Frank MachinYağmur Hasbioğlu Friedrich Kragler

In: Benitez-Alfonso Y., Heinlein M. (eds) Plasmodesmata, 2022. Methods in Molecular Biology


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.

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.

Lei Yang, Yuan Zhou, Shuangfeng Wang, Ying Xu, Steffen Ostendorp, Melissa Tomkins, Julia Kehr, Richard J. Morris, and Friedrich Kragler

2022 Preprint, biorxiv

Heat shock proteins of the HSC70/HSP70 family are evolutionarily conserved chaperones that are 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 its function has remained an open question. Here, we show that the HSC70.1 chaperone binds its own mRNA via its C-terminal Short Variable Region (SVR) and inhibits its own translation. We propose that this negative protein-transcript feedback loop may establish an on-demand chaperone pool that allows for a rapid response to stress. Furthermore, we show that the SVR encoding RNA 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. In summary, it seems 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 maintaining chaperone homestasis between tissues

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 Pre-proof, 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.

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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).


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.

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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.

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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.

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.

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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.

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.

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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.

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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.

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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.

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Tedesco et al. Figure 2a_Characterisation of grafts

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.

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.

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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.

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.

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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.


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.

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