Hairy Roots In Phytoremediation And Phytomining Studies

The use of living plants to clean up polluted soils and waterways is a rapidly developing technology. Phytoremediation offers a range of advantages compared with existing remediation methods, including low cost, minimal site destruction and destabilization, low environmental impact, and favorable aesthetics. A related application of plants is phytomining. This emerging technology involves growing tolerant species on sites of mineralized soil or surface ore bodies, harvesting and drying the metal-rich crop, then ashing the biomass and treating the resulting bio-ore for metal recovery. The main advantage of phytomining compared with conventional mining is its low cost, allowing the exploitation of mineral deposits that are too metal-poor for direct mining operations.

Roots play a primary role in phytoremediation and phytomining, as they are the plant organs in direct contact with soil pollutants and heavy metals. Accordingly, there is a particular need to understand the biochemical and physiological functioning of roots in contaminated environments. Hairy root cultures are a convenient experimental system for such studies. In contrast to whole plants grown either in soil or hydroponically, hairy roots can be propagated indefinitely so that entire experimental programs can be carried out using tissues derived from the same plant, thus avoiding the effects of variability between individual specimens. Use of axenic conditions in hairy root culture prevents microbial symbiosis disguising the remediative activity of plant tissues, and better control over conditions at the roots can be exercised compared with soil cultivation. Separation of hairy roots from the leaves of plants also allows identification of the properties and functions of the roots without interference from translocation effects (67).

Several phytoremediation studies have been conducted using hairy root systems. Axenic cultures of Catharanthus roseus hairy roots were capable of removing 30 mg L_1 of 2,4,6-trinitrotoluene (TNT) from liquid solution within 5 days (68); disappearance of TNT from the medium was not due to simple accumulation within the biomass, as the roots were shown to facilitate TNT biotransformation into aminated nitrotoluenes and other soluble products. Hairy roots of Armoracia rusticana, Atropa belladonna, Solanum aviculare, and S. nigrum were found to metabolize polychlorinated biphenyls (PCBs); the most effective PCB transformations were correlated with an increase in total peroxidase activity in the roots (69). Daucus carota hairy roots have also been tested for transformation of phenols (70). Although chlorinated phenols were more toxic to root growth than phenol itself, both types of compound elicited peroxidase activity in the biomass. Peroxidase levels remained high in the hairy roots as phenol removal and metabolism took place.

As well as remediation of xenobiotic compounds, hairy roots have been applied to remove and concentrate heavy metals from liquid solutions. Cadmium accumulation has been investigated using several species, including Nicotiana tabacum, Beta vulgaris and Calystegia sepium (71), Solanum nigrum (72), Rubia tinctorum (73), and Daucus carota (74). In these studies, aspects of metal tolerance of the hairy roots, such as induction of phyto-chelatins (73,74), and stress responses such as ethylene production and lipid peroxidation (74) were quantified.

Hairy roots have been applied to investigate heavy metal uptake and detoxification by rare plant species capable of growing in high-metal environments and accumulating elevated levels of specific metal ions. These species, known as "hyperaccumulators," store heavy metals in their tissues at concentrations at least 100 times greater than those found in non-hyper-accumulator plants (75). About 400 hyperaccumulators have been identified, including 300 that hyperaccumulate nickel, 26 cobalt, 24 copper, 19 selenium, 16 zinc, 11 manganese, 1 thallium, and 1 cadmium (75). At the present time, the mechanisms of metal uptake by hyperaccumulating plants and the basis of their metal specificity are poorly understood. Hairy roots of several hyperaccumulators have been applied in metal uptake studies in liquid culture systems (67,76,77); these include Alyssum bertolonii and Thlaspi caerulescens, which were tested for hyperaccumulation of nickel and cadmium, respectively. As shown in Fig. 1, hairy roots of both hyperaccumulator species grew well at elevated metal concentrations that were toxic to hairy roots of the non-hyperaccumulator Nicotiana tabacum. The maximum Ni concentration measured in growing A. bertolonii hairy roots was 7200 ¡jlg g_1 dry weight (77), and the maximum Cd level in growing T. caerulescens was 10,600 /jig g_1 dry weight (67); these metal contents are well above the threshold levels for plant hyperaccumulator status (1000 /xg g~' for Ni; 100 /jig g 1 for Cd: Ref. 75). These results from hairy root cultures demonstrate that neither translocation of metal from roots to leaves nor interactions with soil microorganisms are necessary or responsible for metal hyperaccumulation in plants.

