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Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which - epsilon - is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development.

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., (© 2016 American Society of Plant Biologists. All rights reserved.)

In roots of Arabidopsis (Arabidopsis thaliana), l-lactate is generated by the reduction of pyruvate via l-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative l-lactate-metabolizing enzymes based on their homology to CYB2, the l-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses l-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than l-lactate. The key factor making GOX3 more efficient with l-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize l-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-of-function and overexpressor plants indicate that l-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on l-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes l-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of l-lactate after its formation under normoxia., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Hydrogen peroxide (H2O2) operates as a signaling molecule in eukaryotes, but the specificity of its signaling capacities remains largely unrevealed. Here, we analyzed whether a moderate production of H2O2 from two different plant cellular compartments has divergent effects on the plant transcriptome. Arabidopsis thaliana overexpressing glycolate oxidase in the chloroplast (Fahnenstich et al., 2008; Balazadeh et al., 2012) and plants deficient in peroxisomal catalase (Queval et al., 2007; Inzé et al., 2012) were grown under non-photorespiratory conditions and then transferred to photorespiratory conditions to foster the production of H2O2 in both organelles. We show that H2O2 originating in a specific organelle induces two types of responses: one that integrates signals independently from the subcellular site of H2O2 production and another that is dependent on the H2O2 production site. H2O2 produced in peroxisomes induces transcripts involved in protein repair responses, while H2O2 produced in chloroplasts induces early signaling responses, including transcription factors and biosynthetic genes involved in production of secondary signaling messengers. There is a significant bias towards the induction of genes involved in responses to wounding and pathogen attack by chloroplastic-produced H2O2, including indolic glucosinolates-, camalexin-, and stigmasterol-biosynthetic genes. These transcriptional responses were accompanied by the accumulation of 4-methoxy-indol-3-ylmethyl glucosinolate and stigmasterol., (© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)

Glycolate oxidase (GO) catalyses the oxidation of glycolate to glyoxylate, thereby consuming O(2) and producing H(2)O(2). In this work, Arabidopsis thaliana plants expressing GO in the chloroplasts (GO plants) were used to assess the expressional behavior of reactive oxygen species (ROS)-responsive genes and transcription factors (TFs) after metabolic induction of H(2)O(2) formation in chloroplasts. In this organelle, GO uses the glycolate derived from the oxygenase activity of RubisCO. Here, to identify genes responding to an abrupt production of H(2)O(2) in chloroplasts we used quantitative real-time PCR (qRT-PCR) to test the expression of 187 ROS-responsive genes and 1880 TFs after transferring GO and wild-type (WT) plants grown at high CO(2) levels to ambient CO(2) concentration. Our data revealed coordinated expression changes of genes of specific functional networks 0.5 h after metabolic induction of H(2)O(2) production in GO plants, including the induction of indole glucosinolate and camalexin biosynthesis genes. Comparative analysis using available microarray data suggests that signals for the induction of these genes through H(2)O(2) may originate in the chloroplast. The TF profiling indicated an up-regulation in GO plants of a group of genes involved in the regulation of proanthocyanidin and anthocyanin biosynthesis. Moreover, the upregulation of expression of TF and TF-interacting proteins affecting development (e.g., cell division, stem branching, flowering time, flower development) would impact growth and reproductive capacity, resulting in altered development under conditions that promote the formation of H(2)O(2).

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.

The plant plasma membrane H(+)-ATPase is kept at a low activity level by its C-terminal domain, the inhibitory function of which is thought to be mediated by two regions (region I and II) interacting with cytoplasmic domains essential for the catalytic cycle. The activity of the enzyme is well known to be regulated by 14-3-3 proteins, the association of which requires phosphorylation of the penultimate H(+)-ATPase residue, but can be abolished by phosphorylation of residues close-by. The current knowledge about H(+)-ATPase regulation is briefly summed up here, combined with data that query some of the above statements. Expression of various C-terminal deletion constructs of PMA2, a H(+)-ATPase isoform from Nicotiana plumbaginifolia, in yeast indicates that three regions, which do not correspond to regions I or II, contribute to autoinhibition. Their individual and combined action can be abolished by (mimicking) phosphorylation of three threonine residues located within or close to these regions. With respect to the wild-type PMA2, mimicking phosphorylation of two of these residues increases enzyme activity. However, constitutive activation of wild-type PMA2 requires 14-3-3 association. Altogether, the data suggest that regulation of the plant H(+)-ATPase occurs in progressive steps, mediated by several protein kinases and phosphatases, thus allowing gradual as well as fine-tuned adjustment of its activity. Moreover, mating-based split ubiquitin assays indicate a complex interplay between the C-terminal domain and the rest of the enzyme. Notably, their tight contact does not seem to be the cause of the inactive state of the enzyme., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Members of the eukaryotic 14-3-3 family are highly conserved proteins that have been implicated in the modulation of distinct biological processes by phosphorylation-dependent protein-protein interactions. In plants, 14-3-3 mediated regulation of house-keeping proteins such as nitrate reductase and the plasma membrane localized H(+)-ATPase has been intensely studied. Recent proteome-wide approaches have indicated that the plant 14-3-3 interactome is comparable in size and functional complexity to its animal counterpart and, furthermore, shifted the focus of attention to signal mediators. In this regard, in vivo analyses of certain signaling proteins, such as BRASSINAZOLE-RESISTANT 1, a transcription factor controlling brassinosteroid responsive gene expression, verified an essential role for 14-3-3s in hormonal signal transduction processes.

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 plant plasma membrane H(+)-ATPase contains a C-terminal autoinhibitory domain whose displacement from the catalytic site is caused by binding of regulatory 14-3-3 proteins. Members of the highly conserved 14-3-3 family bind their individual target proteins in a sequence-specific and phosphorylation-dependent manner within a central groove, the latter characterized by the presence of highly invariant residues. However, an atypical binding site for 14-3-3s within the H(+)-ATPase has been identified that does not resemble any other 14-3-3 binding motif. Combination of site-directed mutagenesis with glutathione S-transferase pull-down assays points to the importance of the central 14-3-3 groove for the interaction with the apparently unique site of the H(+)-ATPase. Furthermore, a 14-3-3 dimer is essential for binding such unusual motif.