Update on Leaf Functional Anatomy in Relation to Photosynthesis Leaf Functional Anatomy in Relation to Photosynthesis 1 Ichiro Terashima*, Yuko T. Hanba, Danny Tholen, and U ¨ lo Niinemets Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113–0033, Japan (I.T.); Center for Bioresource Field Science, Kyoto Institute of Technology, Ukyo-ku, Kyoto 616–8354, Japan (Y.T.H.); Plant Systems Biology Group, Chinese Academy of Sciences-Max Planck Institute, Germany Partner Institute for Computational Biology, Shanghai 200031, China (D.T.); and Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Tartu 51014, Estonia (U ¨ .N.) Rubisco is a large enzyme with a molecular mass of approximately 550 kD. The maximum rate of CO 2 fixation (i.e. ribulose-1,5-bisphosphate [RuBP] carbox- ylation) at CO 2 saturation is only 15 to 30 mol CO 2 mol 21 Rubisco protein s 21 at 25°C. Affinity to CO 2 is also low, and the K m , K c , at 25°C in the absence of oxygen is comparable to the CO 2 concentration in water equilibrated with air containing 39 Pa CO 2 (approximately 390 mLL 21 ), 13 mM. Moreover, RuBP carboxylation is competitively inhibited by RuBP oxygenation, which is the primary step of the energy-wasting process, photorespiration. If the CO 2 concentration in the chloroplast stroma is low, the carboxylation rate will decrease while the oxygenation rate will increase. Under such conditions, light energy and other resources, including nitrogen and water, are all wasted, eventually leading to a decrement of fitness of the plants. From these data, we may consider that structural features of the leaf contributing to the maintenance of the high CO 2 concentration in the chloroplast stroma may have been selected during evolution. In this Update, we focus on the key structural features that affect CO 2 concentration in the chloroplast stroma. First, we analyze the conductance for CO 2 diffusion from the substomatal cavity to the chloroplast stroma (mesophyll conductance [g m ], also called internal con- ductance). Because the low g m limits photosynthesis, the mesophyll surface area exposed to the intercellular spaces (S mes , mesophyll surface area exposed to inter- cellular spaces per unit leaf area) should be maximized to increase the area for CO 2 dissolution and the effective pathway for CO 2 diffusion, and thereby photosynthe- sis. Second, we analyze the light environment within a leaf, because, for maximizing photosynthesis, light should be delivered to all the chloroplasts in the leaf, distributing along the cell walls. In relation to the light environment within a leaf, we also point out some technical problems in measuring photosynthetic param- eters. Third, the movement and positioning of cellular organelles are discussed. Finally, we discuss the urgent need for ecologically relevant developmental and cell biological studies that clarify the mechanisms that are responsible for structural and cell biological features in nature. THE NATURE OF MESOPHYLL DIFFUSION CONDUCTANCE During photosynthesis, CO 2 diffuses from ambient air through stomata to the intercellular spaces. Then, the CO 2 dissolves in the cell wall water and diffuses across the cell wall, plasma membrane, cytosol, chlo- roplast envelope, and stroma to Rubisco (Fig. 1). CO 2 concentration in the chloroplast can be estimated in several ways (Pons et al., 2009). One of the most reliable methods is the simultaneous measurement of gas exchange and stable carbon (C) isotope dis- crimination. This method relies on the discrimina- tion against 13 CO 2 by Rubisco. In an open system, with unlimited supply of 12 CO 2 and 13 CO 2 , Rubisco will pre- ferentially fix the lighter molecule, 12 CO 2 . In a closed system, Rubisco will eventually fix both 12 CO 2 and 13 CO 2 until no CO 2 is left. The leaf is intermediate between these two extremes. Thus, the ratio 13 CO 2 / 12 CO 2 of CO 2 fixed by a leaf is considerably greater than the ratio in the ambient air. Technically, the ratios of 13 CO 2 to 12 CO 2 in the air incoming to and outgoing from an assimilation chamber are measured with a mass spectrometer, a tunable laser diode absorption spectro- scope, or a cavity ring-down spectroscope (Pons et al., 2009). It has been established that the photosynthetic lim- itation by g m expressed on a leaf area basis is in the same order as that by stomatal conductance (conduc- tance for CO 2 diffusion from the air outside the leaf to the substomatal cavity; Terashima et al., 2006; Flexas et al., 2008; Evans et al., 2009; Niinemets et al., 2009a). The representative values for CO 2 concentrations in the substomatal cavity (C s ), bulk intercellular space (C i ), and chloroplast stroma (C c ) in actively photo- synthesizing leaves in ambient air containing CO 2 at 39 Pa, expressed as ratios, are C s /C a = 0.60 to 0.85 (even as 1 This work was supported by the Ministry of Education, Culture, Sports, Science and Technology-Japan (Grant-in-Aid for Scientific Research on Innovative Areas no. 3103 to I.T.) and by the Estonian Ministry of Education and Research (grant no. SF1090065s07 to U ¨ .N.). * Corresponding author; e-mail itera@biol.s.u-tokyo.ac.jp. www.plantphysiol.org/cgi/doi/10.1104/pp.110.165472 108 Plant Physiology Ò , January 2011, Vol. 155, pp. 108–116, www.plantphysiol.org Ó 2010 American Society of Plant Biologists