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.

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.

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.

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.

Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus.

Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation.