Short Tandem Target Mimic Research Articles

Coordination of miR319-TaPCF8 with TaSPL14 orchestrates auxin signaling and biosynthesis to regulate plant height in common wheat.

Wheat culms, comprising four to six internodes, are critically involved in determining plant height and lodging resistance, essential factors for field performance and regional adaptability. This study revealed the regulatory function of miR319 in common wheat plant height. Repression of tae-miR319 through short tandem target mimics (STTM) caused an increased plant height, while overexpression (OE) of tae-miR319 had the opposite effect. Overexpressing a miR319-resistant target gene TaPCF8 (rTaPCF8), increased plant height. TaPCF8 acted as a transcription repressor of downstream genes TaIAAs, which interact physically with TaSPL14. The significant differences of indole-3-acetic acid (IAA) contents indicate the involvement of auxin pathway in miR319-mediated plant height regulation. Finally, we identified two TaPCF8 haplotypes in global wheat collections. TaPCF8-5A-Hap2, as per association and evolution examinations, was subjected to strong substantial selection throughout wheat breeding. This haplotype, associated with shorter plant height, aligns with global breeding requirements. Consequently, in high-yield wheat breeding, we proposed a potential molecular marker for marker-assisted selection (MAS). Our findings offer fresh perspectives into the molecular mechanisms that underlie the miR319-TaPCF8 module's regulation of plant height by orchestrating auxin signaling and biosynthesis in wheat.

Overexpression of Sly-miR172a improved quality of tomato fruit by regulating MADS-box family

Fruit development is the key link in the reproductive cycle of plants, which determines the quality, yield and economic value of fruit. The present study investigated the effect of Sly-miR172a on the quality of the tomato fruit. The research results showed that overexpression of Sly-miR172a (OE-miR172a) in tomato fruit efficaciously increased the transverse and longitudinal diameter, weight, pericarp thickness, cell number and cell area. Compared with the wild-type (WT) fruit and the silencing miR172a by short tandem target mimic (STTM-miR172a), the relative expression levels of the MADS-box and the flavonoid biosynthesis synthesis pathway genes were higher in OE-miR172a. The expression level of AP2 and Cytokinin oxidase/dehydrogenase (CKX) family genes in OE-miR172a fruit was decreased, and accumulated more content of soluble sugars, lycopene, carotenoid, total phenol, anthocyanin and total flavonoid. The present study showed that overexpression of Sly-miR172 promoted the development and improved quality of tomato fruit.

Knockdown of microRNA390 Enhances Maize Brace Root Growth.

Brace root architecture is a critical determinant of maize's stalk anchorage and nutrition uptake, influencing root lodging resistance, stress tolerance, and plant growth. To identify the key microRNAs (miRNAs) in control of maize brace root growth, we performed small RNA sequencing using brace root samples at emergence and growth stages. We focused on the genetic modulation of brace root development in maize through manipulation of miR390 and its downstream regulated auxin response factors (ARFs). In the present study, miR167, miR166, miR172, and miR390 were identified to be involved in maize brace root growth in inbred line B73. Utilizing short tandem target mimic (STTM) technology, we further developed maize lines with reduced miR390 expression and analyzed their root architecture compared to wild-type controls. Our findings show that STTM390 maize lines exhibit enhanced brace root length and increased whorl numbers. Gene expression analyses revealed that the suppression of miR390 leads to upregulation of its downstream regulated ARF genes, specifically ZmARF11 and ZmARF26, which may significantly alter root architecture. Additionally, loss-of-function mutants for ZmARF11 and ZmARF26 were characterized to further confirm the role of these genes in brace root growth. These results demonstrate that miR390, ZmARF11, and ZmARF26 play crucial roles in regulating maize brace root growth; the involved complicated molecular mechanisms need to be further explored. This study provides a genetic basis for breeding maize varieties with improved lodging resistance and adaptability to diverse agricultural environments.

ZmmiR398b negatively regulates maize resistance to sugarcane mosaic virus infection by targeting ZmCSD2/4/9.

