diversity, we demonstrate that a common symbiosis signalling pathway co-evolved with intracellular endosymbioses, from the ancestral arbuscular mycorrhiza to the more recent ericoid and orchid mycorrhizae in angiosperms and ericoid-like associations of bryophytes. By contrast, species forming exclusively extracellular symbioses, such as ectomycorrhizae, and those forming associations with cyanobacteria, have lost this signalling pathway. This work unifies intracellular symbioses, revealing conservation in their evolution across 450 million yr of plant diversification.

Parental legacy, demography, and admixture influenced the evolution of the two subgenomes of the tetraploid Capsella bursa-pastoris (Brassicaceae).

Allopolyploidy is generally perceived as a major source of evolutionary novelties and as an instantaneous way to create isolation barriers. However, we do not have a clear understanding of how two subgenomes evolve and interact once they have fused in an allopolyploid species nor how isolated they are from their relatives. Here, we address these questions by analyzing genomic and transcriptomic data of allotetraploid Capsella bursa-pastoris in three differentiated populations, Asia, Europe, and the Middle East. We phased the two subgenomes, one descended from the outcrossing and highly diverse Capsella grandiflora (CbpCg) and the other one from the selfing and genetically depauperate Capsella orientalis (CbpCo). For each subgenome, we assessed its relationship with the diploid relatives, temporal changes of effective population size (Ne), signatures of positive and negative selection, and gene expression patterns. In all three regions, Ne of the two subgenomes decreased gradually over time and the CbpCo subgenome accumulated more deleterious changes than CbpCg. There were signs of widespread admixture between C. bursa-pastoris and its diploid relatives. The two subgenomes were impacted differentially depending on geographic region suggesting either strong interploidy gene flow or multiple origins of C. bursa-pastoris. Selective sweeps were more common on the CbpCg subgenome in Europe and the Middle East, and on the CbpCo subgenome in Asia. In contrast, differences in expression were limited with the CbpCg subgenome slightly more expressed than CbpCo in Europe and the Middle-East. In summary, after more than 100,000 generations of co-existence, the two subgenomes of C. bursa-pastoris still retained a strong signature of parental legacy but their evolutionary trajectory strongly varied across geographic regions.

An Evolutionarily Conserved Abscisic Acid Signaling Pathway Regulates Dormancy in the Liverwort Marchantia polymorpha.

Dormancy is a key process allowing land plants to adapt to changing conditions in the terrestrial habitat, allowing the cessation of growth in response to environmental or physiological cues, entrance into a temporary quiescent state, and subsequent reactivation of growth in more favorable environmental conditions [1-3]. Dormancy may be induced seasonally, sporadically (e.g., in response to drought), or developmentally (e.g., seeds and apical dominance). Asexual propagules, known as gemmae, derived via clonal reproduction in bryophytes, are often dormant until displaced from the parent plant. In the liverwort Marchantia polymorpha, gemmae are produced within specialized receptacles, gemma cups, located on the dorsal side of the vegetative thallus [4]. Mature gemmae are detached from the parent plant but may remain in the cup, with gemma growth suppressed as long as the gemmae remain in the gemma cup and the parental plant is alive [5]. Following dispersal of gemmae from gemma cups by rain, the gemmae germinate in the presence of light and moisture, producing clonal offspring [6]. In land plants, the plant hormone abscisic acid (ABA) regulates many aspects of dormancy and water balance [7]. Here, we demonstrate that ABA plays a central role in the control of gemma dormancy as transgenic M. polymorpha gemmae with reduced sensitivity to ABA fail to establish and/or maintain dormancy. Thus, the common ancestor of land plants used the ABA signaling module to regulate germination of progeny in response to environmental cues, with both gemmae and seeds being derived structures co-opting an ancestral response system.

Early evolution of the land plant circadian clock.

