View source for Fractionation of carbon isotopes in oxygenic photosynthesis

[[Photosynthesis]] converts [[carbon dioxide]] to [[carbohydrate]]s via several [[metabolic pathway]]s that provide energy to an organism and preferentially react with certain [[stable isotopes]] of [[carbon]].<ref name=":0">{{Cite journal|last1=G D Farquhar|last2=J R Ehleringer|last3=Hubick|first3=and K. T.|date=1989|title=Carbon Isotope Discrimination and Photosynthesis|journal=Annual Review of Plant Physiology and Plant Molecular Biology|volume=40|issue=1|pages=503–537|doi=10.1146/annurev.pp.40.060189.002443}}</ref> The selective enrichment of one stable isotope over another creates distinct [[Isotope fractionation|isotopic fractionations]] that can be measured and correlated among oxygenic [[phototroph]]s. The degree of carbon isotope fractionation is influenced by several factors, including the metabolism, anatomy, growth rate, and environmental conditions of the organism. Understanding these variations in carbon fractionation across species is useful for [[Biogeochemistry|biogeochemical]] studies, including the reconstruction of [[paleoecology]], [[plant evolution]], and the characterization of [[food chain]]s.<ref name=":9"/><ref>{{Cite book|title=Stable Isotopes in Ecological Research|last1=Fry|first1=B.|last2=Sherr|first2=E. B.|chapter=δ13C Measurements as Indicators of Carbon Flow in Marine and Freshwater Ecosystems |series=Ecological Studies |date=1989|volume=68 |publisher=Springer New York|isbn=9781461281276|location=New York, NY|pages=196–229|doi=10.1007/978-1-4612-3498-2_12}}</ref>

[[File:Kinetic and Equilibrium Isotope Effects.png|thumb|A simplified model of a chemical reaction with pathways for a light isotope (H) and heavy isotope (D) of hydrogen. The same principle applies for the light isotope <sup>12</sup>C and heavy isotope <sup>13</sup>C of carbon. The positions on the energy wells are based on the [[quantum harmonic oscillator]]. Note the lower energy state of the heavier isotope and the higher energy state of the lighter isotope. Under kinetic conditions, such as an enzymatic reaction with RuBisCO, the lighter isotope is favored because of a lower activation energy.]]
Oxygenic [[photosynthesis]] is a [[metabolic pathway]] facilitated by [[autotroph]]s, including plants, algae, and cyanobacteria. This pathway converts inorganic carbon dioxide from the atmosphere or aquatic environment into [[carbohydrate]]s, using water and energy from light, then releases molecular oxygen as a product.  Organic carbon contains less of the stable isotope [[Carbon-13]], or <sup>13</sup>C, relative to the initial inorganic carbon from the atmosphere or water because photosynthetic carbon fixation involves several fractionating reactions with [[kinetic isotope effect]]s.<ref name=":5">{{Cite journal|last=Hayes|first=John|date=2001-01-01|title=Fractionation of Carbon and Hydrogen Isotopes in Biosynthetic Processes|url=https://www.researchgate.net/publication/246232711|journal=Reviews in Mineralogy & Geochemistry |volume=43|issue=1|pages=225–277|doi=10.2138/gsrmg.43.1.225|bibcode=2001RvMG...43..225H}}</ref> These reactions undergo a kinetic isotope effect because they are [[Rate limiting step|limited]] by overcoming an [[activation energy]] barrier. The lighter isotope has a higher [[energy state]] in the [[quantum well]] of a chemical bond, allowing it to be preferentially formed into products. Different organisms fix carbon through different mechanisms, which are reflected in the varying isotope compositions across photosynthetic pathways (see table below, and explanation of notation in "Carbon Isotope Measurement" section). The following sections will outline the different oxygenic photosynthetic pathways and what contributes to their associated delta values.

[[File:Figure 14 - With this method C3, C4 and CAM plant metabolism are well separated.jpg|thumb|Different photosynthetic pathways (C3, C4, and CAM) yields biomass with different δ<sup>13</sup>C values.]]
{| class="wikitable"
|+
Isotope Delta Values of Photosynthetic Pathways
!Pathway
!'''''δ'''''<sup>'''13'''</sup>'''C (‰)'''
|-
|C3
| -20 to -37<ref name=":9">{{Cite journal|last=Kohn|first=Matthew J.|date=2010-11-16|title=Carbon isotope compositions of terrestrial C3 plants as indicators of (paleo)ecology and (paleo)climate|journal=Proceedings of the National Academy of Sciences|volume=107|issue=46|pages=19691–19695|doi=10.1073/pnas.1004933107|issn=0027-8424|pmid=21041671|pmc=2993332|bibcode=2010PNAS..10719691K|doi-access=free}}</ref>
|-
|C4
| -12 to -16<ref name=":4">{{Cite journal|last=O'Leary|first=Marion H.|date=May 1988|title=Carbon Isotopes in Photosynthesis|journal=BioScience|volume=38|issue=5|pages=328–336|doi=10.2307/1310735|issn=0006-3568|jstor=1310735}}</ref>
|-
|CAM
| -10 to -20<ref name=":1">{{Cite journal|last=O'Leary|first=Marion H.|date=1988|title=Carbon Isotopes in Photosynthesis|jstor=1310735|journal=BioScience|volume=38|issue=5|pages=328–336|doi=10.2307/1310735}}</ref>
|-
|Phytoplankton
| -18 to -25<ref name=":5" /><ref name=":6">{{Cite journal|last1=Popp|first1=Brian N.|last2=Laws|first2=Edward A.|last3=Bidigare|first3=Robert R.|last4=Dore|first4=John E.|last5=Hanson|first5=Kristi L.|last6=Wakeham|first6=Stuart G.|date=January 1998|title=Effect of Phytoplankton Cell Geometry on Carbon Isotopic Fractionation|journal=Geochimica et Cosmochimica Acta|volume=62|issue=1|pages=69–77|doi=10.1016/S0016-7037(97)00333-5|issn=0016-7037|bibcode=1998GeCoA..62...69P}}</ref>
|}

