Carbon isotope composition and leaf anatomy as a tool to characterize the photosynthetic mechanism of Artemisia annua L.

Composição dos isótopos do carbono e anatomia foliar como ferramenta para caracterizar o mecanismo fotossintético de Artemisia annua L.

Abstracts

Leaves of Artemisia annua L. are a plentiful source of artemisinin, a drug with proven effectiveness against malaria. The aim of this study was to classify the photosynthetic mechanism of A. annua through studies of the carbon isotope composition (delta 13C) and the leaf anatomy. A. annua presented a delta 13C value of - 31.76 ± 0.07, which characterizes the plants as a typical species of the C3 photosynthethic mechanism, considering that the average delta 13C values for C3 and C4 species are -28 and -14, respectively. The leaf anatomy studies were consistent with the delta 13C results, where, in spite of the existence of parenchymatic cells forming a sheath surrounding the vascular tissue, the cells do not contain chloroplasts or starch. This characteristic is clearly different from that of the Kranz anatomy found in C4 species.

C3 and C4 plants; isotope discrimination; Kranz anatomy; photosynthesis

As folhas de Artemisia annua L. são fonte abundante de artemisinina, uma droga que apresenta ação efetiva contra a malária. O objetivo deste trabalho foi classificar o mecanismo fotossintético de A. annua mediante estudos da composição dos isótopos do carbono (delta 13C) e da anatomia foliar. A. annua apresentou uma delta 13C = - 31.76 ± 0.07, valor tópico de espécies com mecanismo fotossintético C3, que apresentam, em média, valores de delta 13C = - 28, enquanto espécies C4 apresentam, em média, valores de delta 13C = - 14. Os estudos da anatomia foliar confirmaram os resultados encontrados para a delta 13C, onde, a despeito da existência de células parenquimáticas formando um anel ao redor do feixe vascular, essas não apresentaram cloroplastos e amido. Tal observação descaracteriza a existência de anatomia Kranz, típica de espécies C4, em A. annua.

anatomia Kranz; fotossíntese; discriminação isotópica; plantas C3 e C4

SHORT COMMUNICATION

Carbon isotope composition and leaf anatomy as a tool to characterize the photosynthetic mechanism of Artemisia annua L.

Composição dos isótopos do carbono e anatomia foliar como ferramenta para caracterizar o mecanismo fotossintético de Artemisia annua L.

José Abramo Marchese^{I, III,*}; Fernando Broetto^II; Lin Chau Ming^III; Carlos Ducatti^II; Roberto Antonio Rodella^II; Marília Contin Ventrella^IV; Greice Daiane Rodrigues Gomes^I; Lúcia de Franceschi^I

^ILaboratory of Biochemistry and Plant Physiology, Agronomy College, CEFET-PR, Pato Branco 85503-390, Brazil

^IIInstitute of Biosciences, São Paulo State University, Botucatu 18618-000, Brazil

^IIIAgronomic Sciences College, São Paulo State University, Botucatu 18603-970, Brazil

^IVPlant Biology Department, Viçosa Federal University, Viçosa 36570-000, Brazil

ABSTRACT

Leaves of Artemisia annua L. are a plentiful source of artemisinin, a drug with proven effectiveness against malaria. The aim of this study was to classify the photosynthetic mechanism of A. annua through studies of the carbon isotope composition (d ¹³C) and the leaf anatomy. A. annua presented a d ¹³C value of - 31.76 ± 0.07, which characterizes the plants as a typical species of the C₃ photosynthethic mechanism, considering that the average d ¹³C values for C₃ and C₄ species are -28 and -14, respectively. The leaf anatomy studies were consistent with the d ¹³C results, where, in spite of the existence of parenchymatic cells forming a sheath surrounding the vascular tissue, the cells do not contain chloroplasts or starch. This characteristic is clearly different from that of the Kranz anatomy found in C₄ species.

Key words: C₃ and C₄ plants, isotope discrimination, Kranz anatomy, photosynthesis.

