2024
PREPRINT: Re-analysis of mobile mRNA datasets highlights challenges in the detection of mobile transcripts from short-read RNA-Seq data
Pirita Paajanena, Melissa Tomkins, Franziska Hoerbst, Ruth Veevers, Michelle Heeney, Hannah Rae Thomas, Federico Apelt, Eleftheria Saplaoura, Saurabh Gupta, Margaret Frank, Dirk Walther, Christine Faulkner, Julia Kehr, Friedrich Kragler, and Richard J. Morris
Biorxiv preprint, 2024, https://www.biorxiv.org/content/10.1101/2024.07.25.604588v1
Abstract
Short-read RNA-Seq analyses of grafted plants have led to the proposal that large numbers of mRNAs move over long distances between plant tissues, acting as potential signals. The detection of transported transcripts by RNA-Seq is both experimentally and computationally challenging, requiring successful grafting, delicate harvesting, rigorous contamination controls and data processing approaches that can identify rare events in inherently noisy data. Here, we perform a meta-analysis of existing datasets and examine the associated bioinformatic pipelines. Our analysis reveals that technological noise, biological variation and incomplete genome assemblies give rise to features in the data that can distort the interpretation. Taking these considerations into account, we find that a substantial number of transcripts that are currently annotated as mobile are left without support from the available RNA-Seq data. Whilst several annotated mobile mRNAs have been validated, we cannot exclude that others may be false positives. The identified issues may also impact other RNA-Seq studies, in particular those using single nucleotide polymorphisms (SNPs) to detect variants.
Fig. 1. Grafting coupled with RNA-Seq has been employed to identify transcripts that move from tissue of one geno- type/species/ecotype/cultivar into tissue of another genotype/species/ecotype/cultivar across the graft junction. Shown here is a grafting-based strategy for identification of mRNAs that move from shoot (scion) to root (stock), from genotype 2 to genotype 1, using a scion:stock=genotype 2:genotype 1 heterograft. The same strategy can be used to identify transcripts that move from shoot to root from genotype 1 to genotype 2 using a genotype 1:genotype 2 graft. Transcripts that move root to shoot can be identified by analysing mRNAs in shoot tissue. Natural grafts, such as those established between the parasitic dodder plant and its host plants, can be used in place of artificial grafts. A key challenge in all such approaches is how to assign transcripts to each genotype; methods for doing so are based on (1) SNP (single nucleotide polymorphism) identification or on (2) the alignment to different reference genomes. For grafts from the same species, or similar genotypes, SNPs can be used to distinguish between genotypes and thus identify the source genotype of each transcript (1). For grafts between different species, mapping (2) each RNA-Seq read to the genome assemblies can be an effective method for determining which transcripts are specific to one species.
The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation
Kim Lühmann, Silja Seemann, Nina Martinek, Steffen Ostendorp & Julia Kehr
Nature Scientific Reports, 2024, doi.org/10.1038/s41598-024-66955-5
Abstract
Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7’s RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid–liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.
Figure 2a: Comparison of dissociation constants (Kds) of AtGRP7 for RNAs varying in length, with and without UTRs and Introns, as well as single and double stranded RNA. (a) Kds of AtGRP7 for RNAs varying in length displayed in a bar graph. The length of RNAs is shown on the X-axis, the Y-axis shows the Kd in μM. Kds were compared by one-way ANOVA and Tukeys test (p = 0.01). Similar letters indicate no significant difference.
