Bandana Biswas

Genetic analysis of ethylene regulation of legume nodulation.

The gaseous hormone ethylene has multiple roles in plant development and responses to external cues. Among these is the regulation of ‘Rhizobium’-induced nodulation in legumes. Extensive descriptive literature exists, but has been expanded to allow more mechanistic analysis through the application of genetics. Both mutants and transgenics displaying ethylene insensitivity have now been described, suggesting an intimate interplay of ethylene response, plant development and nodulation.

Capturing the biofuel wellhead and powerhouse: the chloroplast and mitochondrial genomes of the leguminous feedstock tree Pongamia pinnata.

Pongamia pinnata (syn. Millettia pinnata) is a novel, fast-growing arboreal legume that bears prolific quantities of oil-rich seeds suitable for the production of biodiesel and aviation biofuel. Here, we have used Illumina® ‘Second Generation DNA Sequencing (2GS)’ and a new short-read de novo assembler, SaSSY, to assemble and annotate the Pongamia chloroplast (152,968 bp; cpDNA) and mitochondrial (425,718 bp; mtDNA) genomes. We also show that SaSSY can be used to accurately assemble 2GS data, by re-assembling the Lotus japonicus cpDNA and in the process assemble its mtDNA (380,861 bp). The Pongamia cpDNA contains 77 unique protein-coding genes and is almost 60% gene-dense. It contains a 50 kb inversion common to other legumes, as well as a novel 6.5 kb inversion that is responsible for the non-disruptive, re-orientation of five protein-coding genes. Additionally, two copies of an inverted repeat firmly place the species outside the subclade of the Fabaceae lacking the inverted repeat. The Pongamia and L. japonicus mtDNA contain just 33 and 31 unique protein-coding genes, respectively, and like other angiosperm mtDNA, have expanded intergenic and multiple repeat regions. Through comparative analysis with Vigna radiata we measured the average synonymous and non-synonymous divergence of all three legume mitochondrial (1.59% and 2.40%, respectively) and chloroplast (8.37% and 8.99%, respectively) protein-coding genes. Finally, we explored the relatedness of Pongamia within the Fabaceae and showed the utility of the organellar genome sequences by mapping transcriptomic data to identify up- and down-regulated stress-responsive gene candidates and confirm in silico predicted RNA editing sites.

The value of biodiversity in legume symbiotic nitrogen fixation and nodulation for biofuel and food production.

Much of modern agriculture is based on immense populations of genetically identical or near-identical varieties, called cultivars. However, advancement of knowledge, and thus experimental utility, is found through biodiversity, whether naturally-found or induced by the experimenter. Globally we are confronted by ever-growing food and energy challenges. Here we demonstrate how such biodiversity from the food legume crop soybean (Glycine max L. Merr) and the bioenergy legume tree Pongamia (Millettia) pinnata is a great value. Legume plants are diverse and are represented by over 18,000 species on this planet. Some, such as soybean, pea and medics are used as food and animal feed crops. Others serve as ornamental (e.g., wisteria), timber (e.g., acacia/wattle) or biofuel (e.g., Pongamia pinnata) resources. Most legumes develop root organs (nodules) after microsymbiont induction that serve as their habitat for biological nitrogen fixation. Through this, nitrogen fertiliser demand is reduced by the efficient symbiosis between soil Rhizobium-type bacteria and the appropriate legume partner. Mechanistic research into the genetics, biochemistry and physiology of legumes is thus strategically essential for future global agriculture. Here we demonstrate how molecular plant science analysis of the genetics of an established food crop (soybean) and an emerging biofuel P. pinnata feedstock contributes to their utility by sustainable production aided by symbiotic nitrogen fixation.

The Role of Symbiotic Nitrogen Fixation in Sustainable Production of Biofuels

With the ever-increasing population of the world (expected to reach 9.6 billion by 2050), and altered life style, comes an increased demand for food, fuel and fiber. However, scarcity of land, water and energy accompanied by climate change means that to produce enough to meet the demands is getting increasingly challenging. Today we must use every avenue from science and technology available to address these challenges. The natural process of symbiotic nitrogen fixation, whereby plants such as legumes fix atmospheric nitrogen gas to ammonia, usable by plants can have a substantial impact as it is found in nature, has low environmental and economic costs and is broadly established. Here we look at the importance of symbiotic nitrogen fixation in the production of biofuel feedstocks; how this process can address major challenges, how improving nitrogen fixation is essential, and what we can do about it.

Genetic and genomic analysis of the tree legume Pongamia pinnata as a feedstock for biofuels.

The tree legume Pongamia {Pongamia pinnata (L.) Pierre [syn. Millettia pinnata (L.) Panigrahi]} is emerging as an important biofuels feedstock. It produces about 30 kg per tree per year of seeds, containing up to 55% oil (w/v), of which approximately 50% is oleic acid (C18:1). The capacity for biological N fixation places Pongamia in a more sustainable position than current nonlegume biofuel feedstocks. Also due to its drought and salinity tolerance, Pongamia can grow on marginal land not destined for production of food. As part of the effort to domesticate Pongamia our research group at The University of Queensland has started to develop specific genetic and genomic tools. Much of the preliminary work to date has focused on characterizing the genetic diversity of wild populations. This diversity is reflective of the outcrossing reproductive biology of Pongamia and necessitates the requirement to develop clonal propagation protocols. Both the chloroplast and mitochondrial genomes of Pongamia have been sequenced and annotated (152,968 and 425,718 bp, respectively), with similarities to previously characterized legume organelle genomes. Many nuclear genes associated with oil biosynthesis and nodulation in Pongamia have been characterized. The continued application of genetic and genomic tools will support the deployment of Pongamia as a sustainable biofuel feedstock.

Scanning the effects of ethyl methanesulfonate on the whole genome of lotus japonicus using second-generation sequencing analysis

Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm.

Nodulation Control in Legumes

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, nodulation reception systems and autoregulation of nodulation (AON; Kinkema et al., 2006). The original discovery of the AON-controlling LRR receptor kinases, GmNARK/ LjHAR1/MtSUNN, which is active in leaf tissue of several legu-mes, now has led to an analysis of the mechanism underlying the signal transduction.

Functional Genomics of the Regulation of Nodule Number in Legumes

Peter M. Gresshoff, Gustavo Gualtieri, Titeki Laniya, Arief Indrasumunar, Akira Miyahara, Sureeporn Nontachaiyapoom, Tim Wells, Bandana Biswas, Pick Kuen Chan, Paul Scott, M. Kinkema, M. Djordjevic, Dana Hoffmann, Lisette Pregelj, Diana M. Buzas, Dong Xi Li, Artem Men, Qunyi Jiang, Cheol-Ho Hwang and Bernard J. Carroll ARC Centre of Excellence for Integrative Legume Research; School of Life Sciences, and School of Molecular and Microbial Sciences and LAFS, The University of Queensland, St. Lucia, Brisbane QLD 4072, AGRF; Genome Interaction Group, RSBS, ANU, Canberra, ACT, Australia.