The CUL3A-LFH1-UBC15 ubiquitin ligase complex mediates SHORT VEGETATIVE PHASE degradation to accelerate flowering at high ambient temperature.

Ambient temperature affects flowering time in plants, and the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a crucial role in the response to changes in ambient temperature. SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature, but the details of SVP degradation are unclear. Here, we show that SVP degradation at high ambient temperature is mediated by the CULLIN3-RING E3 ubiquitin ligase (CRL3) complex in Arabidopsis thaliana. We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain. We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1 (LFH1). Single mutants of LFH1 or CULLIN3A (CUL3A) showed late flowering specifically at 27°C. LFH1 protein levels increased at high ambient temperature. We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP-CUL3A complex formation. Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature, suggesting that the CUL3-LFH1 complex functions in SVP degradation. Screening E2 ubiquitin-conjugating enzymes (UBCs) using RING-BOX PROTEIN 1 (RBX1), a component of the CRL3 complex, as bait identified UBC15. ubc15 mutants also showed late flowering at high ambient temperature. In vitro and in vivo ubiquitination assays using recombinant CUL3A, LFH1, RBX1, and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner. Collectively, these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A, LFH1, and UBC15.

Chloroplasts prevent precocious flowering through a GOLDEN2-LIKE-B-BOX DOMAIN PROTEIN module.

The timing of flowering is tightly controlled by signals that integrate environmental and endogenous cues. Sugars produced by carbon fixation in the chloroplast are a crucial endogenous cue for floral initiation. Chloroplasts also convey information directly to the nucleus through retrograde signaling to control plant growth and development. Here, we show that mutants defective in chlorophyll biosynthesis and chloroplast development flowered early, especially under long-day conditions, although low sugar accumulation was seen in some mutants. Plants treated with the bleaching herbicide norflurazon also flowered early, suggesting that chloroplasts have a role in floral repression. Among retrograde signaling mutants, the golden2-like 1 (glk1) glk2 double mutants showed early flowering under long-day conditions. This early flowering was completely suppressed by constans (co) and flowering locus t (ft) mutations. Leaf vascular-specific knockdown of both GLK1 and GLK2 phenocopied the glk1 glk2 mutants. GLK1 and GLK2 repress flowering by directly activating the expression of B-BOX DOMAIN PROTEIN 14 (BBX14), BBX15, and BBX16 via CCAATC cis-elements in the BBX genes. BBX14/15/16 physically interact with CO in the nucleus, and expression of BBXs hampered CO-mediated FT transcription. Simultaneous knockdown of BBX14/15/16 by artificial miRNA (35S::amiR-BBX14/15/16) caused early flowering with increased FT transcript levels, whereas BBX overexpression caused late flowering. Flowering of glk1/2 and 35S::amiR-BBX14/15/16 plants was insensitive to norflurazon treatment. Taking these observations together, we propose that the GLK1/2-BBX14/15/16 module provides a novel mechanism explaining how the chloroplast represses flowering to balance plant growth and reproductive development.

Corrigendum: Polymerase II-associated factor 1 complex-regulated FLOWERING LOCUS C-Clade genes repress flowering in response to chilling.

[This corrects the article DOI: 10.3389/fpls.2022.817356.].

FLOWERING LOCUS M isoforms differentially affect the subcellular localization and stability of SHORT VEGETATIVE PHASE to regulate temperature-responsive flowering in Arabidopsis.

Temperature is an important environmental cue that affects flowering time in plants. The MADS-box transcription factor FLOWERING LOCUS M (FLM) forms a heterodimeric complex with SHORT VEGETATIVE PHASE (SVP) and controls ambient temperature-responsive flowering in Arabidopsis. FLM-ß and FLM-δ, two major splice variants produced from the FLM locus, exert opposite effects on flowering, but the molecular mechanism by which the interaction between FLM isoforms and SVP affects temperature-responsive flowering remains poorly understood. Here, we show that FLM-ß and FLM-δ play important roles in modulating the temperature-dependent behavior, conformation, and stability of SVP. Nuclear localization of SVP decreases as temperature increases. FLM-ß is required for SVP nuclear translocation at low temperature, whereas SVP interacts with FLM-δ mainly in the cytoplasm at high temperature. SVP preferentially binds to FLM-ß at low temperature in tobacco leaf cells. SVP shows high binding affinity to FLM-ß at low temperature and to FLM-δ at high temperature. SVP undergoes similar structural changes in the interactions with FLM-ß and FLM-δ; however, FLM-δ likely causes more pronounced conformational changes in the SVP structure. FLM-δ causes rapid degradation of SVP at high temperature, compared with FLM-ß, possibly via ubiquitination. Mutation of lysine 53 or lysine 165 in SVP causes increased abundance of SVP due to reduced ubiquitination of SVP and thus delays flowering at high temperature. Our findings suggest that temperature-dependent differential interactions between SVP and FLM isoforms modulate the temperature-responsive induction of flowering in Arabidopsis.

