The miR165/166-PHABULOSA module promotes thermotolerance by transcriptionally and posttranslationally regulating HSFA1.

Heat stress (HS) adversely affects plant growth and productivity. The Class A1 HS transcription factors (HSFA1s) act as master regulators in the plant response to HS. However, how HSFA1-mediated transcriptional reprogramming is modulated during HS remains to be elucidated. Here, we report that a module formed by the microRNAs miR165 and miR166 and their target transcript, PHABULOSA (PHB), regulates HSFA1 at the transcriptional and translational levels to control plant HS responses. HS-triggered induction of MIR165/166 in Arabidopsis thaliana led to decreased expression of target genes including PHB. MIR165/166 overexpression lines and mutations in miR165/166 target genes enhanced HS tolerance, whereas miR165/166 knockdown lines and plants expressing a miR165/166-resistant form of PHB were sensitive to HS. PHB directly repressed the transcription of HSFA1s and globally modulated the expression of HS-responsive genes. PHB and HSFA1s share a common target gene, HSFA2, which is essential for activation of plant responses to HS. PHB physically interacted with HSFA1s and exerted an antagonistic effect on HSFA1 transcriptional activity. PHB and HSFA1s co-regulated transcriptome reprogramming upon HS. Together, these findings indicate that heat-triggered regulation of the miR165/166-PHB module controls HSFA1-mediated transcriptional reprogramming and plays a critical role during HS in Arabidopsis.

The DEAD-box helicase RCF1 plays roles in miRNA biogenesis and RNA splicing in Arabidopsis.

RCF1 is a highly conserved DEAD-box RNA helicase found in yeast, plants, and mammals. Studies about the functions of RCF1 in plants are limited. Here, we uncovered the functions of RCF1 in Arabidopsis thaliana as a player in pri-miRNA processing and splicing, as well as in pre-mRNA splicing. A mutant with miRNA biogenesis defects was isolated, and the defect was traced to a recessive point mutation in RCF1 (rcf1-4). We show that RCF1 promotes D-body formation and facilitates the interaction between pri-miRNAs and HYL1. Finally, we show that intron-containing pri-miRNAs and pre-mRNAs exhibit a global splicing defect in rcf1-4. Together, this work uncovers roles for RCF1 in miRNA biogenesis and RNA splicing in Arabidopsis.

The miR156/SPL12 module orchestrates fruit colour change through directly regulating ethylene production pathway in blueberry.

Colour change is an important event during fruit ripening in blueberry. It is well known that miR156/SPLs act as regulatory modules mediating anthocyanin biosynthesis and ethylene plays critical roles during colour change, but the intrinsic connections between the two pathways remain poorly understood. Previously, we demonstrated that blueberry VcMIR156a/VcSPL12 affects the accumulation of anthocyanins and chlorophylls in tomato and Arabidopsis. In this study, we first showed that VcMIR156a overexpression in blueberry led to enhanced anthocyanin biosynthesis, decreased chlorophyll accumulation, and, intriguingly, concomitant elevation in the expression of ethylene biosynthesis genes and the level of the ethylene precursor ACC. Conversely, VcSPL12 enhanced chlorophyll accumulation and suppressed anthocyanin biosynthesis and ACC synthesis in fruits. Moreover, the treatment with ethylene substitutes and inhibitors attenuated the effects of VcMIR156a and VcSPL12 on pigment accumulation. Protein-DNA interaction assays indicated that VcSPL12 could specifically bind to the promoters and inhibit the activities of the ethylene biosynthetic genes VcACS1 and VcACO6. Collectively, our results show that VcMIR156a/VcSPL12 alters ethylene production through targeting VcACS1 and VcACO6, therefore governing fruit colour change. Additionally, VcSPL12 may directly interact with the promoter region of the chlorophyll biosynthetic gene VcDVR, thereby activating its expression. These findings established an intrinsic connection between the miR156/SPL regulatory module and ethylene pathway.

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.

Plant cytoplasmic ribosomal proteins: an update on classification, nomenclature, evolution and resources.

Standardized naming systems are essential to integrate and unify distinct research fields, and to link multi-species data within and across kingdoms. We conducted a comprehensive survey of cytoplasmic ribosomal proteins (CRPs) in the dicot model Arabidopsis thaliana and the monocot model rice, noting that the standardized naming system has not been widely adopted in the plant community. We generated a database linking the old classical names to their updated and compliant names. We also explored the sequences, molecular evolution, and structural and functional characteristics of all plant CRP families, emphasizing evolutionarily conserved and plant-specific features through cross-kingdom comparisons. Unlike fungal CRP paralogs that were mainly created by whole-genome duplication (WGD) or retroposition under a concerted evolution mode, plant CRP genes evolved primarily through both WGD and tandem duplications in a rapid birth-and-death process. We also provide a web-based resource (http://www.plantcrp.cn/) with the aim of sharing the latest knowledge on plant CRPs and facilitating the continued development of a standardized framework across the entire community.

