Carbon Isotope Composition as a Tool to Characterize the

Carbon Isotope Composition as a Tool to Characterize
Photosynthetic Mechanism of Herbs, Spices, and Medicinal Plants.
the
José Abramo Marchese1,2*; Lin Chau Ming2, Carlos Ducatti3; Fernando Broetto4;
Evandro Tadeu da Silva3; Marcelo Leonardo2; Juliano Toniato3
CEFET-PR, Agronomy College, Laboratory of Biochemistry and Plant Physiology, Pato Branco 85503-390,
Brazil; 2São Paulo State University, Agronomic Sciences College, Botucatu 18603-970, Brazil; 3São Paulo
State University, Isotopes Stables Center, Botucatu 18618-000, Brazil. 4São Paulo State University,
Chemistry and Biochemistry Department, Botucatu 18618-000, Brazil.
* Corresponding author: [email protected]
1
RESUMO:
Composição isotópica de carbono como ferramenta para caracterizar o mecanismo
fotossintético de plantas medicinais, aromáticas e condimentares. O fracionamento
isotópico é um teste simples para caracterizar vias fotossintéticas. Informações dos
mecanismos fotossintéticos das plantas são importantes na introdução e cultivo de
qualquer espécie, e neste caso específico, para espécies medicinais e aromáticas
exóticas e selvagens. Um uso potencial para o fracionamento isotópico está no controle
de qualidade dos fitomedicamentos, possibilitando a descoberta de fraudes através da
composição dos isótopos estáveis na biomassa das plantas medicinais. O objetivo deste
trabalho foi classificar o mecanismo fotossintético de algumas plantas medicinais e
aromáticas através de estudos da composição isotópica de carbono (13C). A 13C de 54
espécies foi investigada para estudos de 13C (13C‰ = [R (amostra)/R (padrão) – 1] x 103
), folhas secas e finamente moídas em moinho criogênico foram analisadas em um
espectrômetro de massa acoplado a um analisador elemental para a determinação da
razão R (R = 13CO2/12CO2). Como resultado, 23 famílias botânicas e 43 espécies
apresentaram mecanismo fotossintético C3; 6 espécies pertencentes as famílias
botânicas Apocynaceae e Poaceae apresentaram um mecanismo fotossintético de planta
C4 e 5 pertencentes as famílias botânicas Agavaceae, Euphorbiaceae e Liliaceae
apresentaram um mecanismo fotossintético de planta CAM.
Palavras-chave: plantas C3, C4 e CAM, discriminação isotópica; fotossíntese.
ABSTRACT: Carbon isotope composition as a tool to characterize the
photosynthetic mechanism of herbs, spices, and medicinal plants. Isotopic screening
has been a simple test for determining the photosynthetic pathway when it is unknown.
Information on the photosynthetic mechanism is important when one wants to introduce
and to cultivate any species, and in this specify case, the exotic and wild herbs, spices,
and medicinal plants. A potential use of the isotopic fractionation this in the quality control
of the Phytomedicines, allowing the discovery of frauds through of stable isotope
composition of biomass of the medicinal plants. The scope of this work was to classify the
photosynthetic mechanism of some herbs, spices, and medicinal plants through studies of
the carbon isotope composition (13C). 13C of 54 species were investigated. For studies of
13C (13C‰ = [R (sample)/R (standard) – 1] x 10-3), dry leaves and powdered in cryogenic
mill were analyzed in a mass spectrometer coupled in an analyser elemental for
determination of the ratio R (R = 13CO2/12CO2). As results, 23 botanical families and 43
species presented photosynthetic mechanism C3; 6 species found among the botanical
families Apocynaceae and Poaceae presented photosynthetic C4 species, and 5 species
found among the botanical families Agavaceae, Euphorbiaceae, and Liliaceae presented
photosynthetic CAM species.
Keywords: C3, C4 and CAM plants, isotope discrimination, photosynthesis.
