Biofortification is a general concept consisting in the amelioration of food quality through conventional breeding or genetic engineering of crops to increase their nutritional value. One aim of biofortification is to make plant foods more nutritious as the plants are growing by themselves without further treatment. One of the

Fig. 3 (continued) and Thlaspi caerulescens (7c). Sequences were aligned with the MAFFT v. 6 software. The trees were obtained with the Neighbor-Joining analysis. The distances are not significant. (a) Phylogenetic tree including all 205 HMA sequences (Cluster I = 23 HMA1, II = 47 HMA2-4; III = 54 HMA5; IV = 36 HMA7; V = 22 HMA6; VI = 21 HMA8) and two S. cerevisiae sequences. For a simplified representation, the individual branches were collapsed when possible. (b) Phylogenetic tree of 48 HMA2, HMA3 and HMA4 sequences well-known examples is the Golden Rice in which two genes have been inserted by genetic engineering leading in the production and accumulation of p-carotene, a precursor of vitamin A, in the grains (Ye et al. 2000). Micronutrient deficiencies are widespread in humans, mainly concerning iron and zinc (White and Broadley 2009). Concerning Fe, it has been shown that a specific targeting of ferritin and nicotianamine synthase was able to increase the iron content in rice endosperm (Wirth et al. 2009). Zn2+ deficiencies are widespread, estimated to be at least 25% of the world population (Maret and Sandstead 2006). A primary reason is that many soils of calcareous or alkaline nature lack sufficient phyto-available Zn2+ (White and Broadley 2009) and because of the narrow food base of a large part of the world population relies on starch-rich staples that generally contain a suboptimal Zn2+ content. Secondary, concerning the rice, for example, the husk and the embryo containing typically 50% of the Zn2+ in the grain and is discarded during the milling and polishing processes. Identification of Zn2+ transporters able to increase the grain filling in Zn2+ is a first step before their manipulation in plants to optimize the Zn2+ grain content.

The pioneering work of Hussain et al. (2004) on Arabidopsis has clearly demonstrated that AtHMA2 and AtHMA4 are in charge of most of Zn2+ translocation from roots to shoot, since a double hma2, hma4 mutant line exhibited a dwarf and sterile phenotype that was overcome by feeding the plants with 3 mM Zn2+ in the nutrient solution. In agreement, it was found that overexpression of AtHMA4 in Arabidopsis using the strong Cauliflower Mosaic Virus promoter (CaMV 35S) was able to increase the shoot Zn2+ content by 60% at 3 p.M Zn2+ in the nutrient solution and by 52% at 200 mM Zn2+ (Verret et al. 2004). These results demonstrate that manipulation of AtHMA4 is a pertinent strategy to increase Zn2+ content in the edible parts of the plant. However, an adverse effect is that this increased translocation of Zn2+ was accompanied by a concomitant increase in Cd2+ content in the leaves (Verret et al. 2004). Thus, it is essential that we gain a better understanding of the molecular basis of HMA selectivity in order to select transporters with a higher selectivity for Zn2+ than Cd2+. Molecular engineering of the transmembrane domains 6-8 exhibiting metal binding sites forming the pore and a survey of the biodiversity of HMA alleles in plants could allow the attainment of such a goal. A deregulation of the activity of the transporter may also be a strategy to enhance Zn2+ translocation in plants. Previous experiments using heterologous expression in yeast or measurements of ATP hydrolysis have shown that the C-terminal extension of these transporters has regulatory properties. Some results point to a total or partial inactivation of the transporter after a deletion of the C-terminus. Verret et al. (2004) reported an inactivation of AtHMA4 expressed in yeast after deletion of its terminal His-stretch. In agreement, Eren et al. (2006) observed that removal of the 244 amino acid C-terminus of AtHMA2 leads to a 43% reduction in the enzyme turnover without significant effect on the Zn2+ Ki/2 for enzyme activation. In contrast, another study reports a strong increase in Cd2+ tolerance conferred on the Cd2+-sensitive yeast ycf1 after deletion of its C-terminus (Mills et al., 2005). More recently, Baekgaard et al. (2010) observed that sequential removal of the His-stretch and the cystein pairs confers a gradual increase in Zn2+ and Cd2+ tolerance of sensitive yeasts through a decrease in their Zn2+ and Cd2+ content. While these observations are difficult to reconcile, expression of a deletion mutant lacking the C-terminal 244 amino acids rescued most of the hma2, hma4 Zn2+-deficiency phenotypes with the exception of embryo or seed development (Wong et al. 2009b). The GFP-tagged protein also appeared partially mis-localized in the root pericycle cells. Thus, manipulation of the C-terminus of AtHMA4 did not at present offer an obvious way to enhance Zn2+ translocation in plants. Tentative expression or overexpression of heterologous genes from metallophytes did not result in an increased translocation of Zn2+ (Barabasz et al. 2010). This may be attributed to the fact that HMAs from metallophytes do not display a higher activity when expressed in yeast than those from non-metallophyte origin (Papoyan and Kochian 2004; Bernard et al. 2004). There are no formal data whether HMAs need to form multimeric complexes to be active but this is likely from what is known from well described P-ATPases such as the H+-ATPases. Thus, another explanation of the lack of efficiency of heterologous expression in plants could be related to the formation of inactive heterologous multimers.

