Transcriptome and hormone profiling reveals Eucalyptus grandis defence responses against Chrysoporthe austroafricana

BACKGROUND : Eucalyptus species and interspecific hybrids exhibit valuable growth and wood properties that make them a highly desirable commodity. However, these trees are challenged by a wide array of biotic stresses during their lifetimes. The Eucalyptus grandis reference genome sequence provides a resource to study pest and pathogen defence mechanisms in long-lived woody plants. E. grandis trees are generally susceptible to Chrysoporthe austroafricana, a causal agent of stem cankers on eucalypts. The aim of this study was to characterize the defence response of E. grandis against C. austroafricana. RESULTS : Hormone profiling of susceptible and moderately resistant clonal E. grandis genotypes indicated a reduction in salicylic acid and gibberellic acid levels at 3 days post inoculation. We hypothesized that these signaling pathways may facilitate resistance. To further investigate other defence mechanisms at this time point, transcriptome profiling was performed. This revealed that cell wall modifications and response to oxidative stress form part of the defence responses common to both genotypes, whilst changes in the hormone signaling pathways may contribute to resistance. Additionally the expression of selected candidate defence response genes was induced earlier in moderately resistant trees than in susceptible trees, supporting the hypothesis that a delayed defence response may occur in the susceptible interaction. CONCLUSION : The ability of a host to fine-tune its defence responses is crucial and the responses identified in this study extends our understanding of plant defence, gained from model systems, to woody perennials.Additional file 1: Table S1. Summary of statistics obtained for transcriptome profiling of TAG5 and ZG14 challenged with C. austroafricana.Additional file 2: Table S2. Summary of significantly differentially expressed genes and their annotations identified from Eucalyptus grandis TAG5 and ZG14.Additional file 3: Figure S1. Molecular function GO terms that are over-represented in TAG5 and ZG14. a – GO terms within the upregulated dataset. b – GO terms within the down-regulated dataset (all terms for this dataset are shown). The y-axis represents the –log2(q-value) and the x-axis represents the GO terms within the datasets. Light and dark grey bars are ZG14 and TAG5 respectively.Additional file 4: Figure S2. Cellular component GO terms that are over-represented in TAG5 and ZG14. a – GO terms within the upregulated dataset. b – GO terms within the down-regulated dataset. The y-axis represents the –log2(q-value) and the x-axis represents the GO terms within the datasets. Light and dark grey bars are ZG14 and TAG5 respectively.Additional file 5: Table S3. List of differentially expressed genes that are common between the susceptible (ZG14) and moderately resistant (TAG5) host.This work was supported by the Genomics Research Institute (GRI) at the University of Pretoria; the National Research Foundation of South Africa (Grant number NBIG 86936); Thuthuka funding (UID:76225); the Forest Molecular Genetics Programme by Mondi and Sappi and the Technology and Human Resources for Industry Programme (UID:80118).http://www.biomedcentral.com/bmcgenomicsam201

Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

DATA AVAILABILITY : All data supporting the findings of this study are available within the paper and its Supplementary Information.Please read abstract in the article.SAFCOL Learning and Development Bursary Division. Open access funding provided by Stellenbosch University.https://link.springer.com/journal/11240hj2024BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologySDG-15:Life on lan

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.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-8137nf201

Horsetails are ancient polyploids : evidence from Equisetum giganteum

Horsetails represent an enigmatic clade within the land plants. Despite consisting only of one genus (Equisetum) that contains 15 species, they are thought to represent the oldest extant genus within the vascular plants dating back possibly as far as the Triassic. Horsetails have retained several ancient features and are also characterized by a particularly high chromosome count (n = 108). Whole-genome duplications (WGDs) have been uncovered in many angiosperm clades and have been associated with the success of angiosperms, both in terms of species richness and biomass dominance, but remain understudied in nonangiosperm clades. Here, we report unambiguous evidence of an ancient WGD in the fern linage, based on sequencing and de novo assembly of an expressed gene catalog (transcriptome) from the giant horsetail (Equisetum giganteum). We demonstrate that horsetails underwent an independent paleopolyploidy during the Late Cretaceous prior to the diversification of the genus but did not experience any recent polyploidizations that could account for their high chromosome number. We also discuss the specific retention of genes following the WGD and how this may be linked to their long-term survival

