Quantitative detection of changes in the leaf-mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd2+), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd2+ can be transported as phytochelatin-Cd2+ by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd2+ detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 mu M), and high (200 mu M) Cd2+ conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ (TM)). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low-Cd2+ conditions, an inorganic pyrophosphatase and a gamma-tonoplast intrinsic protein (gamma-TIP) were up-regulated, indicating changes in energization and water fluxes. in addition, the protein ratio of a CAX1a and a natural resistance-associated macrophage protein (NRAMP), responsible for vacuolar Fe2+ export was increased. CAX1a might play a role in vacuolar Cd2+ transport. An increase in NRAMP activity leads to a higher cytosolic Fe2+ concentration, which may prevent the exchange of Fe2+ by toxic Cd2+. Additionally, an ABC transporter homolog to AtMRP3 showed up-regulation. Under high Cd2+ conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd2+ conditions, was up-regulated. Interestingly, AtMRP3 is able to partially rescue a Cd2+-sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd2+ detoxification.