Journal of Plant Research

, Volume 131, Issue 6, pp 907–914 | Cite as

Mesophyll conductance to CO2 in leaves of Siebold’s beech (Fagus crenata) seedlings under elevated ozone

  • Makoto WatanabeEmail author
  • Yu Kamimaki
  • Marino Mori
  • Shigeaki Okabe
  • Izumi Arakawa
  • Yoshiyuki Kinose
  • Satoshi Nakaba
  • Takeshi Izuta
JPR Symposium Physiological Ecology of Woody Species in Response to Air Pollution and Climate Changes


Ozone is an air pollutant that negatively affects photosynthesis in woody plants. Previous studies suggested that ozone-induced reduction in photosynthetic rates is mainly attributable to a decrease of maximum carboxylation rate (Vcmax) and/or maximum electron transport rate (Jmax) estimated from response of net photosynthetic rate (A) to intercellular CO2 concentration (Ci) (A/Ci curve) assuming that mesophyll conductance for CO2 diffusion (gm) is infinite. Although it is known that Ci-based Vcmax and Jmax are potentially influenced by gm, its contribution to ozone responses in Ci-based Vcmax and Jmax is still unclear. In the present study, therefore, we analysed photosynthetic processes including gm in leaves of Siebold’s beech (Fagus crenata) seedlings grown under three levels of ozone (charcoal-filtered air or ozone at 1.0- or 1.5-times ambient concentration) for two growing seasons in 2016–2017. Leaf gas exchange and chlorophyll fluorescence were simultaneously measured in July and September of the second growing season. We determined the A, stomatal conductance to water vapor and gm, and analysed A/Ci curve and A/Cc curve (Cc: chloroplast CO2 concentration). We also determined the Rubisco and chlorophyll contents in leaves. In September, ozone significantly decreased Ci-based Vcmax. At the same time, ozone decreased gm, whereas there was no significant effect of ozone on Cc-based Vcmax or the contents of Rubisco and chlorophyll in leaves. These results suggest that ozone-induced reduction in Ci-based Vcmax is a result of the decrease in gm rather than in carboxylation capacity. The decrease in gm by elevated ozone was offset by an increase in Ci, and Cc did not differ depending on ozone treatment. Since Cc-based Vcmax was also similar, A was not changed by elevated ozone. We conclude that gm is an important factor for reduction in Ci-based Vcmax of Siebold’s beech under elevated ozone.


Chlorophyll fluorescence Mesophyll conductance Ozone Photosynthesis Siebold’s beech Vcmax 



The authors are greatly indebted to Dr. Kazuhide Matsuda and Mr. Hiroyuki Ozawa (Tokyo University of Agriculture and Technology) for their technical support. The authors would like to thank the editor and two anonymous reviewers for providing valuable comments on an earlier version of the manuscript. This study was supported by JSPS KAKENHI, Young Scientists B (15K16136 to MW), Type B (18H03410 to TI and MW) and Challenging Exploratory Research (15K12217 to TI and MW).


