Search Results (88)

The jujube tree fruit remains a primary fruit in northern China, yet its geographical distribution and yield are significantly constrained by freezing stress during winter. Numerous studies have highlighted the pivotal regulatory function of microRNAs (miRNAs) in plant responses to low-temperature stress. Nevertheless, the specific miRNAs involved in the response to low temperatures and their associated gene networks in Ziziphus jujuba Mill are not well understood. In this investigation, we utilized high-throughput sequencing to analyze small RNA libraries from branches subjected to temperatures of 4 °C and −30 °C. Our analysis identified a total of 342 miRNAs, comprising 123 known miRNAs and 219 novel miRNAs. The differential expression analysis revealed that under low-temperature conditions, 177 miRNAs underwent significant changes. Among them, specific upregulation of miR319 in the less cold-resistant variety and miR6483 in sensitive variety was observed. By employing degradome sequencing, we identified a total of 1551 target genes corresponding to 3059 unique miRNA target interaction pairs involving 299 miRNAs. Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that these target genes are primarily associated with transcriptional regulation, metabolic pathways, and genetic information processing. Through a comprehensive analysis, we pinpointed 11 genes corresponding to 9 miRNAs that are implicated in jujube tree cold stress, and 7 target genes of 7 miRNAs were confirmed by 5′-RACE analysis. These miRNAs are likely to exert crucial regulatory functions in the context of jujube tree cold stress. This study is the first to systematically identify miRNAs and their target genes in the response of Ziziphus jujuba Mill to low-temperature stress, which provides important resources for in-depth analysis of the molecular mechanism of jujube tree cold resistance and for cold-resistant breeding. Full article

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through a sequence-specific recognition of their targets, leading to degradation or inhibition of translation. In the case of plant miRNAs, they have been involved in a multitude of biological processes, from developmental processes to environmental stress responses. Massive sequencing by RNA-seq is becoming a widely used technique to discover plant miRNAs. However, if costs are considered, bioinformatics prediction is a valuable tool for miRNA discovery in plants. Among the tools available, ShortStack is the best for comprehensive prediction and annotation of miRNAs. Therefore, ShortStack was used in this study to predict miRNAs of common bean (Phaseolus vulgaris), one of the most important legumes in the world. Briefly, a high-stringency, homology-based in silico pipeline was followed to systematically predict miRNAs of P. vulgaris without the dependency on high-throughput experimental sequencing infrastructure; meaning that this study was based exclusively on computationally generated miRNA datasets derived from miRbase. Therefore, no biological small RNA-seq, degradome sequencing, or expression validation were performed in this study. In total, 57 distinct, non-redundant clusters were predicted, from which 35 miRNAs showed unique characteristic sequences. Comparative genomic cross-referencing against historical seminal common bean datasets supported our predicted loci, finding that 18 out of the 35 miRNA sequences were located at the same genomic coordinates as the previously reported loci. In addition, functional categorization of all the potential target genes revealed their putative roles in plant development and other cellular processes. It is important to emphasize that, although the predicted loci in P. vulgaris represent candidate miRNAs that require independent experimental validation, this work serves as a foundational approach to be applied for the prediction of miRNAs in underexplored plant species or those with limited genomic resources. Full article

Background/Objectives: Barley malting is an agro-industrial process that produces malt, an essential ingredient for the brewing and distilling industries. Previously, tran-scriptome profiling has revealed mRNA changes during malting but less is known about their regulation. Methods: The spring 2-row barley variety ‘Conrad’ was sampled at five stages of malt-ing. Using small RNA (sRNA)-sequencing and degradome-sequencing data from these malting stages, de novo discovery of mature microRNA (miRNA), as well as cognate mRNAs targeted for slicing, was identified. ShortStack v4.1.0 was used to map sRNA reads to the Hordeum vulgare Morex V3 genome. Results: In total, 33 expressed MIRs were identified, six of which may be novel. Using the degradome-sequencing data from the same malting stages, CleaveLand4 v4.5 pre-dicted 64 sliced mRNA targets, predominantly transcription factors associated with root development. Conclusions: This study provides an overview of post-transcriptional modulations of miRNAs-cognate mRNA targets, as well as plausible interactions between miRNAs during barley malting. Full article