MicroRNAs (miRNAs) are widely involved in various biological processes of plants and contribute to plant resistance against various pathogens. In this study, upon sugarcane mosaic virus (SCMV) infection, the accumulation of maize (Zea mays) miR398b (ZmmiR398b) was significantly reduced in resistant inbred line Chang7-2, while it was increased in susceptible inbred line Mo17. Degradome sequencing analysis coupled with transient co-expression assays revealed that ZmmiR398b can target Cu/Zn-superoxidase dismutase2 (ZmCSD2), ZmCSD4, and ZmCSD9 invivo, of which the expression levels were all upregulated by SCMV infection in Chang7-2 and Mo17. Moreover, overexpressing ZmmiR398b (OE398b) exhibited increased susceptibility to SCMV infection, probably by increasing reactive oxygen species (ROS) accumulation, which were consistent with ZmCSD2/4/9-silenced maize plants. By contrast, silencing ZmmiR398b (STTM398b) through short tandem target mimic (STTM) technology enhanced maize resistance to SCMV infection and decreased ROS levels. Interestingly, copper (Cu)-gradient hydroponic experiments demonstrated that Cu deficiency promoted SCMV infection while Cu sufficiency inhibited SCMV infection by regulating accumulations of ZmmiR398b and ZmCSD2/4/9 in maize. These results revealed that manipulating the ZmmiR398b-ZmCSD2/4/9-ROS module provides a prospective strategy for developing SCMV-tolerant maize varieties.

The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in Populus.

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees.

Cucumber mosaic virus-induced gene and microRNA silencing in water dropwort (Oenanthe javanica (Blume) DC)

Water dropwort (Oenanthe javanica (Blume) DC), an aquatic perennial plant from the Apiaceae family, rich in dietary fibert, vitamins, and minerals. It usually grows in wet soils and water. Despite accumulating the transcriptomic data, gene function research on water dropwort is still far behind than that of the other crops. The cucumber mosaic virus (CMV) induced gene silencing was established to study the functions of gene and microRNA (miRNA) in the water dropwort. CMV Fast New York strain (CMV-Fny) genomic RNAs 1, 2, and 3 were individually cloned into pCB301 vectors. We deleted part of the ORF 2b region and introduced recognition sites. A CMV-induced gene silencing vector was employed to suppress the expression of endogenous genes, including phytoene desaturase (PDS). In order to assess the efficacy of gene silencing, we also cloned conserved sequence of gibberellin insensitive dwarf (GID1) cDNA sequences into the vector and inoculated the water dropwort. The height of CMV-GID1-infected plants was marginally reduced as a result of GID1 gene silencing, and their leaves were noticeably longer and thinner. Additionally, we also used a CMV-induced silencing vector to analyze the roles of endogenous miRNAs. We used a short tandem target mimic approach to clone miR319 and miR396 from water dropwort into the CMV vector. Plants with CMV-miRNA infection were driven to exhibit the distinctive phenotypes. We anticipate that functional genomic research on water dropwort will be facilitated by the CMV-induced gene silencing technique.

MiR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

MicroRNA171a regulates plant development and enhances drought stress tolerance

MicroRNAs (miRNAs) play an important role in plant development, and are involved in biotic and abiotic stress resistance. Previously, we identified the miR171a being as a drought-responsive miRNA in rice by small RNA sequencing. However, the mechanisms underlying the involvement of miR171a in drought stress response remains unknown. Therefore, in this study, we aimed to elucidate the molecular mechanisms miR171a in drought stress response. miR171a-overexpression (OX-miR171a) and miR171a-blocking (STTM171, a short tandem target mimic) lines were generated and characterized. The results indicated that overexpression of miR171a affected rice yield related traits, including primary panicles, 1000–grain weight, and root development trait by regulating primordial differentiation and cell size in the meristematic zone. Furthermore, miR171a overexpression significantly enhanced drought tolerance. In addition miR171a gene knockout significantly reduced this trait in both rice and Arabidopsis. We confirmed that miR171a overexpression confers plant abscisic acid-dependent drought stress resistance. In conclusion, we found that miR171a plays a key role in modulating plant growth development and drought response. Therefore, miR171a serves as a potential target for engineering improved plant drought resistance and yield.