Xylan in the middle : understanding xylan biosynthesis and its metabolic dependencies toward improving wood fiber for industrial processing

Lignocellulosic biomass, encompassing cellulose, lignin and hemicellulose in plant secondary cell walls (SCWs), is the most abundant source of renewable materials on earth. Currently, fast-growing woody dicots such as Eucalyptus and Populus trees are major lignocellulosic (wood fiber) feedstocks for bioproducts such as pulp, paper, cellulose, textiles, bioplastics and other biomaterials. Processing wood for these products entails separating the biomass into its three main components as efficiently as possible without compromising yield. Glucuronoxylan (xylan), the main hemicellulose present in the SCWs of hardwood trees carries chemical modifications that are associated with SCW composition and ultrastructure, and affect the recalcitrance of woody biomass to industrial processing. In this review we highlight the importance of xylan properties for industrial wood fiber processing and how gaining a greater understanding of xylan biosynthesis, specifically xylan modification, could yield novel biotechnology approaches to reduce recalcitrance or introduce novel processing traits. Altering xylan modification patterns has recently become a focus of plant SCW studies due to early findings that altered modification patterns can yield beneficial biomass processing traits. Additionally, it has been noted that plants with altered xylan composition display metabolic differences linked to changes in precursor usage. We explore the possibility of using systems biology and systems genetics approaches to gain insight into the coordination of SCW formation with other interdependent biological processes. Acetyl-CoA, s-adenosylmethionine and nucleotide sugars are precursors needed for xylan modification, however, the pathways which produce metabolic pools during different stages of fiber cell wall formation still have to be identified and their co-regulation during SCW formation elucidated. The crucial dependence on precursor metabolism provides an opportunity to alter xylan modification patterns through metabolic engineering of one or more of these interdependent pathways. The complexity of xylan biosynthesis and modification is currently a stumbling point, but it may provide new avenues for woody biomass engineering that are not possible for other biopolymers.Table S1. List of enzymes which catalyze the biosynthesis of sugar nucleotides, s-adenosyl methionine and acetyl-CoA. (a) The numbering of the enzymes corresponds to Figures 4–6. (b) Name of the enzyme. (c) Not all accessions were provided for each reaction, just one representative from each cellular compartment. Accessions and cellular localisation were obtained from TAIR and SUBA3, respectively.File S1. References pertaining to numbered items in Table 1.File S2. Biotechnology approaches that have scaled from Arabidopsis to Populus.This work was funded in part by the National Research Foundation (NRF) of South Africa – Bioinformatics and Functional Genomics Programme (BFG Grant UID 86936 and 97911), the Technology and Human Resources for Industry Programme (THRIP Grant UID 96413), the Department of Science and Technology (DST, Strategic Grant for the Eucalyptus Genomics Platform) and by Sappi Forest Research through the Forest Molecular Genetics (FMG) Programme at the University of Pretoria (UP). MW acknowledges postgraduate scholarship support from the NRF. VM acknowledges a postdoctoral fellowship support from UP.http://www.frontiersin.org/Plant_Scienceam2019BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Assessment of genome-wide DArT-seq markers for tea Camellia sinensis (L.) O. Kuntze germplasm analysis

