The fasciclin-like arabinogalactan protein family of Eucalyptus grandis contains members that impact wood biology and biomechanics.

Fasciclin-like arabinogalactan protein (FLA) families have been identified and characterised in key plant species, with some members exhibiting functional specialization. Here we identify the FLA family of Eucalyptus grandis, and investigate the roles of three single-FAS domain FLAs, with particular focus on secondary cell-wall formation and wood properties. We use various in-silico approaches to identify and characterise E. grandis genome FLAs, and perform phylogenetic comparisons with other species. For three key FLAs, we perform functional testing including promoter-reporter and overexpression transgenic approaches using eucalypts, poplar and tobacco. Of the 18 eucalypt FLAs identified, several were specifically and highly expressed in stems. The specificity to stem xylem vessel and fibre development was demonstrated with EniFLA1promoter:GUS studies in several species. Testing of select eucalypt FLAs resulted in altered wood development and properties, for example 35S:EgrFLA2 led to a 3 degree reduction in cellulose microfibril angle in eucalypt xylem fibres, and 35S:EgrFLA3 to a reduction in tobacco stem flexural strength. These results indicate that the eucalypt FLA family contains diverse members, and particular members with single FAS domains that are functionally specialized for secondary cell wall growth and properties.

Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection.

BACKGROUND: Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. RESULTS: We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with differential gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the 287 positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. CONCLUSIONS: Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study.

Identification of putative candidate genes for juvenile wood density in Pinus radiata.

Wood formation is a complex developmental process driven by the annual activity of the vascular cambium. Conifers usually produce juvenile wood at young ages followed by mature wood for the rest of their lifetime. Juvenile wood exhibits poorer wood quality (i.e., lower density) compared with mature wood and can account for up to 50% of short-rotation harvested logs, thus representing a major challenge for commercial forestry globally. Wood density is an important quality trait for many timber-related products. Understanding the molecular mechanisms involved in the regulation of juvenile wood density is critical for the improvement of juvenile wood quality via marker-aided selection. A previous study has identified several candidate genes affecting mature wood density in Picea sitchensis (Bong.) Carr.; however, genes associated with juvenile wood density in conifers remain poorly characterized. Here, cDNA microarrays containing 3320 xylem unigenes were used to investigate genes differentially transcribed in juvenile wood with high (HD) and low density (LD) in Pinus radiata D.Don. In total, 814 xylem unigenes with differential transcription were identified in at least one of two microarray experiments and 73 genes (45 for HD, 28 for LD) were identified in both experiments, thus representing putative candidate genes for juvenile wood density. Interestingly, cellulose synthases (PrCesA3, PrCesA11) and sucrose synthase (SuSy), which are involved in secondary cell wall formation, had stronger transcription in juvenile wood with HD, while genes functioning in primary wall formation (pectin synthesis, cell expansion and other modifications) were more transcribed in LD wood. Cell wall genes encoding monolignol biosynthesis enzymes, arabinogalactan proteins, actins and tubulins were differentially transcribed in either HD or LD juvenile wood; however, the latter had exclusively greater transcription of genes involved in monolignol polymerization (laccase and peroxidase). The identified candidate genes also included many non-cell-wall genes (transcription factors, environmental-responsive genes, hormone signalling, etc.) and genes with unknown functions, suggesting complex gene pathways in the regulation of juvenile wood density. Interestingly, 19 out of 73 candidate genes for wood density were among the 108 candidate genes previously identified for microfibril angle, and 16 genes appeared to influence both traits in a synergistic manner for wood stiffness.

Association genetics in Corymbia citriodora subsp. variegata identifies single nucleotide polymorphisms affecting wood growth and cellulosic pulp yield.

Wood is an important biological resource which contributes to nutrient and hydrology cycles through ecosystems, and provides structural support at the plant level. Thousands of genes are involved in wood development, yet their effects on phenotype are not well understood. We have exploited the low genomic linkage disequilibrium (LD) and abundant phenotypic variation of forest trees to explore allelic diversity underlying wood traits in an association study. Candidate gene allelic diversity was modelled against quantitative variation to identify SNPs influencing wood properties, growth and disease resistance across three populations of Corymbia citriodora subsp. variegata, a forest tree of eastern Australia. Nine single nucleotide polymorphism (SNP) associations from six genes were identified in a discovery population (833 individuals). Associations were subsequently tested in two smaller populations (130-160 individuals), 'validating' our findings in three cases for actin 7 (ACT7) and COP1 interacting protein 7 (CIP7). The results imply a functional role for these genes in mediating wood chemical composition and growth, respectively. A flip in the effect of ACT7 on pulp yield between populations suggests gene by environment interactions are at play. Existing evidence of gene function lends strength to the observed associations, and in the case of CIP7 supports a role in cortical photosynthesis.

