A systems genetic analysis identifies putative mechanisms and candidate genes regulating vessel traits in poplar wood.

Wood is the water conducting tissue of tree stems. Like most angiosperm trees, poplar wood contains water-conducting vessel elements whose functional properties affect water transport and growth rates, as well as susceptibility to embolism and hydraulic failure during water stress and drought. Here we used a unique hybrid poplar pedigree carrying genomically characterized chromosomal insertions and deletions to undertake a systems genomics analysis of vessel traits. We assayed gene expression in wood forming tissues from clonal replicates of genotypes covering dosage quantitative trait loci with insertions and deletions, genotypes with extreme vessel trait phenotypes, and control genotypes. A gene co-expression analysis was used to assign genes to modules, which were then used in integrative analyses to identify modules associated with traits, to identify putative molecular and cellular processes associated with each module, and finally to identify candidate genes using multiple criteria including dosage responsiveness. These analyses identified known processes associated with vessel traits including stress response, abscisic acid and cell wall biosynthesis, and in addition identified previously unexplored processes including cell cycle and protein ubiquitination. We discuss our findings relative to component processes contributing to vessel trait variation including signaling, cell cycle, cell expansion, and cell differentiation.

Overcoming the field-of-view to diameter trade-off in microendoscopy via computational optrode-array microscopy.

High-resolution microscopy of deep tissue with large field-of-view (FOV) is critical for elucidating organization of cellular structures in plant biology. Microscopy with an implanted probe offers an effective solution. However, there exists a fundamental trade-off between the FOV and probe diameter arising from aberrations inherent in conventional imaging optics (typically, FOV < 30% of diameter). Here, we demonstrate the use of microfabricated non-imaging probes (optrodes) that when combined with a trained machine-learning algorithm is able to achieve FOV of 1x to 5x the probe diameter. Further increase in FOV is achieved by using multiple optrodes in parallel. With a 1 × 2 optrode array, we demonstrate imaging of fluorescent beads (including 30 FPS video), stained plant stem sections and stained living stems. Our demonstration lays the foundation for fast, high-resolution microscopy with large FOV in deep tissue via microfabricated non-imaging probes and advanced machine learning.

Facile Labeling of Sieve Element Phloem-Protein Bodies Using the Reciprocal Oligosaccharide Probe OGA 488.

Sieve elements of many angiosperms contain structural phloem proteins (P-proteins) that can interact to create large P-protein bodies. P-protein bodies can occlude sieve plates upon injury but the range of functional and physiological roles of P-proteins remains uncertain, in part because of challenges in labeling and visualization methods. Here, we show that a reciprocal oligosaccharide probe, OGA488, can be used in rapid and sensitive labeling of P-protein bodies in Arabidopsis, poplar, snap bean and cucumber in histological sections. OGA488 labeling of knockouts of the two Arabidopsis P-protein-encoding genes, AtSEOR1 and AtSEOR2, indicated that labeling is specific to AtSEOR2. That protein bodies were labeled and visible in Atseor1 knockouts indicates that heterodimerization of AtSEOR1 and AtSEOR2 may not be necessary for P-protein body formation. Double labeling with a previously characterized stain for P-proteins, sulphorhodamine 101, confirmed P-protein labeling and also higher specificity of OGA488 for P-proteins. OGA488 is thus robust and easily used to label P-proteins in histological sections of multiple angiosperm species.

Genetic Regulation of Vessel Morphology in Populus.

During secondary growth, forest trees can modify the anatomy of the wood produced by the vascular cambium in response to environmental conditions. Notably, the trees of the model angiosperm genus, Populus, reduce the risk of cavitation and hydraulic failure under water stress by producing water-conducting vessel elements with narrow lumens, which are more numerous and more interconnected with each other. Here, we determined the genetic architecture of vessel traits affecting hydraulic physiology and resilience to water stress. Vessel traits were measured for clonally replicated genotypes of a unique Populus deltoides x nigra population carrying genomically defined insertions and deletions that create gene dosage variation. We found significant phenotypic variation for all traits measured (mean vessel diameter, height-corrected mean vessel diameter, vessel frequency, height-corrected vessel frequency, vessel grouping index, and mean vessel circularity), and that all traits were under genetic control and showed moderate heritability values, ranging from 0.32 to 0.53. Whole-genome scans of correlations between gene dosage and phenotypic traits identified quantitative trait loci for tree height, mean vessel diameter, height-corrected mean vessel diameter, height-corrected vessel frequency, and vessel grouping index. Our results demonstrate that vessel traits affecting hydraulic physiology are under genetic control, and both pleiotropic and trait-specific quantitative trait loci are found for these traits.

