Amy M. Brunner

FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa

BackgroundAuxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors are key regulators of auxin responses in plants. We identified the suites of genes in the two gene families in Populus and performed comparative genomic analysis with Arabidopsis and rice.ResultsA total of 35 Aux/IAA and 39 ARF genes were identified in the Populus genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted between the two dicotyledonous plants. Activator ARF genes were found to be two fold-overrepresented in the Populus genome. PoptrIAA and PoptrARF gene families appear to have expanded due to high segmental and low tandem duplication events. Furthermore, expression studies showed that genes in the expanded PoptrIAA3 subgroup display differential expression.ConclusionThe present study examines the extent of conservation and divergence in the structure and evolution of Populus Aux/IAA and ARF gene families with respect to Arabidopsis and rice. The gene-family analysis reported here will be useful in conducting future functional genomics studies to understand how the molecular roles of these large gene families translate into a diversity of biologically meaningful auxin effects.

Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus

Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss-of-function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)-mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1-RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1-RNAi trees contained fewer flowers than did wild-type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age-related changes that occur over years of growth.

Genes for control of plant stature and form.

Here we summarize progress in identification of three classes of genes useful for control of plant architecture: those affecting hormone metabolism and signaling; transcription and other regulatory factors; and the cell cycle. We focus on strong modifiers of stature and form that may be useful for directed modification of plant architecture, rather than the detailed mechanisms of gene action. Gibberellin (GA) metabolic and response genes are particularly attractive targets for manipulation because many act in a dose-dependent manner; similar phenotypic effects can be readily achieved in heterologous species; and induced pleiotropic effects--such as on nitrogen assimilation, photosynthesis, and lateral root production--are usually positive with respect to crop performance. Genes encoding transcription factors represent strong candidates for manipulation of plant architecture. For example, AINTEGUMENTA, ARGOS (auxin-regulated gene controlling organ size), and growth-regulating factors (GRFs) are strong modifiers of leaf and/or flower size. Plants overexpressing these genes had increased organ size and did not display negative pleiotropic effects in glasshouse environments. TCP-domain genes such as CINCINNATA, and the associated regulatory miRNAs such as miRJAW, may provide useful means to modulate leaf curvature and other foliage properties. There are considerable opportunities for comparative and translational genomics in nonmodel plant systems.

Genetic containment of forest plantations

Dispersal of pollen, seeds, or vegetative propagules from intensively bred, exotic, or recombinant DNA modified forest plantations may cause detrimental or beneficial ecological impacts on wild or managed ecosystems. Insertion of genes designed to prevent or substantially reduce dispersal could reduce the risk and extent of undesired impacts. Containment measures may also be required by law or marketplace constraints, regardless of risks or benefits. We discuss: (1) the context for when genetic containment or mitigation systems may be needed; (2) technology approaches and mechanisms; (3) the state of knowledge on genes/genomics of sexual reproduction in forest trees; (4) stability of transgene expression during vegetative growth; (5) simulation studies to define the level of containment needed; and (6) needed research to deliver effective containment technologies. We illustrate progress with several examples from our research on recombinant DNA modified poplars. Our simulations show that even partial sterility can provide very substantial reductions in gene flow into wild trees. We conclude that it is impossible to define the most effective containment approaches, nor their reliability, based on current genomic knowledge and technological tools. Additional genomic and technological studies of a wide variety of options are needed. Studies in field environments are essential to provide data relevant to ecological analysis and regulatory decisions and need to be carried out in phylogenetically diverse representatives of the economically most important taxa of forest trees.

Genome-wide transcriptome analysis of the transition from primary to secondary stem development in Populus trichocarpa

BackgroundWith its genome sequence and other experimental attributes, Populus trichocarpa has become the model species for genomic studies of wood development. Wood is derived from secondary growth of tree stems, and begins with the development of a ring of vascular cambium in the young developing stem. The terminal region of the developing shoot provides a steep developmental gradient from primary to secondary growth that facilitates identification of genes that play specialized functions during each of these phases of growth.ResultsUsing a genomic microarray representing the majority of the transcriptome, we profiled gene expression in stem segments that spanned primary to secondary growth. We found 3,016 genes that were differentially expressed during stem development (Q-value ≤ 0.05; >2-fold expression variation), and 15% of these genes encode proteins with no significant identities to known genes. We identified all gene family members putatively involved in secondary growth for carbohydrate active enzymes, tubulins, actins, actin depolymerizing factors, fasciclin-like AGPs, and vascular development-associated transcription factors. Almost 70% of expressed transcription factors were upregulated during the transition to secondary growth. The primary shoot elongation region of the stem contained specific carbohydrate active enzyme and expansin family members that are likely to function in primary cell wall synthesis and modification. Genes involved in plant defense and protective functions were also dominant in the primary growth region.ConclusionOur results describe the global patterns of gene expression that occur during the transition from primary to secondary stem growth. We were able to identify three major patterns of gene expression and over-represented gene ontology categories during stem development. The new regulatory factors and cell wall biogenesis genes that we identified provide candidate genes for further functional characterization, as well as new tools for molecular breeding and biotechnology aimed at improvement of tree growth rate, crown form, and wood quality.

