Deeper roots associated with cooler canopies, higher normalized difference vegetation index, and greater yield in three wheat populations grown on stored soil water.

Simple and repeatable methods are needed to select for deep roots under field conditions. A large-scale field experiment was conducted to assess the association between canopy temperature (CT) measured by airborne thermography and rooting depth determined by the core-break method. Three wheat populations, C306×Westonia (CW), Hartog×Drysdale (HD), and Sundor×Songlen (SS), were grown on stored soil water in NSW Australia in 2017 (n=196-252). Cool and warm CT extremes ('tails') were cored after harvest (13-32% of each population). Rooting depth was significantly correlated with CT at late flowering (r= -0.25, -0.52, and -0.23 for CW, HD, and SS, respectively, P<0.05 hereafter), with normalized difference vegetation index (NDVI) at early grain filling (r=0.30-0.39), and with canopy height (r=0.23-0.48). The cool tails showed significantly deeper roots than the respective warm tails by 8.1 cm and 6.2 cm in CW and HD, and correspondingly, greater yields by an average 19% and 7%, respectively. This study highlighted that CT measured rapidly by airborne thermography or NDVI at early grain filling could be used to guide selection of lines with deeper roots to increase wheat yields. The remote measurement methods in this study were repeatable and high throughput, making them well suited to use in breeding programmes.

Rhizosheaths on wheat grown in acid soils: phosphorus acquisition efficiency and genetic control.

Rhizosheaths comprise soil bound to roots, and in wheat (Triticum aestivum L.) rhizosheath size correlates with root hair length. The aims of this study were to determine the effect that a large rhizosheath has on the phosphorus (P) acquisition by wheat and to investigate the genetic control of rhizosheath size in wheat grown on acid soil.Near-isogenic wheat lines differing in rhizosheath size were evaluated on two acid soils. The soils were fertilized with mineral nutrients and included treatments with either low or high P. The same soils were treated with CaCO3 to raise the pH and detoxify Al(3+) Genotypic differences in rhizosheath size were apparent only when soil pH was low and Al(3+) was present. On acid soils, a large rhizosheath increased shoot biomass compared with a small rhizosheath regardless of P supply. At low P supply, increased shoot biomass could be attributed to a greater uptake of soil P, but at high P supply the increased biomass was due to some other factor. Generation means analysis indicated that rhizosheath size on acid soil was controlled by multiple, additive loci. Subsequently, a quantitative trait loci (QTL) analysis of an F6 population of recombinant inbred lines identified five major loci contributing to the phenotype together accounting for over 60% of the total genetic variance. One locus on chromosome 1D accounted for 34% of the genotypic variation. Genetic control of rhizosheath size appears to be relatively simple and markers based on the QTL provide valuable tools for marker assisted breeding.

Early vigour improves phosphate uptake in wheat.

Quantitative trait loci (QTLs) for shoot biomass were identified in wheat grown on a soil high in total phosphorus (P) but low in plant-available P. The two populations screened included recombinant inbred lines (RILs) from Chuan-Mai 18/Vigour 18 and doubled-haploid lines from Kukri/Janz. Glasshouse-grown plants were harvested at the five-leaf stage. Seven QTLs for shoot biomass were identified in the RILs, with the largest on chromosome 7A accounting for 7.4% of the phenotypic variance. RILs from the upper tail had larger embryos than RILs from the lower tail. Tail lines were then grown in non-limiting P and the results indicated that early vigour and the capacity to access P contributed to the initial distribution. The influence of early vigour on P nutrition was examined further with advanced vigour lines (AVLs). The AVLs accumulated more shoot biomass, maintained lower shoot P concentrations, and showed greater P-acquisition efficiency than Vigour 18. Nine QTLs for shoot biomass were identified in the Kukri/Janz population. Two on chromosomes 4B and 4D accounted for 24.8% of the variance. Candidates underlying these QTLs are the Rht genes. We confirmed the influence of these genes using near-isogenic lines with different Rht alleles. The dwarf and semi-dwarf alleles affected shoot and root biomass at high and low P but not the efficiency of P acquisition. We conclude that early vigour contributed to the distributions in both populations. Early vigour can increase plant growth at suboptimal P and some sources can also improve the efficiency of P acquisition.

