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.

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.

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.

A System for Dosage-Based Functional Genomics in Poplar.

Altering gene dosage through variation in gene copy number is a powerful approach to addressing questions regarding gene regulation, quantitative trait loci, and heterosis, but one that is not easily applied to sexually transmitted species. Elite poplar (Populus spp) varieties are created through interspecific hybridization, followed by clonal propagation. Altered gene dosage relationships are believed to contribute to hybrid performance. Clonal propagation allows for replication and maintenance of meiotically unstable ploidy or structural variants and provides an alternative approach to investigating gene dosage effects not possible in sexually propagated species. Here, we built a genome-wide structural variation system for dosage-based functional genomics and breeding of poplar. We pollinated Populus deltoides with gamma-irradiated Populus nigra pollen to produce >500 F1 seedlings containing dosage lesions in the form of deletions and insertions of chromosomal segments (indel mutations). Using high-precision dosage analysis, we detected indel mutations in ∼55% of the progeny. These indels varied in length, position, and number per individual, cumulatively tiling >99% of the genome, with an average of 10 indels per gene. Combined with future phenotype and transcriptome data, this population will provide an excellent resource for creating and characterizing dosage-based variation in poplar, including the contribution of dosage to quantitative traits and heterosis.

The Populus homeobox gene ARBORKNOX2 regulates cell differentiation during secondary growth.

The stem cells of the vascular cambium divide to produce daughter cells, which in turn divide before undergoing differentiation during the radial growth of woody stems. The genetic regulation of these developmental events is poorly understood, however. We report here the cloning and functional characterization of a Populus class-I KNOX homeobox gene, ARBORKNOX2 (ARK2), which we show influences terminal cell differentiation and cell wall properties during secondary growth. In the early stages of secondary growth, ARK2 is expressed broadly in the cambial zone and in terminally differentiating cell types, before becoming progressively restricted to the cambium. ARK2 overexpression and synthetic miRNA-suppression transgenics reveal positive correlations between ARK2 expression level and the timing of cambium formation, the width of the cambial zone and inhibition of cambial daughter cell differentiation. These phenotypes in turn correlate with changes in the expression of genes affecting transcription, cell division, auxin and cell wall synthesis. Notably, wood properties associated with secondary cell wall synthesis are negatively associated with ARK2 expression, including lignin and cellulose content. Together, our results suggest that ARK2 functions primarily to regulate a complex suite of genes that together influence cell differentiation during secondary growth. We propose that ARK2 may represent a co-evolved transcriptional module that influences complex, adaptive wood properties.

Will genomics guide a greener forest biotech?

Forest biotechnology has been increasingly associated with wood production using plantation forestry, and has stressed applications that use pedigreed material and transgenic trees. Reasons for this emphasis include limitations of available technologies to conform to underlying genetic features of undomesticated forest tree populations. More recently, genomic technologies have rapidly begun to expand the scope of forest biotechnology. Genomic technologies are well suited to describe and make use of the abundant genetic variation present in undomesticated forest tree populations. Genomics thus enables new research and applications for conservation and management of natural forests, and is a primary technological driver for new research addressing the use of forests trees for carbon sequestration, biofuels feedstocks, and other 'green' applications.

The Populus homeobox gene ARBORKNOX1 reveals overlapping mechanisms regulating the shoot apical meristem and the vascular cambium.

Secondary growth is supported by a dividing population of meristematic cells within the vascular cambium whose daughter cells are recruited to differentiate within secondary phloem and xylem tissues. We cloned a Populus Class 1 KNOX homeobox gene, ARBORKNOX1 (ARK1), which is orthologous to Arabidopsis SHOOT MERISTEMLESS (STM). ARK1 is expressed in the shoot apical meristem (SAM) and the vascular cambium, and is down-regulated in the terminally differentiated cells of leaves and secondary vascular tissues that are derived from these meristems. Transformation of Populus with either ARK1 or STM over-expression constructs results in similar morphological phenotypes characterized by inhibition of the differentiation of leaves, internode elongation, and secondary vascular cell types in stems. Microarray analysis showed that 41% of genes up-regulated in the stems of ARK1 over-expressing plants encode proteins involved in extracellular matrix synthesis or modification, including proteins involved in cell identity and signaling, cell adhesion, or cell differentiation. These gene expression differences are reflected in alterations of cell wall biochemistry and lignin composition in ARK1 over-expressing plants. Our results suggest that ARK1 has a complex mode of action that may include regulating cell fates through modification of the extracellular matrix. Our findings support the hypothesis that the SAM and vascular cambium are regulated by overlapping genetic programs.