Tea is the world’s most consumed beverage, next to water. This makes it an economically important crop. However, the genetic relationship between the cultivars at the Tea Research Foundation of Central Africa (TRFCA) have not been classified. This study assessed 56 tea accessions from the TRFCA. The 56 accessions were split into five groups, namely, Cambod type, China type, TRFCA, Tea Research Institute (TRI), and unknown. A total of 16,382 genome-wide genetic markers were developed using a next-generation sequencing (NGS) platform at Diversity Array Technology (DArT). The current study also explored the usefulness of the DArT-seq markers for tea germplasm analysis. The genetic relationships amongst the cultivars were analysed using the neighbour-joining method and UPGMA; this was successful in clustering the different cultivars into groups of origin. Principal coordinate analysis (PCoA) showed that 33 TRFCA-derived cultivars were distributed in all four quadrants. Analysis of molecular variance (AMOVA) revealed that there was a higher proportion of genetic variation (94%) within the groups than there was amongst the groups (6%). Nei’s unbiased genetic distances amongst groups suggested that the 33 TRFCA-derived cultivars had a low genetic distance from the other four groups. This confirms the PCoA inference that the 33 TRFCA had a high genetic admixture. The genetic structure was utilised to assign three cultivars of unknown origin, to the Cambod-type group. Two cultivars were closely related to the China-type group. Our findings are a useful guide for future tea breeding programmes in Southern Africa.The Carnegie Regional Initiative in Science and Education (Carnegie-RISE) through the Southern African Biochemistry and Informatics for Natural Products (SABINA) network. and the National Research Foundation (NRF).http://link.springer.com/journal/11295hj2020BiochemistryGeneticsMicrobiology and Plant Patholog

Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem

BACKGROUND : Histone modifications play an integral role in plant development, but have been poorly studied in woody plants. Investigating chromatin organization in wood-forming tissue and its role in regulating gene expression allows us to understand the mechanisms underlying cellular differentiation during xylogenesis (wood formation) and identify novel functional regions in plant genomes. However, woody tissue poses unique challenges for using high-throughput chromatin immunoprecipitation (ChIP) techniques for studying genome-wide histone modifications in vivo. We investigated the role of the modified histone H3K4me3 (trimethylated lysine 4 of histone H3) in gene expression during the early stages of wood formation using ChIP-seq in Eucalyptus grandis, a woody biomass model. RESULTS : Plant chromatin fixation and isolation protocols were optimized for developing xylem tissue collected from field-grown E. grandis trees. A “nano-ChIP-seq” procedure was employed for ChIP DNA amplification. Over 9 million H3K4me3 ChIP-seq and 18 million control paired-end reads were mapped to the E. grandis reference genome for peak-calling using Model-based Analysis of ChIP-Seq. The 12,177 significant H3K4me3 peaks identified covered ~1.5% of the genome and overlapped some 9,623 protein-coding genes and 38 noncoding RNAs. H3K4me3 library coverage, peaking ~600 - 700 bp downstream of the transcription start site, was highly correlated with gene expression levels measured with RNA-seq. Overall, H3K4me3-enriched genes tended to be less tissue-specific than unenriched genes and were overrepresented for general cellular metabolism and development gene ontology terms. Relative expression of H3K4me3-enriched genes in developing secondary xylem was higher than unenriched genes, however, and highly expressed secondary cell wall-related genes were enriched for H3K4me3 as validated using ChIP-qPCR. CONCLUSIONS : In this first genome-wide analysis of a modified histone in a woody tissue, we optimized a ChIP-seq procedure suitable for field-collected samples. In developing E. grandis xylem, H3K4me3 enrichment is an indicator of active transcription, consistent with its known role in sustaining pre-initiation complex formation in yeast. The H3K4me3 ChIP-seq data from this study paves the way to understanding the chromatin landscape and epigenomic architecture of xylogenesis in plants, and complements RNA-seq evidence of gene expression for the future improvement of the E. grandis genome annotation.Additional file 1: Supplementary Note S1.Additional file 2: Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S6, Figure S7, Figure S8, Figure S9, Figure S10, Figure S11, Figure S12, Figure S13, Figure S14, Figure S15, Figure S16.Additional file 3: Table S1, Table S2, Table S3, Table S4, Table S5, Table S6, Table S7.Additional file 4: Genomic locations and fragment coverage of significant H3K4me3 peaks.Additional file 5: Genomic locations of annotated genes overlapping with significant H3K4me3 peaks.Additional file 6: Genomic locations of low-confidence gene models overlapping with significant H3K4me3 peaks.SH, EM and AM acknowledge funding from the Department of Science and Technology (DST), South Africa, the National Research Foundation of South Africa (NRF) Incentive Funding for Rated Researchers Grant (UID 81111) and NRF Bioinformatics and Functional Genomics Program (UID 71255, UID 86936), Sappi and Mondi through the Forest Molecular Genetics (FMG) Program at the University of Pretoria (UP), and the Technology and Human Resources for Industry Program (THRIP) (UID 80118). AG acknowledges funding from USDA National Institute of Food and Agriculture and the Office of Science (BER), US Department of Energy.http://www.biomedcentral.com/bmcplantbiolam201

