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ABSTRACT: Abstract Lignocellulosic biomass from fast-growing plantation
trees is composed of carbohydrate-rich materials deposited
into plant cell walls in a coordinated manner during
wood formation. The diversity and evolution of the transcriptional
networks regulating this process have not been
studied extensively.We investigated patterns of species-level
nucleotide diversity in the promoters of cellulose synthase
(CesA) genes from different Eucalyptus tree species and
assessed the possible roles of DNA sequence polymorphism
in the gain or loss of cis-elements harboured within the
promoters. Promoter regions of three primary and three
secondary cell wall-associated CesA genes were isolated
from 13 Eucalyptus species and were analysed for nucleotide
and cis-element diversity. Species-level nucleotide diversity
(π) ranged from 0.014 to 0.068, and different CesA promoters
exhibited distinct patterns of sequence conservation.
A set of 22 putative cis-elements were mapped to the CesA
promoters using in silico methods. Forty-two percent of the
mapped cis-element occurrences contained singleton polymorphisms
which resulted in either gain or loss of a ciselement
in a particular Eucalyptus species. The promoters of
Eucalyptus CesA genes contained regions that are highly
conserved at the species (Eucalyptus) and genus (with
Arabidopsis and Populus) level, suggesting the presence of
regulatory modules imposing functional constraint on such
regions. Nucleotide polymorphisms in the CesA promoters
more frequently created new cis-element occurrences than
disrupted existing cis-element occurrences, a process which
may be important for the maintenance and evolution of
cellulose gene regulation in plants.
Tree Genetics & Genomes 02/2013; · 2.34 Impact Factor
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ABSTRACT: Two important role players in plant defence response are the phytohormones salicylic acid (SA) and jasmonic acid (JA); both of which have been well described in model species such as Arabidopsis thaliana. Several pathogenesis related (PR) genes have previously been used as indicators of the onset of SA and JA signaling in Arabidopsis. This information is lacking in tree genera such as Eucalyptus. The aim of this study was to characterize the transcriptional response of PR genes (EgrPR2, EgrPR3, EgrPR4, EgrPR5, and EgrLOX) identified in Eucalyptus grandis to SA and methyl jasmonate (MeJA) treatment as well as to qualify them as diagnostic for the two signaling pathways. Using the genome sequence of E. grandis, we identified candidate Eucalyptus orthologs EgrPR2, EgrPR3, EgrPR4, EgrPR5, and EgrLOX based on a co-phylogenetic approach. The expression of these genes was investigated after various doses of SA and MeJA (a derivative of JA) treatment as well as at various time points. The transcript levels of EgrPR2 were decreased in response to high concentrations of MeJA whereas the expression of EgrPR3 and EgrLOX declined as the concentrations of SA treatment increased, suggesting an antagonistic relationship between SA and MeJA. Our results support EgrPR2 as potentially diagnostic for SA and EgrPR3, EgrPR4, and EgrLOX as indicators of MeJA signaling. To further validate the diagnostic potential of the PR genes we challenged E. grandis clones with the fungal necrotrophic pathogen Chrysoporthe austroafricana. The tolerant clone showed high induction of EgrPR2 and decreased transcript abundance of EgrPR4. Pre-treatment of the susceptible genotype with 5 mM SA resulted in lesion lengths comparable to the tolerant genotype after artificial inoculation with C. austroafricana. Thus expression profiling of EgrPR2 and EgrPR4 genes could serve as a useful diagnostic approach to determine which of the two signaling pathways are activated against various pathogens in Eucalyptus.
Frontiers in plant science. 01/2013; 4:43.
