An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that "mitochondrial translation factors" mTRAN1 and mTRAN2 are land plant-specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)-like RNA binding proteins of the mitoribosomal "small" subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5' regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

MicroRNAs from Holarrhena pubescens stems: Identification by small RNA Sequencing and their Potential Contribution to Human Gene Targets.

Holarrhena pubescens is an effective medicinal plant from the Apocynaceae family, widely distributed over the Indian subcontinent and extensively used by Ayurveda and ethno-medicine systems without apparent side effects. We postulated that miRNAs, endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level, may, after ingestion into the human body, contribute to the medicinal properties of plants of this species by inducing regulated human gene expression to modulate. However, knowledge is scarce about miRNA in Holarrhena. In addition, to test the hypothesis on the potential pharmacological properties of miRNA, we performed a high-throughput sequencing analysis using the Next Generation Sequencing Illumina platform; 42,755,236 raw reads have been generated from H. pubescens stems from a library of small RNA isolated, identifying 687 known and 50 new miRNAs led. The novel H. pubescens miRNAs were predicted to regulate specific human genes, and subsequent annotations of gene functions suggested a possible role in various biological processes and signaling pathways, such as Wnt, MAPK, PI3K-Akt, and AMPK signaling pathways and endocytosis. The association of these putative targets with many diseases, including cancer, congenital malformations, nervous system disorders, and cystic fibrosis, has been demonstrated. The top hub proteins STAT3, MDM2, GSK3B, NANOG, IGF1, PRKCA, SNAP25, SRSF1, HTT, and SNCA show their interaction with human diseases, including cancer and cystic fibrosis. To our knowledge, this is the first report of uncovering H. pubescens miRNAs based on high-throughput sequencing and bioinformatics analysis. This study has provided new insight into a potential cross-species control of human gene expression. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for the beneficial properties of this valuable species.

RETINOBLASTOMA-RELATED interactions with key factors of the RNA-directed DNA methylation (RdDM) pathway and its influence on root development.

MAIN CONCLUSION: Our study presents evidence for a novel mechanism for RBR function in transcriptional gene silencing by interacting with key players of the RdDM pathway in Arabidopsis and several plant clades. Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double-stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs serve as guides to direct AGO4-siRNA complexes to chromatin-bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. The Arabidopsis Retinoblastoma protein homolog (RBR) is a master regulator of the cell cycle, stem cell maintenance, and development. We in silico predicted and explored experimentally the protein-protein interactions (PPIs) between RBR and members of the RdDM pathway. We found that the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2, and SUVR2 contain canonical and non-canonical RBR binding motifs and several of them are conserved since algae and bryophytes. We validated experimentally PPIs between Arabidopsis RBR and several of the RdDM pathway proteins. Moreover, seedlings from loss-of-function mutants in RdDM and RBR show similar phenotypes in the root apical meristem. We show that RdDM and SUVR2 targets are up-regulated in the 35S:AmiGO-RBR background.

A chaperonin containing T-complex polypeptide-1 facilitates the formation of the PbWoxT1-PbPTB3 ribonucleoprotein complex for long-distance RNA trafficking in Pyrus betulaefolia.

Numerous plant endogenous mRNAs move via phloem and thus affect the growth and development of long-distant organs. mRNAs are transported with RNA-binding proteins forming a ribonucleoprotein complex. However, it remains elusive how such RNP complex assembles and facilitates mRNA trafficking. Protease digestion and RNA immunoprecipitation were used to investigate the RNP assembly function of the complete Chaperonin Containing T-complex Polypeptide-1. In situ hybridization, hairy root transformation, microprojectile bombardment, and grafting experiments demonstrate the role of CCT complex in the transport of a PbWoxT1-PbPTB3 RNP complex in Pyrus betulaefolia. PbCCT5 silenced caused defective movement of GFP-PbPTB3 and GFP-PbWoxT1 from hairy roots to new leaves via the phloem. PbCCT5 is shown to interact with PbPTB3. PbCCT complex enhanced PbPTB3 stabilization and permitted assembly of PbWoxT1 and PbPTB3 into an RNP complex. Furthermore, silencing of individual CCT subunits inhibited the intercellular movement of GFP-PbPTB3 and long-distance trafficking of PbWoxT1 and PbPTB3 in grafted plants. Taken together, the CCT complex assembles PbPTB3 and PbWoxT1 into an RNP complex in the phloem in order to facilitate the long-distance trafficking of PbWoxT1 in P. betulaefolia. This study therefore provides important insights into the mechanism of RNP complex formation and transport.

Cotton (Gossypium hirsutum) VIRMA as an N6-Methyladenosine RNA Methylation Regulator Participates in Controlling Chloroplast-Dependent and Independent Leaf Development.

