Comparative physiological, biochemical, metabolomic, and transcriptomic analyses reveal the formation mechanism of heartwood for Acacia melanoxylon.

Acacia melanoxylon is well known as a valuable commercial tree species owing to its high-quality heartwood (HW) products. However, the metabolism and regulatory mechanism of heartwood during wood development remain largely unclear. In this study, both microscopic observation and content determination proved that total amount of starches decreased and phenolics and flavonoids increased gradually from sapwood (SW) to HW. We also obtained the metabolite profiles of 10 metabolites related to phenolics and flavonoids during HW formation by metabolomics. Additionally, we collected a comprehensive overview of genes associated with the biosynthesis of sugars, terpenoids, phenolics, and flavonoids using RNA-seq. A total of ninety-one genes related to HW formation were identified. The transcripts related to plant hormones, programmed cell death (PCD), and dehydration were increased in transition zone (TZ) than in SW. The results of RT-PCR showed that the relative expression level of genes and transcription factors was also high in the TZ, regardless of the horizontal or vertical direction of the trunk. Therefore, the HW formation took place in the TZ for A. melanoxylon from molecular level, and potentially connected to plant hormones, PCD, and cell dehydration. Besides, the increased expression of sugar and terpenoid biosynthesis-related genes in TZ further confirmed the close connection between terpenoid biosynthesis and carbohydrate metabolites of A. melanoxylon. Furthermore, the integrated analysis of metabolism data and RNA-seq data showed the key transcription factors (TFs) regulating flavonoids and phenolics accumulation in HW, including negative correlation TFs (WRKY, MYB) and positive correlation TFs (AP2, bZIP, CBF, PB1, and TCP). And, the genes and metabolites from phenylpropanoid and flavonoid metabolism and biosynthesis were up-regulated and largely accumulated in TZ and HW, respectively. The findings of this research provide a basis for comprehending the buildup of metabolites and the molecular regulatory processes of HW formation in A. melanoxylon.

Physiological, Biochemical, and Molecular Analyses Reveal Dark Heartwood Formation Mechanism in Acacia melanoxylon.

Acacia melanoxylon is highly valued for its commercial applications, with the heartwood exhibiting a range of colors from dark to light among its various clones. The underlying mechanisms contributing to this color variation, however, have not been fully elucidated. In an effort to understand the factors that influence the development of dark heartwood, a comparative analysis was conducted on the microstructure, substance composition, differential gene expression, and metabolite profiles in the sapwood (SW), transition zone (TZ), and heartwood (HW) of two distinct clones, SR14 and SR25. A microscopic examination revealed that heartwood color variations are associated with an increased substance content within the ray parenchyma cells. A substance analysis indicated that the levels of starches, sugars, and lignin were more abundant in SP compared to HW, while the concentrations of phenols, flavonoids, and terpenoids were found to be higher in HW than in SP. Notably, the dark heartwood of the SR25 clone exhibited greater quantities of phenols and flavonoids compared to the SR14 clone, suggesting that these compounds are pivotal to the color distinction of the heartwood. An integrated analysis of transcriptome and metabolomics data uncovered a significant accumulation of sinapyl alcohol, sinapoyl aldehyde, hesperetin, 2', 3, 4, 4', 6'-peptahydroxychalcone 4'-O-glucoside, homoeriodictyol, and (2S)-liquiritigenin in the heartwood of SR25, which correlates with the up-regulated expression of CCRs (evm.TU.Chr3.1751, evm.TU.Chr4.654_667, evm.TU.Chr4.675, evm.TU.Chr4.699, and evm.TU.Chr4.704), COMTs (evm.TU.Chr13.3082, evm.TU.Chr13.3086, and evm.TU.Chr7.1411), CADs (evm.TU.Chr10.2175, evm.TU.Chr1.3453, and evm.TU.Chr8.1600), and HCTs (evm.TU.Chr4.1122, evm.TU.Chr4.1123, evm.TU.Chr8.1758, and evm.TU.Chr9.2960) in the TZ of A. melanoxylon. Furthermore, a marked differential expression of transcription factors (TFs), including MYBs, AP2/ERFs, bHLHs, bZIPs, C2H2s, and WRKYs, were observed to be closely linked to the phenols and flavonoids metabolites, highlighting the potential role of multiple TFs in regulating the biosynthesis of these metabolites and, consequently, influencing the color variation in the heartwood. This study facilitates molecular breeding for the accumulation of metabolites influencing the heartwood color in A. melanoxylon, and offers new insights into the molecular mechanisms underlying heartwood formation in woody plants.

