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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. This article is protected by copyright. All rights reserved.

Three MAPK cascade components in Arabidopsis, YDA (MAPKKK4) and MAPKKK3/5, function redundantly in multiple developmental processes and immunity and regulate floral organ abscission.

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 took place, induced by gravitropic response, or both, to generate a significant auxin gradient that drives the full development of apical hook. This article is protected by copyright. All rights reserved.

Pathogen infection induces massive reprogramming of host primary metabolism. Lipid and fatty acid metabolism is generally disrupted by pathogens and co-opted for their proliferation. Lipid droplets (LDs) that play important roles in regulating cellular lipid metabolism are utilized by a variety of pathogens in mammalian cells. However, the function of LDs during pathogenic infection in plants remains unknown. We show here that infection by rice black streaked dwarf virus (RBSDV) affects the lipid metabolism of maize, which causes elevated accumulation of C18 polyunsaturated fatty acids (PUFAs) leading to viral proliferation and symptom development. Overexpression of one of the two novel LD-associated proteins (LDAPs) of maize (ZmLDAP1 and ZmLDAP2) induces LD clustering. The core capsid protein P8 of RBSDV interacts with ZmLDAP2 and prevents its degradation through the ubiquitin-proteasome system mediated by a UBX domain-containing protein, PUX10. In addition, silencing of theZmLDAP2down-regulates expression of fatty acid desaturase genes in maize, leading to a decrease in C18 PUFAs levels and suppression of RBSDV accumulation. Our findings reveal that the plant virus recruits LDAP to regulate cellular fatty acid metabolism to promote viral multiplication and infection. These results expand the knowledge of the LD functions and viral infection mechanism in plants.One Sentence SummaryRice black streaked dwarf virus recruits a lipid droplet-associated protein to regulate cellular fatty acid metabolism for promoting 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.

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. NAD+ is one of the noncanonical nucleotides recently found to cap the 5′ end of RNAs. This Protocol Extension describes procedures for genome-wide analysis of NAD+-capped RNAs by direct RNA sequencing on an Oxford Nanopore platform.

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.

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.

The 5′ end of a eukaryotic mRNA transcript generally has a 7-methylguanosine (m 7 G) 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 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 m 7 G-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.

As the most common RNA cap in eukaryotes, the 7-methylguanosine (m 7 G) cap impacts nearly all processes that a messenger RNA undergoes, such as splicing, polyadenylation, nuclear export, translation, and degradation. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD + ), can be used as an initiating nucleotide in RNA synthesis to result in NAD + -capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD + cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD + -capped RNAs are widespread in Arabidopsis thaliana . NAD + -capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes, but not the chloroplast genome. NAD + -capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD + -capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms whereby the RNA NAD + cap interfaces with RNA metabolic processes as well as translation initiation. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, or be regulated by, mRNA NAD + capping.

Significance Some RNAs in both prokaryotes and eukaryotes were recently found to contain the NAD+ cap, indicating a novel mechanism in gene regulation through noncanonical RNA capping. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has been used to label NAD+-capped RNAs (NAD-RNAs) for their identification. However, copper-caused RNA fragmentation/degradation interferes with the analysis. We developed the NAD tagSeq II method for transcriptome-wide NAD-RNA analysis based on copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. This method was used to compare NAD-RNA and total transcriptome profiles in Escherichia coli. Our study reveals genome-wide alterations in E. coli RNA NAD+ capping in different growth phases and indicates that NAD-RNAs could be the primary form of transcripts from some genes under certain environments., 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.

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 m(7)G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m(7)G-capped RNAs (m(7)G-RNAs). In addition, the Cu(2+) 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 m(7)G-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 m(7)G-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 m(7)G-RNA depletion, in eukaryotes.

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.

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.

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 m(7)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.

