James Stangoulis

Constitutive Overexpression of the OsNAS Gene Family Reveals Single-Gene Strategies for Effective Iron- and Zinc-Biofortification of Rice Endosperm

Background Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g−1 Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g−1 Fe in endosperm. Methodology/Principal Findings Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g−1 Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g−1 Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm. Conclusions The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.

High-selenium wheat: biofortification for better health

The metalloid Se is ubiquitous in soils, but exists mainly in insoluble forms in high-Fe, low-pH and certain leached soils, and hence is often of limited availability to plants. Consequently, it is often supplied by plants to animals and human consumers at levels too low for optimum health. Se deficiency and suboptimality are manifested in populations as increased rates of thyroid dysfunction, cancer, severe viral diseases, cardiovascular disease and various inflammatory conditions. Se deficiency probably affects at least a billion individuals. Optimal cancer protection appears to require a supra-nutritional Se intake, and involves several mechanisms, which include promotion of apoptosis and inhibition of neo-angiogenesis. Evidence suggests that in some regions Se is declining in the food chain, and new strategies to increase its intake are required. These could include education to increase consumption of higher-Se foods, individual supplementation, food fortification, supplementation of livestock, Se fertilisation of crops and plant breeding for enhanced Se accumulation. Se levels in Australian residents and wheat appear to be above the global estimated mean. Wheat is estimated to supply nearly half the Se utilised by most Australians. Increasing the Se content of wheat represents a food systems approach that would increase population intake, with consequent probable improvement in public health and large health cost savings. The strategies that show most promise to achieve this are biofortification by Se fertilisation and breeding wheat varieties that are more efficient at increasing grain Se density. Research is needed in Australia to determine the most cost-effective fertilisation methods, and to determine the extent of genetic variability for grain Se accumulation. Before recommending large-scale fortification of the food supply with Se, it will be necessary to await the results of current intervention studies with Se on cancer, HIV and AIDS, and asthma.

Selenium concentration in wheat grain: Is there sufficient genotypic variation to use in breeding?

Selenium (Se) is an essential micronutrient for humans and animals, with antioxidant, anti-cancer and anti-viral effects, and wheat is an important dietary source of this element. In this study, surveys of Se concentration in grain of ancestral and wild relatives of wheat, wheat landrace accessions, populations, and commercial cultivars grown in Mexico and Australia were conducted. Cultivars were also grown under the same conditions to assess genotypic variation in Se density. Eleven data sets were reviewed with the aim of assessing the comparative worth of breeding compared with fertilising as a strategy to improve Se intake in human populations. Surveys and field trials that included diverse wheat germplasm as well as other cereals found grain Se concentrations in the range 5–720μgkg−1, but much of this variation was associated with spatial variation in soil selenium. This study detected no significant genotypic variation in grain Se density among modern commercial bread or durum wheat, triticale or barley varieties. However, the diploid wheat, Aegilops tauschii and rye were 42% and 35% higher, respectively, in grain Se concentration than other cereals in separate field trials, and, in a hydroponic trial, rye was 40% higher in foliar Se content than two wheat landraces. While genotypic differences may exist in modern wheat varieties, they are likely to be small in comparison with background soil variation, at least in Australia and Mexico. Field sites that are spatially very uniform in available soil Se would be needed to allow comparison of grain Se concentration and content in order to assess genotypic variation.

Selenium increases seed production in Brassica

Selenium (Se) is essential for humans and animals but is not considered to be essential for higher plants. Although researchers have found increases in vegetative growth due to fertiliser Se, there has been no definitive evidence to date of increased reproductive capacity, in terms of seed production and seed viability. The aim of this study was to evaluate seed production and growth responses to a low dose of Se (as sodium selenite, added to solution culture) compared to very low-Se controls in fast-cycling Brassica rapa L. Although there was no change in total biomass, Se treatment was associated with a 43% increase in seed production. The Se-treated Brassica plants had higher total respiratory activity in leaves and flowers, which may have contributed to higher seed production. This study provides additional evidence for a beneficial role for Se in higher plants.

Kinetic analysis of boron transport in Chara

Abstract. The permeability of biological membranes to boric acid was investigated using the giant internodal cells of the charophyte alga Chara corallina (Klein ex Will. Esk. R.D. Wood). The advantage of this system is that it is possible to distinguish between membrane transport of boron (B) and complexing of B by plant cell walls. Influx of B was found to be rapid, with equilibrium between the intracellular and extracellular phases being established after approximately 24 h when the external concentration was 50 μM. The intracellular concentration at equilibrium was 55 μM, which is consistent with passive distribution of B across the membrane along with a small amount of internal complexation. Efflux of B occurred with a similar half-time to influx, approximately 3 h, which indicates that the intracellular B was not tightly complexed. The concentration dependence of short-term influx measured with 10B-enriched boric acid was biphasic. This was tentatively attributed to the operation of two separate transport systems, a facilitated system that saturates at 5 μM, and a linear component due to simple diffusion of B through the membrane. Vmax and Km for the facilitated transport system were 135 pmol m−2 s−1 and 2 μM, respectively. The permeability coefficient for boric acid in the Chara plasmalemma estimated from the slope of the linear influx component was 4.4 × 10−7 cm s−1 which is an order of magnitude lower than computed from the ether:water partition coefficient for B.