Rhizobium acaciae sp. nov., a new nitrogen-fixing symbiovar isolated from root nodules of Acacia saligna in Tunisia.

Three bacterial strains, 1AS11T, 1AS12 and 1AS13, members of the new symbiovar salignae and isolated from root nodules of Acacia saligna grown in Tunisia, were characterized using a polyphasic approach. All three strains were assigned to the Rhizobium leguminosarum complex on the basis of rrs gene analysis. Phylogenetic analysis based on 1734 nucleotides of four concatenated housekeeping genes (recA, atpD, glnII and gyrB) showed that the three strains were distinct from known rhizobia species of the R. leguminosarum complex and clustered as a separate clade within this complex. Phylogenomic analysis of 92 up-to-date bacterial core genes confirmed the unique clade. The digital DNA-DNA hybridization and blast-based average nucleotide identity values for the three strains and phylogenetically related Rhizobium species ranged from 35.9 to 60.0% and 87.16 to 94.58 %, which were lower than the 70 and 96% species delineation thresholds, respectively. The G+C contents of the strains were 60.82-60.92 mol% and the major fatty acids (>4 %) were summed feature 8 (57.81 %; C18 : 1 ω7c) and C18 : 1 ω7c 11-methyl (13.24%). Strains 1AS11T, 1AS12 and 1AS13 could also be differentiated from their closest described species (Rhizobium indicum, Rhizobium laguerreae and Rhizobium changzhiense) by phenotypic and physiological properties as well as fatty acid content. Based on the phylogenetic, genomic, physiological, genotypic and chemotaxonomic data presented in this study, strains 1AS11T, 1AS12 and 1AS13 represent a new species within the genus Rhizobium and we propose the name Rhizobium acaciae sp. nov. The type strain is 1AS11T (=DSM 113913T=ACCC 62388T).

Integrative analysis of physiology and genomics provides insights into freeze tolerance adaptations of Acacia koa along an elevational cline.

Natural selection for plant species in heterogeneous environments creates genetic variation for traits such as cold tolerance. While physiological or molecular analyses have been used to evaluate stress tolerance adaptations, combining these approaches may provide deeper insight. Acacia koa (koa) occurs from sea level to 2300 m in Hawai'i, USA. At high elevations, natural koa populations have declined due to deforestation, and freeze tolerance is a limiting factor for tree regeneration. We used physiology and molecular analyses to evaluate cold tolerance of koa populations from low (300-750 m), middle (750-1500 m), and high elevations (1500-2100 m). Half of the seedlings were cold acclimated by exposure to progressively lowered air temperatures for eight weeks (from 25.6/22.2°C to 8/4°C, day/night). Using the whole plant physiology-freezing test and koa C-repeat Binding Factor CBF genes, our results indicated that koa can be cold-acclimated when exposed to low, non-freezing temperatures. Seedlings from high elevations had consistently higher expression of Koa CBF genes associated with cold tolerance, helping to explain variation in cold-hardy phenotypes. Evaluation of the genetic background of 22 koa families across the elevations with low coverage RNA sequencing indicated that high elevation koa had relatively low values of heterozygosity, suggesting that adaptation is more likely to arise in the middle and low elevation sources. This physiology and molecular data for cold tolerance of koa across the elevation gradient of the Hawaiian Islands provides insights into natural selection processes and may help to support guidelines for conservation and seed transfer in forest restoration efforts.

Phenotypic, molecular, and symbiotic characterization of the rhizobial symbionts isolated from Acacia saligna grown in different regions in Morocco: a multivariate approach.

