Moving copper in plants

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Eukaryotic cells utilize copper (Cu) transporter (CTR) family proteins to transport Cu+ ions into the cytosol (Peñarrubia et al., 2010). The CTR-like transporters in plants are called COPT (Copper Transporter) (Kampfenkel et al., 1995), and there may be up to six members of the COPT family (Sancenon et al., 2003; Peñarrubia et al., 2010). Until recently, the only functionally characterized COPT transporter was COPT1 (Sancenon et al., 2004). COPT5 has been detected in the tonoplast using proteomics approaches (Carter et al., 2004; Jaquinod et al., 2007) and in this issue of New Phytologist, Klaumann et al. (pp. 393–404) report the functional characterization of COPT5 in Arabidopsis thaliana. The tonoplast localization was confirmed by confocal microscopy analysis of a COPT5-GFP fusion expressed in protoplasts. The tonoplast-localized COPT5 was found to complement a yeast mutant that lacks not only the plasma membrane-localized CTR1 and CTR3 but also the vacuolar CTR2 proteins. COPT5 mRNA was found throughout the plant, with elevated expression in flowers and siliques and reduced expression in rosettes. Three copt5 T-DNA insertion alleles were characterized in which COPT5 mRNA was absent. Only for seedlings under severe Cu depletion in tissue culture were growth defects observed.

‘With the functional characterization of COPT5, a more complete understanding of Cu transport systems in plants has emerged.’

The report by Klaumann et al. corroborates and expands on the findings reported by Garcia-Molina et al. (2011), who investigated the phenotypes of seedlings of copt5-KO (Knock-out) plants more extensively. When grown in tissue culture with a Cu chelator, the KO lines showed reduced vegetative growth, impaired root elongation, chlorosis and strong defects in photosynthetic electron transfer because of reduced accumulation of the essential Cu protein plastocyanin. A tagged version of the COPT5 protein co-migrated with vacuolar markers in a sucrose density gradient. However, a GFP fusion construct, which complemented the root growth phenotype, was found to localize to intracellular structures that closely resemble the prevacuolar compartment (Garcia-Molina et al., 2011). Histochemical staining of transgenic plants in which a COPT5 promoter fragment drives glucuronidase production revealed expression throughout the plant except in pollen, with clearly elevated activity in the vasculature, especially in roots (Garcia-Molina et al., 2011).

Surprisingly, soil-grown copt5 mutants showed no visible change in phenotype, but nevertheless Klaumann et al. performed a detailed analysis for 12-wk-old soil-grown plants, including elemental analysis of isolated vacuoles. Based on normalization to mannosidase activities it could be calculated that c. 15% of the total leaf Cu was recovered in vacuoles in the wild type. The Cu content of vacuoles of the copt5 mutants was c. 1.5–1.8-fold higher compared with the wild type, whereas the content of other minerals was unchanged, supporting the idea that COPT5 affects Cu efflux from vacuoles. The Cu contents of whole leaves for the copt5 mutants and the wild type were comparable. Loss of COPT5 resulted in increased Cu accumulation in roots, while the Cu content of siliques and seeds was lower compared with the wild type, which suggests a physiological role for COPT5 in moving Cu from roots and to reproductive tissue. Although the two studies (Klaumann et al.; Garcia-Molina et al., 2011) did not conclude that COPT5 has exactly the same subcellular location, it is clear that COPT5 functions in a vacuole-related intracellular compartment and serves to mobilize Cu to the cytoplasm, a task that is most critical under Cu deficiency.

An emerging picture of Cu transport in plants

With the functional characterization of COPT5, a more complete understanding of Cu transport systems in plants has emerged (Fig. 1). The trimeric members of the CTR/COPT family transport Cu+ and probably act together with a reductase. CTR/COPT proteins scavenge free Cu+ using extracytoplasmic N-terminal methionine-rich regions; Cu+ is then transferred to the cytoplasm, which requires conserved sequence motifs including a crucial methionine residue (for a review, see Peñarrubia et al., 2010). The driving force for Cu transport is probably the high Cu-chelating capacity of the cytoplasm. COPT1, 2, 3 and 5 all complemented, at least partially, a yeast ctr1/ctr3 deletion mutant by facilitating cellular Cu uptake, measured as 64Cu accumulation (Sancenon et al., 2003). COPT1 and COPT2 showed the highest Cu uptake rates, consistent with a proposed function of these proteins as PM (Plasma membrane) transporters; COPT4 lacks the critical methionine residue and failed to complement a yeast ctr1/ctr3 mutant (Sancenon et al., 2003). For COPT6, no experimental data are available. COPT1 is expressed in most tissues but is especially abundant in root tips and pollen (Sancenon et al., 2004). At times of low Cu availability, COPT1 and COPT2 are up-regulated via the master Cu-responsive transcription factor SPL7 (Squamosa Promoter binding protein-Like 7) (Yamasaki et al., 2009). Antisense repression lines for copt1 showed reduced Cu uptake, and defects in root elongation and pollen development. Over-expression from a constitutive promoter of COPT1 or of the otherwise uncharacterized COPT3 resulted in over-accumulation of Cu and sensitivity to Cu excess (Andrés-Colás et al., 2010). Additional Cu import capacity might be provided by ZIP2 (Zrt, Irt-like Protein 2), which is a member of the ZIP (Zrt, Irt-like Protein) family of mostly Zn2+/Fe2+ transporters. The ZIP2 transcript level is up-regulated by Cu deficiency via SPL7 (Yamasaki et al., 2009). As its expression is regulated by SPL7, it can be speculated – in the absence of in planta data – that ZIP2 really functions in Cu acquisition, perhaps with the capacity to transport Cu2+.

