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Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden
* Corresponding author (maria.greger{at}botan.su.se).
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ABSTRACT |
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INTRODUCTION |
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Much of the Cd that reaches humans from food is derived from wheat. According to Jorhem and Sundström (1993), wheat flour and potatoes (Solanum tuberosum L.) are the main contributors to the average daily dietary intake of Cd in Sweden. Hellstrand and Landner (1998) calculated that 43% of the total human Cd intake from food in Sweden is caused by wheat flour consumption. It is important to decrease the levels of Cd in wheat grains because of the risk of health effects resulting from current exposure to Cd and because the content in wheat seem to be increasing. This is dependent on a better understanding of what regulates Cd accumulation in wheat grains.
A high Cd level in the wheat grains may be caused by a high Cd uptake in the roots, a high translocation of Cd from roots to shoot, and/or a high translocation of Cd within the shoot to the grains. Root uptake of Cd from soil is dependent on the Cd concentration in the soil, the soil pH, and the level of organic matter and zinc in the soil (Eriksson et al., 1996; Eriksson and Söderström, 1996). While these soil factors mainly regulate the amount of soil Cd that is available to the plant, the plant itself regulates the uptake. This is obvious since the uptake of Cd varies among wheat cultivars (Oliver et al., 1995; Wenzel et al., 1996; Li et al., 1997).
After taken up, Cd is partly accumulated in the roots and partly translocated into the xylem and further to the shoot. Within the xylem, Cd moves with the transpiration stream and this movement is affected by interaction of Cd with the negative charges of the cell walls of the vessels (Wolterbeek, 1987). As in the case of Cd uptake in roots, the translocation of Cd from root to shoot has also been shown to differ between and within wheat cultivars. Hart et al. (1998) found that the proportion of Cd translocated to shoots was 1.5 to 4.5 times higher in a bread wheat than in a durum wheat cultivar.
Cadmium is probably either translocated directly via xylem to the grains during maturity or is translocated as a result of the bulk stream of photosynthate from source to sink, i.e., from leaves to the grains via the phloem. According to Mengel and Kirkby (1982), the flag leaf is the most important provider of photosynthates to the grain in the later stage of the grain-filling period, contributing to 70 to 80% of the grain filling. The remainder of assimilate mainly comes from the ear itself (Mengel and Kirkby, 1982). Herren and Feller (1997) suggested that the xylem-to-phloem transfer is important for the Cd accumulation in the maturing grains of wheat. It is possible that Cd follows the photosynthate bulk stream from the flag leaf to the grains. Thus, from the root to the grain, there are many processes that may regulate the accumulation of Cd in wheat grains. Most of the mechanisms behind these processes are still unknown.
The aim of this study was to investigate to what extent the following processes correspond to the accumulation of Cd in the grains: (i) the root uptake of Cd, (ii) the translocation of Cd from roots to shoot, and (iii) the concentration of Cd in shoot, flag leaf, and grain coat. Different Cd-accumulating cultivars of bread and durum wheat as well as plants of different ages were studied. The investigation was conducted as two experiments. In a short-term experiment, young seedlings were cultivated for 19 d in a Cd-containing nutrient solution to measure Cd uptake and Cd translocation from root to shoot for every cultivar during the vegetation phase. In a long-term experiment, the plants were cultivated in the same nutrient solution until maturity and the distribution of Cd among roots, flag leaves, grain coats, and grains was analyzed.
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MATERIALS AND METHODS |
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The grains of spring bread wheat and durum wheat were germinated in the climate chamber in vermiculite moistened with redistilled water. After 4 to 7 d, the seedlings were mounted 4 by 4 in styrofoam disks and transferred to 1-L plastic pots with aerated nutrient solution. During the first week of hydroponic cultivation, the strength of the Hoagland solution was 25%.