The introduced species Acacia saligna is a very promiscuous host as it can be efficiently nodulated with a wide range diversity of rhizobia taxa, including both fast and slow-growing strains. Fourteen nitrogen (N)-fixing bacteria were isolated from root nodules of wild Acacia saligna growing in distinct geographic locations in Morocco and were examined for their symbiotic efficiency and phenotypic properties. Multivariate tools, such as principal component analysis (PCA) and hierarchical clustering analysis (HCA), were used to study the correlation between phenotypic and symbiotic variables and discriminate and describe the similarities between different isolated bacteria with respect to all the phenotypic and symbiotic variables. Phenotypic characterization showed a variable response to extreme temperature, salinity and soil pH. At the plant level, the nodulation, nitrogen fixation, and the shoot and root dry weights were considered. The obtained results show that some of the tested isolates exhibit remarkable tolerances to the studied abiotic stresses while showing significant N2 fixation, indicating their usefulness as effective candidates for the inoculation of acacia trees. The PCA also allowed showing the isolates groups that present a similarity with evaluated phenotypic and symbiotic parameters. The genotypic identification of N2-fixing bacteria, carried out by the 16S rDNA approach, showed a variable genetic diversity among the 14 identified isolates, and their belonging to three different genera, namely Agrobacterium, Phyllobacterium and Rhizobium.

Development and evolution of age-dependent defenses in ant-acacias.

Age-dependent changes in plant defense against herbivores are widespread, but why these changes exist remains a mystery. We explored this question by examining a suite of traits required for the interaction between swollen thorn acacias (genus Vachellia) and ants of the genus Pseudomyrmex In this system, plants provide ants with refuge and food in the form of swollen stipular spines, protein-lipid-rich "Beltian" bodies, and sugar-secreting extrafloral nectaries-the "swollen thorn syndrome." We show that this syndrome develops at a predictable time in shoot development and is tightly associated with the temporal decline in the microRNAs miR156 and miR157 and a corresponding increase in their targets-the SPL transcription factors. Growth under reduced light intensity delays both the decline in miR156/157 and the development of the swollen thorn syndrome, supporting the conclusion that these traits are controlled by the miR156-SPL pathway. Production of extrafloral nectaries by Vachellia sp. that do not house ants is also correlated with a decline in miR156/157, suggesting that this syndrome evolved by co-opting a preexisting age-dependent program. Along with genetic evidence from other model systems, these findings support the hypothesis that the age-dependent development of the swollen thorn syndrome is a consequence of genetic regulation rather than a passive developmental pattern arising from developmental constraints on when these traits can develop.

Highly diverse and highly successful: invasive Australian acacias have not experienced genetic bottlenecks globally.

BACKGROUND AND AIMS: Invasive species may undergo rapid evolution despite very limited standing genetic diversity. This so-called genetic paradox of biological invasions assumes that an invasive species has experienced (and survived) a genetic bottleneck and then underwent local adaptation in the new range. In this study, we test how often Australian acacias (genus Acacia), one of the world's most problematic invasive tree groups, have experienced genetic bottlenecks and inbreeding. METHODS: We collated genetic data from 51 different genetic studies on Acacia species to compare genetic diversity between native and invasive populations. These studies analysed 37 different Acacia species, with genetic data from the invasive ranges of 11 species, and data from the native range for 36 species (14 of these 36 species are known to be invasive somewhere in the world, and the other 22 are not known to be invasive). KEY RESULTS: Levels of genetic diversity are similar in native and invasive populations, and there is little evidence of invasive populations being extensively inbred. Levels of genetic diversity in native range populations also did not differ significantly between species that have and that do not have invasive populations. CONCLUSION: We attribute our findings to the impressive movement, introduction effort and human usage of Australian acacias around the world.

Genetic diversity and population structure of six South African Acacia mearnsii breeding populations based on SSR markers.

Black wattle (Acacia mearnsii) has great economic value as a commercial source of tannins, timber and a source of firewood for local and international markets. It has been suggested that to maximize the genetic gain of A. mearnsii plantations in South Africa, the gene pool that exist within ICFR needs to be broadened via introduction of new genotypes with diverse traits. In this work, 282 A. mearnsii samples sourced from the ICFR breeding program were genotyped using 11 cross-species SSR markers. Our results showed low to moderate genetic differentiation (FST) among the six breeding subpopulations, with positive inbreeding (FIS) values that could be attributed to an historical inbreeding event. Low levels of relatedness could however indicate some mechanism of inbreeding avoidance. The effects from a recent supplementation of genetic material from two native Australian populations were observed through genetic structuring analyses. Analysis of molecular variance (AMOVA) revealed that significant genetic variation was mainly distributed within populations (75%) and among individuals (23%). The results provide significant information on A. mearnsii population genetic diversity and structure, which can be used for conservation of the current subpopulations and future tree improvement programs.