PREPRINT: Targeted engineering of the phase separating PARCL protein
Ruth Veevers, Steffen Ostendorp, Anna Ostendorp, Julia Kehr, Richard J. Morris
Biorixiv Preprint, 2024, https://biorxiv.org/content/10.1101/2024.06.21.600009v1
Abstract:
PARCL is a plant-specific RNA-binding protein (RBP) that exhibits chaperone activity, is abundant in the phloem, intrinsically disordered, and contains a prion-like domain (PLD). PARCL proteins have been observed to form large biomolecular condensates in vivo and in vitro. Biomolecular condensates are membraneless compartments, wherein biomolecules become partitioned from their surrounding liquid environment into liquid droplets with their own composition, dynamics, and function. Which molecular properties drive phase separation is of great interest for targeted engineering efforts. Here, we present results on residue interactions derived from simulations of PARCL using course-grained molecular dynamics with the HPS-Urry model. We adjust the parameters of the simulations to allow the inclusion of folded eYFP tags, since fluorescent tags are often used in phase separation experiments for visualising droplets, yet have not been included in simulations to date. While still simulating phase separation, these trajectories suggest minor changes to droplet and network structure when proteins contain eYFP. By analysing the residues of the PARCL molecules that come within contact distance in the simulations, we identify which individual residues drive phase separation. To experimentally validate these findings, we introduced mutations of the most contacted residues and could indeed confirm that these mutations prevent the formation of condensate droplets. To investigate the RNA-binding of PARCL, we added microRNA to the simulation and find a short region of PARCL consistently making contact with the miRNA, which is also in agreement with predictions and experiments. We discuss the implications of our findings in terms of model-guided engineering of biomolecular condensates.
Fig 1 shows the steps we took to produce and analyse the simulated MD trajectories of multiple interacting units of the same protein, starting from a given protein sequence. We further explain these steps in the following sections.
PREPRINT: Systemic mRNA transport depends on m5C methylation, nuclear mRNA export factors and developmental phase changes
Ying Xu, András Székely, Steffen Ostendorp, Saurabh Gupta, Melissa Tomkins, Lei Yang, Federico Apelt, Yan Zhao, Eleni Mavrothalassiti, Linda Wansing, Julia Kehr, Eleftheria Saplaoura and Friedrich Kragler
Biorxiv preprint, 2024, https://www.biorxiv.org/content/10.1101/2024.05.30.596576v1
Abstract
In Arabidopis a high number of distinct mRNAs move from shoot to root. We previously reported on the correlation of m5C-methylation and lack of mRNA transport in juvenile plants depending on the RNA methyltransferases DNMT2 NSUN2B. However, to our surprise we uncovered that lack of DNMT2 NSUN2B (writer) activity did not abolished transport of TCTP1 and HSC70.1 transcripts in flowering plants. We uncovered that transport of both transcripts is reinstated in dnmt2 nsun2b mutants after commitment to flowering. This finding suggests that additional factors are seemingly involved in regulating / mediating mRNA transport. In search of such candidates, we identified the two ALY2 and ALY4 nuclear mRNA export factors belonging to the ALYREF family as bona fide m5C readers mediating mRNA transport. We show that both proteins are allocated along the phloem and that they bind preferentially to mobile mRNAs. MST measurements indicate that ALY2 and ALY4 bind to mobile mRNAs with relative high affinity with ALY4 showing higher affinity towards m5C-methylated mobile mRNAs. An analysis of the graft-mobile transcriptome of juvenile heterografted-grafted wild type, dnmt2 nsun2b, aly2 and aly4 mutants revealed that the nuclear export factors are key regulators of mRNA transport. We suggest that depending on the developmental stage m5C methylation has a negative and positive regulatory function in mRNA transport and acts together with ALY2 and ALY4 to facilitate mRNA transport in both juvenile and flowering plants.
2023
Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstock
Lei Yang*, Frank Machin*, Shuangfeng Wang, Eleftheria Saplaoura and Friedrich Kragler
* These authors contributed equally
Nature Biotech, 2023, doi: 10.1038/s41587-022-01585-8
Abstract:
Generation of stable gene edited plant lines using CRISPR/Cas9 requires a lengthy process of outcrossing to eliminate CRISPR/Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA (gRNA) transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.
This methodology is also summed up in a short animation.
Figure 1a: Scheme of CRISPR/Cas9 mediated transgene-free gene editing by grafting
Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA
Lei Yang, Yuan Zhou, Shuangfeng Wang, Ying Xu, Steffen Ostendorp, Melissa Tomkins, Julia Kehr, Richard J. Morris, and Friedrich Kragler
New Phytologist, 2022 doi: 10.1111/nph.18703
Abstract
- The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones involved in protein folding, protein transport, and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth- related pathways. Whether Arabidopsis HSC70 binds RNA and whether this interaction is functional has remained an open question.
