Harnessing spatial transcriptomics for advancing plant regeneration research.

Song et al. utilized spatial transcriptomics to study the molecular characteristics of various cells - such as shoot primordia and chlorenchyma cells - in tomato callus during shoot regeneration. This research enhances our knowledge of shoot regeneration and demonstrates the potential of spatial transcriptomics in advancing plant biology.

DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction.

Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD+) and 3'-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA caps. Although dpCoA has been known to cap RNAs in various species, the identities of its capped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developed a method called dpCoA tagSeq, which utilized a thiol-reactive maleimide group to label dpCoA cap with a tag RNA serving as the 5' barcode. The barcoded RNAs were isolated using a complementary DNA strand of the tag RNA prior to direct sequencing by nanopore technology. Our validation experiments with model RNAs showed that dpCoA-RNA was efficiently tagged and captured using this protocol. To confirm that the tagged RNAs are capped by dpCoA and no other thiol-containing molecules, we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosine monophosphate (AMP) moiety before performing the tagSeq protocol. We identified 44 genes that transcribe dpCoA-RNAs in mouse liver, demonstrating the method's effectiveness in identifying and characterizing the capped RNAs. This strategy provides a viable approach to identifying dpCoA-RNAs that allows for further functional investigations of the cap.

Arabidopsis DXO1 activates RNMT1 to methylate the mRNA guanosine cap.

Eukaryotic messenger RNA (mRNA) typically contains a methylated guanosine (m7G) cap, which mediates major steps of mRNA metabolism. Recently, some RNAs in both prokaryotic and eukaryotic organisms have been found to carry a non-canonical cap such as the NAD cap. Here we report that Arabidopsis DXO family protein AtDXO1, which was previously known to be a decapping enzyme for NAD-capped RNAs (NAD-RNA), is an essential component for m7G capping. AtDXO1 associates with and activates RNA guanosine-7 methyltransferase (AtRNMT1) to catalyze conversion of the guanosine cap to the m7G cap. AtRNMT1 is an essential gene. Partial loss-of-function mutations of AtRNMT1 and knockout mutation of AtDXO1 reduce m7G-capped mRNA but increase G-capped mRNAs, leading to similar pleiotropic phenotypes, whereas overexpression of AtRNMT1 partially restores the atdxo1 phenotypes. This work reveals an important mechanism in m7G capping in plants by which the NAD-RNA decapping enzyme AtDXO1 is required for efficient guanosine cap methylation.

Arabidopsis EXTRA-LARGE G PROTEIN 1 (XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity.

Pattern-triggered immunity (PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI. Of the three non-canonical EXTRA-LARGE G PROTEINs (XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity, but XLG1 was not considered to function in defense, based on the analysis of a weak xlg1 allele. In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern (PAMP)-triggered activation of mitogen-activated protein kinases (MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.

Growth asymmetry precedes differential auxin response during apical hook initiation in Arabidopsis.

The development of a hook-like structure at the apical part of the soil-emerging organs has fascinated botanists for centuries, but how it is initiated remains unclear. Here, we demonstrate with high-throughput infrared imaging and 2-D clinostat treatment that, when gravity-induced root bending is absent, apical hook formation still takes place. In such scenarios, hook formation begins with a de novo growth asymmetry at the apical part of a straightly elongating hypocotyl. Remarkably, such de novo asymmetric growth, but not the following hook enlargement, precedes the establishment of a detectable auxin response asymmetry, and is largely independent of auxin biosynthesis, transport and signaling. Moreover, we found that functional cortical microtubule array is essential for the following enlargement of hook curvature. When microtubule array was disrupted by oryzalin, the polar localization of PIN proteins and the formation of an auxin maximum became impaired at the to-be-hook region. Taken together, we propose a more comprehensive model for apical hook initiation, in which the microtubule-dependent polar localization of PINs may mediate the instruction of growth asymmetry that is either stochastically taking place, induced by gravitropic response, or both, to generate a significant auxin gradient that drives the full development of the apical hook.

Arabidopsis PUB2 and PUB4 connect signaling components of pattern-triggered immunity.

Plants use pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI). Precise regulation of information from PRRs to downstream signaling components is vital to mounting an appropriate immune response and requires dynamic interactions of these PTI components. We used transcriptome profiling, phenotypic analysis, molecular genetics, and protein-protein interaction analysis to understand the roles of the Arabidopsis plant U-box (PUB) proteins PUB2 and PUB4 in disease resistance and PTI signaling. Loss of function of both PUB2 and PUB4 diminishes the PAMP-triggered oxidative bursts and dampens mitogen-activated protein kinase signaling, resulting in a severe compromise in resistance to not only pathogenic but also nonpathogenic strains of Pseudomonas syringae. Within PUB4, the E3 ligase activity is dispensable, but the armadillo repeat region is essential and sufficient for its function in immunity. PUB2 and PUB4 interact with PTI signaling components, including FLS2, BIK1, PBL27, and RbohD, and enhance FLS2-BIK1 and BIK1-RbohD interactions. Our study reveals that PUB2 and PUB4 are critical components of plant immunity and connect PTI components to positively regulate defense responses.

AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana.

Pericentromeric DNA, consisting of high-copy-number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal integrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericentromeric silencing, the mechanism is still yet unclear. Here, using whole genome bisulfite sequencing (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 pericentromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyltransferases as previously thought. DNA demethylases can recognize H3K18ac mark and then be recruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an 'eraser' enzyme for H3K18ac mark to prevent DNA demethylation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethylation were shown to have transcriptionally active pericentromeric DNA.

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts.

Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5' cap in Escherichia coli, yeast, mammals, and Arabidopsis Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD+ capping.

Recent findings regarding nicotinamide adenine dinucleotide (NAD+)-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ noncanonical RNA capping to regulate gene expression. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to label NAD-RNAs. However, copper ions can fragment/degrade RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) for labeling NAD-RNAs, followed by identification of tagged RNA by single-molecule direct RNA sequencing. We used this method to compare NAD-RNA and total transcript profiles of Escherichia coli cells in the exponential and stationary phases. We identified hundreds of NAD-RNA species in E. coli and revealed genome-wide alterations of NAD-RNA profiles in the different growth phases. Although no or few NAD-RNAs were detected from some of the most highly expressed genes, the transcripts of some genes were found to be primarily NAD-RNAs. Our study suggests that NAD-RNAs play roles in linking nutrient cues with gene regulation in E. coli.

A novel pathogenicity determinant hijacks maize catalase 1 to enhance viral multiplication and infection.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

NAD tagSeq for transcriptome-wide identification and characterization of NAD+-capped RNAs.

Several noncanonical initial nucleotides (NCINs) have been found to cap RNAs and possibly regulate RNA stability, transcription and translation. NAD+ is one of the NCINs that has recently been discovered to cap RNAs in a wide range of species. Identification of the NAD+-capped RNAs (NAD-RNAs) could help to unveil the cap-mediated regulation mechanisms. We previously reported a method termed NAD tagSeq for genome-wide analysis of NAD-RNAs. NAD tagSeq is based on the previously published NAD captureSeq protocol, which applies an enzymatic reaction and a click chemistry reaction to label NAD-RNAs with biotin followed by enrichment with streptavidin resin and identification by RNA sequencing. In the current NAD tagSeq method, NAD-RNAs are labeled with a synthetic RNA tag and identified by direct RNA sequencing based on Oxford Nanopore technology. Compared to NAD captureSeq, NAD tagSeq provides a simpler procedure for direct sequencing of NAD-RNAs and noncapped RNAs and affords information on the whole sequence organization of NAD-RNAs and the ratio of NAD-RNAs to total transcripts. Furthermore, NAD-RNAs can be enriched by hybridizing a complementary DNA probe to the RNA tag, thus increasing the sequencing coverage of NAD-RNAs. The strategy of tagging RNAs with a synthetic RNA tag and identifying them by direct RNA sequencing might be employed in analyzing other NCIN-capped RNAs. The experimental procedure of NAD tagSeq, including RNA extraction, RNA tagging and direct RNA sequencing, takes ~5 d, and initial data analysis can be completed within 2 d.

New insights into Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs.

Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current 'RNA-sequencing' technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00-6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.

Emerging Roles of microRNAs in Plant Heavy Metal Tolerance and Homeostasis.

Heavy metal stress is a major growth- and yield-limiting factor for plants. Heavy metals include essential metals (copper, iron, zinc, and manganese) and non-essential metals (cadmium, mercury, aluminum, arsenic, and lead). Plants use complex mechanisms of gene regulation under heavy metal stress. MicroRNAs are 21-nucleotide non-coding small RNAs as important modulators of gene expression post-transcriptionally. Recently, high-throughput sequencing has led to the identification of an increasing number of heavy-metal-responsive microRNAs in plants. Metal-regulated microRNAs and their target genes are part of a complex regulatory network that controls various biological processes, including heavy metal uptake and transport, protein folding and assembly, metal chelation, scavenging of reactive oxygen species, hormone signaling, and microRNA biogenesis. In this review, we summarize the recent molecular studies that identify heavy-metal-regulated microRNAs and their roles in the regulation of target genes as part of the microRNA-associated regulatory network in response to heavy metal stress in plants.

Arabidopsis DXO1 possesses deNADding and exonuclease activities and its mutation affects defense-related and photosynthetic gene expression.