Biofortified indica rice attains iron and zinc nutrition dietary targets in the field

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g−1 iron (Fe) and 16 μg g−1 zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g−1 Fe and 28 μg g−1 Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g−1 in glasshouse settings; in contrast, under field conditions, 4 μg g−1 was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g−1 Fe and 45.7 μg g−1 Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Selenium distribution in wheat grain, and the effect of postharvest processing on wheat selenium content

Selenium (Se) is an essential micronutrient for animals and humans, and wheat is a major dietary source of this element. It is improtant that postharvest processing losses of grain Se are minimized. This study, using grain dissection, milling with a Quadrumat mill, and baking and toasting studies, investigated the distribution of Se and other mineral nutrients in wheat grain and the effect of postharvest processing on their retention. The dissection study, although showing Se concentration to be highest in the embryo, confirmed (along with the milling study) previous findings that Se (which occurs mostly as selenomethionine in wheat grain) and S are more evenly distributed throughout the grain when compared to other mineral nutrients, and hence, lower proportions are removed in the milling residue. Postmilling processing did not affect Se concentration or content of wheat products in this study.No genotypic variability was observed for grain distribution of Se in the dissection and milling studies, in contrast to Cu, Fe., Mn, and Zn. This variability could be exploited in breeding for higher proportions of these nutrients in the endosperm to make white flour more nutritious. Further research could include grain dissection and milling studies using larger numbers of cultivars that have been grown together and a flour, extraction rate of around 70%

Quantitative trait loci for grain fructan concentration in wheat (Triticum aestivum L.)

Fructans (fructo-oligosaccharides) are prebiotics that are thought to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health. Fructans are present in the grain of wheat, a staple food crop. In the research reported here, we aimed to detect and map loci affecting grain fructan concentration in wheat using a doubled-haploid population derived from a cross between a high-fructan breeding line, Berkut, and a low-fructan cultivar, Krichauff. Fructan concentration was measured in grain samples grown at two locations in Australia and one in Kazakhstan. Fructan concentration varied widely within the population, ranging from 0.6 to 2.6% of grain dry weight, and was quite repeatable, with broad-sense heritability estimated as 0.71. With a linkage map of 528 molecular markers, quantitative trait loci (QTLs) were detected on chromosomes 2B, 3B, 5A, 6D and 7A. Of these, the QTLs on chromosomes 6D and 7A had the largest effects, explaining 17 and 27% of the total phenotypic variance, respectively, both with the favourable (high-fructan concentration) alleles contributed from Berkut. These chromosome regions had similar effects in another mapping population, Sokoll/Krichauff, with the favourable alleles contributed from Sokoll. It is concluded that grain fructan concentration of wheat can be improved by breeding and that molecular markers could be used to select effectively for favourable alleles in two regions of the wheat genome.

Temporal dynamics in wheat grain zinc distribution: is sink limitation the key?

BACKGROUND AND AIMS Enhancing the zinc (Zn) concentration in wheat (Triticum aestivum) grain is a breeding objective in order to improve human Zn nutrition. At enhanced plant Zn uptake, grain Zn levels do not increase proportionally and within the grain the endosperm Zn levels remain below grain Zn levels. This study analysed the temporal dynamics of Zn concentrations in grain tissues during grain filling to find major bottlenecks. METHODS Plants of two cultivars were grown at 1 and 5 mg Zn kg(-1) soil. Individual panicles were harvested 7, 14, 24 or 34 d after their flowering or at maturity and seeds were dissected into constituting tissues, which were analysed for Zn and other minerals. KEY RESULTS The Zn concentration of the crease was found to increase five- to nine-fold between 7 and 34 d after anthesis, while that of the endosperm decreased by 7 and 45 % when grown at 1 or 5 mg Zn kg(-1), respectively. The Zn turnover rate (d(-1)) in the crease tissues was either independent of the Zn application level or higher at the lower Zn application level, and the Zn concentration increased in the crease tissues with time during grain filling while the turnover rate gradually decreased. CONCLUSIONS There is significant within-seed control over Zn entering the seed endosperm. While the seed crease Zn concentration can be raised to very high levels by increasing external Zn supply, the endosperm Zn concentrations will not increase correspondingly. The limited transfer of Zn beyond the crease requires more research to provide further insight into the rate-determining processes and their location along the pathway from crease to the deeper endosperm.

Boron efficiency in oilseed rape: I. genotypic variation demonstrated in field and pot grown Brassica napus L. and Brassica juncea L.

Boron (B) efficiency of oilseed rape (Brassica napus) and mustard (B. juncea) genotypes was determined on a low B soil at Mt. Compass, South Australia. B efficiency was observed in oilseed rape genotypes, Zhongyou 821, Dunkeld and Zheyou 2, and in mustard genotypes Pusa Bold and CSIRO 6. Genotypes grown in the field were also grown under glass-house conditions, in pots filled with pre-washed sand extracted from the Mt. Compass field site. Two B treatments, one B adequate (0.25 mg B kg−1 soil) and one B deficient (imposed by omission) were used to indicate whether vegetative response to B could predict final yield response and provide a more convenient selection criterion for identifying B-efficient germplasm. Vegetative response of 35 d old (D35) genotypes grown in pot culture closely reflected field response, indicating the expression of B efficiency traits in early growth, and its potential use in selection.