  1. Agathokleous E, Kitao M, Kinose Y (2018) A review study on O3 phytotoxicity metrics for setting critical levels in Asia. Asian J Atmos Environ 12:1–16CrossRefGoogle Scholar
  2. Akimoto H, Mori Y, Sasaki K, Nakanishi H, Ohizumi T, Itano Y (2015) Analysis of monitoring data of ground-level ozone in Japan for long-term trend during 1990–2010: causes of temporal and spatial variation. Atmos Environ 102:302–310CrossRefGoogle Scholar
  3. Barnes JD, Balaguer L, Manrique E, Elvira S, Davison (1992) A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environ Exp Bot 32:85–100CrossRefGoogle Scholar
  4. Bernacchi CJ, Singsaas EL, Pimentel C, Portis AR, Long SP (2001) Improved temperature response functions for models of Rubisco-limited photosynthesis. Plant Cell Environ 24:253–259CrossRefGoogle Scholar
  5. Bernacchi CJ, Portis AR, Nakano H, von Caemmerer S, Long SP (2002) Temperature response of mesophyll conductance. Implications for the determination of Rubisco enzyme kinetics and for limitations to photosynthesis in vivo. Plant Physiol 130:1992–1998CrossRefGoogle Scholar
  6. Bytnerowicz A, Arbaugh M, Schilling S, Frączek W, Alexander D (2008) Ozone distribution and phytotoxic potential in mixed conifer forests of the San Bernardino Mountains, southern California. Environ Pollut 155:398–408CrossRefGoogle Scholar
  7. Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–431CrossRefGoogle Scholar
  8. Egert M, Tevini M (2002) Influence of drought on some physiological parameters symptomatic for oxidative stress in leaves of chives (Allium schoenoprasum). Environ Exp Bot 48:43–49CrossRefGoogle Scholar
  9. Epron D, Godard D, Cornic G, Genty B (1995) Limitation of net CO2 assimilation rate by internal resistance to CO2 transfer in the leaves of two tree species (Fagus sylvatica L. and Castanea sativa Mill.). Plant Cell Environ 18:43–51CrossRefGoogle Scholar
  10. Farquhar GD, von Caemmerer S, Berry JA (1980) A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149:78–90CrossRefGoogle Scholar
  11. Feng Z, Paoletti E, Bytnerowicz A, Harmens H (2015) Ozone and plants. Environ Pollut 202:215–216CrossRefGoogle Scholar
  12. Flexas J, Díaz-Espejo A, Galmés J, Kaldenhoff R, Medrano H, Ribas-Carbo M (2007) Rapid variations of mesophyll conductance in response to changes in CO2 concentration around leaves. Plant Cell Environ 30:1284–1298CrossRefGoogle Scholar
  13. Flexas J, Barbour MM, Brendel O, Cabrera HM, Carriqui M, Díaz-Espejo A, Douthe C, Dreyer E, Ferrio JP, Gago J, Galmés J, Kodama N, Medrano H, Niinemets Ü, Peguero-Pina J, Pou A, Ribas-Carbó M, Tomás M, Tosens T, Warren CR (2012) Mesophyll diffusion conductance to CO2: an unappreciated central player in photosynthesis. Plant Sci 193:70–84CrossRefGoogle Scholar
  14. Flowers MD, Fiscus EL, Burkey KO, Booker FL, Dubois J-JB (2007) Photosynthesis, chlorophyll fluorescence, and yield of snap bean (Phaseolus vulgaris L.) genotypes differing in sensitivity to ozone. Environ Exp Bot 61:190–198CrossRefGoogle Scholar
  15. Gao F, Calatayud V, García-Breijo F, Reig-Armiñana J, Feng Z (2016) Effects of elevated ozone on physiological, anatomical and ultrastructural characteristics of four common urban tree species in China. Ecol Indic 67:367–379CrossRefGoogle Scholar
  16. Genty B, Briantais J, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta Gen Sub 990:87–92CrossRefGoogle Scholar
  17. Harley PC, Loreto F, Di Marco G, Sharkey TD (1992) Theoretical considerations when estimating the mesophyll conductance to CO2 flux by analysis of the response of photosynthesis to CO2. Plant Physiol 98:1429–1436CrossRefGoogle Scholar
  18. Hartmann DL, Klein Tank AMG, Rusticucci M, Alexander LV, Brönnimann S, Charabi Y, Dentener FJ, Dlugokencky EJ, Easterling DR, Kaplan A, Soden BJ, Thorne PW, Wild M, Zhai PM (2013) Observations: atmosphere and surface. In: Stocker TF, Qin D, Plattner G-K, Tignor M, Allen SK, Boschung J, Nauels A, Xia Y, Bex V, Midgley PM (eds) Climate change 2013. The physical science basis. Contribution of working group I to the fifth assessment report of the intergovernmental panel on climate change. Cambridge University Press, Cambridge, pp 159–254Google Scholar
  19. Hikosaka K, Shigeno A (2009) The role of Rubisco and cell walls in the interspecifc variation in photosynthetic capacity. Oecologia 160:443–451CrossRefGoogle Scholar
  20. Hoshika Y, Watanabe M, Inada N, Mao Q, Koike T (2013) Photosynthetic response of early and late leaves of white birch (Betula platyphylla var. japonica) grown under free-air ozone exposure. Environ Pollut 182:242–247CrossRefGoogle Scholar
