LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates.

The transcription factor LEAFY (LFY) plays crucial roles in flower development by activating floral homeotic genes. Activation of LFY targets requires the combined action of LFY and the E3 ubiquitin ligase UFO, although the precise underlying mechanism remains unclear. Here, we show that LFY accumulates in biomolecular condensates within the cytoplasm, while recombinant LFY forms condensates with similar properties in vitro. UFO interacts with LFY within these condensates and marks it for degradation. LFY levels in the nucleus are buffered against changes in total LFY levels induced by proteasome inhibition, UFO overexpression, or mutation of lysine residues in a disordered region of LFY. Perturbation of cytoplasmic LFY condensates by 1,6-hexanediol treatment induces the relocalization of LFY to the nucleus and the subsequent activation of the LFY target AP3 in flowers. Our data suggest that nucleocytoplasmic partitioning, condensation, and ubiquitin-dependent degradation regulate LFY levels in the nucleus to control its activity.

FIONA1-mediated methylation of the 3'UTR of FLC affects FLC transcript levels and flowering in Arabidopsis.

Adenosine bases of RNA can be transiently modified by the deposition of a methyl-group to form N6-methyladenosine (m6A). This adenosine-methylation is an ancient process and the enzymes involved are evolutionary highly conserved. A genetic screen designed to identify suppressors of late flowering transgenic Arabidopsis plants overexpressing the miP1a microProtein yielded a new allele of the FIONA1 (FIO1) m6A-methyltransferase. To characterize the early flowering phenotype of fio1 mutant plants we employed an integrative approach of mRNA-seq, Nanopore direct RNA-sequencing and meRIP-seq to identify differentially expressed transcripts as well as differentially methylated RNAs. We provide evidence that FIO1 is the elusive methyltransferase responsible for the 3'-end methylation of the FLOWERING LOCUS C (FLC) transcript. Furthermore, our genetic and biochemical data suggest that 3'-methylation stabilizes FLC mRNAs and non-methylated FLC is a target for rapid degradation.

A microProtein repressor complex in the shoot meristem controls the transition to flowering.

MicroProteins are potent post-translational regulators. In Arabidopsis (Arabidopsis thaliana), the miP1a/b microProteins delay floral transition by forming a complex with CONSTANS (CO) and the co-repressor protein TOPLESS. To better understand the function of the miP1a microProtein in floral repression, we performed a genetic suppressor screen to identify suppressors of miP1a (sum) function. One mutant, sum1, exhibited strong suppression of the miP1a-induced late-flowering phenotype. Mapping of sum1 identified another allele of the gene encoding the histone H3K4 demethylase JUMONJI14 (JMJ14), which is required for miP1a function. Plants carrying mutations in JMJ14 exhibit an early flowering phenotype that is largely dependent on CO activity, supporting an additional role for CO in the repressive complex. We further investigated whether miP1a function involves chromatin modification, performed whole-genome methylome sequencing studies with plants ectopically expressing miP1a, and identified differentially methylated regions (DMRs). Among these DMRs is the promoter of FLOWERING LOCUS T (FT), the prime target of miP1a that is ectopically methylated in a JMJ14-dependent manner. Moreover, when aberrantly expressed at the shoot apex, CO induces early flowering, but only when JMJ14 is mutated. Detailed analysis of the genetic interaction among CO, JMJ14, miP1a/b, and TPL revealed a potential role for CO as a repressor of flowering in the shoot apical meristem (SAM). Altogether, our results suggest that a repressor complex operates in the SAM, likely to maintain it in an undifferentiated state until leaf-derived florigen signals induce SAM conversion into a floral meristem.

Advanced Cataloging of Lysine-63 Polyubiquitin Networks by Genomic, Interactome, and Sensor-Based Proteomic Analyses.