The barley anion channel, HvALMT1, has multiple roles in guard cell physiology and grain metabolism.

The barley (Hordeum vulgare) gene HvALMT1 encodes an anion channel in guard cells and in certain root tissues indicating that it may perform multiple roles. The protein localizes to the plasma membrane and facilitates malate efflux from cells when constitutively expressed in barley plants and Xenopus oocytes. This study investigated the function of HvALMT1 further by identifying its tissue-specific expression and by generating and characterizing RNAi lines with reduced HvALMT1 expression. We show that transgenic plants with 18-30% of wild-type HvALMT1 expression had impaired guard cell function. They maintained higher stomatal conductance in low light intensity and lost water more rapidly from excised leaves than the null segregant control plants. Tissue-specific expression of HvALMT1 was investigated in developing grain and during germination using transgenic barley lines expressing the green fluorescent protein (GFP) with the HvALMT1 promoter. We found that HvALMT1 is expressed in the nucellar projection, the aleurone layer and the scutellum of developing barley grain. Malate release measured from isolated aleurone layers prepared from imbibed grain was significantly lower in the RNAi barley plants compared with control plants. These data provide molecular and physiological evidence that HvALMT1 functions in guard cells, in grain development and during germination. We propose that HvALMT1 releases malate and perhaps other anions from guard cells to promote stomatal closure. The likely roles of HvALMT1 during seed development and grain germination are also discussed.

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.

Aluminium tolerance of root hairs underlies genotypic differences in rhizosheath size of wheat (Triticum aestivum) grown on acid soil.

We found significant genetic variation in the ability of wheat (Triticum aestivum) to form rhizosheaths on acid soil and assessed whether differences in aluminium (Al(3+) ) tolerance of root hairs between genotypes was the physiological basis for this genetic variation. A method was developed to rapidly screen rhizosheath size in a range of wheat genotypes. Backcrossed populations were generated from cv Fronteira (large rhizosheath) using cv EGA-Burke (small rhizosheath) as the recurrent parent. A positive correlation existed between rhizosheath size on acid soil and root hair length. In hydroponic experiments, root hairs of the backcrossed lines with large rhizosheaths were more tolerant of Al(3+) toxicity than the backcrossed lines with small rhizosheaths. We conclude that greater Al(3+) tolerance of root hairs underlies the larger rhizosheath of wheat grown on acid soil. Tolerance of the root hairs to Al(3+) was largely independent of the TaALMT1 gene which suggests that different genes encode the Al(3+) tolerance of root hairs. The maintenance of longer root hairs in acid soils is important for the efficient uptake of water and nutrients.

Major genes for Na+ exclusion, Nax1 and Nax2 (wheat HKT1;4 and HKT1;5), decrease Na+ accumulation in bread wheat leaves under saline and waterlogged conditions.

Two major genes for Na(+) exclusion in durum wheat, Nax1 and Nax2, that were previously identified as the Na(+) transporters TmHKT1;4-A2 and TmHKT1;5-A, were transferred into bread wheat in order to increase its capacity to restrict the accumulation of Na(+) in leaves. The genes were crossed from tetraploid durum wheat (Triticum turgidum ssp. durum) into hexaploid bread wheat (Triticum aestivum) by interspecific crossing and marker-assisted selection for hexaploid plants containing one or both genes. Nax1 decreased the leaf blade Na(+) concentration by 50%, Nax2 decreased it by 30%, and both genes together decreased it by 60%. The signature phenotype of Nax1, the retention of Na(+) in leaf sheaths resulting in a high Na(+) sheath:blade ratio, was found in the Nax1 lines. This conferred an extra advantage under a combination of waterlogged and saline conditions. The effect of Nax2 on lowering the Na(+) concentration in bread wheat was surprising as this gene is very similar to the TaHKT1;5-D Na(+) transporter already present in bread wheat, putatively at the Kna1 locus. The results indicate that both Nax genes have the potential to improve the salt tolerance of bread wheat.