  21. Inada H, Kondo T, Akhtar N, Hoshino D, Yamaguchi M, Izuta T (2012) Relationship between cultivar difference in the sensitivity of net photosynthesis to ozone and reactive oxygen species scavenging system in Japanese winter wheat (Triticum aestivum). Physiol Plant 146:217–227CrossRefGoogle Scholar
  22. Kinose Y, Azuchi F, Uehara Y, Kanomata T, Kobayashi A, Yamaguchi M, Izuta T (2014) Modeling of stomatal conductance to estimate stomatal ozone uptake by Fagus crenata, Quercus serrata, Quercus mongolica var. crispula and Betula platyphylla. Environ Pollut 194:235–245CrossRefGoogle Scholar
  23. Kinose Y, Fukamachi Y, Okabe S, Hiroshima H, Watanabe M, Izuta T (2017a) Nutrient supply to soil offsets the ozone-induced growth reduction in Fagus crenata seedlings. Trees 31:259–272CrossRefGoogle Scholar
  24. Kinose Y, Fukamachi Y, Okabe S, Hiroshima H, Watanabe M, Izuta T (2017b) Photosynthetic responses to ozone of upper and lower canopy leaves of Fagus crenata Blume seedlings grown under different soil nutrient conditions. Environ Pollut 223:213–222CrossRefGoogle Scholar
  25. Koike T, Watanabe M, Hoshika Y, Kitao M, Matsumura H, Funada R, Izuta T (2013) Effects of ozone on forest ecosystems in East and Southeast Asia. In: Matyssek R, Clarke N, Cudlin P, Mikkelsen TN, Tuovinen J-P, Wieser G, Paoletti E (eds) Climate change, air pollution and global challenges: understanding and perspectives from forest research. Elsevier, Oxford, pp 371–390CrossRefGoogle Scholar
  26. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  27. Li P, Feng Z, Catalayud V, Yuan X, Xu Y, Paoletti E (2017) A meta-analysis on growth, physiological, and biochemical responses of woody species to ground-level ozone highlights the role of plant functional types. Plant Cell Environ 40:2369–2380CrossRefGoogle Scholar
  28. Long SP, Bernacchi CJ (2003) Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error. J Exp Bot 54:2393–2401CrossRefGoogle Scholar
  29. Matyssek R, Sandermann H (2003) Impact of ozone on trees: an ecophysiological perspective. Prog Bot 64:349–404CrossRefGoogle Scholar
  30. Mizokami Y, Noguchi K, Kojima M, Sakakibara H, Terashima I (2015) Mesophyll conductance decreases in the wild type but not in an ABA-deficient mutant (aba1) of Nicotiana plumbaginifolia under drought conditions. Plant Cell Environ 38:388–398CrossRefGoogle Scholar
  31. Moualeu-Ngangue DP, Chen T, Stűtzel H (2017) A new method to estimate photosynthetic parameters through net assimilation rate–intercellular space CO2 concentration (AC i) curve and chlorophyll fluorescence measurements. New Phytol 213:1543–1554CrossRefGoogle Scholar
  32. Nakaji T, Izuta T (2001) Effects of ozone and/or excess soil nitrogen on growth, needle gas exchange rates and Rubisco contents of Pinus densiflora seedlings. Water Air Soil Pollut 130:971–976CrossRefGoogle Scholar
  33. Nakashizuka T, Iida S (1995) Composition, dynamics and disturbance regime of temperate deciduous forests in Monsoon Asia. Vegetatio 121:23–30CrossRefGoogle Scholar
  34. Niu J, Feng Z, Zhang W, Zhao P, Wang X (2014) Non-stomatal limitation to photosynthesis in Cinnamomum camphora seedings exposed to elevated O3. PLoS One 9:e98572CrossRefGoogle Scholar
  35. Nunn AJ, Reiter IM, Häberle K-H, Langebartels C, Bahnweg G, Pretzsch H, Sandermann H, Matyssek R (2005) Response patterns in adult forest trees to chronic ozone stress: identification of variations and consistencies. Environ Pollut 136:365–369CrossRefGoogle Scholar
  36. Paoletti E, Schaub M, Matyssek R, Wieser G, Augustaitis A, Bastrup-Birk AM, Bytnerowicz A, Günthardt-Goerg MS, Müller-Starck G, Serengil Y (2010) Advances of air pollution science: from forest decline to multiple-stress effects on forest ecosystem services. Environ Pollut 158:1986–1989CrossRefGoogle Scholar
  37. Paoletti E, De Marco A, Beddows DCS, Harrison RM, Manning WJ (2014) Ozone levels in European and USA cities are increasing more than at rural sites, while peak values are decreasing. Environ Pollut 192:295–299CrossRefGoogle Scholar
  38. Pell EJ, Eckardt N, Enyedi AJ (1992) Timing of ozone stress and resulting status of ribulose bisphosphate carboxylase/oxygenase and associated net photosynthesis. New Phytol 120:397–405CrossRefGoogle Scholar
  39. Pell EJ, Schlagnhaufer CD, Arteca RN (1997) Ozone-induced oxidative stress: mechanisms of action and reaction. Physiol Plant 100:264–273CrossRefGoogle Scholar