== Carbon isotope measurement ==
Carbon on Earth naturally occurs in two stable isotopes, with 98.9% in the form of [[Carbon-12|<sup>12</sup>C]] and 1.1% in [[Carbon-13|<sup>13</sup>C]].<ref name=":0" /><ref name=":2">{{Cite journal|last=Gonfiantini|first=Roberto|date=April 1984|title=I.A.E.A. advisory group meeting on stable isotope reference samples for geochemical and hydrological investigations|journal=Chemical Geology|volume=46|issue=1|pages=85|doi=10.1016/0009-2541(84)90167-0|bibcode=1984ChGeo..46...85G|issn=0009-2541}}</ref> The ratio between these isotopes varies in biological organisms due to metabolic processes that selectively use one carbon isotope over the other, or "fractionate" carbon through kinetic or thermodynamic effects.<ref name=":0" /> Oxygenic [[photosynthesis]] takes place in plants and microorganisms through different chemical pathways, so various forms of organic material reflect different ratios of [[Carbon-13|<sup>13</sup>C]] isotopes. Understanding these variations in carbon fractionation across species is applied in [[isotope geochemistry]] and [[Isotope analysis|ecological isotope studies]] to understand biochemical processes, establish food chains, or model the carbon cycle through geological time.<ref name=":4" />

Carbon isotope fractionations are expressed in using delta notation of [[Δ13C|''δ''<sup>13</sup>C]] ("delta thirteen C"), which is reported in parts per thousand ([[per mille]], ‰).<ref>{{Cite journal|last=McKone|first=Harold T.|date=September 1992|title=An Introduction to Marine Biogeochemistry (Libes, Susan M.)|journal=Journal of Chemical Education|volume=69|issue=9|pages=A251|doi=10.1021/ed069pa251.2|bibcode=1992JChEd..69..251M|issn=0021-9584|doi-access=free}}</ref>  [[Δ13C|''δ''<sup>13</sup>C]] is defined in relation to the Vienna Pee Dee [[Belemnitida|Belemnite]] (VPDB, <sup>13</sup>C/<sup>12</sup>C = 0.01118) as an established [[Reference materials for stable isotope analysis|reference standard]].<ref name=":2" /><ref>{{Cite journal|date=March 1977|title=Biological Oceanography|journal=The American Biology Teacher|volume=39|issue=3|pages=186|doi=10.2307/4445858|issn=0002-7685|jstor=4445858|last1=McClintock|first1=Barbara M.|hdl=2027/umn.31951d01800724l|hdl-access=free}}</ref> This is called a "delta value" and can be calculated from the formula below:

<math display="block"> \delta^{13}\mathrm{C} = \left ( \frac{\left ( \frac{^{13}\mathrm{C}}{^{12}\mathrm{C}} \right )_\mathrm{sample}}{\left ( \frac{^{13}\mathrm{C}}{^{12}\mathrm{C}} \right )_\mathrm{standard}} -1 \right)\times1000</math>

== Photosynthesis reactions ==
The chemical pathway of oxygenic photosynthesis [[Carbon fixation|fixes carbon]] in two stages: the light-dependent reactions and the light-independent reactions.

The light-dependent reactions capture light energy to transfer electrons from water and convert [[NADP+|NADP<sup>+</sup>]], [[Adenosine diphosphate|ADP]], and inorganic phosphate into the energy-storage molecules [[Nicotinamide adenine dinucleotide phosphate|NADPH]] and [[Adenosine triphosphate|ATP]]. The overall equation for the light-dependent reactions is generally:<ref name=":7">{{Cite book|title=Biology of plants|last=H.|first=Raven, Peter|date=2005|publisher=W.H. Freeman and Co|others=Evert, Ray Franklin., Eichhorn, Susan E.|isbn=978-0716710073|edition= 7th|location=New York|oclc=56051064}}</ref>[[File:Calvin-cycle4.svg|thumb|Overview of the [[Calvin cycle]] and [[carbon fixation]] C3 Pathway]]<blockquote>2 H<sub>2</sub>O + 2 NADP<sup>+</sup> + 3 ADP + 3 P<sub>i</sub> + light → 2 NADPH + 2 H<sup>+</sup> + 3 ATP + O<sub>2</sub></blockquote>The [[Light-independent reactions|light-independent]] reactions undergo the [[Calvin-Benson cycle]], in which the energy from NADPH and ATP is used to convert [[carbon dioxide]] and water into organic compounds via the [[enzyme]] [[RuBisCO]].