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ABSTRACT: The increasing focus on plantation forestry as a renewable source of cellulosic biomass has emphasized the need for tools to study the unique biology of woody genera such as Eucalyptus, Populus and Pinus. The domestication of these woody crops is hampered by long generation times, and breeders are now looking to molecular approaches such as marker-assisted breeding and genetic modification to accelerate tree improvement. Much of what is known about genes involved in the growth and development of plants has come from studies of herbaceous models such as Arabidopsis and rice. However, transferring this information to woody plants often proves difficult, especially for genes expressed in woody stems. Here we report the use of induced somatic sector analysis (ISSA) for characterization of promoter expression patterns directly in the stems of Populus and Eucalyptus trees. As a case study, we used previously characterized primary and secondary cell wall-related cellulose synthase (CesA) promoters cloned from Eucalyptus grandis. We show that ISSA can be used to elucidate the phloem and xylem expression patterns of the CesA genes in Eucalyptus and Populus stems and also show that the staining patterns differ in Eucalyptus and Populus stems. These findings show that ISSA is an efficient approach to investigate promoter function in the developmental context of woody plant tissues and raise questions about the suitability of heterologous promoters for genetic manipulation in plant species.
Planta 11/2012; · 3.00 Impact Factor
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Forest Ecology and Management 09/2012; · 2.49 Impact Factor
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ABSTRACT: 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.
New Phytologist 04/2012; 194(1):54-62. · 6.64 Impact Factor
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ABSTRACT: NAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored.
We overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area.
This study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.
BMC Plant Biology 12/2011; 11:173. · 3.45 Impact Factor
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ABSTRACT: ABSTRACT:
Microarray technology has matured over the past fifteen years into a cost-effective solution with established data analysis protocols for global gene expression profiling. The Agilent-016047 maize 44 K microarray was custom-designed from EST sequences, but only reporter sequences with EST accession numbers are publicly available. The following information is lacking: (a) reporter - gene model match, (b) number of reporters per gene model, (c) potential for cross hybridization, (d) sense/antisense orientation of reporters, (e) position of reporter on B73 genome sequence (for eQTL studies), and (f) functional annotations of genes represented by reporters. To address this, we developed a strategy to annotate the Agilent-016047 maize microarray, and built a publicly accessible annotation database.
Genomic annotation of the 42,034 reporters on the Agilent-016047 maize microarray was based on BLASTN results of the 60-mer reporter sequences and their corresponding ESTs against the maize B73 RefGen v2 "Working Gene Set" (WGS) predicted transcripts and the genome sequence. The agreement between the EST, WGS transcript and gDNA BLASTN results were used to assign the reporters into six genomic annotation groups. These annotation groups were: (i) "annotation by sense gene model" (23,668 reporters), (ii) "annotation by antisense gene model" (4,330); (iii) "annotation by gDNA" without a WGS transcript hit (1,549); (iv) "annotation by EST", in which case the EST from which the reporter was designed, but not the reporter itself, has a WGS transcript hit (3,390); (v) "ambiguous annotation" (2,608); and (vi) "inconclusive annotation" (6,489). Functional annotations of reporters were obtained by BLASTX and Blast2GO analysis of corresponding WGS transcripts against GenBank.The annotations are available in the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/, as well as through a GBrowse annotation file that can be uploaded to the MaizeGDB genome browser as a custom track.The database was used to re-annotate lists of differentially expressed genes reported in case studies of published work using the Agilent-016047 maize microarray. Up to 85% of reporters in each list could be annotated with confidence by a single gene model, however up to 10% of reporters had ambiguous annotations. Overall, more than 57% of reporters gave a measurable signal in tissues as diverse as anthers and leaves.
The Maize Microarray Annotation Database will assist users of the Agilent-016047 maize microarray in (i) refining gene lists for global expression analysis, and (ii) confirming the annotation of candidate genes before functional studies.