Impact of Varying Light and Dew on Ground Cover Estimates from Active NDVI, RGB, and LiDAR.

Canopy ground cover (GC) is an important agronomic measure for evaluating crop establishment and early growth. This study evaluates the reliability of GC estimates, in the presence of varying light and dew on leaves, from three different ground-based sensors: (1) normalized difference vegetation index (NDVI) from the commercially available GreenSeeker®; (2) RGB images from a digital camera, where GC was determined as the portion of pixels from each image meeting a greenness criterion (i.e., (Green - Red)/(Green + Red) > 0); and (3) LiDAR using two separate approaches: (a) GC from LiDAR red reflectance (whereby red reflectance less than five was classified as vegetation) and (b) GC from LiDAR height (whereby height greater than 10 cm was classified as vegetation). Hourly measurements were made early in the season at two different growth stages (tillering and stem elongation), among wheat genotypes highly diverse for canopy characteristics. The active NDVI showed the least variation through time and was particularly stable, regardless of the available light or the presence of dew. In addition, between-sample-time Pearson correlations for NDVI were consistently high and significant (P < 0.0001), ranging from 0.89 to 0.98. In comparison, GC from LiDAR and RGB showed greater variation across sampling times, and LiDAR red reflectance was strongly influenced by the presence of dew. Excluding times when the light was exceedingly low, correlations between GC from RGB and NDVI were consistently high (ranging from 0.79 to 0.92). The high reliability of the active NDVI sensor potentially affords a high degree of flexibility for users by enabling sampling across a broad range of acceptable light conditions.

New phenotyping methods for screening wheat and barley for beneficial responses to water deficit.

This review considers stomatal conductance as an indicator of genotypic differences in the growth response to water stress. The benefits of using stomatal conductance are compared with photosynthetic rate and other indicators of genetic variation in water stress tolerance, along with the use of modern phenomics technologies. Various treatments for screening for genetic diversity in response to water deficit in controlled environments are considered. There is no perfect medium: there are pitfalls in using soil in pots, and in using hydroponics with ionic and non-ionic osmotica. Use of mixed salts or NaCl is recommended over non-ionic osmotica. Developments in infrared thermography provide new and feasible screening methods for detecting genetic variation in the stomatal response to water deficit in controlled environments and in the field.

Evaluation of the Phenotypic Repeatability of Canopy Temperature in Wheat Using Continuous-Terrestrial and Airborne Measurements.

Infrared canopy temperature (CT) is a well-established surrogate measure of stomatal conductance. There is ample evidence showing that genotypic variation in stomatal conductance is associated with grain yield in wheat. Our goal was to determine when CT repeatability is greatest (throughout the season and within the day) to guide CT deployment for research and wheat breeding. CT was measured continuously with ArduCrop wireless infrared thermometers from post-tillering to physiological maturity, and with airborne thermography on cloudless days from manned helicopter at multiple times before and after flowering. Our experiments in wheat, across two years contrasting for water availability, showed that repeatability for CT was greatest later in the season, during grain-filling, and usually in the afternoon. This was supported by the observation that repeatability for ArduCrop, and more so for airborne CT, was significantly associated (P < 0.0001) with calculated clear-sky solar radiation and to a lesser degree, vapor pressure deficit. Adding vapor pressure deficit to a model comprising either clear-sky solar radiation or its determinants, day-of-year and hour-of-day, made little to no improvement to the coefficient of determination. Phenotypic correlations for airborne CT afternoon sampling events were consistently high between events in the same year, more so for the year when soil water was plentiful (r = 0.7 to 0.9) than the year where soil water was limiting (r = 0.4 to 0.9). Phenotypic correlations for afternoon airborne CT were moderate between years contrasting in soil water availability (r = 0.1 to 0.5) and notably greater on two separate days following irrigation or rain in the drier year, ranging from r = 0.39 to 0.53 (P < 0.0001) for the midday events. For ArduCrop CT the pattern of phenotypic correlations, within a given year, was similar for both years: phenotypic correlations were higher during the grain-filling months of October and November and for hours-of-day from 11 onwards. The lowest correlations comprised events from hours-of-day 8 and 9 across all months. The capacity for the airborne method to instantaneously sample CT on hundreds of plots is more suited to large field experiments than the static ArduCrop sensors which measure CT continuously on a single experimental plot at any given time. Our findings provide promising support for the reliable deployment of CT phenotyping for research and wheat breeding, whereby the high repeatability and high phenotypic correlations between afternoon sampling events during grain-filling could enable reliable screening of germplasm from only one or two sampling events.

Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion.

Sodium exclusion from leaves is an important mechanism for salt tolerance in durum wheat. To characterize possible control points for Na(+) exclusion, quantitative cryo-analytical scanning electron microscopy was used to determine cell-specific ion profiles across roots of two durum wheat genotypes with contrasting rates of Na(+) transport from root to shoot grown in 50 mm NaCl. The Na(+) concentration in Line 149 (low transport genotype) declined across the cortex, being highest in the epidermal and sub-epidermal cells (48 mm) and lowest in the inner cortical cells (22 mm). Na(+) was high in the pericycle (85 mm) and low in the xylem parenchyma (34 mm). The Na(+) profile in Tamaroi (high transport genotype) had a similar trend but with a high concentration (130 mm) in the xylem parenchyma. The K(+) profiles were generally inverse to those of Na(+). Chloride was only detected in the epidermis. These data suggest that the epidermal and cortical cells removed most of the Na(+) and Cl(-) from the transpiration stream before it reached the endodermis, and that the endodermis is not the control point for salt uptake by the plant. The pericycle as well as the xylem parenchyma may be important in the control of net Na(+) loading of the xylem.

High Throughput Determination of Plant Height, Ground Cover, and Above-Ground Biomass in Wheat with LiDAR.

Crop improvement efforts are targeting increased above-ground biomass and radiation-use efficiency as drivers for greater yield. Early ground cover and canopy height contribute to biomass production, but manual measurements of these traits, and in particular above-ground biomass, are slow and labor-intensive, more so when made at multiple developmental stages. These constraints limit the ability to capture these data in a temporal fashion, hampering insights that could be gained from multi-dimensional data. Here we demonstrate the capacity of Light Detection and Ranging (LiDAR), mounted on a lightweight, mobile, ground-based platform, for rapid multi-temporal and non-destructive estimation of canopy height, ground cover and above-ground biomass. Field validation of LiDAR measurements is presented. For canopy height, strong relationships with LiDAR (r2 of 0.99 and root mean square error of 0.017 m) were obtained. Ground cover was estimated from LiDAR using two methodologies: red reflectance image and canopy height. In contrast to NDVI, LiDAR was not affected by saturation at high ground cover, and the comparison of both LiDAR methodologies showed strong association (r2 = 0.92 and slope = 1.02) at ground cover above 0.8. For above-ground biomass, a dedicated field experiment was performed with destructive biomass sampled eight times across different developmental stages. Two methodologies are presented for the estimation of biomass from LiDAR: 3D voxel index (3DVI) and 3D profile index (3DPI). The parameters involved in the calculation of 3DVI and 3DPI were optimized for each sample event from tillering to maturity, as well as generalized for any developmental stage. Individual sample point predictions were strong while predictions across all eight sample events, provided the strongest association with biomass (r2 = 0.93 and r2 = 0.92) for 3DPI and 3DVI, respectively. Given these results, we believe that application of this system will provide new opportunities to deliver improved genotypes and agronomic interventions via more efficient and reliable phenotyping of these important traits in large experiments.

Tissue tolerance: an essential but elusive trait for salt-tolerant crops.