A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called "two-hit" amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional "one-hit" amiRNAs. The authors also demonstrated the effectiveness of "two-hit" amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, "two-hit" amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare "two-hit" amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.

Prevalence and predictors of sexual dysfunction in females with type 1 diabetes: a systematic review and meta-analysis.

BACKGROUND: Several observational studies have explored the prevalence and predictors of female sexual dysfunction (FSD) among females with type 1 diabetes. However, no systematic review and meta-analysis of pooled data provide reliable estimates of FSD prevalence among females with type 1 diabetes. AIM: To investigate the global prevalence of FSD, analyze the association between FSD risk and type 1 diabetes, and evaluate the predictors of FSD among females with type 1 diabetes. METHODS: The study search of the present systematic review was conducted through the Wanfang Database, China National Knowledge Infrastructure, PubMed, and Embase from the inception date to February 28, 2023. Heterogeneity among the studies was analyzed with the Q and I2 tests. The sources of heterogeneity were detected through subgroup analyses and meta-regression. OUTCOMES: Outcomes included the pooled prevalence of FSD among females with type 1 diabetes, the association between FSD risk and type 1 diabetes, and the predictors of FSD among females with type 1 diabetes. RESULTS: The pooled prevalence of FSD among females with type 1 diabetes was 38.5% (95% CI, 32.1%-45.0%). The risk of FSD was higher in patients with type 1 diabetes than in healthy controls (odds ratio [OR], 3.77; 95% CI, 2.24-6.35). The significant predictors of FSD among females with type 1 diabetes were depression status (OR, 2.77; 95% CI, 1.29-5.93) and longer diabetes duration (OR, 1.19; 95% CI, 1.06-1.34). CLINICAL IMPLICATIONS: Females with type 1 diabetes had a significantly increased prevalence of FSD, indicating that clinicians should be concerned about FSD among females with type 1 diabetes. STRENGTHS AND LIMITATIONS: The strength of the present study is that it is the first systematic review and meta-analysis to investigate the global prevalence and predictors of FSD among females with type 1 diabetes. The limitation is that the results revealed significant heterogeneity after pooling the articles. CONCLUSIONS: The present systematic review and meta-analysis revealed that the overall prevalence of FSD among females with type 1 diabetes was 38.5%, demonstrating a significant association between FSD risk and type 1 diabetes among females. Furthermore, we found that the significant predictors for FSD among females with type 1 diabetes were depression and a longer duration of diabetes.

Mechanism for the genomic and functional evolution of the MIR2118 family in the grass lineage.

The MIR2118 family has undergone tremendous expansion in the grass lineage, in which the miRNA targets numerous noncoding PHAS loci to produce 21-nt phased small interfering RNAs (phasiRNAs) involved in male fertility. However, the evolutionary trajectory of the grass MIR2118 genes and the functions of phasiRNAs have not yet been fully elucidated. We conducted comparative genomic, molecular evolution, expression and parallel analysis of RNA ends (PARE) analyses of MIR2118 and the miR2118-mediated regulatory pathway in grasses, focusing on Oryza sativa. In total, 617 MIR2118 and eight MIR1859 novel members were identified. Phylogenetic analyses showed that grass MIR2118 genes form a distinct clade from the MIR482/2118 genes of nongrass species. We reconstructed hypothetical evolutionary histories of the grass MIR2118 clusters and its MIR1859 variants, and examined the polycistronic composition and the differential expression of the osa-MIR2118 clusters. PARE data showed that osa-miR2118 might also direct the cleavage of some protein-coding gene transcripts. Importantly, we found that PARE analysis is inherently prone to false-positive target predictions when a large number of small RNAs, such as phasiRNAs, are analysed. Our results revealed the evolution and diversification of the MIR2118 family, and provide new insights into the functions of phasiRNAs in the grasses.

Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits.

Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.

Maize microRNA166 Inactivation Confers Plant Development and Abiotic Stress Resistance.

