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

<p>Abstract</p> <p>Background</p> <p>With its genome sequence and other experimental attributes, <it>Populus trichocarpa </it>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.</p> <p>Results</p> <p>Using 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.</p> <p>Conclusion</p> <p>Our 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.</p

Validating internal controls for quantitative plant gene expression studies

BACKGROUND: Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. RESULTS: Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. CONCLUSION: Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments

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

<p>Abstract</p> <p>Background</p> <p>Auxin/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 <it>Populus </it>and performed comparative genomic analysis with <it>Arabidopsis </it>and rice.</p> <p>Results</p> <p>A total of 35 <it>Aux/IAA </it>and 39 <it>ARF </it>genes were identified in the <it>Populus </it>genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted between the two dicotyledonous plants. Activator <it>ARF </it>genes were found to be two fold-overrepresented in the <it>Populus </it>genome. <it>PoptrIAA </it>and <it>PoptrARF </it>gene families appear to have expanded due to high segmental and low tandem duplication events. Furthermore, expression studies showed that genes in the expanded <it>PoptrIAA3 </it>subgroup display differential expression.</p> <p>Conclusion</p> <p>The present study examines the extent of conservation and divergence in the structure and evolution of <it>Populus Aux/IAA </it>and <it>ARF </it>gene families with respect to <it>Arabidopsis </it>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.</p

Strategies for Engineering Reproductive Sterility in Plantation Forests

A considerable body of research exists concerning the development of technologies to engineer sterility in forest trees. The primary driver for this work has been to mitigate concerns arising from gene flow from commercial plantings of genetically engineered (GE) trees to non-GE plantations, or to wild or feral relatives. More recently, there has been interest in the use of sterility technologies as a means to mitigate the global environmental and socio-economic damage caused by the escape of non-native invasive tree species from planted forests. The current sophisticated understanding of the molecular processes underpinning sexual reproduction in angiosperms has facilitated the successful demonstration of a number of control strategies in hardwood tree species, particularly in the model hardwood tree Poplar. Despite gymnosperm softwood trees, such as pines, making up the majority of the global planted forest estate, only pollen sterility, via cell ablation, has been demonstrated in softwoods. Progress has been limited by the lack of an endogenous model system, long timescales required for testing, and key differences between softwood reproductive pathways and those of well characterized angiosperm model systems. The availability of comprehensive genome and transcriptome resources has allowed unprecedented insights into the reproductive processes of both hardwood and softwood tree species. This increased fundamental knowledge together with the implementation of new breeding technologies, such as gene editing, which potentially face a less oppressive regulatory regime, is making the implementation of engineered sterility into commercial forestry a realistic possibility

Exploring the Relationship Between Fidelity of Implementation and Academic Achievement in a Third-Grade Gifted Curriculum: A Mixed-Methods Study

This study used sequential mixed-methods analyses to investigate the effectiveness of a research-based language arts curriculum for gifted third graders. Using analytic induction, researchers found that teachers’ beliefs and expectations (time, sense of autonomy, expectations for students, professional expertise) influenced the degree to which they implemented the research intervention with fidelity to its design. Next, maximum variation sampling and quantitative analysis of student outcomes determined that postassessment achievement test scores are higher for students in classrooms with teachers who show high fidelity or adherence to the intervention

Contrasting patterns of evolution following whole genome versus tandem duplication events in Populus

Comparative analysis of multiple angiosperm genomes has implicated gene duplication in the expansion and diversification of many gene families. However, empirical data and theory suggest that whole-genome and small-scale duplication events differ with respect to the types of genes preserved as duplicate pairs. We compared gene duplicates resulting from a recent whole genome duplication to a set of tandemly duplicated genes in the model forest tree Populus trichocarpa. We used a combination of microarray expression analyses of a diverse set of tissues and functional annotation to assess factors related to the preservation of duplicate genes of both types. Whole genome duplicates are 700 bp longer and are expressed in 20% more tissues than tandem duplicates. Furthermore, certain functional categories are over-represented in each class of duplicates. In particular, disease resistance genes and receptor-like kinases commonly occur in tandem but are significantly under-retained following whole genome duplication, while whole genome duplicate pairs are enriched for members of signal transduction cascades and transcription factors. The shape of the distribution of expression divergence for duplicated pairs suggests that nearly half of the whole genome duplicates have diverged in expression by a random degeneration process. The remaining pairs have more conserved gene expression than expected by chance, consistent with a role for selection under the constraints of gene balance. We hypothesize that duplicate gene preservation in Populus is driven by a combination of subfunctionalization of duplicate pairs and purifying selection favoring retention of genes encoding proteins with large numbers of interactions

Efficiency of gene silencing in \u3ci\u3eArabidopsis\u3c/i\u3e: direct inverted repeats vs. transitive RNAi vectors

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1 , ETTIN and TTG1 ). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required