Ghosts from the past: even comprehensive sampling of the native range may not be enough to unravel the introduction history of invasive species-the case of Acacia dealbata invasions in South Africa.

PREMISE OF THE STUDY: Knowledge about the introduction history (source(s), number and size of introduction events) of an invasive species is a crucial prerequisite to understand invasion success and to facilitate effective and sustainable management approaches, especially for effective biological control. We investigated the introduction history of the Australian legume tree Acacia dealbata in South Africa. Results of this study will not only provide critical information for the management of this species in South Africa, but will also broaden our overall knowledge on the invasion ecology of this globally important invasive tree. METHODS: We used nuclear microsatellite markers to compare the genetic diversity and structure between 42 native Australian and 18 invasive South African populations and to test different and competing introduction scenarios using Approximate Bayesian Computation analyses. KEY RESULTS: Australian populations were characterized by two distinct genetic clusters, while South African populations lacked any clear genetic structure and showed significantly lower levels of genetic diversity compared to native range populations. South African populations were also genetically divergent from native populations and the most likely introduction scenario indicated an unknown source population. CONCLUSIONS: Although we cannot definitely prove the cause of the observed genetic novelty/diversification in South African Acacia dealbata populations, it cannot be attributable to insufficient sampling of native populations. Our study highlights the complexity of unravelling the introduction histories of commercially important alien species.

Isolation and Lack of Potential Mates may Threaten an Endangered Arid-Zone Acacia.

Clonality may provide reproductive assurance for many threatened plants while limiting sexual reproductive success either through energetic tradeoffs or because clones are self-incompatible. Most stands of the Australian arid-zone plant Acacia carneorum, flower annually but low seed set and an absence of sexual recruitment now suggest that this species and other, important arid-zone ecosystem engineers may have low genotypic diversity. Indeed, our recent landscape-scale genetic study revealed that stands are typically monoclonal, with genets usually separated by kilometers. An inability to set sexually produced seed or a lack of genetically diverse mates may explain almost system-wide reproductive failure. Here, using microsatellite markers, we genotyped 100 seeds from a rare fruiting stand (Middle-Camp), together with all adult plants within it and its 4 neighboring stands (up to 5 km distant). As expected, all stands surveyed were monoclonal. However, the Middle-Camp seeds were generated sexually. Comparing seed genotypes with the single Middle-Camp genotype and those of genets from neighboring and other regional stands (n = 26), revealed that 73 seeds were sired by the Middle-Camp genet. Within these Middle-Camp seeds we detected 19 genotypes in proportions consistent with self-fertilization of that genet. For the remaining 27 seeds, comprising 8 different genotypes, paternity was assigned to the nearest neighboring stands Mallee and Mallee-West, approximately 1 km distant. Ironically, given this species' vast geographic range, a small number of stands with reproductively compatible near neighbors may provide the only sources of novel genotypes.

Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia.

Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation or slow genome evolution has resulted in limited sequence variation. However, the integration of these types of data for large scale phylogenetic studies has rarely been investigated. Here we conduct a phylogenetic analysis of the whole chloroplast genome and two nuclear ribosomal loci for 65 Acacia species from across the most recent Acacia phylogeny. We then combine this data with previously generated amplicon sequences (four chloroplast loci and two nuclear ribosomal loci) for 508 Acacia species. We use several phylogenetic methods, including maximum likelihood bootstrapping (with and without constraint) and ExaBayes, in order to determine the success of combining a dataset of 4000bp with one of 189,000bp. The results of our study indicate that the inclusion of whole genome data gave a far better resolved and well supported representation of the phylogenetic relationships within Acacia than using only amplicon sequences, with the greatest support observed when using a whole genome phylogeny as a constraint on the amplicon sequences. Our study therefore provides methods for optimal integration of genomic and amplicon sequences.

Detailed Chemical Composition of Condensed Tannins via Quantitative (31)P NMR and HSQC Analyses: Acacia catechu, Schinopsis balansae, and Acacia mearnsii.