MicroRNAs are important regulators in plant developmental processes and stress responses. In this study, we generated a series of maize STTM166 transgenic plants. Knock-down of miR166 resulted in various morphological changes, including rolled leaves, enhanced abiotic stress resistance, inferior yield-related traits, vascular pattern and epidermis structures, tassel architecture, as well as abscisic acid (ABA) level elevation and indole acetic acid (IAA) level reduction in maize. To profile miR166 regulated genes, we performed RNA-seq and qRT-PCR analysis. A total of 178 differentially expressed genes (DEGs) were identified, including 118 up-regulated and 60 down-regulated genes. These DEGs were strongly enriched in cell and intercellular components, cell membrane system components, oxidoreductase activity, single organism metabolic process, carbohydrate metabolic process, and oxidation reduction process. These results indicated that miR166 plays important roles in auxin and ABA interaction in monocots, yet the specific mechanism may differ from dicots. The enhanced abiotic stress resistance is partly caused via rolling leaves, high ABA content, modulated vascular structure, and the potential changes of cell membrane structure. The inferior yield-related traits and late flowering are partly controlled by the decreased IAA content, the interplay of miR166 with other miRNAs and AGOs. Taken together, the present study uncovered novel functions of miR166 in maize, and provide insights on applying short tandem target mimics (STTM) technology in plant breeding.

miR1432-OsACOT (Acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice.

Rice grain filling rate contributes largely to grain productivity and accumulation of nutrients. MicroRNAs (miRNAs) are key regulators of development and physiology in plants and become a novel key target for engineering grain size and crop yield. However, there is little studies, so far, showing the miRNA regulation of grain filling and rice yield, in consequence. Here, we show that suppressed expression of rice miR1432 (STTM1432) significantly improves grain weight by enhancing grain filling rate and leads to an increase in overall grain yield up to 17.14% in a field trial. Molecular analysis identified rice Acyl-CoA thioesterase (OsACOT), which is conserved with ACOT13 in other species, as a major target of miR1432 by cleavage. Moreover, overexpression of miR1432-resistant form of OsACOT (OXmACOT) resembled the STTM1432 plants, that is, a large margin of an increase in grain weight up to 46.69% through improving the grain filling rate. Further study indicated that OsACOT was involved in biosynthesis of medium-chain fatty acids. In addition, RNA-seq based transcriptomic analyses of transgenic plants with altered expression of miR1432 demonstrated that downstream genes of miR1432-regulated network are involved in fatty acid metabolism and phytohormones biosynthesis and also overlap with the enrichment analysis of co-expressed genes of OsACOT, which is consistent with the increased levels of auxin and abscisic acid in STTM1432 and OXmACOT plants. Overall, miR1432-OsACOT module plays an important role in grain filling in rice, illustrating its capacity for engineering yield improvement in crops.

Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease.

MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs.IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in Arabidopsis thaliana.

The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis.

MicroRNA 399 as a potential integrator of photo-response, phosphate homeostasis, and sucrose signaling under long day condition.

BACKGROUND: Photoperiod-sensitivity is a critical endogenous regulatory mechanism for plant growth and development under specific environmental conditions, while phosphate and sucrose signaling processes play key roles in cell growth and organ initiation. MicroRNA399 is phosphate-responsive, but, whether it has roles in other metabolic processes remains unknown. RESULTS: MicroRNA399 was determined to be sucrose-responsive through a microRNA array assay. High levels of sucrose inhibited the accumulation of microRNA399 family under phosphate starvation conditions in Arabidopsis thaliana. Similarly, exogenous sucrose supplementation also reduced microRNA399 expression in maize at developmental transition stages. RNA sequencing of a near-isogenic line(photoperiod-sensitive) line and its recurrent parent Huangzao4, a photoperiod-insensitive line, was conducted at various developmental stages. Members of microRNA399 family were down-regulated under long-day conditions in the photoperiod-sensitive near-isogenic line that accumulated more sucrose in vivo compared with the control line Huangzao4. CONCLUSION: MicroRNA399s may play central roles in the integration of sucrose sensing and photoperiodic responses under long day conditions in maize.

Small but powerful: function of microRNAs in plant development.

MicroRNAs (miRNAs) are a group of endogenous noncoding small RNAs frequently 21 nucleotides long. miRNAs act as negative regulators of their target genes through sequence-specific mRNA cleavage, translational repression, or chromatin modifications. Alterations of the expression of a miRNA or its targets often result in a variety of morphological and physiological abnormalities, suggesting the strong impact of miRNAs on plant development. Here, we review the recent advances on the functional studies of plant miRNAs. We will summarize the regulatory networks of miRNAs in a series of developmental processes, including meristem development, establishment of lateral organ polarity and boundaries, vegetative and reproductive organ growth, etc. We will also conclude the conserved and species-specific roles of plant miRNAs in evolution and discuss the strategies for further elucidating the functional mechanisms of miRNAs during plant development.

Genome-wide identification of soybean microRNA responsive to soybean cyst nematodes infection by deep sequencing.

