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Matrix attachment regions (MARs) are thought to buffer transgenes from the influence of surrounding chromosomal sequences, and therefore to reduce transgene silencing and variation in expression. The statistical properties of more than 400 independent transgenic events produced in Populus, with and without flanking MAR elements from the tobacco root gene RB7, were analysed. The expression of two reporter genes in two poplar clones during three phases of vegetative growth, and the association of T-DNA characteristics with expression, was examined. It was found that MARs did not show a consistent effect on transgene expression levels; they had no effect on the green fluorescent protein ( GFP) reporter gene, but reduced expression in the Basta resistance ( BAR) reporter gene by 23%. The presence of MARs reduced expression variability within transformant populations, apparently by reducing the number of silenced or weakly expressing events. Transgene expression was highly stable over vegetative growth cycles that spanned 3 years of growth in the glasshouse and field, but MARs showed no association with the strength of correlations in expression over the years. Nonetheless, MARs increased the correlation in expression between a p35S:: GFP and p rbcS:: BAR transgene linked on the same vector, but the effect was small and varied between the years. The presence of MARs had no effect on the transgene copy number, but was positively associated with T-DNA truncations, as well as with the formation of direct over inverted repeats at the same chromosomal locus. [ABSTRACT FROM AUTHOR]

The recent release of the Populus trichocarpa genome sequence will dramatically enhance the efficiency of functional and comparative genomics research in trees . This provides researchers studying various developmental processes related to the perennial and tree life strategies with a completely new set of tools. Intimately associated with the life strategy of trees are their abilities to maintain juvenile or nonflowering phases for years to decades, and once reproductively competent, to alternate between the production of vegetative and reproductive shoots. Most of what we know about the regulation of the floral transition comes from research on Arabidopsis thaliana, a small, herbaceous, rapid-cycling, annual plant. In this review, we discuss the similarities and differences between Arabidopsis and tree flowering, and how recent findings in Arabidopsis, coupled to comparative and functional genomics in poplars, will help answer the question of how tree maturation and floral initiation is regulated. New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01165.x © New Phytologist (2004) [ABSTRACT FROM AUTHOR]

In addition to their value for wood products, members of the genus Populus (poplars) provide a range of ecological services, including carbon sequestration, bioremediation, nutrient cycling, biofiltration and diverse habitats. They are also widely used model organisms for tree molecular biology and biotechnology. The sequencing of the poplar genome to an approximately 6× depth adds to a long list of important attributes for research. These include facile transformation, vegetative propagation, rapid growth, modest genome size and extensive expressed sequence tags. Here, we discuss how the genome sequence and transformability of poplar, together with its high levels of genetic and ecological diversity, are enabling new insights into the genetic programs controlling ontogeny, ecological adaptation and environmental physiology of trees. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Summary: The role of the floral homeotic gene AGAMOUS (AG) and its close homologues in development of anemophilous, unisexual catkins has not previously been studied.We transformed two RNA interference (RNAi) constructs, PTG and its matrix‐attachment‐region flanked version MPG, into the early‐flowering female poplar clone 6K10 (Populus alba) to suppress the expression of its two duplicate AG orthologues.By early 2018, six out of 22 flowering PTG events and 11 out of 12 flowering MPG events showed modified floral phenotypes in a field trial in Oregon, USA. Flowers in catkins from modified events had 'carpel‐inside‐carpel' phenotypes. Complete disruption of seed production was observed in seven events, and sterile anther‐like organs in 10 events. Events with strong co‐suppression of both the two AG and two SEEDSTICK (STK) paralogues lacked both seeds and associated seed hairs. Alterations in all of the modified floral phenotypes were stable over 4 yr of study. Trees from floral‐modified events did not differ significantly (P < 0.05) from nonmodified transgenic or nontransgenic controls in biomass growth or leaf morphology.AG and STK genes show strong conservation of gene function during poplar catkin development and are promising targets for genetic containment of exotic or genetically engineered trees. [ABSTRACT FROM AUTHOR]

Vegetative and reproductive phase change and phenology are economically and ecologically important traits. Trees typically require several years of growth before flowering and, once mature, seasonal control of the transition to flowering and flower development is necessary to maintain vegetative meristems and for reproductive success. Members of two related gene subfamilies, FLOWERING LOCUST (FT) and TERMINAL FLOWER1 (TFL1)/ CENTRORADIALIS (CEN)/ BROTHER OF FT AND TFL1 (BFT), have antagonistic roles in flowering in diverse species and roles in vegetative phenology in trees, but many details of their functions in trees have yet to be resolved. Here, we used CRISPR/Cas9 to generate single and double mutants involving the five Populus FT and TFL1/CEN/BFT genes. The ft1 mutants exhibited wild-type-like phenotypes in long days and short days, but after chilling, to release dormancy, they showed delayed bud flush and GA3 could compensate for the ft1 mutation. After rooting and generating some phytomers in tissue culture, both cen1 and cen1ft1 mutants produced terminal as well as axillary flowers, indicating that the cen1 flowering phenotype is independent of FT1. The CEN1 showed distinct circannual expression patterns in vegetative and reproductive tissues and comparison with the expression patterns of FT1 and FT2 suggests that the relative levels of CEN1 compared with FT1 and FT2 regulate multiple phases of vegetative and reproductive seasonal development. [ABSTRACT FROM AUTHOR]

We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment ( 35SBP), 35SBP fused to the TMV omega element ( 35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees. [ABSTRACT FROM AUTHOR]

Several xylem-associated regulatory genes have been identified that control processes associated with wood formation in poplar. Prominent among these are the NAC domain transcription factors (NACs). Here, the putative involvement of Populus NAC154 , a co-ortholog of the Arabidopsis gene SND2 , was evaluated as a regulator of “secondary” biosynthetic processes in stem internode tissues by interrogating aqueous methanolic extracts from control and transgenic trees. Comprehensive untargeted metabolite profiling was accomplished with a liquid chromatography–mass spectrometry platform that utilized two different chromatographic supports (HILIC and reversed phase) and both positive and negative ionization modes. Evaluation of current and previous year tissues provided datasets for assessing the effects of NAC154 overexpression in wood maturation processes. Phenolic glycoside levels as well as those of oligolignols, sucrose and arginine were modulated with phenotypic and chemotypic traits exhibiting similar trends. Specifically, increased levels of arginine in the NAC154 overexpressing tissues supports a role for the transcription factor in senescence/dormancy-associated processes. [ABSTRACT FROM AUTHOR]