How to measure respiration responses to temperature using a series of covered leaves using a LI-6400

Martijn Slot, Kaoru Kitajima

Author Affliations

University of Florida


Respiration is measured as the release of CO2 from a portion of a leaf enclosed in a leaf cuvette. By measuring a series of leaves of comparable age and exposition to light, each at a different ambient temperature, a temperature response curve can be constructed, providing information on the temperature sensitivity of respiration in the target species/treatment. The temperature sensitivity of respiration can for example be expressed as the Q10.

For more details, see the upper level summary for Gas Exchange and Chlorophyll Fluorescence, Respiration, and the Gas Exchange Protocol For Li-COR 6400.


Leaf dark respiration (Rdark) increases with temperature, but species, or plants in different experimental conditions, may differ in their sensitivity. The instantaneous temperature sensitivity of Rdark can be calculated from measurements at just two different temperatures, but greater accuracy of the estimate can be achieved by constructing response curves with measurements at multiple temperatures. The temperature in the LI-6400 cuvette can be controlled (ambient ∓ several C), but previous research has shown that warming of a single leaf or a leaf portion yields different estimates of the respiratory temperature sensitivity than when the whole plant (Atkin et al. 2000) or the whole stand (Griffin et al. 2002) is warmed. In this protocol we describe how to determine Rdark temperature-response curves in situ, making use of changes in ambient temperature between pre-dawn and mid-morning. This method works well in climates with considerable diurnal temperature variation. While it may be possible to use ambient temperature changes at other times during the day, the morning is preferred. For example, determining the temperature sensitivity from repeated measurements of Rdark during the early night, while ambient temperature gradually drops, could be confounded by a decreasing contribution of respiration associated with phloem loading as the night progresses. Rdark of pre-darkened leaves measured at the end of the night can be assumed to represent maintenance respiration only. A further advantage of using pre-darkened leaves at the end of the night, as opposed to darkening leaves for a short period at another time of the day, is that the metabolite status of the leaves is not affected by previous sun exposure (Florez-Sarasa et al. 2012).


  • LI-COR 6400 (LI-COR, Lincoln, NE, USA) with CO2 mixer, or buffer volume
  • CO2 cartridges (not needed when using buffer volume)
  • Drierite – a desiccant
  • Soda lime for CO2 scrub
  • Standard RS-232 cable – to download data from LI-COR 6400 (serial-to-USB Converter cable necessary when downloading LI-6400 to computer without COM port. Not necessary when using LI-6400 XT)
  • LI-6400 software – to download data
  • Razor blade
  • ziplock bag
  • cooler bag
  • LI-6020 battery charger
  • Thin aluminum foil

Units, terms, definitions

CO2 concentration – μmol CO2 mol-1 air

Drierite – Desiccant

IRGA – Infra Red Gas Analyzer

Rdark – Dark respiration – μmol CO2 m2 s-1


  1. Just before sunset on the day before Rdark measurements, cover fully expanded, recently matured leaves with thin aluminum foil, so that they will not be exposed to sunlight before the measurements early the following morning. The abaxial side should not be completely covered to allow free gas exchange during the night.
  2. The following morning each leaf is measured once between pre-dawn and mid-morning at ambient temperature. Leaf R is measured as CO2 release rates with a portable infrared gas analyzer (LI-6400), at ambient humidity and a stable ambient CO2 concentration of a buffer volume, or controlled by the built-in CO2 mixer of the LI-6400. For details on the use of the LI-6400, see the Gold Leaf protocol “Gas Exchange For LI-COR 6400“. Thus, during the Rdark measurements, the leaf portion inside the gas exchange cuvette has the same temperature as the whole leaf and branch.
  3. Because the signal-to-noise ratio is rather poor when using the standard LI-6400 leaf cuvette (especially for leaves of inherently slow-growing species or when working in the shade) it is important to match frequently (-matching’ is used to make sure that the two infrared gas analysers read the same CO2 concentration when exposed to the same air). Use an auto program to log multiple values for each leaf. Go to menu 5, select AUTO PROG, pick “autolog”, and append to file (Y). Record at least 5 logs. Match after each. Matching may take 45 seconds or longer, so allow for enough time between logs for matching to be completed.
  4. After the measurement at a single temperature point, cut the leaf with a razor blade and store it in a zip-lock bag on ice until return to the lab for additional trait measurements.
  5. Download Data. Once the data are transferred to an excel file (see for details the Gas Exchange Protocol For LI-COR 6400), re-calculate the respiration rates for the true leaf area if the leaf area enclosed in the default leaf cuvette differed from the maximum of 6 cm2. Respiration rates the Li-COR 6400 outputs are based on the assumption that the measured CO2 flux comes from 6 cm2 of leaf; when less leaf area was enclosed in the cuvette, multiply the respiration rates by (6/(true leaf area)) to get the area-adjusted respiration rates. Average the multiple logs per leaf area.
  6. Fit a temperature response curve. The most common shape for the temperature response to take is an exponential increase. An exponential curve can be linearized by log-transforming the y-axis (Rdark). The slope of this line represents the temperature sensitivity (e.g. expressed as the Q10, the proportional increase of Rdark with a 10�K temperature rise: Q10=e(10*slope)). The confidence intervals of the slope can be used to compare the temperature sensitivities/Q10s of different species, or plants from different experimental treatments.

Other resources

This protocol was developed for a study published here ( in the journal Tree Physiology.

Notes and troubleshooting tips

  • In humid climates, bringing the LI-6400 from an air-conditioned room to the field for measurements can result in condensation problems and the system will give high-humidity alerts. It is recommended to allow the instrument to warm up slowly while still in its case to minimize condensation issues. Alternatively, starting up the instrument and allowing it to warm up before moving into the humidity would also work.

Links to resources and suppliers

LI-6400 operation manual:

Literature references

Atkin OK, Evans JR, Ball MC, Lambers H, Pons TL (2000) Leaf respiration of snow gum in the light and dark; interactions between temperature and irradiance. Plant Physiology 122:915-923. doi: link)

Florez-Sarasa I, Araújo WL, Wallström SV, Rasmusson AG, Fernie AR, Ribas-Carbo M (2012) Light-responsive metabolite and transcript levels are maintained following a dark-adaptation period in leaves of Arabidopsis thaliana. New Phytologist 195: 136-148. doi: 10.1111/j.1469-8137.2012.04153.x

Griffin KL, Turnbull MH, Murthy R, Lin GH, Adams J, Farnsworth B, Mahato T, Bazin G, Potasnak M. Berry JA (2002) Leaf respiration is differentially affected by leaf vs. stand-level night-time warming. Global Change Biology 8:479-485doi: 10.1046/j.1365-2486.2002.00487.x

Slot M, Wright SJ, Kitajima K (2013) Foliar respiration and its temperature sensitivity in trees and lianas: in situ measurements in the upper canopy of a tropical forest. Tree Physiology doi: 10.1093/treephys/tpt026

Health, safety & hazardous waste disposal considerations


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