In temperate and boreal regions, perennials adapt their annual growth cycle to the change of seasons. These adaptations ensure survival in harsh environmental conditions, allowing growth at different latitudes and altitudes, and are therefore tightly regulated. Populus tree species cease growth and form terminal buds in autumn when photoperiod falls below a certain threshold.1 This is followed by establishment of dormancy and cold hardiness over the winter. At the center of the photoperiodic pathway in Populus is the gene FLOWERING LOCUS T2 (FT2), which is expressed during summer and harbors significant SNPs in its locus associated with timing of bud set.1–4 The paralogous gene FT1, on the other hand, is hyper-induced in chilling buds during winter.3,5 Even though its function is so far unknown, it has been suggested to be involved in the regulation of flowering and the release of winter dormancy.3,5 In this study, we employ CRISPR-Cas9-mediated gene editing to individually study the function of the FT-like genes in Populus trees. We show that while FT2 is required for vegetative growth during spring and summer and regulates the entry into dormancy, expression of FT1 is absolutely required for bud flush in spring. Gene expression profiling suggests that this function of FT1 is linked to the release of winter dormancy rather than to the regulation of bud flush per se. These data show how FT duplication and sub-functionalization have allowed Populus trees to regulate two completely different and major developmental control points during the yearly growth cycle.
Many functionally distinct plant tissues have relatively low numbers of cells that are embedded within complex tissues. For example, the shoot apical meristem (SAM) consists of a small population of pluripotent stem cells surrounded by developing leaves and/or flowers at the growing tip of the plant. It is technically challenging to collect enough high-quality SAM samples for molecular analyses. Isolation of Nuclei Tagged in specific Cell Types (INTACT) is an easily reproducible method that allows the enrichment of biotin-tagged cell-type-specific nuclei from the total nuclei pool using biotin-streptavidin affinity purification. Here, we provide a detailed INTACT protocol for isolating nuclei from the Arabidopsis SAM. One can also adapt this protocol to isolate nuclei from other tissues and cell types for investigating tissue/cell-type-specific transcriptome and epigenome and their changes during developmental programs at a high spatiotemporal resolution. Furthermore, due to its low cost and simple procedures, INTACT can be conducted in any standard molecular laboratory.
Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.
Recent findings suggest that alternative splicing has a critical role in controlling the responses of plants to temperature variations. However, alternative splicing factors in plants are largely uncharacterized. Here we establish the putative splice regulator, PORCUPINE (PCP), as temperature-specific regulator of development in Arabidopsis thaliana. Our findings point to the misregulation of WUSCHEL and CLAVATA3 as the possible cause for the meristem defects affecting the pcp-1 loss-of-function mutants at low temperatures.
Developmental plasticity enables plants to respond rapidly to changing environmental conditions, such as temperature fluctuations. Understanding how plants measure temperature and integrate this information into developmental programs at the molecular level will be essential to breed thermo-tolerant crop varieties. Recent studies identified alternative splicing (AS) as a possible 'molecular thermometer', allowing plants to quickly adjust the abundance of functional transcripts to environmental perturbations. In this review, recent advances regarding the effects of temperature-responsive AS on plant development will be discussed, with emphasis on the circadian clock and flowering time control. The challenge for the near future will be to understand the molecular mechanisms by which temperature can influence AS regulation.
The timing of flowering is a crucial decision in the life cycle of plants since favourable conditions are needed to maximize reproductive success and, hence, the survival of the species. It is therefore not surprising that plants constantly monitor endogenous and environmental signals, such as day length (photoperiod) and temperature, to adjust the timing of the floral transition. Temperature in particular has been shown to have a tremendous effect on the timing of flowering: the effect of prolonged periods of cold, called the vernalization response, has been extensively studied and the underlying epigenetic mechanisms are reasonably well understood in Arabidopsis thaliana. In contrast, the effect of moderate changes in ambient growth temperature on the progression of flowering, the thermosensory pathway, is only starting to be understood on the molecular level. Several genes and molecular mechanisms underlying the thermosensory pathway have already been identified and characterized in detail. At a time when global temperature is rising due to climate change, this knowledge will be pivotal to ensure crop production in the future.
The transition to flowering is a crucial step in the plant life cycle that is controlled by multiple endogenous and environmental cues, including hormones, sugars, temperature, and photoperiod. Permissive photoperiod induces the expression of FLOWERING LOCUS T (FT) in the phloem companion cells of leaves. The FT protein then acts as a florigen that is transported to the shoot apical meristem, where it physically interacts with the Basic Leucine Zipper Domain transcription factor FD and 14-3-3 proteins. However, despite the importance of FD in promoting flowering, its direct transcriptional targets are largely unknown. Here, we combined chromatin immunoprecipitation sequencing and RNA sequencing to identify targets of FD at the genome scale and assessed the contribution of FT to DNA binding. We further investigated the ability of FD to form protein complexes with FT and TERMINAL FLOWER1 through interaction with 14-3-3 proteins. Importantly, we observed direct binding of FD to targets involved in several aspects of plant development. These target genes were previously unknown to be directly related to the regulation of flowering time. Our results confirm FD as a central regulator of floral transition at the shoot meristem and provide evidence for crosstalk between the regulation of flowering and other signaling pathways, such as pathways involved in hormone signaling.
