# Measurement of Diurnal Variation in Needle PRI and Shoot Photosynthesis in a Boreal Forest
## Materials and Methods
### Shoot Chamber Data  
Carbon fluxes of individual pine shoots were measured using a dynamic gas exchange measurement system consisting of chambers, sample tubing, analysers, and a control unit (Figure 1). The chambers were made from PMMA (polymethyl methacrylate) and the inner surfaces were coated with FEP (fluorinated ethylene propylene) film. Needle growth was prevented by de-budding the shoots before installing the chambers. Thus only fully-expanded previous-year needles were included. The chambers were installed in top whorls on topmost, fully-exposed shoots, and were pointing towards the south. A small fan was mixing air inside the chambers. One shoot chamber was used in 2014 and two chambers in 2015. One of the chambers used in 2015 contained a flattened shoot: the needles were carefully spread inside the thin chamber to avoid self-shading. Other shoots were contained in cylindrical chambers which did not deform the shoot shape.  
During operation the chambers remained open and the gas concentrations were close to ambient. The chambers closed automatically 48 to 72 times per day for 1.5 to 3 min for determining the gas exchange rates of the shoots enclosed inside the chamber. During measurements, sample air flow (with a flow rate of 3-4 clpm) was drawn to an infra-red gas analyser (LI-840A, LI-COR, Lincoln, NE, USA) which recorded the CO\({}_{2}\) and H\({}_{2}\)O concentration change during each closure with 5-s interval. Photosynthetic photon flux density (PPFD) and temperature inside the chamber were recorded with the same frequency, using an LI-190 quantum sensor (LI-COR, Lincoln, NE, USA) and a t-type thermocouple or PT100 resistance thermometer, respectively. The needle areas were determined after the measurement period for calculating the exchange rates per needle area units. A detailed description of the system and gas-exchange rate calculation can be found in [29; 30].  
The measurements were used to determine shoot net assimilation \((A)\) in units of grams of CO\({}_{2}\). Nighttime measurements were used to determine the shoot respiration \((R)\). We used nighttime measurements to determine the relationship between temperature and respiration (Appendix A). Based on the high dispersion of the relationship, we assumed the simplest linear relationship between temperature and respiration and calculated shoot respiration for the time of each measurement. Added respiration to net assimilation yielded the shoot photosynthetic assimilation:  
\[P=A+R \tag{2}\]  
Finally, we calculated the shoot LUE, \(\varepsilon\), as:  
\[\varepsilon=P/\text{PPFD} \tag{3}\]  
where PPFD is measured in umol m\({}^{-2}\) s\({}^{-1}\). Due to the natural variation in shoot structure (e.g., in the ratio of projected to total needle area for different illumination angles, [31]), between the flat and cylindrical chambers, the results were not directly comparable between the shoots with different surface areas. However, as we were only interested of the correlations of \(P\) and \(\varepsilon\) with PRI, an absolute scale is not required. Hence, we normalized \(P\) and \(\varepsilon\) by their maximum daily values.  
To achieve compatibility with needle PRI measurements (i.e., to obtain a sunlit shoot data value for the time of each needle PRI data point), the data were first visually filtered for the presence of clouds using the PPFD values. We averaged data from all days during which needle PRI was measured for each campaign (i.e., excluding the days with heavy cloud cover) were averaged, and resampled to a regular grid and smoothed with a Savitzky-Golay filter. Thus, we obtained the typical mid-summer daily cycles for the sunlit shoot normalised photosynthetic assimilation \(P\) and light use efficiency \(\varepsilon\) for the two campaigns.