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.

The Rise of the CRISPR/Cpf1 System for Efficient Genome Editing in Plants.

Cpf1, an endonuclease of the class 2 CRISPR family, fills the gaps that were previously faced in the world of genome engineering tools, which include the TALEN, ZFN, and CRISPR/Cas9. Other simultaneously discovered nucleases were not able to carry out re-engineering at the same region due to the loss of a target site after first-time engineering. Cpf1 acts as a dual nuclease, functioning as an endoribonuclease to process crRNA and endodeoxyribonuclease to cleave target sequences and generate double-stranded breaks. Additionally, Cpf1 allows for multiplexed genome editing, as a single crRNA array transcript can target multiple loci in the genome. The CRISPR/Cpf1 system enables gene deletion, insertion, base editing, and locus tagging in monocot as well as in dicot plants with fewer off-target effects. This tool has been efficiently demonstrated into tobacco, rice, soybean, wheat, etc. This review covers the development and applications of Cpf1 mediated genome editing technology in plants.

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.