Transcriptome profiling of Pinus radiata juvenile wood with contrasting stiffness identifies putative candidate genes involved in microfibril orientation and cell wall mechanics.

BACKGROUND: The mechanical properties of wood are largely determined by the orientation of cellulose microfibrils in secondary cell walls. Several genes and their allelic variants have previously been found to affect microfibril angle (MFA) and wood stiffness; however, the molecular mechanisms controlling microfibril orientation and mechanical strength are largely uncharacterised. In the present study, cDNA microarrays were used to compare gene expression in developing xylem with contrasting stiffness and MFA in juvenile Pinus radiata trees in order to gain further insights into the molecular mechanisms underlying microfibril orientation and cell wall mechanics. RESULTS: Juvenile radiata pine trees with higher stiffness (HS) had lower MFA in the earlywood and latewood of each ring compared to low stiffness (LS) trees. Approximately 3.4 to 14.5% out of 3, 320 xylem unigenes on cDNA microarrays were differentially regulated in juvenile wood with contrasting stiffness and MFA. Greater variation in MFA and stiffness was observed in earlywood compared to latewood, suggesting earlywood contributes most to differences in stiffness; however, 3-4 times more genes were differentially regulated in latewood than in earlywood. A total of 108 xylem unigenes were differentially regulated in juvenile wood with HS and LS in at least two seasons, including 43 unigenes with unknown functions. Many genes involved in cytoskeleton development and secondary wall formation (cellulose and lignin biosynthesis) were preferentially transcribed in wood with HS and low MFA. In contrast, several genes involved in cell division and primary wall synthesis were more abundantly transcribed in LS wood with high MFA. CONCLUSIONS: Microarray expression profiles in Pinus radiata juvenile wood with contrasting stiffness has shed more light on the transcriptional control of microfibril orientation and the mechanical properties of wood. The identified candidate genes provide an invaluable resource for further gene function and association genetics studies aimed at deepening our understanding of cell wall biomechanics with a view to improving the mechanical properties of wood.

Transcriptome profiling of wood maturation in Pinus radiata identifies differentially expressed genes with implications in juvenile and mature wood variation.

Trees usually produce wood with distinct properties at different developmental stages. Juvenile wood (JW) formed in younger trees has poorer properties than mature wood (MW) formed in later years. We used cDNA microarrays to compare the xylem transcriptomes of Pinus radiata trees synthesising JW and MW respectively. JW and MW formation involved considerable transcriptome flux, with the greatest change occurring in spring (19.3%) compared to autumn (9.2%). We identified 147 candidate genes in response to wood maturation, of which 34.0% were implicated in cell wall formation and 19.7% were functional unknowns. Majority of the candidate genes were identified from MW and JW in spring (127) with fewer genes in autumn (30). Many genes involved in secondary wall formation (cellulose synthesis and lignification) and cytoskeleton development were more transcribed in MW in spring, while in JW in spring most genes functioned in primary wall synthesis, signalling and stress responses. Some identified genes may play roles in sensing environmental signals during the transition from JW to MW and in controlling distinct tracheids and wood traits between JW and MW.

Comparative genomics reveals conservative evolution of the xylem transcriptome in vascular plants.

