Table of Contents:
Root-shoot translocation of heavy metals
Contrary to the non hyperaccumulators which store heavy metals in their below ground parts, hyperaccumulators store the absorbed heavy metals in their above ground parts through bulk flow of metals via xylem, from roots to shoots (Grenan 2009). Storage of heavy metals in above ground parts is the characteristic feature of the hyperaccumulators (Kupper and Kroneck 2005). Lory et al. (2013) have shown that in the Cd/Zn hyperaccumulator Noccaea caerulescens, 86% of the absorbed Cd is translocated to the shoots.
The first step towards the allocation of the heavy metals to shoots is the translocation of the absorbed metals from root symplast to xylem apoplast (Lietenmaier and Kupper 2013). This necessitates the availability ofheavy metals for xylem loading, which is facilitated through their low sequestration and easy efflux out of the vacuoles of the root cells (Rascio and Navari-Izzo 2011). In the Cd/Zn hyperaccumulator Аосслея caerulescens, compared to their related non hyperaccumulator Noccaea an’ense, the hyperaccumulator showed reduced sequestration into the root vacuoles, which was associated with higher root to shoot translocation efficiency of Noccaea caerulescens (Zhao et al. 2006). Similar observations were also recorded for hyperaccumulator and non hyperaccumulator relatives of Sedum alfredii growing on highly Cd/Zn contaminated soils (Lu et al. 2013). A lower sequestration into root vacuoles accounted for the enhanced As translocation in hyperaccumulator compared with non hyperaccumulator species of Pteris (Poynton et al. 2004).
The feature of enhanced xylem loading in the hyperaccumulators is attributed to the over expression of certain genes which are also present in non hyperaccumulators but only in their normal or down regulated forms (Kotrba et al. 2009, Rascio and Navari-Izzo 2011). The P-1S type ATPases, HMAs, MATE, YSL are few of the important genes coding for xylem loading and heavy metal transport (Axelsen and Palmgren 1998). It was reported that HMA4 is strongly over expressed in the roots of Cd/Zn hyperaccumulator plants Arabidopsis halleri and Noccaea caerulescens (Bernard et al. 2004, Weber et al. 2004). HMA4 expression is up regulated when these plants are exposed to high levels of Cd and Zn and is down regulated in non hyperaccumulator species (Leitexnnaier et al. 2011). The up regulation of HMA4 is responsible for efflux ofCd and Zn from root symplast to xylem vessels (Willems et al. 2010). In addition, HMA4 enhances the expression of genes belonging to ZIP family, thus enhancing the metal uptake by the root cells (Willems et al. 2007, 2010). Over expression of HMA4 in shoot cells indicates its involvement in xylem unloading as well (Cracium et al. 2012). It has been observed that overexpression of HMA4 in non hyperaccumulators results in their poisoning due to lack of any specific mechanism to detoxify Cd and Zn reaching the shoots (Hanikeime et al. 2008). HMA4 plays an important role in transport of Cd and Zn in case of rice (Опт sativa) as well (Takahashi et al. 2012). FDR3 genes belonging to MATE family is constitutively overexpressed in the roots of Thlaspi caerulescens and Arabidopsis halleri (Talke et al. 2006). TcYSL3, TcYSL5 and YSL7 genes ofYSL family mediate loading and unloading ofNi complexed with nicotinamine in Thlaspi caerulescens (Rascio and Navari-Izzo 2011).
Detoxiflcation/sequestration of heavy metals
After translocating the absorbed heavy metals to the above ground parts, hyperaccumulators have to store them in such a way that it does not interfere with the normal metabolic activities of the plants (Figure 2). Greater efficiency in detoxification and sequestration are the important traits of hyperaccumulators, which allow them to accumulate high concentration of heavy metals in their cells without any phytotoxic effect (Rascio and Navari-Izzo 2011). Another interesting feature is that the accumulation of heavy metals usually occurs in leaves which are the sites of photosynthesis (Rascio and Navar-Izzo 2011). Hyperaccumulation avoids the phytotoxic effects of heavy metals by following any of the two strategies.
Sequestration in shoot vacuoles
The main purpose of sequestration of heavy metals is to remove them from their metabolically active cytoplasm and moving them to inactive compartments, mainly cell wall and vacuoles (Leitenmaier and Kupper 2013). Vacuoles are the most preferred sites for heavy metal sequestration because they contain enzymes like phosphatases, lipases, proteinases, which are not targeted by heavy metals (Carter et al. 2004). Further, the large vacuoles of epidermal cells are preferred as those cells do not harbor chloroplasts, thus minimizing the probability of inhibiting photosynthesis (Kupper et al. 2000, Frey et al. 2000). Preferential storage of hyperaccumulated metals in epidermis has been shown for a majority of hyperaccumulator species and for elements as chemically diverse as Al, As, Cd, Ni, Se and Zn (Freeman et al. 2006, Cosio et al. 2005). If the storage capacity of epidermis is exceeded, storage occurs in mesophyll cells (Kupper et al. 2009). Cell wall too is metabolically active and is the first site at which heavy metal encounters the cell, but its role in hyperaccumulation is still controversial (Fonia et al. 2017). However, Can' et al. (2003) have shown that in Camellia sinensis (tea), Al is accumulated mostly in cell walls, with a very low concentration inside the cells.
Comparative transcriptome analysis between hyperaccumulator and non hyperaccumulator species has demonstrated that the process of sequestration is also dependent upon the constitutive expression of genes that encodes proteins operating in heavy metal transfer across tonoplast and in excluding them from cytoplasm (Rascio and Navari-Izzo 2011). Protein families involved in vacuolar sequestration are Nramps (Natural resistance associated macrophage proteins), CDFs (Cation diffusion facilitator), HMA (Heavy metal ATPase), CaCA (Ca2+/cation antiportor), ABC (ATP binding cassatte), etc. (Verbruggen et al. 2009).