NAD tagSeq has recently been developed for the identification and characterization of NAD+-capped RNAs (NAD-RNAs). This method adopts a strategy of chemo-enzymatic reactions to label the NAD-RNAs with a synthetic RNA tag before subjecting to the Oxford Nanopore direct RNA sequencing. A computational tool designed for analyzing the sequencing data of tagged RNA will facilitate the broader application of this method. Hence, we introduce TagSeqTools as a flexible, general pipeline for the identification and quantification of tagged RNAs (i.e., NAD+-capped RNAs) using long-read transcriptome sequencing data generated by NAD tagSeq method. TagSeqTools comprises two major modules, TagSeek for differentiating tagged and untagged reads, and TagSeqQuant for the quantitative and further characterization analysis of genes and isoforms. Besides, the pipeline also integrates some advanced functions to identify antisense or splicing, and supports the data reformation for visualization. Therefore, TagSeqTools provides a convenient and comprehensive workflow for researchers to analyze the data produced by the NAD tagSeq method or other tagging-based experiments using Oxford nanopore direct RNA sequencing. The pipeline is available at https://github.com/dorothyzh/TagSeqTools, under Apache License 2.0.

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.

Background Homeostasis of the proteome is critical to the development of chloroplasts and also affects the expression of certain nuclear genes. CLPC1 facilitates the translocation of chloroplast pre-proteins and mediates protein degradation. Results We found that proteins involved in photosynthesis are dramatically decreased in their abundance in the clpc1 mutant, whereas many proteins involved in chloroplast transcription and translation were increased in the mutant. Expression of the full-length CLPC1 protein, but not of the N-terminus-deleted CLPC1 (ΔN), in the clpc1 mutant background restored the normal levels of most of these proteins. Interestingly, the ΔN complementation line could also restore some proteins affected by the mutation to normal levels. We also found that that the clpc1 mutation profoundly affects transcript levels of chloroplast genes. Sense transcripts of many chloroplast genes are up-regulated in the clpc1 mutant. The level of SVR7, a PPR protein, was affected by the clpc1 mutation. We showed that SVR7 might be a target of CLPC1 as CLPC1-SVR7 interaction was detected through co-immunoprecipitation. Conclusion Our study indicates that in addition to its role in maintaining proteome homeostasis, CLPC1 and likely the CLP proteasome complex also play a role in transcriptome homeostasis through its functions in maintaining proteome homeostasis. Electronic supplementary material The online version of this article (10.1186/s12870-018-1396-0) contains supplementary material, which is available to authorized users.

Background Disulfide bonds are traditionally considered to play only structural roles. In recent years, increasing evidence suggests that the disulfide proteome is made up of structural disulfides and reversible disulfides. Unlike structural disulfides, reversible disulfides are usually of important functional roles and may serve as redox switches. Interestingly, only specific disulfide bonds are reversible while others are not. However, whether reversible disulfides can be predicted based on structural information remains largely unknown. Methods In this study , two datasets with both types of disulfides were compiled using independent approaches. By comparison of various features extracted from the local structural signatures, we identified several features that differ significantly between reversible and structural disulfides, including disulfide bond length, along with the number, amino acid composition, secondary structure and physical-chemical properties of surrounding amino acids. A SVM-based classifier was developed for predicting reversible disulfides. Results By 10-fold cross-validation, the model achieved accuracy of 0.750, sensitivity of 0.352, specificity of 0.953, MCC of 0.405 and AUC of 0.751 using the RevSS_PDB dataset. The robustness was further validated by using RevSS_RedoxDB as independent testing dataset. This model was applied to proteins with known structures in the PDB database. The results show that one third of the predicted reversible disulfide containing proteins are well-known redox enzymes, while the remaining are non-enzyme proteins. Given that reversible disulfides are frequently reported from functionally important non-enzyme proteins such as transcription factors, the predictions may provide valuable candidates of novel reversible disulfides for further experimental investigation. Conclusions This study provides the first comparative analysis between the reversible and the structural disulfides. Distinct features remarkably different between these two groups of disulfides were identified, and a SVM-based classifier for predicting reversible disulfides was developed accordingly. A web server named RevssPred can be accessed freely from: http://biocomputer.bio.cuhk.edu.hk/RevssPred. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3668-8) contains supplementary material, which is available to authorized users.