Figure 1.

Copper (Cu) transport systems. Only well-characterized Cu transporters are shown in their most likely locations with the proposed direction of transport. Open symbols, COPT (Copper Transporter) transporters; closed symbols, ATP-driven Cu pumps. Metallochaperones that might deliver Cu to HMA (Heavy Metal transporting ATPase)5 and HMA7 are omitted. Arrows, vesicular transport routes; block arrows with question marks, possible Cu-recycling pathways for apoplastic proteins or organelles. The porous outer membranes of plastids and mitochondria are indicated by dashed lines. Abundant Cu proteins are in grey boxes: Cu/ZnSOD, Cu/Zn superoxide dismutase; Cyt-c OX, cytochrome-c oxidase; ETR, ethylene receptors; PC, plastocyanin.

Four heavy metal-transporting P-type ATPases (HMA (Heavy Metal transporting ATPase)5, HMA6/PAA (P-type ATPase of Arabidopsis)1, HMA7/RAN (Responsive to ANtagonist)1 and HMA8/PAA2) move Cu into organelles (see Burkhead et al., 2009). HMA7 functions to deliver Cu to the ethylene receptors, probably in an early secretory compartment. HMA5 is the only Cu pump reported to be regulated by Cu at the transcript level; it is highly expressed in roots and induced by Cu excess (Andrés-Colás et al., 2006). HMA5 functions to remove Cu from the symplast, consistent with a role in xylem loading. However, soil-grown hma5 mutants are phenotypically like the wild type. Yeast two-hybrid studies have suggested that HMA5 and HMA7 might acquire Cu from cytoplasmic Cu chaperones (Andrés-Colás et al., 2006). HMA6 and HMA8 function in the chloroplast envelope and thylakoids to deliver Cu to stromal Cu/Zn superoxide dismutase and plastocyanin. Finally, the yellow stripe-like (YSL) proteins YSL2 and YSL3 might have an as yet incompletely characterized biochemical role in the mobilization of Cu from vegetative tissue for loading into the seeds (see Burkhead et al., 2009).

Linking biosynthetic and homeostatic functions of Cu-transport systems

Cu is a cofactor (Burkhead et al., 2009) and therefore it can be argued that all Cu transporters have a biosynthetic function. A primarily biosynthetic (cofactor delivery) function is evident for the three ATP-driven Cu pumps HMA6, HMA7 and HMA8. Transporter function can also be viewed in a homeostatic context: functioning to maintain appropriate Cu concentrations both in local compartments and globally in the plant over time; this seems to be the primary function of COPT family members. The phenotypes of copt1 and copt5 mutants are evident on low Cu and manifest themselves in the tissues where the transporters are normally expressed. However, copt1 and copt5 phenotypes on low Cu cannot be explained by a lack of cytosolic Cu enzyme function, because the only known cytosolic Cu protein is a Cu/Zn superoxide dismutase whose expression is shut off when Cu becomes limiting. Cu/Zn superoxide dismutase down-regulation occurs via SPL7-dependent up-regulation of miR398 (Yamasaki et al., 2007, 2009), one of at least four Cu microRNAs (Burkhead et al., 2009). Other Cu microRNAs target mRNA for apoplastic Cu enzymes including LAC4 (Lacasse 4) and LAC17 (Abdel-Ghany & Pilon, 2008), two Cu enzymes that were recently shown to be involved in lignification (Berthet et al., 2011). It was proposed that the Cu microRNAs serve to maintain a pool of available Cu that could be used to supply the cofactor to the most essential Cu proteins (Burkhead et al., 2009). However, this hypothesis is thus far untested. One problem is that many Cu enzymes bind their cofactor very strongly, preventing its release, regardless of Cu microRNA action at the transcript level. However, a vacuole with hydrolytic activity could potentially serve to deplete Cu enzymes which are active in extracytoplasmic compartments and the released Cu could then be transported to the cytosol by COPT5 (Fig. 1). Such a mechanism would explain the phenotypes of copt5 mutants on very low Cu when the only ‘available’ Cu is already in the plant. Interestingly, copt5 mutants were shown to have increased lignification under Cu-deficient conditions (Garcia-Molina et al., 2011), which might hint at a defect in recycling the Cu-containing laccases. An implication of the recycling hypothesis is that deficiency induces Cu-protein breakdown via endocytosis or autophagy (Fig. 1).

Most metal homeostasis studies have focused on responses in a steady state: plants are maintained with the micronutrient either at an optimal concentration or at a concentration that is too low or too high, and responses in terms of, for example, gene expression, metal content or enzyme function are compared. However, homeostasis involves responses to change. Now that most components involved in Cu transport have been characterized, new insights into how these components function as a system should come from experiments in which growth conditions are altered from optimal to suboptimal and vice versa. The analysis of responses over time then has the potential to reveal priorities – if these indeed exist – in cofactor delivery throughout the plant as it grows and develops. Furthermore, such experiments could be employed to determine whether Cu-recycling pathways do exist which would require the measurement of dynamics in Cu pools. This approach is particularly promising when used to investigate specific transporter loss-of-function lines.

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