For a plant to persist in saline soil, osmotic adjustment of all plant cells is essential. The more salt-tolerant species accumulate Na+ and Cl- to concentrations in leaves and roots that are similar to the external solution, thus allowing energy-efficient osmotic adjustment. Adverse effects of Na+ and Cl- on metabolism must be avoided, resulting in a situation known as 'tissue tolerance'. The strategy of sequestering Na+ and Cl- in vacuoles and keeping concentrations low in the cytoplasm is an important contributor to tissue tolerance. Although there are clear differences between species in the ability to accommodate these ions in their leaves, it remains unknown whether there is genetic variation in this ability within a species. This viewpoint considers the concept of tissue tolerance, and how to measure it. Four conclusions are drawn: (1) osmotic adjustment is inseparable from the trait of tissue tolerance; (2) energy-efficient osmotic adjustment should involve ions and only minimal organic solutes; (3) screening methods should focus on measuring tolerance, not injury; and (4) high-throughput protocols that avoid the need for control plants and multiple Na+ or Cl- measurements should be developed. We present guidelines to identify useful genetic variation in tissue tolerance that can be harnessed for plant breeding of salt tolerance.

Methodology for High-Throughput Field Phenotyping of Canopy Temperature Using Airborne Thermography.

Lower canopy temperature (CT), resulting from increased stomatal conductance, has been associated with increased yield in wheat. Historically, CT has been measured with hand-held infrared thermometers. Using the hand-held CT method on large field trials is problematic, mostly because measurements are confounded by temporal weather changes during the time required to measure all plots. The hand-held CT method is laborious and yet the resulting heritability low, thereby reducing confidence in selection in large scale breeding endeavors. We have developed a reliable and scalable crop phenotyping method for assessing CT in large field experiments. The method involves airborne thermography from a manned helicopter using a radiometrically-calibrated thermal camera. Thermal image data is acquired from large experiments in the order of seconds, thereby enabling simultaneous measurement of CT on potentially 1000s of plots. Effects of temporal weather variation when phenotyping large experiments using hand-held infrared thermometers are therefore reduced. The method is designed for cost-effective and large-scale use by the non-technical user and includes custom-developed software for data processing to obtain CT data on a single-plot basis for analysis. Broad-sense heritability was routinely >0.50, and as high as 0.79, for airborne thermography CT measured near anthesis on a wheat experiment comprising 768 plots of size 2 × 6 m. Image analysis based on the frequency distribution of temperature pixels to remove the possible influence of background soil did not improve broad-sense heritability. Total image acquisition and processing time was ca. 25 min and required only one person (excluding the helicopter pilot). The results indicate the potential to phenotype CT on large populations in genetics studies or for selection within a plant breeding program.

Infrared thermography in plant phenotyping for salinity tolerance.

The recent advances made in the use of infrared thermal imaging (thermography) as a non-invasive, high-throughput technique for the screening of salinity tolerance in plants is reviewed. Taking wheat seedlings as an example, the methods and protocols used to impose a homogeneous salt stress to a large number of genotypes, as well as capturing infrared images of these genotypes and automatically processing the images are described in detail in this chapter. We also present the source code of the Matlab program applied to automatically identify plants and batch process IR images.

Impact of ancestral wheat sodium exclusion genes Nax1 and Nax2 on grain yield of durum wheat on saline soils.

Nax1 and Nax2 are two genetic loci that control the removal of Na+ from the xylem and thereby help to exclude Na+ from leaves of plants in saline soil. They originate in the wheat ancestral relative Triticum monococcum L. and are not present in modern durum or bread wheat. The Nax1 and Nax2 loci carry TmHKT1;4-A2 and TmHKT1;5-A, respectively, which are the candidate genes for these functions. This paper describes the development of near-isogenic breeding lines suitable for assessing the impact of the Nax loci and their performance in controlled environment and fields of varying salinity. In young plants grown in 150mM NaCl, Nax1 reduced the leaf Na+ concentration by 3-fold, Nax2 by 2-fold and both Nax1 and Nax2 together by 4-fold. In 250mM NaCl, Nax1 promoted leaf longevity and greater photosynthesis and stomatal conductance. In the uppermost leaf, the Na+-excluding effect of the Nax loci was much stronger. In the field, Na+ in the flag leaf was reduced 100-fold by Nax1 and 4-fold by Nax2; however, Nax1 lines yielded 5-10% less than recurrent parent (cv. Tamaroi) in saline soil. In contrast, Nax2 lines had no yield penalty and at high salinity they yielded close to 25% more than Tamaroi, indicating this material is suitable for breeding commercial durum wheat with improved yield on saline soils.

