DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele inArabidopsis thaliana. Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed inNicotiana benthamiana.DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OFphyA-1051 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.

No abstract

Cleavage and polyadenylation at the 3ʹ end of the pre-mRNA is essential for mRNA function, by regulating its translatability, stability, and translocation to the cytoplasm. Cleavage factor I (CFI) is a multi-subunit component of the pre-mRNA 3ʹ end processing machinery in eukaryotes. Here we report that plant CFI 25 subunit of CFI plays an important role in maintaining the diversity of the 3ʹ ends of mRNA. The genome of Arabidopsis thaliana (L.) Heynh. contained four genes encoding three putative CFI subunits (AtCFI 25, AtCFI 59, and AtCFI 68), orthologous to the mammalian CFI subunits. There were two CFI 25 paralogs (AtCFI 25a and AtCFI 25b) that shared homology with the human CFI 25. Two null alleles of AtCFI 25a displayed, smaller rosette leaves, longer stigmatic papilla, smaller anther, earlier flowering and lower fertility, compared to wild-type plants. Null alleles of AtCFI 25b, as well as, plants ectopically expressing full-length cDNA of AtCFI 25a, displayed no obvious morphological defects. AtCFI 25a was shown to interact with AtCFI 25b, AtCFI 68, and itself, suggesting various forms of CFI in plants. Furthermore we show that AtCFI 25a function was essential for maintaining proper diversity of the 3ʹ end lengths of transcripts coding for CFI subunits, suggesting a self-regulation of the CFI machinery in plants. AtCFI 25a was also important to maintain 3ʹ ends for other genes, to different extent. Collectively, AtCFI 25a, but not AtCFI 25b, seemed to play important roles during Arabidopsis development by maintaining proper diversity of the 3ʹ UTR lengths.

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1’s function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.

DELLA transcriptional regulators are central components in the control of plant body form in response to the environment. This is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis.Furthermore, the degradation of DELLA induced by the warmth anticipated changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when co-expressed in N. benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro.We propose that DELLAs are destabilized not only by the canonical GAdependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature. 4 mutation ( Fig. 2A). The degradation of the rga-∆17 also required COP1 (Fig. 1C). Co-expression of COP1 caused 26S proteasome-dependent decrease of HA-RGA and also of HA-(rga-D17) in leaves of long-day grown Nicotiana benthamiana plants, while it had no impact on levels of the unrelated protein HA-GFP ( Fig. 2 B and C). Warm temperature decreased HA-(rga-D17) in a COP1mediated manner (SI Appendix, Fig. S2). This suggests that COP1 mediates the destabilization of RGA by non-canonical mechanisms. To explore this possibility, we first investigated whether COP1 physically interacts with DELLA proteins. COP1 interacts physically with GAI and RGA in yeast.We performed yeast two-hybrid (Y2H) assays between COP1 and the two DELLAs with a major role in light-and temperature-dependent growth, RGA and GIBBERELLIC ACID INSENSITIVE (GAI) (10, 11,30). To avoid the reported strong auto-activation of full-length DELLAs in yeast, we used N-terminal deleted versions named M5GAI and RGA52 (12, 31). COP1 was able to interact with both ( Fig. 3A). SUPRESSOR OF phyA-105 1 (SPA1) and other SPA proteins involved in a functional complex with COP1 (20, 32) were also able to interact with GAI and RGA in Y2H assays (Fig. 3B). COP1 interacts with GAI and RGA in planta.To investigate whether the interaction between DELLAs and COP1 also occurs in plant cells, we first performed co-immunoprecipitation assays in leaves of N. benthamiana coexpressing DsRED-COP1-HA and YFP-M5GAI or YFP-RGA52. While DsRED-COP1-HA was pulled down by anti-GFP antibodies from leaf extracts coexpressing YFP-M5GAI and the interaction appeared to be enhanced in the

No abstract

Tandem affinity purification (TAP) coupled to mass spectrometry has become a powerful approach to identify protein-protein interactions from different biological systems, including plants, in a proteome-wide manner. By using two sequential affinity purification steps, TAP allows for isolation of high-purity TAP-tagged proteins of interest and their associated proteins. Here we describe optimized procedures to use the GS TAP technology for protein complex isolation from Arabidopsis cell suspension cultures.