After the first week, the nutrient solution was replaced with new of 50% strength and CdCl2 was added to a final concentration of 0.5 µM. All test plants at that time had three leaves. In both short- and long-term experiments, controls without Cd for every cultivar were also maintained. This allowed us to access the background concentration of Cd. After 1 wk, the nutrient solution was replaced with one of full strength together with 0.5 µM Cd. After the first addition of Cd and 19 d later, the nutrient solution with 0.5 µM Cd was replaced on three occasions. After 19 d in the presence of Cd, half of the pots of each cultivar were harvested (short-term cultivation), while three plants from the remaining plants were transferred to 2.3-L plastic containers and used in the long-term experiment.
In the long-term experiment, each wheat plant was allowed to develop three shoots. The nutrient solution was replaced with fresh solution (with full-strength Hoagland and 0.5 µM Cd) once a week. In addition, extra copper (0.184 µmol per 2 L) was added twice a week, since a prestudy had shown that the original copper content in the Hoagland formula was insufficient for maturing wheat. Between replacements of the nutrient solution, extra nutrient solution with Cd was added twice a week for a total weekly dose of 200 to 1000 mL, with the smaller dose in the end of the cultivation period. This was due to a slower growth rate and thus a smaller nutrient requirement in the end of the cultivation period.
All plants, randomly distributed, were grown in a climate chamber equipped with fluorescent lamps (Osram HQI-E 400W/D) that gave a photon flux density of 220 to 265 µmol m–2 s–1 during the first days of cultivation. As the plants grew and came closer to the light source, the photon flux density increased and was 530 to 700 µmol m–2 s–1 when the plants had reached their full height.
Harvest and Metal Analysis
The roots were rinsed for 3 s each in two pots with redistilled water, one pot with 20 mM EDTA and two more pots with redistilled water. The shoots were rinsed with redistilled water. The roots and shoots were then separated and the fresh and dry weight (105°C for 24 h) was recorded.
In the short-term experiment, the roots and shoots were analyzed separately. In the long-term experiment the roots, flag leaves, grains, and grain coats (glumes, lemma, palea, and awn) were analyzed separately. The dried material was wet digested in HNO3:HClO4 (7:3 v/v). Analyses of Cd concentration in the different plant parts were performed with an atomic absorption spectrometer (SpectrAA-100, Varian Springvale, Australia).
Calculations and Statistical Treatment
The Cd concentration of the controls was subtracted from that of the Cd treated plants to get the Cd concentration resulting from the Cd treatment. Cadmium net uptake via roots was calculated as Cd content in whole plant in relation to root dry weight. Cadmium translocation to the shoot was calculated as content of Cd in the shoot in relation to the Cd content in the whole plant.
Four replicates, i.e., pots, were used for each experiment. Each replicate contained four plants for the short-term experiment of spring wheat while three plants in the other cases. Statistical test for significant differences was calculated by the Student's t test, Tukey-Kramer, and correlation tests which were performed by the computer programs MS Excel-98, JMP 2.02, and Cricket Graph III, respectively. The level of significance, P = 0.05 for r values, was according to Spearman's ranking test.
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RESULTS |
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The translocation of Cd from root to shoot, calculated as the amount of Cd in the shoot in relation to the amount of Cd in the whole plant, was between 150 and 450 µg mg–1 (Fig. 1D). The translocation among the low-Cd accumulators tended to be lowest for winter bread wheat, followed by spring bread wheat, which in turn had lower Cd translocation than the durum wheat. In general, the translocation seemed to be higher among the durum wheat cultivars, 350 to 450 µg mg–1. There was a tendency within the winter bread wheats and durum wheats for the low-Cd accumlators to translocate Cd to the shoot at a lower rate than the high Cd accumulators of the same wheat type. Significant differences among each type were only found among the winter bread wheat, where Stava had lower translocation than Mjölner.
