U.S. Department of Energy

Pacific Northwest National Laboratory

Direct Real-Time Monitoring and Assessment of Single-Leaf Carbon Fixation and Respiration Rates for Arabidopsis thalianaby Mass Spectrometry

Introduction 

While most metabolic changes which result from genetic alterations are assessed using LC-MS and GC-MS, less emphasis historically has been placed on direct real-time analysis of important processes involved in gas (e.g., oxygen and carbon dioxide) uptake and exchange, which can indicate changes in specific metabolic processes within a biological system.  We demonstrate here how atmospheric mass spectrometry provides a means to look at how important processes such as carbon fixation and photorespiration occur within plants whose photosynthesis rates have been altered via knock-out (KO) and over-expression (OE) of the enzyme arogenate dehydratase (ADT). We report here on the utility of atmospheric Real-Time High Definition Mass spectrometry (RTHD-MS) as a new tool to characterize, in vivo, genetically modified plant strains.

Methods 

All KO, OE and wild-type (WT) lines were grown in soil in a greenhouse (16-h days, 27–28 °C; 8-h nights, 24–26 °C) until 4 wks. Selected plants were isolated from light for ~24 hrs. in a sealed exposure chamber which incorporated a down draft flow system of ~4 L/min. Single leaves were isolated for direct gas-phase sampling by a Shimadzu QP2010 mass spectrometer (MS) configured with a patented inlet technology. A sampling rate of 2.0 mL/min was found to provide high resolution of metabolic processes. Each plant was light and dark cycled as follows: initial off, 30 min-on, 50 min-off, 65 min-on, 79.5 min-off, 85 min-on, 96 min-off.

Preliminary data

Each 100 minute sampling period represented 22 comparable points of interest. All subject plants were acclimated in darkness using natural atmosphere for exposure and monitoring. The darkness acclimated state served two purposes: 1) to deplete the plant of all starch reserves, and 2) it established the initial baseline concentration of carbon dioxide (ppm) by combining the atmospheric CO2 value with the CO2 produced by the subject leaf via photorespiration.  For each cycle period, data was collected showing the rate of total carbon dioxide uptake by each plant per leaf mass.  Rate data was collected at each of the six light cycle periods and calculated as slopes to represent rate of total carbon fixation. Further gravimetric data was calculated to show part per million, per milligram of plant leaf, carbon dioxide fixation and production capacity. Corresponding proteomics data show that the KO ADT plant lines contain significantly more photosynthesis and photorespiration pathway enzymes than WT, while OE lines show the converse effect being that OE lines show a significant reduction in photosynthesis and photorespiration pathway machinery.  Here our gas exchange data support our proteomic observations in that WT and KO lines have similar rates of total carbon dioxide uptake, while OE lines show a significant lessened rate of carbon dioxide uptake.  We are currently performing the same experiments with 13CO2 and 18O2 gases to further quantify the amount of CO2 fixed by the plants via photosynthesis and how much CO2 is lost back to the environment through the photorespiration pathway.  All qualitative and quantitative comparisons are related back to the natural wild type (WT) for possible insight into the differential affects that ADT composition has on photosynthesis and photorespiration.

Novel aspect 

High resolution monitoring of genetically modified plant respiratory processes using direct, real-time gas phase sampling coupled with mass spectrometry.

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