INTRODUCTION
The photosynthetic mechanism or biochemical pathways of photosynthesis are highly
conserved. Most plants are C3 plants, in which the first product of photosynthesis is the
three-carbon compound posphoglyceric acid. A second biochemical pathway that allows
the concentration of CO2 in leaves has evolved in C4 plants, which initially fix inorganic
carbon in mesophyll cells, through the enzyme PEP carboxylase, into four-carbon
compound oxaloacetic acid. In C4 plants, oxaloacetate is converted into malate or
aspartate, which then diffuses into bundle sheath cells that surround the vascular bundles
where descarboxylation supplies high concentration of CO2 to Rubisco. In higher plants
the C4 pathway involves both biochemical and anatomical modifications, but it is not clear
which of these modifications evolved first. Some plants that possess characteristics of
both C3 and C4 plants have been classified as C3-C4 intermediates, and these may
represent transitional stages in the evolution of C4 photosynthesis from C3 photosynthesis
(von Caemmerer, 1992; Lawlor, 2001; Hibberd & Quick, 2002). Finally, some plants
present the Crassulacean Acid Metabolism (CAM). CAM plants assimilate atmospheric
CO2 into C4 acids, through the enzyme PEP carboxylase, predominantly at night and
subsequently refix this CO2, through the enzyme Rubisco, to the level of carbohydrates
during the following day (Griffiths, 1992; Cushman & Bohnert, 1997; Lambers et al., 1998;
Lawlor, 2001).
The carbon dioxide in the earth’s atmosphere is composed of different carbon
isotopes. The majority is 12CO2 (98.9%); only approximately 1.1% of the total amount of
CO2 in the atmosphere is 13CO2; an even smaller fraction (10-10%,) is the radioactive
species 14CO2. Modern ecophysiological research makes abundant use of the fact that the
isotope composition of plant biomass differs from that of the atmosphere. It is of special
interest that isotope composition differs between plants which differ in photosynthetic
pathway (Lambers et al., 1998).
The chemical properties of 13CO2 are identical to those of 12CO2, but because of the
slight difference in mass (2.3%), plants use less 13CO2 than 12CO2. C3 plants (13C about –
28 ‰) discriminate more 13CO2 than the C4 plants (13C about –14 ‰). The largest isotope
discrimination step is the carboxilation reaction catalyzed by Rubisco, the primary CO2
fixation enzyme of C3 plants, which has an intrinsic discrimination value (13C) of –30 ‰.
By contrast, PEP carboxylase, the primary CO2 fixation enzyme of C4 plants, has a much
smaller isotope discrimination effect (13C = –2 to 5.7 ‰) (Sternberg et al., 1984; Farquhar
et al., 1989; O’Leary et al., 1992; O’Leary, 1993; Lambers et al., 1998; Condon et al.,
2002). C3 species represent approximately 85% of all plant species, C4 species accounting
for about 5%, and CAM species for 10% (Lambers et al., 1998).
Isotopic screening has been a simple test for determining the photosynthetic pathway
when it is unknown (Farquhar et al., 1989). Information on the photosynthetic mechanism
is important when one wants to introduce and to cultivate any species, and in this specify
case, the exotic and wild herbs, spices, and medicinal plants. In general, plants with
photosynthetic mechanism of the type C4 adapt better in hot climates, while the C3 are
going better in less hot climates (Loomis & Connor, 1992; Hall & Rao, 1995; Körner &
Bazzaz, 1996; Lambers et al., 1998; Lawlor, 2001). A potential use of the isotopic
fractionation this in the quality control of the Phytomedicines, allowing the discovery of
frauds through of stable isotope composition of biomass of the medicinal plants.
The scope of this work was to classify the photosynthetic mechanism of some herbs,
spices, and medicinal plants through studies of the carbon isotope composition (13C).
MATERIAL AND METHODS
Leaves samples of medicinal plants collection of the Horticulture Section, Agronomic
Sciences College, São Paulo State University, Brazil, were used in the experiment. The
plants species were identified for the Dr. Lin Chau Ming and the first author.
The leaves of plants were analysed in the Isotopes Stables Center of São Paulo State
University. Leaves were dried at 800C overnight and powdered in cryogenic mill (-1960C).
In triplicate, were analysed in a mass spectrometer coupled in an analyser elemental for
determination of the ratio R (R = 13CO2/12CO2). The standard ratio is that of Pee Dee
belemnite. Carbon isotope composition (13C) is a measured of the 13C/12C ratio in a
sample of plant relative to the value of the same ratio in an accepted international
standard, the limestone Pee Dee belemnite. Thus,
13C (‰) = (Rp/Rs – 1) x 1000
where Rp is the 13C/12C ratio measures in plant material and Rs is the ratio of standard and
‰ is per mil. Carbon isotope composition provides a means of relating samples of diverse
origin for carbon isotope content. Samples of contemporary plant material have negative
values of 13C because the 13C/12C ratio in the atmosphere is less than in Pee Dee
belemnite and because there is a net discrimination against 13C by plants during uptake
and fixation of CO2 into plant dry matter (O’Leary et al., 1992; O’Leary, 1993; Condon et
al., 2002; Dawson et al., 2002).