The overall general equation for the light-independent reactions is the following:<ref name=":7" /><blockquote>3 CO<sub>2</sub> + 9 ATP + 6 NADPH + 6 H<sup>+</sup> → C<sub>3</sub>H<sub>6</sub>O<sub>3</sub>-phosphate + 9 ADP + 8 P<sub>i</sub> + 6 NADP<sup>+</sup> + 3 H<sub>2</sub>O</blockquote>The 3-carbon products (C<sub>3</sub>H<sub>6</sub>O<sub>3</sub>-phosphate) of the Calvin cycle are later converted to [[glucose]] or other carbohydrates such as [[starch]], [[sucrose]], and [[cellulose]].

== Fractionation via RuBisCO ==
[[File:RuBisCO reaction.svg|thumb|Carboxylation of ribulose-1,5-bisphosphate (1) into two molecules of 3-phosphoglycerate (4) by RuBisCO. The intermediate molecule at (3) is 3-keto-2-carboxyarabinitol-1,5-bisphosphate, which decays almost instantaneously into 3-phosphoglycerate.]]

The large fractionation of <sup>13</sup>C in photosynthesis is due to the carboxylation reaction, which is carried out by the enzyme ribulose-1,5-bisphosphate carboxylase oxygenase, or [[RuBisCO]].<ref name=":4" /> RuBisCO catalyzes the reaction between a five-carbon molecule, [[Ribulose 1,5-bisphosphate|ribulose-1,5-bisphosphate]] (abbreviated as RuBP) and CO<sub>2</sub>  to form two molecules of [[3-Phosphoglyceric acid|3-phosphoglyceric acid]] (abbreviated as PGA). PGA reacts with NADPH to produce [[3-phosphoglyceraldehyde]].<ref name=":5" />

Isotope fractionation due to Rubisco (form I) carboxylation alone is predicted to be a 28‰ depletion, on average.<ref>{{Cite journal|last1=Tabita|first1=F. R.|last2=Satagopan|first2=S.|last3=Hanson|first3=T. E.|last4=Kreel|first4=N. E.|last5=Scott|first5=S. S.|date=2007-06-19|title=Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships|journal=Journal of Experimental Botany|volume=59|issue=7|pages=1515–1524|doi=10.1093/jxb/erm361|pmid=18281717|issn=0022-0957|doi-access=free}}</ref><ref name=":4" /> However, fractionation values vary between organisms, ranging from an 11‰ depletion observed in [[Coccolithophore|coccolithophorid]] algae to a 29‰ depletion observed in [[spinach]].<ref>{{Cite journal|last1=Scott|first1=Kathleen M.|last2=Schwedock|first2=Julie|last3=Schrag|first3=Daniel P.|last4=Cavanaugh|first4=Colleen M.|date=December 2004|title=Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum|journal=Environmental Microbiology|volume=6|issue=12|pages=1210–1219|doi=10.1111/j.1462-2920.2004.00642.x|pmid=15560819|issn=1462-2912}}</ref><ref>{{Cite journal|last1=Guy|first1=R. D.|last2=Fogel|first2=M. L.|last3=Berry|first3=J. A.|date=1993-01-01|title=Photosynthetic Fractionation of the Stable Isotopes of Oxygen and Carbon|journal=Plant Physiology|volume=101|issue=1|pages=37–47|doi=10.1104/pp.101.1.37|issn=0032-0889|pmid=12231663|pmc=158645}}</ref> [[Intrinsic KIE of RuBisCO|RuBisCO causes a kinetic isotope effect]] because <sup>12</sup>CO<sub>2</sub> and <sup>13</sup>CO<sub>2</sub> compete for the same active site and <sup>13</sup>C has an intrinsically lower reaction rate.<ref>{{Cite journal|last1=McNevin|first1=Dennis B.|last2=Badger|first2=Murray R.|last3=Whitney|first3=Spencer M.|last4=Caemmerer|first4=Susanne von|last5=Tcherkez|first5=Guillaume G. B.|last6=Farquhar|first6=Graham D.|date=2007-12-07|title=Differences in Carbon Isotope Discrimination of Three Variants of D-Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Reflect Differences in Their Catalytic Mechanisms|journal=Journal of Biological Chemistry|volume=282|issue=49|pages=36068–36076|doi=10.1074/jbc.M706274200|issn=0021-9258|pmid=17925403|doi-access=free}}</ref>

== <sup>13</sup>C fractionation model ==

In addition to the discriminating effects of enzymatic reactions, the diffusion of CO<sub>2</sub> gas to the carboxylation site within a plant cell also influences isotopic fractionation.<ref name=":8">{{Cite journal|last1=Farquhar|first1=Graham|last2=O'Leary|first2=M.H.|last3=Berry|first3=Joseph|date=1982-01-01|title=On the Relationship Between Carbon Isotope Discrimination and the Intercellular Carbon Dioxide Concentration in Leaves|url=https://www.researchgate.net/publication/230890521|journal=Australian Journal of Plant Physiology|volume=13|issue=2|pages=281–292|doi=10.1071/PP9820121}}</ref> Depending on the type of plant (see sections below), external CO<sub>2</sub> must be transported through the [[boundary layer]] and [[Stomatal conductance|stomata]] and into the internal gas space of a plant cell, where it dissolves and diffuses to the chloroplast.<ref name=":4" /> The [[Mass diffusivity|diffusivity]] of a gas is inversely proportional to the square root of its molecular [[reduced mass]] (relatively to air), causing <sup>13</sup>CO<sub>2</sub> to be 4.4‰ less diffusive than <sup>12</sup>CO<sub>2</sub>.

