International Journal of Molecular Sciences

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19 pages, 2620 KiB

Open AccessArticle

Genome-Wide Discovery of miRNAs with Differential Expression Patterns in Responses to Salinity in the Two Contrasting Wheat Cultivars

by Muhammad Zeeshan, Cheng-Wei Qiu, Shama Naz, Fangbin Cao and Feibo Wu

Int. J. Mol. Sci. 2021, 22(22), 12556; https://doi.org/10.3390/ijms222212556 - 21 Nov 2021

Cited by 19 | Viewed by 2733

Abstract

Salinity is a serious environmental issue. It has a substantial effect on crop yield, as many crop species are sensitive to salinity due to climate change, and it impact is continuing to increase. Plant microRNAs (miRNAs) contribute to salinity stress response in bread [...] Read more.

Salinity is a serious environmental issue. It has a substantial effect on crop yield, as many crop species are sensitive to salinity due to climate change, and it impact is continuing to increase. Plant microRNAs (miRNAs) contribute to salinity stress response in bread wheat. However, the underlying molecular mechanisms by which miRNAs confer salt tolerance in wheat are unclear. We conducted a genome-wide discovery study using Illumina high throughput sequencing and comprehensive in silico analysis to obtain insight into the underlying mechanisms by which small RNAs confer tolerance to salinity in roots of two contrasting wheat cvv., namely Suntop (salt-tolerant) and Sunmate (salt-sensitive). A total of 191 microRNAs were identified in both cultivars, consisting of 110 known miRNAs and 81 novel miRNAs; 181 miRNAs were shared between the two cultivars. The known miRNAs belonged to 35 families consisted of 23 conserved and 12 unique families. Salinity stress induced 43 and 75 miRNAs in Suntop and Sunmate, respectively. Among them, 14 and 29 known and novel miRNAs were expressed in Suntop and 37 and 38 in Sunmate. In silico analysis revealed 861 putative target mRNAs for the 75 known miRNAs and 52 putative target mRNAs for the 15 candidate novel miRNAs. Furthermore, seven miRNAs including tae-miR156, tae-miR160, tae-miR171a-b, tae-miR319, tae-miR159a-b, tae-miR9657 and novel-mir59 that regulate auxin responsive-factor, SPL, SCL6, PCF5, R2R3 MYB, and CBL-CIPK, respectively, were predicted to contribute to salt tolerance in Suntop. This information helps further our understanding of how the molecular mechanisms of salt tolerance are mediated by miRNAs and may facilitate the genetic improvement of wheat cultivars. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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18 pages, 3669 KiB

Open AccessArticle

Polycistronic Artificial microRNA-Mediated Resistance to Cucumber Green Mottle Mosaic Virus in Cucumber

by Shuo Miao, Chaoqiong Liang, Jianqiang Li, Barbara Baker and Laixin Luo

Int. J. Mol. Sci. 2021, 22(22), 12237; https://doi.org/10.3390/ijms222212237 - 12 Nov 2021

Cited by 13 | Viewed by 2869

Abstract

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted [...] Read more.

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic trans-acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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15 pages, 2630 KiB

Open AccessArticle

Distinct Evolutionary Profiles and Functions of microRNA156 and microRNA529 in Land Plants

by Qi Xie, Xufeng Wang, Juan He, Ting Lan, Jiayu Zheng, Yupeng Li, Jinkang Pan, Ling Lin, Junyi Zhao, Jing Li, Yu Yu, Beixin Mo, Xuemei Chen, Lei Gao and Lin Liu

Int. J. Mol. Sci. 2021, 22(20), 11100; https://doi.org/10.3390/ijms222011100 - 14 Oct 2021

Cited by 7 | Viewed by 2601

Abstract

MicroRNA156 (miR156) and miR529 have high sequence similarity and recognize overlapping sites in the same target genes, SQUAMOSA promoter binding protein-like (SPL or SBP box) genes, making it difficult to accurately distinguish their roles in regulatory networks that affect numerous biological functions. [...] Read more.