- We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C-terminal Short Variable Region (SVR) and inhibits its own translation.
- The SVR encoding mRNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants.We propose that this negative protein-transcript feedback loop may establish an on-demand that allows for a rapid response to stress.
- In summary, our data suggest that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a non-cell-autonomous manner, potentially maintaining chaperone homeostasis between tissues.
Figure 8. Speculative model of HSC70 functioning as a non-cell-autonomous mobile chaperone regulating its own translation.
HSC70 can block its own translation by binding its mRNA (negative feedback). In non-stressed cells (with regular chaperone activity demands) this will result in a balanced pool of complexes of HSC70 bound to its own mRNA and of translated HSC70 protein available for folding of client proteins. HSC70 transcript and protein, possibly as HSC70 transcript-protein complexes, can move between cells and to distant tissues. The function of this movement remains to be determined, but potentially plays a role in maintaining homeostatic chaperone levels between tissues/cells over longer time periods. Under stress conditions, the HSC70 transcript-protein complexes may dissociate, freeing up both active HSC70 protein and relieving the auto-inhibitory activity on its own translation, thus allowing for a rapid (minutes) response to sudden increases in chaperone demand. After longer time periods (hours) HSC70 gene expression can be induced in a stressed tissue/cell producing more of HSC70 protein and RNA. As HSC70 transcript and protein can move they would be delivered to non-stressed tissues/cells. In recipient cells imported additional HSC70 protein might interfere with translation and lower chaperone activity in distant tissues resulting in coordinated decreased growth in e.g., a locally stressed plant. In parallel, recipient cells/tissues might be able to adopt faster to anticipated stress by additionally received HSC70 mRNA. Both features, HSC70 protein – transcript auto-inhibitory activity and transcript/protein transport between cells, may enable a multicellular organism to establish chaperone homeostasis between e.g., stressed and non-stressed tissues over long time periods providing additional robustness under stress conditions to ensure coordinated growth within and between tissues.
Arabidopsis callus grafts: a system to study symplasmic intercellular macromolecular transport and gene silencing spread
Yağmur Hasbioğlu, Stephanie Weber, Friedrich Kragler, Frank Qasim Machin
The Plant Journal, 2023 doi: 10.1111/tpj.16326
Abstract
In the present study, we present callus grafting, comprising a method for reproducibly generating tissue chimeras from callus cultures of Arabidopsis thaliana. In this way, callus cultures of different genetic backgrounds may be co-cultivated such that cell-to-cell connectivity is achieved as a chimeric tissue is formed. To track intercellular connectivity and transport between non-clonal callus cells, we used transgenic lines expressing fluorescently tagged mobile and non-mobile fusion constructs. Using fluorescently-labelled reporter lines that label plasmodesmata, we show that secondary complex plasmodesmata are present at the cell walls of connected cells. We use this system to investigate cell-to-cell transport across the callus graft junction and show that different proteins and RNAs are mobile between non-clonal callus cells. Finally, we take advantage of the callus culture system to probe intercellular connectivity of grafted leaf and root calli and the effect of different light regimes of cell-to-cell transport. Taking advantage of the ability of callus to be cultivated in the complete absence of light, we show that the rate of silencing spread is significantly decreased in chimeric calli cultivated in total darkness. We propose that callus grafting is a fast and reliable method for analysing the capacity of a macromolecule to be exchanged between cells independent of the vasculature.
Figure 3: Fixation and sectioning allows images to be taken of cell walls with several PD complexes as revealed by PD-specific proteins.
(a) CLSM images of cryosections of 35S:MP17-GFP callus. Green indicates MP17-GFP and blue is cell walls stained with Calcofluor. Scale bar = 0.5 μm.
