Zinc

The micronutrient Zn is required for proper cell functioning (Berg and Shi 1996, Salgueiro et al., 2000, Sinclair and Krämer 2012), as it is a highly effective cofactor for hundreds of enzymes, the structural Zn-finger domains that mediate DNA-binding of transcription factors, and protein–protein interactions (Coleman 1998, Shahzad et al.,2014). In plants, Zn is taken up at the root-soil interface, predominantly as free ions (Guerinot 2000, Milner et al., 2013, Shahzad et al.,2014). In recent years, significant progress has been made in our knowledge of the regulation of Zn acquisition in plants, and this phenomenon has been documented in many research publications and elegantly summarised in multiple reviews (e.g. (Sinclair and Krämer 2012). Many Zn uptake transporters have been identified and belong to the Zrt/IRT-like protein (ZIP) family of zinc transporters. In A. thaliana, the ZIP family contains 15 members (Mäseret al.,2001), including the AtIRT1 which localizes preferentially to the plasma membrane of root epidermal cells (Vert et al.,2002, Barberon et al.,2011). It has been reported that irt1 mutant accumulates less Zn as compared to wild type revealing its implication in Zn uptake (Henriques et al.,2002). For ample information on the regulation of Zn uptake in Arabidopsis readers are referred to (Sinclair and Krämer 2012). After their acquisition at the root periphery, Zn can be fixed into the root via transport into vacuoles. Alternatively, their symplastic journey, thought to be mediated by the plasmodesmata, ends with their loading into root xylem. Zn transport, two members of the Arabidopsis P_1B-ATPase subfamily: HMA2 and its most closely related sequence in the HMAcluster, HMA4 play crucial role in Zn loading into xylem (Hussainet al., 2004, Verret et al., 2004, Hanikenne et al., 2008, Wong et al., 2009, Siemianowski et al., 2011). The most recent reports on their biological functions and the molecular mechanisms of their regulation in A. thalianawill be presented below.

1. Zinc root-to-shoot translocation in Arabidopsis.

In Arabidopsis, HMA4 and HMA2 gene encodes for a metal transporter located in the plasma membrane, with a very similar structure. HMA2 and HMA4 proteins play key roles in Zn loading into the root xylem, even though the signalling pathways that regulate their expression and activity remain poorly understood.

The Arabidopsis genes HMA2 (At4g30110) and HMA4 (At2g19110) are found in the Zn/Co/Cd/Pb subclass of the P_1B-ATPases, and have well-documented roles in Zn loading into xylem (Hussain et al., 2004, Verret et al., 2004). In Arabidopsis, none of the individual hma mutants exhibit an observable or distinctive morphological phenotype when grown in soil, as

compared to the wild-type (Hussain et al., 2004). The mutation of both genes results in a drastic Zn reduction in the shoot. This severely compromises the plant life cycle through visible morphological alterations, a stunted phenotype, and the formation of sterile flowers that lack pollen (decreasing plant fertility). This severe phenotype can be alleviated by supplying double mutants with high Zn.

HMA4: Arabidopsis thaliana HMA4 was originally identified as a gene with increased expression in the Zn hyperaccumulator Arabidopsis halleri, as compared to its non-hyperaccumulator relative A. thaliana (Hanikenne et al., 2008). The Zn content in the aerial parts of hma4 mutant plants is GHFUHDVHG DW D =Q FRQFHQWUDWLRQ RI ȝ0 DOWKRXJK LW is not VLJQLILFDQWO\ DIIHFWHG DW KLJKHU FRQFHQWUDWLRQV ȝ0Overexpression of AtHMA4 in A.

thaliana resulted in a 2-fold increase in Zn content in leaves, whereas no significant change was observed in root Zn content (Verretet al.,2004).

HMA2: Expression of HMA2 promoter was observed in vascular bundles, and appeared to be expressed in components of both the xylem and the phloem (Hussainet al.,2004). No decrease in the Zn content is observed in the single mutant hma2. The A. thaliana hma2hma4 double mutant shows a 2-fold Zn increase in root pericycle cells, which causes a 2-fold decrease in shoots. This hma2hma4 double mutant Zn-deficiency phenotype can be restored with the expression of full-length AtHMA2 (Wonget al.,2009).

In Arabidopsis roots, HMA4expression levels appeared to be enhanced by Zn (Hanikenne et al.,2008), but it is still unclear whether the transcript level is modulated directly or indirectly by Zn. The specific mRNA activity of HMA4 in A. halleriand A. thaliana was observed in root pericycle and xylem parenchyma (Hanikenne et al., 2008):KHQWKHȕ-glucuronidase (GUS) reporter gene was placed under the control of the HMA4 promoter in A. thaliana, expression was predominantly observed in root stellar cells located at the periphery of the xylem (Verretet al.,2004). This expression pattern is consistent with the involvement of AtHMA4in Zn loading into the xylem (Hussain et al., 2004). A possible role for AtHMA4 in Zn efflux from the cytoplasm is also supported by its ability to restore growth to the zntA mutant in S. cerevisiae, which is defective in its endogenous Zn efflux pump at high Zn concentrations (Rensinget al., 1997, Millset al.,2003, Verretet al.,2004, Millset al.,2005).

In future, it will be interesting to identify the signalling pathways that regulate HMA2 and HMA4 at transcriptional and protein levels. Zinc sensing and signaling in Arabidopsis.

The Zn deficiency appears to be first sensed in shoots; the signal is then transmitted to the roots, where these cation transporters function (Assunçãoet al., 2010b, Assunçaoet al., 2013).

This suggests the presence of long-distance Zn deficiency signalling molecules (which are yet to be identified). A recent working model of Zn deficiency signalling (Assunçao et al., 2013) proposes that the Arabidopsis transcription factors bZIP19 and bZIP23 play important roles in the response to Zn deficiency by regulating downstream genes, including ZIP members (i.e.the Zrt/Irt-like proteins, candidates that mediate root Zn uptake and transport) (Guerinot 2000, Assunçãoet al.,2010b). (Figure I.4).

Figure I.4. Overview of current understanding of Zn transporters &

accumulation. Those adaptations are highlighted in red. (I) Enhanced Zn uptake into root cells is thought to be driven by ZIP4 as Zn-nicotianamine complex by YSL proteins. (III) In the leaves, favored by ZIP6 in A. halleri. (IV) Detoxification is assumed to be operated by chelation of metals. <

> refers to chelation. Possible ligands of Zn in the cytoplasm are histidine and nicotianamine. (V) Vacuolar sequestration in the leaves is the main pathway of detoxification of metals. Zn is mainly stored in vacuoles of mesophyll (A. halleri) and/or epidermal cells (Thlaspi caerulescens), through the activity of MTP1, and possibly HMA3 and MHX. In the vacuoles, a large pool of malate favors the formation of Zn-malate complexes. (VI) Other adaptive processes include homeostasis of other nutrients, in particular Fe, P (PHT1-4), enhanced stress responses/protection (higher Glutathione GSH level, etc.).

(Verbruggenet al.,2009).