MicroRNA156 (miR156) and miR529 have high sequence similarity and recognize overlapping sites in the same target genes, SQUAMOSA promoter binding protein-like (SPL or SBP box) genes, making it difficult to accurately distinguish their roles in regulatory networks that affect numerous biological functions. Here, we collected data about miR156 and miR529 family members from representative land plants and performed sequence comparisons, phylogenetic analysis, small RNA sequencing, and parallel analysis of RNA ends (PARE) analysis to dissect their evolutionary and functional differences. Although miR156 and miR529 are highly similar, there are differences in their mismatch-sensitive regions, which are essential for target recognition. In land plants, miR156 precursors are conserved mainly within the hairpin region, whereas miR529 precursors are conserved outside the hairpin region, including both the 5’ and 3’ arms. Phylogenetic analysis showed that MIR156 and MIR529 evolved independently, through divergent evolutionary patterns. The two genes also exhibit different expression patterns, with MIR529 preferentially expressed in reproductive tissues and MIR156 in other tissues. PARE analysis revealed that miR156 and miR529 possess specific targets in addition to common targets in maize, pointing to functional differences between them. Based on our findings, we developed a method for the rapid identification of miR529 and miR156 family members and uncovered the evolutionary divergence of these families, providing insights into their different regulatory roles in plant growth and development. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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8 pages, 935 KiB

Open AccessCommunication

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection

by Timo Schlemmer, Patrick Barth, Lisa Weipert, Christian Preußer, Martin Hardt, Anna Möbus, Tobias Busche and Aline Koch

Int. J. Mol. Sci. 2021, 22(13), 7212; https://doi.org/10.3390/ijms22137212 - 5 Jul 2021

Cited by 12 | Viewed by 3796

Abstract

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA [...] Read more.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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22 pages, 46838 KiB

Open AccessArticle

Role of Long Noncoding RNAs ZlMSTRG.11348 and UeMSTRG.02678 in Temperature-Dependent Culm Swelling in Zizania latifolia

by Zheng-Hong Wang, Ning Yan, Xi Luo, Sai-Sai Guo, Shu-Qin Xue, Jiang-Qiong Liu, Shen-Shen Zhang, Li-Wen Zheng, Jing-Ze Zhang and De-Ping Guo

Int. J. Mol. Sci. 2021, 22(11), 6020; https://doi.org/10.3390/ijms22116020 - 2 Jun 2021

Cited by 2 | Viewed by 2715

Abstract

Temperature influences the physiological processes and ecology of both hosts and endophytes; however, it remains unclear how long noncoding RNAs (lncRNAs) modulate the consequences of temperature-dependent changes in host–pathogen interactions. To explore the role of lncRNAs in culm gall formation induced by the [...] Read more.

Temperature influences the physiological processes and ecology of both hosts and endophytes; however, it remains unclear how long noncoding RNAs (lncRNAs) modulate the consequences of temperature-dependent changes in host–pathogen interactions. To explore the role of lncRNAs in culm gall formation induced by the smut fungus Ustilago esculenta in Zizania latifolia, we employed RNA sequencing to identify lncRNAs and their potential cis-targets in Z. latifolia and U. esculenta under different temperatures. In Z. latifolia and U. esculenta, we identified 3194 and 173 lncRNAs as well as 126 and four potential target genes for differentially expressed lncRNAs, respectively. Further function and expression analysis revealed that lncRNA ZlMSTRG.11348 regulates amino acid metabolism in Z. latifolia and lncRNA UeMSTRG.02678 regulates amino acid transport in U. esculenta. The plant defence response was also found to be regulated by lncRNAs and suppressed in Z. latifolia infected with U. esculenta grown at 25 °C, which may result from the expression of effector genes in U. esculenta. Moreover, in Z. latifolia infected with U. esculenta, the expression of genes related to phytohormones was altered under different temperatures. Our results demonstrate that lncRNAs are important components of the regulatory networks in plant-microbe-environment interactions, and may play a part in regulating culm swelling in Z. latifolia plants. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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15 pages, 2550 KiB

