Development of the Channelized Optical System II for In Situ, High-Frequency Measurements of Dissolved Inorganic Carbon in Seawater.

This study describes the development of the CHANnelized Optical System II (CHANOS II), an autonomous, in situ sensor capable of measuring seawater dissolved inorganic carbon (DIC) at high frequency (up to ∼1 Hz). In this sensor, CO2 from acidified seawater is dynamically equilibrated with a pH-sensitive indicator dye encapsulated in gas-permeable Teflon AF 2400 tubing. The pH in the CO2 equilibrated indicator is measured spectrophotometrically and can be quantitatively correlated to the sample DIC. Ground-truthed field data demonstrate the sensor's capabilities in both time-series measurements and surface mapping in two coastal sites across tidal cycles. CHANOS II achieved an accuracy and precision of ±5.9 and ±5.5 µmol kg-1. The mean difference between traditional bottle and sensor measurements was -3.7 ± 10.0 (1σ) µmol kg-1. The sensor can perform calibration in situ using Certified Reference Materials (CRMs) to ensure measurement quality. The coastal time-series measurements highlight high-frequency variability and episodic biogeochemical shifts that are difficult to capture by traditional methods. Surface DIC mapping shows multiple endmembers in an estuary and highlights fine-scale spatial variabilities of DIC. The development of CHANOS II demonstrates a significant technological advance in seawater CO2 system sensing, which enables high-resolution, subsurface time-series, and profiling deployments.

Different Post-Sowing Nitrogen Management Approaches Required to Improve Nitrogen and Water Use Efficiency of Canola and Mustard.

Strategic use of nitrogen (N) may improve N use efficiency, but there is limited information on the influence of N supply at crucial growth stages on N accumulation, water use, and water use efficiency of canola and mustard. In this study, we hypothesize that genetic variation among canola and mustard can alter the response of timing and rate of post-sowing N application at targeted phenological growth stages by improving N and water use and their efficiencies. Field experiments were conducted in South Australia during two growing seasons with contrasting water availabilities. Two mustard and four canola cultivars, including two triazine tolerant (TT) and two non-TT cultivars were evaluated under different post-sowing N application strategies comprising three N rates and different timings of application. Mustard used more water than canola in the season with higher rainfall, but canola and mustard used similar amounts of water in the drier season. Nitrogen increased the water use efficiency (WUE) of canola and mustard cultivars. Nitrogen rate and timing did not influence the total water use of canola and mustard but influenced the partitioning of pre- and post-flowering water use. Even though, highest N uptake was observed in the treatment with continuous supply of N with 200 kg N ha-1 in five splits it did not influence the N efficiencies parameters which indicate that yield of canola and mustard are limited by N rate in these environments. In treatment with limited N supply, targeting N at the rosette stage improve N use efficiency of canola and mustard. However, the limited N uptake potential of mustard makes timing of N application the most important consideration whereas correct N rate should be main consideration for canola.

Estimating Biomass and Canopy Height With LiDAR for Field Crop Breeding.

Above-ground biomass (AGB) is a trait with much potential for exploitation within wheat breeding programs and is linked closely to canopy height (CH). However, collecting phenotypic data for AGB and CH within breeding programs is labor intensive, and in the case of AGB, destructive and prone to assessment error. As a result, measuring these traits is seldom a priority for breeders, especially at the early stages of a selection program. LiDAR has been demonstrated as a sensor capable of collecting three-dimensional data from wheat field trials, and potentially suitable for providing objective, non-destructive, high-throughput estimates of AGB and CH for use by wheat breeders. The current study investigates the deployment of a LiDAR system on a ground-based high-throughput phenotyping platform in eight wheat field trials across southern Australia, for the non-destructive estimate of AGB and CH. LiDAR-derived measurements were compared to manual measurements of AGB and CH collected at each site and assessed for their suitability of application within a breeding program. Correlations between AGB and LiDAR Projected Volume (LPV) were generally strong (up to r = 0.86), as were correlations between CH and LiDAR Canopy Height (LCH) (up to r = 0.94). Heritability (H2) of LPV (H2 = 0.32-0.90) was observed to be greater than, or similar to, the heritability of AGB (H2 = 0.12-0.78) for the majority of measurements. A similar level of heritability was observed for LCH (H2 = 0.41-0.98) and CH (H2 = 0.49-0.98). Further to this, measurements of LPV and LCH were shown to be highly repeatable when collected from either the same or opposite direction of travel. LiDAR scans were collected at a rate of 2,400 plots per hour, with the potential to further increase throughput to 7,400 plots per hour. This research demonstrates the capability of LiDAR sensors to collect high-quality, non-destructive, repeatable measurements of AGB and CH suitable for use within both breeding and research programs.