Several noncanonical initial nucleotides (NCINs) have been found to cap RNAs and possibly regulate RNA stability, transcription and translation. NAD

Significance The 5′ end of a eukaryotic messenger RNA generally contains an 7-methylguanosine (m7G) cap, which has an essential role in regulating gene expression. Recent discoveries of RNAs with a noncanonical NAD+ moiety indicate the existence of a previously unknown mechanism for controlling gene expression. We have developed a method termed NAD tagSeq for the accurate identification and quantification of NAD+-capped RNAs and for revealing the complete sequences of NAD-RNAs using single-molecule RNA sequencing. Using this method, we found that NAD-RNAs in Arabidopsis were mostly derived from protein-coding genes and that they have essentially the same overall sequence structures as the canonical m7G-RNAs. The identification of NAD-RNAs and their sequence structures facilitates the elucidation of their possible molecular and physiological functions., 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

As the most common RNA cap in eukaryotes, the 7-methylguanosine (m(7)G) cap impacts nearly all processes that a messenger RNA undergoes, such as splicing, polyadenylation, nuclear export, translation, and degradation. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD(+)), can be used as an initiating nucleotide in RNA synthesis to result in NAD(+)-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD(+) cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD(+)-capped RNAs are widespread in Arabidopsis thaliana. NAD(+)-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes, but not the chloroplast genome. NAD(+)-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD(+)-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms whereby the RNA NAD(+) cap interfaces with RNA metabolic processes as well as translation initiation. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, or be regulated by, mRNA NAD(+) capping.

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.

Sexual dimorphism like dioecy (separate male and female individuals) have evolved in diverse multicellular eukaryotes while the molecular mechanisms underlying the development of such a key biological trait remains elusive (1). The living fossil Ginkgo biloba represents an early diverged lineage of land plants with dioecy. However, its sex-determination system and molecular basis have long been controversial or unknown. In the present research, we assembled the first and largest to date chromosome-level genome of a non-model tree species using Hi-C data. With this reference genome, we addressed both questions using genome resequencing data gathered from 97 male and 265 female trees of ginkgo, as well as transcriptome data from three developmental stages for both sexes. Our results support vertebrate-like XY chromosomes for ginkgo and five potential sex-determination genes, which may originate ~14 million years ago. This is the earliest diverged sex determination region in all reported plants as yet. The present research resolved a long-term controversy, lay a foundation for future studies on the origin and evolution of plant sexes, and provide genetic markers for sex identification of ginkgo which will be valuable for both nurseries and field ecology of ginkgo.

Oxidative burst is one of the earliest plant cellular responses triggered by pathogen infection. Reactive oxygen species can cause oxidative modifications of redox-sensitive proteins to mediate the defense responses. Identification and characterization of proteins that undergo oxidative modifications in these processes is an important step toward understanding molecular mechanisms of plant defense responses. In this study, an in vivo 15N metabolic labeling method combined with a cysteine-containing peptide enrichment technique was applied to identify and quantify proteins and their redox states in Arabidopsis in response to infection by Pseudomonas syringae pv tomato DC3000 (Pst). Changes of peptide redox states were compared and corrected with the changes of protein levels. A total of forty peptides representing thirty-six non-redundant proteins showed significantly redox state changes in response to the infection by the virulent Pst strain and the avirulent Pst strain (Pst avrRpm1), of which 23 had previously not been recognized to undergo oxidative PTMs. The differentially expressed redox-sensitive proteins are involved in cell wall organization, primary metabolism, photosynthesis and stress responses. Interestingly, proteins located at extracellular were more susceptible to be regulated on the redox PTMs level. These findings provide a foundation for further investigation into the redox signaling during plant defense responses.

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.