A prevailing model for fractionation of atmospheric CO<sub>2</sub> in plants combines the isotope effects of the carboxylation reaction with the isotope effects from [[Stomatal conductance|gas diffusion into the plant]] in the following equation:<ref name=":8" />

<math display="block"> \delta^{13}C_\text{sample} = \delta^{13}C_\text{atm} - a - (b-a)(c_{i}/c_{a})</math>

Where:

* δ<sup>13</sup>C<sub>sample</sub> is the delta-value of the organism for <sup>13</sup>C composition
*δ<sup>13</sup>C<sub>atm</sub> is the delta-value of atmospheric CO<sub>2</sub>, which is = -7.8‰
* the discrimination due to diffusion ''a'' = 4.4‰
* the carboxylation discrimination ''b'' = 30‰
* c<sub>a</sub> is the partial pressure of CO<sub>2</sub> in the external atmosphere, and 
* c<sub>i</sub> is the partial pressure of CO<sub>2</sub> in the intercellular spaces.

This model, derived ''[[ab initio]]'', generally describes fractionation of carbon in the majority of plants, which facilitate [[C3 carbon fixation]]. Modifications have been made to this model with empirical findings.<ref>{{Cite journal|last1=Schubert|first1=Brian A.|last2=Jahren|first2=A. Hope|date=November 2012|title=The effect of atmospheric CO2 concentration on carbon isotope fractionation in C3 land plants|journal=Geochimica et Cosmochimica Acta|volume=96|pages=29–43|doi=10.1016/j.gca.2012.08.003|issn=0016-7037|bibcode=2012GeCoA..96...29S|doi-access=free}}</ref> However, several additional factors, not included in this general model, will increase or decrease <sup>13</sup>C fractionation across species. Such factors include the competing oxygenation reaction of RuBisCO, anatomical and temporal adaptations to enzyme activity, and variations in cell growth and geometry. The isotopic fractionations of different photosynthetic pathways are uniquely characterized by these factors, as described below.

== In C3 plants ==
[[File:C3 c4 d13C comparison.png|thumb|Histograms of the carbon isotope ratios from modern grasses. Note that C3 plants are roughly 14‰ depleted in <sup>13</sup>C relative to C4 plants.]]
A C3 plant uses [[C3 carbon fixation]], one of the three metabolic photosynthesis pathways which also include [[C4 carbon fixation|C4]] and [[Crassulacean acid metabolism|CAM]] (described below). These plants are called "C3" due to the three-carbon compound ([[3-Phosphoglyceric acid]], or 3-PGA) produced by the CO<sub>2</sub> fixation mechanism in these plants. This C3 mechanism is the first step of the Calvin-Benson cycle, which converts CO<sub>2</sub> and [[RuBP]] into [[3-Phosphoglyceric acid|3-PGA]].

C3 plants are the most common type of plant, and typically thrive under moderate sunlight intensity and temperatures, CO<sub>2</sub> concentrations above 200 ppm, and abundant groundwater.<ref>{{Cite book|last=Whitehead|first=Mark|title=International Encyclopedia of Geography |date=2017-03-06|chapter=Environment and the State|journal=International Encyclopedia of Geography: People, the Earth, Environment and Technology|location=Oxford, UK|publisher=John Wiley & Sons, Ltd|pages=1–11|isbn=9780470659632|doi=10.1002/9781118786352.wbieg0920}}</ref> C3 plants do not grow well in very hot or arid regions, in which C4 and CAM plants are better adapted.

The isotope fractionations in C3 carbon fixation arise from the combined effects of CO<sub>2</sub> gas diffusion through the [[stomata]] of the plant, and the carboxylation via [[RuBisCO]].<ref name=":0" /> [[Stomatal conductance]] discriminates against the heavier <sup>13</sup>C by 4.4‰.<ref name=":0" /> RuBisCO carboxylation contributes a larger discrimination of 27‰.<ref name=":0" />

RuBisCO enzyme catalyzes the carboxylation of CO<sub>2</sub> and the 5-carbon sugar, [[Ribulose 1,5-bisphosphate|RuBP]], into [[3-phosphoglycerate]], a 3-carbon compound through the following reaction:

<math>CO_2+H_2O+RuBP \xrightarrow[RuBisCO]{} 2(3 \mbox{-} \text{phosphoglycerate}) </math>

The product [[3-phosphoglycerate]] is depleted in <sup>13</sup>C due to the [[kinetic isotope effect]] of the above reaction. The overall <sup>13</sup>C fractionation for C3 photosynthesis ranges between -20 and -37‰.<ref name=":9" />