BACKGROUND: Wood is a valuable natural resource and a major carbon sink. Wood formation is an important developmental process in vascular plants which played a crucial role in plant evolution. Although genes involved in xylem formation have been investigated, the molecular mechanisms of xylem evolution are not well understood. We use comparative genomics to examine evolution of the xylem transcriptome to gain insights into xylem evolution. RESULTS: The xylem transcriptome is highly conserved in conifers, but considerably divergent in angiosperms. The functional domains of genes in the xylem transcriptome are moderately to highly conserved in vascular plants, suggesting the existence of a common ancestral xylem transcriptome. Compared to the total transcriptome derived from a range of tissues, the xylem transcriptome is relatively conserved in vascular plants. Of the xylem transcriptome, cell wall genes, ancestral xylem genes, known proteins and transcription factors are relatively more conserved in vascular plants. A total of 527 putative xylem orthologs were identified, which are unevenly distributed across the Arabidopsis chromosomes with eight hot spots observed. Phylogenetic analysis revealed that evolution of the xylem transcriptome has paralleled plant evolution. We also identified 274 conifer-specific xylem unigenes, all of which are of unknown function. These xylem orthologs and conifer-specific unigenes are likely to have played a crucial role in xylem evolution. CONCLUSIONS: Conifers have highly conserved xylem transcriptomes, while angiosperm xylem transcriptomes are relatively diversified. Vascular plants share a common ancestral xylem transcriptome. The xylem transcriptomes of vascular plants are more conserved than the total transcriptomes. Evolution of the xylem transcriptome has largely followed the trend of plant evolution.

Seasonal reorganization of the xylem transcriptome at different tree ages reveals novel insights into wood formation in Pinus radiata.

*Seasonal wood development produces earlywood (EW) and latewood (LW) with distinct properties. The molecular mechanisms controlling EW and LW formation at different tree ages are poorly understood. *Seasonal reorganization of the xylem transcriptome was investigated in Pinus radiata at four tree ages using cDNA microarrays. Transcriptome profiles were compared with seasonal wood variation measured by SilviScan (CSIRO, Clayton, Australia). *The xylem transcriptome was considerably reorganized during seasonal change, and this reorganization showed a maturation-related pattern. The greater reorganization occurred at the transition (30%) and juvenile (21%) stages, but it declined with tree maturity (11-13%). However, this pattern does not correlate well with maturation-related patterns of seasonal wood variation. In total, 319 seasonal-responsive xylem candidate genes were identified. Many transcripts involved in primary and secondary wall biosynthesis were preferentially accumulated in EW and LW, respectively. A large proportion (45-81%) of the candidate genes are preferentially regulated at a single age and their transcript abundance may influence maturation-related patterns of seasonal wood variation. *Seasonal reorganization of the xylem transcriptome is significantly affected by tree age. Physiological changes at the transition stage may contribute to its greater seasonal transcriptome reorganization. Identified stage-preferential xylem transcripts could influence seasonal wood variation at different tree ages.

Fasciclin-like arabinogalactan proteins: specialization for stem biomechanics and cell wall architecture in Arabidopsis and Eucalyptus.

The ancient cell adhesion fasciclin (FAS) domain is found in bacteria, fungi, algae, insects and animals, and occurs in a large family of fasciclin-like arabinogalactan proteins (FLAs) in higher plants. Functional roles for FAS-containing proteins have been determined for insects, algae and vertebrates; however, the biological functions of the various higher-plant FLAs are not clear. Expression of some FLAs has been correlated with the onset of secondary-wall cellulose synthesis in Arabidopsis stems, and also with wood formation in the stems and branches of trees, suggesting a biological role in plant stems. We examined whether FLAs contribute to plant stem biomechanics. Using phylogenetic, transcript abundance and promoter-GUS fusion analyses, we identified a conserved subset of single FAS domain FLAs (group A FLAs) in Eucalyptus and Arabidopsis that have specific and high transcript abundance in stems, particularly in stem cells undergoing secondary-wall deposition, and that the phylogenetic conservation appears to extend to other dicots and monocots. Gene-function analyses revealed that Arabidopsis T-DNA knockout double mutant stems had altered stem biomechanics with reduced tensile strength and a reduced tensile modulus of elasticity, as well as altered cell-wall architecture and composition, with increased cellulose microfibril angle and reduced arabinose, galactose and cellulose content. Using materials engineering concepts, we relate the effects of these FLAs on cell-wall composition with stem biomechanics. Our results suggest that a subset of single FAS domain FLAs contributes to plant stem strength by affecting cellulose deposition, and to the stem modulus of elasticity by affecting the integrity of the cell-wall matrix.

Identification of a Cis-acting regulatory polymorphism in a Eucalypt COBRA-like gene affecting cellulose content.

