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Lignin is a highly abundant aromatic biopolymer deposited during the final stages of secondary cell wall formation in plants and it constitutes a substantial proportion of the dry weight of woody plant stems. Lignin contributes structural support to xylem cell walls and hydrophobisity to water-conducting vessels and forms a defence mechanism against pathogen invasion. Although being an essential part of normal plant cell development, lignin content and composition are targets for tree improvement, because residual lignin in paper pulp has negative effects on paper quality and lignin therefore has to be removed using treatments that are expensive and often detrimental to the environment. At present, little is known about the amount of allelic diversity in lignin biosynthetic genes and whether such diversity may be associated with variation in lignin content and composition. However, the identification of alleles associated with desirable lignin phenotypes is dependent on a detailed understanding of the molecular evolution and population genetics of these genes. This M. Sc. study was aimed at analysing nucleotide and allelic diversity in two lignin biosynthetic genes of Eucalyptus trees. Additionally, the study aimed to develop single nucleotide polymorphism (SNP) markers that could be used to assay allelic diversity for these genes in populations of two target species, E. grandis and E. smithii. Orthologues of the tobacco LIM-domain1 (NtLIM1) transcription factor gene involved in the regulation of lignin biosynthesis were isolated from E. grandis and E. smithii. Approximately 3 kb of genomic sequence including the promoter and full-length gene regions were isolated for the two orthologues, respectively labeled EgrLIM1 and EsLIM1. The predicted amino acid sequences of EgrLIM1 and EsLIM1 were 99.4% identical to each other and indicated that LIM1 is a small protein of only 188 residues in eucalypt trees and has a predicted molecular weight of 21.0 kDa. Quantitative, real-time RT-PCR analysis confirmed the expression of LIM1 in wood-forming tissues undergoing lignification. Ten putative cis-regulatory elements were observed in the promoter regions of EgrLIM1 and EsLIM1including a GA-dinucleotide microsatellite that appears to be specific to LIM1 promoters of Eucalyptus tree species. The full-length LIM1 gene sequences could subsequently be used in the assessment of nucleotide and allelic diversity, together with the full-length CAD2 sequences that were already available in the public domain. The level of nucleotide and allelic diversity and the distribution and decay of linkage disequilibrium (LD) were surveyed in 5 and 3 derived gene fragments of CAD2 and LIM1 obtained from 20 E. grandis and 20 E. smithii individuals. Each gene displayed a unique genetic diversity profile, but for the most part, nucleotide diversity () was estimated at approximately 0.0010 except for the E. grandis LIM1 gene where lower than 0.0040 was observed. Generally, except for the high amounts of LD observed in the CAD2 gene of E. grandis (> 2.5 kb), LD decayed within 500 bp. A large number (13 to 45) of SNP sites (defined as single nucleotide changes with minor allele frequencies of at least 0.10 in each species) were observed in each gene of each species. The high SNP density (ranging from one per 45 to one per 155 bp) observed in the two genes facilitated the efficient development of SNP markers to be used in future aspects of LD mapping, association genetics and marker-assisted breeding. The allele sequences obtained for the CAD2 and LIM1 genes were used as templates for the development of SNP marker panels (a series of six or seven SNP markers analysed together) for the analysis (tagging) of SNP haplotype diversity in species-wide reference populations (100 E. grandis and 137E. smithii individuals) of the two species. Each tag SNP was assayed using a single base extension assay and capillary gel electrophoresis. High polymorphism information content (average PIC of 0.836) was observed for the SNP marker panels. Four SNPs in the CAD2 and two in the LIM1 genes were found to be polymorphic in E. grandis and E. smithii (i.e. trans-specific SNPs), suggesting a possible ancestral origin for these polymorphisms. Assessment of candidate gene variation in the genomes of forest trees is of importance to ultimately be able to predict the amount and structure of nucleotide diversity available for the future design of SNP assays at the whole-genome level. Such assays will be useful to study differentiation among tree species and populations, to associate nucleotide polymorphisms with desirable phenotypes and to increase the efficiency of tree improvement approaches.
Eucalyptus trees are an important source of wood and fibre. The wood (secondary xylem) of this genus is widely used for pulp and papermaking. However, our understanding of the mechanisms which control the wood formation process (xylogenesis) in Eucalyptus and other woody species is far from complete. One reason is that xylogenesis is a very complex developmental process. The major components of wood are lignin and cellulose. Many genes involved in lignin and cellulose biosynthesis have been characterized. For example, Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two important lignin biosynthesis genes. Plant cellulose is synthesized by cellulose synthase enzymes with the aid of some other proteins, such as sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). Another factor which makes it difficult to analyze the function of Eucalyptus wood formation genes in vivo, is the long generation times of Eucalyptus trees and the difficulty to obtain transgenic Eucalyptus plants. Therefore, in this study, we investigated the use of Arabidopsis thaliana as a model system for functional analysis of wood formation genes. We transformed a lignin and a cellulose biosynthesis gene isolated from Eucalyptus to wild-type and mutant genetic backgrounds of Arabidopsis in order to test our ability to modify the cell wall chemistry of Arabidopsis thaliana using tree genes. The Eucalyptus CCR (EUCCR) gene was transformed into wild-type Arabidopsis (Col-0) and irregular xylem 4 (irx4) mutant plants, in which the homolog of EUCCR is mutated. A Eucalyptus cellulose synthase gene (EgCesA1) was also transformed into irregular xylem 1 (irx1) mutant plants, in which the homolog of EgCesA1 is mutated. Transgenics were only obtained from wild-type Col-0 transformed with EUCCR and from irx1 transformed with EgCesA1. We studied the cell wall chemistry of wild-type Arabidopsis plants overexpressing the Eucalyptus CCR gene. Chemical analysis of inflorescence stems revealed the modification of lignin and cellulose content in transgenic plants. Total lignin content was increased in T2 (5%) and T3 (12%) lines as revealed by micro-Klason lignin and thioglycolic acid quantification methods, respectively. High Pressure Liquid Chromatography (HPLC) analysis revealed that cellulose content was significantly decreased (10%) in T2 transgenic plants. This suggested the reallocation of carbon from cellulose to lignin as a result of overexpression of EUCCR in transgenic plants. Interestingly, thioacidolysis analysis revealed that in T2 plants, monomethoxylated guaiacyl (G) monomer was increased (16%) and bimethoxylated syringyl (S) monomer was decreased (21%). Therefore, the S/G lignin monomer ratio was significant decreased (32%). This implied that EUCCR might be specific to G monomer biosynthesis. The results described above confirmed that Arabidopsis thaliana can be used to model the function of wood formation genes isolated from Eucalyptus. Two novel full-length Eucalyptus sucrose synthase (SuSy) genes, EgSuSy1 and EgSuSy3, and one putative pseudogene, EgSuSy2, were also isolated in this study. Degenerate PCR was used to amplify Eucalyptus SuSy fragments from cDNA and genomic DNA. 3 RACE was used to amplify the 3 ends of two Eucalyptus SuSy genes. Genome walking was performed to obtain the 5 ends of EgSuSy1 and EgSuSy2 whereas 5 RACE technology was used to isolate the 5 end of EgSuSy3. However, 3 RACE analysis failed when we tried to identify the 3 end of EgSuSy2. Sequencing results from the genome walking product of EgSuSy2 further revealed that the start codon of this gene was missing, and we hypothesize that this is a psuedogene in the Eucalyptus genome. The EgSuSy1 cDNA was 2498 bp in length with an open reading frame of 2418 bp encoding 805 amino acids with a predicted molecular mass of 92.3 kDa. The 2528 bp full-length EgSuSy3 cDNA contained the same length of open reading frame as EgSuSy1, but encoded a polypeptide with a predicted molecular mass of 92.8 kDa. The results of quantitative real-time RT-PCR, phylogenetic analysis and gene structure of the two genes revealed that both genes might be involved in cellulose biosynthesis in primary and secondary cell walls of Eucalyptus. These two genes, EgSuSy1 and EgSuSy3, could therefore be useful targets for genetic engineering of wood properties in Eucalyptus.