A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference.

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.

Ldi-miR396-LdPMaT1 enhances reactive oxygen species scavenging capacity and promotes drought tolerance in Lilium distichumNakai autotetraploids.

Drought is a key environmental stress that inhibits plant growth, development, yield and quality. Whole-genome replication is an effective method for breeding drought resistant cultivars. Here, we evaluated the tolerance of Lilium distichum Nakai diploids (2n = 2× = 24) and artificially induced autotetraploids (2n = 4× = 48) to drought simulated by polyethylene glycol (PEG) stress. Autotetraploids showed stronger drought tolerance than diploids, and high-throughput sequencing during PEG stress identified five differentially expressed miRNAs. Transcriptome analysis revealed significantly different reactive oxygen species (ROS)-scavenger expression levels between diploids and autotetraploids, which increased the drought tolerance of autotetraploids. Specifically, we identified ldi-miR396b and its only target gene (LdPMaT1) for further study based on its expression level and ROS-scavenging ability in response to drought stress (DS). Autotetraploids showed higher expression of LdPMaT1 and significantly downregulated expression of ldi-miR396b under DS compared with diploids. Through a short tandem target mimic (STTM) in transgenic lilies, functional studies revealed that miR396b silencing promotes LdPMaT1 expression and the DS response. Under PEG stress, STTM393 transgenic lines showed improved drought resistance mediated by lowered MDA content but exhibited high antioxidant enzyme activity, consistent with the autotetraploid results. Collectively, these findings suggest that ldi-miR396b-LdPMaT1 potentially enhances ROS-scavenging ability, which contributes to improved stress adaptation in autotetraploid lilies.

Function identification of miR159a, a positive regulator during poplar resistance to drought stress

Abstract Drought seriously affects the growth and development of plants. MiR159 is a highly conserved and abundant microRNA family that plays a crucial role in plant growth and stress responses. However, studies of its function in woody plants are still lacking. Here, the expression of miR159a was significantly upregulated after drought treatment in poplar, and the overexpression of miR159a (OX159a) significantly reduced the open area of the stomata and improved water-use efficiency in poplar. After drought treatment, OX159a lines had better scavenging ability of reactive oxygen species and damage of the membrane system was less than that in wild-type lines. MYB was the target gene of miR159a, as verified by psRNATarget prediction, RT–qPCR, degradome sequencing, and 5′ rapid amplification of cDNA ends (5′ RACE). Additionally, miR159a–short tandem target mimic suppression (STTM) poplar lines showed increased sensitivity to drought stress. Transcriptomic analysis comparing OX159a lines with wild-type lines revealed upregulation of a series of genes related to response to water deprivation and metabolite synthesis. Moreover, drought-responsive miR172d and miR398 were significantly upregulated and downregulated respectively in OX159a lines. This investigation demonstrated that miR159a played a key role in the tolerance of poplar to drought by reducing stomata open area, increasing the number and total area of xylem vessels, and enhancing water-use efficiency, and provided new insights into the role of plant miR159a and crucial candidate genes for the molecular breeding of trees with tolerance to drought stress.

Blocking miR528 function promotes tillering and regrowth in switchgrass.

MiRNAs have been reported to be the key regulators involving a wide range of biological processes in diverse plant species, but their functions in switchgrass, an important biofuel and forage crop, are largely unknown. Here, we reported the novel function of miR528, which has expanded to four copies in switchgrass, in controlling biomass trait of tillering number and regrowth rate after mowing. Blocking miR528 activity by expressing short tandem target mimic (STTM) increased tiller number and regrowth rate after mowing. The quadruple pvmir528 mutant lines derived from genome editing also showed such improved traits. Degradome and RNA-seq analysis, combined with in situ hybridization assay revealed that up-regulation of two miR528 targets coding for Cu/Zn-SOD enzymes, might be responsible for the improved traits of tillering and regrowth in pvmir528 mutant. Additionally, natural variations in the miR528-SOD interaction exist in C3 and C4 monocot species, implying the distinct regulatory strength of the miR528-SOD module during monocot evolution. Overall, our data illuminated a novel role of miR528 in controlling biomass traits and provided a new target for genetic manipulation-mediated crop improvement.

