Non-coding RNA
In response to phosphate (Pi) deficiency, it has been shown that micro-RNAs (miRNAs) and mRNAs are transported through the phloem for delivery to sink tissues. Growing evidence also indicates that long non-coding RNAs (lncRNAs) are critical regulators of Pi homeostasis in plants. However, whether lncRNAs are present in and move through the phloem, in response to Pi deficiency, remains to be established. Here, using cucumber as a model plant, we show that lncRNAs are enriched in the phloem translocation stream and respond, systemically, to an imposed Pi-stress. A well-known lncRNA, IPS1, the target mimic (TM) of miRNA399, accumulates to a high level in the phloem, but is not responsive to early Pi deficiency. An additional 24 miRNA TMs were also detected in the phloem translocation stream; among them miRNA171 TMs and miR166 TMs were induced in response to an imposed Pi stress. Grafting studies identified 22 lncRNAs which move systemically into developing leaves and root tips. A CU-rich PTB motif was further identified in these mobile lncRNAs. Our findings revealed that lncRNAs respond to Pi deficiency, non-cell-autonomously, and may act as systemic signaling agents to coordinate early Pi deficiency signaling, at the whole-plant level.
Inflorescence architecture is a major determinant of spikelet numbers per panicle, a key component of grain yield in rice. In this study, Short Panicle 3 (SP3) was identified from a short panicle 3 (sp3) mutant in which T‐DNA was inserted in the promoter of SP3, resulting in a knockdown mutation. SP3 encodes a DNA binding with one finger (Dof) transcriptional activator. Quantitative real time (qRT)‐PCR and RNA in situ hybridization assays confirmed that SP3 is preferentially expressed in the young rice inflorescence, specifically in the branch primordial regions. SP3 acts as a negative regulator of inflorescence meristem abortion by upregulating APO2/RFL. SP3 both up‐ and downregulates expression of genes involved in cytokinin biosynthesis and catabolism, respectively. Consequently, cytokinin concentrations are decreased in young sp3 panicles, thereby leading to small panicles having fewer branches and spikelets. Our findings support a model in which SP3 regulates panicle architecture by modulating cytokinin homeostasis. Potential applications to rice breeding, through gene‐editing of the SP3 promoter are assessed.
TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) proteins, a family of plant‐specific transcription factors, play important roles in many developmental processes. However, genetic and functional redundancy among class I TCP limits the analysis of their biological roles. Here, we identified a dominant‐negative mutant of Arabidopsis thaliana TCP7 named leaf curling‐upward (lcu), which exhibits smaller leaf cells and shorter hypocotyls than the wild type, due to defective endoreplication. A septuple loss‐of‐function mutant of TCP7, TCP8, TCP14, TCP15, TCP21, TCP22, and TCP23 displayed similar developmental defects to those of lcu. Genome‐wide RNA‐sequencing showed that lcu and the septuple mutant share many misexpressed genes. Intriguingly, TCP7 directly targets the CYCLIN D1;1 (CYCD1;1) locus and activates its transcription. We determined that the C‐terminus of TCP7 accounts for its transcriptional activation activity. Furthermore, the mutant protein LCU exhibited reduced transcriptional activation activity due to the introduction of an EAR‐like repressive domain at its C‐terminus. Together, these observations indicate that TCP7 plays important roles during leaf and hypocotyl development, redundantly, with at least six class I TCPs, and regulates the expression of CYCD1;1 to affect endoreplication in Arabidopsis.
Mimicry target‐directed microRNA degradation is widespread and highly conserved among eukaryotes. However, little is known about its mechanism of action. In this letter, by using STTM160 (target mimic of miR160) as a reporter, we show that dysfunction of HAWAIIAN SKIRT (HWS) suppresses the pleiotropic phenotype of STTM160. Small RNA sequencing and Northern blot analyses suggested that HWS only affects a subset of microRNAs. Intriguingly, we identified a stable coexistence of miR160/miR399 and their mimicry targets within the AGO1 complex when HWS is compromised, pointing to a possible role of HWS in the clearance of RNA‐induced silencing complexes associated with mimicry target.