(b) CLSM images of cryosection of 35S:PDLP5-Cherry callus. Red indicates PDLP5-Cherry and blue indicates cell walls stained with Calcofluor. Scale bar = 0.5 μm.
(c–f) CLSM image of a cryosection of a callus graft between 35S:MP17-GFP (c) and 35S:PDLP5-Cherry (d), stained with Calcofluor (e) and merged (f). Scale bars = 0.5 μm.
Structural and functional analysis of a plant nucleolar RNA chaperone‑like protein
Rita Fernandes, Anna Ostendorp, Steffen Ostendorp, Judith Mehrmann, Sven Falke, Melissa Ann Graewert, Magdalena Weingartner, Julia Kehr, Stefan Hoth
Scientific Reports, 2023, doi: 10.1038/s41598-023-36426-4
Abstract
Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE‑LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC‑SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC‑MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre‑rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone‑like activity. Our data suggested that NURC1 may have a function in pre‑rRNA processing and thus ribosome biogenesis.
Figure 1. NURC1 and other plant RBFs are localized in the nucleolus. Subcellular localization of GFP-tagged NURC1, At5g05210 and EBP2 (At3g22660) transiently co-expressed with the nuclear marker MAIL1-mCherry in N. benthamiana leaf cells. Fluorescence of the respective fusion proteins was analyzed by using confocal microscopy. The merged pictures indicated the localization of all GFP fusion proteins in the nucleolus.
2022
Exact Bayesian inference for the detection of graft-mobile transcripts from sequencing data
Melissa Tomkins*, Franziska Hoerbst*, Saurabh Gupta, Federico Apelt, Julia Kehr, Friedrich Kragler and Richard J. Morris; *these authors contributed equally
R. Soc. Interface, 2022, doi: https://doi.org/10.1098/rsif.2022.0644
Abstract: The long-distance transport of messenger RNAs (mRNAs) has been shown to be important for several developmental processes in plants. A popular method for identifying travelling mRNAs is to perform RNA-Seq on grafted plants. This approach depends on the ability to correctly assign sequenced mRNAs to the genetic background from which they originated. The assign- ment is often based on the identification of single-nucleotide polymorphisms (SNPs) between otherwise identical sequences. A major challenge is there- fore to distinguish SNPs from sequencing errors. Here, we show how Bayes factors can be computed analytically using RNA-Seq data over all the SNPs in an mRNA. We used simulations to evaluate the performance of the proposed framework and demonstrate how Bayes factors accurately identify graft-mobile transcripts. The comparison with other detection methods using simulated data shows how not taking the variability in read depth, error rates and multiple SNPs per transcript into account can lead to incorrect classification. Our results suggest experimental design cri- teria for successful graft-mobile mRNA detection and show the pitfalls of filtering for sequencing errors or focussing on single SNPs within an mRNA.
Figure 1: Workflow of the inference steps for determining the evidence that a transcript has travelled across a graft junction. Two plants with different genetic back- grounds are grafted in different combinations. The grafted parts are known as stock and scion. Homograft data can be used to infer the error rates per SNP in stock and scion for each genetic background. The heterograft data can then be evaluated in light of the homograft data. If the evidence for a transcript having traversed the graft junction is greater than the evidence for the data being explainable by sequencing errors, then the transcript is a candidate for being long-distance mobile.