Increasing shoot Zn2+ content is interesting for some crops, but the major goal is an enhanced Zn2+ content in grains. In a test using five different Arabidopsis transgenic lines overexpressing the endogenous gene AtHMA4, the Zn2+ content measured in the seeds was on average 14% lesser than in the wild type (A Vavasseur, A. Chevalier and P. Richaud, unpublished results). This unexpected result means that the constitutive overexpression of AtHMA4 leads to a spreading of the metal in all the shoot tissues to the detriment of the initial vectorization of metal trafficking. Thus, in order to increase the Zn2+ content in specific edible parts of the plant, a precise vectorisation of the transport of the micronutrient is essential. This could be achieved by (1) the introduction of multi copies of the genes of interest, mimicking what is observed in metallophytes (Hanikenne et al. 2008; Lochlainn et al. 2011), (2) the use of promoters from metallophytes that have been shown extremely active (Hanikenne et al. 2008; Lochlainn et al. 2011), (3) a modification of native promoters to enhance their activity, and (4) the use of promoters strongly expressed in the pericycle (Dembinsky et al. 2007). In any case, while AtHMA4 and orthologs stay interesting biotechnological targets to enhance Zn2+ translocation, enhancing Zn2+ content in grain will certainly require manipulation of other genes involved in Zn2+ trafficking from the phloem to the different tissues of the seeds. A pioneering study from Tauris et al. (2009) using laser capture micro dissection in barley grain indicated that HvHMA2 and HvHMA4 were detected in transfer cells but not in the aleurone and endosperm tissues. Transporters from other families such as ZIP, YSL, MTP and CAX were detected in these specific tissues and could be in charge of Zn2+ trafficking in these tissues. Such studies should allow the discovery of new candidate genes for Zn2+ biofortification.

The biofortification concept aimed at an amelioration of food quality also includes a diminution of toxics or anti-nutrient compounds such as phytate in food (Raboy 2007). In this domain, AtHMA3 was initially identified as a Cd2+/ Pb2+ vacuolar pump when expressed in yeast, able to rescue the metal-sensitive phenotype of ycf1 mutant lines (Gravot et al. 2004). In plants, AtHMA3 also localized at the tonoplast and is expressed at a low level in roots and shoot vascular tissues (Morel et al. 2009). In accordance with the function determined in yeast, a hma3 knock-out mutant was found slightly more sensitive to Zn2+, Cd2+, Pb2+ and Co2+ while overexpression of the protein in Arabidopsis gave a strong tolerance to these metals (Morel et al. 2009). Interestingly, very recently, a major QTL explaining more than 80% of the phenotypic variance in the shoot Cd2+ content of rice was detected in different mapping populations (Ueno et al. 2009; Ishikawa et al. 2010; Tezuka et al. 2010). These QTL identified OsHMA3 as a vacuolar transporter able to sequester Cd2+ at the level of the roots (Ishikawa et al. 2010; Tezuka et al. 2010). Identification of OsHMA3 as an essential transporter for sequestration of Cd2+ in the roots provides an efficient way to breed rice and other crops with low Cd2+ accumulation.

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