High-Throughput Field Imaging and Basic Image Analysis in a Wheat Breeding Programme.

Visual assessment of colour-based traits plays a key role within field-crop breeding programmes, though the process is subjective and time-consuming. Digital image analysis has previously been investigated as an objective alternative to visual assessment for a limited number of traits, showing suitability and slight improvement to throughput over visual assessment. However, easily adoptable, field-based high-throughput methods are still lacking. The aim of the current study was to produce a high-throughput digital imaging and analysis pipeline for the assessment of colour-based traits within a wheat breeding programme. This was achieved through the steps of (i) a proof-of-concept study demonstrating basic image analysis methods in a greenhouse, (ii) application of these methods to field trials using hand-held imaging, and (iii) developing a field-based high-throughput imaging infrastructure for data collection. The proof of concept study showed a strong correlation (r = 0.95) between visual and digital assessments of wheat physiological yellowing (PY) in a greenhouse environment, with both scores having similar heritability (H2 = 0.85 and 0.76, respectively). Digital assessment of hand-held field images showed strong correlations to visual scores for PY (r = 0.61 and 0.78), senescence (r = 0.74 and 0.75) and Septoria tritici blotch (STB; r = 0.76), with greater heritability of digital scores, excluding STB. Development of the high-throughput imaging infrastructure allowed for images of field plots to be collected at a rate of 7,400 plots per hour. Images of an advanced breeding trial collected with this system were analysed for canopy cover at two time-points, with digital scores correlating strongly to visual scores (r = 0.88 and 0.86) and having similar or greater heritability. This study details how high-throughput digital phenotyping can be applied to colour-based traits within field trials of a wheat breeding programme. It discusses the logistics of implementing such systems with minimal disruption to the programme, provides a detailed methodology for the basic image analysis methods utilized, and has potential for application to other field-crop breeding or research programmes.

QTL mapping of root traits in phosphorus-deficient soils reveals important genomic regions for improving NDVI and grain yield in barley.

KEY MESSAGE: Major QTLs for root rhizosheath size are not correlated with grain yield or yield response to phosphorus. Important QTLs were found to improve phosphorus efficiency. Root traits are important for phosphorus (P) acquisition, but they are often difficult to characterize and their breeding values are seldom assessed under field conditions. This has shed doubts on using seedling-based criteria of root traits to select and breed for P efficiency. Eight root traits were assessed under controlled conditions in a barley doubled-haploid population in soils differing in P levels. The population was also phenotyped for grain yield, normalized difference vegetation index (NDVI), grain P uptake and P utilization efficiency at maturity (PutEGY) under field conditions. Several quantitative traits loci (QTLs) from the root screening and the field trials were co-incident. QTLs for root rhizosheath size and root diameter explained the highest phenotypic variation in comparison to QTLs for other root traits. Shared QTLs were found between root diameter and grain yield, and total root length and PutEGY. A common major QTL for rhizosheath size and NDVI was mapped to the HvMATE gene marker on chromosome 4H. Collocations between major QTLs for NDVI and grain yield were detected on chromosomes 6H and 7H. When results from BIP and MET were combined, QTLs detected for grain yield were also those QTLs found for NDVI. QTLs qGY5H, qGY6H and qGY7Hb on 7H were robust QTLs in improving P efficiency. A selection of multiple loci may be needed to optimize the breeding outcomes due to the QTL x Environment interaction. We suggest that rhizosheath size alone is not a reliable trait to predict P efficiency or grain yield.

QTL mapping of grain yield and phosphorus efficiency in barley in a Mediterranean-like environment.

KEY MESSAGE: Key QTLs were identified for P efficiency in barley. Phosphorus efficiency and grain yield can be improved simultaneously in breeding. An important breeding goal for many crop species is improved phosphorus (P) efficiency. As in many other crops, selection for P efficient barley varieties has been slow because of inconsistent definitions of P efficiency and unknown genetic controls of P efficiency. We used two criteria to assess P efficiency in a doubled haploid Commander/Fleet population: P responsiveness (estimated as the deviation from the regression of yield with added P against yield with no added P treatment) and PUE (relative yield). Phosphorus responsiveness, PUE and grain yield were phenotyped at 0 and 30 kg P/ha in five environments. Lines consistently responsive to 30 kg P/ha across environments had the highest yield at the two P rates, and P responsiveness showed significantly higher broad sense heritability than PUE in the materials we studied. Genotyping of the population was subjected to a 9,000 single nucleotide polymorphism array and quantitative trait loci (QTLs) for P responsiveness were mapped with yield at 30 kg P/ha, which are common QTLs for yield when P was not limiting growth. The largest QTL for P responsiveness was mapped to 7HL in 2 years. PUE varied from 31 to 124 % across environments and one of the QTLs for PUE was mapped with yield at 0 kg P/ha. Our results demonstrate P responsiveness and grain yield can be improved simultaneously under high-input agricultural systems, but breeding for high PUE varieties may need to explore landrace or wild barley germplasm for low P tolerant alleles.