In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions.

Protein-lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical interaction between a protein of interest and a panel of phospholipids provide crucial information for predicting the functionality of these interactions in vivo. In this protocol, which we developed in the context of evaluating protein-lipid binding of the Arabidopsis thaliana florigen FLOWERING LOCUS T, we describe four independent in vitro experiments to determine the interaction of a protein with phospholipids: lipid-protein overlay assays, liposome binding assays, biotin-phospholipid pull-down assays, and fluorescence polarization assays. These complementary assays allow the researcher to test whether the protein of interest interacts with lipids in the test panel, identify the relevant lipids, and assess the strength of the interaction.

Polymerase II-Associated Factor 1 Complex-Regulated FLOWERING LOCUS C-Clade Genes Repress Flowering in Response to Chilling.

RNA polymerase II-associated factor 1 complex (PAF1C) regulates the transition from the vegetative to the reproductive phase primarily by modulating the expression of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M [FLM, also known as MADS AFFECTING FLOWERING1 (MAF1)] at standard growth temperatures. However, the role of PAF1C in the regulation of flowering time at chilling temperatures (i.e., cold temperatures that are above freezing) and whether PAF1C affects other FLC-clade genes (MAF2-MAF5) remains unknown. Here, we showed that Arabidopsis thaliana mutants of any of the six known genes that encode components of PAF1C [CELL DIVISION CYCLE73/PLANT HOMOLOGOUS TO PARAFIBROMIN, VERNALIZATION INDEPENDENCE2 (VIP2)/EARLY FLOWERING7 (ELF7), VIP3, VIP4, VIP5, and VIP6/ELF8] showed temperature-insensitive early flowering across a broad temperature range (10°C-27°C). Flowering of PAF1C-deficient mutants at 10°C was even earlier than that in flc, flm, and flc flm mutants, suggesting that PAF1C regulates additional factors. Indeed, RNA sequencing (RNA-Seq) of PAF1C-deficient mutants revealed downregulation of MAF2-MAF5 in addition to FLC and FLM at both 10 and 23°C. Consistent with the reduced expression of FLC and the FLC-clade members FLM/MAF1 and MAF2-MAF5, chromatin immunoprecipitation (ChIP)-quantitative PCR assays showed reduced levels of the permissive epigenetic modification H3K4me3/H3K36me3 and increased levels of the repressive modification H3K27me3 at their chromatin. Knocking down MAF2-MAF5 using artificial microRNAs (amiRNAs) in the flc flm background (35S::amiR-MAF2-5 flc flm) resulted in significantly earlier flowering than flc flm mutants and even earlier than short vegetative phase (svp) mutants at 10°C. Wild-type seedlings showed higher accumulation of FLC and FLC-clade gene transcripts at 10°C compared to 23°C. Our yeast two-hybrid assays and in vivo co-immunoprecipitation (Co-IP) analyses revealed that MAF2-MAF5 directly interact with the prominent floral repressor SVP. Late flowering caused by SVP overexpression was almost completely suppressed by the elf7 and vip4 mutations, suggesting that SVP-mediated floral repression required a functional PAF1C. Taken together, our results showed that PAF1C regulates the transcription of FLC and FLC-clade genes to modulate temperature-responsive flowering at a broad range of temperatures and that the interaction between SVP and these FLC-clade proteins is important for floral repression.

Florigen sequestration in cellular membranes modulates temperature-responsive flowering.

Plants respond to temperature changes by modulating florigen activity to optimize the timing of flowering. We show that the Arabidopsis thaliana mobile florigen FLOWERING LOCUS T (FT) interacts with the negatively charged phospholipid phosphatidylglycerol (PG) at cellular membranes and binds the lipid bilayer. Perturbing PG biosynthesis in phloem companion cells leads to temperature-insensitive early flowering. Low temperatures facilitate FT sequestration in the cellular membrane of the companion cell, thus reducing soluble FT levels and delaying flowering. A mutant in PHOSPHATIDYLGLYCEROLPHOSPHATE SYNTHASE 1 accumulates more soluble FT at lower temperatures and exhibits reduced temperature sensitivity. Thus, cellular membranes sequester FT through their ability to bind the phospholipid PG, and this sequestration modulates the plant's response to temperature changes.