The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.S1 Fig. Phylogenetic and bioinformatics analysis of all members of the Arabidopsis domain of unknown function 1218 (DUF1218) family, and expression profiling of the candidate members, MODIFYING WALL LIGNIN-1 (MWL-1, At1g31720) and MWL-2 (At4g19370). (A) Neighbor-joining phylogenetic tree of Arabdiopsis DUF1218-containing proteins. ClustalW was used to align protein sequences from TAIR and the alignment thereafter used to construct the tree using p-distance and pairwise deletion with 1000 bootstrap replicates in MEGA5 [16]. Prediction of subcellular localization, signal peptide and number of transmembrane domains was done using SUBA3 [31], Signal-3L [18] and TMHMM[19] respectively, with default settings. Highlighted in pink are the related MWL-1 and 2 sequences. (B) Arabidopsis expression profiles for MWL-1 and MWL-2 across different tissues during development, exctracted from The Bio-Analytic Resource for Plant Biology (http://bar. utoronto.ca/welcome.htm) [20]. Preferential expression is seen at distinct developmental stages, however, there is overlap in the secondary cell wall depositing, 2nd internode region. (DOCX)S2 Fig. Gene ontology enrichment of MWL-1 top 300 co-expressed genes in Arabidopsis. Co-expressed genes were extracted from ATTED-II [10]. GO-full was conducted in Cytoscape 2.8.2 [22] using BiNGO 2.44 [21], while overrepresentation summary enrichment was performed with the REVIGO server [23]. (DOCX)S3 Fig. Phenotypic analysis of At1g31720 (MWL-1) single T-DNA knockout line mutants and MWL-1 overexpression lines. (A) RT-PCR detection of endogenous MWL-1 transcript in the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative RT-PCR analysis of MWL-1 overexpression lines 1 to 3 showing detection of MWL-1 transgene in the transgenic lines. Actin2 was used as a control gene and RT-PCR was performed on cDNA from stem tissue. Actin2 and MWL-1 gene-specific oligonucleotide sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-1 single T-DNA knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative (F) stem length of MWL-1 single T-DNA knockout line and overexpression lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66. Error bars indicate the standard error. Scale bar, 3 cm. Based on a two-tailed Student’s t-test (P-value 0.05) no significant differences were seen in the growth and development of the single mutant and transgenic OE lines in comparison to the WT controls.S4 Fig. Phenotypic analysis of At4g19370 (MWL-2) single T-DNA knockout line mutants and MWL-2 overexpression lines. (A) RT-PCR detection of endogenous MWL-2 transcript in the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative RT-PCR analysis of MWL-2 overexpression lines 1 to 3 showing detection of MWL-2 transgene in the transgenic lines except for OE1 which could be indicative of positional effect (position in the genome), or co-suppression dominant repression. Actin2 was used as a control gene and RT-PCR was performed on stem tissue. Actin2 and MWL-2 gene-specific oligonucleotide sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-2 single T-DNA knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative (F) stem length of MWL-2 single T-DNA knockout line and overexpression lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66. Error bars indicate the standard error while significant difference from the WT based on a two-tailed Student’s t-test (P-value 0.05) is indicated by . Scale bar, 3 cm. No significant differences were seen in the growth and development of the single mutant and transgenic OE lines in comparison to the WT controls except for OE-Line 2. (DOCX)S5 Fig. Transverse sections of six-week-old stem tissue stained with phloroglucinol from At1g31720 (MWL-1) and At4g19370 (MWL-2) T-DNA knockout mutant and overexpression (OE) lines. Transverse sections from wildtype (WT) (A), At1g31720 mutant (B), At4g19370 mutant (C), At1g31720 x At4g19370 double knockdown mutant (D), OEAt1g31720 line 1 (E), line 2 (F), line 3 (G), OEAt4g19370 line 1(H), line 2 (I), line 3 (J). Scale bar, 100μm (indicated in red). No discernible differences were seen in the transverse sections of the single and double mutant as well as the transgenic OE lines in comparison to the WT controls. (DOCX)S1 Table. List of oligonucleotides used in the study. (DOCX)S2 Table. Top 300 Arabidopsis co-expressed genes for MWL-1 (At1g31720) from ATTED-II represented as MR value. (DOCX)S3 Table. Top 300 Arabidopsis co-expressed genes for MWL-2 (At4g19370) from ATTED-II represented as MR value. (DOCX)S4 Table. Structural cell wall carbohydrates and lignin content from MWL-1 and MWL-2 overexpression, single and double knockout lines compared to its respective wildtype (WT) control. (DOCX)Sappi through the Forest Molecular Genetics (FMG) Programme, the Technology and Human Resources for Industry Programme (THRIP, UID 80118), and the National Research Foundation (NRF, UID 71255 and 86936) of South Africa. RM acknowledges an NRF Ph.D. Prestige and Equity Scholarship.http://www.plosone.orgam2016Chemical EngineeringGenetic