Increased expression of six ZIP family genes by zinc (Zn) deficiency is associated with enhanced uptake and root-to-shoot translocation of Zn in barley (Hordeum vulgare).

Low zinc (Zn) in soils reduces yield and grain Zn content. Regulation of ZRT/IRT-like protein (ZIP) family genes is a major mechanism in plant adaptation to low and fluctuating Zn in soil. Although several Zn deficiency-inducible ZIP genes are identified in cereals, there has been no systematic study on the association of Zn deficiency-induced uptake and root-to-shoot translocation with expression of ZIP family genes. We measured Zn deficiency-induced uptake and root-to-shoot translocation of Zn in barley (Hordeum vulgare) plants by resupplying 0.5 µM Zn, and quantified the transcripts of thirteen HvZIP genes. Subcellular localization and tissue-specific expression were also determined for Zn deficiency-inducible HvZIP genes. Zn deficiency enhanced the capacity of uptake and root-to-shoot translocation of Zn, and sustained the enhanced capacity for 6 d after Zn resupply. Six HvZIP genes were highly induced in roots of Zn-deficient plants, and their proteins were localized in the plasma membrane. Tissue-specific expression in roots supports their roles in uptake and root-to-shoot translocation of Zn under low Zn conditions. Our results provide a comprehensive view on the physiological roles of ZIP genes in plant adaptation to low and fluctuating Zn in soil, and pave the way for development of new strategies to improve Zn-deficiency tolerance and biofortification in cereals.

Can citrate efflux from roots improve phosphorus uptake by plants? Testing the hypothesis with near-isogenic lines of wheat.

Phosphorus (P) deficiency in some plant species triggers the release of organic anions such as citrate and malate from roots. These anions are widely suggested to enhance the availability of phosphate for plant uptake by mobilizing sparingly-soluble forms in the soil. Carazinho is an old wheat (Triticum aestivum) cultivar from Brazil, which secretes citrate constitutively from its root apices, and here we show that it also produces relatively more biomass on soils with low P availability than two recent Australian cultivars that lack citrate efflux. To test whether citrate efflux explains this phenotype, we generated two sets of near-isogenic lines that differ in citrate efflux and compared their biomass production in different soil types and with different P treatments in glasshouse experiments and field trials. Citrate efflux improved relative biomass production in two of six glasshouse trials but only at the lowest P treatments where growth was most severely limited by P availability. Furthermore, citrate efflux provided no consistent advantage for biomass production or yield in multiple field trials. Theoretical modeling indicates that the effectiveness of citrate efflux in mobilizing soil P is greater as the volume of soil into which it diffuses increases. As efflux from these wheat plants is restricted to the root apices, the potential for citrate to mobilize sufficient P to increase shoot biomass may be limited. We conclude that Carazinho has other attributes that contribute to its comparatively good performance in low-P soils.

HvZIP7 mediates zinc accumulation in barley (Hordeum vulgare) at moderately high zinc supply.

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport. The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport. HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport. The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals.

A comparison of hydroponic and soil-based screening methods to identify salt tolerance in the field in barley.

