ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

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 phosphate-sufficient 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.

Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis.

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 ABA-mediated 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.

COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability.

Seasonal changes in day length are perceived by plant photoreceptors and transmitted to the circadian clock to modulate developmental responses such as flowering time. Blue-light-sensing cryptochromes, the E3 ubiquitin-ligase COP1, and clock-associated proteins ELF3 and GI regulate this process, although the regulatory link between them is unclear. Here we present data showing that COP1 acts with ELF3 to mediate day length signaling from CRY2 to GI within the photoperiod flowering pathway. We found that COP1 and ELF3 interact in vivo and show that ELF3 allows COP1 to interact with GI in vivo, leading to GI degradation in planta. Accordingly, mutation of COP1 or ELF3 disturbs the pattern of GI cyclic accumulation. We propose a model in which ELF3 acts as a substrate adaptor, enabling COP1 to modulate light input signal to the circadian clock through targeted destabilization of GI.

Mapping of STS markers obtained from oat resistance gene analog sequences.

Two previously isolated resistance gene analogs (RGAs) of oat have been located as RFLPs in the reference map of Avena byzantina 'Kanota' x Avena sativa 'Ogle' in regions either homologous or homoeologous to loci for resistance to Puccinia coronata, the causal agent of crown rust. In this study, the RGAs were mapped in two recombinant inbred line (RIL) populations that segregate for crown rust resistance: the diploid Avena strigosa x Avena wiestii RIL population (Asw), which has been used for mapping the complex locus PcA, and the hexaploid MN841801-1 x Noble-2 RIL population (MN), in which QTLs have been located. To obtain single-locus markers, RGAs were converted to sequence tagged site (STS) markers using a procedure involving extension of the original RGA sequence lengths by PCR genome walking, amplification and cloning of the parental fragments, and identification of single nucleotide polymorphisms. The procedure successfully obtained STSs from different members of the L7M2 family of sequences, the initial NBS of which have nucleotide similarities of >83%. However, for RGA III2.18, the parental lines were not polymorphic for the STSs assayed. A sequence characterized amplified region (SCAR) marker with features of an RGA had been previously identified for gene Pc94. This marker was also mapped in the above RIL populations. Markers based on RGA L7M2 co-localized with markers defining the QTL Prq1a in linkage group MN3, and were located 15.2 cM from PcA in linkage group AswAC. The SCAR marker for Pc94 was also located in the QTL Prq1a but at 39.5 cM from PcA in AswAC, indicating that the NBS-LRR sequence represented by this marker is not related to PcA. L7M2 was also excluded as a member of the PcA cluster, although it could be an appropriate marker for the Prq1a cluster if chromosome rearrangements are postulated.

Plant hormones and nutrient signaling.

Plants count on a wide variety of metabolic, physiological, and developmental responses to adapt their growth to variations in mineral nutrient availability. To react to such variations plants have evolved complex sensing and signaling mechanisms that allow them to monitor the external and internal concentration of each of these nutrients, both in absolute terms and also relatively to the status of other nutrients. Recent evidence has shown that hormones participate in the control of these regulatory networks. Conversely, mineral nutrient conditions influence hormone biosynthesis, further supporting close interrelation between hormonal stimuli and nutritional homeostasis. In this review, we summarize these evidences and analyze possible transcriptional correlations between hormonal and nutritional responses, as a means to further characterize the role of hormones in the response of plants to limiting nutrients in soil.

LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-box protein involved in light-dependent development and gene expression, undergoes COP1-mediated ubiquitination.

B-box containing proteins play an important role in light signaling in plants. Here, we identify LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), a B-box encoding gene that genetically interacts with two key regulators of light signaling, ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). STH3 physically interacts with HY5 in vivo and shows a COP1-dependent localization to nuclear speckles when coexpressed with COP1 in plant cells. A T-DNA insertion mutant, sth3, is hyposensitive to high fluence blue, red, and far-red light and has elongated hypocotyls under short days. Analyses of double mutants between sth3, sth2, and hy5 suggest that they have partially overlapping functions. Interestingly, functional assays in protoplasts suggest that STH3 can activate transcription both independently and together with STH2 through the G-box promoter element. Furthermore, sth3 suppresses the cop1 hypocotyl phenotype in the dark as well as the anthocyanin accumulation in the light. Finally, COP1 ubiquitinates STH3 in vitro, suggesting that STH3 is regulated by COP1. In conclusion, we have identified STH3 as a positive regulator of photomorphogenesis acting in concert with STH2 and HY5, while also being a target of COP1-mediated ubiquitination.