Characterization of NAC domain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus

Wood has a wide variety of uses and is arguably the most important renewable raw material. The composition of xylem cell types in wood determines the utility of different types of wood for distinct commercial applications. Using expression profiling and phylogenetic analysis, we identified many xylem-associated regulatory genes that may control the differentiation of cells involved in wood formation in Arabidopsis and poplar. Prominent among these are NAC domain transcription factors (NACs). We studied NACs with putative involvement as negative (XND1 from Arabidopsis and its poplar orthologs PopNAC118, PopNAC122, PopNAC128, PopNAC129), or positive (SND2 and SND3 from Arabidopsis and their poplar orthologs PopNAC105, PopNAC154, PopNAC156, PopNAC157) regulators of secondary cell wall synthesis. Using quantitative PCR and in situ hybridization, we evaluated expression of these Populus NACs in a developmental gradient and in association with reaction wood and found that representatives from both groups were associated with wood-forming tissue and phloem fibers. Additionally, XND1 orthologs were expressed in mesophyll cells of developing leaves. We prepared transgenic Arabidopsis and poplar plants for overexpression of selected NACs. XND1 overexpression in poplar resulted in severe stunting. Additionally, poplar XND1 overexpressors lacked phloem fibers and showed reductions in cell size and number, vessel number, and frequency of rays in the xylem. Overexpression of PopNAC122, an XND1 ortholog, yielded an analogous phenotype in Arabidopsis. Overexpression of PopNAC154 in poplar reduced height growth and increased the relative proportion of bark versus xylem.

Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons

High stability of transgene expression is essential for functional genomics studies using transformation approaches and for application of genetic engineering to commercial forestry. We quantified expression of two reporter genes, green fluorescent protein (GFP) and the herbicide bialaphos resistance gene (BAR), in 2256 transgenic poplar trees derived from 404 primary events, and in 106 in vitro-redifferentiated subevents, over 3 years in the greenhouse and in the field. No gene silencing (complete breakdown of expression) was observed for GFP or BAR expression in any of the primary transgenic events during the course of the study. Transgenic cassettes were physically eliminated in four subevents (2.5%) derived from three different primary events during re-organogenesis. Transgene copy number was positively correlated with transgene expression level; however, a majority of transformants (85%) carried single-copy transgenes. About one-third of the events containing two-copy inserts had repeats formed at the same chromosomal position, with direct repeats being the main type observed (87%). All events containing more than two transgene copies showed repeat formation at least at one locus, with direct repeats again dominant (77%). Loci with two direct repeats had substantially greater transgene expression level than other types of two-copy T-DNA configurations, but insert organization was not associated with stability of transgene expression. Use of the poplar rbcS promoter, which drove BAR in the transgenic constructs, had no adverse effect on transgene expression levels or stability compared with the heterologous CaMV 35S promoter, which directed GFP expression.

Vernalization and the chilling requirement to exit bud dormancy: shared or separate regulation?

Similarities have long been recognized between vernalization, the prolonged exposure to cold temperatures that promotes the floral transition in many plants, and the chilling requirement to release bud dormancy in woody plants of temperate climates. In both cases the extended chilling period occurring during winter is used to coordinate developmental events to the appropriate seasonal time. However, whether or not these processes share common regulatory components and molecular mechanisms remain largely unknown. Both gene function and association genetics studies in Populus are beginning to answer this question. In Populus, studies have revealed that orthologs of the antagonistic flowering time genes FT and CEN/TFL1 might have central roles in both processes. We review Populus seasonal shoot development related to dormancy release and the floral transition and evidence for FT/TFL1-mediated regulation of these processes to consider the question of regulatory overlap. In addition, we discuss the potential for and challenges to integrating functional and population genomics studies to uncover the regulatory mechanisms underpinning these processes in woody plant systems.

Options for Genetic Engineering of Floral Sterility in Forest Trees

Engineering of genetic sterility in transgenically modified trees destined for commercial uses will simplify compliance with regulatory guidelines and mitigate ecological concerns of transgene dispersal. It could also be a critical technology for reducing the rate of escape and invasive mobility of exotic plantation species. Added benefits may include increased biomass production by redirecting energy normally expended on reproduction, and elimination of nuisance pollen and fruits. We discuss the two basic strategies for genetically engineering reproductive sterility; 1) suppression of reproductive gene expression and 2) genetic cell ablation of floral structures through the use of cytotoxins or gene products whose overexpression is detrimental to cell function. We also discuss various cytotoxins and inhibitors thereof that could be used to reverse sterility and enable traditional breeding.