Photosynthetic pathway




This article is modified from Perez-Harguindeguy et al (2013).”New handbook for standardised measurement of plant functional traits worldwide” is a product of and is hosted by Nucleo Diversus (with additional Spanish translation). For more on this trait and on its context as part of the entire trait handbook visit its primary site Nucleo DiverSus at


Three main photosynthetic pathways operate in terrestrial plants, each with their particular biochemistry, including C3, C4 and CAM. These pathways have important consequences for optimum temperatures of photosynthesis (higher in C4 than in C3 plants), water- and nutrient-use efficiencies, and responsiveness to elevated CO2 Compared with C3 plants, C4 plants tend to perform well in warm, sunny and relatively dry and/or salty environments (e.g. in tropical savannah-like ecosystems), whereas CAM plants are generally very conservative with water and occur predominantly in dry and warm ecosystems. Some submerged aquatic plants have CAM too. There are obligate CAM species and also facultative ones, which may switch between C3 and CAM, depending on environmental factors (e.g. epiphytic orchids in high-elevation Australian rainforests). Two main identification methods are available, namely C-isotope composition and anatomical observations. CAM can be inexpensively confirmed by verifying that stomata are open at night and closed during the day, or by measuring diurnal patterns of organic acids or leaf pH values. Which method to choose (a combination would be the most reliable) depends on facilities or funding, as well as on the aim of the work (e.g. to contrast C4v. C3, or CAM v. C3). Although C-isotope composition can be affected by environmental factors, intraspecific genetic differences and/or phenological conditions, intraspecific variability is small enough not to interfere with the distinction between C4 and C3 photosynthetic pathways. In many plant families, only C3 metabolism has been found. It is useful to know in which families C4 and CAM have been found, so that species from those families can be screened systematically as potential candidates for these pathways (see Table 1). Below we describe two methods which in combination provide good contrast between pathway types.

Table 1. C4 and CAM families. Families in which C4 (Osmond et al. 1980; Sage 2004) or CAM photosynthesis (Kluge and Ting 1978; Zotz et al. 1997; Crayn et al. 2001; Reinert and Blankenship 2010) has been reported; in parentheses genera in which both C3 and C4 metabolism occur.
Acanthaceae, Aizoaceae (Mollugo), Amaranthaceae (Alternanthera), Asteraceae (Flaveria), Boraginaceae (Heliotropium), Capparidaceae, Caryophyllaceae, Chenopodiaceae (Atriplex, Bassia, Kochia, Suaeda), Cyperaceae (Cyperus, Scirpus), Euphorbiaceae, (Euphorbia), Gisekiaceae, Hydrocharitaceae, Molluginaceae, Nyctaginaceae (Boerhavia), Poaceae (Alloteropsis, Panicum), Polygonaceae, Portulacaceae, Scrophulariaceae, Zygophyllaceae (Kalistromia, Zygophyllum). Agavaceae, Aizoaceae, Aloaceae, Amarylllidaceae, Araceae, Asclepidiaceae, Asteraceae, Bromeliaceae, Bromeliaceae, Cactaceae, Clusiaceae, Commelinaceae, Crassulaceae, Cucurbitaceae, Cycadaceae, Didieraceae, Didieraceae, Dracaenaceae, Euphorbiaceae, Geraniaceae, Gesneriaceae, Isoetaceae, Lamiaceae, Lamiaceae, Liliacea, Orchidaceae (photosynthetic roots), Oxalidaceae, Piperaceae, Piperaceae, Plantaginaceae, Polypodiaceae, Portulacaceae, Rapataceae, Rubiaceae, Salvadoraceae, Vitaceae, Vittariaceae, Welwitschiaceae.


What and how to collect

Collect the fully expanded leaves or analogous photosynthetic structures of adult, healthy plants growing in full sunlight or as close to full sunlight as possible. We recommend sampling at least three leaves from each of three individual plants. If conducting anatomical analysis (see under (B) Anatomical analysis below), store at least part of the samples fresh (see SLA).