The chemical composition of Acacia catechu, Schinopsis balansae, and Acacia mearnsii proanthocyanidins has been determined using a novel analytical approach that rests on the concerted use of quantitative (31)P NMR and two-dimensional heteronuclear NMR spectroscopy. This approach has offered significant detailed information regarding the structure and purity of these complex and often elusive proanthocyanidins. More specifically, rings A, B, and C of their flavan-3-ol units show well-defined and resolved absorbance regions in both the quantitative (31)P NMR and HSQC spectra. By integrating each of these regions in the (31)P NMR spectra, it is possible to identify the oxygenation patterns of the flavan-3-ol units. At the same time it is possible to acquire a fingerprint of the proanthocyanidin sample and evaluate its purity via the HSQC information. This analytical approach is suitable for both the purified natural product proanthocyanidins and their commercial analogues. Overall, this effort demonstrates the power of the concerted use of these two NMR techniques for the structural elucidation of natural products containing labile hydroxy protons and a carbon framework that can be traced out via HSQC.

Tolerance, arsenic uptake, and oxidative stress in Acacia farnesiana under arsenate-stress.

Acacia farnesiana is a shrub widely distributed in soils heavily polluted with arsenic in Mexico. However, the mechanisms by which this species tolerates the phytotoxic effects of arsenic are unknown. This study aimed to investigate the tolerance and bioaccumulation of As by A. farnesiana seedlings exposed to high doses of arsenate (AsV) and the role of peroxidases (POX) and glutathione S-transferases (GST) in alleviating As-stress. For that, long-period tests were performed in vitro under different AsV treatments. A. farnesiana showed a remarkable tolerance to AsV, achieving a half-inhibitory concentration (IC50) of about 2.8 mM. Bioaccumulation reached about 940 and 4380 mg As·kg(-1) of dry weight in shoots and roots, respectively, exposed for 60 days to 0.58 mM AsV. Seedlings exposed to such conditions registered a growth delay during the first 15 days, when the fastest As uptake rate (117 mg kg(-1) day(-1)) occurred, coinciding with both the highest rate of lipid peroxidation and the strongest up-regulation of enzyme activities. GST activity showed a strong correlation with the As bioaccumulated, suggesting its role in imparting AsV tolerance. This study demonstrated that besides tolerance to AsV, A. farnesiana bioaccumulates considerable amounts of As, suggesting that it may be useful for phytostabilization purposes.

PhyloJIVE: Integrating biodiversity data with the Tree of Life.

MOTIVATION: Rich collections of biodiversity information such as spatial distributions, species descriptions and trait data are now synthesized in publicly available online sources such as GBIF. Also phylogenetic knowledge now provides a sound understanding of the origin of organisms and their place in the tree of life. We demonstrate with PhyloJIVE that any phylogenetic tree can be linked to online biodiversity data in the browser. This evolutionary view of biodiversity data is demonstrated in a case study that suggests that this approach may be useful to scientists and non-experts users.

The critical role of ants in the extensive dispersal of Acacia seeds revealed by genetic parentage assignment.

Ants are prominent seed dispersal agents in many ecosystems, and dispersal distances are small in comparison with vertebrate dispersal agents. However, the distance and distribution of ant-mediated dispersal in arid/semi-arid environments remains poorly explored. We used microsatellite markers and parentage assignment to quantify the distance and distribution of dispersed seeds of Acacia karina, retrieved from the middens of Iridomyrmex agilis and Melophorus turneri perthensis. From parentage assignment, we could not distinguish the maternal from each parent pair assigned to each seed, so we applied two approaches to estimate dispersal distances, one conservative (CONS), where the parent closest to the ant midden was considered to be maternal, and the second where both parents were deemed equally likely (EL) to be maternal, and used both distances. Parentage was assigned to 124 seeds from eight middens. Maximum seed dispersal distances detected were 417 m (CONS) and 423 m (EL), more than double the estimated global maximum. Mean seed dispersal distances of 40 m (±5.8 SE) (CONS) and 79 m (±6.4 SE) (EL) exceeded the published global average of 2.24 m (±7.19 SD) by at least one order of magnitude. For both approaches and both ant species, seed dispersal was predominantly (44-84% of all seeds) within 50 m from the maternal source, with fewer dispersal events at longer distances. Ants in this semi-arid environment have demonstrated a greater capacity to disperse seeds than estimated elsewhere, which highlights their important role in this system, and suggests significant novel ecological and evolutionary consequences for myrmecochorous species in arid/semi-arid Australia.

