Characterising standard genetic parts and establishing common principles for engineering legume and cereal roots

Plant synthetic biology and cereal engineering depends on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimisation, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multi-genic traits where the expression of each transgene needs to be precisely regulated. Here we present data on the characterisation of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimisation and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimisation and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use, and also propose a general set of principles and guidelines for those engineering cereal species

Zinc mediates control of nitrogen fixation via transcription factor filamentation

20 Pág.Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.This work was supported by the project Enabling Nutrient Symbioses in Agriculture (ENSA), which is funded by Bill & Melinda Gates Agricultural Innovations (INV- 57461), the Bill & Melinda Gates Foundation and the Foreign, Commonwealth and Development Office (INV-55767), a Carlsberg Foundation grant (CF21-0139) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 834221).Open access funding provided by La Trobe University.Peer reviewe

Selenium and Lung Cancer: A Systematic Review and Meta Analysis

Selenium is a natural health product widely used in the treatment and prevention of lung cancers, but large chemoprevention trials have yielded conflicting results. We conducted a systematic review of selenium for lung cancers, and assessed potential interactions with conventional therapies.Two independent reviewers searched six databases from inception to March 2009 for evidence pertaining to the safety and efficacy of selenium for lung cancers. Pubmed and EMBASE were searched to October 2009 for evidence on interactions with chemo- or radiation-therapy. In the efficacy analysis there were nine reports of five RCTs and two biomarker-based studies, 29 reports of 26 observational studies, and 41 preclinical studies. Fifteen human studies, one case report, and 36 preclinical studies were included in the interactions analysis. Based on available evidence, there appears to be a different chemopreventive effect dependent on baseline selenium status, such that selenium supplementation may reduce risk of lung cancers in populations with lower baseline selenium status (serum<106 ng/mL), but increase risk of lung cancers in those with higher selenium (≥ 121.6 ng/mL). Pooling data from two trials yielded no impact to odds of lung cancer, OR 0.93 (95% confidence interval 0.61-1.43); other cancers that were the primary endpoints of these trials, OR 1.51 (95%CI 0.70-3.24); and all-cause-death, OR 0.93 (95%CI 0.79-1.10). In the treatment of lung cancers, selenium may reduce cisplatin-induced nephrotoxicity and side effects associated with radiation therapy.Selenium may be effective for lung cancer prevention among individuals with lower selenium status, but at present should not be used as a general strategy for lung cancer prevention. Although promising, more evidence on the ability of selenium to reduce cisplatin and radiation therapy toxicity is required to ensure that therapeutic efficacy is maintained before any broad clinical recommendations can be made in this context

Soybean nodulation regulatory peptides and methods of use

Peptide fragments of CLAVATA3/ESR-related (CLE) proteins of soybean are provided which are activators of autoregulation of nodulation in soybean. CLE30 and CLE80 peptide fragments may act systemically while CLE60 peptides may act locally in the plant root. CLE30 and CLE80 are normally induced in response to Rhizobium inoculation of the plant root, while CLE60 acts as a nitrate or ammonium sensor. Also provided are interfering mutant CLE peptides and proteins, encoding nucleic acids and constructs for genetic modification of leguminous plants as a means for control of NARK-dependent nodulation and/or nitrogen fixation, particularly with regard to major leguminous crop plants such as soybean and common bean

Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation

Systemic autoregulation of nodulation in legumes involves a root-derived signal (Q) that is perceived by a CLAVATA1-like leucine-rich repeat receptor kinase (e.g. GmNARK). Perception of Q triggers the production of a shoot-derived inhibitor that prevents further nodule development. We have identified three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) in soybean (Glycine max) that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to Bradyrhizobium-sp.-produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by Bradyrhizobium sp. inoculation and, unlike the other two GmCLE peptides, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas Bradyrhizobium sp.-induced regulation was contingent on GmNARK function in the shoot

Nitrate sensing and signaling during soybean nodulation

Legume nodulation and subsequent nitrogen fixation represent fascinating biological systems involving development, signaling and sensing (1) . We have cloned the responsible genes for two induced non-nodulation mutations (nod49/rj1 and nod139/nn5) in soybean. They control the synthesis of the two components of the putative nodulation (Nod) factor receptor in legumes, namely two divergent LysM receptor kinases (2,3). Gene assignment was confirmed by complementation. Gene duplication and functional divergence between duplicated genes was analysed. Significantly we discovered that axenic overexpression of one component (namely GmNFR1a) led to increased acid-soil tolerance for nodulation. This supports the conclusion that Nod factor recognition and signalling are the key target for acid soil, a globally significant problem. We also recognised that localised inhibition of nodulation by nitrate results from expression of a CLV3-like CLE peptide (NIC1). Transgenic overexpression of the gene in soybean led to substantial inhibition of local nodulation, but not distant nodulation on non-transgenic roots (4). This inhibition did not occur when tested in a NARK-deficient (supernodulating) background, suggesting that NARK functions in both roots and shoots, using related peptide ligands, but subject to different regulatory clues (see diagram in refs 1 and 4.) These discoveries may have significance for future soybean improvement