BACKGROUND: The soybean cyst nematode (SCN), Heterodera glycines, is one of the most devastating diseases limiting soybean production worldwide. It is known that small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in regulating plant growth and development, defense against pathogens, and responses to environmental changes. RESULTS: In order to understand the role of soybean miRNAs during SCN infection, we analyzed 24 small RNA libraries including three biological replicates from two soybean cultivars (SCN susceptible KS4607, and SCN HG Type 7 resistant KS4313N) that were grown under SCN-infested and -noninfested soil at two different time points (SCN feeding establishment and egg production). In total, 537 known and 70 putative novel miRNAs in soybean were identified from a total of 0.3 billion reads (average about 13.5 million reads for each sample) with the programs of Bowtie and miRDeep2 mapper. Differential expression analyses were carried out using edgeR to identify miRNAs involved in the soybean-SCN interaction. Comparative analysis of miRNA profiling indicated a total of 60 miRNAs belonging to 25 families that might be specifically related to cultivar responses to SCN. Quantitative RT-PCR validated similar miRNA interaction patterns as sequencing results. CONCLUSION: These findings suggest that miRNAs are likely to play key roles in soybean response to SCN. The present work could provide a framework for miRNA functional identification and the development of novel approaches for improving soybean SCN resistance in future studies.

Suppression of microRNA159 impacts multiple agronomic traits in rice (Oryza sativa L.).

BACKGROUND: microRNAs (miRNAs) are important regulators in plant growth and development. miR159 is a conserved miRNA among different plant species and has various functions in plants. Studies on miR159 are mostly done on model plant, Arabidopsis thaliana. In rice, studies on miR159 were either based upon genome-wide expression analyses focused upon responses to different nitrogen forms and abiotic stress or upon phenotypic studies of transgenic plants overexpressing its precursor. STTM (Short Tandem Target Mimic) is an effective tool to block the activity of endogenous mature miRNA activity in plant. Therefore, specific roles of miR159 in rice could be explored by down regulating miR159 through STTM. RESULTS: In this study, expression of mature miR159 was successfully suppressed by STTM which resulted in the increased expressions of its two targets genes, OsGAMYB and OsGAMYBL1 (GAMYB-LIKE 1). Overall, STTM159 plants exhibited short stature along with smaller organ size and reduction in stem diameter, length of flag leaf, main panicle, spikelet hulls and grain size. Histological analysis of stem, leaf and mature spikelet hull showed the reduced number of small vascular bundles (SVB), less number of small veins (SV) between two big veins (LV) and less cell number in outer parenchyma. Gene Ontology (GO) enrichment analysis of differentially expressed genes between wild type plants and STTM159 transgenic plants showed that genes involved in cell division, auxin, cytokinin (CK) and brassinosteroids (BRs) biosynthesis and signaling are significantly down-regulated in STTM159 plants. CONCLUSION: Our data suggests that in rice, miR159 positively regulates organ size, including stem, leaf, and grain size due to the promotion of cell division. Further analysis from the RNA-seq data showed that the decreased cell divisions in STTM159 transgenic plants may result, at least partly from the lower expression of the genes involved in cell cycle and hormone homeostasis, which provides new insights of rice miR159-specific functions.

GSK3-like kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis.

Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-of-function mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3(T180A) had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.

microRNA-dependent gene regulatory networks in maize leaf senescence.

BACKGROUND: Maize grain yield depends mainly on the photosynthetic efficiency of functional leaves, which is controlled by an array of gene networks and other factors, including environmental conditions. MicroRNAs (miRNAs) are small RNA molecules that play important roles in plant developmental regulation. A few senescence-associated miRNAs (SA-miRNAs) have been identified as important participants in regulating leaf senescence by modulating the expression levels of their target genes. RESULTS: To elucidate miRNA roles in leaf senescence and their underlying molecular mechanisms in maize, a stay-green line, Yu87-1, and an early leaf senescence line, Early leaf senescence-1 (ELS-1), were selected as experimental materials for the differential expression of candidate miRNAs. Four small RNA libraries were constructed from ear leaves at 20 and 30 days after pollination and sequenced by Illumina deep sequencing technology. Altogether, 81 miRNAs were detected in both lines. Of these, 16 miRNAs of nine families were differentially expressed between ELS-1 andYu87-1. The phenotypic and chlorophyll content analyses of both lines identified these 16 differentially expressed miRNAs as candidate SA-miRNAs. CONCLUSIONS: In this study, 16 candidate SA-miRNAs of ELS-1 were identified through small RNA deep sequencing technology. Degradome sequencing results indicated that these candidate SA-miRNAs may regulate leaf senescence through their target genes, mainly transcription factors, and potentially control chlorophyll degradation pathways. The results highlight the regulatory roles of miRNAs during leaf senescence in maize.