J Plant Res (2015) 128:777–789 DOI 10.1007/s10265-015-0736-5 REGULAR PAPER Rapid response of leaf photosynthesis in two fern species Pteridium aquilinum and Thelypteris dentata to changes in CO2 measured by tunable diode laser absorption spectroscopy Keisuke Nishida1 · Naomi Kodama2,3,4 · Seiichiro Yonemura2 · Yuko T. Hanba5 Received: 6 November 2014 / Accepted: 20 April 2015 / Published online: 3 June 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract We investigated stomatal conductance (gs) angiosperms. Together, these results suggest that regu- and mesophyll conductance (gm) in response to atmos- lation of gm to [CO2] may differ between angiosperms pheric CO2 concentration [CO2] in two primitive land and ferns. plants, the fern species Pteridium aquilinum and The- lypteris dentata, using the concurrent measurement of Keywords CO2 response · Ferns · Mesophyll leaf gas exchange and carbon isotope discrimination. conductance · Pteridophytes · Photosynthesis [CO2] was initially decreased from 400 to 200 μmol 1 1 mol− , and then increased from 200 to 700 μmol mol− , 1 and finally decreased from 700 to 400 μmol mol− . Introduction Analysis by tunable diode laser absorption spectroscopy (TDLAS) revealed a rapid and continuous response Atmospheric CO2 is a substrate for leaf photosynthesis in in gm within a few minutes. In most cases, both ferns land plants, and thus CO2 availability at the carboxylation showed rapid and significant responses of gm to changes site is one of the most important limiting factors for leaf in [CO2]. The largest changes (quote % decrease) were photosynthesis. In the process of leaf photosynthesis in C3 obtained when [CO2] was decreased from 400 to 200 land plants, CO2 diffuses from the atmosphere through sto- 1 μmol mol− . This is in contrast to angiosperms where mata, intercellular air spaces, and the leaf mesophyll to the an increase in gm is commonly observed at low [CO2]. site of carboxylation in the chloroplasts. CO2 concentra- Similarly, fern species observed little or no response tion in the chloroplast is lower than that in the atmosphere of gs to changes in [CO2] whereas, a concomitant because of significant resistance to CO2 diffusion through decline of gm and gs with [CO2] is often reported in this diffusional pathway, i.e., limitations in CO2 diffusion strongly reduce leaf photosynthesis. There are two major * Keisuke Nishida CO2 diffusional limitations; CO2 conductance though sto- nishida‑[email protected] mata, gs, and that from substomatal cavities to the chloro- plast, termed g . 1 m The Graduate School of Science, Kyoto Institute of Technology, Atmospheric CO levels have changed substantially Matsugasaki, Sakyo‑ku, Kyoto 606‑8585, Japan 2 over the evolutionary history of land plants. It is estimated 2 Agro‑Meteorology Division, National Institute that atmospheric CO levels were approximately 10 times for Agro-Environmental Sciences, 3‑1‑3 Kannondai, 2 Tsukuba 305‑8604, Japan higher than the present when land plants started to evolve 360–480 million years ago (Royer et al. 2004). Ferns are 3 Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Zuricherstrasse 111, 8903 Birmensdorf, Switzerland a major component of the fossil flora, and although they are primitive, non-seed plants, they are closely related 4 Present Address: School of Human Science and Environment, Hyogo University, 1‑1‑12 Shinzaike‑honcho, to seed plants (Pryer et al. 2001). Atmospheric CO2 lev- Himeji 607‑0092, Japan els fell abruptly during the Cretaceous period (Kuypers 5 Department of Applied Biology, Kyoto Institute of Technology, et al. 1999), which coincides with a major diversification Matsugasaki, Sakyo‑ku, Kyoto 606‑8585, Japan in the fern group (Pryer et al. 2004). On the other hand, 1 3 778 J Plant Res (2015) 128:777–789 angiosperms, which are currently the dominant group of et al. 2013). For angiosperms, there is conflicting evidence seed plants, emerged during a period when atmospheric as to how gm responds to [CO2]. Some studies reported CO2 level was only two- to three-fold higher than the pre- insignificant effects of [CO2] on gm (Harley et al. 1992; sent (Haworth et al. 2011). This implies that ferns and angi- Tazoe et al. 2009), whereas other studies reported a decline osperms evolved under different selection pressures, which in gm at high [CO2] (Bunce 2010; Douthe et al. 2011; may have resulted in different mechanisms of CO2 diffu- Hassiotou et al. 2009; Loreto et al. 1992, Tazoe et al. 2011), sion between these two plant groups (Carriquí et al. 2015). or showed curved responses to changes in [CO2] (Flexas Changes in the mechanisms of CO2 diffusion in plant evo- et al. 2007; Vrábl et al. 2009). Gago et al. (2013) obtained lutionary history have been suggested because stomatal fre- a curved response in gm with changes in [CO2] for three quency in fossil plants, which strongly affects gs, has been fern species. However, the observed decline in gm at low shown to track