Abstract
Cultivated cassava (Manihot esculenta) has a higher rate of photosynthesis than is usual for C3 plants and photosynthesis is not light saturated. For these reasons it has been suggested that cultivated cassava could be derived from wild species possessing C4 photosynthesis. The natural abundance of 13C and activities of phosphoenolpyruvate carboxylase and phosphoglycolate phosphatase were measured in leaves of 20 wild cassava species to test this hypothesis. All the species studied, including M. flabellifolia the potential wild progenitor of cultivated cassava, clearly exhibited C3 not C4 characteristics.
Key words: Cassava, 13C discrimination, phosphoenolpyruvate carboxylase (PEP Case), phosphoglycolate phosphatase (PGP)
INTRODUCTION
Cassava (Manihot esculenta Crantz, Euphorbiaceae), a native to tropical America, is a major source of energy for more than 500 million people in tropical countries of Africa, Asia and Latin America (Cock, 1985). Cultivated cassava (M. esculenta) may be derived from a wild progenitor M. flabellifolia (Fregeneet al., 1994; Roaet al., 1997).
Cassava endures several months of water stress during its seasonal cycle which, despite its extreme drought tolerance, strongly limits crop production (El‐Sharkawy, 1993; Calatayudet al., 2000). In plants with C4 photosynthesis, water use efficiency is increased and photorespiration is suppressed, thus C4 plants are often more competitive than C3 plants in drought‐prone areas and in hot environments (Edwards and Ku, 1987). Although M. esculenta displays C3 biochemistry (Edwardset al., 1990), it has several other characteristics typical of C4 not C3 species. For instance, photosynthesis does not saturate at high irradiance; photosynthetic rates are very high; photosynthesis has a broad temperature optimum ranging from 20 to 45 °C; and cassava leaves also have distinct green bundle sheath cells (Angelovet al., 1993), as do C4 species. These features suggest that cultivated cassava (M. esculenta) may be derived from wild species possessing C4 photosynthesis (Angelovet al., 1993). It would be of interest to know whether ancestors of cultivated cassava possess C4 features; if so, it would suggest that this characteristic has been selected out during domestication of the plant, and that the potential for re‐introduction of C4‐like features exists. This could be valuable for breeding more drought‐tolerant and productive cultivars.
The objective of the present work was to determine whether selected wild species of Manihot display C4 photosynthesis. As indicators of C4 characteristics, the natural abundance of 13C (routinely used to determine the mode of CO2 fixation in plants), and activities of phosphoenolpyruvate carboxylase (PEP Case) and phosphoglycolate phosphatase (PGP) (key enzymes of the C4 pathway and photorespiration, respectively) were examined in 20 wild species of Manihot.
MATERIALS AND METHODS
Twenty wild species of Manihot [listed in Table 1 with their genotype (accession) number] were used in this study. The plants were obtained from the in vitro germplasm collection of the International Centre for Tropical Agriculture (CIAT, its Spanish acronym) in Cali (Colombia) and propagated by in vitro culture according to Roca and Mroginski (1991). For M. crassisepala, plants were obtained directly from the field germplasm collection at CIAT and propagated by stem cuttings. Plants of Arachis pintoi Krapovickus & Gregory (Leguminosae) and Brachiaria dictyoneura Figari & Notaris (Gramineae), grown from seed, were used as C3 and C4 controls, respectively.
Table 1.
Young leaf carbon isotope composition (13C) and activities of phosphoenolpyruvate carboxylase (PEP Case) and phosphoglycolate phosphatase (PGP) in 20 wild species of Manihot and two control C3 and C4 photosynthetic plants (means ± s.d., n = 3 individuals)
| Plant species | 13C (‰) | PEP Case [µmol (mgChl)–1 min–1] | PGP [nmol (mgChl)–1 min–1] |
| M. aesculifolia (1) | –24·8 ± 1·2 | 0·10 ± 0·09 | nd |
| M. alutacea (11) | –31·3 ± 2·4 | 1·83 ± 0·64 | 138·7 ± 48·4 |
| M. anomala (3) | –30·9 ± 1·0 | 2·13 ± 0·60 | 153·5 ± 19·4 |
| M. caerulescens (13) | –31·6 ± 0·5 | 1·75 ± 0·45 | 61·4 ± 28·6 |
| M. carthaginensis (297) | –30·7 ± 1·9 | 1·55 ± 0·54 | 275·6 ± 127·8 |
| M. cecropiaefolia (3) | –30·8 ± 0·9 | 0·46 ± 0·01 | 114·0 ± 84·6 |
| M. chlorosticta (10) | –27·2 ± 1·7 | 1·96 ± 0·30 | 119·4 ± 18·9 |
| M. crassisepala (4) | –23·7 ± 0·2 | 2·07 ± 1·00 | 234·4 ± 12·6 |
| M. epruinosa (1) | –29·8 ± 0·4 | 1·71 ± 0·62 | 158·4 ± 128·3 |
| M. flabellifolia (36) | –29·3 ± 2·2 | 2·28 ± 1·20 | 159·8 ± 58·5 |
| M. fructiculosa (6) | –30·0 ± 0·7 | 2·12 ± 0·85 | 185·8 ± 109·2 |
| M. guaranitica (6) | –31·2 ± 0·8 | 3·27 ± 0·64 | 249·8 ± 81·3 |
| M. irwinii (6) | –30·7 ± 1·6 | 1·25 ± 0·24 | 117·7 ± 5·7 |
| M. jacobinensis (9) | –31·2 ± 1·9 | 1·49 ± 0·40 | 94·7 ± 20·4 |
| M. orbicularis (18) | –30·7 ± 0·5 | 1·48 ± 0·60 | 308·5 ± 44·2 |
| M. purpureo–costata (4) | –29·1 ± 2·8 | 1·35 ± 0·08 | 88·0 ± 1·3 |
| M. rubricaulis (19) | –31·9 ± 0·2 | 5·53 ± 0·13 | 317·2 ± 65·8 |
| M. triphylla (21) | –32·2 ± 0·2 | 0·64 ± 0·42 | 21·8 ± 0·8 |
| M. tristis (12) | –29·6 ± 0·7 | 1·35 ± 0·14 | 110·1 ± 12·7 |
| M. violacea (3) | –31·4 ± 0·6 | 1·04 ± 0·50 | 175·8 ± 14·5 |
| Arachis pintoi (C3) | –29·5 ± 1·2 | 1·62 ± 0·55 | 111·0 ± 62·2 |
| Brachiaria dictyoneura (C4) | –13·1 ± 0·5 | 37·35 ± 6·04 | nd |
Genotype number (accession) given in parentheses. nd, Not detectable.