While angiosperm clocks can be described as an intricate network of interlocked transcriptional feedback loops, clocks of green algae have been modelled as a loop of only two genes. To investigate the transition from a simple clock in algae to a complex one in angiosperms, we performed an inventory of circadian clock genes in bryophytes and charophytes. Additionally, we performed functional characterization of putative core clock genes in the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis. Phylogenetic construction was combined with studies of spatiotemporal expression patterns and analysis of M. polymorpha clock gene mutants. Homologues to core clock genes identified in Arabidopsis were found not only in bryophytes but also in charophytes, albeit in fewer copies. Circadian rhythms were detected for most identified genes in M. polymorpha and A. agrestis, and mutant analysis supports a role for putative clock genes in M. polymorpha. Our data are in line with a recent hypothesis that adaptation to terrestrial life occurred earlier than previously expected in the evolutionary history of charophyte algae. Both gene duplication and acquisition of new genes was important in the evolution of the plant circadian clock, but gene loss has also contributed to shaping the clock of bryophytes.

Association mapping in Salix viminalis L. (Salicaceae) - identification of candidate genes associated with growth and phenology.

Willow species (Salix) are important as short-rotation biomass crops for bioenergy, which creates a demand for faster genetic improvement and breeding through deployment of molecular marker-assisted selection (MAS). To find markers associated with important adaptive traits, such as growth and phenology, for use in MAS, we genetically dissected the trait variation of a Salix viminalis (L.) population of 323 accessions. The accessions were sampled throughout northern Europe and were established at two field sites in Pustnäs, Sweden, and at Woburn, UK, offering the opportunity to assess the impact of genotype-by-environment interactions (G × E) on trait-marker associations. Field measurements were recorded for growth and phenology traits. The accessions were genotyped using 1536 SNP markers developed from phenology candidate genes and from genes previously observed to be differentially expressed in contrasting environments. Association mapping between 1233 of these SNPs and the measured traits was performed taking into account population structure and threshold selection bias. At a false discovery rate (FDR) of 0.2, 29 SNPs were associated with bud burst, leaf senescence, number of shoots or shoot diameter. The percentage of accession variation (Radj2) explained by these associations ranged from 0.3% to 4.4%, suggesting that the studied traits are controlled by many loci of limited individual impact. Despite this, a SNP in the EARLY FLOWERING 3 gene was repeatedly associated (FDR < 0.2) with bud burst. The rare homozygous genotype exhibited 0.4-1.0 lower bud burst scores than the other genotype classes on a five-grade scale. Consequently, this marker could be promising for use in MAS and the gene deserves further study. Otherwise, associations were less consistent across sites, likely due to their small Radj2 estimates and to considerable G × E interactions indicated by multivariate association analyses and modest trait accession correlations across sites (0.32-0.61).

The influence of population structure on gene expression and flowering time variation in the ubiquitous weed Capsella bursa-pastoris (Brassicaceae).

Population structure is a potential problem when testing for adaptive phenotypic differences among populations. The observed phenotypic differences among populations can simply be due to genetic drift, and if the genetic distance between them is not considered, the differentiation may be falsely interpreted as adaptive. Conversely, adaptive and demographic processes might have been tightly associated and correcting for the population structure may lead to false negatives. Here, we evaluated this problem in the cosmopolitan weed Capsella bursa-pastoris. We used RNA-Seq to analyse gene expression differences among 24 accessions, which belonged to a much larger group that had been previously characterized for flowering time and circadian rhythm and were genotyped using genotyping-by-sequencing (GBS) technique. We found that clustering of accessions for gene expression retrieved the same three clusters that were obtained with GBS data previously, namely Europe, the Middle East and Asia. Moreover, the three groups were also differentiated for both flowering time and circadian rhythm variation. Correction for population genetic structure when analysing differential gene expression analysis removed all differences among the three groups. This may suggest that most differences are neutral and simply reflect population history. However, geographical variation in flowering time and circadian rhythm indicated that the distribution of adaptive traits might be confounded by population structure. To bypass this confounding effect, we compared gene expression differentiation between flowering ecotypes within the genetic groups. Among the differentially expressed genes, FLOWERING LOCUS C was the strongest candidate for local adaptation in regulation of flowering time.

Auxin Produced by the Indole-3-Pyruvic Acid Pathway Regulates Development and Gemmae Dormancy in the Liverwort Marchantia polymorpha.