Alternative splicing occurs in all eukaryotic organisms. Since the first description of multiexon genes and the splicing machinery, the field has expanded rapidly, especially in animals and yeast. However, our knowledge about splicing in plants is still quite fragmented. Though eukaryotes show some similarity in the composition and dynamics of their splicing machinery, observations of unique plant traits are only starting to emerge. For instance, plant alternative splicing is closely linked to their ability to perceive various environmental stimuli. Due to their sessile lifestyle, temperature is a central source of information, allowing plants to adjust their development to match current growth conditions. Hence, seasonal temperature fluctuations and day-night cycles can strongly influence plant morphology across developmental stages. Here we discuss available data on temperature-dependent alternative splicing in plants. Given its fragmented state, it is not always possible to fit specific observations into a coherent picture, yet it is sufficient to estimate the complexity of this field and the need for further research. Better understanding of alternative splicing as a part of plant temperature response and adaptation may also prove to be a powerful tool for both fundamental and applied sciences.
Plant life-long organogenesis involves sequential, time and tissue specific expression of developmental genes. This requires activities of Polycomb Group (PcG) and trithorax Group complexes (trxG), respectively responsible for repressive Histone 3 trimethylation at lysine 27 (H3K27me3) and activation-related Histone 3 trimethylation at lysine 4 (H3K4me3). However, the genome-wide dynamics in histone modifications that occur during developmental processes have remained elusive. Here, we report the distributions of H3K27me3 and H3K4me3 along with expression changes, in a developmental series including Arabidopsis thaliana leaf and three stages of flower development. We found that chromatin mark levels are highly dynamic over the time series on nearly half of all Arabidopsis genes. Moreover, during early flower morphogenesis, changes in H3K4me3 prevail over changes in H3K27me3 and quantitatively correlate with expression changes, while H3K27me3 changes occur later. Notably, we found that H3K4me3 increase during the early activation of PcG target genes while H3K27me3 level remain relatively constant at the locus. Our results reveal that H3K4me3 predicts changes in gene expression better than H3K27me3, unveil unexpected chromatin mechanisms at gene activation and underline the relevance of tissue-specific temporal epigenomics.
TERMINAL FLOWER1 acts in the shoot apical meristem as a mobile cell-non-autonomous transcriptional cofactor that associates with DNA to regulate meristem indeterminacy and flowering. The floral transition is a critical step in the life cycle of flowering plants, and several mechanisms control this finely orchestrated process. TERMINAL FLOWER1 (TFL1) is a floral repressor and close relative of the florigen, FLOWERING LOCUS T (FT). During the floral transition, TFL1 expression is up-regulated in the inflorescence apex to maintain the indeterminate growth of the shoot apical meristem (SAM). Both TFL1 and FT are mobile proteins, but they move in different ways. FT moves from the leaves to the SAM, while TFL1 appears to move within the SAM. The importance of TFL1 movement for its function in the regulation of flowering time and shoot indeterminacy and its molecular function are still largely unclear. Our results using Arabidopsis (Arabidopsis thaliana) indicate that TFL1 moves from its place of expression in the center of the SAM to the meristem layer L1 and that the movement in the SAM is required for the regulation of the floral transition. Chromatin immunoprecipitation sequencing and RNA sequencing demonstrated that TFL1 functions as a cotranscription factor that associates with and regulates the expression of hundreds of genes. These newly identified direct TFL1 targets provide the possibility to discover new roles for TFL1 in the regulation of floral transition and inflorescence development.
The transition from vegetative to reproductive growth is a key event in the plant life cycle. Plants therefore use a variety of environmental and endogenous signals to determine the optimal time for flowering to ensure reproductive success. These signals are integrated at the shoot apical meristem (SAM), which subsequently undergoes a shift in identity and begins producing flowers rather than leaves, while still maintaining pluripotency and meristematic function. Gibberellic acid (GA), an important hormone associated with cell growth and differentiation, has been shown to promote flowering in many plant species including Arabidopsis thaliana , but the details of how spatial and temporal regulation of GAs in the SAM contribute to floral transition are poorly understood. In this study, we show that the gene GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2 ), which encodes a GA‐inactivating enzyme, is significantly upregulated at the SAM during floral transition and contributes to the regulation of flowering time. Loss of GAMT2 function leads to early flowering, whereas transgenic misexpression of GAMT2 in specific regions around the SAM delays flowering. We also found that GAMT2 expression is independent of the key floral regulator LEAFY but is strongly increased by the application of exogenous GA. Our results indicate that GAMT2 is a repressor of flowering that may act as a buffer of GA levels at the SAM to help prevent premature flowering.
