Age-dependent seasonal growth cessation in Populus.

In temperate and boreal regions, perennial plants adapt their annual growth cycle to the change of seasons. In natural forests, juvenile seedlings usually display longer growth seasons compared to adult trees to ensure their establishment and survival under canopy shade. However, how trees adjust their annual growth according to their age is not known. In this study, we show that age-dependent seasonal growth cessation is genetically controlled and found that the miR156-SPL3/5 module, a key regulon of vegetative phase change (VPC), also triggers age-dependent growth cessation in Populus trees. We show that miR156 promotes shoot elongation during vegetative growth, and its targets SPL3/5s function in the same pathway but as repressors. We find that the miR156-SPL3/5s regulon controls growth cessation in both leaves and shoot apices and through multiple pathways, but with a different mechanism compared to how the miR156-SPL regulon controls VPC in annual plants. Taken together, our results reveal an age-dependent genetic network in mediating seasonal growth cessation, a key phenological process in the climate adaptation of perennial trees.

Whole-genome resequencing facilitates the development of a 50K single nucleotide polymorphism genotyping array for Scots pine (Pinus sylvestris L.) and its transferability to other pine species.

Scots pine (Pinus sylvestris L.) is one of the most widespread and economically important conifer species in the world. Applications like genomic selection and association studies, which could help accelerate breeding cycles, are challenging in Scots pine because of its large and repetitive genome. For this reason, genotyping tools for conifer species, and in particular for Scots pine, are commonly based on transcribed regions of the genome. In this article, we present the Axiom Psyl50K array, the first single nucleotide polymorphism (SNP) genotyping array for Scots pine based on whole-genome resequencing, that represents both genic and intergenic regions. This array was designed following a two-step procedure: first, 192 trees were sequenced, and a 430K SNP screening array was constructed. Then, 480 samples, including haploid megagametophytes, full-sib family trios, breeding population, and range-wide individuals from across Eurasia were genotyped with the screening array. The best 50K SNPs were selected based on quality, replicability, distribution across the draft genome assembly, balance between genic and intergenic regions, and genotype-environment and genotype-phenotype associations. Of the final 49 877 probes tiled in the array, 20 372 (40.84%) occur inside gene models, while the rest lie in intergenic regions. We also show that the Psyl50K array can yield enough high-confidence SNPs for genetic studies in pine species from North America and Eurasia. This new genotyping tool will be a valuable resource for high-throughput fundamental and applied research of Scots pine and other pine species.

Threatened forests: As the Northern forests suffer from the effects of climate change, genomics has great potential to help them adapt: As the Northern forests suffer from the effects of climate change, genomics has great potential to help them adapt.

Climate change is having dramatic effects on forest health and growth - tree genomics provides tools for understanding and mitigating these effects.

Systems genetic analysis of lignin biosynthesis in Populus tremula.

The genetic control underlying natural variation in lignin content and composition in trees is not fully understood. We performed a systems genetic analysis to uncover the genetic regulation of lignin biosynthesis in a natural 'SwAsp' population of aspen (Populus tremula) trees. We analyzed gene expression by RNA sequencing (RNA-seq) in differentiating xylem tissues, and lignin content and composition using Pyrolysis-GC-MS in mature wood of 268 trees from 99 genotypes. Abundant variation was observed for lignin content and composition, and genome-wide association study identified proteins in the pentose phosphate pathway and arabinogalactan protein glycosylation among the top-ranked genes that are associated with these traits. Variation in gene expression and the associated genetic polymorphism was revealed through the identification of 312 705 local and 292 003 distant expression quantitative trait loci (eQTL). A co-expression network analysis suggested modularization of lignin biosynthesis and novel functions for the lignin-biosynthetic CINNAMYL ALCOHOL DEHYDROGENASE 2 and CAFFEOYL-CoA O-METHYLTRANSFERASE 3. PHENYLALANINE AMMONIA LYASE 3 was co-expressed with HOMEOBOX PROTEIN 5 (HB5), and the role of HB5 in stimulating lignification was demonstrated in transgenic trees. The systems genetic approach allowed linking natural variation in lignin biosynthesis to trees´ responses to external cues such as mechanical stimulus and nutrient availability.

Identification of growth regulators using cross-species network analysis in plants.