Intrinsically disordered plant protein PARCL co-localizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD
Anna Ostendorp, Steffen Ostendorp, Yuan Zhou, Zoé Chaudron, Lukas Wolffram, Khadija Rombi, Linn von Pein, Sven Falke, Cy M. Jeffries, Dmitri I. Svergun, Christian Betzel, Richard J. Morris, Friedrich Kragler and Julia Kehr
JBC, 2022, doi: 10.1016/j.jbc.2022.102631
In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins (RBPs) facilitate RNA translocation. In the present study, we characterized two orthologs of the phloem-associated RNA chaperone-like (PARCL) protein from Arabidopsis thaliana and Brassica napus at functional and structural levels. Microscale thermophoresis (MST) showed that these phloem-abundant proteins can bind a broad spectrum of RNAs and show RNA chaperone activity in FRET-based in vitro assays. Our SAXS experiments revealed a high degree of disorder, typical for RNA-binding proteins. In agroinfiltrated tobacco plants, eYFP-PARCL proteins mainly accumulated in nuclei and nucleoli and formed cytosolic and nuclear condensates. We found that formation of these condensates was impaired by tyrosine-to-glutamate mutations in the predicted prion-like domain (PLD), while C-terminal serine-to-glutamate mutations did not affect condensation but reduced RNA binding and chaperone activity. Furthermore, our in vitro experiments confirmed phase separation of PARCL and co-localization of RNA with the condensates, while mutation as well as phosphorylation of the PLD reduced phase separation. Together, our results suggest that RNA binding and condensate formation of PARCL can be regulated independently by modification of the C-terminus and/ or the PLD.
Fig. 3E: Spatially aligned ensemble model representatives of PARCL at pH 7.5 (ribbons) superposed with the anisotropic ‘shape volume’ of the ensemble obtained from dummy atom modelling (transparent spheres).
Protocol: Arabidopsis Callus Grafting Method to Test Cell-to-Cell Mobility of Proteins
Frank MachinYağmur Hasbioğlu Friedrich Kragler
In: Benitez-Alfonso Y., Heinlein M. (eds) Plasmodesmata, 2022. Methods in Molecular Biology
https://doi.org/10.1007/978-1-0716-2132-5_20
A callus is a semi-disorganized tissue that can be induced to develop from diverse tissues by the addition of exogenous hormones. The fast growth and ease of propagation have made callus cultures useful for creating a wide variety of different experimental systems.
Here, we describe a detailed and simple procedure by which different, non-clonal calli from transgenic and wild-type A. thaliana plants can be co-cultured such that they form symplasmic connections via plasmodesmata (PD). We show that callus cultures can be used to study both PD formation and transport of macromolecules between non-clonal cells via PD in a tissue lacking a vasculature. Further, we include a simple protocol for a method by which calli can be sectioned to image cells and PD by confocal laser scanning microscopy.
3D Printable files: Moulds for callus co-cultivation
Frank Machin, Yağmur Hasbioğlu, Friedrich Kragler
2021, availabe from Zenodo: https://doi.org/10.5281/zenodo.4446896
3D Printable files to create moulds for Arabidopsis callus co-cultivation and for callus cryosectioning.
3 Files are available: one for callus cryosectioning, and two callus moulds.
2021
The Impact of Metabolic Scion–Rootstock Interactions in Different Grapevine Tissues and Phloem Exudates
Sara Tedesco, Alexander Erban, Saurabh Gupta, Joachim Kopka, Pedro Fevereiro, Friedrich Kragler and Ana Pina
Metabolites, 2021, doi: 10.3390/metabo11060349
In viticulture, grafting is used to propagate Phylloxera-susceptible European grapevines, thereby using resistant American rootstocks. Although scion–rootstock reciprocal signaling is essential for the formation of a proper vascular union and for coordinated growth, our knowledge of graft partner interactions is very limited. In order to elucidate the scale and the content of scion–rootstock metabolic interactions, we profiled the metabolome of eleven graft combination in leaves, stems, and phloem exudate from both above and below the graft union 5–6 months after grafting. We compared the metabolome of scions vs. rootstocks of homografts vs. heterografts and investigated the reciprocal effect of the rootstock on the scion metabolome. This approach revealed that (1) grafting has a minor impact on the metabolome of grafted grapevines when tissues and genotypes were compared, (2) heterografting affects rootstocks more than scions, (3) the presence of a heterologous grafting partner increases defense-related compounds in both scion and rootstocks in shorter and longer distances from the graft, and (4) leaves were revealed as the best tissue to search for grafting-related metabolic markers. These results will provide a valuable metabolomics resource for scion–rootstock interaction studies and will facilitate future efforts on the identification of metabolic markers for important agronomic traits in grafted grapevines.