Scenedesmus quadricauda, a local freshwater microalga, was used to treat primary settled and filtrate wastewater and to produce algal lipid. For the primary settled wastewater, the maximum biomass concentrations of acclimated and unacclimated microalgae were 0.995 g/L and 0.940 g/L, respectively. Over 90% orthophosphate and 95% ammonia nitrogen in the acclimated and unacclimated cultures, respectively, were removed after five days. The lipid contents of the microalgae were higher than 13% in all cultures. The highest growth rate occurred in the 25% filtrate culture. Over 80% phosphate was removed under the 25% and 50% filtrate cultures within six days, while over 90% ammonia nitrogen was removed within five days under both conditions. The lipid content was the highest (18.1%) under the 50% filtrate condition. C16:0, C18:2n6, and C18:3n3 were dominant fatty acids. In conclusion, S. quadricauda is a viable candidate for wastewater treatment and lipid production.

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein-protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/.

In recent years, concerns about using Bisphenol A (BPA) in daily consume products and its effects in many chronic human diseases have prompted the removal of BPA. However, the widely used BPA alternatives, including Bisphenol S (BPS), have a high structural similarity with BPA, suggesting that they may have similar biological effects towards human beings. Indeed, BPS was also found to have endocrine-disrupting effects. Epigenetic mechanism was reported to be involved in BPA-induced biological effects in both in vitro and in vivo models. However, there is no assessment on whether BPS could cause epigenetic changes. In this work, we investigated the possible epigenetic effects of BPS that might induce in human breast cancer cell line MCF-7. We found that BPS could change DNA methylation level of transposons. Besides, methylation status in promoter of breast cancer related genes CDH1, SFN, TNFRSF10C were also changed, which implied that BPS might play a role in the development of breast cancer. Gene expression profiling showed that some genes related to breast cancer progression were upregulated, including THBS4, PPARGC1A, CREB5, COL5A3. Gene ontology (GO) analysis of the differentially expressed genes revealed the significantly changes in PI3K-Akt signaling pathway and extracellular matrix, which were related to the proliferation, migration and invasion of breast cancer cells. These results illustrated that BPS exposure might play roles in the progression of breast cancer.

Cuticular waxes are complex mixtures of very long chain fatty acids and their derivatives that cover plant surfaces. Mutants of the ECERIFERUM2 {cer2) gene of Arabidopsis condition bright green stems and siliques, indicative of the relatively low abundance of the cuticular wax crystals that comprise the wax bloom on wild-type plants. We cloned the CER2 gene via chromosome walking. Three lines of evidence establish that the cloned sequence represents the CER2 gene: (1) this sequence is capable of complementing the cer2 mutant phenotype in transgenic plants; (2) the corresponding DNA sequence isolated from plants homozygous for the cer2-2 mutant allele contains a sequence polymorphism that generates a premature stop codon; and (3) the deduced CER2 protein sequence exhibits sequence similarity to that of a maize gene (glossy2) that is also involved in cuticular wax •Reprinted with permission of The Plant Cell, 1996, 8:1291-1304. 2Graduate student and primary researcher and author. Interdepartmental Genetics Program, Iowa State University. ^Associate Professor, Department of Biochemistry and Biophysics, Iowa State University. ^Associate Professor, Departments of Agronomy, and Zoology and Genetics, Iowa State University. ^Author for correspondence

Table S1. Proteases accumulation in seedlings of different genotypes. Table S2. Primers used in the study. Table S3. Peptides identified of CLPC1 in SVR7-GFP CO-IP experiment. Figure S1. Relative expression levels of sense transcripts in the clpc1 mutant and its complementation lines. Figure S2. Over-expressing CLPC2 in clpc1 mutant partially or fully restored the chloroplast RNA level. Figure S3. Schematic diagram to show the plant materials we used. Supplementary dataset 1. Spectral examples of 4 proteins from our iTRAQ-based proteomics analysis. (PDF 2601 kb)

