Plants produce secondary metabolites that serve various functions, including defense against biotic and abiotic stimuli. Many of these secondary metabolites possess valuable applications in diverse fields, including medicine, cosmetic, agriculture, and food and beverage industries, exhibiting their importance in both plant biology and various human needs. Small RNAs (sRNA), such as microRNA (miRNA) and small interfering RNA (siRNA), have been shown to play significant roles in regulating the metabolic pathways post-transcriptionally by targeting specific key genes and transcription factors, thus offering a promising tool for enhancing plant secondary metabolite biosynthesis. In this review, we summarize current approaches for manipulating sRNAs to regulate secondary metabolite biosynthesis in plants. We provide an overview of the latest research strategies for sRNA manipulation across diverse plant species, including the identification of potential sRNAs involved in secondary metabolite biosynthesis in non-model plants. We also highlight the potential future research directions, focusing on the manipulation of sRNAs to produce high-value compounds with applications in pharmaceuticals, nutraceuticals, agriculture, cosmetics, and other industries. By exploring these advanced techniques, we aim to unlock new potentials for biotechnological applications, contributing to the production of high-value plant-derived products.

Long intergenic non-coding RNAs (lincRNAs) are emerging as regulators of protein-coding genes (PCGs) in many plant and animal developmental processes and stress responses. In this study, we characterize the genome-wide lincRNAs in potatoes responsive to a vascular bacterial disease presumably caused by Candidatus Liberibacter solanacearum (CLso). Approximately 4397 lincRNAs were detected in healthy and infected potato plants at various stages of zebra chip (ZC) disease progression. Of them, ~65% (2844) were novel lincRNAs, and less than 1% (9) were orthologs of Arabidopsis and rice based on reciprocal BLAST analysis, suggesting species-specific expansion. Among the proximal lincRNAs within 50 kbp from a PCG, ~49% were transcribed from the same strand, while ~39% and ~15% followed convergent (head-to-head) and divergent (tail-to-tail) orientations, respectively. Approximately 30% (1308) were differentially expressed following CLso infection, with substantial changes occurring 21 days after infection (DAI). Weighted Gene Co-expression Network Analysis (WGCNA) of lincRNAs and PCGs identified 46 highly correlated lincRNA-PCG pairs exhibiting co-up or co-downregulation. Furthermore, overexpression of selected lincRNAs in transgenic potato hairy roots resulted in perturbation of neighboring PCG expression and conferred tolerance to CLso infection. Our results provide novel insights into potato lincRNAs\' identity, expression dynamics, and functional relevance to CLso infection.

Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small RNAs (sRNAs) mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in the sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its likely paternal imprinted nature. These important features are modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines, we observe defects in endosperm development and a loss of around 70% of all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms are also dependent on OsCLSY3. We observed that many imprinted genes and seed development-associated genes are under the control of OsCLSY3. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction.

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1-244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes.

Wheat leaf rust, caused by the fungus Puccinia triticina (Pt), severely affects the grain quality and quantity of bread wheat (Triticum aestivum L.). Hairpin small(s)RNAs, like micro(mi)RNAs and their variants [including isomiRNAs (isomiRs) and microRNA-like RNAs (milRNAs)], along with their corresponding target genes, bestow leaf rust disease resistance, development and progression from both interacting species. However, the regulatory networks remain inadequately understood. Thirteen differentially expressed novel miRNAs, including two isomiRs and three milRNAs were discerned from induced reads of wheat sRNA libraries, and a further 5,393 and 1,275 candidate target genes were predicted in wheat and Pt, respectively. Functional annotation divulged that wheat-originated miRNAs/isomiRs were involved in resistance, while Pt-derived milRNAs imparted pathogenesis. The identified milRNAs- Tae-Pt-milR5, Tae-Pt-milR12, and Tae-Pt-milR14b and their cleavage sites on Pt target gene MEP5 were confirmed through degradome library screening, suggesting cross-kingdom translocation of Pt virulent genes in wheat host. Co-expression analysis of miRNAs/isomiRs-target genes provided insights into combating leaf rust disease, while co-expression analysis of milRNAs-target gene pairs reflected the extent of pathogenicity exerted by Pt with varied expression levels at the analyzed time points. The analysis pinpointed leaf rust-responsive candidate hairpin sRNAs- Tae-miR8, Tae-Pt-miR12, Tae-Pt-miR14a, and Tae-Pt-miR14b in wheat and Tae-Pt-milR12 in Pt. This study provides new insights into the hairpin sRNAs involved in the resistance and pathogenesis of wheat and Pt, respectively. Furthermore, crucial hairpin sRNAs and their promising targets for future biotechnological interventions to augment stress resilience have been identified.

