Improved techniques for measurement of nanolitre volumes of phloem exudate from aphid stylectomy

Background: When conducting aphid stylectomy, measuring accurate rates of phloem exudation is difficult because the volumes collected are in the nanolitre (nl) range. In a new method, exudate volume was calculated from optical measurement of droplet diameter as it forms on the tip of a severed aphid stylet. Evaporation was shown to decrease the accuracy of the measurement but was countered with the addition of water-saturated mineral oil. Volume measurements by optical estimation of the volume of a sphere suspended in oil was affected by the curvature of the oil surface. In contrast, measuring the exudate volume from optical measurement of droplet-diameter as formed on the tip of a severed aphid stylet, removes any inaccuracies due to oil surface curvature. A modified technique is proposed for measuring exudate volumes without oil by estimating the flow rate from photo-sequences of the collection period; a correction for evaporation is applied later. Results: A change in oil volume of ±1.75% from an optimum volume of 285 μl had a statistically significant effect on droplet measurement, under or over-estimating droplet volume due to optical effects caused by the oil surface. Using microscope image capture and measurement software, a modified method for measuring phloem volume in air was developed, by reducing air exposure during measurement to approximately 5 s for each measurement. Phloem volumes were measured using both techniques with measurements in air being on average 19.9 nl less (SD 18.87, p<0.001) than those made in oil, and there was a strong linear relationship (R2=0.942) between the techniques. This linear relationship enabled the development of a correction equation with no significant difference at the 5% level between corrected volumes and actual volumes measured under oil. Conclusions: This study showed that oil has a significant role in countering evaporation but oil volume must be carefully optimised for optical measurement of droplets to ensure measurement accuracy. A linear correction factor was generated to correct the volumes measured in air for loss due to evaporation and the method provides for a much simpler alternative to previous approaches for measuring exudation rates and volumes from a cut aphid stylet.Lachlan J Palmer, Lyndon T Palmer, Jeremy Pritchard, Robin D Graham and James CR Stangouli

High-throughput measurement methodologies for developing nutrient-dense crops

With the development of nutrient-dense crops comes the need for analytical methodologies to enable rapid and accurate analysis of the micronutrients of interest. The analysis of provitamin A carotenoids (pVACs) and the minerals iron (Fe) and zinc (Zn) are the focus of this chapter with the considerations and commonly employed methods discussed. When analyzing samples there are various considerations to minimise analyte degradation (in the case of provitamin A) and reduce possible contamination from external sources (for Fe and Zn). Spectroscopic and chromatographic analyses are the most common analysis approaches utilised when screening for carotenoids. Spectroscopic analyses including near-infrared spectroscopy (NIRS) and iCheck are rapid and require minimal samples preparation and provide fast analysis times. The carotenoids present in the sample is dependent on the crop analyzed and resulting number and concentration of carotenoids present will impact the final decision on suitable analysis techniques. For example, in crops with high concentrations of non-pVACs, chromatographic analysis is necessary in order to accurately quantify the micronutrients. This process is able to accurately identify and quantify individual carotenoids, but requires extensive sample preparation and often long chromatographic separation analysis. When analyzing the minerals Fe and Zn, these same techniques are not suitable, but it is still important to ensure careful sample preparation to deliver accurate analytical results. Degradation of these micronutrients is not a concern, however, possible contamination from soil/ dust/ insects can lead to inaccurate results. Commonly employed analysis such as atomic absorption spectroscopy (AAS) and Inductively Coupled Plasma-Optical Emission Spectrometry ICP-OES or Inductively Coupled Plasma-Mass spectrometry (ICP-MS) require sample digestion prior to analysis and highly pure reagents and gases. These techniques are able to analyze multiple elements and have high accuracy and sensitivity but require specialised facilities and highly trained staff. The use of high-throughput analyses to complement these high-accuracy methods include colorimetric and X-ray flourescence (XRF) technologies. These approaches enable much higher throughput with simple sample preparation and enable screening for micronutrient concentration without the need for specialised facilities.Keywords: Screening, Analysis, Carotenoid, Provitamin A, Iron, Zinc, Micronutrient analysi

Characterisation of ethylene pathway components in non-climacteric capsicum

Background: Climacteric fruit exhibit high ethylene and respiration levels during ripening but these levels are limited in non-climacteric fruit. Even though capsicum is in the same family as the well-characterised climacteric tomato (Solanaceae), it is non-climacteric and does not ripen normally in response to ethylene or if harvested when mature green. However, ripening progresses normally in capsicum fruit when they are harvested during or after what is called the ‘Breaker stage’. Whether ethylene, and components of the ethylene pathway such as 1-aminocyclopropane 1-carboxylate (ACC) oxidase (ACO), ACC synthase (ACS) and the ethylene receptor (ETR), contribute to non-climacteric ripening in capsicum has not been studied in detail. To elucidate the behaviour of ethylene pathway components in capsicum during ripening, further analysis is therefore needed. The effects of ethylene or inhibitors of ethylene perception, such as 1-methylcyclopropene, on capsicum fruit ripening and the ethylene pathway components may also shed some light on the role of ethylene in non-climacteric ripening. Results: The expression of several isoforms of ACO, ACS and ETR were limited during capsicum ripening except one ACO isoform (CaACO4). ACS activity and ACC content were also low in capsicum despite the increase in ACO activity during the onset of ripening. Ethylene did not stimulate capsicum ripening but 1-methylcyclopropene treatment delayed the ripening of Breaker-harvested fruit. Some of the ACO, ACS and ETR isoforms were also differentially expressed upon treatment with ethylene or 1-methylcyclopropene. Conclusions: ACS activity may be the rate limiting step in the ethylene pathway of capsicum which restricts ACC content. The differential expression of several ethylene pathway components during ripening and upon ethylene or 1-methylclopropene treatment suggests that the ethylene pathway may be regulated differently in non-climacteric capsicum compared to the climacteric tomato. Ethylene independent pathways may also exist in non-climacteric ripening as evidenced by the up-regulation of CaACO4 during ripening onset despite being negatively regulated by ethylene exposure. However, some level of ethylene perception may still be needed to induce ripening especially during the Breaker stage. A model of capsicum ripening is also presented to illustrate the probable role of ethylene in this non-climacteric fruit.Wan M Aizat, Jason A Able, James CR Stangoulis and Amanda J Abl