Cucumber Fusarium wilt, which is induced by the soil-borne pathogen Fusarium oxysporum f. sp. Cucumerinum (FOC), represents a highly destructive disease. Cucumber seedling grafted onto figleaf gourd (Cucurbita ficifolia Bouché) rootstock (CFC) demonstrated better resistance to FOC. However, the molecular mechanism underlying this enhanced disease resistance capability is largely unknown. To elucidate this, we performed transcriptome, small RNA, and degradome sequencing for leaves from CFC and self-grafted cucumbers (SGC) as controls, with and without FOC infections, respectively. Our results indicated that three NAC genes, all predicted as targets of csa-miR164, were significantly up-regulated in CFC after FOC infection. Co-transformation assay in Nicotiana benthamiana confirmed that csa-miR164f directly inhibits NAC2, and transient overexpression of NAC2 in cucumber enhanced resistance to FOC, supporting its positive role in defense. Therefore, our results suggest that three NACs, upregulated in CFC, as an alternative pathway, enhance the reactive oxygen species burst and hypersensitive response, which further elevates the resistance to FOC infection. These results provide new insights into the molecular basis for improved FOC resistance in CFC. Full article

Abiotic stress of cold is one of the limitation factors that hinder the production of alfalfa (Medicago sativa). Although there are a large number of studies suggesting that non-coding RNAs (ncRNAs) play an important role in plant response to abiotic stress, the mechanism by which ncRNAs and competing endogenous RNAs (ceRNAs) influence the low-temperature tolerance of alfalfa remains understudied. In this study, we integrated whole-transcriptome RNA-seq and genome-wide association studies (GWASs) to identify cold stress-related metabolic pathways and candidate genes, differentially expressed (DE) mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Degradome sequencing was used to verify the ceRNA network under cold stress. A total of 46,936 DEmRNAs were identified. Ribosome (ko03010), amino sugar and nucleotide sugar metabolism (ko00520), ribosome biogenesis in eukaryotes (ko03008), circadian rhythm–plant (ko00270), and starch and sucrose metabolism (ko00500) were the top five KEGG terms with the highest p-value, enriching the most number of DEmRNAs. MS.gene53818 (MsUAM1) was considered to be the critical candidate gene for alfalfa response to cold stress by conjoint analysis of GWASs and DEmRNAs. A total of 223 DEmiRNAs, 1852 DElncRNAs, and 13 DEcircRNAs were identified under cold stress. Functional analysis indicates that they play important roles in GO terms such as leaf development (GO:0048366), DNA-binding transcription factor activity (GO:0003700), central vacuole (GO:0042807), response to auxin (GO:0009733), and water channel activity (GO:0015250), as well as in KEGG pathways such as plant hormone signal transduction, starch and sucrose metabolism, and flavone and flavonol biosynthesis (ko00944). A ceRNA network comprising 28 DElncRNAs, 8 DEcircRNAs, 11 DEmiRNAs, and 23 DEmRNA triplets was constructed. In this study, mRNAs and ncRNAs were identified that may be involved in alfalfa’s response to cold stress, and a ceRNA regulatory network related to cold stress was established, providing valuable genic resources for further research on the molecular mechanisms underlying alfalfa cold stress. Full article

Heat threatens male fertility in crops, yet the regulatory basis of anther dehiscence under high temperatures remains unclear. We compared a heat-sensitive pepper cultivar (DL) with a heat-tolerant landrace (B021) across two anther stages using integrated transcriptome, small-RNA, degradome, co-expression, and enzymatic assays. DL showed a collapse of anther dehiscence above 34–38 °C, whereas B021 retained normal dehiscence at 39 °C, and histology revealed tapetal enlargement, premature degeneration, and locule contraction only in DL. RNA-seq indicated genotype- and stage-dependent reprogramming, with DL suppressing phenylpropanoid/cell-wall, transport, and proteostasis pathways, while B021 maintained reproductive and stress-integration programs. Small-RNA profiling and degradome sequencing identified conserved miRNA families with in vivo target cleavage, and notably, miR397 targeting a laccase gene showed stronger evidence in B021, which is consistent with controlled lignification. Functional organization of differentially expressed miRNA targets highlighted modules in respiration/redox, hormone and terpenoid metabolism, vascular–cell-wall programs, and proteostasis/osmotic buffering. WGCNA modules correlated with heat-tolerance traits converged on the same processes. Enzyme assays corroborated multi-omics predictions, with SOD, CAT, and POD activities consistently induced in B021 and limited MDA accumulation. Together, the data supports a model in which tolerant anthers sustain dehiscence under heat by coordinating secondary-wall formation, auxin/jasmonate/gibberellin crosstalk, respiratory and reactive oxygen species buffering, and protein/membrane quality control, providing tractable targets for breeding heat-resilient peppers. Full article