What genes make a tree a tree?

Woody growth is evolutionarily ancient, yet has been gained and lost multiple times in plant evolution and is readily enhanced or minimized in eudicot speciation. New molecular genetic and genomic studies in Populus and Arabidopsis that are defining the genes responsible for cambium function and woody growth suggest that the genes regulating woody growth are not unique to woody plants. Surprisingly, key genetic mechanisms originally characterized as regulating the meristematic cells of the shoot apical meristem are also expressed in the vascular cambium during woody growth. This has important implications for the development of Populus as a model species and illustrates why forest trees constitute a contrived group of plants that have more in common with herbaceous relatives than we foresters like to admit.

Phyllotactic pattern and stem cell fate are determined by the Arabidopsis homeobox gene BELLRINGER.

Lateral organs in plants arise from the meristem in a stereotypical pattern known as phyllotaxy. Spiral patterns result from initiation of successive organs at a fixed angle of divergence but variable patterns of physical contact. Such patterns ultimately give rise to individual leaves and flowers at positions related to each other by consecutive terms in the mathematical series first described by Leonardo Fibonacci. We demonstrate that a BELL1 related homeodomain protein in Arabidopsis, BELLRINGER, maintains the spiral phyllotactic pattern. In the absence of BELLRINGER, the regular pattern of organ initiation is disturbed and lateral organs are initiated more frequently. BELLRINGER is also required for maintenance of stem cell fate in the absence of the regulatory genes SHOOT MERISTEMLESS and ASYMMETRIC LEAVES1. We propose a model whereby BELLRINGER coordinates the maintenance of stem cells with differentiation of daughter cells in stem cell lineages.

Secretion trap tagging of secreted and membrane-spanning proteins using Arabidopsis gene traps.

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a beta-glucuronidase reporter enzyme that is inhibited by N-linked glycosylation specific to the secretory pathway. Treatment of seedlings with tunicamycin inhibits glycosylation, resulting in increased activity of secreted beta-glucuronidase fusions that result from gene trap integration downstream of exons encoding signal peptides. In the 2,059 gene trap lines that we screened, 32 secretion trap expression patterns were identified in a wide variety of tissues including embryos, meristems, and the developing vasculature. Genes disrupted by the secretion traps encode putative extracellular signaling proteins, membrane transport proteins, and novel secreted proteins of unknown function missed by conventional mutagenesis and gene prediction. Secretion traps provide a unique reagent for gene expression studies and can guide the genetic combination of loss of function alleles in related genes.

IAA8 expression during vascular cell differentiation.

We report the characterization of a member of the auxin-induced IAA gene family from zinnia, designated zIAA8, which is expressed by mesophyll cells differentiating as tracheary elements in vitro. Transcription of zIAA8 is up-regulated within 3 h after cell isolation in inductive medium, indicating that cells perceive and respond to growth factor stimulus early in culture. Transcript levels of zIAA8 remain high through 72 h of culture in medium containing auxin and cytokinin or auxin alone, but low in medium containing only cytokinin or control medium lacking growth factors, demonstrating auxin-specific induction and consistent with lack of desensitization to prolonged auxin stimulation. In situ localization shows zIAA8 is localized to primary vasculature, root tips, and nascent leaves in zinnia seedlings. The observation that zIAA8 is expressed during vascular development in planta supports the hypothesis that expression early in culture reflects early events during normal vascular differentiation. The promoter of Arabidopsis IAA8 drives expression of the GUS reporter in a pattern in Arabidopsis similar to that for zIAA8 in zinnia, suggesting conservation of cis regulatory elements between the species and confirming the results from in situ localization. The vascular expression pattern of the IAA8 promoter in leaves mirrors the developmentally regulated auxin gradient in expanding leaf blades. The expression patterns of zIAA8 and IAA8 yield new insight into vascular development in vitro and in planta, and provide much needed markers for early vascular differentiation.