Genetic engineering of superior wood properties and exploiting natural genetic variation found within commercially important trees, such as Eucalyptus spp., promise to increase cellulose biomass production. It is therefore essential to understand the molecular genetics of wood formation. Digital Gene Expression (DGE) profiling is adept in not only assessing the expression level of genes transcriptome-wide, but also in characterising alternative splice forms of transcripts and identifying novel transcripts. Tension wood is a specialised type of wood which functions in the response to mechanical stress in trees and is formed on the upper side of a branch or a bent stem. The characteristics of tension wood differ from normal wood by increased cellulose and xyloglucan content and decreased lignin and xylan content. During tension wood formation, transcriptome-wide changes in the expression of genes involved in secondary cell wall formation underlie changes in cell wall composition. Most notably is an increase in fasciclin-like arabinogalactan protein (FLA) and xyloglucan endotransglucosylase (XTH) and a decrease in lignin biosynthesis gene expression. Differential expression patterns are shown by cellulose synthase (CesA) genes, which have been found to be either up- or down-regulated during tension wood formation. No previous study has profiled gene expression during early as well as late tension wood formation. The aim of this M. Sc study was to identify genes that are differentially expressed during early tension wood induction and late tension wood formation in the immature xylem tissues of Eucalyptus grandis x Eucalyptus urophylla hybrid trees. DGE profiling is a transcriptome-wide expression profiling technique based on ultra-high throughput second generation DNA sequencing technology. The processing, analysis and interpretation of DGE data has not yet been standardised. To address this problem, a case study was performed of DGE data mapping to seven well characterised Eucalyptus grandis CesA (EgCesA) genes. The DGE data processing guidelines developed based on this case study produced EgCesA expression profiles in normal wood that were comparable to the profiles of these genes determined with other technologies. A possible alternative splice variant occurring during tension wood formation was identified for the secondary cell wall gene EgCesA3. However future work is needed for the validation of this observation. Early tension wood induction and late tension wood formation was investigated by sampling differentiating xylem from ramets of a Eucalyptus grandis x Eucalyptus urophylla clone induced to form tension wood for 6 hours, 24 hours, 1 week, 2 weeks and 6 months. Up to 2,654 transcripts were found to be significantly differentially expressed during tension wood formation. FLA transcripts were the highest expressed transcripts and were, along with XTH genes, highly up-regulated in early and late tension wood formation. Genes differentially regulated during early tension wood formation reflected a general stress response and hormone signalling pathways. Late tension wood formation was marked by the differential regulation of secondary cell wall biosynthetic genes, which reflected the chemical composition of tension wood. Two secondary cell wall CesA genes were significantly up-regulated, while genes involved in lignin and xylan biosynthesis were significantly down-regulated. Observations suggest that the eucalypt trees used in this study formed tension wood to stabilise the bent stem, while apical dominance was transferred to new side branches which showed signs of extra secondary growth.
The long-held tenets of the energy sector are being rewritten in the twenty-first century. The rise of unconventional oil and gas and of renewables is transforming our economies and improving our understanding of the distribution of the world's energy resources and their impacts. A complete knowledge of the dynamics underpinning energy markets is n
This book is a printed edition of the Special Issue "Forest Pathology and Plant Health" that was published in Forests
Lignin - Trends and Applications consists of 11 chapters related to the lignin structure, modification, depolymerization, degradation process, computational modeling, and applications. This is a useful book for readers from diverse areas, such as physics, chemistry, biology, materials science, and engineering. It is expected that this book may expand the reader's knowledge about this complex natural polymer.
Marking the change in focus of tree genomics from single species to comparative approaches, this book covers biological, genomic, and evolutionary aspects of angiosperm trees that provide information and perspectives to support researchers broadening the focus of their research. The diversity of angiosperm trees in morphology, anatomy, physiology and biochemistry has been described and cataloged by various scientific disciplines, but the molecular, genetic, and evolutionary mechanisms underlying this diversity have only recently been explored. Excitingly, advances in genomic and sequencing technologies are ushering a new era of research broadly termed comparative genomics, which simultaneously exploits and describes the evolutionary origins and genetic regulation of traits of interest. Within tree genomics, this research is already underway, as the number of complete genome sequences available for angiosperm trees is increasing at an impressive pace and the number of species for which RNAseq data are available is rapidly expanding. Because they are extensively covered by other literature and are rapidly changing, technical and computational approaches—such as the latest sequencing technologies—are not a main focus of this book. Instead, this comprehensive volume provides a valuable, broader view of tree genomics whose relevance will outlive the particulars of current-day technical approaches. The first section of the book discusses background on the evolution and diversification of angiosperm trees, as well as offers description of the salient features and diversity of the unique physiology and wood anatomy of angiosperm trees. The second section explores the two most advanced model angiosperm tree species (poplars and eucalypts) as well as species that are soon to emerge as new models. The third section describes the structural features and evolutionary histories of angiosperm tree genomes, followed by a fourth section focusing on the genomics of traits of biological, ecological, and economic interest. In summary, this book is a timely and well-referenced foundational resource for the forest tree community looking to embrace comparative approaches for the study of angiosperm trees.
This book describes the scientific principles that are used throughout the world to ensure the rapid, healthy growth of forest plantations. As the population of the world increases so does the amount of wood people use. Large areas of natural forests are being cleared every year and converted to other uses. Almost as large an area of plantation forests is being established annually to replace those lost natural forests. Eventually, plantations will produce a large proportion of the wood used around the world for firewood, building, the manufacture of paper and bioenergy. Forest plantations can also provide various environmental benefits including carbon storage, rehabilitation of degraded land, serving as disposal sites for various forms of industrial or agricultural waste and enhancing biodiversity in regions that have been largely cleared for agriculture. Whatever their motivation, plantation forest growers want their plantations to be healthy and grow rapidly to achieve their purpose as soon as possible. This book discusses how this is done. It is written for a worldwide audience, from forestry professionals and scientists through to small plantation growers, and describes how plantations may be grown responsibly and profitably.
2. IMPORTANCE OF NITROGEN METABOLISM 2. 1. Range of naturally occurring nitrogenous components in forest trees 2. 2. Gene expression and mapping 2. 3. Metabolic changes in organized and unorganized systems 2. 4. Nitrogen and nutrition 2. 5. Aspects of intermediary nitrogen metabolism 3. NITROGEN METABOLISM IN GROWTH AND DEVELOPMENT 3. 1. Precultural factors 3. 2. Callus formation 3. 3. Cell suspensions 3. 3. 1. Conifers 3. 3. 2. Acer 3. 4. Morphogenesis 3. 4. 1. Nitrogen metabolism of natural embryos 3. 4. 2. Somatic embryogenesis 3. 4. 2. 1. Sweetgum (Liquidambar styraciflua) 3. 4. 2. 2. Douglar-fir and loblolly pine 3. 4. 3. Organogenesis 4. OUTLOOK 11. CARBOHYDRATE UTILIZATION AND METABOLISM - T. A. Thorpe 325 1. INTRODUCTION 2. NUTRITIONAL ASPECTS 3. CARBOHYDRATE UPTAKE 4. CARBOHYDRATE METABOLISM 4. 1. Sucrose degradation 4. 2. Metabolism of other carbon sources 4. 3. Hexose mobilization and metabolism 4. 3. 1. Cell cycle studies 4. 3. 2. Growth studies 4. 3. 3. Organized development 4. 4. Cell wall biogenesis 4. 4. 1. Primary cell walls 4. 4. 2. Cell wall turnover 4. 4. 3. Secondary cell walls 4. 5. Carbon skeleton utilization 5. OSMOTIC ROLE 6. CONCLUDING THOUGHTS 369 12. THE USE OF IN VITRO TECHNIQUES FOR GENETIC MODIFICATIO~FOREST TREES - E. G. Kirby 1. INTRODUCTION 2. IN VITRO SELECTION 2. 1. Natural variation 2. 2. Induction of variation 2. 3. Selection techniques 2. 4. Plant regeneration 2 . • 5. Applications x 3. SOMATIC HYBRIDIZATION 3. 1.