Primary carbohydrate metabolism genesparticipate in heat-stress memory at the shootapical meristem ofArabidopsis thaliana
Justyna Jadwiga Olas, Federico Apelt, Maria Grazia Annunziata, Sheeba John, Sarah Isabel Richard, Saurabh Gupta, Friedrich Kragler, Salma Balazadeh and Bernd Mueller-Roeber
Molecular Plant, 2021, doi: 10.1016/j.molp.2021.05.024
In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, lit- tle is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana displays an autonomous heat-stress (HS) memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA sequencing, we iden- tified genes participating in establishing the SAM’s HS transcriptional memory, including the stem cell (SC) regulators CLAVATA1 (CLV1) and CLV3, HEAT SHOCK PROTEIN 17.6A (HSP17.6A), and the primary carbo- hydrate metabolism gene FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We demonstrate that sugar availability is essential for survival of plants at high temperature. HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2A) directly regulates the expression of HSP17.6A and FBA6 by binding to the heat-shock ele- ments in their promoters, indicating that HSFA2 is required for transcriptional activation of SAM memory genes. Collectively, these findings indicate that plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.
Figure 7: A simplified model for the regula- tion of heat-stress memory in the SAM. Thermopriming affects sugar availability and in- duces the expression of specific HSFs in the SAM, including the master regulator HSFA2. HSFA2 binds to heat shock elements in the 50 upstream regulatory regions of memory genes, including FBA6 and HSP17.6A, at the SAM. Solid lines, direct interactions; dashed lines, indirect in- teractions.
Shoot and Root Single Cell Sequencing Reveals Tissue- and Daytime-Specific Transcriptome Profiles
Federico Apelt, Eleni Mavrothalassiti, Saurabh Gupta, Frank Machin, Justyna Jadwiga Olas, Maria Grazia Annunziata, Dana Schindelasch, Friedrich Kragler
Plant Physiology, 2021, doi: 10.1093/plphys/kiab537
Although several large-scale single-cell RNA (scRNAseq) studies addressing the root of Arabidopsis (Arabidopsis thaliana) have been published, there is still need for a de novo reference map for both root and especially above-ground cell types. As the plants’ transcriptome substantially changes throughout the day, shaped by the circadian clock, we performed scRNAseq on both Arabidopsis root and above-ground tissues at defined times of the day. For the root scRNAseq analysis we used tissue-specific reporter lines grown on plates and harvested at the end of the day (ED). In addition, we submitted above-ground tissues from plants grown on soil at ED and end of the night (EN) to scRNAseq, which allowed us to identify common cell types/markers between root and shoot and uncover transcriptome changes to above-ground tissues depending on the time of the day. The dataset was also exploited beyond the traditional scRNAseq analysis to investigate non-annotated and di-cistronic transcripts. We experimentally confirmed the predicted presence of some of these transcripts and also addressed the potential function of a previously unidentified marker gene for dividing cells. In summary, this work provides insights into the spatial control of gene expression from nearly 70,000 cells of Arabidopsis for below- and whole above-ground tissue at single-cell resolution at defined time points.
Figure 2D: Schematic representation of rosette cell types of Arabidopsis plants. Upper panel represents longitudinal section through the meristem, whereas lower panel shows longitudinal and cross section of the leaf. SAM, shoot apical meristem.
AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana
Qianwen Wang, Xiucong Bao, Shengjie Chen, Huan Zhong, Yaqin Liu, Li Zhang, Yiji Xia, Friedrich Kragler, Ming Luo, Xiang David Li, Hon-Ming Lam, Shoudong Zhang
Nucleic Acids Research, 2021, https://doi.org/10.1093/nar/gkab706
Pericentromeric DNA, consisting of high-copy- number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal in- tegrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericen- tromeric silencing, the mechanism is still yet un- clear. Here, using whole genome bisulfite sequenc- ing (WGBS) and chromatin immunoprecipitation- sequencing (ChIP-Seq), we mapped the genome- wide patterns of differential DNA methylation and histone H3 lysine 18 acetylation (H3K18ac) in wild- type and hda6 mutant strains. Results show pericen- tromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyl- transferases as previously thought. DNA demethy- lases can recognize H3K18ac mark and then be re- cruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an ‘eraser’ enzyme for H3K18ac mark to prevent DNA demethy- lation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethyla- tion were shown to have transcriptionally active peri- centromeric DNA.