Nonsense-mediated mRNA decay modulates Arabidopsis flowering time via the SET DOMAIN GROUP 40-FLOWERING LOCUS C module.

The nonsense-mediated mRNA decay (NMD) surveillance system clears aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. Although loss of the core NMD proteins UP-FRAMESHIFT1 (UPF1) and UPF3 leads to late flowering in Arabidopsis, the underlying mechanism remains elusive. Here, we showed that mutations in UPF1 and UPF3 cause temperature- and photoperiod-independent late flowering. Expression analyses revealed high FLOWERING LOCUS C (FLC) mRNA levels in upf mutants; in agreement with this, the flc mutation strongly suppressed the late flowering of upf mutants. Vernalization accelerated flowering of upf mutants in a temperature-independent manner. FLC transcript levels rose in wild-type plants upon NMD inhibition. In upf mutants, we observed increased enrichment of H3K4me3 and reduced enrichment of H3K27me3 in FLC chromatin. Transcriptome analyses showed that SET DOMAIN GROUP 40 (SDG40) mRNA levels increased in upf mutants, and the SDG40 transcript underwent NMD-coupled alternative splicing, suggesting that SDG40 affects flowering time in upf mutants. Furthermore, NMD directly regulated SDG40 transcript stability. The sdg40 mutants showed decreased H3K4me3 and increased H3K27me3 levels in FLC chromatin, flowered early, and rescued the late flowering of upf mutants. Taken together, these results suggest that NMD epigenetically regulates FLC through SDG40 to modulate flowering time in Arabidopsis.

Profiling Protein-DNA Interactions by Chromatin Immunoprecipitation in Arabidopsis.

In plant cells, transcription factors play an important role in the regulation of gene expression, which eventually leads to the formation of complex phenotypes. Although chromatin immunoprecipitation (ChIP) involves a lengthy process that requires up to 4 days to complete, it is a powerful technique to investigate the interactions between transcription factors and their target sequences in vivo. Here, we describe a detailed ChIP protocol, focusing on ChIP-qPCR, from material collection to data analyses. Moreover, we explain multiple checkpoints for the quality control of ChIP-qPCR data to ensure the success of this protocol. As this protocol is robust, it can be adapted to other plant materials and plant species, and it can be used for genome-wide profiling experiments, including ChIP-chip and ChIP-seq analyses. We believe that our ChIP-qPCR protocol facilitates research on the interactions between plant transcription factors and their target sequences in vivo.

Regulation of flowering time by ambient temperature: repressing the repressors and activating the activators.

Plants display remarkable developmental flexibility as they continuously sense and respond to changes in their environment. This flexibility allows them to select the optimal timing for critical developmental decisions such as when to flower. Ambient temperature is a major environmental factor that influences flowering; the mechanisms involved in ambient temperature-responsive flowering have attracted particular attention as a consequence of the effects of global climate change on temperature. PHYTOCHROME INTERACTING FACTOR 4 and alternative splicing of FLOWERING LOCUS M affect temperature-responsive flowering; however, the exact temperature-sensing mechanism in plants remains elusive. Further study of these molecular mechanisms will contribute to our understanding of how plants sense ambient temperature and respond via diverse biological signaling cascades.

Evolution and functional diversification of FLOWERING LOCUS T/TERMINAL FLOWER 1 family genes in plants.

Plant growth and development, particularly the induction of flowering, are tightly controlled by key regulators in response to endogenous and environmental cues. The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family of phosphatidylethanolamine-binding protein (PEBP) genes is central to plant development, especially the regulation of flowering time and plant architecture. FT, the long-sought florigen, promotes flowering and TFL1 represses flowering. The balance between FT and TFL1 modulates plant architecture by switching the meristem from indeterminate to determinate growth, or vice versa. Recent studies in a broad range of plant species demonstrated that, in addition to their roles in flowering time and plant architecture, FT/TFL1 family genes participate in diverse aspects of plant development, such as bamboo seed germination and potato tuber formation. In this review, we briefly summarize the evolution of the FT/TFL1 family and highlight recent findings on their conserved and divergent functions in different species.

PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 modulates flowering in a florigen-independent manner by regulating SVP.

PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 (PECT1) regulates phosphatidylethanolamine biosynthesis and controls the phosphatidylethanolamine:phosphatidylcholine ratio in Arabidopsis thaliana Previous studies have suggested that PECT1 regulates flowering time by modulating the interaction between phosphatidylcholine and FLOWERING LOCUS T (FT), a florigen, in the shoot apical meristem (SAM). Here, we show that knockdown of PECT1 by artificial microRNA in the SAM (pFD::amiR-PECT1) accelerated flowering under inductive and even non-inductive conditions, in which FT transcription is almost absent, and in ft-10 twin sister of ft-1 double mutants under both conditions. Transcriptome analyses suggested that PECT1 affects flowering by regulating SHORT VEGETATIVE PHASE (SVP) and GIBBERELLIN 20 OXIDASE 2 (GA20ox2). SVP misexpression in the SAM suppressed the early flowering of pFD::amiR-PECT1 plants. pFD::amiR-PECT1 plants showed increased gibberellin (GA) levels in the SAM, concomitant with the reduction of REPRESSOR OF GA1-3 levels. Consistent with this, GA treatment had little effect on flowering time of pFD::amiR-PECT1 plants and the GA antagonist paclobutrazol strongly affected flowering in these plants. Together, these results suggest that PECT1 also regulates flowering time through a florigen-independent pathway, modulating SVP expression and thus regulating GA production.

Role of AT1G72910, AT1G72940, and ADR1-LIKE 2 in Plant Immunity under Nonsense-Mediated mRNA Decay-Compromised Conditions at Low Temperatures.

Nonsense-mediated mRNA decay (NMD) removes aberrant transcripts to avoid the accumulation of truncated proteins. NMD regulates nucleotide-binding, leucine-rich repeat (NLR) genes to prevent autoimmunity; however, the function of a large number of NLRs still remains poorly understood. Here, we show that three NLR genes (AT1G72910, AT1G72940, and ADR1-LIKE 2) are important for NMD-mediated regulation of defense signaling at lower temperatures. At 16 °C, the NMD-compromised up-frameshift protein1 (upf1) upf3 mutants showed growth arrest that can be rescued by the artificial miRNA-mediated knockdown of the three NLR genes. mRNA levels of these NLRs are induced by Pseudomonas syringae inoculation and exogenous SA treatment. Mutations in AT1G72910, AT1G72940, and ADR1-LIKE 2 genes resulted in increased susceptibility to Pseudomonas syringae, whereas their overexpression resulted in severely stunted growth, which was dependent on basal disease resistance genes. The NMD-deficient upf1 upf3 mutants accumulated higher levels of NMD signature-containing transcripts from these NLR genes at 16 °C. Furthermore, mRNA degradation kinetics showed that these NMD signature-containing transcripts were more stable in upf1 upf3 mutants. Based on these findings, we propose that AT1G72910, AT1G72940, and ADR1-LIKE 2 are directly regulated by NMD in a temperature-dependent manner and play an important role in modulating plant immunity at lower temperatures.

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis.

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft-10 tsf-1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper-vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1-20 mutants. The terminal flower formed in tfl1-20 mutants converted to a hyper-vegetative shoot in ft-10 tsf-1 mutants. Grafting ft-10 tsf-1 or ft-10 tsf-1 tfl1-20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild-type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft-10 tsf-1 mutants the vasculature-specific misexpression of FT converted the hyper-vegetative shoot to a normal inflorescence, and in the ft-10 tsf-1 tfl1-20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.

Psychometric Functions of Vowel Detection and Identification in Long-Term Speech-Shaped Noise.

Purpose The goal of this study was to investigate vowel detection and identification in noise and provide baseline data regarding how vowel perception changed with signal-to-noise ratios. Psychometric functions of vowel detection and identification for 12 American English isolated vowels in long-term speech-shaped noise were examined for young listeners with normal hearing in this study. Method Vowel detection was measured at sensation levels from -10 to +5 dB (re: thresholds of vowel detection from the study of Liu and Eddins, 2008a ) with a 4-interval forced-choice procedure. Thresholds of vowel detection were computed for each listener as the speech level at which 70.7% correct performance was reached. Vowel identification was then examined at sensation levels from 0 to 12 dB relative to detection thresholds for each listener. Thresholds of vowel identification were calculated as the speech level with vowel identifiability ( d') equals to 1. Results Thresholds of vowel detection and identification were significantly affected by vowel category. Slopes of psychometric functions of vowel identification were significantly dependent on vowel category, whereas slopes of psychometric functions of vowel detection were not. Conclusions These results suggest that, given the same sensation levels, especially at low sensation levels, vowel sounds are not equally perceivable in terms of identifiability.