Hairy root cultures were used to investigate the role of the root cell wall and the distribution of metal between the apoplasm and symplasm of root cells during metal hyperaccumulation (67). As indicated in Fig. 2, there were significant differences in the patterns of metal uptake for the two species A. bertolonii and T. caerulescens compared with the non-hyperaccumulator N. tabacum. Over a period of 28 days, the proportion of Ni in the biomass that was retained in the cell wall fractions of A. bertolonii and N. tabacum hairy roots was relatively low, averaging only 17% for A. bertolonii and 24% for N. tabacum (Fig. 2a). In other words, most of the Ni taken up by both species quickly entered into the symplasm of the root cells. As Ni was toxic to N. tabacum but not to A. bertolonii (Fig. la), A. bertolonii must possess intracellular mechanisms for Ni detoxification that are not available to N. tabacum. In contrast, as shown in Fig. 2b, almost all of the Cd found in the T. caerulescens hairy roots was located in the cell walls during the first 7 days of contact with the metal, suggesting that association

Cleansing Biotope Phytoremediation

Figure 1 Growth of hairy roots of hyperaccumulator and non-hyperaccu-mulator species in medium with and without heavy metals, (a) Effect of Ni at an initial concentration of 20 ppm. (o) A. bertolonii without Ni; (•) A. bertolonii with Ni; (□) N. tabacum without Ni; and (■) N. tabacum with Ni. Nickel did not affect growth of A. bertolonii hairy roots but was very toxic to N. tabacum. (Data from Ref. 77.) (b) Effect of Cd at an initial concentration of 20 ppm. (o) T. caerulescens without Cd; (•) T. caerulescens with Cd; (□) N. tabacum without Cd; and (■) N. tabacum with Cd. Cadmium was particularly detrimental to growth of N. tabacum hairy roots. (Data from Ref. 67.)

Figure 1 Growth of hairy roots of hyperaccumulator and non-hyperaccu-mulator species in medium with and without heavy metals, (a) Effect of Ni at an initial concentration of 20 ppm. (o) A. bertolonii without Ni; (•) A. bertolonii with Ni; (□) N. tabacum without Ni; and (■) N. tabacum with Ni. Nickel did not affect growth of A. bertolonii hairy roots but was very toxic to N. tabacum. (Data from Ref. 77.) (b) Effect of Cd at an initial concentration of 20 ppm. (o) T. caerulescens without Cd; (•) T. caerulescens with Cd; (□) N. tabacum without Cd; and (■) N. tabacum with Cd. Cadmium was particularly detrimental to growth of N. tabacum hairy roots. (Data from Ref. 67.)

10 15 20 Time (days)

Figure 2 Percentage of heavy metal in the biomass associated with the cell walls, (a) Nickel was added to (•) A. bertolonii and (o) N. tabacum hairy roots at an initial concentration of 20 ppm. Most of the Ni in the biomass of both species was located in the symplasm of the cells, (b) Cadmium was added to (•) T. caerulescens and (o) N. tabacum hairy roots at an initial concentration of 20 ppm. Virtually all of the Cd in the T. caerulescens roots was retained in the cell walls for the first 7 days, whereas most of the Cd in N. tabacum roots was initially transferred into the symplasm. (Data from Ref. 67.)

10 15 20 Time (days)

Figure 2 Percentage of heavy metal in the biomass associated with the cell walls, (a) Nickel was added to (•) A. bertolonii and (o) N. tabacum hairy roots at an initial concentration of 20 ppm. Most of the Ni in the biomass of both species was located in the symplasm of the cells, (b) Cadmium was added to (•) T. caerulescens and (o) N. tabacum hairy roots at an initial concentration of 20 ppm. Virtually all of the Cd in the T. caerulescens roots was retained in the cell walls for the first 7 days, whereas most of the Cd in N. tabacum roots was initially transferred into the symplasm. (Data from Ref. 67.)

with the cell walls is a primary mechanism of Cd accumulation in this species, at least during the early stages of metal exposure. The proportion of Cd in the walls decreased after 7 days as some Cd passed into the sym-plasm of the cells; however, most of the Cd (about 60%) continued to remain with the wall fraction.

The pattern of Cd uptake by N. tabacum hairy roots was the reverse of that with T. caerulescens. Initially, the cell walls accounted for only a minor proportion of the Cd in the N. tabacum biomass, as most of the Cd entered directly into the cells. Cell wall Cd levels in N. tabacum increased with time and eventually accounted for more than 80% of the biomass Cd; this increase was possibly due to cell lysis during the culture, as Cd had a severe detrimental effect on the growth (Fig. lb) and integrity of N. tabacum roots. The ability of T. caerulescens hairy roots to hold virtually all of their Cd in the cell walls for several days could represent a critical defensive strategy, providing time for the development of intracellular mechanisms for Cd complexation or compartmentation. By the time Cd entered the sym-plasm of the T. caerulescens cells, the metal could be effectively detoxified so that growth of the roots was maintained (Fig. lb).

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