RESULTS AND DISCUSSION
The most of the analysed plants, 23 botanical families and 43 species, presented
photosynthetic mechanism C3, that possess on the average a 13C‰ = - 28 (O’Leary,
1993; Farquhar et al., 1989). The ratio of 13C/12C in dry matter of C3 plants is the result of
discrimination against 13C during several processes. These processes include
discrimination that occurs during diffusion of CO2 through the stomata; discrimination by
Rubisco during the process of carboxilation of CO2 into first product of photosynthesis;
and some downstream fractionations associated with metabolism and (possibly)
respiration (O’leary et al., 1992; O’leary, 1993; Condon et al., 2002).
The 6 species found among the botanical families Apocynaceae and Poaceae
presented photosynthetic C4 species, that possess on the average a 13C‰ = - 14. In C4
plants most of the 13CO2 that is discriminated against by Rubisco does not diffuse back to
the atmosphere. This is prevented first by the diffusion barrier between the vascular
bundle sheath and the mesophyll cells. Second, mesophyll cells contain large quantities
of carbonic anhydrase and of PEP carboxylase, which discriminate less against the 13CO2,
and scavenge most of the CO2 that might escape from the bundle sheath (O’leary, 1993;
Lambers et al., 1998, Farquhar et al., 1989).
The 5 species found among the botanical families Agavaceae, Euphorbiaceae, and
Liliaceae presented photosynthetic CAM species. Species of these genders are known for
they present photosynthesis CAM (Martin et al., 1990; Luo et al., 1991; Griffiths, 1992;
Winter & Holtum, 2002). Like Rubisco from C3 and C4 plants, the enzyme from CAM
plants discriminates against 13CO2. The fractionation is considerably less than that of C3
plants and similar to that of C4 species (Sternberg et al., 1983; Lambers et al., 1998). This
is expected, as the stomata are closed during malate descarboxylation and fixation of CO2
by Rubisco (Lambers et al., 1998).
Table 1. Carbon isotope composition and photosynthetic mechanism of some herbs,
spices, and medicinal plants from collection of Horticulture Section of the São Paulo State
University. Botucatu-SP, Brazil, 2004. Legends: 13C = carbon isotope composition; NT =
native; EX = exotic; HB = herbaceous; SB = shrub; SS = subshrub; AR = arboreous; PC =
plant creeper.
Botanical name
Agavaceae
Sansevieria zeylanica Willd.
Amaranthaceae
Pfaffia glomerata [Spreng] Pedersen
Local name
[13C‰] PDB
Photosynthetic
Mechanism
Origin/growin
g habit
espada-de-são-jorge
-15,29  0,04
CAM
EX/HB
fáfia
-27,55  0,92
C3
NT/SB
terramicina
-27,88  0,09
C3
NT/EB
aroeira
-28,01  0,03
C3
NT/AR
janaguba
-17,45  0,05
C4
NT/AR
Asteraceae
Baccharis trimera Less.
carqueja
-31,72  0,16
C3
NT/SS
Artemisia camphorata L.
artemisia
-28,76  0,09
EX/EB
Tanacetum vulgare L.
catinga-de-mulata
-29,32  0,07
C3
C3
Achillea millefolium L.
mil-folhas
-28,97  0,52
C3
EX/EB
guaco
-30,71  0,03
C3
NT/PC
carobinha
-28,41  0,12
C3
NT/SS
Boraginaceae
Symphytum officinalis L.
confrei
-28,64  0,10
C3
EX/EB
Cactaceae
Peireskia aculeata Mill.
ora-pro-nobis
-30,88  0,05
C3
EX/SB
Caesalpiniaceae
Bauhinia forficata Link.
pata-de-vaca
-27,84  0,22
C3
NT/SB
embaúba
-29,75  0,23
C3
NT/AR
espinheira-santa
-27,84  0,03
C3
NT/AR
espinheira-santa
-30,27  0,16
C3
NT/AR
erva-de-santa-maria
-28,19  0,14
C3
NT/SS
Equisetaceae
Equisetum hiemale L.
cavalinha
-28,42  0,08
C3
NT/EB
Euphorbiaceae
Euphorbia tirucalli L.