All plants were grown in individual 15‐l plastic pots containing mixed peat and sand in a growth chamber. The environmental conditions were 30/25 °C (day/night) with a photoperiod of 12 h and an irradiance of 860 µmol m–2 s–1 PAR (Lowmounp, Luminaire non‐Wallasped Hood; General Electric, Hendersonville, NC, USA) at the top of the plants, and a relative humidity of 70–80 %. Plants were watered with 1200 ml distilled water (sufficient for full growth; Calatayudet al., 2000) three times weekly, and with a complete nutrient solution each month. Four‐month‐old plants (three replicates per genotype) were used for the experiment. For each plant, a young leaf (2 weeks old) was sampled at midday for analyses.
Approximately 100 mg of the leaf blade was dried and ground to a fine, homogeneous powder. Heavy isotope contents were determined by mass spectrometry (ROBOPRED‐CN) calibrated for natural abundance analyses on pure CO2 obtained by combustion in quartz sealed tubes in the presence of CuO (Cliquetet al., 1990). Results are expressed in δ units vs. PDB (Pee Dee Belemnite) for C.
To measure enzyme activity, the remaining leaf blade was homogenized at 5 °C with an Ultraturax Omni 2000 grinder in 1 ml buffer containing 50 mol m–3 Hepes‐KOH, pH 7·4, 12 mol m–3 MgCl2, 1 mol m–3 EGTA, 1 mol m–3 EDTA, 1 mol m–3 DTT, 10 % glycerol, 2 mol m–3 benzamidine and 2 mol m–3 ϵ‐amino‐n‐caproic acid, according to Siegl and Stitt (1990). After 10 s mixing with vortex, the suspension was centrifuged at 16 000 g for 2 min. Chlorophyll in the pellet was determined using Arnon’s (1949) method. The supernatant was desalted by centrifugal filtration at 5000 g at 5 °C with Sephadex G‐25, equilibrated with the extraction buffer and supplemented with 0·1 % BSA (bovine serum albumin) according to Marqueset al. (1983). All enzyme assays were made on this extract at 30 °C. PEP Case activity was measured spectrophotometrically by coupling the malate producing reaction with NADH‐oxidation mediated by malate dehydrogenase (Van Quyet al., 1991). PGP activity was assayed colorimetrically by determining the orthophosphate released using a modified Fiske‐Subbarow procedure (Baldyet al., 1989).
RESULTS AND DISCUSSION
Most of the wild species studied had 13C values between –25 and –32 ‰ (Table 1), close to those in the C3 A. pintoi and to C3 plants in general (between –25 and –30 ‰; Edwards and Ku, 1987). M. crassisepala and M. aesculifolia had the highest δ13C values (–23·7 and –24·8 ‰ respectively), but these were still far lower than those found in B. dictyoneura (C4) and in C4 plants in general (between –11 and –16 ‰). These results show that wild cassava species do not possess C4 photosynthesis.
Most of the values of PEP Case activity in the wild cassava species were between 1·0 and 2·3 µmol NADH (mg Chl)–1 min–1 (Table 1), similar to the range reported by El‐Sharkawy and Cock (1990) and Edwardset al. (1990) in leaves of cultivated cassava [between 0·4 and 2·3 µmol of NADH (mg Chl)–1 min–1]. In the literature, the range of PEP Case activity is generally between 6·7 and 56 µmol NADH (mg Chl)–1 min–1 in C4 plants and generally between 0·1 and 2·61 µmol of NADH (mg Chl)–1 min–1 in C3 plants (Edwardset al., 1990; El‐Sharkawy and Cock, 1990). Our data for a C3 and a C4 plant were within these ranges. All the wild cassava species displayed PEP Case activity similar to that found in C3 plants (Table 1).
PGP activity in leaves was between 100 and 300 nmol Pi (mg Chl)–1 min–1 for the majority of cassava species analysed, close to that of the C3 plant A. pintoi (Table 1). However, no activity was detected in M. aesculifolia or in the C4 plant B. dictyoneura, suggesting low photorespiration in M. aesculifolia. Since only very low PEP Case activity was detected in this species, there could have been a problem in extracting proteins from M. aesculifolia.
In conclusion, the wild species of cassava studied here, including M. flabellifolia the potential wild progenitor of M. esculenta (Fregeneet al., 1994; Roaet al., 1997), exhibit traits typical of C3 photosynthesis, indicating that cultivated cassava, despite its peculiar photosynthetic characteristics, is not derived from wild C4 species.
Supplementary Material
Received: 28 June 2001; Returned for revision: 28 August 2001; Accepted: 12 October 2001.
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