Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability.

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up-regulate biosynthesis of two low molecular weight metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - that play key roles in metal transport and nutrition. The CE-OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X-ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field-grown CE-OsNAS2 grain and positively correlated with NA and DMA concentrations.

Diurnal Changes in Transcript and Metabolite Levels during the Iron Deficiency Response of Rice.

BACKGROUND: Rice (Oryza sativa L.) is highly susceptible to iron (Fe) deficiency due to low secretion levels of the mugineic acid (MA) family phytosiderophore (PS) 2'-deoxymugineic acid (DMA) into the rhizosphere. The low levels of DMA secreted by rice have proved challenging to measure and, therefore, the pattern of DMA secretion under Fe deficiency has been less extensively studied relative to other graminaceous monocot species that secrete high levels of PS, such as barley (Hordeum vulgare L.). RESULTS: Gene expression and metabolite analyses were used to characterise diurnal changes occurring during the Fe deficiency response of rice. Iron deficiency inducible genes involved in root DMA biosynthesis and secretion followed a diurnal pattern with peak induction occurring 3-5 h after the onset of light; a result consistent with that of other Strategy II plant species such as barley and wheat. Furthermore, triple quadrupole mass spectrometry identified 3-5 h after the onset of light as peak time of DMA secretion from Fe-deficient rice roots. Metabolite profiling identified accumulation of amines associated with metal chelation, metal translocation and plant oxidative stress responses occurring with peak induction 10-12 h after the onset of light. CONCLUSION: The results of this study confirmed that rice shares a similar peak time of Fe deficiency associated induction of DMA secretion compared to other Strategy II plant species but has less prominent daily fluctuations of DMA secretion. It also revealed metabolic changes associated with the remediation of Fe deficiency and mitigation of damage from resulting stress in rice roots. This study complements previous studies on the genetic changes in response to Fe deficiency in rice and constitutes an important advance towards our understanding of the molecular mechanisms underlying the rice Fe deficiency response.

Using Next-Generation Sequencing to Contrast the Diet and Explore Pest-Reduction Services of Sympatric Bird Species in Macadamia Orchards in Australia.

Worldwide, avian communities inhabiting agro-ecosystems are threatened as a consequence of agricultural intensification. Unravelling their ecological role is essential to focus conservation efforts. Dietary analysis can elucidate bird-insect interactions and expose avian pest-reduction services, thus supporting avian conservation. In this study, we used next-generation sequencing to analyse the dietary arthropod contents of 11 sympatric bird species foraging in macadamia orchards in eastern Australia. Across all species and based on arthropod DNA sequence similarities ≥98% with records in the Barcode of Life Database, 257 operational taxonomy units were assigned to 8 orders, 40 families, 90 genera and 89 species. These taxa included 15 insect pests, 5 of which were macadamia pests. Among the latter group, Nezara viridula (Pentatomidae; green vegetable bug), considered a major pest, was present in 23% of all faecal samples collected. Results also showed that resource partitioning in this system is low, as most bird species shared large proportion of their diets by feeding primarily on lepidopteran, dipteran and arachnids. Dietary composition differed between some species, most likely because of differences in foraging behaviour. Overall, this study reached a level of taxonomic resolution never achieved before in the studied species, thus contributing to a significant improvement in the avian ecological knowledge. Our results showed that bird communities prey upon economically important pests in macadamia orchards. This study set a precedent by exploring avian pest-reduction services using next-generation sequencing, which could contribute to the conservation of avian communities and their natural habitats in agricultural systems.

Association of Increased Grain Iron and Zinc Concentrations with Agro-morphological Traits of Biofortified Rice.