The lack of resolution when studying the many different ubiquitin chain types found in eukaryotic cells has been a major hurdle to our understanding of their specific roles. We currently have very little insight into the cellular and physiological functions of Lys-63 (K63)-linked ubiquitin chains, although they are the second most abundant forms of ubiquitin in plant cells. To overcome this problem, we developed several large-scale approaches to characterize (1) the E2-E3 ubiquitination machinery driving K63-linked ubiquitin chain formation and (2) K63 polyubiquitination targets to provide a comprehensive picture of K63 polyubiquitin networks in Arabidopsis (Arabidopsis thaliana). Our work identified the ubiquitin-conjugating enzymes (E2s) UBC35/36 as the major drivers of K63 polyubiquitin chain formation and highlights the major role of these proteins in plant growth and development. Interactome approaches allowed us to identify many proteins that interact with the K63 polyubiquitination-dedicated E2s UBC35/36 and their cognate E2 variants, including more than a dozen E3 ligases and their putative targets. In parallel, we improved the in vivo detection of proteins decorated with K63-linked ubiquitin chains by sensor-based proteomics, yielding important insights into the roles of K63 polyubiquitination in plant cells. This work strongly increases our understanding of K63 polyubiquitination networks and functions in plants.

Light Triggers the miRNA-Biogenetic Inconsistency for De-etiolated Seedling Survivability in Arabidopsis thaliana.

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1, SERRATE, and HYPONASTIC LEAVES 1, whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels. However, the levels of most miRNAs do not notably increase in response to light. To reconcile this inconsistency, we demonstrated that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, these data suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.

The B-Box-Containing MicroProtein miP1a/BBX31 Regulates Photomorphogenesis and UV-B Protection.

The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) represents a major hub in the light-signaling cascade both under visible and UV-B light. The mode of transcriptional regulation of HY5, especially under UV-B light, is not well characterized. B-BOX (BBX) transcription factors regulate HY5 transcription and also posttranscriptionally modulate HY5 to control photomorphogenesis under white light. Here, we identify BBX31 as a key signaling intermediate in visible and UV-B light signal transduction in Arabidopsis (Arabidopsis thaliana). BBX31 expression is induced by UV-B radiation in a fluence-dependent manner. HY5 directly binds to the promoter of BBX31 and regulates its transcript levels. Loss- and gain-of-function mutants of BBX31 indicate that it acts as a negative regulator of photomorphogenesis under white light but is a positive regulator of UV-B signaling. Genetic interaction studies suggest that BBX31 regulates photomorphogenesis independent of HY5 We found no evidence for a direct BBX31-HY5 interaction, and they primarily regulate different sets of genes in white light. Under high doses of UV-B radiation, BBX31 promotes the accumulation of UV-protective flavonoids and phenolic compounds. It enhances tolerance to UV-B radiation by regulating genes involved in photoprotection and DNA repair in a HY5-dependent manner. Under UV-B radiation, overexpression of BBX31 enhances HY5 transcriptional levels in a UV RESISTANCE LOCUS8-dependent manner, suggesting that BBX31 might regulate HY5 transcription.

Approaches to identify and characterize microProteins and their potential uses in biotechnology.

MicroProteins are small proteins that contain a single protein domain and are related to larger, often multi-domain proteins. At the molecular level, microProteins act by interfering with the formation of higher order protein complexes. In the past years, several microProteins have been identified in plants and animals that strongly influence biological processes. Due to their ability to act as dominant regulators in a targeted manner, microProteins have a high potential for biotechnological use. In this review, we present different ways in which microProteins are generated and we elaborate on techniques used to identify and characterize them. Finally, we give an outlook on possible applications in biotechnology.

Synthetic MicroProteins: Versatile Tools for Posttranslational Regulation of Target Proteins.

MicroProteins are small, single-domain proteins that regulate multidomain proteins by sequestering them into novel, often nonproductive, complexes. Several microProteins have been identified in plants and animals, most of which negatively regulate transcription factors. MicroProtein candidates that potentially target a wide range of different protein classes were recently identified in a computational approach. Here, we classified all Arabidopsis (Arabidopsis thaliana) microProteins and developed a synthetic microProtein approach to target specific protein classes, such as hydrolases, receptors, and lyases, in a proof-of-concept approach. Our findings reveal that microProteins can be used to influence different physiological processes, which makes them useful tools for posttranslational regulation in plants and potentially also in animals.

MicroProtein-Mediated Recruitment of CONSTANS into a TOPLESS Trimeric Complex Represses Flowering in Arabidopsis.

MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition.