Differences in copper accumulation and copper stress between eight populations of Haumaniastrum katangense

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Abstract

Haumaniastrum katangense is well known as the “copper flower” of the Katangan Copper Belt. Few studies, however, are available on the physiology of this remarkable plant, including questions like stress responses and population-dependent differences. In the current study, we compared the response to copper for eight populations of this species in terms of copper accumulation, copper resistance, and various physiological parameters that might change under copper toxicity stress (biophysics of photosynthesis, growth, chlorophylls and carotenoids). Among six populations growing well under experimental conditions, three were found to be copper sensitive in terms of a strong inhibition of growth by 10 μM copper, while the other three were rather resistant. As the most prominent copper tolerance associated difference, copper resistant populations (as judged by their growth, photosynthetic activity and pigmentation) showed a decrease of iron accumulation in response to increased copper supply, while copper sensitive populations increased their Fe accumulation in response under these conditions. Copper sensitive populations showed the expected loss of pigments under copper toxicity stress, while two of the three copper tolerant populations even showed an enhancement of chlorophylls and violaxanthin in response to toxic copper. Also for other pigments population-specific differences in copper response were found, but they did not correlate with copper tolerance. Photosynthesis biophysics was affected by copper stress like in other species, no clearly tolerance/population-specific differences were found.

Highlights

► We compared the Cu-response for 8 Haumaniastrum katangense populations from different habitats. ► Cu-tolerant populations decreased Fe-accumulation in response to increased copper. ► Cu-sensitive populations increased Fe-accumulation in response to elevated Cu. ► Cu-toxicity in sensitive populations led to enhanced loss of pigments. ► In Cu-tolerant populations, chlorophylls & violaxanthin increased at high copper.

Introduction

Copper is an essential micronutrient for growth and development of animals (incl. humans) and plants at its optimal concentrations, but the excess of copper in organisms can lead to toxicity (reviewed e.g. by see Küpper and Kroneck, 2005, Burkhead et al., 2009). Such toxicity can originate from the use of copper in agriculture, e.g. as the “Bordeaux Mixture” for vine downy mildew disease as well as in other pesticides. Runoff from agricultural fields can reach several micromolar copper (Gallagher et al., 2001, Ribolzi et al., 2002, Zhang et al., 2003, He et al., 2009), a concentration that is lethal for many aquatic plants (review by Küpper and Kroneck, 2005). Copper pollution can also result from industrial activities such as copper mining and waste deposition (Ke et al., 2007). As the result of a long term field application, copper residues can cause impacts on soil biota by reducing soil microbial biomass and eliminating earthworms in the orchard (Van-Zwieten et al., 2004). But the excess copper in soils also leads to growth reduction and may even be lethal for terrestrial plants (Rhoads et al., 1989, Flemming and Trevors, 1989).

The symptoms of copper toxicity are a series of physiological alterations which occur at the cellular and molecular level, resulting in functional changes that ultimately lead changes in morphology. For example, significant reductions of root calcium and iron contents, as well as extensive damage to root epidermal cells in maize plants were observed (Ouzounidou et al., 1995). Copper impairs cellular transport processes and changes biochemical metabolism dramatically (Hall, 2002). Already at low concentrations, Cu inhibits photosynthesis by the formation of Cu-chlorophylls, which are unsuitable for photosynthesis – the identified main target is PSII (Küpper et al., 1996, Küpper et al., 1998, Küpper et al., 2002, Mijovilovich et al., 2009; see Küpper et al., 2006 for a comprehensive review).

Therefore urgent need to remediate Cu contaminated soils. With the emerging technology of higher plants for phytoremediation, in order to clean up the Cu-contaminated soil and environments, investigations on the selection of suitable plant species and the understanding of biological mechanisms have been intensively conducted (for recent reviews see Lasat, 2000, Lasat, 2002, Hall, 2002, Küpper and Kroneck, 2005). Phytoremediation of contaminated soil is a both cost-effective and environmental friendly method. Certain species have a high capability of Cu tolerance and/or accumulation, while most others are sensitive to Cu stress. Hyperaccumulators of heavy metals are plant species that actively accumulate metals in their shoots. A commonly used threshold for defining hyperaccumulators is an accumulation more than 100 times higher compared to non-accumulator plants. For Cu, this threshold would be around 1000 mg kg−1 (Lasat, 2002, Küpper et al., 2009a, Paton and Brooks, 1996).

Recent research identified the amphibious water plant Crassula helmsii as a new Cu accumulator for water environments. The shoots of the plant are able to accumulate Cu in a concentration beyond 9000 mg kg−1 in 0.6 mg kg−1 Cu2+ nutrient solution, but they are unsuitable for phytoremediation due to their growth characteristics (Küpper et al., 2009a).

Many plants of the genus Haumaniastrum grow in soils with exceedingly high contents of copper and cobalt. Some of them even grow only or mostly over Cu deposits; notably Haumaniastrum katangense is well known as the “copper flower” of the Katangan Copper Belt (called “la fleur du cuivre” by Duvigneaud, 1958). Those plants colonize mining sites and are widely distributed on the Cu-rich soils of Upper Katanga and western Zambia (Malaisse and Brooks, 1982, Leteinturier et al., 1999, Malaisse et al., 1999). These plants tolerate very high Cu levels, and an earlier study suggested that it may be a Cu hyperaccumulator (Paton and Brooks, 1996). But it remained unclear how much of the metal in these field-collected samples was actually taken up into the plant via the root or how much just became attached to the plants (incl. “uptake” into the stomata) as dust – such dust/aerosol “uptake” was shown to be potentially a large proportion (Faucon et al., 2007). In some studies, Cu levels in this species were clearly not in the hyperaccumulator range (e.g. Brooks, 1977, Chipeng et al., 2010). Further, for other hyperaccumulator species very strong differences in metal accumulation potential were found (as a classical case, see the Cd accumulation differences in Thlaspi caerulescens ecotypes, Lombi et al., 2000), so that the variation in Cu contents found for H. katangense could be soil-based or population-based.