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss- and gain-of-function alleles for auxin synthesis- and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.

Neofunctionalization of duplicated Tic40 genes caused a gain-of-function variation related to male fertility in Brassica oleracea lineages.

Gene duplication followed by functional divergence in the event of polyploidization is a major contributor to evolutionary novelties. The Brassica genus evolved from a common ancestor after whole-genome triplication. Here, we studied the evolutionary and functional features of Brassica spp. homologs to Tic40 (for translocon at the inner membrane of chloroplasts with 40 kDa). Four Tic40 loci were identified in allotetraploid Brassica napus and two loci in each of three basic diploid Brassica spp. Although these Tic40 homologs share high sequence identities and similar expression patterns, they exhibit altered functional features. Complementation assays conducted on Arabidopsis thaliana tic40 and the B. napus male-sterile line 7365A suggested that all Brassica spp. Tic40 homologs retain an ancestral function similar to that of AtTic40, whereas BolC9.Tic40 in Brassica oleracea and its ortholog in B. napus, BnaC9.Tic40, in addition, evolved a novel function that can rescue the fertility of 7365A. A homologous chromosomal rearrangement placed bnac9.tic40 originating from the A genome (BraA10.Tic40) as an allele of BnaC9.Tic40 in the C genome, resulting in phenotypic variation for male sterility in the B. napus near-isogenic two-type line 7365AB. Assessment of the complementation activity of chimeric B. napus Tic40 domain-swapping constructs in 7365A suggested that amino acid replacements in the carboxyl terminus of BnaC9.Tic40 cause this functional divergence. The distribution of these amino acid replacements in 59 diverse Brassica spp. accessions demonstrated that the neofunctionalization of Tic40 is restricted to B. oleracea and its derivatives and thus occurred after the divergence of the Brassica spp. A, B, and C genomes.

Association of FLOWERING LOCUS T/TERMINAL FLOWER 1-like gene FTL2 expression with growth rhythm in Scots pine (Pinus sylvestris).

Understanding the genetic basis of the timing of bud set, an important trait in conifers, is relevant for adaptation and forestry practice. In common garden experiments, both Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) show a latitudinal cline in the trait. We compared the regulation of their bud set biology by examining the expression of PsFTL2, a Pinus sylvestris homolog to PaFTL2, a FLOWERING LOCUS T/TERMINAL FLOWER 1 (FT/TFL1)-like gene, the expression levels of which have been found previously to be associated with the timing of bud set in Norway spruce. In a common garden study, we analyzed the relationship of bud phenology under natural and artificial photoperiods and the expression of PsFTL2 in a set of Scots pine populations from different latitudes. The expression of PsFTL2 increased in the needles preceding bud set and decreased during bud burst. In the northernmost population, even short night periods were efficient to trigger this expression, which also increased earlier under all photoperiodic regimes compared with the southern populations. Despite the different biology, with few limitations, the two conifers that diverged 140 million yr ago probably share an association of FTL2 with bud set, pointing to a common mechanism for the timing of growth cessation in conifers.

Patterns of nucleotide diversity at photoperiod related genes in Norway spruce [Picea abies (L.) Karst].

The ability of plants to track seasonal changes is largely dependent on genes assigned to the photoperiod pathway, and variation in those genes is thereby important for adaptation to local day length conditions. Extensive physiological data in several temperate conifer species suggest that populations are adapted to local light conditions, but data on the genes underlying this adaptation are more limited. Here we present nucleotide diversity data from 19 genes putatively involved in photoperiodic response in Norway spruce (Picea abies). Based on similarity to model plants the genes were grouped into three categories according to their presumed position in the photoperiod pathway: photoreceptors, circadian clock genes, and downstream targets. An HKA (Hudson, Kreitman and Aquade) test showed a significant excess of diversity at photoreceptor genes, but no departure from neutrality at circadian genes and downstream targets. Departures from neutrality were also tested with Tajima's D and Fay and Wu's H statistics under three demographic scenarios: the standard neutral model, a population expansion model, and a more complex population split model. Only one gene, the circadian clock gene PaPRR3 with a highly positive Tajima's D value, deviates significantly from all tested demographic scenarios. As the PaPRR3 gene harbours multiple non-synonymous variants it appears as an excellent candidate gene for control of photoperiod response in Norway spruce.

