MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.

Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo-and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JAregulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response. INTRODUCTIONThe plant hormones jasmonates (JAs) are fatty acid-derived oxylipins required for the regulation of multiple physiological aspects of plant growth, development, and defense (Wasternack, 2007;Kazan and Manners, 2008;Browse, 2009;Pauwels et al., 2009). Thus, JAs are widely recognized as regulators of plant responses to environmental stresses such as pathogen and pest attack, wounding, ozone exposure, and water deficit (Devoto et al., 2005;Browse and Howe, 2008). They are also important regulators of growth and developmental programs such as gamete development, the cell cycle, root growth, tendril coiling, and senescence in many plant species (Pauwels et al., 2008;Zhang and Turner, 2008;Reinbothe et al., 2009;Yoshida et al., 2009). JAs are being recognized as important integrators of developmental and stress signals to modulate the allocation of resources to grow or to defend (Moreno et al., 2009;Robson et al., 2010).Transcription is a major regulatory step in the activation of these responses, and JAs trigger an important transcriptional reprogramming of the cells to switch the basal developmental programs into the necessary stress response program (Reymond et al., 2004;Devoto et al., 2005;Mandaokar et al., 2006;Pauwels et al., 2008). The signaling events that lead to transcriptional reprogramming are starting to be elucidated. Upon elicitation by exogenous or endogenous signals, the hormone (+)-7-iso-jasmonoyl-L-isoleucine [also known as (3R,7S)-jasmonoyl-L-isoleucine or JA-Ile] is synthesized by JAR1 (Fonseca et al., 2009b;Suza et al., 2010;Wasternack and Kombrink, 2010). JA-Ile is perceived by a receptor complex formed by the protein COI1 and the JAZ repressors (Xie et al., 1998;Thines et...

Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF) has an important role in the control of phosphate (Pi) starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1) mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS) in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like) control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide) corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress.

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. PHOSPHATE STARVATION RESPONSE 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis, SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling.phosphate sensor | phosphate starvation signaling S ince the beginning of molecular genetics, phosphate (Pi) starvation rescue systems, especially the Pi starvation rescues systems of bacteria and yeast, have served as emblematic models for studies of regulation of gene activity. In plants, these systems have gained additional interest because of the complexity and multicellular nature of plants (1, 2), and especially due to their potential for improving Pi acquisition and use in crops, a major goal toward sustainable agriculture. Considerable information has been gathered in the past decade on the components of the Pi starvation signaling pathway (reviewed in refs. 3-6). Major findings in plants include (i) identification of PHOSPHATE STARVATION RESPONSE 1 (PHR1) and related transcription factors as master regulators of Pi starvation responses (PSRs) (7-11); (ii) demonstration of the involvement of ubiquitin system components, including PHO2 and NLA, in Pi signaling (12-16); (iii) identification of miRNAs as mobile signals in Pi homeostasis (17, 18); and (iv) identification of Pi starvation-induced (PSI) riboregulators of miRNA activity, based on target mimicry (19) and natural antisense RNA that activates translation of PHO1 mRNA (20). In addition, a singular characteristic of nutrient starvation responses in plants is that several of these responses are at long distance, systemically controlled by plant shoot nutrient status, whereas others are controlled by local nutrient concentration. Transcriptomic a...

ORCID ID: 0000-0002-8800-2400 (V.R.)CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABAmediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.

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ORCID IDs: 0000-0003-2780-4888 (C.R.); 0000-0002-8800-2400 (V.R.)Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphatesufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. INTRODUCTIONTrafficking of cargo proteins coming from the plasma membrane (PM) or the Golgi apparatus (GA) to the vacuole occurs through multivesicular bodies (MVBs) (Winter and Hauser, 2006). These organelles, also termed late endosomes or prevacuolar compartments (PVCs), contain internal vesicles that will be delivered, together with their cargoes, into the lumen of vacuoles/lysosomes upon MVB fusion with the tonoplast (Winter and Hauser, 2006). This process plays a central role in controlling the reutilization, storage, or degradation of membrane components and thus regulates fundamental biological processes including membrane turnover, defense against pathogens, development, hormone transport, nutrient uptake, and cell signaling. In the case of membrane-associated regulatory proteins, the MVB route allows modulation of their function by regulating their abundance at the PM and in other vesicular compartments (e.g., endosomes).The sorting of most integral membrane proteins into intraluminal vesicles (ILVs) is dependent on the attachment of ubiquitin to their cytosolic domains, although ubiquitin-independent sorting mechanisms also exist (McNatt et al., 2007). Selective packaging of protein cargoes into ILVs of MVB is mediated by ESCRT (ENDOSOMAL SORTING COMPLEXES REQUIRED FOR TRANSPORT) protein complexes (Conibear, 2002;Winter and Hauser, 2006;Nickerson et al., 2007;Henne et al., 2011). The latter consist of several cytosolic proteins of the VPS-E (class E Vacuolar Protein Sorting) class that are organized into five complexes: ESCRT-0...

Higher plants have several strategies to perpetuate themselves under adequate ecophysiological conditions. The production of heterogeneous seeds is one such strategy. That is, to ensure the survival of the next generation, an individual plant might produce seeds that are heterogeneous with respect to the extent of dormancy, dispersion and persistence within the seed bank. Heterogeneity can affect not only certain physiological and molecular properties related to seed germination, but also such characteristics as colour, size and shape, parameters commonly used to differentiate morphs within a heterogeneous seed population. In heterogeneous seeds, the above features determine seed behaviour and alter their mechanism of germination. In this work, emphasis is placed on the existence of seed mutants having major alterations in characteristics of the testa and hormonal response. These mutants constitute a valuable tool for elucidating the mechanism of dormancy, germination and perpetuation of seeds. Finally, ontogeny and heterogeneity are reviewed, providing the first data related to the possible hormonal control of heterogeneity in seeds. These results raise the hypothesis that one of the factors triggering differences in germination among heterogeneous seeds may be an alteration in the signalling and action mechanism of ethylene and abscisic acid (ABA).