Navigating the transcriptional roadmap regulating plant secondary cell wall deposition

The current status of lignocellulosic biomass as an invaluable resource in industry, agriculture, and health has spurred increased interest in understanding the transcriptional regulation of secondary cell wall (SCW) biosynthesis. The last decade of research has revealed an extensive network of NAC, MYB and other families of transcription factors regulating Arabidopsis SCW biosynthesis, and numerous studies have explored SCW-related transcription factors in other dicots and monocots. Whilst the general structure of the Arabidopsis network has been a topic of several reviews, they have not comprehensively represented the detailed protein-DNA and protein-protein interactions described in the literature, and an understanding of network dynamics and functionality has not yet been achieved for SCW formation. Furthermore the methodologies employed in studies of SCW transcriptional regulation have not received much attention, especially in the case of non-model organisms. In this review, we have reconstructed the most exhaustive literature-based network representations to date of SCW transcriptional regulation in Arabidopsis. We include a manipulable Cytoscape representation of the Arabidopsis SCW transcriptional network to aid in future studies, along with a list of supporting literature for each documented interaction. Amongst other topics, we discuss the various components of the network, its evolutionary conservation in plants, putative modules and dynamic mechanisms that may influence network function, and the approaches that have been employed in network inference. Future research should aim to better understand network function and its response to dynamic perturbations, whilst the development and application of genome-wide approaches such as ChIP-seq and systems genetics are in progress for the study of SCW transcriptional regulation in non-model organisms.The Mandela Rhodes Foundation and National Research Foundation of South Africahttp://www.frontiersin.orgam201

Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues

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.This work was supported through funding provided by Mondi and Sappi to the Forest Molecular Genetics (FMG) Programme, the Technology and Human Resources for Industry Programme (THRIP) and the National Research Foundation (NRF) of South Africa as well as a Linkage Grant from the Australian Research Council (LP0776563) to GB, AAM and AVS.http://link.springer.com/journal/425hb201