Populations with low linkage disequilibrium (LD) offer unique opportunities to study functional variants influencing quantitative traits. We exploited the low LD in forest trees to identify functional polymorphisms in a Eucalyptus nitens COBRA-like gene (EniCOBL4A), whose Arabidopsis homolog has been implicated in cellulose deposition. Linkage analysis in a full-sib family revealed that EniCOBL4A is the most strongly associated marker in a quantitative trait locus (QTL) region for cellulose content. Analysis of LD by genotyping 11 common single-nucleotide polymorphisms (SNPs) and a simple sequence repeat (SSR) in an association population revealed that LD declines within the length of the gene. Using association studies we fine mapped the effect of the gene to SNP7, a synonymous SNP in exon 5, which occurs between two small haplotype blocks. We observed patterns of allelic expression imbalance (AEI) and differential binding of nuclear proteins to the SNP7 region that indicate that SNP7 is a cis-acting regulatory polymorphism affecting allelic expression. We also observed AEI in SNP7 heterozygotes in a full-sib family that is linked to heritable allele-specific methylation near SNP7. This study demonstrates the potential to reveal functional polymorphisms underlying quantitative traits in low LD populations.

Generation and analysis of expressed sequence tags from six developing xylem libraries in Pinus radiata D. Don.

BACKGROUND: Wood is a major renewable natural resource for the timber, fibre and bioenergy industry. Pinus radiata D. Don is the most important commercial plantation tree species in Australia and several other countries; however, genomic resources for this species are very limited in public databases. Our primary objective was to sequence a large number of expressed sequence tags (ESTs) from genes involved in wood formation in radiata pine. RESULTS: Six developing xylem cDNA libraries were constructed from earlywood and latewood tissues sampled at juvenile (7 yrs), transition (11 yrs) and mature (30 yrs) ages, respectively. These xylem tissues represent six typical development stages in a rotation period of radiata pine. A total of 6,389 high quality ESTs were collected from 5,952 cDNA clones. Assembly of 5,952 ESTs from 5' end sequences generated 3,304 unigenes including 952 contigs and 2,352 singletons. About 97.0% of the 5,952 ESTs and 96.1% of the unigenes have matches in the UniProt and TIGR databases. Of the 3,174 unigenes with matches, 42.9% were not assigned GO (Gene Ontology) terms and their functions are unknown or unclassified. More than half (52.1%) of the 5,952 ESTs have matches in the Pfam database and represent 772 known protein families. About 18.0% of the 5,952 ESTs matched cell wall related genes in the MAIZEWALL database, representing all 18 categories, 91 of all 174 families and possibly 557 genes. Fifteen cell wall-related genes are ranked in the 30 most abundant genes, including CesA, tubulin, AGP, SAMS, actin, laccase, CCoAMT, MetE, phytocyanin, pectate lyase, cellulase, SuSy, expansin, chitinase and UDP-glucose dehydrogenase. Based on the PlantTFDB database 41 of the 64 transcription factor families in the poplar genome were identified as being involved in radiata pine wood formation. Comparative analysis of GO term abundance revealed a distinct transcriptome in juvenile earlywood formation compared to other stages of wood development. CONCLUSION: The first large scale genomic resource in radiata pine was generated from six developing xylem cDNA libraries. Cell wall-related genes and transcription factors were identified. Juvenile earlywood has a distinct transcriptome, which is likely to contribute to the undesirable properties of juvenile wood in radiata pine. The publicly available resource of radiata pine will also be valuable for gene function studies and comparative genomics in forest trees.

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers.

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.

beta-tubulin affects cellulose microfibril orientation in plant secondary fibre cell walls.

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

Early flowering induction and Agrobacterium transformation of the hardwood tree species Eucalyptus occidentalis.

Investigation of the genes controlling flowering in eucalypts is hindered by the lack of an early-flowering genotype. Induction of early flowering was studied in five provenances of Eucalyptus occidentalis Endl. sourced from throughout its geographic range. Seedlings initiated flowers from 13 weeks after sowing when grown under optimal conditions with a 16-h photoperiod. By 16 weeks, seedlings from four widely dispersed provenances had initiated floral buds, suggesting that competence to flower early is a common characteristic of the species. The different provenances displayed considerable variation in seedling growth rate. Elevated levels of CO2 had no effect on seedling growth rate, but were associated with delayed flowering. Transformation experiments demonstrated that E. occidentalis is susceptible to Agrobacterium-mediated transformation, in common with several other eucalypt species. E. occidentalis may be a valuable experimental species for molecular and other flowering studies in eucalypts.