With the need to increase plant productivity, one of the challenges plant scientists are facing is to identify genes that play a role in beneficial plant traits. Moreover, even when such genes are found, it is generally not trivial to transfer this knowledge about gene function across species to identify functional orthologs. Here, we focused on the leaf to study plant growth. First, we built leaf growth transcriptional networks in Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and aspen (Populus tremula). Next, known growth regulators, here defined as genes that when mutated or ectopically expressed alter plant growth, together with cross-species conserved networks, were used as guides to predict novel Arabidopsis growth regulators. Using an in-depth literature screening, 34 out of 100 top predicted growth regulators were confirmed to affect leaf phenotype when mutated or overexpressed and thus represent novel potential growth regulators. Globally, these growth regulators were involved in cell cycle, plant defense responses, gibberellin, auxin, and brassinosteroid signaling. Phenotypic characterization of loss-of-function lines confirmed two predicted growth regulators to be involved in leaf growth (NPF6.4 and LATE MERISTEM IDENTITY2). In conclusion, the presented network approach offers an integrative cross-species strategy to identify genes involved in plant growth and development.

Cone-setting in spruce is regulated by conserved elements of the age-dependent flowering pathway.

Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

GIGANTEA influences leaf senescence in trees in two different ways.

GIGANTEA (GI) genes have a central role in plant development and influence several processes. Hybrid aspen T89 (Populus tremula x tremuloides) trees with low GI expression engineered through RNAi show severely compromised growth. To study the effect of reduced GI expression on leaf traits with special emphasis on leaf senescence, we grafted GI-RNAi scions onto wild-type rootstocks and successfully restored growth of the scions. The RNAi line had a distorted leaf shape and reduced photosynthesis, probably caused by modulation of phloem or stomatal function, increased starch accumulation, a higher carbon-to-nitrogen ratio, and reduced capacity to withstand moderate light stress. GI-RNAi also induced senescence under long day (LD) and moderate light conditions. Furthermore, the GI-RNAi lines were affected in their capacity to respond to "autumn environmental cues" inducing senescence, a type of leaf senescence that has physiological and biochemical characteristics that differ from those of senescence induced directly by stress under LD conditions. Overexpression of GI delayed senescence under simulated autumn conditions. The two different effects on leaf senescence under LD or simulated autumn conditions were not affected by the expression of FLOWERING LOCUS T. GI expression regulated leaf senescence locally-the phenotype followed the genotype of the branch, independent of its position on the tree-and trees with modified gene expression were affected in a similar way when grown in the field as under controlled conditions. Taken together, GI plays a central role in sensing environmental changes during autumn and determining the appropriate timing for leaf senescence in Populus.

Phytochrome B and PHYTOCHROME INTERACTING FACTOR8 modulate seasonal growth in trees.

The seasonally synchronized annual growth cycle that is regulated mainly by photoperiod and temperature cues is a crucial adaptive strategy for perennial plants in boreal and temperate ecosystems. Phytochrome B (phyB), as a light and thermal sensor, has been extensively studied in Arabidopsis. However, the specific mechanisms for how the phytochrome photoreceptors control the phenology in tree species remain poorly understood. We characterized the functions of PHYB genes and their downstream PHYTOCHROME INTERACTING FACTOR (PIF) targets in the regulation of shade avoidance and seasonal growth in hybrid aspen trees. We show that while phyB1 and phyB2, as phyB in other plants, act as suppressors of shoot elongation during vegetative growth, they act as promoters of tree seasonal growth. Furthermore, while the Populus homologs of both PIF4 and PIF8 are involved in the shade avoidance syndrome (SAS), only PIF8 plays a major role as a suppressor of seasonal growth. Our data suggest that the PHYB-PIF8 regulon controls seasonal growth through the regulation of FT and CENL1 expression while a genome-wide transcriptome analysis suggests how, in Populus trees, phyB coordinately regulates SAS responses and seasonal growth cessation.

Peptide encoding Populus CLV3/ESR-RELATED 47 (PttCLE47) promotes cambial development and secondary xylem formation in hybrid aspen.

The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED (CLE) peptide ligands in connection with their receptors are important players in cell-to-cell communications in plants. Here, we investigated the function of the Populus CLV3/ESR-RELATED 47 (PttCLE47) gene during secondary growth and wood formation in hybrid aspen (Populus tremula × tremuloides) using an RNA interference (RNAi) approach. Expression of PttCLE47 peaks in the vascular cambium. Silencing of the PttCLE47 gene expression affected lateral expansion of stems and decreased apical height growth and leaf size. In particular, PttCLE47 RNAi trees exhibited a narrower secondary xylem zone with less xylem cells/cell file. The reduced radial growth phenotype also correlated with a reduced number of cambial cell layers. In agreement with these results, expression of several cambial regulator genes was downregulated in the stems of the transgenic trees in comparison with controls. Altogether, these results suggest that the PttCLE47 gene is a major positive regulator of cambial activity in hybrid aspen, mainly promoting the production of secondary xylem. Furthermore, in contrast to previously characterized CLE genes expressed in the wood-forming zone, PttCLE47 appears to be active at its site of expression.