Growing evidences indicate that calcium-dependent protein kinases (CDPKs) are involved in many aspects of plant’s growth, development and responses to biotic and abiotic stresses. However, the biological roles of most CDPKs remain unclear. Here we report that CDPK1 is induced by infection with both virulent and avirulent Pseudomonas syringae strains. To understand the biological function of CDPK1, we analyzed phenotypes of the T-DNA insertion line (cpk10) which did not express the CDPK1 gene. The cpk10 mutant exhibited deduced expression of the defense-related genes PR1, PR2, and AIG1 following inoculation with the avirulent pathogen Pst AvrRpt2. In addition, the CDPK1 gene was found to be expressed in guard cells and the cpk10 mutant showed alteration in stomata closure after Pst DC3000 infection. We also found that expression of CDPK1 is induced by plant hormones salicylic acid, methyl jasmonate, and abscisic acid. These results indicate that CDPK1 may be involved in plant defense response.

Highlight DIG6 encodes a large 60S subunit GTPase 1 and affects ribosome biogenesis, and auxin response and homeostasis., The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

Heterotrimeric GTP-binding protein (G-protein)-mediated abscisic acid (ABA) and drought-stress responses have been documented in numerous plant species. However, our understanding of the function of rice G-protein subunits in ABA signalling and drought tolerance is limited. In this study, the function of G-protein subunits in ABA response and drought resistance in rice plants was explored. It was found that the transcription level of qPE9-1 (rice Gγ subunit) gradually decreased with increasing ABA concentration and the lack of qPE9-1 showed an enhanced drought tolerance in rice plants. In contrast, mRNA levels of RGB1 (rice Gβ subunit) were significantly upregulated by ABA treatment and the lack of RGB1 led to reduced drought tolerance. Furthermore, the results suggested that qPE9-1 negatively regulates the ABA response by suppressing the expression of key transcription factors involved in ABA and stress responses, while RGB1 positively regulates ABA biosynthesis by upregulating NCED gene expression under both normal and drought stress conditions. Taken together, it is proposed that RGB1 is a positive regulator of the ABA response and drought adaption in rice plants, whereas qPE9-1 is modulated by RGB1 and functions as a negative regulator in the ABA-dependent drought-stress responses.

Lowland rice (Nipponbare) and upland rice (Gaoshan 1) that are comparable under normal and moderate drought conditions showed dramatic differences in severe drought conditions, both naturally occurring long-term drought and simulated rapid water deficits. We focused on their root response and found that enhanced tolerance of upland rice to severe drought conditions was mainly due to the lower level of ABA in its roots than in those of the lowland rice. We first excluded the effect of ABA biosynthesis and catabolism on root-accumulated ABA levels in both types of rice by monitoring the expression of four OsNCED genes and two OsABA8ox genes. Next, we excluded the impact of the aerial parts on roots by suppressing leaf-biosynthesized ABA with fluridone and NDGA (nordihydroguaiaretic acid), and measuring the ABA level in detached roots. Instead, we proved that upland rice had the ability to export considerably more root-sourced ABA than lowland rice under severe drought, which improved ABA-dependent drought adaptation. The investigation of apoplastic pH in root cells and root anatomy showed that ABA leakage in the root system of upland rice was related to high apoplastic pH and the absence of Casparian bands in the sclerenchyma layer. Finally, taking some genes as examples, we predicted that different ABA levels in rice roots stimulated distinct ABA perception and signaling cascades, which influenced its response to water stress.

