A presentation on how to develop procedures to grow healthy plants under sterile conditions, manipulate root exudation with stress, and quantify total organic carbon in exudates and determine composition of exudates using GC-MS

Bugbee, Bruce

Chard, J.

Hamilton, M.

Henry, A.

Hess, J. R.

Norton, J.

Palmer, C.

Petersen, M.

English

DigitalCommons@USU

Utah State University DigitalCommons@USU Water Stress Research 2002 Water & Nutrient Stress Increase Root Exudation A. Henry Utah State University J. Chard Utah State University J. Norton Utah State University M. Petersen Utah State University Bruce Bugbee Utah State University, bruce.bugbee@usu.edu M. Hamilton INEEL See next page for additional authors Follow this and additional works at: https://digitalcommons.usu.edu/cpl_waterstress  Part of the Plant Sciences Commons Recommended Citation Henry, A.; Chard, J.; Norton, J.; Petersen, M.; Bugbee, Bruce; Hamilton, M.; Palmer, C.; and Hess, J. R., "Water & Nutrient Stress Increase Root Exudation" (2002). Water Stress. Paper 2. https://digitalcommons.usu.edu/cpl_waterstress/2 This Presentation is brought to you for free and open access by the Research at DigitalCommons@USU. It has been accepted for inclusion in Water Stress by an authorized administrator of DigitalCommons@USU. For more information, please contact digitalcommons@usu.edu. Authors A. Henry, J. Chard, J. Norton, M. Petersen, Bruce Bugbee, M. Hamilton, C. Palmer, and J. R. Hess This presentation is available at DigitalCommons@USU: https://digitalcommons.usu.edu/cpl_waterstress/2 Water & Nutrient Stress Increase Root ExudationM. Hamilton, C. Palmer, J.R. HessA. Henry, J. Chard, J. NortonM. Petersen, B. BugbeeDevelop procedures to:• Grow healthy plants under sterile conditions• Manipulate root exudation with stress• Quantify total organic carbon in exudates • Determine composition of exudates using GC-MSObjectivesCo-metabolismIncreased plant uptakeIncreased/decreased contaminant mobility Implications for PhytoremediationOur focus for the qualitative analysis: organic acidsThe chelating properties of these compounds can be useful for phytoremediation, and they are a class of compound most likely to be found in root exudateSystem for sterile cultureFoam plugGround-glass jointAir filterSilicone stoppersAmber vialSeptumOttawa sandHPS lampsLaminar flow hoodTreatments High NH4+ K+ stress Drought  FloodingDays After Planting0 5 10 15 20 25 30 35 40 45 50 55 60 65% Sterile Plants020406080100Trial 3in growth chamberTrial 5enclosed shootTrial 4in laminar flow hoodTrial 6improved planting techniqueAssessing microbial contaminationVerifying Plate Counts:Acridine Orange Stain of LeachateClean sample Root debris10 µmContaminated sampleVerifying Plate Counts: Acridine Orange Stain of Leachatebacteria10 µmClean rootPhenolic Aniline Blue Stain of Root10 µmContaminated rootbacteriaPhenolic Aniline Blue Stain of Root10 µmTreatments startedTreatment Averagesdays after planting20 30 40 50 60 70µg C exuded per day02004006008001000K+ stressflooddroughtcontrolNH4+ treatmentThe K+ stressed plants had the highest amount of carbon exuded at any point in time.  We think this is because the plants were releasing a compound to sequester K+ from the soil. Cumulative carbon exuded per gram dry plantmg C exudedper g dry plant Percent of controlAverage Std. dev.control 2.6 0.4NH4+2.3 0.1 90K+ 3.7 0.6 144flood 3.8 0.9 145drought 4.4 0.5 170Compounds released by rootsSloughed-off cellsPrimary types of exudatesDistribution of CarbonRhizospheresandBulk sandLeachates17 %9 %63 %11 %100 %SolubleInsolubleBased on the distribution of carbon released by the roots (mostly soluble with not much left on the sand), we conclude that the exudates we’re seeing are mostly compounds released directly from the root, not whole cells released from the root.GC-MS Data: ExudatesFumaric AcidDays after planting30 35 40 45 50 55 60 65 70Concentration (mg/L)0246810droughtfloodNH4+ trtmtK+ stresscontrolMalonic AcidDays after planting30 35 40 45 50 55 60 65 70Concentration (mg/L)0246810droughtfloodNH4+ trtmtK+ stresscontrolOxalic AcidDays after planting30 35 40 45 50 55 60 65 70Concentration (mg/L)0246810droughtfloodNH4+ trtmtK+ stresscontrolSuccinic AcidDays after planting30 35 40 45 50 55 60 65 70Concentration (mg/L)0246810droughtfloodNH4+ trtmtK+ stresscontrolUnlike the TOC graph, organic acids were exuded in the largest amounts from the drought and flooding treatments.  This may be due to buildup of osmotic potential to deal with the low water potentials in the drought treatment, or just leakier root cells due to the stress treatments.What’s in the root vs. what’s released by the rootSuccinic AcidRoot (mg/kg)0 100 200 300 400 500Exudate (mg/L)02468101214r2 = 0.55Fumaric AcidRoot (mg/kg)0 500 1000 1500 2000Exudate (mg/L)024681012r2 = 0.54Oxalic AcidRoot (mg/kg)0 50 100 150 200 250 300 350 400Exudate (mg/L)024681012Malonic AcidRoot (mg/kg)0 20 40 60 80 100 120Exudate (mg/L)0123456r2 = ns r2 = nsConclusionsTreatment Averagesdays after planting20 30 40 50 60 70µg C exuded per day02004006008001000K+ stressflooddroughtcontrolNH4+ treatment1. Stress increases root exudation.  Drought and flooding treatments increased release of organic acids.2.  Concentrations of succinic and fumaric acid in the root correlated with amounts released by the root.Succinic AcidDays after planting30 35 40 45 50 55 60 65 70Concentration (mg/L)0246810droughtfloodNH4+ trtmtK+ stresscontrolSuccinic AcidRoot (mg/kg)0 100 200 300 400 500Exudate (mg/L)02468101214r2 = 0.55

Water & Nutrient Stress Increase Root Exudation

https://digitalcommons.usu.edu/cgi/viewcontent.cgi?article=1001&amp;context=cpl_waterstress

Water & Nutrient Stress Increase Root Exudation

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