Open AccessArticle

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

by Eduardo Luján-Soto, Vasti T. Juárez-González, José L. Reyes and Tzvetanka D. Dinkova

Int. J. Mol. Sci. 2021, 22(10), 5310; https://doi.org/10.3390/ijms22105310 - 18 May 2021

Cited by 11 | Viewed by 2715

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. [...] Read more.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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17 pages, 3820 KiB

Open AccessArticle

Genome-Wide Identification of Barley Long Noncoding RNAs and Analysis of Their Regulatory Interactions during Shoot and Grain Development

by Sebastian Gasparis, Mateusz Przyborowski and Anna Nadolska-Orczyk

Int. J. Mol. Sci. 2021, 22(10), 5087; https://doi.org/10.3390/ijms22105087 - 11 May 2021

Cited by 4 | Viewed by 2765

Abstract

Long noncoding RNAs (lncRNAs) are a class of RNA molecules with gene regulatory functions in plant development and the stress response. Although the number of lncRNAs identified in plants is rapidly increasing, very little is known about their role in barley development. In [...] Read more.

Long noncoding RNAs (lncRNAs) are a class of RNA molecules with gene regulatory functions in plant development and the stress response. Although the number of lncRNAs identified in plants is rapidly increasing, very little is known about their role in barley development. In this study, we performed global identification of barley lncRNAs based on 53 RNAseq libraries derived from nine different barley tissues and organs. In total, 17,250 lncRNAs derived from 10,883 loci were identified, including 8954 novel lncRNAs. Differential expression of lncRNAs was observed in the developing shoot apices and grains, the two organs that have a direct influence on the final yield. The regulatory interaction of differentially expressed lncRNAs with the potential target genes was evaluated. We identified 176 cis-acting lncRNAs in shoot apices and 424 in grains, while the number of trans-acting lncRNAs in these organs was 1736 and 540, respectively. The potential target protein-coding genes were identified, and their biological function was annotated using MapMan ontology. This is the first insight into the roles of lncRNAs in barley development on the genome-wide scale, and our results provide a solid background for future functional studies. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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15 pages, 24214 KiB

Open AccessArticle

Genome-Wide Analysis Identified a Set of Conserved lncRNAs Associated with Domestication-Related Traits in Rice

by Huang He, Yan-Fei Zhou, Yu-Wei Yang, Zhi Zhang, Meng-Qi Lei, Yan-Zhao Feng, Yu-Chan Zhang, Yue-Qin Chen, Jian-Ping Lian and Yang Yu

Int. J. Mol. Sci. 2021, 22(9), 4742; https://doi.org/10.3390/ijms22094742 - 29 Apr 2021

Cited by 9 | Viewed by 2835

Abstract

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. [...] Read more.

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. However, limited data shows plant long noncoding RNAs (lncRNAs) are also involved in this biological process. In this study, we performed strand-specific RNA sequencing of cultivated rice Oryza sativa ssp. japonica and O. sativa ssp. indica, and their wild progenitor O. rufipogon. We identified a total of 8528 lncRNAs, including 4072 lncRNAs in O. rufipogon, 2091 lncRNAs in japonica rice, and 2365 lncRNAs in indica rice. The lncRNAs expressed in wild rice were revealed to be shorter in length and had fewer exon numbers when compared with lncRNAs from cultivated rice. We also identified a number of conserved lncRNAs in the wild and cultivated rice. The functional study demonstrated that several of these conserved lncRNAs are associated with domestication-related traits in rice. Our findings revealed the feature and conservation of lncRNAs during rice domestication and will further promote functional studies of lncRNAs in rice. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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17 pages, 8233 KiB

Open AccessArticle

MIR172d Is Required for Floral Organ Identity and Number in Tomato

by Wanping Lin, Suresh Kumar Gupta, Tzahi Arazi and Ben Spitzer-Rimon

Int. J. Mol. Sci. 2021, 22(9), 4659; https://doi.org/10.3390/ijms22094659 - 28 Apr 2021

Cited by 18 | Viewed by 3183

Abstract

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is [...] Read more.