RESUMO

As folhas de Artemisia annua L. são fonte abundante de artemisinina, uma droga que apresenta ação efetiva contra a malária. O objetivo deste trabalho foi classificar o mecanismo fotossintético de A. annua mediante estudos da composição dos isótopos do carbono (d ¹³C) e da anatomia foliar. A. annua apresentou uma d ¹³C = - 31.76 ± 0.07, valor tópico de espécies com mecanismo fotossintético C₃, que apresentam, em média, valores de d ¹³C = - 28, enquanto espécies C₄ apresentam, em média, valores de d ¹³C = - 14. Os estudos da anatomia foliar confirmaram os resultados encontrados para a d ¹³C, onde, a despeito da existência de células parenquimáticas formando um anel ao redor do feixe vascular, essas não apresentaram cloroplastos e amido. Tal observação descaracteriza a existência de anatomia Kranz, típica de espécies C₄, em A. annua.

Palavras-chave: anatomia Kranz, fotossíntese, discriminação isotópica, plantas C₃ e C₄.

A. annua (Asteraceae) is a native Chinese herbaceous plant acclimatized in Brazil. The leaves are a plentiful source of artemisinin, a sesquiterpene lactone, with proven effectiveness against Plasmodium resistant strains, the parasitic causal agent of malaria (Klayman, 1985; Geldre et al., 1997). Chemical synthesis of artemisinin is complex, involving many stages and with low yields (Chan et al., 1995; Geldre et al., 1997). In view of the high costs of chemical synthesis, the isolation of artemisin from the A. annua plant is the preferred way to obtain the drug (Woerdenbag et al., 1991; Ferreira and Janick, 1996; Geldre et al., 1997).

A. annua is considered a short-day plant (Ferreira et al.,1995) either in a qualitative or absolute sense (Marchese et al., 2002) while some genotypes can present a requirement for low temperatures or vernalization to accelerate flowering (Marchese et al., 2002). Apart from its photoperiodic behaviour and the effect of water supply and different temperatures on the artemisinin content (Marchese, 1999; Marchese and Rehder, 2001), little other information is available in the literature relating to the physiology of A. annua. Due to the importance of A. annua as a source of artemisinin, in many countries where malaria occurs efforts are underway to introduce and acclimatize the plant (Mueller et al., 2000).

Information on the photosynthetic mechanism can be useful for the introduction of any species, but especially for an exotic medicinal plant, such as A. annua. In general, plants with the C₄ photosynthetic mechanism are better adapted for hot climates, while C₃ plants are more appropriate for temperate regions (Loomis and Connor, 1992; Rudall, 1994; Hall and Rao, 1995; Körner and Bazzaz, 1996; Lambers et al., 1998; Lawlor, 2001).

The photosynthetic mechanism or biochemical pathways of photosynthesis are highly conserved. The majority of plants are C₃ plants, in which the first product of photosynthesis is the three-carbon compound phosphoglyceric acid. A second biochemical pathway, that leads to the concentration of CO₂ in leaves, is found in C₄ plants, which initially fix inorganic carbon in mesophyll cells into the four-carbon compound oxaloacetic acid. In C₄ plants, oxaloacetate is converted into malate or aspartate, which then diffuses into the bundle sheath cells that surround the vascular system where descarboxylation supplies high concentrations of CO₂ for 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 plants may represent transitional stages in the evolution of C₄ photosynthesis from C₃ photosynthesis (von Caemmerer, 1992; Lawlor, 2001; Hibberd and Quick, 2002).

Plants discriminate carbon isotopes during photosynthesis. The carbon dioxide in the earth's atmosphere is composed of different carbon isotopes. The principal carbon isotope is ¹²CO₂ (98.9 atom %) while only about 1.1 atom % of the total CO₂ in the atmosphere is ¹³CO₂ and an even smaller fraction (10^-10 atom %) 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. Furthermore, isotope composition differs between plants, according to their 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 (d‰¹³C about –28 ‰) discriminate more ¹³CO₂ than the C₄ plants (d‰¹³C about –14 ‰). The largest isotope discrimination step is the carboxylation reaction catalyzed by Rubisco, the primary CO₂ fixation enzyme of C₃ plants, which has an intrinsic discrimination value (D¹³C) of –30 ‰. On the other hand, PEP carboxylase, the primary CO₂ fixation enzyme of C₄ plants, has a much smaller isotope discrimination effect (D¹³C = –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).