Enhancing wood properties, particularly fiber length (FL), represents a critical objective in Populus tomentosa breeding programs. However, the molecular mechanisms regulating these traits remain largely elusive. Here, an integrative analysis of the PtomiR171 family, uncovering substantial functional divergence among PtomiR171 family members and identified a PtomiR171a-PtoGRAS50 regulatory axis that may control cellulose-related gene expression and influence fiber development in P. tomentosa. Single-nucleotide polymorphism (SNP)-based association studies implicated the role of the PtomiR171a-PtoGRAS50 module in modulating FL. Combined with dual-luciferase reporter gene assay, real-time reverse transcription polymerase chain reaction (RT-qPCR), transcriptome and degradome analysis, PtomiR171a exerts a negative regulatory effect on PtoGRAS50, which is a key regulator of early xylem development. DNA affinity purification sequencing (DAP-seq) identified two downstream putative target genes of PtoGRAS50, both of which are involved in cellulose biosynthesis and metabolism. Unlike previous studies about miRNAs in P. tomentosa, this work narrows its scope to miR171 and elucidates the downstream regulatory module. Collectively, these findings elucidate a critical PtomiR171a-PtoGRAS50 regulatory axis, advancing our understanding of the genetic networks that orchestrate wood properties, deepening insights into FL modulation, and laying a foundation for the development of targeted genetic strategies to enhance wood quality in P. tomentosa. Full article

Drought stress severely limits peanut productivity, highlighting the urgent need to understand the molecular mechanisms that underlie drought adaptation. While microRNAs (miRNAs) are known to play essential roles in plant stress responses, their functional contributions in polyploid crops like peanut remain insufficiently explored. This study provides the first integrated transcriptomic analysis of drought-responsive miRNAs in tetraploid peanut (Arachis hypogaea). We performed high-throughput sRNA sequencing on a drought-tolerant cultivar Fenhua 8 under PEG6000-simulated drought stress, identifying 10 conserved drought-responsive miRNAs. Among these, ahy-miR398 and ahy-miR408 were significantly downregulated under drought conditions. Degradome sequencing revealed that ahy-miR398 targets copper chaperones for superoxide dismutase (CCSs), potentially reducing SOD activation and amplifying oxidative stress. In contrast, ahy-miR408 targets laccase 12 (LAC12), P-type ATPase copper transporters (COPAs), and a blue copper protein-like (PCL) gene. These targets are involved in copper homeostasis and the regulation of reactive oxygen species (ROS), suggesting that ahy-miR408 plays a role in oxidative stress management. Functional validation in transgenic Arabidopsis lines overexpressing ahy-miR398 or ahy-miR408 showed significantly reduced drought tolerance, with impaired seed germination, shorter primary roots, and exacerbated growth suppression during water deprivation. Taken together, these findings highlight a novel miRNA-mediated regulatory network in peanut drought adaptation, centered on copper-associated oxidative stress management. This study provides new insights into miRNA-based regulation in polyploid crops and offers potential molecular targets for breeding climate-resilient peanut varieties, especially in arid regions where yield stability is crucial. Full article

Peanut (Arachis hypogaea L.) is an important oil and cash crop, but its growth and productivity are severely constrained by low-temperature stress. Growth-regulating factors (GRFs) are plant-specific transcription factors involved in development and stress responses, yet their roles in peanut remain poorly understood. In this study, we identified AhGRF3b as a direct target of ahy-miR396 using degradome sequencing, which demonstrated precise miRNA-mediated cleavage sites within the AhGRF3b transcript. Expression profiling confirmed that ahy-miR396 suppresses AhGRF3b via post-transcriptional cleavage rather than translational repression. Functional analyses showed that overexpression of AhGRF3b in Arabidopsis thaliana promoted leaf expansion by enhancing cell proliferation. Specifically, leaf length, width, and petiole length increased by 104%, 22%, and 28%, respectively (p < 0.05). Under cold stress (0 °C for 7 days), transgenic lines (OE-2 and OE-6) exhibited significantly better growth than Col-0, with fresh weight increased by 158% and 146%, respectively (p < 0.05). Effect size analysis further confirmed these differences (Cohen’s d = 11.6 for OE-2 vs. Col-0; d = 6.3 for OE-6 vs. Col-0). Protein–protein interaction assays, performed using the yeast two-hybrid (Y2H) system and 3D protein–protein docking models, further supported that AhGRF3b interacts with Catalase 1 (AhCAT1), vacuolar cation/proton exchanger 3 (AhCAX3), probable polyamine oxidase 4 (AhPAO4), and ACT domain-containing protein 11 (AhACR11), which are involved in reactive oxygen species (ROS) scavenging and ion homeostasis. These interactions were associated with enhanced CAT and PAO enzymatic activities, reduced ROS accumulation, and upregulation of stress-related genes under cold stress. These findings suggest that the ahy-miR396/AhGRF3b module plays a potential regulatory role in leaf morphogenesis and cold tolerance, providing valuable genetic resources for breeding cold-tolerant peanut varieties. Full article

