Genetic engineering of reproductive sterility in forest trees

Containment of transgenes inserted into genetically engineered forest trees will probably be necessary before most commercial uses are possible. This is a consequence of (1) high rates of gene dispersal by pollen and seed, (2) proximity of engineered trees in plantations to natural or feral stands of interfertile species, and (3) potentially undesirable ecological effects if certain transgenes become widely dispersed. In addition to gene containment, engineering of complete or male sterility may stimulate faster wood production, reduce production of allergenic pollen, and facilitate hybrid breeding. We review the regulatory and ecological rationale for engineering sterility, potentially useful floral genes, strategies for creating sterility-causing transgenes, and problems peculiar to engineering sterility in forest trees. Each of the two primary options — ablating floral tissuesvia floral promoter-cytotoxin fusions, and disrupting expression of essential floral genes by various methods of gene suppression — has advantages and disadvantages. Because promoters from structural and enzymatic floral-specific genes often work well in heterologous species, ablation methods based on these genes probably will not require cloning of homologs from angiosperm trees. Methods that inhibit gene expression will require cloning of tree genes and may be more prone to epigenetic variability, but should allow assay of transgene efficacy in seedlings. Practical constraints include the requirement for vegetative propagation if complete sterility is engineered and the need for highly stable forms of sterility in long-lived trees. The latter may require suppression of more than one floral gene or employment of more than one genetic mechanism for sterility.     

死皮对橡胶树树皮线粒体超微结构及活性氧代谢的影响

为了解析橡胶树（Hevea brasiliensis）死皮的发生机制,有效进行死皮防治,以健康、轻度死皮、重度死皮橡胶树树皮为材料,研究死皮发生过程中树皮线粒体超微结构变化规律及活性氧（ROS）相关基因的表达模式变化。结果表明：死皮树线粒体超微结构发生不规则形变,膜内基质溶解,嵴消失,内腔空泡化等,且严重程度与死皮严重程度成正比。荧光定量PCR结果表明,过氧化物酶基因HbPOD2和HbPOD3在死皮树中的表达量高于健康树,可作为监测割胶强度、刺激强度和死皮发生的“标志”基因。植物细胞重要ROS清除酶过氧化氢酶基因HbCAT在死皮树中也下调表达,预示ROS产生与清除之间的平衡是影响橡胶树死皮发生的关键因素。橡胶树中重要抗氧化代谢物基因表达结果表明,HbGST1、HbGST2和HbPPO在死皮树中的表达量均高于健康树,可能与死皮发生过程胶乳原位凝固相关。本研究通过揭示死皮发生过程树皮超微结构和ROS相关基因表达模式变化,为阐明橡胶树死皮发生机制提供新观点,同时为进一步开发监测割胶强度、刺激强度和死皮发生的基因“标志”提供理论基础。     

Trees within trees: genes and species, molecules and morphology

The construction and interpretation of gene trees is fundamental in molecular systematics. If the gene is defined in a historical (coalescent) sense, there can be multiple gene trees within the single contiguous set of nucleotides, and attempts to construct a single tree for such a sequence must deal with homoplasy created by conflict among divergent histories. On a larger scale, incongruence is expected among gene tree topologies at different loci of individuals within sexually reproducing species, and it has been suggested that this discordance can be used to delimit species. A practical concern for such topological methods is that polymorphisms may be maintained through numerous cladogenic events; this polymorphism problem is less of a concern for nontopological approaches to species delimitation using molecular data. Although a central theoretical concern in molecular systematics is discordance between a given gene tree and the true "species tree," the primary empirical problem faced in reconstructing taxic phylogeny is incongruence among the trees inferred from different sequences. Linkage relationships limit character independence and thus have important implications for handling multiple data sets in phylogenetic analysis, particularly at the species level, where incongruence among different historically associated loci is expected. Gene trees can also be reconstructed for loci that influence phenotypic characters, but there is at best a tenuous relationship between phenotypic homoplasy and homoplasy in such gene trees. Nevertheless, expression patterns and orthology relationships of genes involved in the expression of phenotypes can in theory provide criteria for homology assessment of morphological characters.     