Plant Methods 10/2011; 7:31. · 2.83 Impact Factor
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ABSTRACT: Fusarium circinatum and Fusarium subglutinans are two distinct species in the Gibberella fujikuroi species complex. A genetic linkage map produced from an interspecific cross between these species was used to identify quantitative trait loci (QTLs) associated with variation in mycelial growth and morphology of colony margins (CMs) in the 94 F(1) progeny. Mycelial growth was assessed by measuring culture size at 25°C and 30°C, while CM morphology was characterized in the parents and assessed in their F(1) progeny. In order to test the pathogenicity of the progeny, Pinus patula seedlings were inoculated and lesion lengths were measured after 3weeks. Seven putative QTLs were associated with mycelial growth, three for growth at 25°C and four at 30°C. One highly significant QTL (P<0.001) was present at both growth temperatures. For CM morphology, a QTL was identified at the same position (P<0.001) as the QTL responsible for growth at the two temperatures. The putative QTLs accounted for 45 and 41% of the total mycelial growth variation at 25°C and 30°C, respectively, and for 21% of the variation in CM morphology. Only one of the 94 F(1) progeny was pathogenic on P. patula seedlings. This observation could be explained by the genetic constitution of this F(1) isolate, namely that ∼96% of its genome originated from the F. circinatum parent. This F(1) individual also grew significantly faster at 25°C than the F. circinatum parent (P<0.05), as well as more rapidly than the average growth for the remaining 93 F(1) progeny (P<0.05). However, no association was found between mycelial growth and pathogenicity at 25°C. The highly significant QTL associated with growth at two temperatures, suggests that this is a principal genomic region involved in mycelial growth at both temperatures, and that the same region is also responsible for CM morphology.
Fungal Biology 09/2011; 115(9):902-8. · 1.43 Impact Factor
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ABSTRACT: De novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species (E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs.
We present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions.
De novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants.
BMC Genomics 01/2010; 11:681. · 4.07 Impact Factor
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ABSTRACT: Higher plants contain a family of cellulose synthase catalytic subunit (CesA) genes that encode components of an enzyme complex embedded in the cell membrane. Recent studies in several higher plant species have demonstrated that two groups of CesA genes exist, associated with either primary or secondary cell wall deposition. We cloned six full-length CesA cDNAs from Eucalyptus grandis W. Hill ex Maiden (EgCesA1 through 6) and determined their expression patterns in a variety of organs from an adult tree. The six EgCesA genes encode predicted proteins of 978 to 1097 amino acid residues, each of which contains all of the key regions and motifs characteristic of functional CESA proteins. The predicted proteins share limited amino acid identity with each other, ranging from 61 to 70%. In contrast, similar CESA proteins from higher plant species exhibit 81 to 90% identity with the six EgCESAs. Gene expression analysis using quantitative reverse-transcription polymerase chain reaction indicated that transcripts of EgCesA1 to 3 were abundant in tissues enriched with cells laying down secondary cell walls (e.g., xylem), but were weakly expressed in tissues undergoing primary growth (e.g., unfolding leaves). Expression of EgCesA4 and EgCesA5 was upregulated in tissues rich in rapidly dividing cells undergoing primary wall synthesis, whereas EgCesA6 was weakly expressed in all tissues analyzed. These results suggest that Eucalyptus, like other higher plants, expresses two contrasting groups of apparently co-regulated CesAs involved in either primary or secondary cell wall biosynthesis.
Tree Physiology 06/2006; 26(5):545-56. · 2.88 Impact Factor
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ABSTRACT: Despite the availability of high-throughput transcript profiling technology, little is known about tissue-specific gene expression patterns in the wood-forming tissues of Eucalyptus plantation tree species. We used cDNA-amplified fragment length polymorphism (AFLP) analysis in combination with infrared fragment detection and semi-automated band quantification to profile gene expression in a 6-year-old, fast- growing Eucalyptus tree. The expression profiles of 6385 transcript-derived fragments (TDFs) were analyzed across four major woody tissues (mature xylem, immature xylem, phloem and cork) collected from two stem positions, to provide a global view of transcript abundance and variability in the Eucalyptus stem. About 21% of the TDFs were differentially expressed and could be grouped into clusters representing co- expressed genes. A total of 71 TDFs representing different gene clusters were isolated and characterized. These included genes implicated in cell fate, signal transduction and cell wall biosynthesis, processes closely associated with xylogenesis. Analysis of the expression levels of selected TDFs by quantitative RT-PCR corroborated the TDF quantification and confirmed that cDNA-AFLP analysis is a highly efficient and accurate tool for transcript profiling and gene discovery in wood-forming tissues of tree species.
Tree Physiology 04/2006; 26(3):365-75. · 2.88 Impact Factor