Highlight AtDsPTP1 was found to regulate ABA accumulation and act as a negative regulator in osmotic stress signalling during Arabidospsis seed germination and seedling establishment., Dual-specificity protein phosphatases (DsPTPs) target both tyrosine and serine/threonine residues and play roles in plant growth and development. We have characterized an Arabidopsis mutant, dsptp1, which shows a higher seed germination rate and better root elongation under osmotic stress than the wild type. By contrast, its overexpression line, DsPTP1-OE, shows inhibited seed germination and root elongation; and its complemented line, DsPTP1-Com, resembles the wild type and rescues DsPTP1-OE under osmotic stress. Expression of AtDsPTP1 is enhanced by osmotic stress in seed coats, bases of rosette leaves, and roots. Compared with the wild type, the dsptp1 mutant shows increased proline accumulation, reduced malondialdehyde (MDA) content and ion leakage, and enhanced antioxidant enzyme activity in response to osmotic stress. AtDsPTP1 regulates the transcript levels of various dehydration-responsive genes under osmotic stress. Abscisic acid (ABA) accumulation in dsptp1 under osmotic stress is reduced with reduced expression of the ABA-biosynthesis gene NCED3 and increased expression of the ABA-catabolism gene CYP707A4. AtDsPTP1 also regulates the expression of key components in the ABA-signalling pathway. In conclusion, AtDsPTP1 regulates ABA accumulation, and acts as a negative regulator in osmotic stress signalling during Arabidospsis seed germination and seedling establishment.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are well-known for their role in controlling membrane fusion, the final, but crucial step, in vesicular transport in eukaryotes. SNARE proteins contribute to various biological processes including pathogen defense and channel activity regulation, as well as plant growth and development. Precise targeting of SNARE proteins to destined compartments is a prerequisite for their proper functioning. However, the underlying mechanism(s) for SNARE targeting in plants remains obscure. Here, we investigate the targeting mechanism of the

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are well-known for their role in controlling membrane fusion, the final, but crucial step, in vesicular transport in eukaryotes. SNARE proteins contribute to various biological processes including pathogen defense and channel activity regulation, as well as plant growth and development. Precise targeting of SNARE proteins to destined compartments is a prerequisite for their proper functioning. However, the underlying mechanism(s) for SNARE targeting in plants remains obscure. Here, we investigate the targeting mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is involved in protein trafficking in the early secretory pathway. Two distinct signal motifs that are required for efficient BET12 ER export were identified. Pulldown assays and in vivo imaging implicated that both the COPI and COPII pathways were required for BET12 targeting. Further studies using an ER-export-defective form of BET12 revealed that the Golgi-localized Qb-SNARE MEMB12, a negative regulator of pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its interacting partner. Ectopic expression of BET12 caused no inhibition in the general ER-Golgi anterograde transport but caused intracellular accumulation of PR1, suggesting that BET12 has a regulatory role in PR1 trafficking in A. thaliana.

Enzyme classification of proteins with predicted reversible disulfides in human (PDF). This figure was modified from the “Enzyme Classification” result obtained from PDB database. Only reprehensive proteins of less than 30% similarity to each other, and of less than 30% similarity to any of the RevssPDB sequences were used for generating this figure. Proteins not assigned to known enzyme groups were labeled as “Unknown”. (PDF 43 kb)

Amino acid composition of the structural signatures for reversible and structural disulfides (PDF). x-axis denotes amino acid types, and y-axis gives the fraction of each amino acid in the structural signatures. Statistical significance was determined by Two-tailed Student's t-test, and Bonferroni-adjusted p-values were denoted by * when p < 0.05, ** when p < 0.01, and *** when p < 0.001. (PDF 101 kb)

Enzyme classification of reversible disulfide containing proteins in the training datasets (PDF). This figure was modified from the “Enzyme Classification” result obtained from PDB database. Only reprehensive proteins of less than 30% similarity to each other are used to generate this figure. Proteins not assigned to known enzyme groups were labeled as “Unknown”. (PDF 46 kb)

Extraction of structural signatures surrounding disulfide in the protein structure (PDF). (a) The three dimensional structure of OxyR (PDB_ID is 1L6A) which is a transcription factor important for oxidative stress response in bacteria. The disulfide bond formed between C199 and C208 is shown as a pink bar. (b) For each Cys, the surrounding segments (within 10Å to disulfide-bonded Cys) are extracted and then combined into the so-called â structural signaturesâ according to their primary sequence, respectively. The signatures from the two disulfide-bonded Cys are then merged together for analysis. (PDF 374 kb)