Wheat grain yield on saline soils is improved by an ancestral Na⁺ transporter gene.

The ability of wheat to maintain a low sodium concentration ([Na(+)]) in leaves correlates with improved growth under saline conditions. This trait, termed Na(+) exclusion, contributes to the greater salt tolerance of bread wheat relative to durum wheat. To improve the salt tolerance of durum wheat, we explored natural diversity in shoot Na(+) exclusion within ancestral wheat germplasm. Previously, we showed that crossing of Nax2, a gene locus in the wheat relative Triticum monococcum into a commercial durum wheat (Triticum turgidum ssp. durum var. Tamaroi) reduced its leaf [Na(+)] (ref. 5). Here we show that a gene in the Nax2 locus, TmHKT1;5-A, encodes a Na(+)-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, which is therefore ideally localized to withdraw Na(+) from the xylem and reduce transport of Na(+) to leaves. Field trials on saline soils demonstrate that the presence of TmHKT1;5-A significantly reduces leaf [Na(+)] and increases durum wheat grain yield by 25% compared to near-isogenic lines without the Nax2 locus.

Characterisation of HvALMT1 function in transgenic barley plants.

HvALMT1 from barley (Hordeum vulgare L.) encodes a protein capable of facilitating the transport of malate and other organic anions when expressed in Xenopus oocytes. The HvALMT1 gene is primarily expressed in guard cells of stomata, in regions behind the root apex and at lateral root junctions. We investigated the function of HvALMT1 in planta by overexpressing it in barley under the control of a constitutive promoter. Transgenic plants expressing HvALMT1 at levels four to 9-fold greater than controls showed reduced growth and plants showing the highest expression failed to set seed. Although measurements of conductance indicated that stomatal function was not totally impaired in the transgenic plants the time taken for the stomata to close in response to low light was significantly longer compared with controls. Elemental and metabolomic analyses of the transgenic barley shoots revealed that the concentration of calcium and levels of ascorbate, serine, threonine and pentanoate were consistently greater (2- to 14-fold) in plants that overexpressed HvALMT1, whereas whole-shoot tissue levels of fumarate were significantly lower (60-85% reduction). Transgenic plants also showed significantly greater efflux of malate and succinate from their roots than control plants. Efflux of these organic anions occurred independently of Al3+ and conferred greater Al3+ resistance in solution culture and in acidic soil. These results are consistent with HvALMT1 contributing to anion homeostasis in the cytosol and osmotic adjustment by transporting organic anions out of the cell or by sequestering them into cytosolic vesicles.

A new screening method for osmotic component of salinity tolerance in cereals using infrared thermography.

A high-throughput, automated image analysis protocol for the capture, identification and analysis of thermal images acquired with a long-wave infrared (IR) camera was developed to quantify the osmotic stress response of wheat and barley to salinity. There was a strong curvilinear relationship between direct measurements of stomatal conductance and leaf temperature of barley grown in a range of salt concentrations. This indicated that thermography accurately reflected the physiological status of salt-stressed barley seedlings. Leaf temperature differences between barley grown at 200 mM NaCl and 0 mM NaCl reached 1.6°C - the sensitivity of the IR signal increasing at higher salt concentrations. Seventeen durum wheat genotypes and one barley genotype, known to vary for osmotic stress tolerance, were grown in control (no salt) and 150 mM NaCl treatments to validate the newly-developed automated thermal imaging protocol. The ranking of the 18 genotypes based on both a growth study and the IR measurements was consistent with previous reports in the literature for these genotypes. This study shows the potential of IR thermal imaging for the screening of large numbers of genotypes varying for stomatal traits, specifically those related to salt tolerance.