CDFs are also called as metal transporter proteins (MTPs) and contain members involved in the transport of bivalent cations such as Zn2+, Fe2+, Cd2+, Co2+ and Mir~ from cytoplasm to vacuoles (Verbruggen et al. 2009). MTP1, a gene encoding for tonoplast specific protein, is highly over expressed in the leaves of Zn/Ni hyperaccumulators and play an important role in enhancing Zn accumulation (Drager et al. 2004, Gustin et al. 2009). It has been observed that Zn transport into vacuoles may initiate Zn deficiency response, which in turn enhances the heavy metal uptake and translocation via increased expression of Zn transporters in hyperaccumulator plants (Gustin et al. 2009). Persant et al. (2001) have shown that MTP members also facilitate the vacuole storage of Ni in Thlaspi goesingense. Kim et al. (2004), however, have shown that TgMTPl is localized in the plasma membrane and mediates the efflux of both Ni and Zn from cytoplasm. Over expression of ShMTP is responsible for vacuolar accumulation of Mn in tropical legume Stylosanthes hamata (Delhaize et al. 2003).
The over expression of HMA3 coding for vacuolar P1B-ATPase was shown to play significant role in Cd accumulation in Noccaea caerulescens (Ueno et al. 2011). MHX protein of CaCA family is shown to play an important role in Zn vacuolar storage in Arabidopsis halleri (Elbaz et al. 2006). The over expression of Nramp’s was associated with Cd accumulation in Noccaea caerulescens (Takahashi et al. 2011).
Sequestration of heavy metals in mesophyll cells is an exceptional phenomenon and has been observed in a few hyperaccumulator species (Leitemnaier and Kupper 2013). In Zn/Cd hyperaccumulator Arabidopsis hallerii, although Zn is sequestered in trichomes and epidermal cells, a major portion of Cd is stored in mesophyll cells (Kupper et al. 2000). Accumulation of Cd in the mesophyll cells is considered to be responsible for the toxic responses shown by Arabidopsis hallerii at much lower concentration as compared to other Cd hyperaccumulators like Noccaea caerulescens. Sedum alfredii, a Cd/Zn accumulator, is yet another example where Cd sequestration occurs in mesophyll cells beside pith and cortex of the stem (Tian et al. 2011). However, the thick and succulent leaves of Sedum alfredii have exceptionally large vacuoles in the mesophyll that allow a large amount of Cd to be stored, without any toxic effect as compared to Arabidopsis hallerii (Leitemnaier and Kupper 2013). Gossia bidwilli, Virotia neurophylla, Macadamia integrifolia and Macadamia tetraphylla are a few Mn hyperaccumulators where Mn sequestration occurs in multiple palisade cell layers (Fernando et al. 2006a, b).
Detoxification of heaty metals
Apart from sequestration, detoxification of heavy metals is another strategy adopted by hyperaccumulators to avoid toxicity. Heavy metal toxicity is avoided by binding them to cextain metal binding proteins or ligands. The association of heavy metals with ligands prevents the persistence of heavy metal as free ions in the cytoplasm (Rascio and Navari-Izzo 2011). The best known ligands for this purpose are thiols including glutathione, phytochellatins and metallothioneins and non thiols such as histidine and nicotinamide. Non thiols are more significant in hyperaccumulators rather than high molecular mass ligands like phytochelatins because of the excessive amount of sulphur and high metabolic cost that this kind of chelation requires (Schat et al. 2002, Rabb et al. 2004).
Histidine is an important amino acid residue forming metal binding sites in metalloprotein and was first shown to bind Ni in Ni hyperaccumulator Alyssum lesbiacum (Kramer et al. 1996), but later it was found to be involved in hyperaccumulation of Zn as well (Kupper et al. 2004). Nicotinamide has a strong affinity for binding Fe, Zn, Cu and Ni, and is found in high concentration in hyperaccumulators like Alyssum halleri and Noccaea caerulescens (Rascio and Navari-Izzo 2011). Small ligands such as organic acids also play a significant role in detoxification of heavy metals. Citrate is the main ligand which binds Ni in the leaves of Thlaspi goesingense (Kramer et al. 2000), while citrate and acetate bind Cd in the leaves of Solatium nigrum (Sim et al. 2006). A significantly high concentration of citrate ligands has been reported in Phaseolus vulgaris, Crotalaria cobalticola, Raufolia serpentine and Silene cucubalis upon their exposure to high levels of Ni and Co (Boyd 2007). Most of the Zn in Arabidopsis halleri and Cd in Thlaspi caerulescens are complexed with malate (Sarret et al. 2002, Salt et al. 1999) and Al with oxalic acids in the roots of buckwheat (Fagopyrum esculentum).
Metal hyperaccumulators generally have a high concentration of glutathione, cysteine and
O-acetylserine as compared to non hyperaccumulators (Rascio and Navari-Izzo 2011). Glutathione can play a direct role in metal chelation and can also be a substrate for biosynthesis of phytochelatins (Rauser 1999). Cysteine also serves as building blocks of phytochelatins and play an important role in detoxification of As and Cd (Leitemnaier and Kupper 2013). The major detoxification strategy in Se hyperaccumulators is to get rid of selenoaminoacids, mainly selenocysteine, derived from selenate assimilation in leaf chloroplasts. Selenoaminoacids are misincorporated in proteins instead of sulphur amino acid, resulting in Se toxicity. The detoxification occurs through methylation of Se-Cys to a harmless non protein amino acidmethyloselenocysteine in the reaction catalysed by selenocysteine methylotransferase, which is constitutively expressed and activated only in leaves of hyperaccumulator species (Sors et al. 2009).