MicroRNAs (miRNAs) are vital regulators that repress gene expression in the cytoplasm in two main ways: mRNA degradation and translational inhibition. Several animal studies have shown that miRNAs also target promoters, thereby activating expression. Whether this miRNA action also occurs in plants is unknown. In this study, we demonstrated that several miRNAs regulate target promoters in Arabidopsis thaliana. For example, miR5658 was predominantly present in the nucleus and activated the expression of AT3G25290 directly by binding to its promoter. Our observations suggest that this mode of action may be a general feature of plant miRNAs, and thus provide insight into the vital roles of plant miRNAs in the nucleus.
MicroRNAs (miRNAs) are key regulators of gene expression in many important biological processes of plants. However, few miRNAs have been shown to regulate seed vigor. Here, we conducted microarray assays to analyze miRNA expression levels in seeds of the rice (Oryza sativa L.) cultivar ZR02. Results showed significant differences in the expression of 11 miRNAs between artificially aged and untreated control seeds. Among these, osa‐miR164c was transcriptionally upregulated, while osa‐miR168a was downregulated in artificially aged seeds; this was verified by quantitative real‐time PCR analysis. Under the same aging condition, osa‐miR164c overexpression in OE164c transgenic seeds and osa‐miR168a silencing in MIM168a transgenic seeds of the rice cultivar Kasalath led to lower germination rates, whereas osa‐miR164c silencing in MIM164c and osa‐miR168a overexpression in OE168a resulted in higher seed germination rates compared with wild‐type seeds. Meanwhile, changes in cytomembrane permeability of seeds and in the expression level of osa‐miR164c target genes (OsPM27 and OsPSK5) and osa‐miR168a target genes (OsAGO1 and OsPTR2) under aging conditions coincided with changes in seed vigor induced by osa‐miR164c and osa‐miR168a. Thus, genetic manipulation of miRNAs has important implications in the development of crop cultivars with high vigor and extended life span of seeds.
Since approximate a century ago, many hybrid crops have been continually developed by crossing two inbred varieties. Owing to heterosis (hybrid vigor) in plants, these hybrids often have superior agricultural performances in yield or disease resistance succeeding their inbred parental lines. Several classical hypotheses have been proposed to explain the genetic causes of heterosis. During recent years, many new genetics and genomics strategies have been developed and used for the identifications of heterotic genes in plants. Heterotic effects of the heterotic loci and molecular functions of the heterotic genes are being investigated in many plants such as rice, maize, sorghum, Arabidopsis and tomato. More and more data and knowledge coming from the molecular studies of heterotic loci and genes will serve as a valuable resource for hybrid breeding by molecular design in future. This review aims to address recent advances in our understanding of the genetic and molecular mechanisms of heterosis in plants. The remaining scientific questions on the molecular basis of heterosis and the potential applications in breeding are also proposed and discussed.
Rejuvenation refers to the process enabling plants to regain physiological and molecular characteristics lost after entering the adult phase. The underlying molecular mechanism is poorly understood. Previous studies have revealed that microRNA156 (miR156) is highly accumulated at juvenile stage and maintains juvenile traits by repressing a group of SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE (SPL) transcription factors. Here, we found that induction of miR156 expression in adult leaves can only restore some aspects of juvenile traits, such as loss of epidermal leaf hairs on the lower side of leaves and absence of serration at the leaf edges, but is incapable of delaying flowering and promoting adventitious root production.
In land plants, cytidine‐to‐uridine (C‐to‐U) editing of organellar transcripts is an important post‐transcriptional process, which is considered to remediate DNA genetic mutations to restore the coding of functional proteins. Pentatricopeptide repeat (PPR) proteins have key roles in C‐to‐U editing. Owing to its large number, however, the biological functions of many PPR proteins remain to be identified. Through characterizing a small kernel4 (smk4 ) mutant, here we report the function of Smk4 and its role in maize growth and development. Null mutation of Smk4 slows plant growth and development, causing small plants, delayed flowering time, and small kernels. Cloning revealed that Smk4 encodes a new E‐subclass PPR protein, and localization indicated that SMK4 is exclusively localized in mitochondria. Loss of Smk4 function abolishes C‐to‐U editing at position 1489 of the cytochrome c oxidase1 (cox1 ) transcript, causing an amino acid change from serine to proline at 497 in Cox1. Cox1 is a core component of mitochondrial complex IV. Indeed, complex IV activity is reduced in the smk4 , along with drastically elevated expression of alternative oxidases (AOX). These results indicate that SMK4 functions in the C‐to‐U editing of cox1 ‐1489, and this editing is crucial for mitochondrial complex IV activity, plant growth, and kernel development in maize.
