Materials and Reagents

1. Arabidopsis plants
   Note: We plated Nossen ecotype seeds that had been surface-sterilized on germination medium (GM) agar plates (Motohashi et al., 2003) containing 1% sucrose, with the appropriate selection agent (antibiotic or herbicide) per specific genotype. Plants were kept at 4 °C for 3 days to improve germination rates and then grown in lighted growth chambers (CF-405, TOMY-Seiko, Tokyo, Japan) with approximately 75 μmol photon/m²/s at 22 °C under a 16 h-light /8-h dark cycle (long-day conditions) for 3 weeks.
   

Equipment

1. Photosynthesis yield analyzer (Walz, MINI- PAM) (the equipment used in this protocol) (Figure 1)
   Compact design and easy operation are the most outstanding features of the MINI-PAM. This device is in particular well-suited for determination of quantum yield and photosynthetic electron transport rate (ETR). A flexible 5.5 mm glass fiberoptic was attached in the system and it can provide considerable high actinic intensities of white light. An optional 2 mm plastic fiberoptic (MINI-PAM/F1) is also used by excellent signal quality and can be attached to the cover of an optional gas-exchange system for measuring both CO₂ and H₂O exchange as well as fluorescence. For an exact measuring quantum flux density and temperature at precisely the fluorescence measuring spot, a useful leaf-clip holder is available as an accessory (Arabidopsis Leaf-Clip Holder, model: 2060-B). This leaf clip holder is especially developed for small leaves like an Arabidopsis leaf. With the help of the leaf clip holder, the photosynthetic active radiation (PAR) can be measured and an apparent electron transport rate (ETR) is calculated.
   A simple explanation of the equipment used can be found at the following URL: http://www.walz.com/downloads/manuals/mini-pam-II/MINI-PAM-II_Broschure.pdf. It should be noted that the current equipment being sold is the MINI-PAM II.
   
   
   Figure 1. Photosynthesis yield analyzer
   
   
2. Pulse-amplitude-modulated (PAM) fluorometer (TEACHING-PAM) (Walz) (alternative equipment which can be used to measure chlorophyll fluorescence)
   Note: It is noted here that the MINI- PAM and TEACHING-PAM were developed for beginners; advanced researchers may utilize the larger PAM-2000 fluorometer (essentially the same instrument) to yield additional and more detailed results.
   

Procedure