A Versatile Agrobacterium-Mediated Transient Gene Expression System for Herbaceous Plants and Trees

Plant transient expression is a powerful method used widely for the functional characterization of genes and protein production. In comparison with stable transformation, it has the advantages of being simple, quick, economical, and effective. In the present study, we developed a novel transient gene expression system based on Agrobacterium-mediated transformation. This system is simple and convenient and allows for high transient expression levels. Hyperosmotic pretreatment of plants significantly improved the transient expression in this system. Furthermore, other factors, including acetosyringone concentration, cocultivation time, and Agrobacterium cell density, significantly influenced transient expression efficiency. The results showed that this method is suitable for use with herbaceous plants (such as tobacco and Arabidopsis) and trees (such as birch, poplar, tamarisk, cork, willow, and aralia), suggesting that it may be applied widely in plant transient expression studies.     

Gene expression profiling in the stem of young maritime pine trees: detection of ammonium stress-responsive genes in the apex

The shoots of young conifer trees represent an interesting model to study the development and growth of conifers from meristematic cells in the shoot apex to differentiated tissues at the shoot base. In this work, microarray analysis was used to monitor contrasting patterns of gene expression between the apex and the base of maritime pine shoots. A group of differentially expressed genes were selected and validated by examining their relative expression levels in different sections along the stem, from the top to the bottom. After validation of the microarray data, additional gene expression analyses were also performed in the shoots of young maritime pine trees exposed to different levels of ammonium nutrition. Our results show that the apex of maritime pine trees is extremely sensitive to conditions of ammonium excess or deficiency, as revealed by the observed changes in the expression of stress-responsive genes. This new knowledge may be used to precocious detection of early symptoms of nitrogen nutritional stresses, thereby increasing survival and growth rates of young trees in managed forests.     

Control of pollen-mediated gene flow in transgenic trees

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

AffyTrees: facilitating comparative analysis of Affymetrix plant microarray chips

Microarrays measure the expression of large numbers of genes simultaneously and can be used to delve into interaction networks involving many genes at a time. However, it is often difficult to decide to what extent knowledge about the expression of genes gleaned in one model organism can be transferred to other species. This can be examined either by measuring the expression of genes of interest under comparable experimental conditions in other species, or by gathering the necessary data from comparable microarray experiments. However, it is essential to know which genes to compare between the organisms. To facilitate comparison of expression data across different species, we have implemented a Web-based software tool that provides information about sequence orthologs across a range of Affymetrix microarray chips. AffyTrees provides a quick and easy way of assigning which probe sets on different Affymetrix chips measure the expression of orthologous genes. Even in cases where gene or genome duplications have complicated the assignment, groups of comparable probe sets can be identified. The phylogenetic trees provide a resource that can be used to improve sequence annotation and detect biases in the sequence complement of Affymetrix chips. Being able to identify sequence orthologs and recognize biases in the sequence complement of chips is necessary for reliable cross-species microarray comparison. As the amount of work required to generate a single phylogeny in a nonautomated manner is considerable, AffyTrees can greatly reduce the workload for scientists interested in large-scale cross-species comparisons.     

Mechanic defences and reproduction in desert trees under different habitat management

Herbivory can have deleterious effects on plant reproduction by limitation of photosynthates that are either lost by consumption, used to re-growth or invested in defences. In addition, herbivores can also exert direct impact on plant reproduction by consuming flowers. Spine length can act as an inducible defence in plants, because it tends to increase with increasing herbivore pressure. I hypothesized that almost 40 years of different habitat management (cattle exclusion within a protected area vs. cattle grazing in adjacent areas) could affect inflorescence abundance, spine length and fruit production in Prosopis flexuosa trees. The study area was located in the Central Monte desert of Argentina. I estimated differences in spine length, number of inflorescences and fruit production in trees inside the Man and Biosphere Reserve of Ñacuñán and in the adjacent cattle ranches surrounding the protected area. Inflorescence abundance in the tree canopy was similar in cattle grazed and protected sites, but the presence of large herbivores was associated with limited fruit production. Spines were 37% shorter and fruit production three times higher in trees inside the reserve than in trees in cattle ranches. A negative exponential model was used to describe the relationship between reproduction and spine length in trees. The results indicate that after almost four decades of cattle exclusion, trees inside the protected area show higher reproduction and shorter spines than cattle-browsed trees in surrounding areas. The negative association between defence and reproduction may be due to competition for photosynthates. The present results could be useful and relevant in conservation because they provide evidence on how anthropogenic habitat use can affect plant phenotypes and fitness, which in turn can affect the long-term ecological and evolutionary dynamics of plant populations.     