There are also differences in leaf anatomy between C₄ and C₃ plants. A cross section, of a typical C₃ leaf reveals one major cell type with chloroplasts, the mesophyll. In contrast, a typical C₄ leaf has two distinct chloroplast-containing cell types: mesophyll and bundle sheath cells (Mauseth, 1988; Rudall, 1994; Lawlor, 2001). The aim of this investigation was to classify the photosynthetic mechanism of A. annua through studies of carbon isotope composition (d‰¹³C) and leaf anatomy.

Plants of a genotype of A. annua originating from Vietnam plant population (CPQBA 2/39x1V) and developed by the Breeding Program of the Centre of Chemical, Biological and Agricultural Research of the State University at Campinas (CPQBA/UNICAMP) were used in this study. For carbon isotope composition (d‰¹³C) measurements the leaves were dried at 80ºC overnight and powdered in a cryogenic mill (-196ºC). They were then analysed, in triplicate, in a mass spectrometer coupled to an elemental analyser for the determination of the ratio R (R = ¹³CO₂/¹²CO₂). The standard ratio is that of Pee Dee belemnite (PDB). Carbon isotope composition (d‰¹³C) is a measure 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 (PDB). Thus,

d‰¹³C = [(R_p/R_s) – 1] x 1000

where R_p is the ¹³C/¹²C ratio measured in plant material and R_s is the ratio of standard (PDB). Carbon isotope composition provides a means of relating samples of diverse origin. Samples of contemporary plant material have negative values of d‰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).

For leaf anatomical studies, the leaves were fixed in FAA₅₀, and stored in ethanol 70% (Johansen, 1940). The samples were embedded in historesin (Leica^®) according to Gerrits (1991) and stained with toluidine blue (O'Brien et al., 1964). Sections for light microscopy were obtained using a rotatory microtome at a thickness of 8 µm, and treated with PAS (Periodic acid/Shiff reagent) (Pearse, 1968). This general test for carbohydrate gives a rose to purple coloration for starch or structural wall carbohydrates. The sections were covered with synthetic resin (Permount^®) and a cover -slip.

A. annua produced values for d‰¹³C of - 31.76 ± 0.07 (table 1), a result that characterizes the plant as a typical species of the C₃ photosynthetic mechanism, considering that the average d‰¹³C values for C₃ and C₄ species are -28 and – 14 respectively (Sternberg et al., 1984; Farquhar et al., 1989; O'Leary et al., 1992; O'Leary, 1993; Lambers et al., 1998; Condon et al., 2002). The ratio of ¹³C/¹²C in dry matter of C₃ plants is the result of discrimination against ¹³C during several processes. These include: during diffusion of CO₂ through the stomata; at Rubisco during the process of CO₂ fixation; and at some downstream metabolic steps and (possibly) respiration (Condon et al., 2002).

The leaf anatomy studies (figure 1) were in agreement with the results of D‰¹³C. The parenchyma cells which form a bundle sheath surrounding the vascular tissue contain no chloroplasts or starch. The leaf is amphistomatic and the mesophyll is dorsiventral, with little differentiation into palisade and spongy parenchyma. Starch (figure 1) is present throughout the mesophyll, indicating no specific areas for its production. Such leaf characteristics are not found in the Kranz anatomy of a C₄ species, but are typical of C₃ species (Mauseth, 1988; Rudall, 1994; Lawlor, 2001).

The existence of an evident parenchyma bundle sheath with little differentiation of the mesophyll cells may represent a transitional stage in the evolution of C₄ photosynthesis from C₃ photosynthesis (Lawlor, 2001; Condon et al., 2002), but, in general, the Kranz anatomy bundle sheath consists of a single layer of large cells containing large chloroplasts and starch (Mauseth, 1988; Rudall, 1994). The test with PAS produced a negative reaction for the vascular bundle sheath cells to starch, indicating the absence of chloroplasts in these cells. The results of d‰¹³C together with the absence of chloroplasts and starch in parenchyma vascular bundle sheath cells suggest that leaves of A. annua have a C₃ photosynthetic mechanism.

Received: 21/10/2004, Accepted: 03/01/2005

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Publication Dates

• Publication in this collection
  03 June 2005
• Date of issue
  Mar 2005

History

• Accepted
  03 Jan 2005
• Received
  21 Oct 2004