aveloz; coroa-de-cristo
-14,97  0,18
CAM
EX/SB
malva-cheirosa
-29,29  0,05
C3
EX/SS
palmeirinha
-29,36  0,20
C3
NT/HB
hortelã
-29,13  0,09
C3
EX/EB
alfavaca-cheiro-de-anis
-29,05  0,53
C3
EX/EB
orelha-de-coelho
-27,66  0,77
C3
EX/EB
boldo-rasteiro
-29,42  0,21
C3
EX/EB
-28,53  0,01
C3
EX/SS
Alternanthera brasiliana [L.] O. Kuntze
Anacardiaceae
Myracrodruon urundeuva Allemão
Apocynaceae
Himatanthus drasticus [Martius] Plumel
Mikania glomerata Spreng.
Bignoniaceae
Jacaranda decurrens Cham.
Cecropiaceae
Cecropia glaziovii Snethlage
Celastraceae
Maytenus aquifolium Mart.
Maytenus ilicifolia Reissek
Chenopodiaceae
Chenopodium ambrosioides L.
Geraniaceae
Pelargonium graveolens Art.
Iridaceae
Eleutherine bulbosa [Mill.] Urb.
Lamiaceae
Menhta sp.
Ocimum selloi Benth.
Stachys lanata L.
Plectranthus neochylus Schlechter
Tetradenia riparia [Hochst.] N.E.Br.
incenso
EX/EB
Table 1. (Continued)
Origin/growin
g habit
-27,83  0,11
Photosynthetic
Mechanism
C3
-29,73  0,07
C3
EX/HB
falso-boldo
-28,49  0,23
C3
EX/HB
Ocimum basilicum L.
manjericão, alfavaca
-31,73  0,01
C3
EX/SS
Liliaceae
Aloe arborescens Mill.
babosa
-18,35  0,04
CAM
EX/HB
EX/HB
Botanical name
Local name
[13C‰] PDB
Rosmarinus officinalis L.
alecrim
Origanum vulgare L.
orégano
Plectranthus barbatus Andr.
EX/SS
Aloe sp.
babosa
-12,44  0,25
CAM
Aloe Vera [L.] Burm. F.
babosa
-15,63  0,03
CAM
EX/HB
sete-sangrias
-29,86  0,04
C3
NT/HB
pitanga
-29,16  0,04
C3
NT/AR
Lythraceae
Cuphea carthagenensis [Jacq.] Macbr.
Myrtaceae
Eugenia uniflora L.
Piperaceae
Potomorphe umbellata [L.] Miq.
Piper aduncum L.
Poaceae
Cymbopogon citratus [DC.] Stapf.
Cymbopogon flexuosus [DC.] Stapf.
Eleonurus sp.
Vetiveria zizanioides Stapf.
pariparoba
-28,19  0,28
C3
NT/EB
falso-jaborandi
-29,49  0,05
C3
NT/SB
capim-limão
-14,63  0,46
C4
EX/EB
EX/EB
-13,93  0,10
C4
barba-de-bode
-13,99  0,09
C4
NT/EB
vetiver
-14,83  0,11
C4
EX/EB
EX/EB
capim-santo
Cymbopogon winterianus Jowitt
citronela
-14,32  0,20
C4
Portulacaceae
Talinum triangulare [Jacq.] Willd.
maria-gorda
-25,26  0,52
C3
NT/EB
arruda
-28,79  0,04
C3
EX/EB
coentro-de-caboclo
-28,93  0,37
C3
NT/EB
funcho
-29,65  0,00
C3
EX/HB
erva-cidreira-brasileira
-29,40  0,67
C3
NT/SS
cidró
-27,53  0,03
C3
EX/SS
NT/SB
Rutaceae
Ruta graveolens L.
Umbelliferae
Eryingium foetidum L.
Foeniculum vulgare [Mill.] Gaertner
Verbenaceae
Lippia alba [Mill.] N.E. Br.
Aloysia triphylla [L' Hérit] Britt.
Lippia sidoides Cham.
alecrim-pimenta
-30,11  0,15
C3
Lippia alba [Mill.]N.E.Br.
cidreira-brasileira
-30,29  0,16
C3
NT/SS
Vitaceae
Cissus verticillata [L.] Nich. & C.E. Jarvis
insulina
-27,73  0,06
C3
NT/ PC
Zingiberaceae
Curcuma longa L.
açafrão
-27,77  0,63
C3
EX/HB
-26,44  0,54
C3
NT/HB
Costus spicatus [Jacq.] Sw.
cana-do-brejo
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