MicroRNA397 regulates tolerance to drought and fungal infection by regulating lignin deposition in chickpea root.

Plants deposit lignin in the secondary cell wall as a common response to drought and pathogen attacks. Cell wall localised multicopper oxidase family enzymes LACCASES (LACs) catalyse the formation of monolignol radicals and facilitate lignin formation. We show an upregulation of the expression of several LAC genes and a downregulation of microRNA397 (CamiR397) in response to natural drought in chickpea roots. CamiR397 was found to target LAC4 and LAC17L out of twenty annotated LACs in chickpea. CamiR397 and its target genes are expressed in the root. Overexpression of CamiR397 reduced expression of LAC4 and LAC17L and lignin deposition in chickpea root xylem causing reduction in xylem wall thickness. Downregulation of CamiR397 activity by expressing a short tandem target mimic (STTM397) construct increased root lignin deposition in chickpea. CamiR397-overexpressing and STTM397 chickpea lines showed sensitivity and tolerance, respectively, towards natural drought. Infection with a fungal pathogen Macrophomina phaseolina, responsible for dry root rot (DRR) disease in chickpea, induced local lignin deposition and LAC gene expression. CamiR397-overexpressing and STTM397 chickpea lines showed more sensitivity and tolerance, respectively, to DRR. Our results demonstrated the regulatory role of CamiR397 in root lignification during drought and DRR in an agriculturally important crop chickpea.

Endoplasmic reticulum stress-responsive microRNAs are involved in the regulation of abiotic stresses in wheat.

ER stress-responsive miRNAs, tae-miR164, tae-miR2916, and tae-miR396e-5p, are essential in response to abiotic stress. Investigating ER stress-responsive miRNAs is necessary to improve plant tolerance to environmental stress. MicroRNAs (miRNAs) play vital regulatory roles in plant responses to environmental stress. Recently, the endoplasmic reticulum (ER) stress pathway, an essential signalling pathway in plants in response to adverse conditions, has been widely studied in model plants. However, miRNAs associated with ER stress response remain largely unknown. Using high-throughput sequencing, three ER stress-responsive miRNAs, tae-miR164, tae-miR2916, and tae-miR396e-5p were identified, and their target genes were confirmed. These three miRNAs and their target genes actively responded to dithiothreitol, polyethylene glycol, salt, heat, and cold stresses. Furthermore, in some instances, the expression patterns of the miRNAs and their corresponding target genes were contrasting. Knockdown of tae-miR164, tae-miR2916, or tae-miR396e-5p using a barley stripe mosaic virus-based miRNA silencing system substantially enhanced the tolerance of wheat plants to drought, salt, and heat stress. Under conditions involving these stresses, inhibiting the miR164 function by using the short tandem target mimic approach in Arabidopsis thaliana resulted in phenotypes consistent with those of miR164-silenced wheat plants. Correspondingly, overexpression of tae-miR164 in Arabidopsis resulted in a decreased tolerance to drought stress and, to some extent, a decrease in tolerance to salt and high temperature. These results revealed that tae-miR164 plays a negative regulatory role in wheat/Arabidopsis in response to drought, salt, and heat stress. Taken together, our study provides new insights into the regulatory role of ER stress-responsive miRNAs in abiotic stress responses.