(A) C-isotope analysis

Storing and processing

Dry the samples immediately after collecting. Once dry, the sample can be stored for long periods of time without affecting its isotope composition. If this is not possible, the sample should first be stored moist and cool (see SLA) or killed by using a micro-wave and then be dried as quickly as possible at 70-80C, to avoid changes caused by loss of organic matter (through leaf respiration or microbial decomposition). Although not the preferred procedure, samples can also be collected from a portion of a herbarium specimen. Be aware that insecticides or other sprays that may have been used to preserve the specimen, can affect its isotope composition.

Bulk the replicate leaves or tissues for each plant, then grind the dried tissues thoroughly to pass through a 40-μm-mesh or finer screen. It is often easier with small samples to grind all of the material with mortar and pestle. Only small amounts of tissue are required for a C-isotope-ratio analysis. In most cases, less than 3 mg of dried organic material is used.


Carbon isotope ratios of organic material ( 13Cleaf) are measured using an isotope ratio mass spectrometer (IRMS, precision between 0.03 ‰ and 0.3 ‰, dependent on the IRMS used) and are traditionally expressed relative to the Pee Dee Belemnite (PDB) standard as 13C in units of per mil (‰), i.e. parts per thousand. After isotopic analysis, the photosynthetic pathway of the species can be determined on the basis of the following (see graphic explanation in Material S4; Fig. 1):

C3 photosynthesis 13C: -21‰ to -35‰,

C4 photosynthesis: -10‰ to -14‰,

Facultative CAM: -15‰ to -20‰ and

Obligate CAM: -10‰ to -15‰.

Separating C3 or C4 from CAM plants is difficult on the basis of 13C alone (for facultative CAM plants, 13C values have been found to range as widely as from -14‰ to -23‰). However, as a rule of thumb, if 13C is between -10‰ and -23 ‰, and the photosynthetic tissue is succulent or organic acid concentrations are high during the night, but low during the day, then the plant is CAM. In such cases, anatomical observations and diurnal measurements of gas exchange or biochemical analysis would be decisive (see (B)Anatomical analysis in the present Section).

(B)Anatomical analysis

C3 and C4 plants typically show consistent differences in leaf anatomy, best seen in a cross-section. Using a razor blade or microtome, make cross-sections of leaf blades of at least three plants per species, making sure to include some regular veins (particularly thick and protruding veins, including the midrib and major laterals, are not relevant). C3 plants have leaves in which all chloroplasts are essentially similar in appearance and spread over the entire mesophyll (photosynthetic tissues). The mesophyll cells are not concentrated around the veins and are usually organised into -palisade’ and -spongy’ layers parallel to, and respectively adjacent to, the upper to lower epidermis (see Material S4; Fig. 2) (vertically held C3 leaves often have a palisade layer adjacent to each epidermis and a spongy layer between the two palisades). The cells directly surrounding the veins (transport structures with thin-walled phloem and generally thicker-walled xylem cells), called bundle sheath cells, normally contain no chloroplasts. C4 plants, in contrast, typically exhibit -Kranz anatomy’, viz., the veins are surrounded by a distinct layer of bundle-sheath cells (Material S4; Fig. 2) that are often thick-walled, and possess abundant, often enlarged chloroplasts that contain large starch granules. The mesophyll cells are usually concentrated around the bundle-sheath cells, often as a single layer whose cells are radially oriented relative to the centre of the vein, and contain smaller chloroplasts with no starch grains. These differences can usually be identified easily under an ordinary light microscope. Many plant physiology and anatomy textbooks give further illustrations of Kranz v. typical C3 leaf anatomy (see More on methods below).

Figure 2. Comparison of leaf anatomy of (a) a typical C3 plant and (b) a typical C4 plant.

If Kranz anatomy is observed, the species is C4 If not, it is likely to be C3 unless the plant is particularly succulent and belongs to one of the families with CAM occurrence. In the latter case, it could be classified as (possible) CAM. Many CAM leaves do not have typical C3 palisade or spongy mesophyll layers, but only a thin layer of more or less isodiametric, chloroplast-containing cells just under their epidermis, with the entire centre of the leaf consisting of large, thin-walled, colourless parenchyma cells that store water and organic acids. If living plants are within easy reach, an additional check could be to determine the pH of the liquid obtained by crushing fresh leaf samples in the afternoon (see pH of Green Leaves), and again (with new, fresh samples from the same leaf population) at pre-dawn. Because in a CAM plant, organic (mostly malic) acids build up during the night, and are broken down during the day to supply CO2 for the photosynthesis in the leaf, CAM species show a distinctly lower pH after the night than they do in the afternoon. In addition, C-isotope ratios can provide further evidence to distinguish between CAM and C3 or C4 metabolism (see (A)C-isotope analysis above).