Genetic architecture of spring and autumn phenology in Salix.

BACKGROUND: In woody plants from temperate regions, adaptation to the local climate results in annual cycles of growth and dormancy, and optimal regulation of these cycles are critical for growth, long-term survival, and competitive success. In this study we have investigated the genetic background to growth phenology in a Salix pedigree by assessing genetic and phenotypic variation in growth cessation, leaf senescence and bud burst in different years and environments. A previously constructed linkage map using the same pedigree and anchored to the annotated genome of P. trichocarpa was improved in target regions and used for QTL analysis of the traits. The major aims in this study were to map QTLs for phenology traits in Salix, and to identify candidate genes in QTL hot spots through comparative mapping with the closely related Populus trichocarpa. RESULTS: All traits varied significantly among genotypes and the broad-sense heritabilities ranged between 0.5 and 0.9, with the highest for leaf senescence. In total across experiment and years, 80 QTLs were detected. For individual traits, the QTLs explained together from 21.5 to 56.5% of the variation. Generally each individual QTL explained a low amount of the variation but three QTLs explained above 15% of the variation with one QTL for leaf senescence explaining 34% of the variation. The majority of the QTLs were recurrently identified across traits, years and environments. Two hotspots were identified on linkage group (LG) II and X where narrow QTLs for all traits co-localized. CONCLUSIONS: This study provides the most detailed analysis of QTL detection for phenology in Salix conducted so far. Several hotspot regions were found where QTLs for different traits and QTLs for the same trait but identified during different years co-localised. Many QTLs co-localised with QTLs found in poplar for similar traits that could indicate common pathways for these traits in Salicaceae. This study is an important first step in identifying QTLs and candidate genes for phenology traits in Salix.

No time for spruce: rapid dampening of circadian rhythms in Picea abies (L. Karst).

The identification and cloning of full-length homologs of circadian clock genes from Picea abies represent a first step to study the function and evolution of the circadian clock in gymnosperms. Phylogenetic analyses suggest that the sequences of key circadian clock genes are conserved between angiosperms and gymnosperms. though fewer homologous copies were found for most gene families in P. abies. We detected diurnal cycling of circadian clock genes in P. abies using quantitative real-time PCR; however, cycling appeared to be rapidly dampened under free-running conditions. Given the unexpected absence of transcriptional cycling during constant conditions, we employed a complementary method to assay circadian rhythmic outputs and measured delayed fluorescence in seedlings of Norway spruce. Neither of the two approaches to study circadian rhythms in Norway spruce could detect robust ∼24 h cycling behavior under constant conditions. These data suggest gene conservation but fundamental differences in clock function between gymnosperms and other plant taxa.

FLOWERING LOCUS T/TERMINAL FLOWER1-like genes affect growth rhythm and bud set in Norway spruce.

The timing of bud set, as one determinant of the annual growth rhythm, is critical for local adaptation of the conifer Norway spruce (Picea abies). Previous gene expression and population genetic studies have suggested a role for P. abies FLOWERING LOCUS T/TERMINAL FLOWER1-Like2 (PaFTL2) in the control of growth cessation and bud set in Norway spruce as well as in local adaptation resulting in clinal variation for timing of bud set. Using transgenic plants with PaFTL2 driven by an inducible promoter, we found that PaFTL2 indeed induces bud set and most probably also growth cessation. PaFTL2 shows high expression around the procambium and vascular tissue and in the crown region in buds of both seedlings and older trees. Furthermore, PaFTL2 expression is induced in vegetative shoots and all bud types in late summer, when growth cessation occurs. This supports the notion that PaFTL2 is involved in growth cessation. A close paralog to PaFTL2, PaFTL1, is strongly expressed in meristems during the summer, possibly to repress meristem activity and the formation of needle primordia during this period. The temporal and spatial expression of PaFTL1 and PaFTL2 largely complement each other, which suggests that they act in concert to control perennial growth in Norway spruce.