Success in breeding crops for yield and other quantitative traits depends on the use of methods to evaluate genotypes accurately under field conditions. Although many screening criteria have been suggested to distinguish between genotypes for their salt tolerance under controlled environmental conditions, there is a need to test these criteria in the field. In this study, the salt tolerance, ion concentrations, and accumulation of compatible solutes of genotypes of barley with a range of putative salt tolerance were investigated using three growing conditions (hydroponics, soil in pots, and natural saline field). Initially, 60 genotypes of barley were screened for their salt tolerance and uptake of Na(+), Cl(-), and K(+) at 150 mM NaCl and, based on this, a subset of 15 genotypes was selected for testing in pots and in the field. Expression of salt tolerance in saline solution culture was not a reliable indicator of the differences in salt tolerance between barley plants that were evident in saline soil-based comparisons. Significant correlations were observed in the rankings of genotypes on the basis of their grain yield production at a moderately saline field site and their relative shoot growth in pots at EC(e) 7.2 [Spearman's rank correlation (rs)=0.79] and EC(e) 15.3 (rs=0.82) and the crucial parameter of leaf Na(+) (rs=0.72) and Cl(-) (rs=0.82) concentrations at EC(e) 7.2 dS m(-1). This work has established screening procedures that correlated well with grain yield at sites with moderate levels of soil salinity. This study also showed that both salt exclusion and osmotic tolerance are involved in salt tolerance and that the relative importance of these traits may differ with the severity of the salt stress. In soil, ion exclusion tended to be more important at low to moderate levels of stress but osmotic stress became more important at higher stress levels. Salt exclusion coupled with a synthesis of organic solutes were shown to be important components of salt tolerance in the tolerant genotypes and further field tests of these plants under stress conditions will help to verify their potential utility in crop-improvement programmes.

Additive effects of Na+ and Cl- ions on barley growth under salinity stress.

Soil salinity affects large areas of the world's cultivated land, causing significant reductions in crop yield. Despite the fact that most plants accumulate both sodium (Na(+)) and chloride (Cl(-)) ions in high concentrations in their shoot tissues when grown in saline soils, most research on salt tolerance in annual plants has focused on the toxic effects of Na(+) accumulation. It has previously been suggested that Cl(-) toxicity may also be an important cause of growth reduction in barley plants. Here, the extent to which specific ion toxicities of Na(+) and Cl(-) reduce the growth of barley grown in saline soils is shown under varying salinity treatments using four barley genotypes differing in their salt tolerance in solution and soil-based systems. High Na(+), Cl(-), and NaCl separately reduced the growth of barley, however, the reductions in growth and photosynthesis were greatest under NaCl stress and were mainly additive of the effects of Na(+) and Cl(-) stress. The results demonstrated that Na(+) and Cl(-) exclusion among barley genotypes are independent mechanisms and different genotypes expressed different combinations of the two mechanisms. High concentrations of Na(+) reduced K(+) and Ca(2+) uptake and reduced photosynthesis mainly by reducing stomatal conductance. By comparison, high Cl(-) concentration reduced photosynthetic capacity due to non-stomatal effects: there was chlorophyll degradation, and a reduction in the actual quantum yield of PSII electron transport which was associated with both photochemical quenching and the efficiency of excitation energy capture. The results also showed that there are fundamental differences in salinity responses between soil and solution culture, and that the importance of the different mechanisms of salt damage varies according to the system under which the plants were grown.

High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress.

Despite the fact that most plants accumulate both sodium (Na(+)) and chloride (Cl(-)) ions to high concentration in their shoot tissues when grown in saline soils, most research on salt tolerance in annual plants has focused on the toxic effects of Na(+) accumulation. There have also been some recent concerns about the ability of hydroponic systems to predict the responses of plants to salinity in soil. To address these two issues, an experiment was conducted to compare the responses to Na(+) and to Cl(-) separately in comparison with the response to NaCl in a soil-based system using two varieties of faba bean (Vicia faba), that differed in salinity tolerance. The variety Nura is a salt-sensitive variety that accumulates Na(+) and Cl(-) to high concentrations while the line 1487/7 is salt tolerant which accumulates lower concentrations of Na(+) and Cl(-). Soils were prepared which were treated with Na(+) or Cl(-) by using a combination of different Na(+) salts and Cl(-) salts, respectively, or with NaCl. While this method produced Na(+)-dominant and Cl(-)-dominant soils, it unavoidably led to changes in the availability of other anions and cations, but tissue analysis of the plants did not indicate any nutritional deficiencies or toxicities other than those targeted by the salt treatments. The growth, water use, ionic composition, photosynthesis, and chlorophyll fluorescence were measured. Both high Na(+) and high Cl(-) reduced growth of faba bean but plants were more sensitive to Cl(-) than to Na(+). The reductions in growth and photosynthesis were greater under NaCl stress and the effect was mainly additive. An important difference to previous hydroponic studies was that increasing the concentrations of NaCl in the soil increased the concentration of Cl(-) more than the concentration of Na(+). The data showed that salinity caused by high concentrations of NaCl can reduce growth by the accumulation of high concentrations of both Na(+) and Cl(-) simultaneously, but the effects of the two ions may differ. High Cl(-) concentration reduces the photosynthetic capacity and quantum yield due to chlorophyll degradation which may result from a structural impact of high Cl(-) concentration on PSII. High Na(+) interferes with K(+) and Ca(2+) nutrition and disturbs efficient stomatal regulation which results in a depression of photosynthesis and growth. These results suggest that the importance of Cl(-) toxicity as a cause of reductions in growth and yield under salinity stress may have been underestimated.

