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.

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.