Friedrich Kragler

Single-cell RNA sequencing (scRNAseq) has become an essential tool in plant biology to gain high-resolution information on cell type-specific gene expression, developmental dynamics, and tissue-specific responses to environmental factors. A major technical challenge in applying scRNAseq to plants is the efficient and reproducible isolation of viable single cells without cell walls, representing the heterogeneity of plant tissues. Protoplast isolation, which removes the cell wall enzymatically to yield single, living plant cells, is a widely used approach for this purpose. However, successful protoplast isolation requires careful optimization to maintain cell viability and preserve cell-type-specific transcriptome profiles. Here, we present a robust and reproducible protocol for isolating high-quality protoplasts from multiple Arabidopsis thaliana tissues, including above-ground (leaves, stems, flowers) and root tissues. This protocol accounts for the unique physiological properties of tissue types, optimizing enzymatic digestion conditions and purification steps to ensure high yield and broad representation of diverse cell types. The resulting protoplasts are suitable for applications such as scRNAseq, flow cytometry (FACS), and transient gene expression analysis. It allows for the generation of single-cell suspensions from multiple tissue types. The presented method supports exploration of cellular heterogeneity and enhances the reliability of single-cell approaches in plant research.

A plant's stem cell population in the shoot apical meristem (SAM) is maintained by WUSCHEL (WUS) and CLAVATA3 (CLV3). SAM size is dynamic and undergoes a more than 2-fold expansion upon transition to reproductive growth. The mechanism controlling this doming is largely unknown; however, coinciding increased trehalose 6-phosphate (T6P) levels suggest a participation of the T6P pathway in Arabidopsis (Arabidopsis thaliana). Moreover, lines misexpressing or with reduced expression of TREHALOSE PHOSPHATE SYNTHASE1 (TPS1) have smaller and larger SAMs, respectively. Here, we show that TREHALOSE PHOSPHATE PHOSPHATASEJ (TPPJ) is directly regulated by WUS. Changing TPPJ transcript levels in the outer layer affects SAM size and flowering time, and its reduction in the late-flowering clv3 mutant restores wild-type flowering. This is associated with altered mature microRNA156 abundance and expression of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes SPL3, SPL4, SPL5, and SPL9. Furthermore, SPL4 is controlled by WUS, while SPL4 directly represses WUS, establishing negative feedback regulation. This feedback loop is important for age pathway-induced flowering involving the T6P pathway and suggests dynamic feedback regulations between central meristem maintenance and flowering time regulators with sugar signaling throughout development.

Short-read RNA-seq studies of grafted plants have led to the proposal that thousands of messenger RNAs (mRNAs) move over long distances between plant tissues^1–7, potentially acting as signals^8–12. Transport of mRNAs between cells and tissues has been shown to play a role in several physiological and developmental processes in plants, such as tuberization¹³, leaf development¹⁴ and meristem maintenance¹⁵; yet for most mobile mRNAs, the biological relevance of transport remains to be determined^16–19. Here we perform a meta-analysis of existing mobile mRNA datasets and examine the associated bioinformatic pipelines. Taking technological noise, biological variation, potential contamination and incomplete genome assemblies into account, we find that a high percentage of currently annotated graft-mobile transcripts are left without statistical support from available RNA-seq data. This meta-analysis challenges the findings of previous studies and current views on mRNA communication.

Grafting techniques have been used for centuries for trait improvement in agriculture and horticulture. In recent years, technical progress with the grafting of species previously thought to be recalcitrant to the formation of a graft union, and the discovery of new types of information exchange between grafted plants, have stirred renewed interest in the use of grafting as a research tool. In this Review, we describe our current understanding of the molecular and cellular processes involved in the establishment of successful grafts between plants of the same genotype (homografts) or different genotypes (heterografts). We also highlight recent progress with the elucidation of the mechanisms underlying the exchange of macromolecules (small RNAs, messenger RNAs and proteins) across graft junctions as well as the transfer of cell organelles and its role in horizontal gene and genome transfer. Finally, we discuss novel applications of grafting, including new opportunities for transgene-free genetic engineering, and the relevance of grafting in plant evolution.

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.

In Arabidopis a high number of distinct mRNAs move from shoot to root. We previously reported on the correlation of m ⁵ C-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 m ⁵ C 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 m ⁵ C-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 m ⁵ C 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.

Summary: 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 chaperone pool 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 noncell‐autonomous manner, potentially maintaining chaperone homeostasis between tissues.

Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (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 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.