Programmable gene regulators that can modulate the activity of selected targets in trans are a useful tool for probing and manipulating gene function. CRISPR technology provides a convenient method for gene targeting that can also be adapted for multiplexing and other modifications to enable strong regulation by a range of different effectors. We generated a vector toolbox for CRISPR/dCas9-based targeted gene regulation in plants, modified with the previously described MS2 system to amplify the strength of regulation, and using Golden Gate-based cloning to enable rapid vector assembly with a high degree of flexibility in the choice of promoters, effectors and targets. We tested the system using the floral regulator FLOWERING LOCUS T (FT) as a target and a range of different effector domains including the transcriptional activator VP64, the H3K27 acetyltransferase p300 and the H3K9 methyltransferase KRYPTONITE. When transformed into Arabidopsis thaliana, several of the constructs caused altered flowering time phenotypes that were associated with changes in FT expression and/or epigenetic status, thus demonstrating the effectiveness of the system. The MS2-CRISPR/dCas9 system can be used to modulate transcriptional activity and epigenetic status of specific target genes in plants, and provides a versatile tool that can easily be used with different targets and types of regulation for a range of applications.
Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae.
Plants undergo transcriptome reprograming to adapt to daily and seasonal fluctuations in light and temperature conditions. While most efforts have focused on the role of master transcription factors, the importance of splicing factors modulating these processes is now emerging. Efficient pre-mRNA splicing depends on proper spliceosome assembly, which in plants and animals requires the methylosome complex. Ion Chloride nucleotide-sensitive protein (PICLN) is part of the methylosome complex in both humans and Arabidopsis (Arabidopsis thaliana), and we show here that the human PICLN ortholog rescues phenotypes of Arabidopsis picln mutants. Altered photomorphogenic and photoperiodic responses in Arabidopsis picln mutants are associated with changes in pre-mRNA splicing that partially overlap with those in PROTEIN ARGININE METHYL TRANSFERASE5 (prmt5) mutants. Mammalian PICLN also acts in concert with the Survival Motor Neuron (SMN) complex component GEMIN2 to modulate the late steps of UsnRNP assembly, and many alternative splicing events regulated by PICLN but not PRMT5, the main protein of the methylosome, are controlled by Arabidopsis GEMIN2. As with GEMIN2 and SM PROTEIN E1/PORCUPINE (SME1/PCP), low temperature, which increases PICLN expression, aggravates morphological and molecular defects of picln mutants. Taken together, these results establish a key role for PICLN in the regulation of pre-mRNA splicing and in mediating plant adaptation to daily and seasonal fluctuations in environmental conditions.
In contrast to animals, plants cannot avoid unfavorable temperature conditions. Instead, plants have evolved intricate signaling pathways that enable them to perceive and respond to temperature. General acclimation processes that prepare the plant to respond to stressful heat and cold usually occur throughout the whole plant. More specific temperature responses, however, are limited to certain tissues or cell types. While global responses are amenable to epigenomic analyses, responses that are highly localized are more problematic as the chromatin in question is not easily accessible. Here we review current knowledge of the epigenetic regulation of FLOWERING LOCUS C and FLOWERING LOCUS T as examples of temperature-responsive flowering time regulator genes that are expressed broadly throughout the plants and in specific cell types, respectively. While this work has undoubtedly been extremely successful, we reason that future analyses would benefit from higher spatiotemporal resolution. We conclude by reviewing methods and successful applications of tissue-and cell type-specific epigenomic analyses and provide a brief outlook on future single-cell epigenomics.
Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17-and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain-and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development.
In Arabidopsis, various cell type-specific whole-genome expression analyses have been conducted. However, the vast majority of these were performed with cellular RNA from root tissues or other easily accessible cell types [1]. Nuclear RNA was neglected for a long time as not being representative for transcriptomic studies. In recent years, however, there have been reports describing the validity of nuclear RNA for these types of studies [2] and [3]. Here we describe the generation, quality assessment and analysis of nuclear transcriptomic data from Arabidopsis embryos published by Slane et al. (2014) [4]. Comparison of nuclear with cellular gene expression demonstrated the usefulness of nuclear transcriptomics.
Intron splicing increases proteome complexity, promotes RNA stability, and enhances transcription. However, introns and the concomitant need for splicing extend the time required for gene expression and can cause an undesirable delay in the activation of genes. Here, we show that the plant microRNA processing factor SERRATE (SE) plays an unexpected and pivotal role in the regulation of intronless genes. Arabidopsis SE associated with more than 1000, mainly intronless, genes in a transcription-dependent manner. Chromatin-bound SE liaised with paused and elongating polymerase II complexes and promoted their association with intronless target genes. Our results indicate that stress-responsive genes contain no or few introns, which negatively affects their expression strength, but that some genes circumvent this limitation via a novel SE-dependent transcriptional activation mechanism. Transcriptome analysis of a Drosophila mutant defective in ARS2, the metazoan homologue of SE, suggests that SE/ARS2 function in regulating intronless genes might be conserved across kingdoms.
Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.