Cellulose factories: advancing bioenergy production from forest trees

Fast-growing, short-rotation forest trees, such as Populus and Eucalyptus, produce large amounts of cellulose-rich biomass that could be utilized for bioenergy and biopolymer production. Major obstacles need to be overcome before the deployment of these genera as energy crops, including the effective removal of lignin and the subsequent liberation of carbohydrate constituents from wood cell walls. However, significant opportunities exist to both select for and engineer the structure and interaction of cell wall biopolymers, which could afford a means to improve processing and product development. The molecular underpinnings and regulation of cell wall carbohydrate biosynthesis are rapidly being elucidated, and are providing tools to strategically develop and guide the targeted modification required to adapt forest trees for the emerging bioeconomy. Much insight has already been gained from the perturbation of individual genes and pathways, but it is not known to what extent the natural variation in the sequence and expression of these same genes underlies the inherent variation in wood properties of field-grown trees. The integration of data from next-generation genomic technologies applied in natural and experimental populations will enable a systems genetics approach to study cell wall carbohydrate production in trees, and should advance the development of future woody bioenergy and biopolymer crops.     

Assessing the performance of single-copy genes for recovering robust phylogenies

Phylogenies involving nonmodel species are based on a few genes, mostly chosen following historical or practical criteria. Because gene trees are sometimes incongruent with species trees, the resulting phylogenies may not accurately reflect the evolutionary relationships among species. The increase in availability of genome sequences now provides large numbers of genes that could be used for building phylogenies. However, for practical reasons only a few genes can be sequenced for a wide range of species. Here we asked whether we can identify a few genes, among the single-copy genes common to most fungal genomes, that are sufficient for recovering accurate and well-supported phylogenies. Fungi represent a model group for phylogenomics because many complete fungal genomes are available. An automated procedure was developed to extract single-copy orthologous genes from complete fungal genomes using a Markov Clustering Algorithm (Tribe-MCL). Using 21 complete, publicly available fungal genomes with reliable protein predictions, 246 single-copy orthologous gene clusters were identified. We inferred the maximum likelihood trees using the individual orthologous sequences and constructed a reference tree from concatenated protein alignments. The topologies of the individual gene trees were compared to that of the reference tree using three different methods. The performance of individual genes in recovering the reference tree was highly variable. Gene size and the number of variable sites were highly correlated and significantly affected the performance of the genes, but the average substitution rate did not. Two genes recovered exactly the same topology as the reference tree, and when concatenated provided high bootstrap values. The genes typically used for fungal phylogenies did not perform well, which suggests that current fungal phylogenies based on these genes may not accurately reflect the evolutionary relationships among species. Analyses on subsets of species showed that the phylogenetic performance did not seem to depend strongly on the sample. We expect that the best-performing genes identified here will be very useful for phylogenetic studies of fungi, at least at a large taxonomic scale. Furthermore, we compare the method developed here for finding genes for building robust phylogenies with previous ones and we advocate that our method could be applied to other groups of organisms when more complete genomes are available.     

NMR-based metabolomic analysis of the seasonal and regional variability of phytochemical compounds in Curtisia dentata stem bark

Traditional medicine markets are provided with medicinal plant material throughout the year, however, internal (e.g. plant age, genetic variability and differential expression of genes) and external factors (e.g. water and nutrient availability, rainfall, photoperiod and herbivory), affect secondary metabolite production in plants. In this study, seasonal variability in metabolite production in Curtisia dentata trees from two geographically separated regions in South Africa are compared. NMR analysis of C. dentata stem bark samples yielded spectral data which were processed in MestReNova to perform multivariate data analysis using Soft Independent Modeling of Class Analogy (SIMCA) software. This study shows that there are not only seasonal, regional and yearly differences in secondary metabolite production in C. dentata trees, but that production patterns of hydrophilic and lipophilic chemical compounds in individual trees also vary. Sucrose, isoeugenol and betulinic acid have been used in a targeted analysis to show the variation in individual compounds in the individual trees as a response to seasonal and geographical differences. Therefore, the season and year, as well as the region, harvesting site and specific trees from which plant material is collected affect the concentrations of chemical compounds extracted from C. dentata stem bark for the preparation of remedies.     

