Metals such as iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) are required as essential micronutrients for all organisms because they fulfil structural and catalytic roles in large numbers of proteins. However, these ions are also cytotoxic and thus tightly regulated mechanisms are required to maintain the availability of these nutrients within specific limits. As sessile organisms, plants must possess efficient mechanisms to extract or exclude metals from the soil and deliver ions to required tissues, cells and subcellular compartments throughout the plant while maintaining the concentration of ‘free’, including weakly bound, ions below the threshold to induce toxicity. This is achieved through regulated coordination of processes for transmembrane transport for cellular uptake, export and subcellular compartmentalization, and chelation of free ions by small organic molecules, peptides and proteins (Clemens et al., 2002). Understanding these processes is important for improving global crop production because large areas of agricultural soils are deficient in available micronutrients or have been contaminated by industrial activities. Of critical importance in understanding these mechanisms is the ability to measure the concentration of free metal ions in vivo. In this issue of New Phytologist, Lanquar et al. (2014, pp. 198–208) report a major step forward in understanding Zn homeostasis in plants by establishing a toolset to determine cytosolic free Zn2+ concentrations in Arabidopsis thaliana roots.
‘In Zn sufficient conditions, the authors determined a cytosolic free [Zn2+] of c. 0.4 nM. This is the first experimental estimation for cytosolic free [Zn2+] in plant cells.’
The state-of-the-art for measuring metals
A range of techniques have been applied to measure the concentration and distribution of metals in plant tissues and each of them has made significant contributions to the field of plant metal homeostasis research, but also has significant drawbacks. The application of inductively coupled plasma atomic emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS) permits simultaneous measurement of a wide selection of elements in plant samples with high sensitivity, accuracy and relatively high throughput and ushered in the plant ‘ionomics’ era (Lahner et al., 2003). These techniques are now standard practice within the field to measure total metal concentrations within various tissue samples. However, major limitations arise when information about compartmentalization beyond organ-level is required. ICP techniques have been applied to measure metal concentrations in vacuoles and chloroplasts in plant cells by fractionating leaf samples (Lanquar et al., 2010) but these protocols are time-consuming and it is difficult to obtain pure samples or to entirely prevent movement of ions across organelle membranes during sample preparation. X-ray fluorescence and x-ray absorption based techniques can provide information on metal distribution with high spatial resolution but with low sensitivity. These techniques depend on access to specialized equipment and are low throughput but have been useful in describing metal distribution in metal hyperaccumulator species and metal-rich tissues in nonhyperaccumulators (Küpper et al., 1999, 2000; Kim et al., 2006; Sarret et al., 2006). Fluorescent dyes such as zinquin or zinypr have been used to detect Zn2+ by confocal microscopy in intact plant samples. These have been applied to identify Zn-enriched compartments in plants and to describe changes in Zn distribution in mutant or transgenic lines (Sarret et al., 2006; Sinclair et al., 2007; Helmersson et al., 2008; Haydon et al., 2012). These probes are inexpensive and relatively straightforward to use for a skilled microscopist but the information is only qualitative. Therefore, there is great need for better tools to understand metal ion distribution in plant tissues.
Fluorescent reporters for biological research
A range of genetically-encodable fluorescent reporters or biosensors for small molecules has been developed which has transformed the respective fields to various degrees (comprehensively reviewed by Newman et al., 2011). Biosensors are available to measure pH, reactive oxygen species, ions, small metabolites and other biologically relevant molecules. These reporters are most commonly based on green fluorescent protein (GFP) or GFP variants that either change wavelength for excitation or emission in response to the stimulus, or are Förster (or fluorescence) resonance energy transfer (FRET)-based sensors. FRET sensors comprise two fluorescent proteins (FPs), whereby energy emitted from a donor FP excites an acceptor FP. The two proteins are bridged by a linker region containing a substrate-binding domain, which induces a conformational change upon binding to the molecule of interest. Substrate binding leads to a change in energy transfer from the donor to the acceptor FP and a detectable shift in the ratio of fluorescence emission from each FP (Fig. 1). In general, these biosensors have not been developed specifically with plant research in mind and therefore have been applied more extensively in animal or yeast cells. Adoption of these sensors in plant systems often requires adaptations to overcome the challenges of working in a multicellular context. Several fluorescent biosensors have been successfully applied in plants cells including redox sensitive GFP (roGFP), and FRET-based sensors for calcium ions (Ca2+) and hexoses. These biosensors have been used to measure their substrates in specific cell types (Allen et al., 2001) and subcellular compartments (Schwarzländer et al., 2009; Rosenwasser et al., 2011; Krebs et al., 2012) and identify novel transporters (Chen et al., 2010, 2012).
