The Plant Cell, Vol. 26: 4566, December 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.

IN BRIEF

Membrane Bound: C2-Domain Abscisic Acid-Related Proteins Help Abscisic Acid Receptors Get Where They Need to Go We’ve learned so much about abscisic acid (ABA) since its discovery in 1963. This phytohormone, whose levels increase under osmotic stress conditions and fluctuate during seed development, plays a vital role in regulating plant stress responses, transpiration, seed germination, and embryogenesis. In order to function, ABA must bind to soluble PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL) ABA receptors. Following perception of ABA, these receptors form ternary complexes with clade A PP2C phosphatases (such as ABA INSENSITIVE1 and HYPERSENSITIVE TO ABA1), thereby inactivating them (Park et al., 2009) and unleashing a cascade of ABA responses. These ABA responses involve ion transporters and membrane-associated enzymes, which generate second messengers that function in ABA signaling. How do the cytosolic PYR/ PYL ABA receptors get to these membraneassociated enzymes? Diffusion alone is unlikely to accomplish this task. In general, cytosolic proteins can reside on the periphery of vesicles, and they can transiently interact with membranes for trafficking or signaling purposes; these associations are driven by protein modules that recognize specific features of proteins or membranes (Cho and Stahelin, 2005). The identities of the proteins that help PYR/PYL receptors arrive at their destination in the membrane have only recently been revealed. In their search for interacting partners of PYR/PYL receptors in Arabidopsis thaliana, Rodriguez et al. (2014) discovered a family of proteins that interact with PYR/PYLs and positively regulate ABA sensitivity. The authors began their study by performing yeast two-hybrid screening in the absence of ABA using the highly expressed PYL4 as bait. This analysis led to the discovery of the 10-member family of C2-domain ABA-related (CAR) proteins. CAR proteins interact with several PYR/PYLs and are also found in crops such as tomato (Solanum lycopersicum) and rice (Oryza sativa). The presence of

www.plantcell.org/cgi/doi/10.1105/tpc.114.134411

a C2-domain in these proteins is intriguing, as these calcium binding motifs are involved in targeting proteins to cell membranes. Indeed, high local calcium levels are found at the cell membrane in response to various stimuli (ABA, abiotic stress, and pathogen attack), suggesting that the C2-domain in CAR proteins enables them to translocate to cell membranes in response to calcium oscillations. Bimolecular fluorescence complementation assays, transient expression analysis in tobacco (Nicotiana benthamiana) leaves, and coimmunoprecipitation experiments revealed that CAR1 and PYL4 interact in plant cells. The authors transiently expressed various combinations of CAR proteins fused to green fluorescent protein in tobacco cells, which they coexpressed with a plasma membrane marker. This analysis, as well as biochemical experiments, revealed that CAR proteins localize to the plasma membrane, while further analysis revealed that membrane recruitment of PYL4 by CAR1 affects only a fraction of the total receptor pool. Crystallographic analysis demonstrated that, in addition to the C2-domain, CAR4 contains a specific CAR signature that is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. The authors then used gain-of-function and loss-of function approaches to investigate whether CAR genes affect ABA signaling. While CAR1 overexpressors were hypersensitive to the inhibition of seedling establishment and shoot growth by ABA, triple car mutant seedlings were less sensitive to ABA-mediated inhibition of seedling establishment and root growth than the wild type (see figure). Moreover, car5 car9 pyr1 pyl4 pyl8 mutants exhibited reduced sensitivity to ABA-mediated inhibition of primary root growth and salt-induced inhibition of lateral root growth, indicating that CAR and PYR/ PYL proteins additively regulate root sensitivity to ABA, as both are needed for a proper ABA response. After all, PYR/PYL

Wild-type and triple car mutant seedlings 10 d after transfer from Murashige and Skoog medium to Murashige and Skoog medium supplemented with 10 mM ABA. (Reprinted from Rodriguez et al. [2014], Figure 5D.)

ABA receptors can’t get where they need to go without CAR proteins.

Jennifer Lockhart Science Editor [email protected] ORCID ID: 0000-0002-1394-8947

REFERENCES Cho, W., and Stahelin, R.V. (2005). Membraneprotein interactions in cell signaling and membrane trafficking. Annu. Rev. Biophys. Biomol. Struct. 34: 119–151. Park, S.Y., et al. (2009). Abscisic acid inhibits type 2C protein phosphatases via the PYR/ PYL family of START proteins. Science 324: 1068–1071. Rodriguez, L., et al. (2014). C2-domain abscisic acid-related proteins mediate the interaction of PYR/PYL/RCAR abscisic acid receptors with the plasma membrane and regulate abscisic acid sensitivity in Arabidopsis. Plant Cell 26: 4802–4820.

Membrane Bound: C2-Domain Abscisic Acid-Related Proteins Help Abscisic Acid Receptors Get Where They Need to Go Jennifer Lockhart Plant Cell 2014;26;4566; originally published online December 2, 2014; DOI 10.1105/tpc.114.134411 This information is current as of May 31, 2015 References

This article cites 3 articles, 2 of which can be accessed free at: http://www.plantcell.org/content/26/12/4566.full.html#ref-list-1

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Membrane bound: C2-domain abscisic acid-related proteins help abscisic acid receptors get where they need to go.

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