changes in atmospheric CO2 level (Wood- [CO2] in angiosperms and ferns (sub-stomatal CO2 concen- 1 ward 1998). As such, it’s suggested that stomatal function tration, Ci < 50 µmol mol− ) may be an artifact related to developed to enhance CO2 diffusion to cope with decreases partially photorespired CO2 (Tholen et al. 2012). Further- in CO2 level. However, changes in gm in land plant history more, the chlorophyll fluorescence technique used can lead cannot be determined through similar anatomical imprints to errors in the estimation of gm in conditions of changing in the fossil record. Although the difference in gm is pos- [CO2] (Gilbert et al. 2011). Because of these potential arti- sibly still partially reflected in extant plants of angiosperms facts and errors, it is necessary to confirm previous studies and ferns. Leaf mesophyll anatomy affecting gm, including on the response of gm to [CO2] in angiosperms and ferns chloroplast surface area facing the intercellular airspaces through the use of complimentary methods. We chose spe- and cell wall thickness, could have changed from ferns to cifically to look at ferns because of the limited information angiosperms (Carriquí et al. 2015), which may be affected published on CO2 responses and to determine if like sto- by the decrease in atmospheric CO2 level. A comparison of matal responses to CO2, ferns also differed in gm responses CO2 diffusional limitations in extant ferns with extant angi- compared with angiosperms. osperms could provide crucial information to estimate how The purposes of this study were to determine: (1) the photosynthesis traits have evolved in land plants. If atmos- photosynthetic traits of ferns, including gm and gs at the 1 pheric CO2 levels can influence selection pressure, phylo- present [CO2] (400 µmol mol− ) for comparison against genetically distant fern groups may also vary in internal published values for ferns and angiosperms, and (2) the morphology and gm. rapid and continuous response of gm, gs and photosynthetic In the present atmospheric CO2 conditions, fern species rate of ferns to changing [CO2]. For these purposes, we have much lower photosynthetic capacity than angiosperms developed a custom-designed gas exchange system using a (Wright et al. 2005). In ferns, both gs and gm are lower than concurrent measurement of gas exchange and carbon iso- in angiosperms. A lower gm is suggested to be the major tope ratio using tunable diode laser absorption spectros- mechanism underlying the lower photosynthetic capacity copy (TDLAS), to quantify the rapid, continuous responses of fern species (Carriquí et al. 2015). However, there are in gm in fern species in response to changes in [CO2] with a only three published determinations of gm of fern species to time resolution of a few minutes (Tazoe et al. 2009, 2011). the best of our knowledge (Carriquí et al. 2015; Gago et al. O2 gas was used at a level of 2 % for gas exchange meas- 2013; Volkova et al. 2009). Anatomical and physiologi- urements in order to minimize the effect of photorespira- cal mechanisms underlying the low gm of fern species still tion on carbon isotope measurements. To the best of our remain to be confirmed. knowledge, this is the first study to examine continuous The response of gs to atmospheric CO2 concentra- responses in gm in fern species in response to changes in tion [CO2] is different between angiosperms and ferns. [CO2]. We also determined leaf anatomical traits using Extensive studies on angiosperms have shown that gs typi- light micrographs and calculated photosynthetic parameters cally increases with a decrease in [CO2] (e.g., Brodribb using the light–response curve and A/Ci curve, to compare et al. 2009; Messinger et al. 2006). However, three ferns, the photosynthetic traits of ferns with those of angiosperms Osmunda regalis, Blechnum gibbum and Nephrolepis exal- reported previously. tata, showed small responses to changes in [CO2] (Gago We selected two fern species from order Polypodi- et al. 2013). The averaged gs for six ferns and lycophytes ales, Pteridium aquilinum and Thelypteris dentata. From showed no response to an increase in [CO2] above ambi- recent phylogenetic studies, Polypodiales is the most ent, while they showed a slight increase with a decrease in modern order among the seven fern orders in Polypo- [CO2] (Brodribb et al. 2009). Studies for the response of gm diopsida (Smith et al. 2006). The estimated divergence to [CO2] are limited compared with gs in angiosperms, and time of Pteridium (Dennstaedtiaceae family) and Thelyp- to the best of our knowledge, there is only one published teris (Thelypteridiaceae family, Eupolipods II) is ~90 and study on the response of gm to [CO2] in fern species (Gago ~65 million years, respectively (Pryer et al. 2004), when 1 3 J Plant Res (2015) 128:777–789 779 atmospheric CO2 levels decreased with time from ~2,000 The gas exchange experiment was carried out in October to ~500 ppm (Bice and Norris 2002).