Relatedness defies biogeography: the tale of two island endemics (Acacia heterophylla and A. koa).

Despite the normally strong link between geographic proximity and relatedness of recently diverged taxa, truly puzzling biogeographic anomalies to this expectation exist in nature. Using a dated phylogeny, population genetic structure and estimates of ecological niche overlap, we tested the hypothesis that two geographically very disjunct, but morphologically very similar, island endemics (Acacia heterophylla from Réunion Island and A. koa from the Hawaiian archipelago) are the result of dispersal between these two island groups, rather than independent colonization events from Australia followed by convergent evolution. Our genetic results indicated that A. heterophylla renders A. koa paraphyletic and that the former colonized the Mascarene archipelago directly from the Hawaiian Islands ≤ 1.4 million yr ago. This colonization sequence was corroborated by similar ecological niches between the two island taxa, but not between A. melanoxylon from Australia (a sister, and presumed ancestral, taxon to A. koa and A. heterophylla) and Hawaiian A. koa. It is widely accepted that the long-distance dispersal of plants occurs more frequently than previously thought. Here, however, we document one of the most exceptional examples of such dispersal. Despite c. 18 000 km separating A. heterophylla and A. koa, these two island endemics from two different oceans probably represent a single taxon as a result of recent extreme long-distance dispersal.

Extensive long-distance pollen dispersal and highly outcrossed mating in historically small and disjunct populations of Acacia woodmaniorum (Fabaceae), a rare banded iron formation endemic.

BACKGROUND AND AIMS: Understanding patterns of pollen dispersal and variation in mating systems provides insights into the evolutionary potential of plant species and how historically rare species with small disjunct populations persist over long time frames. This study aims to quantify the role of pollen dispersal and the mating system in maintaining contemporary levels of connectivity and facilitating persistence of small populations of the historically rare Acacia woodmaniorum. METHODS: Progeny arrays of A. woodmaniorum were genotyped with nine polymorphic microsatellite markers. A low number of fathers contributed to seed within single pods; therefore, sampling to remove bias of correlated paternity was implemented for further analysis. Pollen immigration and mating system parameters were then assessed in eight populations of varying size and degree of isolation. KEY RESULTS: Pollen immigration into small disjunct populations was extensive (mean minimum estimate 40 % and mean maximum estimate 57 % of progeny) and dispersal occurred over large distances (≤1870m). Pollen immigration resulted in large effective population sizes and was sufficient to ensure adaptive and inbreeding connectivity in small disjunct populations. High outcrossing (mean tm = 0·975) and a lack of apparent inbreeding suggested that a self-incompatibility mechanism is operating. Population parameters, including size and degree of geographic disjunction, were not useful predictors of pollen dispersal or components of the mating system. CONCLUSIONS: Extensive long-distance pollen dispersal and a highly outcrossed mating system are likely to play a key role in maintaining genetic diversity and limiting negative genetic effects of inbreeding and drift in small disjunct populations of A. woodmaniorum. It is proposed that maintenance of genetic connectivity through habitat and pollinator conservation will be a key factor in the persistence of this and other historically rare species with similar extensive long-distance pollen dispersal and highly outcrossed mating systems.

Isolation and characterization of CCoAOMT in interspecific hybrid of Acacia auriculiformis x Acacia mangium--a key gene in lignin biosynthesis.

This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.

Chromosome-level genome of the transformable northern wattle, Acacia crassicarpa.

The genus Acacia is a large group of woody legumes containing an enormous amount of morphological diversity in leaf shape. This diversity is at least in part the result of an innovation in leaf development where many Acacia species are capable of developing leaves of both bifacial and unifacial morphologies. While not unique in the plant kingdom, unifaciality is most commonly associated with monocots, and its developmental genetic mechanisms have yet to be explored beyond this group. In this study, we identify an accession of Acacia crassicarpa with high regeneration rates and isolate a clone for genome sequencing. We generate a chromosome-level assembly of this readily transformable clone, and using comparative analyses, confirm a whole-genome duplication unique to Caesalpinoid legumes. This resource will be important for future work examining genome evolution in legumes and the unique developmental genetic mechanisms underlying unifacial morphogenesis in Acacia.