Quantitative plant biology—Old and new
Richard J. Morris, Kirsten H. ten Tusscher
Quantitative Plant Biology, 2021, https://dx.doi.org/10.1017/qpb.2021.4
Quantitative approaches in plant biology have a long history that have led to several ground- breaking discoveries and given rise to new principles, new paradigms and new methodologies. We take a short historical trip into the past to explore some of the many great scientists and influences that have led to the development of quantitative plant biology. We have not been constrained by historical fact, although we have tried not to deviate too much. We end with a forward look, expressing our hopes and ambitions for this exciting interdisciplinary field.
Validation of a novel associative transcriptomics pipeline in Brassica oleracea: identifying candidates for vernalisation response
Shannon Woodhouse, Zhesi He, Hugh Woolfenden, Burkhard Steuernagel, Wilfried Haerty, Ian Bancroft, Judith A. Irwin, Richard J. Morris and Rachel Wells
BMC Genomics, 2021, https://doi.org/10.1186/s12864-021-07805-w
Background: Associative transcriptomics has been used extensively in Brassica napus to enable the rapid identification of markers correlated with traits of interest. However, within the important vegetable crop species, Brassica oleracea, the use of associative transcriptomics has been limited due to a lack of fixed genetic resources and the difficulties in generating material due to self-incompatibility. Within Brassica vegetables, the harvestable product can be vegetative or floral tissues and therefore synchronisation of the floral transition is an important goal for growers and breeders. Vernalisation is known to be a key determinant of the floral transition, yet how different vernalisation treatments influence flowering in B. oleracea is not well understood.
Results: Here, we present results from phenotyping a diverse set of 69 B. oleracea accessions for heading and flowering traits under different environmental conditions. We developed a new associative transcriptomics pipeline, and inferred and validated a population structure, for the phenotyped accessions. A genome-wide association study identified miR172D as a candidate for the vernalisation response. Gene expression marker association identified variation in expression of BoFLC.C2 as a further candidate for vernalisation response.
Conclusions: This study describes a new pipeline for performing associative transcriptomics studies in B. oleracea. Using flowering time as an example trait, it provides insights into the genetic basis of vernalisation response in B. oleracea through associative transcriptomics and confirms its characterisation as a complex G x E trait. Candidate leads were identified in miR172D and BoFLC.C2. These results could facilitate marker-based breeding efforts to produce B. oleracea lines with more synchronous heading dates, potentially leading to improved yields.
2020
A Phenotypic Search on Graft Compatibility in Grapevine
Sara Tedesco, Ana Pina, Pedro Fevereiro, Friedrich Kragler
Agronomy, 2020, doi: 10.3390/agronomy10050706
Grafting is the most used propagation method in viticulture and is the unique control strategy against Phylloxera. Nevertheless, its practice remains limited mainly due to inconsistent graft success and difficulties in predicting graft compatibility responses of proposed scion–rootstock combinations, slowing down the selection of elite rootstocks. Aiming to identify optimal phenotypic parameters related to graft (in)compatibility, we used four clones of two grapevine cultivars that show different compatibility behavior when grafted onto the same rootstock. Several physiological parameters, internal anatomy of the graft union, chlorophyll fluorescence, and pigment contents of homo- and heterografts were monitored in a nursery-grafting context. The measurements highlighted enhanced performance of the heterografts due to rooting difficulties of Vitis vinifera homografts. This suggests that in viticulture, homografts should only be used as compatibility controls regarding qualitative attributes. By observing the internal anatomy of the union, we found that grapevines might require longer times for graft healing than anticipated. While Affinity Coefficients were not informative to assess incompatibility, leaf chlorophyll concentration analysis proved to be a more sensitive indicator of stress than the analysis of chlorophyll fluorescence. Overall, we conclude that graft take correlated best with callus formation at the graft junction three weeks after grafting.