Reassessment of tissue Na(+) concentration as a criterion for salinity tolerance in bread wheat.

Wheat is the most important crop grown on many of world's saline and sodic soils, and breeding for improved salinity tolerance (ST) is the only feasible way of improving yield and yield stability under these conditions. There are a number of possible mechanisms by which cereals can tolerate high levels of salinity, but these can be considered in terms of Na(+) exclusion and tissue tolerance. Na(+) exclusion has been the focus of much of the recent work in wheat, but with relatively little progress to date in developing high-yielding, salt-tolerant genotypes. Using a diverse collection of bread wheat germplasm, the present study was conducted to assess the value of tissue Na(+) concentration as a criterion for ST, and to determine whether ST differs with growth stage. Two experiments were conducted, the first with 38 genotypes and the second with 21 genotypes. A wide range of Na(+) concentrations within the roots and shoots as well as in ST were observed in both experiments. However, maintenance of growth and yield when grown with 100 mM NaCl was not correlated with the ability of a genotype to exclude Na(+) either from an individual leaf blade or from the whole shoot. The K(+) : Na(+) ratio also showed a wide range among the genotypes, but it did not explain the variation in ST among the genotypes. The results suggested that Na(+) exclusion and tissue tolerance varied independently, and there was no significant relationship between Na(+) exclusion and ST in bread wheat. Consequently, similar levels of ST may be achieved through different combinations of exclusion and tissue tolerance. Breeding for improved ST in bread wheat needs to select for traits related to both exclusion and tissue tolerance.

Drought-adaptive attributes in the Seri/Babax hexaploid wheat population.

Agronomic and physiological traits associated with drought adaptation were assessed within the Seri/Babax recombinant inbred line population, which was derived from parents similar in height and maturity but divergent in their sensitivity to drought. Field trials under different water regimes were conducted over 3 years in Mexico and under rainfed conditions in Australia. Under drought, canopy temperature (CT) was the single-most drought-adaptive trait contributing to a higher performance (r2 = 0.74, P < 0.0001), highly heritable (h2 = 0.65, P < 0.0001) and consistently associated with yield phenotypically (r = -0.75, P < 0.0001) and genetically [R (g) = -0.95, P < 0.0001]. CT epitomises a mechanism of dehydration avoidance expressed throughout the cycle and across latitudes, which can be utilised as a selection criteria to identify high-yielding wheat genotypes or as an important predictor of yield performance under drought. Early response under drought, suggested by a high association of CT with estimates of biomass at booting (r = -0.44, P < 0.0001), leaf chlorophyll (r = -0.22 P < 0.0001) and plant height (r = -0.64, P < 0.0001), contrast with the small relationships with anthesis and maturity (averaged, r = -0.10, P < 0.0001), and with osmotic potential (r = -0.20, P < 0.0001). Results suggest that the ability to extract water from the soil under increasing soil water deficit is a major attribute of drought adaptation. The genetic variation and transgressive segregation suggest further genomic and transcriptomic studies for unravelling the complex relationship between drought adaptation and performance under drought.

Relaxin increases ubiquitin-dependent degradation of fibronectin in vitro and ameliorates renal fibrosis in vivo.

Fibronectin, a large adhesive glycoprotein, is a prominent constituent of the extracellular matrix. Abnormalities in fibronectin homeostasis occur in numerous disease states, ranging from primary fibrosing conditions to neoplastic transformation. We demonstrate that fibronectin is a target protein substrate for ubiquitin-dependent degradation. Coimmunoprecipitation experiments and confocal microscopy demonstrated ubiquitin-fibronectin interaction. In an in vitro model of renal fibrosis, relaxin, an insulin-like growth factor, increased ubiquitin-dependent fibronectin degradation. Relaxin also was evaluated in an anti-glomerular basement membrane model of renal fibrosis. Animals treated with relaxin experienced renoprotection, manifested by decreased serum creatinine and proteinuria. Histological evaluation of kidney sections from animals treated with relaxin showed decreased glomerulosclerosis and interstitial fibrosis. We conclude that relaxin might be developed as a useful agent for the treatment of renal fibrosis.