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-d^CR). The slmir172c-d^CR plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172c^CR loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172d^CR alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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20 pages, 3364 KiB

Open AccessArticle

Barley Seeds miRNome Stability during Long-Term Storage and Aging

by Marta Puchta, Jolanta Groszyk, Magdalena Małecka, Marek D. Koter, Maciej Niedzielski, Monika Rakoczy-Trojanowska and Maja Boczkowska

Int. J. Mol. Sci. 2021, 22(9), 4315; https://doi.org/10.3390/ijms22094315 - 21 Apr 2021

Cited by 10 | Viewed by 2893

Abstract

Seed aging is a complex biological process that has been attracting scientists’ attention for many years. High-throughput small RNA sequencing was applied to examine microRNAs contribution in barley seeds senescence. Unique samples of seeds that, despite having the same genetic makeup, differed in [...] Read more.

Seed aging is a complex biological process that has been attracting scientists’ attention for many years. High-throughput small RNA sequencing was applied to examine microRNAs contribution in barley seeds senescence. Unique samples of seeds that, despite having the same genetic makeup, differed in viability after over 45 years of storage in a dry state were investigated. In total, 61 known and 81 novel miRNA were identified in dry seeds. The highest level of expression was found in four conserved miRNA families, i.e., miR159, miR156, miR166, and miR168. However, the most astonishing result was the lack of significant differences in the level of almost all miRNAs in seed samples with significantly different viability. This result reveals that miRNAs in dry seeds are extremely stable. This is also the first identified RNA fraction that is not deteriorating along with the loss of seed viability. Moreover, the novel miRNA hvu-new41, with higher expression in seeds with the lowest viability as detected by RT-qPCR, has the potential to become an indicator of the decreasing viability of seeds during storage in a dry state. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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9 pages, 1596 KiB

Open AccessArticle

Uptake of MicroRNAs from Exosome-Like Nanovesicles of Edible Plant Juice by Rat Enterocytes

by Yuko Ito, Kohei Taniguchi, Yuki Kuranaga, Nabil Eid, Yosuke Inomata, Sang-Woong Lee and Kazuhisa Uchiyama

Int. J. Mol. Sci. 2021, 22(7), 3749; https://doi.org/10.3390/ijms22073749 - 3 Apr 2021

Cited by 31 | Viewed by 4882

Abstract

MicroRNAs (miRNAs) are small RNAs present in extracellular vesicles (EVs) that, when transferred to a target cell, affect its biological functions. Plant miRNAs regulate the expression of certain mammalian genes. Here, we characterized EVs in fruit and vegetable juice, and their miRNA cargo, [...] Read more.

MicroRNAs (miRNAs) are small RNAs present in extracellular vesicles (EVs) that, when transferred to a target cell, affect its biological functions. Plant miRNAs regulate the expression of certain mammalian genes. Here, we characterized EVs in fruit and vegetable juice, and their miRNA cargo, and investigated whether such miRNA-containing EVs could be taken up by mammalian enterocytes in vitro. Using filtration and ultra-centrifugation methods, EVs were purified from commercially available and manually squeezed plant juice. EV morphological features and subcellular localization were analyzed using the NanoSight tracking system and electron microscopy. Plant EV miRNA levels were evaluated using quantitative reverse transcription PCR. For the in vitro EV uptake experiments, rat intestinal epithelial cells (IEC6) were used. Plant EVs shared morphological features with mammalian EVs and contained miR156a-5p, miR166a-3p, and miR168a-5p. EVs were present in the cell sap-filled central vacuoles and were taken up by IEC6 cells. Edible plant cells produce EVs that contain various miRNAs and release them into the central vacuole. The exogenous plant EVs are taken up by mammalian enterocytes in vitro. These findings suggest the possibility that exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract. Full article

(This article belongs to the Special Issue Plant Non-coding RNAs)

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