The wide range of variation in delta values expressed in C3 plants is modulated by the [[stomatal conductance]], or the rate of CO<sub>2</sub> entering, or water vapor exiting, the small pores in the epidermis of a leaf.<ref name=":0" /> The δ<sup>13</sup>C of C3 plants depends on the relationship between stomatal conductance and photosynthetic rate, which is a good proxy of water use efficiency in the leaf.<ref name=":11">{{Cite journal|last1=Moreno-Gutiérrez|first1=Cristina|last2=Dawson|first2=Todd E.|last3=Nicolás|first3=Emilio|last4=Querejeta|first4=José Ignacio|date=2012-08-23|title=Isotopes reveal contrasting water use strategies among coexisting plant species in a Mediterranean ecosystem|journal=New Phytologist|volume=196|issue=2|pages=489–496|doi=10.1111/j.1469-8137.2012.04276.x|pmid=22913668|issn=0028-646X|doi-access=}}</ref> C3 plants with high water-use efficiency tend to be less fractionated in <sup>13</sup>C  (i.e., δ<sup>13</sup>C is relatively less negative) compared to C3 plants with low water-use efficiency.<ref name=":11" />

== In C4 plants ==
[[File:C4 Plant Anatomy.svg|thumb|In the C4 pathway, a layer of mesophyll cells encircles bundle sheath cells that have large chloroplasts necessary for the Calvin cycle.

A: Mesophyll Cell B: Chloroplast C: Vascular Tissue D: Bundle Sheath Cell E: Stroma F: Vascular Tissue, provides continuous source of water.  

1) Carbon is fixed to produce oxaloacetate by PEP carboxylase. 2) The four carbon molecule then exits the cell and enters the chloroplasts of bundle sheath cells.  3) It is then broken down releasing carbon dioxide and producing pyruvate. Carbon dioxide combines with ribulose bisphosphate and proceeds to the Calvin Cycle.]]
C4 plants have developed the [[C4 carbon fixation]] pathway to conserve water loss, thus are more prevalent in hot, sunny, and dry climates.<ref name=":3" /> These plants differ from C3 plants because CO<sub>2</sub> is initially converted to a four-carbon molecule, [[malate]], which is shuttled to bundle sheath cells, released back as CO<sub>2</sub> and only then enters the Calvin Cycle. In contrast, C3 plants directly perform the Calvin Cycle in mesophyll cells, without making use of a CO<sub>2</sub> concentration method. Malate, the four-carbon compound is the namesake of "C4" photosynthesis. This pathway allows C4 photosynthesis to efficiently shuttle CO<sub>2</sub> to the RuBisCO enzyme and maintain high concentrations of CO<sub>2</sub> within [[bundle sheath cells]]. These cells are part of the characteristic ''kranz leaf anatomy'', which spatially separates photosynthetic cell-types in a concentric arrangement to accumulate CO<sub>2</sub> near RuBisCO.<ref>{{Cite journal|last=Kennedy|first=Robert A.|date=April 23, 1976|title=Photorespiration in C3 and C4 Plant Tissue Cultures|journal=Plant Physiology|volume=58|issue=4|pages=573–575|doi=10.1104/pp.58.4.573|pmid=16659720|pmc=543284}}</ref>