Long-Term Cultivation
The Cd concentration in grains did not significantly differ between the three types of wheat, i.e., among winter bread wheat (2.31 µg Cd (gDW)–1 ± [SE]), spring bread wheat (1.77 ± 0.27) and durum wheat (2.57 ± 0.42). A comparison of Cd concentration in grains among the different cultivars of winter bread wheat shows that the low-Cd accumulator (Stava) tended to have lower concentrations than the high Cd accumulators (Mjölner and Rental) (Fig. 2A)
. Among the spring bread wheat cultivars, the same tendency was found. However, significant differences in grain Cd levels was found among the cultivars of durum wheat. The low-Cd accumulator Helidur had a lower Cd concentration than did the high Cd accumulators Grandur and Extradur. Furthermore, the low accumulator Astrodur had a lower Cd concentration than Grandur, but did not differ significantly from Extradur. There was also a significant difference between the two low accumulators among the durum type; Astrodur had a higher concentration in the grain than did Helidur.
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As in the case with the short-term cultivated roots (Fig. 1A), no significant differences could be found either between the cultivars or between the different wheat types in the long-term study (Fig. 2D). The highest Cd levels in the analyzed parts of the long-term cultivated plants were found in the roots. Roots had 7.6 to 36 times higher levels than the grains, except for the low accumulators Vinjett and Helidur, which had 66 and 80 times higher Cd level than in grains, respectively (Table 2). The high accumulators among the durum wheat, Grandur and Extradur, had the lowest difference in Cd concentration between roots and grains; the levels were only 8 and 7.5 times higher in roots, respectively, than in grains for these cultivars.
Correlation Tests
The relationship between Cd level in roots and Cd level in grains was also analyzed. No correlation was found between either the short-term cultivated roots and grain Cd levels or the long-term cultivated roots and grain levels (not shown). Nor did any correlation exist between the total net uptake via roots of Cd in the short-term cultivated plants and the concentration in the grain in any wheat type (not shown).
A significant positive correlation was found among the cultivars of all three wheat types between the Cd level in grains and the level in the shoots during the vegetation phase (Table 3). A positive correlation was also found between the grain Cd level and the translocation of Cd from roots to shoot. There was also a significant positive correlation between the Cd levels in flag leaves and the levels in grains as well as between Cd level in grain coat and grains for spring bread and durum wheat but not for winter bread wheat. These correlations mean that a high level of Cd in the shoot, in the flag leaves or a high translocation of Cd to the shoot results in a high level of Cd in the grains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reason for the different results found in grain Cd accumulation among wheat types in field studies but not in this study may be the absence of root-soil interaction and concomitant rhizosphere mechanisms. Roots of the various wheat types may have different capacities to release metals from the soil colloids that increase the Cd concentration of the soil solution and thereby the Cd uptake. Such mechanisms could be release of hydrogen ions, which would decrease the pH of the rhizosphere, or excretion of organic acids (Mench and Martin 1991, Murányi et al. 1994).
In this investigation, significant differences within each wheat types were found only for durum wheat and both the highest and the lowest grain Cd accumulation were found among the durum wheat cultivars (Fig. 2A). The difference was about eight times, and was found between the grain levels in Helidur and Grandur (Fig. 2A). Thus, it can be assumed that the greatest differences between cultivars are to be found in durum wheat. In the literature, one can find even greater differences for durum wheat cultivars compared with what was found in this investigation. Cieslinski et al. (1996) investigated the concentration of Cd in four cultivars of durum wheat grown in the same soil under controlled conditions and found a 20-fold difference in grain levels of Cd between two durum wheat cultivars.
If the relative periods of vegetative and reproductive growth of the different cultivars is taken into account, the same pattern of grain Cd accumulation appears (not shown). Thus, neither cultivation time (time from sowing to harvest) nor the grain-filling period (time from development of ears to harvest) seem to be the reason for different Cd levels in the grains of various cultivars.