Genetic dissection of zinc, iron, copper, manganese and phosphorus in wheat (Triticum aestivum L.) grain and rachis at two developmental stages

The development of high-yielding wheat genotypes containing micronutrient-dense grains are the main prio- rities of biofortification programs. At the International Maize and Wheat Improvement Center, breeders have successfully crossed high zinc progenitors including synthetic hexaploid wheat, T. dicoccum, T. spelta and landraces to generate high-zinc varieties. In this study, we report a genome-wide association using a wheat diversity panel to dissect the genetics controlling zinc, iron, copper, manganese and phosphorus concentrations in the grain and rachis during grain development and at physiological maturity. Significant marker-trait associations (MTAs) were identified for each nutrient using multi-locus mixed model methodologies. For mature grain, markers that showed significant pleiotropic effects were found on chromo- somes 1A, 3B and 5B, of which those on chromosome 5B at ∼95.5 cM were consistent over two growing seasons. Co-located MTAs were identified for the nutrient concentrations in developing grain, rachis and mature grain on multiple chromosomes. The identified genomic regions included putative candidate genes involved in metal uptake and transport and storage protein processing. These findings add to our understanding of the genetics of the five important nutrients in wheat grain and provide information on genetic markers for selecting high mi- cronutrient genotypes.Suong T. Cua, Georgia Guilda, Alison Nicolsona, Govindan Velub, Ravi Singhb, James Stangouli

Genome wide association mapping of grain arsenic, copper, molybdenum and zinc in rice (Oryza sativa L.) grown at four international field sites

Peer reviewedPublisher PD

Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat 3 wild emmer wheat RIL population

Mineral nutrient malnutrition, and particularly deficiency in zinc and iron, afflicts over 3 billion people worldwide. Wild emmer wheat, Triticum turgidum ssp. dicoccoides, genepool harbors a rich allelic repertoire for mineral nutrients in the grain. The genetic and physiological basis of grain protein, micronutrients (zinc, iron, copper and manganese) and macronutrients (calcium, magnesium, potassium, phosphorus and sulfur) concentration was studied in tetraploid wheat population of 152 recombinant inbred lines (RILs), derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16). Wide genetic variation was found among the RILs for all grain minerals, with considerable transgressive effect. A total of 82 QTLs were mapped for 10 minerals with LOD score range of 3.2–16.7. Most QTLs were in favor of the wild allele (50 QTLs). Fourteen pairs of QTLs for the same trait were mapped to seemingly homoeologous positions, reflecting synteny between the A and B genomes. Significant positive correlation was found between grain protein concentration (GPC), Zn, Fe and Cu, which was supported by significant overlap between the respective QTLs, suggesting common physiological and/or genetic factors controlling the concentrations of these mineral nutrients. Few genomic regions (chromosomes 2A, 5A, 6B and 7A) were found to harbor clusters of QTLs for GPC and other nutrients. These identified QTLs may facilitate the use of wild alleles for improving grain nutritional quality of elite wheat cultivars, especially in terms of protein, Zn and Fe

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.Alexander A. T. Johnson, Bianca Kyriacou, Damien L. Callahan, Lorraine Carruthers, James Stangoulis, Enzo Lombi and Mark Teste

Variation in grain Zn concentration, and the grain ionome, in field-grown Indian wheat

Wheat is an important dietary source of zinc (Zn) and other mineral elements in many countries. Dietary Zn deficiency is widespread, especially in developing countries, and breeding (genetic biofortification) through the HarvestPlus programme has recently started to deliver new wheat varieties to help alleviate this problem in South Asia. To better understand the potential of wheat to alleviate dietary Zn deficiency, this study aimed to characterise the baseline effects of genotype (G), site (E), and genotype by site interactions (GxE) on grain Zn concentration under a wide range of soil conditions in India. Field experiments were conducted on a diverse panel of 36 Indian-adapted wheat genotypes, grown on a range of soil types (pH range 4.5–9.5), in 2013–14 (five sites) and 2014–15 (six sites). Grain samples were analysed using inductively coupled plasma-mass spectrometry (ICP-MS). The mean grain Zn concentration of the genotypes ranged from 24.9–34.8 mg kg-1, averaged across site and year. Genotype and site effects were associated with 10% and 6% of the overall variation in grain Zn concentration, respectively. Whilst G x E interaction effects were evident across the panel, some genotypes had consistent rankings between sites and years. Grain Zn concentration correlated positively with grain concentrations of iron (Fe), sulphur (S), and eight other elements, but did not correlate negatively with grain yield, i.e. no yield dilution was observed. Despite a relatively small contribution of genotype to the overall variation in grain Zn concentration, due to experiments being conducted across many contrasting sites and two years, our data are consistent with reports that biofortifying wheat through breeding is likely to be effective at scale given that some genotypes performed consistently across diverse soil types. Notably, all soils in this study were probably Zn deficient and interactions between wheat genotypes and soil Zn availability/management (e.g. the use of Zn-containing fertilisers) need to be better-understood to improve Zn supply in food systems