These chemical and anatomical mechanisms improve the ability of RuBisCO to fix carbon, rather than perform its wasteful [[oxygenase]] activity. The RuBisCO oxygenase activity, called [[photorespiration]], causes the RuBP substrate to be lost to oxygenation, and consumes energy in doing so. The adaptations of C4 plants provide an advantage over the C3 pathway, which loses efficiency due to photorespiration.<ref name=":10">{{Cite book|title=C4 plant biology|date=1999|publisher=Academic Press|others=Sage, Rowan Frederick., Monson, R. K. (Russell K.), 1954-|isbn=9780080528397|location=San Diego|oclc=176630229}}</ref> The ratio of photorespiration to photosynthesis in a plant varies with environmental conditions, since decreased CO<sub>2</sub> and elevated O<sub>2</sub> concentrations would increase the efficiency of photorespiration.<ref name=":3">{{Cite journal|date=1991-03-01|title=Climate change and the evolution of C4 photosynthesis|journal=Trends in Ecology & Evolution|volume=6|issue=3|pages=95–99|doi=10.1016/0169-5347(91)90183-X|pmid=21232434|issn=0169-5347|last1=Ehleringer|first1=James R.|last2=Sage|first2=Rowan F.|last3=Flanagan|first3=Lawrence B.|last4=Pearcy|first4=Robert W.}}</ref> Atmospheric CO<sub>2</sub> on Earth decreased abruptly at a point between 32 and 25 million years ago. This gave a selective advantage to the evolution of the C4 pathway, which can limit photorespiration rate despite the reduced ambient CO<sub>2</sub>.<ref>{{Cite journal|title=Photorespiration and the Evolution of C4 Photosynthesis {{!}} Annual Review of Plant Biology|journal=Annual Review of Plant Biology|volume=63|issue=1|pages=19–47|doi=10.1146/annurev-arplant-042811-105511|pmid=22404472|year=2012|last1=Sage|first1=Rowan F.|last2=Sage|first2=Tammy L.|last3=Kocacinar|first3=Ferit|s2cid=24199852}}</ref> Today, C4 plants represent roughly 5% of plant biomass on Earth, but about 23% of terrestrial carbon fixation.<ref>{{Cite journal|last1=Bond|first1=W. J.|last2=Woodward|first2=F. I.|last3=Midgley|first3=G. F.|date=2004-11-12|title=The global distribution of ecosystems in a world without fire|journal=New Phytologist|volume=165|issue=2|pages=525–538|doi=10.1111/j.1469-8137.2004.01252.x|pmid=15720663|s2cid=4954178 |issn=0028-646X}}</ref><ref>{{Cite journal|last1=Osborne|first1=C. P.|last2=Beerling|first2=D. J.|date=2006-01-29|title=Nature's green revolution: the remarkable evolutionary rise of C4 plants|journal=Philosophical Transactions of the Royal Society B: Biological Sciences|volume=361|issue=1465|pages=173–194|doi=10.1098/rstb.2005.1737|pmid=16553316|pmc=1626541|issn=0962-8436}}</ref><ref>{{Cite journal|last=Kellogg|first=Elizabeth A.|date=July 2013|title=C4 photosynthesis|journal=Current Biology|volume=23|issue=14|pages=R594–R599|doi=10.1016/j.cub.2013.04.066|pmid=23885869|issn=0960-9822|doi-access=free}}</ref> Types of plants which use C4 photosynthesis include [[Poaceae|grasses]] and economically important crops, such as [[Zea mays|maize]], [[sugar cane]], [[millet]], and [[sorghum]].<ref name=":10" /><ref>{{Cite journal|last1=Zhu|first1=Xin-Guang|last2=Long|first2=Stephen P|last3=Ort|first3=Donald R|date=April 2008|title=What is the maximum efficiency with which photosynthesis can convert solar energy into biomass?|journal=Current Opinion in Biotechnology|volume=19|issue=2|pages=153–159|doi=10.1016/j.copbio.2008.02.004|pmid=18374559|issn=0958-1669}}</ref>

Isotopic fractionation differs between [[C4 carbon fixation]] and C3, due to the spatial separation in C4 plants of CO<sub>2</sub> capture (in the mesophyll cells) and the Calvin cycle (in the bundle sheath cells). In C4 plants, carbon is converted to [[bicarbonate]], fixed into [[oxaloacetate]] via the enzyme [[phosphoenolpyruvate carboxylase|phosphoenolpyruvate (PEP) carboxylase]], and is then converted to [[malate]].<ref name=":5" /> The malate is transported from the [[mesophyll]] to [[Bundle-sheath|bundle sheath]] cells, which are impermeable to CO<sub>2</sub>. The internal CO<sub>2</sub> is concentrated in these cells as malate is reoxidized then decarboxylated back into CO<sub>2</sub> and pyruvate. This enables RuBisCO to perform catalysis while internal CO<sub>2</sub> is sufficiently high to avoid the competing photorespiration reaction. The delta value in the C4 pathway is -12 to -16‰ depleted in <sup>13</sup>C due to the combined effects of PEP carboxylase and RuBisCO.

The isotopic discrimination in the C4 pathway varies relative to the C3 pathway due to the additional chemical conversion steps and activity of PEP carboxylase. After diffusion into the stomata, the conversion of CO<sub>2</sub> to bicarbonate concentrates the heavier <sup>13</sup>C. The subsequent fixation via PEP carboxylase is thereby less depleted in <sup>13</sup>C than that from Rubisco: about 2‰ depleted in PEP carboxylase, versus 29‰ in RuBisCO.<ref name=":0" /><ref name=":4" /> However, a portion of the isotopically heavy carbon that is fixed by PEP carboxylase leaks out of the bundle sheath cells. This limits the carbon available to RuBisCO, which in turn lowers its fractionation effect.<ref name=":5" /> This accounts for the overall delta value in C4 plants to be -12 to -16 ‰.<ref name=":5" />

