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.

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.

Classical ethylene insensitive mutants of the Arabidopsis EIN2 orthologue lack the expected 'hypernodulation' response in Lotus japonicus.

Three independent ethylene insensitive mutants were selected from an EMS- mutagenized population of Lotus japonicus MG-20 (Miyakojima). The mutants, called 'Enigma', were mutated in the LjEIN2a gene from Lotus chromosome 1, sharing significant homology with Arabidopsis EIN2 (ethylene-insensitive2). All three alleles showed classical ethylene insensitivity phenotypes (e.g., Triple Response), but lacked the increased nodulation phenotype commonly associated with ethylene insensitivity. Indeed, all showed a marginal reduction in nodule number per plant, a phenotype that is enigmatic to sickle, an ethylene-insensitive EIN2 mutant in Medicago truncatula. In contrast to wild type, but similar to an ETR1-1 ethylene ethylene-insensitive transgenic of L. japonicus, enigma mutants formed nodules in between the protoxylem poles, demonstrating the influence of ethylene on radial positioning. Suppression of nodule numbers by nitrate and colonisation by mycorrhizal fungi in the enigma-1 mutant were indistinguishable from the wild-type MG-20. However, reflecting endogenous ethylene feedback, the enigma-1 mutant released more than twice the wild-type amount of ethylene. enigma-1 had a moderate reduction in growth, greater root mass (and lateral root formation), delayed flowering and ripening, smaller pods and seeds. Expression analysis of ethylene-regulated genes, such as ETR1, NRL1 (neverripe-like 1), and EIL3 in shoots and roots of enigma-1 and MG-20 illustrated that the ethylene-insensitive mutation strongly affected transcriptional responses in the root. These mutants open the possibility that EIN2 in L. japonicus, a determinate nodulating legume, acts in a more complex fashion possibly through the presence of a duplicated copy of LjEIN2.

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.

Unusual presentation of metastasis in adenocarcinoma cervix.

Metastatic carcinoma in an abdominal wall incision from carcinoma cervix is an uncommon and often a preterminal event. It has been reported mostly in advanced cases, often previously treated with radiotherapy. Here a case of cervical cancer with subcutaneous and rectus sheath recurrence 4 months after abdominal hysterectomy in a previously unsuspected case of adenocarcinoma cervix is reported. The patient was treated with excision of the metastatic masses followed by chemoradiation and is doing well at 5 months since presentation.

Role of oral amino acid supplementation in intra-uterine growth restriction and its outcome.

To compare the perinatal outcome in intra-uterine growth restriction (IUGR) with or without oral amino acid supplementation, a randomised control trial was conducted. Out of 50 mothers having IUGR, 25 received amino acid supplementation, treated as cases (group I) and rest 25 mothers with IUGR who had not received amino acid supplementation treated as controls (group II). The outcome of this study is compared in respect of (a) period of gestation at the time of delivery, (b) birth weight, (c) Apgar score, (d) neonatal intensive care unit (NICU) admission. In group I 88% continued pregnancy beyond 36 weeks and in comparison 48% delivered in between 34 and 36 weeks in group II. In group I 80% babies had birth weight between 2.5 and < 3kg, in comparison 84% babies weighing between 2 and < 2.5kg in group II. Eighty per cent of babies having Apgar score in between 7 and 10 in group I and 92% of group I cases not required any NICU admission. Oral amino acid supplementation commencing from second trimester of pregnancy significantly improves the perinatal outcome in IUGR cases.

Tree legumes as feedstock for sustainable biofuel production: Opportunities and challenges.

Concerns about future fossil fuel supplies and the environmental effects of their consumption have prompted the search for alternative sources of liquid fuels, specifically biofuels. However, it is important that the sources of such biofuel have minimal impact on global food supplies, land use, and commodity prices. Many legume trees can be grown on so-called marginal land with beneficial effects to the environment through their symbiotic interaction with "Rhizobia" and the associated process of root nodule development and biological nitrogen fixation. Once established legume trees can live for many years and some produce an annual yield of oil-rich seeds. For example, the tropical and sub-tropical legume tree Pongamia pinnata produces large seeds (∼1.5-2g) that contain about 40% oil, the quality and composition of which is regarded as highly desirable for sustainable biofuel production. Here we consider the benefits of legume trees as future energy crops, particularly in relation to their impact on nitrogen inputs and the net energy balance for biofuel production, and also ways in which these as yet fully domesticated species may be further improved for optimal use as biofuel feedstock.

A novel ABA insensitive mutant of Lotus japonicus with a wilty phenotype displays unaltered nodulation regulation.

An ABA insensitive mutant, Beyma, was isolated in Lotus japonicus MG-20 from an EMS mutagenesis population using root growth inhibition to applied ABA as the screening criterion. (The name 'Beyma' was taken from the Australian Aboriginal language, Wagiman, beyma, meaning 'drying up'.) The stable mutant that segregates as a dominant Mendelian mutation is insensitive to ABA induced inhibition of germination, vegetative growth, stomatal opening, as well as nodulation. Tissue ABA levels were normal, suggesting a sensitivity rather than biosynthesis mutation. It is slow-growing (50-70% of wild-type MG-20) and has a near-constitutive wilty phenotype associated with its inability to regulate stomatal opening. Whilst showing a wide range of ABA insensitive phenotypes, Beyma did not show alteration of nodule number control, as, in the absence of added ABA, the number and patterning (but not size) of nodules formed in the mutant were similar to that of MG-20. Split root experiments on MG-20 showed that application of ABA on one side of the root inhibited nodulation locally but not systemically. We propose that ABA is not involved directly in systemic autoregulation of nodulation (AON).

Malignant melanoma of vagina--a case report.

Malignant melanoma of vagina called mucosal lentigenous melanomas are extremely malignant. Malignant malanoma is a very rare tumour, it accounts for less than 3% of malignant tumours of vagina. Vaginal malanomas are aggressive and according to some authors they are less suitable for radical excision. Therefore wide local excision may be preferred. Multidisciplinary treatment modalities are required for more advanced lesions particularly in patients who have vaginal melanoma lesion more than 4 mm (AJCC stage IIB) in depth and high risk group.

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.