Fig. 2a: Internal characterization of the graft union. (a) Example images of the category A to E charactering the internal graft unions. Category A represents a perfect union in which the graft line is almost invisible. Category B shows few structural imperfections and/or slight discontinuities between wood and bark or cambial invaginations. Category C is characterized by bark discontinuities and D by wood discontinuities. Category E includes broken/unattached unions and/or unions with dead tissue in proximity of the union line.
Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis
Eleftheria Saplaoura, Valentina Perrera, Vincent Colot, Friedrich Kragler
Journal of visualized experiments – JoVE, 2020, doi:10.3791/61231
Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.
Fig. 1: RNA samples are incubated with an antibody for 5-methylated cytosines and the complexes are pulled down with protein G magnetic beads that capture the antibodies along with the bound RNA. The eluted RNA samples are analyzed by deep sequencing and qRT-PCR.
Physiological Profiling of Embryos and Dormant Seeds in Two Arabidopsis Accessions Reveals a Metabolic Switch in Carbon Reserve Accumulation
Catalina Moreno Curtidor, Maria Grazia Annunziata, Saurabh Gupta, Federico Apelt, Sarah Isabel Richard, Friedrich Kragler, Bernd Mueller-Roeber and Justyna Jadwiga Olas
Frontiers in Plant Science, 2020, 10.3389/fpls.2020.588433
In flowering plants, sugars act as carbon sources providing energy for developing embryos and seeds. Although most studies focus on carbon metabolism in whole seeds, knowledge about how particular sugars contribute to the developmental transitions during embryogenesis is scarce. To develop a quantitative understanding of how carbon composition changes during embryo development, and to determine how sugar status contributes to final seed or embryo size, we performed metabolic profiling of hand-dissected embryos at late torpedo and mature stages, and dormant seeds, in two Arabidopsis thaliana accessions with medium [Columbia-0 (Col-0)] and large [Burren-0 (Bur-0)] seed sizes, respectively. Our results show that, in both accessions, metabolite profiles of embryos largely differ from those of dormant seeds. We found that developmental transitions from torpedo to mature embryos, and further to dormant seeds, are associated with major metabolic switches in carbon reserve accumulation. While glucose, sucrose, and starch predominantly accumulated during seed dormancy, fructose levels were strongly elevated in mature embryos. Interestingly, Bur-0 seeds contain larger mature embryos than Col-0 seeds. Fructose and starch were accumulated to significantly higher levels in mature Bur-0 than Col-0 embryos, suggesting that they contribute to the enlarged mature Bur-0 embryos. Furthermore, we found that Bur-0 embryos accumulated a higher level of sucrose compared to hexose sugars and that changes in sucrose metabolism are mediated by sucrose synthase (SUS), with SUS genes acting non-redundantly, and in a tissue-specific manner to utilize sucrose during late embryogenesis.
Figure 7. Simplified model describing carbohydrate signatures of embryos during late torpedo and mature stages as well as dormant seeds of A. thaliana Col-0 and Bur-0 accessions. Note that Bur-0 embryos accumulate much higher carbon reserves during late embryogenesis than Col-0 embryos. AA, amino acids.
2019
m5C Methylation Guides Systemic Transport of Messenger RNA over Graft Junctions in Plants
Lei Yang, Valentina Perrera, Eleftheria Saplaoura, Federico Apelt, Mathieu Bahin, Amira Kramdi, Justyna Olas, Bernd Mueller-Roeber, Ewelina Sokolowska, Wenna Zhang, Runsheng Li, Nicolas Pitzalis, Manfred Heinlein, Shoudong Zhang, Auguste Genovesio, Vincent Colot, Friedrich Kragler
Current Biology, 2019, doi: 10.1016/j.cub.2019.06.042
In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thaliana mRNAs harboring the modified base 5-methylcytosine (m5C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m5C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.