H. katangense is presently known to occur in Angola, Dem. Rep. Congo, Tanzania and Zambia. In Tanzania, three districts are concerned, namely Ufipa, Nkansi and Sumbawanga districts; for these sites no heavy metal contents in soil are reported. In Zambia, H. katangense populations exist on Cu sites of the Copperbelt, and other places where no Cu rocks have been reported. The first group includes at least Chililabombwe, Chingola, Kalulushi, Luanshya, Mufulira. The last group includes occurrences (a) in Brachystegia woodlands or miombo open forest, notably in Abercorn District, as well as (b) ruderal sites (Kawinbe, Kapata, Chilongowelo, Kapiri Mposhi, Mpulungu). In Dem. Rep. Congo, H. katangense is known to occur on at least 18 Cu sites of the Katangan Copper Belt (Etoile, Kambove, Kamoya I, Kasombo, Kasonta, Kinsevere, Luishia, Luiswishi, Lukuni, Lupoto, Mambilima, Menda, Niamumenda, Ruashi, Shangolowe, Shinkolobwe, Shituru, Sokoroshe I, Tantara) as well as 5 sites in the Katangan part of the Zambian Copperbelt (Kamikuba, Kinsenda, Kipapila, Kishiba, Musoshi). Several other sites are related to man made pollution:

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    Old indigene smelters furnaces (Kasungami, Mwanamumba, Luishia).

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    Road verges (a) near Cu mining sites (Etoile, Fungurume II, Kavifwafwaulu, Kasombo, Kazinyanga, Kela, Kipushi, TFM left, Ruashi).

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    Ruderal sites in the area of the Katangan Copper Belt (Baya, Lubumbashi town, Katanga village, Katuba, Karavia).

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    Down stream of rivers receiving polluted waters of Cu industry (Buluo stream, Lubumbashi valley, Lubudi valley).

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    Railway joining Cu site to Cu processing plant (Kamalondo).

Further, in Congo there are H. katangense populations without any clear occurrence of human activities related to Cu industry:

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    Ruderal sites outside Katangan Copper Belt (Baya, Dilolo, Kando, Lukafu).

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    Dembo (Katete).

In this way, presently H. katangense is no more strictly related to Cu anomalies. This reality differs from the meaning when the plant was described.

Therefore, in the current study we compared Cu accumulation and Cu-induced inhibition of physiology in nutrient solution for eight populations of H. katangense that were collected from sites with different local ecology in the Katangan Copper Belt (Dem. Rep. of Congo). The aim of this comparison was to see how much variability in the physiological response to Cu exists between populations, i.e. in how far Cu metabolism of these populations adapted (in a genetically fixed way so that it persists when all these populations are treated under identical laboratory conditions) to their local environment. For this comparison, we chose physiological parameters that are known to respond to Cu stress, i.e. growth of roots and shoots, metal uptake (copper, plus iron because interferences between metabolism of these two metals are known), biophysics of photosynthesis (measured here by Chl fluorescence kinetics) and pigmentation (chlorophylls and carotenoids).

Section snippets

Plant seeds

Eight populations of H. katangense were used for the Cu stress treatment. The seeds were collected in several expeditions by Prof. Francois Malaisse from the South-Eastern part (“Katangan Copper Belt”) of the Democratic Republic of Congo in Africa. The various collecting sites differ in their soil conditions e.g. due to the presence or absence of past mining activities or more or less recent man made Cu pollutions. The populations are listed in Table 1.

Plant cultivation

H. katangense seeds were incubated at 27 °C

Growth

This study was started with eight populations of H. katangense as described in the methods, but only six could be cultivated long-term under experimental conditions and were used for all further analyses. During the whole hypodronic experiments, there was no visible symptom of nutrient deficiency. Among these, populations 2, 23 and 23a tolerated high Cu concentrations (10 μM) better than average in terms of showing good growth of both roots and shoots (Fig. 1a and b). Damaged plants often had

Discussion

This study was originally aimed at finding copper accumulating ecotypes of H. katangense, based on previous reports that some populations could be copper hyperaccumulators (Paton and Brooks, 1996). The experiments did not reveal, however, any population that could be called a hyperaccumulator. In all investigated populations, Cu levels generally stayed in the range of Cu-stressed non-accumulators (less than 50 mg kg−1 in shoot dry weight) compared to almost 10,000 mg kg−1 in C. helmsii that was

Acknowledgements

The authors would like to thank Peter Kroneck for many fruitful discussions about this project. This work was financially supported by the Fundamental Research Funds for the Central Universities, the National Natural Science Foundation of China (NSFC, 20307008) and the Zhejiang Provincial Planned Project for Science and Technology of China (No. 2007C23039) to HP, by a grant of the Fonds der Chemischen Industrie (FCI) to HK and by the Ausschuss für Forschungsfragen of the Universität Konstanz to

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    Current address: ETH Zurich, Department of Biology, Plant Biotechnology, Universitätsstrasse 2, 8092 Zurich, Switzerland.

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