Regulatory 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray crystal structure of 14-3-3 in complex with the entire binding motif, revealing a previously unidentified mode of interaction. A 14-3-3 dimer simultaneously binds two H(+)-ATPase peptides, each of which forms a loop within the typical 14-3-3 binding groove and therefore exits from the center of the dimer. Several H(+)-ATPase mutants support this structure determination. Accordingly, 14-3-3 binding could result in H(+)-ATPase oligomerization. Indeed, by using single-particle electron cryomicroscopy, the 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrates a hexameric arrangement. Fitting of 14-3-3 and H(+)-ATPase atomic structures into the 3D reconstruction map suggests the spatial arrangement of the holocomplex.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

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14-3-3 Dimers are well known to interact with diverse target proteins throughout eukaryotes. Most notably, association of 14-3-3s commonly requires phosphorylation of a serine or threonine residue within a specific sequence motif of the client protein. Studies with a focus on individual target proteins have unequivocally demonstrated 14-3-3s to be the crucial factors modifying the client’s activity state upon phosphorylation and, thus, finishing the job initiated by a kinase. In this respect, a recent in-depth analysis of the rice transcription factor FLOWERING LOCUS D1 (OsFD1) revealed 14-3-3s to be essential players in floral induction. Such fascinating discoveries, however, can often be ascribed to the random identification of 14-3-3 as an interaction partner of the favorite protein. In contrast, our understanding of 14-3-3 function in higher organisms is frustratingly limited, mainly due to an overwhelming spectrum of putative targets in combination with the existence of a multigene 14-3-3 family. In this review we will discuss our current understanding of the function of plant 14-3-3 proteins, taking into account recent surveys of the Arabidopsis 14-3-3 interactome.

SummaryOur research shows that there is isoform specificity and redundancy among 6 out of 13 14-3-3 members in root growth under control and abiotic stress conditions.