Higher Photochemical Quenching and Better Maintenance of Carbon Dioxide Fixation Are Key Traits for Phosphorus Use Efficiency in the Wheat Breeding Line, RAC875.

Maintaining carbohydrate biosynthesis and C assimilation is critical under phosphorus (P) deficiency as inorganic P (Pi) is essential for ATP synthesis. Low available P in agricultural soils occurs worldwide and fertilizer P sources are being depleted. Thus, identifying biosynthetic traits that are favorable for P use efficiency (PUE) in crops is crucial. This study characterized agronomic traits, gas exchange, and chlorophyll traits of two wheat genotypes that differ in PUE. RAC875 was a P efficient genotype and Wyalkatchem was a P inefficient genotype. The plants were grown in pots under growth room conditions at two P levels; 10 mg P kg-1 soil (low P) and 30 mg P kg-1 soil (adequate P) and gas exchange and chlorophyll fluorescence were measured at the vegetative and booting stages using a portable photosynthesis system (LI-6800, LI-COR, United States). Results showed significant differences in some agronomic traits between the two wheat genotypes, i.e., greater leaf size and area, and a higher ratio of productive tillers to total tillers in RC875 when compared with Wyalkatchem. The CO2 response curve showed Wyalkatchem was more severely affected by low P than RAC875 at the booting stage. The relative ratio of the photosynthetic rate at low P to adequate P was also higher in RAC875 at the booting stage. Photochemical quenching (qP) in RAC875 was significantly higher when compared with Wyalkatchem at the booting stage. Maintaining CO2 fixation capacity under low P and higher qP would be associated with P efficiency in RAC875 and measuring qP could be a potential method to screen for P efficient wheat.

Genotypic Variation in the Root and Shoot Metabolite Profiles of Wheat (Triticum aestivum L.) Indicate Sustained, Preferential Carbon Allocation as a Potential Mechanism in Phosphorus Efficiency.

Changes in the levels of plant metabolites in response to nutrient deficiency is indicative of how plants utilize scarce resources. In this study, changes in the metabolite profile of roots and shoots of wheat genotypes differing in phosphorus use efficiency (PUE) was investigated. Under low P supply and at 28 days after sowing (DAS), the wheat breeding line, RAC875 (P efficient) produced 42% more shoot biomass than the wheat variety, and Wyalkatchem (P inefficient). Significant changes in the metabolite profile in leaves and roots were observed under low P supply and significant genotypic variation was evident. Under low P supply, an increase in raffinose and 1-kestose was evident in roots of both wheat genotypes, with RAC875 accumulating more when compared to Wyalkatchem. There was no significant increase in raffinose and 1-kestose in leaves when plants were grown under P deficiency. P deficiency had no significant impact on the levels of sucrose, maltose, glucose and fructose in both genotypes, and while phosphorylated sugars (glucose-6-P and fructose-6-P) remained unchanged in RAC875, in Wyalkatchem, glucose-6-P significantly decreased in roots, and fructose-6-P significantly decreased in both leaves and roots. Glycerol-3-P decreased twofold in roots of both wheat genotypes in response to low P. In roots, RAC875 exhibited significantly lower levels of fumarate, malate, maleate and itaconate than Wyalkatchem, while low P enhanced organic acid exudation in RAC875 but not in Wyalkatchem. RAC875 showed greater accumulation of aspartate, glutamine and ß-alanine in leaves than Wyalkatchem under low P supply. Greater accumulation of raffinose and 1-kestose in roots and aspartate, glutamine and ß-alanine in leaves appears to be associated with enhanced PUE in RAC875. Glucose-6-P and fructose-6-P are important for glycolysis, thus maintaining these metabolites would enable RAC875 to maintain carbohydrate metabolism and shoot biomass under P deficiency. The work presented here provides evidence that differences in metabolite profiles can be observed between wheat varieties that differ in PUE and key metabolic pathways are maintained in the efficient genotype to ensure carbon supply under P deficiency.

Changes in the Elemental and Metabolite Profile of Wheat Phloem Sap during Grain Filling Indicate a Dynamic between Plant Maturity and Time of Day.

The long distance transport of Fe and Zn in the phloem sap of wheat (Triticum aestivum L.) is the key route for seed supply, due to wheat having a xylem discontinuity. To date, our knowledge is limited on Fe and Zn homeostasis in the phloem sap during the reproductive and grain filling stages. With the use of aphid stylectomy to collect samples of phloem sap, we explored maturity and morning versus afternoon (within-day) changes in nutrient and metabolite profiles. Phloem exudate was collected from a wheat breeding line, SAMNYT16, at three times during the grain filling period and at both midday and mid-afternoon. There were significant changes in the concentration of Mg, K, Fe and Zn during the course of grain loading and there were also significant within-day differences for Fe and K concentrations in the phloem exudate during the early phases of grain development. We found that, for K and Fe, there was an increase of 1.1- and 1.4-fold, respectively, for samples taken prior to midday to those from mid-afternoon. There was also a significant decrease in K, Fe and Zn phloem sap concentration of 1.5-, 1.4- and 1.1-fold, respectively, from the start of peak grain loading to the end of grain loading. Of the 79 metabolites detected within samples of phloem exudate, 43 had significant maturity differences and 38 had significant within-day variability. Glutamine was found to increase by 3.3⁻5.9-fold from midday to mid-afternoon and citric acid was found to decrease by 1.6-fold from the start of grain loading to the end of grain loading. These two metabolites are of interest as they can complex metal ions and may play a role in long distance transport of metal ions. The work presented here gives further insight into the complex composition of the phloem sap and variability that can occur during the day and also with increasing maturity.