RNA capping and decapping tightly coordinate with transcription, translation, and RNA decay to regulate gene expression. Proteins in the DXO/Rai1 family have been implicated in mRNA decapping and decay, and mammalian DXO was recently found to also function as a decapping enzyme for NAD+ -capped RNAs (NAD-RNA). The Arabidopsis genome contains a single gene encoding a DXO/Rai1 protein, AtDXO1. Here we show that AtDXO1 possesses both NAD-RNA decapping activity and 5'-3' exonuclease activity but does not hydrolyze the m7 G cap. The atdxo1 mutation increased the stability of NAD-RNAs and led to pleiotropic phenotypes, including severe growth retardation, pale color, and multiple developmental defects. Transcriptome profiling analysis showed that the atdxo1 mutation resulted in upregulation of defense-related genes but downregulation of photosynthesis-related genes. The autoimmunity phenotype of the mutant could be suppressed by either eds1 or npr1 mutation. However, the various phenotypes associated with the atdxo1 mutant could be complemented by an enzymatically inactive AtDXO1. The atdxo1 mutation apparently enhances post-transcriptional gene silencing by elevating levels of siRNAs. Our study indicates that AtDXO1 regulates gene expression in various biological and physiological processes through its pleiotropic molecular functions in mediating RNA processing and decay.

Redox-sensitive bZIP68 plays a role in balancing stress tolerance with growth in Arabidopsis.

Perturbation of the cellular redox state by stress conditions is sensed by redox-sensitive proteins so that the cell can physiologically respond to stressors. However, the mechanisms linking sensing to response remain poorly understood in plants. Here we report that the transcription factor bZIP68 underwent in vivo oxidation in Arabidopsis cells under oxidative stress which is dependent on its redox-sensitive Cys320 residue. bZIP68 is primarily localized to the nucleus under normal growth conditions in Arabidopsis seedlings. Oxidative stress reduces its accumulation in the nucleus and increases its cytosolic localization. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that bZIP68 primarily binds to promoter regions containing the core G-box (CACGTG) or G-box-like motif of the genes involved in abiotic and biotic stress responses, photosynthesis, biosynthetic processes, and transcriptional regulation. The bzip68 mutant displayed slower growth under normal conditions but enhanced tolerance to oxidative stress. The results from the ChIP-seq and phenotypic and transcriptome comparison between the bzip68 mutant and wildtype indicate that bZIP68 normally suppresses expression of stress tolerance genes and promotes expression of growth-related genes, whereas its inactivation enhances stress tolerance but suppresses growth. bZIP68 might balance stress tolerance with growth through the extent of its oxidative inactivation according to the environment.

Analyzing and Predicting Phloem Mobility of Macromolecules with an Online Database.

Phloem, a specialized plant tissue, serves as a superhighway for macromolecular exchanges between different organs or tissues in plants. These mobile macromolecules may function as signaling molecules to sense intrinsic developmental cues or environmental inputs. Among these mobile molecules, RNAs generally need non-cell-autonomous pathway proteins (NCAPPs) to bind to and help them move along the symplasmic passage (through plasmodesmata) into the phloem stream. Grafting experiments combined with next-generation sequencing discovered that around 11.4% of identified Arabidopsis mobile mRNAs have a tRNA-like structure (TLS) motif. Adding an artificial tRNA-like structure at the 5' end of cell-autonomous RNAs (e.g., GUS transcript) can trigger its mobility and movement across a grafting junction to distant organs. Based on the accumulated data and the role of the TLS motif in RNA mobility, we built a web server in our database PLaMoM (a database for plant mobile macromolecules) to enable plant biologists to predict and analyze the transcripts they are interested in. In this chapter, we describe how to use our built-in web server to investigate RNA mobility.

NAD tagSeq reveals that NAD+-capped RNAs are mostly produced from a large number of protein-coding genes in Arabidopsis.

The 5' end of a eukaryotic mRNA transcript generally has a 7-methylguanosine (m7G) cap that protects mRNA from degradation and mediates almost all other aspects of gene expression. Some RNAs in Escherichia coli, yeast, and mammals were recently found to contain an NAD+ cap. Here, we report the development of the method NAD tagSeq for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq can allow more accurate identification and quantification of NAD-RNAs, as well as reveal the sequences of whole NAD-RNA transcripts using single-molecule RNA sequencing. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis were produced by at least several thousand genes, most of which are protein-coding genes, with the majority of these transcripts coming from <200 genes. For some Arabidopsis genes, over 5% of their transcripts were NAD capped. Gene ontology terms overrepresented in the 2,000 genes that produced the highest numbers of NAD-RNAs are related to photosynthesis, protein synthesis, and responses to cytokinin and stresses. The NAD-RNAs in Arabidopsis generally have the same overall sequence structures as the canonical m7G-capped mRNAs, although most of them appear to have a shorter 5' untranslated region (5' UTR). The identification and quantification of NAD-RNAs and revelation of their sequence features can provide essential steps toward understanding the functions of NAD-RNAs.