This makes the underestimation of the true F V′/F M′ value light intensity dependent as well, since a higher light intensity induces more non-photochemical quenching. Question 4. Which part of the leaf is probed and analyzed by a fluorescence measurement? The leaf is optically complex. In a dorsiventral #RSL3 mw randurls[1|1|,|CHEM1|]# leaf, the palisade parenchyma cells have been shown to act as light guides, keeping the light more or less focused (Vogelmann and Martin 1993; Vogelmann et al. 1996). The lobed cells of the spongy mesophyll and the spaces that surround these cells, on the other hand, disperse the light (Vogelmann and Martin 1993). At the

same time, there is a strong light gradient within the leaf (Vogelmann 1989, 1993). This means that the light intensity decreases rapidly as light penetrates into the leaf. As a consequence, illuminating and probing Chl a fluorescence emission on

the adaxial surface of the leaf, chloroplasts located deep in the leaf will be excited Barasertib by a much lower photon flux density than those located close to the adaxial side of the leaf (Terashima and Saeki 1985; Fukshansky and Martinez von Remisowsky 1992). At the same time, the spectral distribution of the light changes as well: as light penetrates the mesophyll, the relative contribution of green and far-red (FR) light progressively increases, because the absorption of these wavelengths by the leaf is less efficient (Sun et al. 1998; Rappaport et al. 2007). The chloroplasts located deeper in the leaf, i.e., those of the spongy tissue, acclimate to these lower, FR-enriched light intensities by increasing the antenna size of PSII, reducing the number of RCs, and decreasing the PSI/PSII ratio (Terashima et al. 1986; Evans 1999; Fey et al. 2005; Pantaleoni et al. 2009). Since the emitted fluorescence is a linear function of the light intensity (Vogelmann and Evans 2002; cf. Schansker et al. 2006), chloroplasts located deeper in the leaf will contribute to a lesser extent to

the detected fluorescence signal. In practice, fluorescence measurements will probe mainly chloroplasts in the palisade parenchyma cells (Vogelmann and Evans 2002). The assumption that not all chloroplasts are crotamiton assayed is supported by the observation that a fivefold decrease in the chlorophyll content of the leaf does not affect the detected F O and F M values (Dinç et al. 2012). In fact, since the total amount of fluorescence emitted by the leaf does not change, it suggests that the light beam probes deeper in the leaf as more chlorophyll is lost. The optical properties of the leaf also mean that measurements made on the abaxial (bottom) side of the leaf have characteristics that differ considerably from those made on the adaxial (top) side of the leaf (Schreiber et al. 1977).