AspWood: High-Spatial-Resolution Transcriptome Profiles Reveal Uncharacterized Modularity of Wood Formation in Populus tremula.

Trees represent the largest terrestrial carbon sink and a renewable source of ligno-cellulose. There is significant scope for yield and quality improvement in these largely undomesticated species, and efforts to engineer elite varieties will benefit from improved understanding of the transcriptional network underlying cambial growth and wood formation. We generated high-spatial-resolution RNA sequencing data spanning the secondary phloem, vascular cambium, and wood-forming tissues of Populus tremula The transcriptome comprised 28,294 expressed, annotated genes, 78 novel protein-coding genes, and 567 putative long intergenic noncoding RNAs. Most paralogs originating from the Salicaceae whole-genome duplication had diverged expression, with the exception of those highly expressed during secondary cell wall deposition. Coexpression network analyses revealed that regulation of the transcriptome underlying cambial growth and wood formation comprises numerous modules forming a continuum of active processes across the tissues. A comparative analysis revealed that a majority of these modules are conserved in Picea abies The high spatial resolution of our data enabled identification of novel roles for characterized genes involved in xylan and cellulose biosynthesis, regulators of xylem vessel and fiber differentiation and lignification. An associated web resource (AspWood, http://aspwood.popgenie.org) provides interactive tools for exploring the expression profiles and coexpression network.

Transcriptional Roadmap to Seasonal Variation in Wood Formation of Norway Spruce.

Seasonal cues influence several aspects of the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. We investigated seasonal changes in cambial activity, secondary cell wall formation, and tracheid cell death in woody tissues of Norway spruce (Picea abies) throughout one seasonal cycle. RNA sequencing was performed simultaneously in both the xylem and cambium/phloem tissues of the stem. Principal component analysis revealed gradual shifts in the transcriptomes that followed a chronological order throughout the season. A notable remodeling of the transcriptome was observed in the winter, with many genes having maximal expression during the coldest months of the year. A highly coexpressed set of monolignol biosynthesis genes showed high expression during the period of secondary cell wall formation as well as a second peak in midwinter. This midwinter peak in expression did not trigger lignin deposition, as determined by pyrolysis-gas chromatography/mass spectrometry. Coexpression consensus network analyses suggested the involvement of transcription factors belonging to the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES and MYELOBLASTOSIS-HOMEOBOX families in the seasonal control of secondary cell wall formation of tracheids. Interestingly, the lifetime of the latewood tracheids stretched beyond the winter dormancy period, correlating with a lack of cell death-related gene expression. Our transcriptomic analyses combined with phylogenetic and microscopic analyses also identified the cellulose and lignin biosynthetic genes and putative regulators for latewood formation and tracheid cell death in Norway spruce, providing a toolbox for further physiological and functional assays of these important phase transitions.

LEAFY activity is post-transcriptionally regulated by BLADE ON PETIOLE2 and CULLIN3 in Arabidopsis.

The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence and identity. LFY interacts genetically and physically with UNUSUAL FLORAL ORGANS, a substrate adaptor of CULLIN1-RING ubiquitin ligase complexes (CRL1). The functionally redundant genes BLADE ON PETIOLE1 (BOP1) and -2 (BOP2) are potential candidates to regulate LFY activity and have recently been shown to be substrate adaptors of CULLIN3 (CUL3)-RING ubiquitin ligases (CRL3). We tested the hypothesis that LFY activity is controlled by BOPs and CUL3s in plants and that LFY is a substrate for ubiquitination by BOP-containing CRL3 complexes. When constitutively expressed, LFY activity is fully dependent on BOP2 as well as on CUL3A and B to regulate target genes such as APETALA1 and to induce ectopic flower formation. We also show that LFY and BOP2 proteins interact physically and that LFY-dependent ubiquitinated species are produced in vitro in a reconstituted cell-free CRL3 system in the presence of LFY, BOP2 and CUL3. This new post-translational regulation of LFY activity by CRL3 complexes makes it a unique transcription factor subjected to a positive dual regulation by both CRL1 and CRL3 complexes and suggests a novel mechanism for promoting flower development.

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.

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.

Class I KNOX transcription factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl.