Small noncoding RNAs are recognized as crucial regulators of seed germination, but their role in seed aging remains unclear. To address this, we performed RNA sequencing (RNA-seq) on barley (Hordeum vulgare L.) seeds with varying viability levels after long-term storage in hermetically sealed containers since the 1972 harvest. This globally unique material, characterized by genetic homogeneity and contrasting germination capacities, enabled an in-depth analysis of microtranscriptomic changes during germination. We identified 62 known miRNAs from 11 families and 234 novel miRNAs, with miR159, miR168, and miR166 showing consistently high expression across all germination stages and viability groups. Differential expression analysis revealed 28 miRNAs whose abundance varied significantly with seed viability and germination phase. Functional predictions supported by quantitative reverse transcription PCR (qRT–PCR) and degradome-based target identification indicated that these miRNAs regulate key developmental and metabolic pathways. Several isomiRs exhibited sample-specific expression, suggesting the viability-dependent activation of distinct molecular mechanisms. Gene Ontology analysis highlighted processes related to nucleic acid binding, nuclear organization, and cytoplasmic metabolism as central during germination. We propose that miRNA profiles may reflect an “epigenetic inheritance”—a molecular memory of aging stored in seeds—rather than solely a response to current conditions. This concept may help explain aging-related phenotypes such as delayed germination and reduced vigor, warranting further investigation. Full article

Ogura cytoplasmic male sterility (CMS) in Chinese cabbage (Brassica rapa) is characterized by complete pollen abortion, wherein stamens fail to produce viable pollen while pistils retain normal fertility. This maternally inherited trait is valuable for hybrid breeding. This study employed integrated analysis of miRNA, transcriptome, and degradome sequencing data aligned to the Chinese cabbage reference genome to elucidate the molecular function of bra-miR9569 in Ogura CMS pollen fertility and explore its associated pathways. Subsequently, a bra-miR9569 overexpression vector was constructed and transformed into Arabidopsis thaliana. Phenotypic characterization of transgenic Arabidopsis lines, combined with anther viability assessment and quantification of ATP content and reactive oxygen species (ROS) levels in Chinese cabbage, was performed to analyze the effects of bra-miR9569. Our findings demonstrate that mutation of the mitochondrial gene orf138 in Ogura CMS lines leads to upregulation of bra-miR9569. This microRNA negatively regulates the expression of the ATP-related gene AHA6, resulting in reduced H⁺-ATPase activity. The consequent energy deficiency triggers cellular content degradation, ultimately causing failure of pollen wall formation and pollen abortion. Full article

One of the key elements in the analysis of gene expression and its post-translational regulation is miRNAs. Degradome-seq analyses are performed to analyze the cleavage of target RNAs in the transcriptome. This work presents the first degradome-seq library preparation protocol that enables successful construction of libraries, even from highly degraded RNA samples with RIN below 3, thus significantly expanding the possibilities for research when working with low-quality material. The developed protocol improves the efficiency of library preparation in degradome-seq analysis used to identify miRNA targets, reduces library preparation time, and lowers the cost of purchasing reagents by using reagents from the RNA-seq library preparation kit and proprietary-designed primers. A crucial feature of this new protocol is optimizing the purification step for short library fragments, which increases the yield of correctly sized fragments compared to previously used methods. This is achieved by implementing an original method involving tube-spin purification with gauze and precipitation using sodium acetate with glycogen, greatly enhancing recovery efficiency—a factor especially critical when working with degraded RNA. Cloning to a plasmid and sequencing of the inserted fragment verified the correctness of the library preparation using the developed protocol. This protocol represents a groundbreaking tool for degradome research, enabling the construction and sequencing of degradome libraries, even from degraded samples previously considered unsuitable for such analyses. This is due to the use of residues from the sRNA-seq library kit. It noticeably reduces the cost of library construction. The precision of the excised fragment after electrophoresis was performed during the procedure to isolate fragments of the correct length, which was improved using additional size markers. Compared to previously used methods, optimizing the purification method of degradome-seq libraries allowed an increase in the yield of fragments obtained. Full article