Overexpression of Sly-miR167a delayed postharvest chilling injury of tomato fruit under low temperature storage

Chilling injury (CI) of tomato fruit leads to the deterioration of fruit quality, thereby shortening the shelf life of tomato fruit. The present experiment investigates the effect of Sly-miR167a on postharvest cold tolerance of tomato fruit. Results demonstrated that overexpression of Sly-miR167a in tomato fruit effectively reduced electrolyte leakage (EL), hydrogen peroxide (H2O2) content, malondialdehyde (MDA) content and CI index during low temperature storage. Compared with wild-type (WT) and the silencing miR167a by short tandem target mimic (miR167a-STTM) fruit, overexpression of Sly-miR167a (miR167a-OE) fruit accumulated more ascorbic acid (AsA), proline, lycopene, and phenolics, and also maintained higher activities of antioxidant enzymes. At low temperatures, miR167a-OE fruit sustained better quality, and had lower expression levels of ARF6a, higher levels of ethylene production and higher expression levels of CBF1. Overexpression of Sly-miR167a delayed the appearance of CI symptoms, improving the chilling tolerance of postharvest tomato fruit.

Expressing a Short Tandem Target Mimic (STTM) of miR164b/e-3p enhances poplar leaf serration by co-regulating the miR164–NAC module

MicroRNAs (miRNAs) are short non-coding RNAs (21–24 nt) that play important roles in plant growth and development. The miR164 family is highly conserved in plants and the miR164–NAM/ATAF/CUC (NAC) module is validated to regulate leaf and flower development, lateral root initiation and stress response. However, our knowledge of its role in Populus remains limited. In this study, two mature miRNA species, miR164e-5p and miR164e-3p, were identified in Populus deltoides. Their nucleotide sequences were identical to those of miR164a/b/c/d/e-5p and miR164b/e-3p in P. tremula × P. alba clone 717-1B4 (hereinafter poplar 717), respectively. Transgenic plants of poplar 717, including overexpression lines (35S::pri-miR164e) and Short Tandem Target Mimic lines (STTM-miR164a-d,e-5p and STTM-miR164b/e-3p), were generated to study the roles of miR164e-5p and miR164e-3p in poplar. Compared with poplar 717, the leaf margins of 35S::pri-miR164e lines were smoother, the leaves of STTM-miR164b/e-3p line were more serrated, while the leaf morphology of STTM-miR164a-d,e-5p lines had no obvious change. In addition, both 35S::pri-miR164e and STTM-miR164b/e-3p plants had a dwarf phenotype. Expressions of miR164a-d,e-5p target genes, including PtaCUC2a, PtaCUC2b and PtaORE1, was significantly reduced in the apex of 35S::pri-miR164e lines. Green fluorescent protein (GFP) reporter assay showed that PtaCUC2a/2b and PtaORE1 were cleaved by miR164a-d,e-5p, and the cleavage was inhibited by STTM-miR164b/e-3p. Therefore, miR164b/e-3p may cooperate with miR164a-d,e-5p to regulate certain NAC members, such as PtaCUC2a/2b and PtaORE1, thereby regulating leaf development and plant growth in poplar. Our findings add new insights into the mechanisms by which the miR164–NAC module regulates plant development.

The MdmiR156n Regulates Drought Tolerance and Flavonoid Synthesis in Apple Calli and Arabidopsis.

Drought is the major abiotic stress that limits apple productivity and quality. To date, many important and divergent regulatory functions of miR156/SBP genes in plant growth and development have been well understood. However, little is known about the role of apple miR156 in response to abiotic stress. To better understand the functions of MdmiR156 in abiotic stress tolerance, we constructed the overexpression (OE) and short tandem target mimic (STTM) vector of MdmiR156n and performed its functional analysis through the characterization of transgenic apple calli and Arabidopsis thaliana plants. In this study, MdmiR156n overexpression significantly increased the length of primary roots and the number of lateral roots in transgenic Arabidopsis plants under drought stress. In addition, MdmiR156n transgenic Arabidopsis and apple calli had a lower electrolyte leakage rate and less cell membrane damage than WT and STTM156 after drought stress. Further studies showed that MdmiR156n overexpression promoted the accumulation of flavonoids and scavenging of reactive oxygen species (ROS) under drought conditions in transgenic apple calli and A. thaliana plants. Taken together, overexpression MdmiR156n enhances drought tolerance by regulating flavonoid synthesis and ROS signaling cascades in apple calli and A. thaliana.