The class I KNOX transcription factors SHOOT MERISTEMLESS (STM) and KNAT1 are important regulators of meristem maintenance in shoot apices, with a dual role of promoting cell proliferation and inhibiting differentiation. We examined whether they control stem cell maintenance in the cambium of Arabidopsis hypocotyls, a wood-forming lateral meristem, in a similar fashion as in the shoot apical meristem. Weak loss-of-function alleles of KNAT1 and STM led to reduced formation of xylem fibers - highly differentiated cambial derivatives - whereas cell proliferation in the cambium was only mildly affected. In a knat1;stm double mutant, xylem fiber differentiation was completely abolished, but residual cambial activity was maintained. Expression of early and late markers of xylary cell differentiation was globally reduced in the knat1;stm double mutant. KNAT1 and STM were found to act through transcriptional repression of the meristem boundary genes BLADE-ON-PETIOLE 1 (BOP1) and BOP2 on xylem fiber differentiation. Together, these data indicate that, in the cambium, KNAT1 and STM, contrary to their function in the shoot apical meristem, promote cell differentiation through repression of BOP genes.

WUSCHEL-RELATED HOMEOBOX4 (WOX4)-like genes regulate cambial cell division activity and secondary growth in Populus trees.

Plant secondary growth derives from the meristematic activity of the vascular cambium. In Arabidopsis thaliana, cell divisions in the cambium are regulated by the transcription factor WOX4, a key target of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED 41 (CLE41) signaling pathway. However, function of the WOX4-like genes in plants that are dependent on a much more prolific secondary growth, such as trees, remains unclear. Here, we investigate the role of WOX4 and CLE41 homologs for stem secondary growth in Populus trees. In Populus, PttWOX4 genes are specifically expressed in the cambial region during vegetative growth, but not after growth cessation and during dormancy, possibly involving a regulation by auxin. In PttWOX4a/b RNAi trees, primary growth was not affected whereas the width of the vascular cambium was severely reduced and secondary growth was greatly diminished. Our data show that in Populus trees, PttWOX4 genes control cell division activity in the vascular cambium, and hence growth in stem girth. This activity involves the positive regulation of PttWOX4a/b through PttCLE41-related genes. Finally, expression profiling suggests that the CLE41 signaling pathway is an evolutionarily conserved program for the regulation of vascular cambium activity between angiosperm and gymnosperm tree species.

NorWood: a gene expression resource for evo-devo studies of conifer wood development.

The secondary xylem of conifers is composed mainly of tracheids that differ anatomically and chemically from angiosperm xylem cells. There is currently no high-spatial-resolution data available profiling gene expression during wood formation for any coniferous species, which limits insight into tracheid development. RNA-sequencing data from replicated, high-spatial-resolution section series throughout the cambial and woody tissues of Picea abies were used to generate the NorWood.conGenIE.org web resource, which facilitates exploration of the associated gene expression profiles and co-expression networks. Integration within PlantGenIE.org enabled a comparative regulomics analysis, revealing divergent co-expression networks between P. abies and the two angiosperm species Arabidopsis thaliana and Populus tremula for the secondary cell wall (SCW) master regulator NAC Class IIB transcription factors. The SCW cellulose synthase genes (CesAs) were located in the neighbourhoods of the NAC factors in A. thaliana and P. tremula, but not in P. abies. The NorWood co-expression network enabled identification of potential SCW CesA regulators in P. abies. The NorWood web resource represents a powerful community tool for generating evo-devo insights into the divergence of wood formation between angiosperms and gymnosperms and for advancing understanding of the regulation of wood development in P. abies.

FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus.

Eucalyptus trees are among the most important species for industrial forestry worldwide. However, as with most forest trees, flowering does not begin for one to several years after planting which can limit the rate of conventional and molecular breeding. To speed flowering, we transformed a Eucalyptus grandis × urophylla hybrid (SP7) with a variety of constructs that enable overexpression of FLOWERING LOCUS T (FT). We found that FT expression led to very early flowering, with events showing floral buds within 1-5 months of transplanting to the glasshouse. The most rapid flowering was observed when the cauliflower mosaic virus 35S promoter was used to drive the Arabidopsis thaliana FT gene (AtFT). Early flowering was also observed with AtFT overexpression from a 409S ubiquitin promoter and under heat induction conditions with Populus trichocarpa FT1 (PtFT1) under control of a heat-shock promoter. Early flowering trees grew robustly, but exhibited a highly branched phenotype compared to the strong apical dominance of nonflowering transgenic and control trees. AtFT-induced flowers were morphologically normal and produced viable pollen grains and viable self- and cross-pollinated seeds. Many self-seedlings inherited AtFT and flowered early. FT overexpression-induced flowering in Eucalyptus may be a valuable means for accelerating breeding and genetic studies as the transgene can be easily segregated away in progeny, restoring normal growth and form.

Insights into conifer giga-genomes.

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world's forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20-30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes.

Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2, and -3 are the dominant paralogs.

Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA(12) is converted to GA(9). GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:ß-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.