Biofortification of rice (Oryza sativa L.) with micronutrients is widely recognized as a sustainable strategy to alleviate human iron (Fe) and zinc (Zn) deficiencies in developing countries where rice is the staple food. Constitutive overexpression of the rice nicotianamine synthase (OsNAS) genes has been successfully implemented to increase Fe and Zn concentrations in unpolished and polished rice grain. Intensive research is now needed to couple this high-micronutrient trait with high grain yields. We investigated associations of increased grain Fe and Zn concentrations with agro-morphological traits of backcross twice second filial (BC2F2) transgenic progeny carrying OsNAS1 or OsNAS2 overexpression constructs under indica/japonica and japonica/japonica genetic backgrounds. Thirteen agro-morphological traits were evaluated in BC2F2 transgenic progeny grown under hydroponic conditions. Concentrations of eight mineral nutrients (Fe, Zn, copper, manganese, calcium, magnesium, potassium, and phosphorus) in roots, stems/sheaths, non-flag leaves, flag leaves, panicles, and grain were also determined. A distance-based linear model (DistLM) was utilized to extract plant tissue nutrient predictors accounting for the largest variation in agro-morphological traits differing between transgenic and non-transgenic progeny. Overall, the BC2F2 transgenic progeny contained up to 148% higher Fe and 336% higher Zn concentrations in unpolished grain compared to non-transgenic progeny. However, unpolished grain concentrations surpassing 23 µg Fe g-1 and 40 µg Zn g-1 in BC2F2indica/japonica progeny, and 36 µg Fe g-1 and 56 µg Zn g1 in BC2F2japonica/japonica progeny, were associated with significant reductions in grain yield. DistLM analyses identified grain-Zn and panicle-magnesium as the primary nutrient predictors associated with grain yield reductions in the indica/japonica and japonica/japonica background, respectively. We subsequently produced polished grain from high-yield BC2F2 transgenic progeny carrying either the OsNAS1 or OsNAS2 overexpression constructs. The OsNAS2 overexpressing progeny had higher percentages of Fe and Zn in polished rice grain compared to the OsNAS1 overexpressing progeny. Results from this study demonstrate that genetic background has a major effect on the development of Fe and Zn biofortified rice. Moreover, our study shows that high-yielding rice lines with Fe and Zn biofortified polished grain can be developed by OsNAS2 overexpression and monitoring for Zn overaccumulation in the grain.

A low dose of an organophosphate insecticide causes dysbiosis and sex-dependent responses in the intestinal microbiota of the Japanese quail (Coturnix japonica).

Organophosphate insecticides have been directly or indirectly implicated in avian populations declining worldwide. Birds in agricultural environments are commonly exposed to these insecticides, mainly through ingestion of invertebrates after insecticide application. Despite insecticide exposure in birds occurring mostly by ingestion, the impact of organophosphates on the avian digestive system has been poorly researched. In this work we used the Japanese quail (Coturnix japonica) as an avian model to study short-term microbial community responses to a single dose of trichlorfon at low concentration in three sample origins of the gastrointestinal tract (GIT): caecum, large intestine and faeces. Using next-generation sequencing of 16S rRNA gene amplicons as bacterial markers, the study showed that ingestion of insecticide caused significant changes in the GIT microbiome. Specifically, microbiota composition and diversity differed between treated and untreated quail. Insecticide-associated responses in the caecum showed differences between sexes which did not occur with the other sample types. In caecal microbiota, only treated females showed significant shifts in a number of genera within the Lachnospiraceae and the Enterobacteriaceae families. The major responses in the large intestine were a significant reduction in the genus Lactobacillus and increases in abundance of a number of Proteobacteria genera. All microbial shifts in faeces occurred in phylotypes that were represented at low relative abundances. In general, changes in microbiota possibly resulted from contrasting responses towards the insecticide, either positive (e.g., biodegrading bacteria) or negative (e.g., insecticide-susceptible bacteria). This study demonstrates the significant impact that organophosphate insecticides have on the avian gut microbiota; showing that a single small dose of trichlorfon caused dysbiosis in the GIT of the Japanese quail. Further research is necessary to understand the implications on birds' health, especially in females.