MicroRNAs (miRNAs) are known to fine‐tune growth, development, and stress‐induced responses. Osa‐miR1873 is a rice‐specific miRNA targeting LOC_Os05g01790 . Here, we show that Osa‐miR1873 fine‐tunes rice immunity against Magnaporthe oryzae and yield traits via LOC_Os05g01790 . Osa‐miR1873 was significantly upregulated in a susceptible accession but downregulated in a resistance accession at 24 h post‐inoculation (hpi) of M. oryzae . Overexpressing Osa‐miR1873 enhanced susceptibility to M . oryzae and compromised induction of defense responses. In contrast, blocking Osa‐miR1873 through target mimicry compromised susceptibility to M . oryzae and enhanced induction of defense responses. Altered expression of Osa‐miR1873 also resulted in some defects in yield traits, including grain numbers and seed setting rate. Moreover, overexpression of the target gene LOC_Os05g01790 increased rice blast disease resistance but severely penalized growth and yield. Taken together, we demonstrate that Osa‐miR1873 fine‐tunes the rice immunity‐growth trade‐off via LOC_Os05g01790 , and blocking Osa‐miR1873 could improve blast disease resistance without significant yield penalty. Thus, the Osa‐miR1873‐LOC_Os05g01790 regulatory module is valuable in balancing yield traits and blast resistance.
The juvenile‐to‐adult transition in plants involves changes in vegetative growth and plant architecture; the timing of this transition has important implications for agriculture. The microRNA miR156 regulates this transition and shoot maturation in plants. In Arabidopsis thaliana, deposition of histone H3 trimethylation on lysine 27 (H3K27me3, a repressive mark) at the MIR156A/C loci is regulated by Polycomb Repressive Complex 1 (PRC1) or PRC2, depending on the developmental stage. The levels of miR156 progressively decline during shoot maturation. The amount of H3K27me3 at MIR156A/C loci affects miR156 levels; however, whether this epigenetic regulation is conserved remains unclear. Here, we found that in rice (Oryza sativa), the putative PRC1 subunit LIKE HETEROCHROMATIN PROTEIN 1 (OsLHP1), with the miR156–SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE (SPL) module, affects developmental phase transitions. Loss of OsLHP1 function results in ectopic expression of MIR156B/C/I/E, phenocopy of miR156 overexpression, and reduced H3k27me3 levels at MIR156B/C/I/E. This indicates that OsLHP1 has functionally diverged from Arabidopsis LHP1. Genetic and transcriptome analyses of wild‐type, miR156b/c‐overexpression, and Oslhp1‐2 mutant plants suggest that OsLHP1 acts upstream of miR156 and SPL during the juvenile‐to‐adult transition. Therefore, modifying the OsLHP1–miR156–SPL pathway may enable alteration of the vegetative period and plant architecture.
N6‐methyladenosine (m6A) is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA metabolism. However, the occurrence of the m6A modification in plant circular RNAs has not been reported. A widely used method to identify m6A modifications relies on m6A‐specific antibodies followed by next‐generation sequencing of precipitated RNAs (MeRIP‐Seq). However, one limitation of MeRIP‐Seq is that it does not provide the precise location of m6A at single‐nucleotide resolution. Although more recent sequencing techniques such as Nanopore‐based direct RNA sequencing (DRS) can overcome such limitations, the technology does not allow sequencing of circular RNAs, as these molecules lack a poly(A) tail. Here, we developed a novel method to detect the precise location of m6A modifications in circular RNAs using Nanopore DRS. We first enriched our samples for circular RNAs, which we then fragmented and sequenced on the Nanopore platform with a customized protocol. Using this method, we identified 470 unique circular RNAs from DRS reads based on the back‐spliced junction region. Among exonic circular RNAs, about 10% contained m6A sites, which mainly occurred around acceptor and donor splice sites. This study demonstrates the utility of our antibody‐independent method in identifying total and methylated circular RNAs using Nanopore DRS. This method has the additional advantage of providing the exact location of m6A sites at single‐base resolution in circular RNAs or linear transcripts from non‐coding RNA without poly(A) tails.
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