Postdoctoral associate University of Maryland, College Park
The gaseous hormone ethylene regulates plant growth, development, and stress responses throughout the entire life cycle. Components in the ethylene signaling pathway have been identified, but many underlying mechanisms of ethylene signal transduction remain unknown. Ethylene receptors, CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), and ETHYLENE INSENSITIVE2 (EIN2) form a complex that is localized at the endoplasmic reticulum (ER) membrane. In the presence of ethylene, CTR1 no longer phosphorylates EIN2, and the C-terminal EIN2 domain is cleaved from the N-terminal domain, which consists of 12 transmembrane domains in the ER membrane. The cleaved C-terminal EIN2 domain then regulates downstream signaling in the processing-bodies and nucleus. The N-terminal EIN2 domain has high sequence similarity to the NRAMP transporters that are known to transport divalent metal ions, such as Fe2+ and Mn2+. However, whether EIN2 transports a metal has been elusive. Here, we present evidence that the Arabidopsis N-terminal EIN2 domain is capable of transporting Ca2+ against the Ca2+ gradient across the ER membrane into the ER lumen. We monitored cytosolic Ca2+ levels using a yellow cameleon YC3.6 sensor in mammalian cells. Following addition of thapsigargin, an inhibitor of the ER Ca2+ ATPase, control cells showed elevated cytosolic Ca2+ levels whereas cells expressing the N-terminal EIN2 domain showed only a slight increase. We then assayed a yeast mutant that is susceptible to Ca2+ depletion and found that expressing the Arabidopsis N-terminal EIN2 domain restored yeast growth in Ca2+-depleted media. Interestingly, EIN2 is the only known NRAMP-like protein with the ability to transport Ca2+. Lastly, we find that ethylene insensitivity is correlated with inhibition of Ca2+ transport by an ein2 missense allele that encodes an amino acid substitution in a highly conserved residue in the NRAMP metal binding pocket. Therefore, we conclude that Ca2+ transport by EIN2 is required for ethylene signaling.
Coauthors: Erwan Michard – University of Maryland, College Park;Jennifer Shemansky – University of Maryland, College Park;José Feijó – University of Maryland, College Park;Caren Chang – University of Maryland, College Park