Overview of Repressive miRNA Regulation by Short Tandem Target Mimic (STTM): Applications and Impact on Plant Biology.

The application of miRNA mimic technology for silencing mature miRNA began in 2007. This technique originated from the discovery of the INDUCED BY PHOSPHATE STARVATION 1 (IPS1) gene, which was found to be a competitive mimic that prevents the cleavage of the targeted mRNA by miRNA inhibition at the post-transcriptional level. To date, various studies have been conducted to understand the molecular mimic mechanism and to improve the efficiency of this technology. As a result, several mimic tools have been developed: target mimicry (TM), short tandem target mimic (STTM), and molecular sponges (SPs). STTM is the most-developed tool due to its stability and effectiveness in decoying miRNA. This review discusses the application of STTM technology on the loss-of-function studies of miRNA and members from diverse plant species. A modified STTM approach for studying the function of miRNA with spatial-temporal expression under the control of specific promoters is further explored. STTM technology will enhance our understanding of the miRNA activity in plant-tissue-specific development and stress responses for applications in improving plant traits via miRNA regulation.

Knockdown of Sly-miR160a using short tandem target mimic (STTM) enhanced expression of auxin signaling genes and delayed postharvest ripening of tomato fruit

The miR160 is a highly conserved microRNA (miRNA) that regulates the auxin signaling pathway through negative regulation of auxin response factors (ARFs) and plays pivotal roles in fruit development. In the present study, the knockdown of miR160a using short tandem target mimic (STTM 160a) and overexpression of miR160a (miR160a-OE) transgenic tomato lines were generated to investigate the possible roles of miR160 in postharvest ripening tomato fruit. The results showed that the STTM 160a increased total chlorophyll content, reduced lycopene and total carotenoid accumulation, weight loss, and respiration rate while increasing firmness in tomato fruit compared with the wild-type (WT) and miR160a-OE. Additionally, miR160a-OE lines exhibited a typical auxin hypersensitivity and postharvest ripening acceleration, whereas STTM 160a lines displayed an auxin hyposensitivity and postharvest ripening reduction. Moreover, the relative expression levels of ARF10A (Solyc11g069500.2.1), ARF10B (Solyc06g075150.3.1), ARF17 (Solyc11g013480.2.1), and Aux/IAA genes were up-regulated in STTM 160a lines. The results suggest that the knockdown of Sly-miR160a delays postharvest ripening in tomato fruit, and provide valuable information for genetic engineering in controlling developmental processes.

Glutamate–GABA imbalance mediated by miR-8-5p and its STTM regulates phase-related behavior of locusts

The aggregation of locusts from solitary to gregarious phases is crucial for the formation of devastating locust plagues. Locust management requires research on the prevention of aggregation or alternative and greener solutions to replace insecticide use, and insect-derived microRNAs (miRNAs) show the potential for application in pest control. Here, we performed a genome-wide screen of the differential expression of miRNAs between solitary and gregarious locusts and showed that miR-8-5p controls the γ-aminobutyric acid (GABA)/glutamate functional balance by directly targeting glutamate decarboxylase (Gad). Blocking glutamate-GABA neurotransmission by miR-8-5p overexpression or Gad RNAi in solitary locusts decreased GABA production, resulting in locust aggregation behavior. Conversely, activating this pathway by miR-8-5p knockdown in gregarious locusts induced GABA production to eliminate aggregation behavior. Further results demonstrated that ionotropic glutamate/GABA receptors tuned glutamate/GABA to trigger/hamper the aggregation behavior of locusts. Finally, we successfully established a transgenic rice line expressing the miR-8-5p inhibitor by short tandem target mimic (STTM). When locusts fed on transgenic rice plants, Gad transcript levels in the brain increased greatly, and aggregation behavior was lost. This study provided insights into different regulatory pathways in the phase change of locusts and a potential control approach through behavioral regulation in insect pests.