N6-methyladenosine (m6A) is one of the most abundant internal modifications of mRNA, which plays important roles in gene expression regulation, and plant growth and development. Vir-like m6A methyltransferase associated (VIRMA) serves as a scaffold for bridging the catalytic core components of the m6A methyltransferase complex. The role of VIRMA in regulating leaf development and its related mechanisms have not been reported. Here, we identified and characterized two upland cotton (Gossypium hirsutum) VIRMA genes, named as GhVIR-A and GhVIR-D, which share 98.5% identity with each other. GhVIR-A and GhVIR-D were ubiquitously expressed in different tissues and relatively higher expressed in leaves and main stem apexes (MSA). Knocking down the expression of GhVIR genes by the virus-induced gene silencing (VIGS) system influences leaf cell size, cell shape, and total cell numbers, thereby determining cotton leaf morphogenesis. The dot-blot assay and colorimetric experiment showed the ratio of m6A to A in mRNA is lower in leaves of GhVIR-VIGS plants compared with control plants. Messenger RNA (mRNA) high-throughput sequencing (RNA-seq) and a qRT-PCR experiment showed that GhVIRs regulate leaf development through influencing expression of some transcription factor genes, tubulin genes, and chloroplast genes including photosystem, carbon fixation, and ribosome assembly. Chloroplast structure, chlorophyll content, and photosynthetic efficiency were changed and unsuitable for leaf growth and development in GhVIR-VIGS plants compared with control plants. Taken together, our results demonstrate GhVIRs function in cotton leaf development by chloroplast dependent and independent pathways.

Organellar transcripts dominate the cellular mRNA pool across plants of varying ploidy levels.

Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA sequencing (RNA-seq) to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9 to 82.3%) of messenger RNA (mRNA) transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3 to 3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than 10-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.

Resistance to RNA interference by plant-derived double-stranded RNAs but not plant-derived short interfering RNAs in Helicoverpa armigera.

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Mitochondrion-encoded circular RNAs are widespread and translatable in plants.

Nucleus-encoded circular RNAs (ncircRNAs) have been widely detected in eukaryotes, and most circRNA identification algorithms are designed to identify them. However, using these algorithms, few mitochondrion-encoded circRNAs (mcircRNAs) have been identified in plants, and the role of plant mcircRNAs has not yet been addressed. Here, we developed a circRNA identification algorithm, mitochondrion-encoded circRNA identifier, based on common features of plant mitochondrial genomes. We identified 7,524, 9,819, 1,699, 1,821, 1,809, and 5,133 mcircRNAs in maize (Zea mays), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and grape (Vitis vinifera), respectively. These mcircRNAs were experimentally validated. Plant mcircRNAs had distinct characteristics from ncircRNAs, and they were more likely to be derived from RNA degradation but not intron backsplicing. Alternative circularization was prevalent in plant mitochondria, and most parental genomic regions hosted multiple mcircRNA isoforms, which have homogenous 5' termini but heterogeneous 3' ends. By analysis of mitopolysome and mitoribosome profiling data, 1,463 mcircRNAs bound to ribosomes were detected in maize and Arabidopsis. Further analysis of mass spectrometry-based proteomics data identified 358 mcircRNA-derived polypeptides. Overall, we developed a computational pipeline that efficiently identifies plant mcircRNAs, and we demonstrated mcircRNAs are widespread and translated in plants.

The elemental defense effect of cadmium on Alternaria brassicicola in Brassica juncea.

BACKGROUND: The elemental defense hypothesis states a new defensive strategy that hyperaccumulators defense against herbivores or pathogens attacks by accumulating heavy metals. Brassica juncea has an excellent ability of cadmium (Cd) accumulation. However, the elemental defense effect and its regulation mechanism in B. juncea remain unclear. RESULTS: In this study, we profiled the elemental defense effect and the molecular regulatory mechanism in Cd-accumulated B. juncea after Alternaria brassicicola infection. B. juncea treated with 180 mg Kg- 1 DW CdCl2 2.5H2O exhibited obvious elemental defense effect after 72 h of infection with A. brassicicola. The expression of some defense-related genes including BjNPR1, BjPR12, BjPR2, and stress-related miRNAs (miR156, miR397, miR398a, miR398b/c, miR408, miR395a, miR395b, miR396a, and miR396b) were remarkably elevated during elemental defense in B. juncea. CONCLUSIONS: The results indicate that Cd-accumulated B. juncea may defend against pathogens by coordinating salicylic acid (SA) and jasmonic acid (JA) mediated systemic acquired resistance (SAR) and elemental defense in a synergistic joint effect. Furthermore, the expression of miRNAs related to heavy metal stress response and disease resistance may regulate the balance between pathogen defense and heavy metal stress-responsive in B. juncea. The findings provide experimental evidence for the elemental defense hypothesis in plants from the perspectives of phytohormones, defense-related genes, and miRNAs.

Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA-sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco.

KEY MESSAGE: Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA-gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos. While gene-editing methodologies, such as CRISPR-Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA-sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.

Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte.

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector.

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues.

Acquiring high-quality RNA in sufficient amounts is crucial in plant molecular biology and genetic studies. Several methods for RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only a few molecular studies are available for the species. One reason is the difficulty in obtaining RNA due to the content of the samples, which are rich in polyphenols, polysaccharides, and secondary metabolites. Furthermore, there are few studies available for the isolation of RNA from guava or Psidium samples, which hampers advances in the study of the genus. Here, quality and yields of RNA isolates were compared using six extraction protocols: two protocols based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, one protocol which uses the TRIzol reagent, one which applies guanidine thiocyanate lysis buffer followed by organic phase extraction, and two commercial kits (PureLink RNA Mini Kit and RNeasy Plant Mini Kit). The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf, and root), genotypes, and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaves was 203.06 µg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-qPCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments. CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they were compatible for downstream RNA-based applications, besides being simple and cost-effective.

LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance.

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

Long noncoding RNA lncRNA354 functions as a competing endogenous RNA of miR160b to regulate ARF genes in response to salt stress in upland cotton.

Long non-coding RNAs (lncRNAs) play important roles in response to biotic and abiotic stress through acting as competing endogenous RNAs (ceRNAs) to decoy mature miRNAs. However, whether this mechanism is involved in cotton salt stress response remains unknown. We report the characterization of an endogenous lncRNA, lncRNA354, whose expression was reduced in salt-treated cotton and was localized at the nucleus and cytoplasm. Using endogenous target mimic (eTM) analysis, we predicted that lncRNA354 had a potential binding site for miR160b. Transient expression in tobacco demonstrated that lncRNA354 was a miR160b eTM and attenuated miR160b suppression of its target genes, including auxin response factors (ARFs). Silencing or overexpressing lncRNA354 affected the expression of miR160b and target ARFs. Silencing lncRNA354 and targets GhARF17/18 resulted in taller cotton plants and enhanced the resistant to salt stress. Overexpression of lncRNA354 and targets GhARF17/18 in Arabidopsis led to dwarf plants, decreased root dry weight and reduced salt tolerance. Our results show that the lncRNA354-miR160b effect on GhARF17/18 expression may modulate auxin signalling and thus affect growth. These results also shed new light on a mechanism of lncRNA-associated responses to salt stress.

N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent manner.

BACKGROUND: Epigenetic mark such as DNA methylation plays pivotal roles in regulating ripening of both climacteric and non-climacteric fruits. However, it remains unclear whether mRNA m6A methylation, which has been shown to regulate ripening of the tomato, a typical climacteric fruit, is functionally conserved for ripening control among different types of fruits. RESULTS: Here we show that m6A methylation displays a dramatic change at ripening onset of strawberry, a classical non-climacteric fruit. The m6A modification in coding sequence (CDS) regions appears to be ripening-specific and tends to stabilize the mRNAs, whereas m6A around the stop codons and within the 3' untranslated regions is generally negatively correlated with the abundance of associated mRNAs. We identified thousands of transcripts with m6A hypermethylation in the CDS regions, including those of NCED5, ABAR, and AREB1 in the abscisic acid (ABA) biosynthesis and signaling pathway. We demonstrate that the methyltransferases MTA and MTB are indispensable for normal ripening of strawberry fruit, and MTA-mediated m6A modification promotes mRNA stability of NCED5 and AREB1, while facilitating translation of ABAR. CONCLUSION: Our findings uncover that m6A methylation regulates ripening of the non-climacteric strawberry fruit by targeting the ABA pathway, which is distinct from that in the climacteric tomato fruit.

Mdm-MIR393b-mediated adventitious root formation by targeted regulation of MdTIR1A expression and weakened sensitivity to auxin in apple rootstock.

Adventitious root (AR) formation is of great significance for apple rootstock breeding. It is widely accepted that miR393 influences AR formation in many plant species; however, the molecular mechanism by which factors regulate AR formation remains insufficient. In this study, the evolutionary relationship of mdm-miR393 and candidate target genes MdTIR1/AFB was systematically identified, and the expression patterns were analysed. Multisequence alignment analysis of miR393 family members suggests that miR393 conservatively evolved between different species. The evolutionary relationship of the TIR1/AFBs can be divided into G1, G2 and G3 subgroups. During AR formation, the expression level of mdm-miR393a/b/c was significantly upregulated at 1 d and 7 d by exogenous auxin treatment. Furthermore, the expression levels of MdTIR1A, MdTIR1D, MdAFB1, MdAFB2, MdAFB3, MdAFB4 and MdAFB8 also appeared to be significantly changed by exogenous auxin induction. Subsequently, tissue-specific expression analysis showed that the expression levels of mdm-miR393 and MdTIR1/AFBs in different tissues exhibited significant differences. The promoter of mdm-miR393 contains multiple elements that respond to ABA, adversity and light signals; auxin treatment can activate the mdm-MIR393b promoter but is obviously inhibited by NPA treatment. The targeting relationship between mdm-MIR393b and MdTIR1A was verified by expression patterns, degradation group data, transient tobacco conversion results, and genes functions experiments. Heterologous overexpression of mdm-MIR393b (35S::mdm-MIR393b) decreased the number of ARs in the phenotype and reduced the expression level of the target gene NtTIR1 in tobacco. Compared to the wild type, the 35S::mdm-MIR393b transgenic plants demonstrated insensitivity to auxin. Furthermore, tir1 mutant exhibited reduced root system structure relative to the control. The above results illustrated that mdm-MIR393b is involved in mediating AR formation by targeted regulation of MdTIR1A expression in apple rootstock.

