<sup>13</sup>C标记测定根际根系分泌物浓度

Laurent  Simon; Feth el Zahar   Haichar

doi:10.21769/BioProtoc.3228

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Peer-reviewed

Determination of Root Exudate Concentration in the Rhizosphere Using ¹³C Labeling

¹³C标记测定根际根系分泌物浓度

LS Laurent Simon

Feth el Zahar Haichar email

发布: 2019年05月05日第9卷第9期 DOI: 10.21769/BioProtoc.3228 浏览次数: 6573

评审: Joëlle SchläpferVenkatasalam ShanmugabalajiLaura Zanin

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Cover of Ecology and Evolution, featuring study using the protocol.

Jul 2018

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Abstract

One of the most remarkable metabolic features of plant roots is their ability to secrete a wide range of compounds into the rhizosphere, defined as the volume of soil around living roots. Around 5%-21% of total photosynthetically fixed carbon is transferred into the rhizosphere through root exudates. Until recently, studies on the quantity and quality of root exudates were conducted mostly under axenic or monoxenic in vitro conditions. Today, in situ assays are required to provide a better understanding of root exudates dynamics and role in plant-microbe interactions. By incubating plants with ¹³CO₂ in situ for one week and quantifying ¹³C enrichment from the root-adhering soil using mass spectrometry, we were able to determine root exudate levels. Indeed, labeled substrate ¹³CO₂ is converted into organic carbon via plant photosynthesis and transferred into the soil through root exudation. We assume that all ¹³C increases above natural abundance are mainly derived from exudates produced by ¹³C-labeled plants.

Keywords: Root exudates (根分泌物)

Plant (植物)

Rhizosphere (根际)

¹³CO₂ labeling (¹³CO₂标记)

Root-adhering soil (根系粘附土壤)

¹³C-content (¹³C-含量)

Isotope Ratio Mass Spectrometry (同位素比值质谱法)

Poaceae (禾本科)

Monocotyledon (单子叶植物)

Legume (豆科植物)

Arabidopsis thaliana (拟南芥)

Background

Through the exudation of a wide variety of compounds, plants communicate with the soil microbial community in their immediate vicinity, cope with herbivores, foster beneficial symbioses, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species. Understanding how plants differ quantitatively in their root exudate patterns, according to their genotypes and traits as well as to environmental parameters and the soil microbial community is an exciting research challenge. This objective could be achieved by quantifying root exudate content using ¹³CO₂ plant-labeling coupled with a ¹³C enrichment measurement from the root-adhering soil using mass spectrometry. This protocol was set up by using different plant species such as Poaceae, legumes (Medicago truncatula), Arabidopsis thaliana, and Monocotyledon plants such as maize and wheat. As rhizodeposition also depends on plant species, the collection period was after 10 weeks for Poaceae and 4 weeks for the rest. This method can be used with other species, for which the collection period must be set up according to what is known about the studied plant.

Materials and Reagents

1 mm mesh size for soil sieving (Fisherbrand tamis, France)
Plastic pots for plant growing (7 x 7 x 6.4, black pots, France)
5 x 8 mm “Ultra Clean” tin capsules (Elemental Microanalysis, Okehampton, UK, catalog number: D1034)
In the case of calcareous soils: 5 x 8 mm silver capsules (Elemental Microanalysis, Okehampton, UK, catalog number: D2009)
Soil and seeds for plant growing
The soil used to set up this protocol was luvisol with no added nitrogen source collected at La Côte Saint-André (Isère, France) and which was continuously harvested with maize. It was a loamy soil composed of 16.2% clay, 45.4% loam and 28.4% sand, with a total nitrogen content of 1.9 g·kg^-1
Reference materials for ¹³C measurements: IAEA-CH3 (cellulose), IAEA-CH6 (sucrose) (Coplen et al., 2006) and partially ¹³C-labelled material [e.g., D-Glucose (3-13C, 99%)] (Eurisotop, Saint-Aubin, France, catalog number: CLM-1393-0.25)
Calibration material for carbon (C) concentration measurements: aspartic acid (Elemental Microanalysis, Okehampton, UK, catalog number: B2042)
Distilled water
Liquid N₂
Pure ¹³CO₂ (> 99% atom ¹³C; purchased from Cortec Net, Paris, France)
In the case of calcareous soils: hydrochloric acid (HCl) 37% (CAS number: 7647-01-0, e.g., Sigma-Aldrich, Saint-Louis, MO, USA, catalog number: 30721-M)

Equipment

50 ml beaker
Spatula and forceps
Desiccator (Freeze-dryer) (Alpha 1-4 LSC, Martin Christ^®, Osterode am Harz, Germany)
Mixer mill grinder (Mixer Mill MM 200, Retsch^®, Haan, Germany)
Laboratory balance with a resolution of 0.01 mg or better (XP6 microbalance, Mettler Toledo, Greifensee, Switzerland)
Isotope ratio mass spectrometer (Isoprime 100, Elementar UK Ltd., Cheadle, UK), coupled in continuous flow with an elemental analyzer (vario PyroCube in CN mode, Elementar Analysensysteme, Lagenselbold, Germany, or FlashEA 1112, ThermoElectron, Waltham, MA, USA)
Growth chamber equipped for automatic control of light, temperature, moisture, evapotranspiration, irrigation and CO₂ concentration

Software

Isoprime 100 isotope ratio mass spectrometer operating software: Ionvantage^® for Isoprime, build 1.6.1.0 (Elementar Analysensysteme, Lagenselbold, Germany)
Elemental analyzer operating software (pyrocube Software v4.0.1 was used in this protocol, Elementar Analysensysteme, Lagenselbold, Germany)

Procedure

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Simon, L. and Haichar, F. E. Z. (2019). Determination of Root Exudate Concentration in the Rhizosphere Using ¹³C Labeling. Bio-protocol 9(9): e3228. DOI: 10.21769/BioProtoc.3228.