  40. R Development Core Team (2017) R: a language and environment for statistical computing. R Foundation for Statistical Computing. Accessed 17 Jan 2018
  41. Schreiber U, Bilger W, Neubauer C (1994) Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Schulze E-D, Caldwell MM (eds) Ecophysiology of photosynthesis. Springer, Berlin, pp 49–70Google Scholar
  42. Sitch S, Cox PM, Collins WJ, Huntingford C (2007) Indirect radiative forcing of climate change through ozone effects on the land-carbon sink. Nature 448:791–794CrossRefGoogle Scholar
  43. Velikova V, Tsonev T, Pinelli P, Alessio GA, Loreto F (2005) Localized ozone fumigation system for studying ozone effects on photosynthesis, respiration, electron transport rate and isoprene emission in field-grown Mediterranean oak species. Tree Physiol 25:1523–1532CrossRefGoogle Scholar
  44. von Caemmerer S (2000) Biochemical models of leaf photosynthesis. CSIRO Publishing, CollingwoodCrossRefGoogle Scholar
  45. Warren CR, Löw M, Matyssek R, Tausz M (2007) Internal conductance to CO2 transfer of adult Fagus sylvatica: variation between sun and shade leaves and due to free-air ozone fumigation. Environ Exp Bot 59:130–138CrossRefGoogle Scholar
  46. Watanabe M, Yonekura T, Honda Y, Yoshidome M, Nakaji T, Izuta T (2005) Effects of ozone and soil water stress, singly and in combination, on leaf antioxidative systems of Fagus crenata seedlings. J Agric Meteorol 60:1105–1108CrossRefGoogle Scholar
  47. Watanabe M, Hoshika Y, Inada N, Wang X, Mao Q, Koike T (2013) Photosynthetic traits of Siebold’s beech and oak saplings grown under free air ozone exposure in northern Japan. Environ Pollut 174:50–56CrossRefGoogle Scholar
  48. Watanabe M, Hoshika Y, Koike T (2014) Photosynthetic responses of Monarch birch seedlings to differing timing of free air ozone fumigation. J Plant Res 127:339–345CrossRefGoogle Scholar
  49. Watanabe M, Hoshika Y, Koike T, Izuta T (2017) Effects of ozone on Japanese trees. In: Izuta T (ed) Air pollution impacts on plant in East Asia. Springer Japan, Tokyo, pp 73–100CrossRefGoogle Scholar
  50. Werner H, Fabian P (2002) Free-air fumigation of mature trees: a novel system for controlled ozone enrichment in grown-up beech and spruce canopies. Environ Sci Pollut Res 9:117–121CrossRefGoogle Scholar
  51. Wittig VE, Ainsworth EA, Long SP (2007) To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the last 3 decades of experiments. Plant Cell Environ 30:1150–1162CrossRefGoogle Scholar
  52. Xiong D, Liu X, Liu L, Douthe C, Li Y, Peng S, Huang J (2015) Rapid responses of mesophyll conductance to changes of CO2 concentration, temperature and irradiance are affected by N supplements in rice. Plant Cell Environ 38:2541–2550CrossRefGoogle Scholar
  53. Yamaguchi M, Watanabe M, Iwasaki M, Tabe C, Matsumura H, Kohno Y, Izuta T (2007) Growth and photosynthetic responses of Fagus crenata seedlings to O3 under different nitrogen loads. Trees 21:707–718CrossRefGoogle Scholar
  54. Yamaguchi M, Watanabe M, Matsumura H, Kohno Y, Izuta T (2011) Experimental studies on the effects of ozone on growth and photosynthetic activity of Japanese forest tree species. Asian J Atmos Environ 5:65–87CrossRefGoogle Scholar
  55. Yonekura T, Dokiya Y, Fukami M, Izuta T (2001) Effects of ozone and/or soil water stress on growth and photosynthesis of Fagus crenata seedlings. Water Air Soil Pollut 130:965–970CrossRefGoogle Scholar
  56. Zhang J, Schaub M, Ferdinand JA, Skelly JM, Steiner KC, Savage JE (2010) Leaf age affects the responses of foliar injury and gas exchange to tropospheric ozone in Prunus serotina seedlings. Environ Pollut 158:2627–2634CrossRefGoogle Scholar
  57. Zhang W, Feng Z, Wang X, Niu J (2014) Impacts of elevated ozone on growth and photosynthesis of Metasequoia glyptostroboides Hu et Cheng. Plant Sci 226:182–188CrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Makoto Watanabe
    • 1
    Email author
  • Yu Kamimaki
    • 1
  • Marino Mori
    • 1
  • Shigeaki Okabe
    • 1
  • Izumi Arakawa
    • 2
  • Yoshiyuki Kinose
    • 3
  • Satoshi Nakaba
    • 1
  • Takeshi Izuta
    • 1
  1. 1.Institute of AgricultureTokyo University of Agriculture and TechnologyFuchuJapan
  2. 2.United Graduate School of Agricultural ScienceTokyo University of Agriculture and TechnologyFuchuJapan
  3. 3.Graduate Faculty of Interdisciplinary ResearchUniversity of YamanashiKofuJapan

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