== In CAM plants ==
Plants that use [[Crassulacean acid metabolism]], also known as CAM photosynthesis, temporally separate their chemical reactions between day and night. This strategy modulates stomatal conductance to increase water-use efficiency, so is well-adapted for arid climates.<ref>{{Cite journal|last=Ting|first=I P|date=June 1985|title=Crassulacean Acid Metabolism|journal=Annual Review of Plant Physiology|volume=36|issue=1|pages=595–622|doi=10.1146/annurev.pp.36.060185.003115|issn=0066-4294|hdl=10150/552219|hdl-access=free}}</ref> During the night, CAM plants open stomata to allow CO<sub>2</sub> to enter the cell and undergo fixation into organic acids that are stored in vacuoles. This carbon is released to the Calvin cycle during the day, when stomata are closed to prevent water loss, and the light reactions can drive the necessary ATP and NADPH production.<ref>{{Cite journal|last=Ting|first=I.|date=1985-01-01|title=Crassulacean Acid Metabolism|journal=Annual Review of Plant Physiology and Plant Molecular Biology|volume=36|issue=1|pages=595–622|doi=10.1146/annurev.pp.36.060185.003115|issn=1040-2519|hdl=10150/552219|hdl-access=free}}</ref> This pathway differs from C4 photosynthesis because CAM plants separate carbon by storing fixed CO<sub>2</sub> in vesicles at night, then transporting it for use during the day. Thus, CAM plants temporally concentrate CO<sub>2</sub> to improve RuBisCO efficiency, whereas C4 plants spatially concentrate CO<sub>2</sub> in bundle sheath cells. The distribution of plants which use CAM photosynthesis includes [[epiphyte]]s (e.g., [[Orchidaceae|orchids]], [[Bromeliaceae|bromeliads]]) and [[xerophyte]]s (e.g., [[Succulent plant|succulents]], [[Cactus|cacti]]).<ref>{{Cite book|title=Crassulacean Acid Metabolism|last1=Smith|first1=J. A. C.|last2=Winter|first2=K.|chapter=Taxonomic Distribution of Crassulacean Acid Metabolism |series=Ecological Studies |date=1996|volume=114 |publisher=Springer Berlin Heidelberg|isbn=9783642790621|location=Berlin, Heidelberg|pages=427–436|doi=10.1007/978-3-642-79060-7_27}}</ref>

In Crassulacean acid metabolism, isotopic fractionation combines the effects of the C3 pathway in the daytime and the C4 pathway in the nighttime. At night, when temperature and [[Evapotranspiration|water loss]] are lower, the CO<sub>2</sub> diffuses through the stomata and produce malate via phosphenolpyruvate carboxylase.<ref name=":5" /><ref name=":1" /> During the following day, stomata are closed, malate is decarboxylated, and CO<sub>2</sub> is fixed by RuBisCO. This process alone is similar to that of C4 plants and yields characteristic C4 fractionation values of approximately -11‰.<ref name=":1" /> However, in the afternoon, CAM plants may open their stomata and perform C3 photosynthesis.<ref name=":1" /> In daytime alone, CAM plants have approximately -28‰ fractionation, characteristic of C3 plants.<ref name=":1" /> These combined effects provide [[Δ13C|''δ''<sup>13</sup>C]] values for CAM plants in the range of -10 to -20‰.

The <sup>13</sup>C to <sup>12</sup>C ratio in CAM plants can indicate the temporal separation of CO<sub>2</sub> fixation, which is the extent of biomass derived from nocturnal CO<sub>2</sub>  fixation relative to diurnal CO<sub>2</sub> fixation.<ref>{{Cite journal|last1=Winter|first1=Klaus|last2=Holtum|first2=Joseph A. M.|date=2002-08-01|title=How Closely Do the δ13C Values of Crassulacean Acid Metabolism Plants Reflect the Proportion of CO2 Fixed during Day and Night?|journal=Plant Physiology|volume=129|issue=4|pages=1843–1851|doi=10.1104/pp.002915|issn=0032-0889|pmid=12177497|pmc=166772}}</ref> This distinction can be made because PEP carboxylase, the enzyme responsible for net CO<sub>2</sub> uptake at night, discriminates <sup>13</sup>C less than RuBisCO, which is responsible to daytime CO<sub>2</sub> uptake.  CAM plants which fix CO<sub>2</sub> primarily at night would be predicted to show [[Δ13C|''δ''<sup>13</sup>C]] values more similar to C4 plants, whereas daytime CO<sub>2</sub> fixation would show [[Δ13C|''δ''<sup>13</sup>C]] values more similar to C3 plants.

== In phytoplankton ==
In contrast to terrestrial plants, where CO<sub>2</sub> diffusion in air is relatively fast and typically not limiting, diffusion of dissolved CO<sub>2</sub> in water is considerably slower and can often limit carbon fixation in phytoplankton.<ref name=":4" /> As gaseous CO<sub>2(g)</sub> is dissolved into aqueous CO<sub>2(aq)</sub>, it is fractionated by both kinetic and [[Equilibrium fractionation|equilibrium effects]] that are temperature-dependent.<ref>{{Cite journal|date=1995-01-01|title=Carbon isotope fractionation during gas-water exchange and dissolution of CO2|journal=Geochimica et Cosmochimica Acta|volume=59|issue=1|pages=107–114|doi=10.1016/0016-7037(95)91550-D|issn=0016-7037|last1=Zhang|first1=J.|last2=Quay|first2=P.D.|last3=Wilbur|first3=D.O.|bibcode=1995GeCoA..59..107Z}}</ref> Relative to plants, the dissolved CO<sub>2</sub> source for phytoplankton can be enriched in <sup>13</sup>C by about 8‰ from atmospheric CO<sub>2</sub>.<ref>{{Cite journal|last1=Köhler|first1=P.|last2=Fischer|first2=H.|last3=Schmitt|first3=J.|date=March 2010|title=Atmosphericδ13CO2and its relation topCO2and deep oceanδ13C during the late Pleistocene|journal=Paleoceanography|volume=25|issue=1|doi=10.1029/2008pa001703|bibcode=2010PalOc..25.1213K|issn=0883-8305|url=http://epic.awi.de/16456/1/Khl2007c.pdf}}</ref>

Isotope fractionation of <sup>13</sup>C by [[phytoplankton]] photosynthesis is affected by the diffusion of extracellular aqueous CO<sub>2</sub> into the cell, the RuBisCO-dependent cell growth rate, and the cell geometry and surface area.<ref name=":6" /> The use of bicarbonate and carbon-concentrating mechanisms in phytoplankton distinguishes the isotopic fractionation from plant photosynthetic pathways.