The objective was to find out whether cultivars with high ability to concentrate Cd in the grains have high uptake in the roots, high translocation from roots to shoot, or high redistribution of Cd to the grains from the shoot. The present study shows that the cultivars with high- and low-Cd accumulation in the grains, which had been determined by field studies, had about the same Cd levels in the roots. Therefore, root uptake of Cd does not appear to control the amount of Cd that is accumulated in the grains. Furthermore, it is unlikely that Cd taken up by the roots is translocated directly to the grains. This is in agreement with an investigation on maize (Zea mays L.) that was performed by Florijn and van Beusichem (1993). They reported that internal redistribution rather than the uptake caused genotypic differences in shoot Cd concentration of maize inbred lines. Also Hart et al. (1998) reported that greater accumulation of Cd in grains of durum wheat compared with bread wheat was not a consequence of differential Cd influx rates in roots.
During the vegetation phase in all three wheat types, relationships were found between the Cd level in grains and the Cd level in the shoots (Table 3); therefore, it may be that translocation to shoot and shoot Cd concentration is important for the accumulation of Cd in the grains. In addition, there was a correlation between the Cd levels in grains and the translocation from roots to shoot that was significant for the spring bread wheat and durum wheat and almost significant for winter bread wheat (Table 3). However, if the translocation from root to shoot were the only parameter that contributed to the grain accumulation of Cd, the distribution pattern between cultivars of shoot Cd concentration (Fig. 1C) and Cd translocation (Fig. 1D) would be the same as for grain Cd concentration (Fig. 2A). That is not the case. More fluctuations were found between the grain Cd levels of different cultivars (Fig. 2A) than between the shoot Cd levels (Fig. 1C) and the Cd translocation from roots to shoot (Fig. 1D).
This means that there must also be redistribution within the shoot that is important for the grain accumulation of Cd. This is supported in the literature. Cakmak et al. (2000) investigated the absorption and translocation of leaf applied Cd in different wheat cultivars. They found that two cultivars of bread wheat with almost the same amount of leaf absorbed Cd, differed by 3.5 and 4 times in the translocation of Cd to the remainder of the shoot and to the roots, respectively. Herren and Feller (1997) reported that the redistribution of Cd within cereal shoots is highly relevant for the accumulation of Cd in the grains. This redistribution is according to one theory believed to work via the flag leaves. The flag leaf is the most important provider of photosynthates to the grain in the later stage of the grain-filling period (Mengel and Kirkby, 1982). In awned wheat, the flag leaf contributes 70% of the grain filling, whereas in cultivars without awns about 80% may originate from the flag leaf. The remainder of assimilates comes mainly from the ear itself. The differences that are seen among the cultivars in this study in grain Cd levels (Fig. 2A) could, if the reasoning above is true, be a function of the flag leaf (Fig. 2C). A correlation between the level of Cd in the grains and in the flag leaves was found among the spring bread wheat and the durum wheat but not among the winter bread wheat (Table 3). According to these results, redistribution via the flag leaves is viable for durum wheat and spring bread wheat but not for winter bread wheat.
If a redistribution via the flag leaves were the only influence on the grain Cd accumulation, the ratio between [Cd]flag leaf: [Cd]grain should be constant for all cultivars, but the cultivars with really low grain Cd levels (Helidur and Vinjett) had a very high ratio (Table 2). Similar trends was also found for [Cd]grain coat: [Cd]grain (Table 2). It is therefore likely that low accumulating cultivars have an as yet undefined mechanism that prevents Cd accumulation in the grain.
In conclusion, this investigation indicates that there are differences between the cultivars in their ability to accumulate Cd in the grains. Although the differences in this study were not great, the largest differences were found among durum wheat cultivars. The differences that were found between the grain accumulations of Cd were due to variation in the translocation of Cd from root to shoot and within the shoot, rather than to Cd uptake in the roots. In addition, it appears that low Cd-accumulating cultivars have a mechanism that prevents Cd accumulation in the grain, and it is possible that this trait could be used in breeding efforts.
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ACKNOWLEDGMENTS |
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Received for publication October 31, 2002.
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