N6 -Methyladenosine mRNA methylation is important for salt stress tolerance in Arabidopsis.

As the most abundant internal modification of mRNA, N6 -methyladenosine (m6 A) methylation of RNA is emerging as a new layer of epitranscriptomic gene regulation in cellular processes, including embryo development, flowering-time control, microspore generation and fruit ripening, in plants. However, the cellular role of m6 A in plant responses to environmental stimuli remains largely unexplored. In this study, we show that m6 A methylation plays an important role in salt stress tolerance in Arabidopsis. All mutants of m6 A writer components, including MTA, MTB, VIRILIZER (VIR) and HAKAI, displayed salt-sensitive phenotypes in an m6 A-dependent manner. The vir mutant, in which the level of m6 A was most highly reduced, exhibited salt-hypersensitive phenotypes. Analysis of the m6 A methylome in the vir mutant revealed a transcriptome-wide loss of m6 A modification in the 3' untranslated region (3'-UTR). We demonstrated further that VIR-mediated m6 A methylation modulates reactive oxygen species homeostasis by negatively regulating the mRNA stability of several salt stress negative regulators, including ATAF1, GI and GSTU17, through affecting 3'-UTR lengthening linked to alternative polyadenylation. Our results highlight the important role played by epitranscriptomic mRNA methylation in the salt stress response of Arabidopsis and indicate a strong link between m6 A methylation and 3'-UTR length and mRNA stability during stress adaptation.

The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status.

RNAs transmit information from DNA to encode proteins that perform all cellular processes and regulate gene expression in multiple ways. From the time of synthesis to degradation, RNA molecules are associated with proteins called RNA-binding proteins (RBPs). The RBPs play diverse roles in many aspects of gene expression including pre-mRNA processing and post-transcriptional and translational regulation. In the last decade, the application of modern techniques to identify RNA-protein interactions with individual proteins, RNAs, and the whole transcriptome has led to the discovery of a hidden landscape of these interactions in plants. Global approaches such as RNA interactome capture (RIC) to identify proteins that bind protein-coding transcripts have led to the identification of close to 2000 putative RBPs in plants. Interestingly, many of these were found to be metabolic enzymes with no known canonical RNA-binding domains. Here, we review the methods used to analyze RNA-protein interactions in plants thus far and highlight the understanding of plant RNA-protein interactions these techniques have provided us. We also review some recent protein-centric, RNA-centric, and global approaches developed with non-plant systems and discuss their potential application to plants. We also provide an overview of results from classical studies of RNA-protein interaction in plants and discuss the significance of the increasingly evident ubiquity of RNA-protein interactions for the study of gene regulation and RNA biology in plants.

Validation of suitable genes for normalization of diurnal gene expression studies in Chenopodium quinoa.

Quinoa depicts high nutritional quality and abiotic stress resistance, attracting strong interest in the last years. To unravel the function of candidate genes for agronomically relevant traits, studying their transcriptional activities by RT-qPCR is an important experimental approach. The accuracy of such experiments strongly depends on precise data normalization. To date, validation of potential candidate genes for normalization of diurnal expression studies has not been performed in C. quinoa. We selected eight candidate genes based on transcriptome data and literature survey, including conventionally used reference genes. We used three statistical algorithms (BestKeeper, geNorm and NormFinder) to test their stability and added further validation by a simulation-based strategy. We demonstrated that using different reference genes, including those top ranked by stability, causes significant differences among the resulting diurnal expression patterns. Our results show that isocitrate dehydrogenase enzyme (IDH-A) and polypyrimidine tract-binding protein (PTB) are suitable genes to normalize diurnal expression data of two different quinoa accessions. Moreover, we validated our reference genes by normalizing two known diurnally regulated genes, BTC1 and BBX19. The validated reference genes obtained in this study will improve the accuracy of RT-qPCR data normalization and facilitate gene expression studies in quinoa.