English Consonant Identification in Multi-Talker Babble: Effects of Chinese-Native Listeners' English Experience.

The identification of English consonants in quiet and multi-talker babble was examined for three groups of young adult listeners: Chinese in China, Chinese in the USA (CNU), and English-native listeners. As expected, native listeners outperformed non-native listeners. The two non-native groups had similar performance in quiet, whereas CNU listeners performed significantly better than Chinese in China listeners in babble. It is concluded that CNU listeners may benefit from English experience, for example, better use of temporal variation in noise and better capacity against informational masking, to perceive English consonants better in babble. Possible explanations regarding the differential noise effect on the three groups are discussed.

TWIN SISTER OF FT (TSF) Interacts with FRUCTOKINASE6 and Inhibits Its Kinase Activity in Arabidopsis.

In flowering plants, the developmental switch to the reproductive phase is tightly regulated and involves the integration of internal and external signals. FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) integrate signals from multiple pathways. FT and TSF function as florigenic substances, and share high sequence similarity with mammalian Raf kinase inhibitor protein (RKIP). Despite their strong similarity to RKIP, the kinase inhibitory activity of FT and TSF remains to be investigated. We performed a yeast two-hybrid screen and found that TSF interacted with FRUCTOKINASE6 (FRK6), which phosphorylates fructose for various metabolic pathways. Among the seven Arabidopsis FRKs, FRK6 and FRK7 have high sequence similarity; therefore, we investigated whether TSF interacts with FRK6 and FRK7. In vitro pull-down assays and bimolecular fluorescence complementation assays revealed that TSF interacts with FRK6 in the nucleus, but not with FRK7. Kinase activity assays suggested that TSF inhibits the kinase activity of FRK6, whereas FT does not. By contrast, neither TSF nor FT inhibits the kinase activity of FRK7. The frk6 and frk7 mutants show slightly delayed flowering, but only under short-day (SD) conditions. Plastochron length is also affected in both frk6 and frk7 mutants under SD conditions. FT expression levels decreased in frk6 mutants, but not in frk7 mutants. Taken together, our findings suggest that TSF physically interacts with FRK6 and affects its kinase activity, whereas FT does not, although these proteins share high sequence similarity.

A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts.

BACKGROUND: Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming. RESULTS: Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence. CONCLUSIONS: The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes.

Base-pair opening dynamics of the microRNA precursor pri-miR156a affect temperature-responsive flowering in Arabidopsis.

Internal and environmental cues, including ambient temperature changes, regulate the timing of flowering in plants. Arabidopsis miR156 represses flowering and plays an important role in the regulation of temperature-responsive flowering. However, the molecular basis of miR156 processing at lower temperatures remains largely unknown. Here, we performed nuclear magnetic resonance studies to investigate the base-pair opening dynamics of model RNAs at 16 °C and investigated the in vivo effects of the mutant RNAs on temperature-responsive flowering. The A9C and A10CG mutations in the B5 bulge of the lower stem of pri-miR156a stabilized the C15∙G98 and U16∙A97 base-pairs at the cleavage site of pri-miR156a at 16 °C. Consistent with this, production of mature miR156 was severely affected in plants overexpressing the A9C and A10CG constructs and these plants exhibited almost no delay in flowering at 16 °C. The A10G and A9AC mutations did not strongly affect C15∙G98 and U16∙A97 base-pairs at 16 °C, and plants overexpressing A10G and A9AC mutants of miR156 produced more mature miR156 than plants overexpressing the A9C and A10CG mutants and showed a strong delay in flowering at 16 °C. Interestingly, the A9AC mutation had distinct effects on the opening dynamics of the C15∙G98 and U16∙A97 base-pairs between 16 °C and 23 °C, and plants expressing the A9AC mutant miR156 showed only a moderate delay in flowering at 16 °C. Based on these results, we propose that fine-tuning of the base-pair stability at the cleavage site is essential for efficient processing of pri-miR156a at a low temperature and for reduced flowering sensitivity to ambient temperature changes.