A systems genetics approach to deciphering the effect of dosage variation on leaf morphology in Populus.

Gene copy number variation is frequent in plant genomes of various species, but the impact of such gene dosage variation on morphological traits is poorly understood. We used a large population of Populus carrying genomically characterized insertions and deletions across the genome to systematically assay the effect of gene dosage variation on a suite of leaf morphology traits. A systems genetics approach was used to integrate insertion and deletion locations, leaf morphology phenotypes, gene expression, and transcriptional network data, to provide an overview of how gene dosage influences morphology. Dosage-sensitive genomic regions were identified that influenced individual or pleiotropic morphological traits. We also identified cis-expression quantitative trait loci (QTL) within these dosage QTL regions, a subset of which modulated trans-expression QTL as well. Integration of data types within a gene co-expression framework identified co-expressed gene modules that are dosage sensitive, enriched for dosage expression QTL, and associated with morphological traits. Functional description of these modules linked dosage-sensitive morphological variation to specific cellular processes, as well as candidate regulatory genes. Together, these results show that gene dosage variation can influence morphological variation through complex changes in gene expression, and suggest that frequently occurring gene dosage variation has the potential to likewise influence quantitative traits in nature.

Evolutionary network genomics of wood formation in a phylogenetic survey of angiosperm forest trees.

Wood formation was present in early angiosperms, but has been highly modified through evolution to generate the anatomical diversity seen in extant angiosperm lineages. In this project, we modeled changes in gene coexpression relationships associated with the evolution of wood formation in a phylogenetic survey of 13 angiosperm tree species. Gravitropic stimulation was used as an experimental treatment to alter wood formation and also perturb gene expression. Gene transcript abundances were determined using RNA sequencing of developing wood tissues from upright trees, and from the top (tension wood) and bottom (opposite wood) tissues of gravistimulated trees. A network-based approach was employed to align gene coexpression networks across species based on orthologous relationships. A large-scale, multilayer network was modeled that identified both lineage-specific gene coexpression modules and modules conserved across multiple species. Functional annotation and analysis of modules identified specific regulatory processes associated with conserved modules, including regulation of hormones, protein phosphorylation, meristem development and epigenetic processes. Our results provide novel insights into the evolution and development of wood formation, and demonstrate the ability to identify biological processes and genes important for the evolution of a foundational trait in nonmodel, undomesticated forest trees.

Woody Plant Evolution: Exceptional Lianas Reveal Rules of Woody Growth.

Sometimes the exceptions prove the rule, and lianas show some of the most exceptional stem anatomical variation in plants. New research describes the evolution and development of liana stem anatomical variants, and reveals new rules of woody growth.

Brassinosteroid regulation of wood formation in poplar.

Brassinosteroids have been implicated in the differentiation of vascular cell types in herbaceous plants, but their roles during secondary growth and wood formation are not well defined. Here we pharmacologically and genetically manipulated brassinosteroid levels in poplar trees and assayed the effects on secondary growth and wood formation, and on gene expression within stems. Elevated brassinosteroid levels resulted in increases in secondary growth and tension wood formation, while inhibition of brassinosteroid synthesis resulted in decreased growth and secondary vascular differentiation. Analysis of gene expression showed that brassinosteroid action is positively associated with genes involved in cell differentiation and cell-wall biosynthesis. The results presented here show that brassinosteroids play a foundational role in the regulation of secondary growth and wood formation, in part through the regulation of cell differentiation and secondary cell wall biosynthesis.

A comprehensive genomic scan reveals gene dosage balance impacts on quantitative traits in Populus trees.

Gene dosage variation and the associated changes in gene expression influence a wide variety of traits, ranging from cancer in humans to yield in plants. It is also expected to affect important traits of ecological and agronomic importance in forest trees, but this variation has not been systematically characterized or exploited. Here we performed a comprehensive scan of the Populus genome for dosage-sensitive loci affecting quantitative trait variation for spring and fall phenology and biomass production. The study population was a large collection of clonally propagated F1 hybrid lines of Populus that saturate the genome 10-fold with deletions and insertions (indels) of known sizes and positions. As a group, the phenotypic means of the indel lines consistently differed from control nonindel lines, with an overall negative effect of both insertions and deletions on all biomass-related traits but more diverse effects and an overall wider phenotypic distribution of the indel lines for the phenology-related traits. We also investigated the correlation between gene dosage at specific chromosomal locations and phenotype, to identify dosage quantitative trait loci (dQTL). Such dQTL were detected for most phenotypes examined, but stronger effect dQTL were identified for the phenology-related traits than for the biomass traits. Our genome-wide screen for dosage sensitivity in a higher eukaryote demonstrates the importance of global genomic balance and the impact of dosage on life history traits.

The auxin receptor TIR1 homolog (PagFBL 1) regulates adventitious rooting through interactions with Aux/IAA28 in Populus.

Adventitious roots occur naturally in many species and can also be induced from explants of some tree species including Populus, providing an important means of clonal propagation. Auxin has been identified as playing a crucial role in adventitious root formation, but the associated molecular regulatory mechanisms need to be elucidated. In this study, we examined the role of PagFBL1, the hybrid poplar (Populus alba × P. glandulosa clone 84K) homolog of Arabidopsis auxin receptor TIR1, in adventitious root formation in poplar. Similar to the distribution pattern of auxin during initiation of adventitious roots, PagFBL1 expression was concentrated in the cambium and secondary phloem in stems during adventitious root induction and initiation phases, but decreased in emerging adventitious root primordia. Overexpressing PagFBL1 stimulated adventitious root formation and increased root biomass, while knock-down of PagFBL1 transcript levels delayed adventitious root formation and decreased root biomass. Transcriptome analyses of PagFBL1 overexpressing lines indicated that an extensive remodelling of gene expression was stimulated by auxin signalling pathway during early adventitious root formation. In addition, PagIAA28 was identified as downstream targets of PagFBL1. We propose that the PagFBL1-PagIAA28 module promotes adventitious rooting and could be targeted to improve Populus propagation by cuttings.

Mosaic modularity: an updated perspective and research agenda for the evolution of vascular cambial growth.

Secondary growth from a vascular cambium, present today only in seed plants and isoetalean lycophytes, has a 400-million-yr evolutionary history that involves considerably broader taxonomic diversity, most of it hidden in the fossil record. Approaching vascular cambial growth as a complex developmental process, we review data from living plants and fossils that reveal diverse modes of secondary growth. These are consistent with a modular nature of secondary growth, when considered as a tracheophyte-wide structural feature. This modular perspective identifies putative constituent developmental modules of cambial growth, for which we review developmental anatomy and regulation. Based on these data, we propose a hypothesis that explains the sources of diversity of secondary growth, considered across the entire tracheophyte clade, and opens up new avenues for exploring the origin of secondary growth. In this hypothesis, various modes of secondary growth reflect a mosaic pattern of expression of different developmental-regulatory modules among different lineages. We outline an approach that queries three information systems (living seed plants, living seed-free plants, and fossils) and integrates data on developmental regulation, anatomy, gene evolution and phylogeny to test the mosaic modularity hypothesis and its implications, and to inform efforts aimed at understanding the evolution of secondary growth.

Transcriptional and temporal response of Populus stems to gravi-stimulation.

Plants modify development in response to external stimuli, to produce new growth that is appropriate for environmental conditions. For example, gravi-stimulation of leaning branches in angiosperm trees results in modifications of wood development, to produce tension wood that pulls leaning stems upright. Here, we use gravi-stimulation and tension wood response to dissect the temporal changes in gene expression underlying wood formation in Populus stems. Using time-series analysis of seven time points over a 14-d experiment, we identified 8,919 genes that were differentially expressed between tension wood (upper) and opposite wood (lower) sides of leaning stems. Clustering of differentially expressed genes showed four major transcriptional responses, including gene clusters whose transcript levels were associated with two types of tissue-specific impulse responses that peaked at about 24-48 h, and gene clusters with sustained changes in transcript levels that persisted until the end of the 14-d experiment. Functional enrichment analysis of those clusters suggests they reflect temporal changes in pathways associated with hormone regulation, protein localization, cell wall biosynthesis and epigenetic processes. Time-series analysis of gene expression is an underutilized approach for dissecting complex developmental responses in plants, and can reveal gene clusters and mechanisms influencing development.

Hardwood Tree Genomics: Unlocking Woody Plant Biology.

Woody perennial angiosperms (i.e., hardwood trees) are polyphyletic in origin and occur in most angiosperm orders. Despite their independent origins, hardwoods have shared physiological, anatomical, and life history traits distinct from their herbaceous relatives. New high-throughput DNA sequencing platforms have provided access to numerous woody plant genomes beyond the early reference genomes of Populus and Eucalyptus, references that now include willow and oak, with pecan and chestnut soon to follow. Genomic studies within these diverse and undomesticated species have successfully linked genes to ecological, physiological, and developmental traits directly. Moreover, comparative genomic approaches are providing insights into speciation events while large-scale DNA resequencing of native collections is identifying population-level genetic diversity responsible for variation in key woody plant biology across and within species. Current research is focused on developing genomic prediction models for breeding, defining speciation and local adaptation, detecting and characterizing somatic mutations, revealing the mechanisms of gender determination and flowering, and application of systems biology approaches to model complex regulatory networks underlying quantitative traits. Emerging technologies such as single-molecule, long-read sequencing is being employed as additional woody plant species, and genotypes within species, are sequenced, thus enabling a comparative ("evo-devo") approach to understanding the unique biology of large woody plants. Resource availability, current genomic and genetic applications, new discoveries and predicted future developments are illustrated and discussed for poplar, eucalyptus, willow, oak, chestnut, and pecan.

Identifying gene coexpression networks underlying the dynamic regulation of wood-forming tissues in Populus under diverse environmental conditions.

Trees modify wood formation through integration of environmental and developmental signals in complex but poorly defined transcriptional networks, allowing trees to produce woody tissues appropriate to diverse environmental conditions. In order to identify relationships among genes expressed during wood formation, we integrated data from new and publically available datasets in Populus. These datasets were generated from woody tissue and include transcriptome profiling, transcription factor binding, DNA accessibility and genome-wide association mapping experiments. Coexpression modules were calculated, each of which contains genes showing similar expression patterns across experimental conditions, genotypes and treatments. Conserved gene coexpression modules (four modules totaling 8398 genes) were identified that were highly preserved across diverse environmental conditions and genetic backgrounds. Functional annotations as well as correlations with specific experimental treatments associated individual conserved modules with distinct biological processes underlying wood formation, such as cell-wall biosynthesis, meristem development and epigenetic pathways. Module genes were also enriched for DNase I hypersensitivity footprints and binding from four transcription factors associated with wood formation. The conserved modules are excellent candidates for modeling core developmental pathways common to wood formation in diverse environments and genotypes, and serve as testbeds for hypothesis generation and testing for future studies.

Transcript profiling of a novel plant meristem, the monocot cambium.

While monocots lack the ability to produce a vascular cambium or woody growth, some monocot lineages evolved a novel lateral meristem, the monocot cambium, which supports secondary radial growth of stems. In contrast to the vascular cambium found in woody angiosperm and gymnosperm species, the monocot cambium produces secondary vascular bundles, which have an amphivasal organization of tracheids encircling a central strand of phloem. Currently there is no information concerning the molecular genetic basis of the development or evolution of the monocot cambium. Here we report high-quality transcriptomes for monocot cambium and early derivative tissues in two monocot genera, Yucca and Cordyline. Monocot cambium transcript profiles were compared to those of vascular cambia and secondary xylem tissues of two forest tree species, Populus trichocarpa and Eucalyptus grandis. Monocot cambium transcript levels showed that there are extensive overlaps between the regulation of monocot cambia and vascular cambia. Candidate regulatory genes that vary between the monocot and vascular cambia were also identified, and included members of the KANADI and CLE families involved in polarity and cell-cell signaling, respectively. We suggest that the monocot cambium may have evolved in part through reactivation of genetic mechanisms involved in vascular cambium regulation.

Immunolocalization in Secondary Xylem of Populus.

Immunolocalization can be used to precisely visualize the location of specific proteins, cell wall components, or any other molecules within cells or tissues for which specific antibodies are available. Here we describe an immunolocalization protocol for tissue sections of woody Populus stems. The protocol includes descriptions of the required sectioning, fixation, probing, detection, and imaging parameters, as well suggested controls useful in interpreting results.

Gravitropisms and reaction woods of forest trees - evolution, functions and mechanisms.

Contents 790 I. 790 II. 792 III. 795 IV. 797 V. 798 VI. 800 VII. 800 800 References 800 SUMMARY: The woody stems of trees perceive gravity to determine their orientation, and can produce reaction woods to reinforce or change their position. Together, graviperception and reaction woods play fundamental roles in tree architecture, posture control, and reorientation of stems displaced by wind or other environmental forces. Angiosperms and gymnosperms have evolved strikingly different types of reaction wood. Tension wood of angiosperms creates strong tensile force to pull stems upward, while compression wood of gymnosperms creates compressive force to push stems upward. In this review, the general features and evolution of tension wood and compression wood are presented, along with descriptions of how gravitropisms and reaction woods contribute to the survival and morphology of trees. An overview is presented of the molecular and genetic mechanisms underlying graviperception, initial graviresponse and the regulation of tension wood development in the model angiosperm, Populus. Critical research questions and new approaches are discussed.