Metabolite profiling of wheat (Triticum aestivum L.) phloem exudate.

BACKGROUND: Biofortification of staple crops with essential micronutrients relies on the efficient, long distance transport of nutrients to the developing seed. The main route of this transport in common wheat (Triticum aestivum) is via the phloem, but due to the reactive nature of some essential micronutrients (specifically Fe and Zn), they need to form ligands with metabolites for transport within the phloem. Current methods available in collecting phloem exudate allows for small volumes (µL or nL) to be collected which limits the breadth of metabolite analysis. We present a technical advance in the measurement of 79 metabolites in as little as 19.5 nL of phloem exudate. This was achieved by using mass spectrometry based, metabolomic techniques. RESULTS: Using gas chromatography-mass spectrometry (GC-MS), 79 metabolites were detected in wheat phloem. Of these, 53 were identified with respect to their chemistry and 26 were classified as unknowns. Using the ratio of ion area for each metabolite to the total ion area for all metabolites, 39 showed significant changes in metabolite profile with a change in wheat reproductive maturity, from 8-12 to 17-21 days after anthesis. Of these, 21 were shown to increase and 18 decreased as the plant matured. An amine group derivitisation method coupled with liquid chromatography MS (LC-MS) based metabolomics was able to quantify 26 metabolites and semi-quantitative data was available for a further 3 metabolites. CONCLUSIONS: This study demonstrates that it is possible to determine metabolite profiles from extremely small volumes of phloem exudate and that this method can be used to determine variability within the metabolite profile of phloem that has occurred with changes in maturity. This is also believed to be the first report of the presence of the important metal complexing metabolite, nicotianamine in the phloem of wheat.

Nutrient variability in phloem: examining changes in K, Mg, Zn and Fe concentration during grain loading in common wheat (Triticum aestivum).

In wheat, nutrients are transported to seeds via the phloem yet access to this vascular tissue for exudate collection and quantitative analysis of elemental composition is difficult. The purest phloem is collected through the use of aphid stylectomy with volumes of exudate collected normally in the range of 20-500 nl. In this work a new method using inductively coupled plasma mass spectroscopy (ICP-MS) was developed to measure the concentration of K, Mg, Zn and Fe in volumes of wheat (Triticum aestivum, genotype Samnyt 16) phloem as small as 15.5 nl. This improved method was used to observe changes in phloem nutrient concentration during the grain loading period. There were statistically significant increases in phloem Mg and Zn concentration and a significant decrease in K over the period from 1-2 days after anthesis (DAA) to 9-12 DAA. During this period, there was no statistically significant change in phloem Fe concentration.

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 (R(2)=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.

The mechanism of boron mobility in wheat and canola phloem.

Low-molecular-weight borate complexes were isolated from canola (Brassica napus) and wheat (Triticum aestivum) phloem exudates, as well as the cytoplasm of the fresh-water alga Chara corallina, and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phloem exudate was collected from field-grown canola inflorescence stalks by shallow incision, while wheat phloem exudate was collected by aphid stylectomy. Chara cytoplasm was collected by careful manual separation of the cell wall, vacuole, and cytosolic compartments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed the presence of isotopic borate complexes, at mass-to-charge ratio of 690.22/691.22 in the canola and wheat phloem and at 300.11/301.11 in canola phloem and Chara cytoplasm. Using reference compounds, the borate complexes with mass-to-charge ratio 690.22/691.22 was identified as a bis-sucrose (Suc) borate complex in which the 4,6-hydroxyl pairs from the two alpha-glucopyranoside moieties formed an [L(2)B](-1) complex. Further investigation using liquid chromatography electrospray ionization triple quadrupole mass spectrometry analysis confirmed the presence of the bis-Suc borate complex in wheat phloem with a concentration up to 220 microm. The 300.11/301.11 complex was putatively identified as a bis-N-acetyl-serine borate complex but its concentration was below the detection limits of the liquid chromatography electrospray ionization triple quadrupole mass spectrometer so could not be quantified. The presence of borate complexes in the phloem provides a mechanistic explanation for the observed phloem boron mobility in canola and wheat and other species that transport Suc as their primary photoassimilate.