The difference between intracellular and extracellular CO<sub>2</sub> concentrations reflects the CO<sub>2</sub> demand of a phytoplankton cell, which is dependent on its growth rate. The ratio of carbon demand to supply governs the diffusion of CO<sub>2</sub> into the cell, and is negatively correlated with the magnitude of the carbon fractionation by phytoplankton.<ref>{{Cite journal|last1=Laws|first1=Edward A.|last2=Popp|first2=Brian N.|last3=Cassar|first3=Nicolas|last4=Tanimoto|first4=Jamie|date=2002|title=13C discrimination patterns in oceanic phytoplankton: likely influence of CO2 concentrating mechanisms, and implications for palaeoreconstructions|journal=Functional Plant Biology|volume=29|issue=3|pages=323–333|doi=10.1071/pp01183|pmid=32689479|issn=1445-4416}}</ref> Combined, these relationships allow the fractionation between CO<sub>2(aq)</sub> and phytoplankton biomass to be used to estimate the phytoplankton growth rates.<ref>{{Cite journal|date=1995-03-01|title=Dependence of phytoplankton carbon isotopic composition on growth rate and [CO2)aq: Theoretical considerations and experimental results|journal=Geochimica et Cosmochimica Acta|volume=59|issue=6|pages=1131–1138|doi=10.1016/0016-7037(95)00030-4|issn=0016-7037|last1=Laws|first1=Edward A.|last2=Popp|first2=Brian N.|last3=Bidigare|first3=Robert R.|last4=Kennicutt|first4=Mahlon C.|last5=Macko|first5=Stephen A.|bibcode=1995GeCoA..59.1131L}}</ref>

However, growth rate alone does not account for observed fractionation. The flux of CO<sub>2(aq)</sub> into and out of a cell is roughly proportional to the cell surface area, and the cell carbon biomass varies as a function of cell volume. Phytoplankton geometry that maximizes surface area to volume should have larger isotopic fractionation from photosynthesis.<ref>{{Cite journal|date=1998-01-01|title=Effect of Phytoplankton Cell Geometry on Carbon Isotopic Fractionation|journal=Geochimica et Cosmochimica Acta|volume=62|issue=1|pages=69–77|doi=10.1016/S0016-7037(97)00333-5|issn=0016-7037|last1=Popp|first1=Brian N.|last2=Laws|first2=Edward A.|last3=Bidigare|first3=Robert R.|last4=Dore|first4=John E.|last5=Hanson|first5=Kristi L.|last6=Wakeham|first6=Stuart G.|bibcode=1998GeCoA..62...69P}}</ref>

The biochemical characteristics of phytoplankton are similar to C3 plants, whereas the gas exchange characteristics more closely resemble the C4 strategy.<ref>{{Cite journal|last1=Laws|first1=Edward A.|last2=Bidigare|first2=Robert R.|last3=Popp|first3=Brian N.|date=November 1997|title=Effect of growth rate and CO2concentration on carbon isotopic fractionation by the marine diatomPhaeodactylum tricornutum|journal=Limnology and Oceanography|volume=42|issue=7|pages=1552–1560|doi=10.4319/lo.1997.42.7.1552|bibcode=1997LimOc..42.1552L|issn=0024-3590|doi-access=free}}</ref> More specifically, phytoplankton improve the efficiency of their primary carbon-fixing enzyme, RuBisCO, with carbon concentrating mechanisms (CCM), just as C4 plants accumulate CO<sub>2</sub> in the bundle sheath cells. Different forms of CCM in phytoplankton include the active uptake of bicarbonate and CO<sub>2</sub> through the cell membrane, the [[active transport]] of inorganic carbon from the cellular membrane to the chloroplasts, and active, unidirectional conversion of CO<sub>2</sub> to bicarbonate.<ref>{{Cite journal|last1=Cassar|first1=Nicolas|last2=Laws|first2=Edward A.|last3=Popp|first3=Brian N.|date=November 2006|title=Carbon isotopic fractionation by the marine diatom Phaeodactylum tricornutum under nutrient- and light-limited growth conditions|journal=Geochimica et Cosmochimica Acta|volume=70|issue=21|pages=5323–5335|doi=10.1016/j.gca.2006.08.024|bibcode=2006GeCoA..70.5323C|issn=0016-7037}}</ref> The parameters affecting <sup>13</sup>C fractionation in phytoplankton contribute to [[Δ13C|''δ''<sup>13</sup>C]] values between -18 and -25‰.<ref name=":5" /><ref name=":6" />

== See also ==

*[[RuBisCO]]
*[[Isotope geochemistry]]
*[[Intrinsic KIE of RuBisCO]]
* [[Paleoclimatology]]

== References ==
{{Reflist}}

[[Category:Isotope separation]]
[[Category:Isotopes of carbon]]