Growth and defence in young pine and spruce and the expression of resistance to a stem-feeding weevil

Defence in young trees has been much less studied than defence in older ones. In conifers, resin within ducts in bark is an important quantitative defence, but its expression in young trees may be influenced by developmental or physical constraints on the absolute size of the resin ducts as well as by differential allocation of resources to growth and resin synthesis. To examine these relationships, we used nitrogen fertilisation of 1- and 2-year-old pine and spruce to produce trees of different sizes and measured the effect on the number and size of resin ducts and the amount of resin they contained. All of these variables tended to increase with stem diameter, indicating a positive relationship between resin-based defence and growth of 1- and 2-year-old trees. In pine, however, the mass of resin flowing from severed ducts was much lower relative to duct area in 1- than in 2-year-old trees, suggesting that the older trees allocated a higher proportion of the carbon budget to resin synthesis. Resin-based defence in 1-year-old pines appears to be both positively related to growth and resource limited. In spruce, resin production was generally lower, and age-related differences were not observed, suggesting that resin-based defence is less important in this species. Bio-assays of 2-year-old trees with the pine weevil, Hylobius abietis, emphasised the importance of resin as a defence against this bark feeding insect. Nitrogen fertilisation had a limited influence on resistance expression. One-year-old trees remained susceptible because of their small size, low resin production and limited response to fertilisation. The strong growth response of 2-year-old trees to fertilisation increased resin-based defence, but most spruce trees remained susceptible, while most pines were resistant at all levels of fertilisation.     

Genomics to tree breeding and forest health

Genomic discovery in forest trees follows paradigms from both agricultural crop and livestock improvement and human medicine. Forest trees in a domesticated state can be improved using genomic-based breeding technologies, whereas the health of trees in a natural and undomesticated state might be managed using those same technologies. These applications begin by first dissecting complex traits in trees to their individual gene components and for that the association genetics approach is quite powerful in trees. This is true for several reasons including large, random mating, and unstructured populations and the rapid decay of linkage disequilibrium in many tree species. Once marker by trait associations are discovered, they can be used in genomic-based breeding and forest health diagnostics. Initial studies in trees have found ample nucleotide diversity in candidate genes to perform association studies and single nucleotide polymorphisms have been associated with economic and adaptive traits. Population genetic neutrality tests have been applied to identify genes probably under natural selection and thus make good candidates for developing forest health diagnostic tools.     

Restoration of threatened species: a noble cause for transgenic trees

Some of the first applications of transgenic trees in North America may be for the conservation or restoration of threatened forest trees that have been devastated by fungal pathogens or insect pests. In some cases, where resistance has yet to be found in the natural population of a tree species, incorporating genes from other organisms may offer the only hope for restoration. In others, transgenics may play a role as part of an integrated approach, along with conventional breeding or biocontrol agents. American chestnut (Castanea dentata) was wiped out as a canopy species by a fungal disease accidentally introduced into the United States around 1900. Similarly, American elm (Ulmus americana) virtually disappeared as a favored street tree from Northeastern U.S. cities after the introduction of the Dutch elm disease fungus in the 1940s. In both cases, progress has been made toward restoration via conventional techniques such as selection and propagation of tolerant cultivars (American elm) or breeding with a related resistant species (American chestnut). Recently, progress has also been made with development of systems for engineering antifungal candidate genes into these “heritage trees.” An Agrobacterium-leaf disk system has been used to produce transgenic American elm trees engineered with an antimicrobial peptide gene that may enhance resistance to Dutch elm disease. Two gene transfer systems have been developed for American chestnut using Agrobacterium-mediated transformation of embryogenic cultures, setting the stage for the first tests of potential antifungal genes for their ability to confer resistance to the chestnut blight fungus. Despite the promise of transgenic approaches for restoration of these heritage trees, a number of technical, environmental, economic, and ethical questions remain to be addressed before such trees can be deployed, and the debate around these questions may be quite different from that associated with transgenic trees developed for other purposes.