Soybean (Glycine max (L.) Merr.) is a critical crop in China, serving as a primary source of food, oil, and animal feed. Drought stress significantly impacts soybean growth and yield. MicroRNAs (miRNAs) play crucial roles in plant drought responses. The miR164 family is highly conserved across plant species and has been shown to participate in drought responses in a range of plants, yet the function of miR164 in soybean remains unclear. In this study, we identified GmNAC35 as a direct target of miR164 through published degradome sequencing data and 5′ RLM-RACE assays. Under drought stress, miR164 members (e.g., MIR164a, MIR164f, and MIR164k) rapidly down-regulated, reaching their lowest expression at 2 h and returning to basal levels within 6 h. Conversely, GmNAC35 showed an inverse pattern, indicating negative regulation by miR164. Overexpression of GmNAC35 enhanced drought tolerance in transgenic soybean plants, as evidenced by higher survival rates and reduced water loss. Transcriptomic analysis revealed that GmNAC35 modulates stress-responsive pathways, including ABA signaling and phenylpropanoid biosynthesis. Our findings indicate that miR164 negatively regulates GmNAC35, a positive regulator of drought tolerance. This enhances our understanding of the molecular mechanisms of drought tolerance in soybean and may inform strategies for breeding drought-resistant varieties. Full article

Small RNAs (sRNAs) are pivotal in regulating gene expression and are involved in a diverse array of biological processes. Among these, microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) have been extensively investigated over the past decades. We conducted an in-depth analysis of deep sequencing data from the gymnosperm Ginkgo biloba, encompassing sRNA, transcriptome, and degradome libraries. Our analysis identified a total of 746 miRNAs and 654 phasiRNA precursor (PHAS) loci, with 526 (80%) of the PHAS loci predicted to be triggered by 515 miRNAs (69%). Several miRNA-PHAS modules, particularly the miR159/miR319-PHAS module, were found to potentially regulate reproductive development by targeting GAMYB genes and triggering phasiRNA biogenesis. The miR390-PHAS module appears to be involved in flavonoid biosynthesis by targeting key enzyme genes such as chalcone synthase (CHS) and anthocyanin synthase (ANS). Through target gene identification and coexpression analysis, we uncovered two distinct models of complex regulatory networks: growth-related factors like ARF and GRF seem to be regulated exclusively by miRNAs (Model 1), while certain disease resistance-related genes are predicted to be regulated by both miRNAs and phasiRNAs (Model 2), indicating diverse regulatory mechanisms across different biological processes. Overall, our study provides a comprehensive annotation of miRNA and PHAS loci in G. biloba and elucidates a post-transcriptional regulatory network, offering novel insights into sRNA research in gymnosperms. Full article

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is one of the main afforestation tree species in southern China. Continuous planting for multiple generations has led to a decrease in the content of available phosphorus in the soil. To adapt to low phosphorus stress, plants develop a series of physiological, biochemical, and developmental responses through self-regulation. Recent studies have shown that miRNAs play a regulatory role in plants’ responses to low phosphorus stress. However, the regulatory mechanism of miRNAs in Chinese fir in response to low phosphorus stress is still unclear. Here, we performed small RNA sequencing on the Chinese fir roots treated with normal phosphorus and low phosphorus and identified a total of 321 miRNAs, including 139 known miRNAs and 182 new miRNAs, with 43 differentially expressed miRNAs (DEMs). Integrative analysis combined with degradome sequencing data revealed that 193 miRNAs (98 known and 95 new) targeted 469 genes, among which 23 DEMs targeted 44 genes. Gene enrichment analysis indicated that under low phosphorus stress, transcription and transcriptional regulation, as well as signal transduction, were significantly activated in Chinese fir. Modules in the miRNA–target pathways, such as miR166/HD-ZIP III, miR169/NFYA7, miR529/SPL, and miR399/UBC23, may be the key regulatory factors in the response to low phosphorus stress in Chinese fir. In addition, we found that PC-3p-1033_8666 was significantly downregulated and that PC-5p-3786_2830 was significantly upregulated, which presumably respond to low phosphorus stress by indirectly affecting phosphorus-related hormone signaling or PSR genes. The identified miRNA–target network and significantly activated pathways in this study provide insights into the post-transcriptional regulatory mechanisms of Chinese fir adapting to low phosphorus environments, which can offer theoretical references for the stress resistance and superior variety breeding of Chinese fir. Full article