Notes and troubleshooting tips

Special cases or extras

(1) Permanent slides or photographs and chloroplast visibility. A range of methods is available for making the microscope slides permanent; however, be aware that some may result in poorer visibility of the chloroplasts. One method for retaining the green colour of the chloroplasts is to soak the plant or leaves in a solution of 100 g CuSO4 in 25 mL of 40% formal alcohol (formaldehyde alcohol), 1000 mL distilled water and 0.3 mL 10% H2SO4 for 2 weeks, then in 4% formal alcohol for 1 week, subsequently rinse with tap water for 1-2 h and store in 4% formal alcohol until use. However, material thus treated can be sectioned only by using a microtome after embedding or freezing it, in contrast to many living, turgid leaves, which can be sectioned free-hand by using a suitable technique such as sectioning a rolled-up leaf or a stack of several leaves. Photomicrographs of freshly prepared sections are an alternative way to keep records for later assessment.

Literature references

References on theory, significance and large datasets:

Earnshaw MJ, Carver KA, Gunn TC, Kerenga K, Harvey V, Griffiths H, Broadmeadow MSJ (1990) Photosynthetic pathway, chilling tolerance and cell sap osmotic potential values of grasses along an altitudinal gradient in Papua New Guinea. Oecologia 84, 280-288.

Ehleringer JR, Cerling TE, Helliker BR (1997) C4 photosynthesis, atmospheric CO2, and climate. Oecologia 112, 285-299. doi:10.1007/s004420050311

Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40, 503-537. doi:10.1146/annurev.pp.40.060189.002443

Hibberd JM, Quick WP (2002) Characteristics of C4 photosynthesis in stems and petioles of C3 flowering plants. Nature 415, 451-454. doi:10.1038/415451a

Lüttge (1997) Physiolgical ecology of tropical plants. Springer-Verlag: Berlin

O’Leary MH (1981) Carbon isotope fractionation in plants. Phytochemistry 20, 553-567. doi:10.1016/0031-9422(81)85134-5

Pyankov VI, Gunin PD, Tsoog S, Black CC (2000) C-4 plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate. Oecologia 123, 15-31. doi:10.1007/s004420050985

Sage RF (2001) Environmental and evolutionary preconditions for the origin and diversification of the C4 photosynthetic syndrome. Plant Biology 3, 202-213. doi:10.1055/s-2001-15206

Wand SJE, Midgley GG, Jones MH, Curtis PS (1999) Responses of wild C4 and C3 grasses (Poaceae) species to elevated atmospheric CO2 concentration: a meta-analytical test of current theories and perceptions. Global Change Biology 5, 723-741. doi:10.1046/j.1365-2486.1999.00265.x

Zotz G, Ziegler H (1997) The occurrence of crassulacean metabolism among vascular epiphytes from central Panama. New Phytologist 137, 223-229. doi:10.1046/j.1469-8137.1997.00800.x

More on methods:

Belea A, Kiss AS, Galbacs Z (1998) New methods for determination of C-3, C-4 and CAM-type plants. Cereal Research Communications 26, 413-418.

Ehleringer JR (1991) 13C/12C fractionation and its utility in terrestrial plantstudies. In Carbon isotopes techniques. Eds DC Coleman, B Fry, pp. 187-200. Academic Press: London

Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40, 503-537. doi:10.1146/annurev.pp.40.060189.002443

Hattersley PW, Watson L (1992) Diversification of photosynthesis. In Grass evolution and domestication. Ed.GP Chapman, pp. 38-116. Cambridge University Press: London

Mohr H, Schopfer P (1995) Plant physiology. 4th edn. (Springer: Berlin)

Pierce S, Winter K, Griffiths H (2002) Carbon isotope ratio and the extent of daily CAM use by Bromeliaceae. New Phytologist 156, 75-83. doi:10.1046/j.1469-8137.2002.00489.x

Taiz L, Zeiger E (2010) Plant physiology. The Benjamin/Cummings Publishing: Redwood City, CA

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