MTP1‐dependent Zn sequestration into shoot vacuoles suggests dual roles in Zn tolerance and accumulation in Zn‐hyperaccumulating plants

Abstract

SummaryThe integral membrane protein Thlaspi goesingense metal tolerance protein 1 (TgMTP1) has been suggested to play an important role in Zn hyperaccumulation in T. goesingense. Here, we show that the TgMTP1 protein is accumulated to high levels at the vacuolar membrane in shoot tissue of T. goesingense. TgMTP1 is likely to act in the transport of Zn into the vacuole, enhancing both Zn accumulation and tolerance. By specifically expressing TgMTP1 in Arabidopsis thaliana shoots, we show that TgMTP1, localized at the vacuolar membrane, can drive the enhanced shoot accumulation of Zn by initiating a systemic Zn deficiency response. The systematic response includes increased expression of Zn transporters (ZIP3, ZIP4, ZIP5 and ZIP9) in both shoot and root tissue. Furthermore, shoot-specific accumulation of TgMTP1 at the vacuolar membrane also leads to increased resistance to Zn in A. thaliana, probably through enhanced Zn compartmentalization in the vacuole. Such evidence leads to the conclusion that the high levels of TgMTP1 at the vacuolar membrane in shoot tissue of the Zn hyperaccumulator T. goesingense play a role in both Zn tolerance and enhanced Zn uptake and accumulation, via the activation of a systemic Zn deficiency response.

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“…CAXs and some MTP transporters catalyze the transport of heavy metals across the vacuolar membrane and are essential for vacuolar sequestration. Increasing the expression of the tonoplast-localized MTP1 can enhance Zn resistance (Blaudez et al, 2003;Gustin et al, 2009;van der Zaal et al, 1999). In yeast, ATP-binding cassette (ABC) transporters sequester heavy metals into vacuoles.…”

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“…Gradients were spun at 100,000g for 4 h at 48C. Eight fractions representing the entire gradient were collected and sucrose removed according to Gustin et al (2009). Each fraction was then centrifuged at 100,000g for 1 h at 48C and the pellets resuspended in microsomal buffer lacking DTT and sucrose, and the protein concentration determined using the BCA protein assay kit according to the manufacturer's instructions (Pierce).…”

“…For sucrose gradient analysis, the microsomal membranes were separated using a sucrose step gradient (22, 32, 38 and 45% [w/v] sucrose with 10 mM BTP-MES, pH 7.8, and 5 mM EDTA) (Gustin et al, 2009). Gradients were spun at 100,000g for 4 h at 48C.…”

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“…Plasma membrane was purified from Arabidopsis root tissue according to Gustin et al (2009). First, 8 to 10 g fresh weight of root was collected from 6-week-old plants grow in soil and thoroughly washed in deionized water.…”