Zinc biosensors provide novel insight into zinc homeostasis in plants
In this issue of New Phytologist, Lanquar et al. (2014) describe the first use in plants of a set of eCALWY Zn2+ biosensors. These FRET-based sensors comprise cerulean, an enhanced cyan fluorescent protein (CFP), and citrine, an enhanced yellow fluorescent protein (YFP), each fused to a copper chaperone Atox1, and a region of the copper transporter ATP7B/Wilsons (WD4), respectively, separated by a flexible linker region (Vinkenborg et al., 2009). In the absence of Zn2+-binding to a metal-binding pocket formed between Atox1 and WD4, FRET between the two weakly self-associating FPs is high, yielding a high citrine to cerulean fluorescence ratio (Fig. 1a). Zn2+-binding to the metal binding pocket induces a conformational change to disrupt the interaction between the two FPs (Fig. 1b), reducing FRET and the citrine to cerulean ratio (Fig. 1c). This biosensor was modified to generate a set of sensors (eCALWY-1 to -6) each with different Zn2+-binding affinity ranging from the low picomolar to micromolar range (Vinkenborg et al., 2009).
Lanquar et al. (2014) successfully expressed five eCALWY sensors in Arabidopsis thaliana. The eCALWY sensors, driven by the strong, cauliflower mosaic virus 35S promoter were transformed into the rdr6 silencing mutant to obtain lines with sufficient levels of fluorescent signal for imaging. Using these lines, an estimation of cytosolic free [Zn2+] was determined in roots of seedlings grown in Zn sufficient, Zn limited and Zn excess media conditions. In Zn sufficient conditions, the authors determined a cytosolic free [Zn2+] of c. 0.4 nM. This is the first experimental estimation for cytosolic free [Zn2+] in plant cells. The value is similar to that calculated with these sensors in animal cells (Vinkenborg et al., 2009), but much higher than estimates in prokaryotes (Outten & O'Halloran, 2001). Across the three Zn conditions, representing several orders of magnitude change in external Zn supply, the cytosolic [Zn2+] was maintained within a five-fold range. This indicates a tremendous buffering capacity for cytosolic [Zn2+] in plant roots across a wide range of external Zn availability.
The authors next employed the eCALWY lines to look at dynamic changes in cytosolic Zn2+ concentration in Arabidopsis roots in response to short-term changes in external Zn conditions. These experiments utilized the RootChip, a recently developed microfluidic system that permits imaging in Arabidopsis roots while manipulating the growth media (Grossmann et al., 2011). These data described several transport processes, which contribute to the buffering of cytosolic free [Zn2+] in response to changes in availability of external Zn. Through careful experimental design, the authors could identify distinct contributions to the cytoplasmic pool of Zn2+ under Zn deficiency from mobilized internal Zn stores, most likely the vacuole, and from a high-affinity uptake system. In addition, Zn resupply experiments suggested the existence of a high capacity, low affinity uptake system. The authors propose candidate transporters that might fulfil these roles (Fig. 2).
A new era for plant zinc homeostasis research?
The work of Lanquar et al. (2014) has laid a solid foundation to answer important questions about plant Zn homeostasis. Having described several Zn transport processes in Arabidopsis roots contributing to cytosolic [Zn2+] buffering, an exciting prospect will be to identify the transporters responsible, revealing the elusive modes for Zn uptake despite the existence of candidates for over 15 yr (Clemens et al., 2002). In addition, the data generated from this new system are well-suited to mathematical modelling approaches to better understand the dynamics of metal homeostasis. There is also great potential to improve the utility of the eCALWY sensors, and therefore the impact to the research community. Driving eCALWY expression from the more modest UBIQUITIN10 promoter, similar to that done for the cameleon Ca2+ sensors (Krebs et al., 2012), would make these sensors more useful for genetic studies. Targeting the eCALWY sensors to subcellular compartments as done for several biosensors in plants (Schwarzländer et al., 2009; Rosenwasser et al., 2011; Krebs et al., 2012), and the eCALWY sensors in animals (Vinkenborg et al., 2009) would permit calculations of free [Zn2+] within organelles to define major internal Zn2+ stores and potentially identify and characterize additional transport processes. Although major hurdles still exist, these tools hopefully open up a new frontier in plant metal homeostasis research.