Canker caused by Lonsdalea populi has seriously reduced the economic and ecological benefits of poplar. MicroRNAs play vital roles in the response of plants to biotic stress. However, there is little research about the regulatory mechanism of miRNAs among different tree varieties upon pathogen infection. To dissect miRNAs involved in L. populi resistance, three poplar varieties, 2025 (susceptible), 107 (moderately resistant) and Populus. tomentosa cv \'henan\' (resistant) were selected to elucidate the expression profiles of miRNAs using small RNA-seq. A total of 227 miRNAs were identified from all varieties. Intriguingly, miR160, miR169, miR171 and miR482b-5p were only identified in the resistant variety P. tomentosa upon pathogen infection, and these miRNAs might be important candidates for future investigation to improve the tolerance of poplar to L. populi. Among all identified miRNAs, 174 were differentially expressed in all varieties. Functional annotation analysis indicated that an array of miRNAs, including miR482, miR472, miR169, miR481, and miR172, should be involved in disease resistance and phytohormone signal transduction. Furthermore, correlation analysis of small RNA-seq and RNA-seq identified a handful of L. populi-responsive miRNAs and target genes, which exhibited that miR159 and miR172 played key roles in resistant variety P. tomentosa by targeting MYB and ERF, while miR6462c-5p and miR828 were related to the susceptibility of 2025 by targeting MYB. The comprehensive integration analysis in this research provides new insights into the regulatory pathways involved in the defence response of poplar to L. populi and offers crucial candidate miRNAs-target genes modules for poplar resistance improvement.

MicroRNA482 (miR482) is a conserved microRNA family in plants, playing critical regulatory roles in different biological activities. Though the members of the miR482 gene family have been identified in plants, a systematic study has not been reported yet. In the present research, 140 mature sequences generated by 106 precursors were used for molecular characterization, phylogenetic analysis, and target gene prediction, and the competing endogenous RNA (ceRNA) network mediated by miR482 was summarized. The length of mature sequences ranged from 17 nt to 25 nt, with 22 nt being the most abundant, and the start and end of the mature sequences had a preference for uracil (U). By sequence multiplex comparison, it was found that the mature sequences of 5p were clustered into one group, and others were clustered into the other group. Phylogenetic analysis revealed that the 140 mature sequences were categorized into six groups. Meanwhile, all the precursor sequences formed a stable hairpin structure, and the 106 precursors were divided into five groups. However, the expression of miR482 varied significantly between different species and tissues. In total, 149 target genes were predicted and their functions focused on single-organism process, cellular process, and cell and cell part. The ceRNA network of miR482 in tomato, cotton, and peanut was summarized based on related publications. In conclusion, this research will provide a foundation for further understanding of the miR482 gene family.

BACKGROUND: The yield of major crops is generally limited by sink capacity and source strength. Cucumber is a typical raffinose family oligosaccharides (RFOs)-transporting crop. Non-coding RNAs and alternative polyadenylation (APA) play important roles in the regulation of growth process in plants. However, their roles on the sink‒source regulation have not been demonstrated in RFOs-translocating species.
RESULTS: Here, whole-transcriptome sequencing was applied to compare the leaves of cucumber under different sink strength, that is, no fruit-carrying leaves (NFNLs) and fruit-carrying leaves (FNLs) at 12th node from the bottom. The results show that 1101 differentially expressed (DE) mRNAs, 79 DE long non-coding RNAs (lncRNAs) and 23 DE miRNAs were identified, which were enriched in photosynthesis, energy production and conversion, plant hormone signal transduction, starch and carbohydrate metabolism and protein synthesis pathways. Potential co-expression networks like, DE lncRNAs-DE mRNAs/ DE miRNAs-DE mRNAs, and competing endogenous RNA (ceRNA) regulation models (DE lncRNAs-DE miRNAs-DE mRNAs) associated with sink‒source allocation, were constructed. Furthermore, 37 and 48 DE genes, which enriched in MAPK signaling and plant hormone signal transduction pathway, exist differentially APA, and SPS (CsaV3_2G033300), GBSS1 (CsaV3_5G001560), ERS1 (CsaV3_7G029600), PNO1 (CsaV3_3G003950) and Myb (CsaV3_3G022290) may be regulated by both ncRNAs and APA between FNLs and NFNLs, speculating that ncRNAs and APA are involved in the regulation of gene expression of cucumber sink‒source carbon partitioning.
CONCLUSIONS: These results reveal a comprehensive network among mRNAs, ncRNAs, and APA in cucumber sink-source relationships. Our findings also provide valuable information for further research on the molecular mechanism of ncRNA and APA to enhance cucumber yield.

Plant microRNAs (miRNA) are regularly consumed orally along with diet, gaining attention for their RNA-based drug potential because of their ability to regulate mammalian gene expression specifically at the post-transcriptional level. Medicinally valued plants are well known for their anti-inflammatory property; however, the contribution of their miRNA in managing inflammation has been less studied. We investigated miRNA from four medicinally valued regularly consumed spices, and validated one of the most potential miRNA \'Clo-miR-14\' for its thermal stability, and absorption in the plasma samples of RA patient\'s by RT-PCR. In vitro and in vivo studies were performed to investigate the effect of Clo-miR-14 in ameliorating rheumatoid arthritis (RA) like symptoms. Our results suggest that \'Clo-miR-14,\' an exogenous miRNA present in Curcuma longa, absorbed through regular diet, has robust thermal stability at 100°C in humans. It significantly reduced pro-inflammatory cytokines (TNF, IL-1β, IL-6) and RA-like symptoms, suggesting that plant-based miRNA could be a promising candidate as an RNA-based drug for RA pathogenesis.