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Prediction of Plant microRNA Targets
Abstract
We predict regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity. Complementary sites within predicted targets are conserved in rice. Of the 49 predicted targets, 34 are members of transcription factor gene families involved in developmental patterning or cell differentiation. The near-perfect complementarity between plant miRNAs and their targets suggests that many plant miRNAs act similarly to small interfering RNAs and direct mRNA cleavage. The targeting of developmental transcription factors suggests that many plant miRNAs function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages.
... Micro RNA is a class of unique small endogenous, noncoding RNA that is involved in regulation of gene expression by binding to the target mRNA leading to mRNA cleavage, translational repression, mRNA de-adenylation or transcriptional silencing thereby controlling the expression of the translated product (Jones- Rhoades et al., 2006;Winter et al., 2012). The control for gene expression is based on complementary pairing at specific positions for the target (Rhoades et al., 2002). These microRNAs are responsible for specific temporal and spatial control of their gene targets. ...
... MicroRNAs (miRNAs), are long noncoding RNAs, that regulate gene expression by binding to the target mRNAs, resulting in mRNA cleavage or inhibition of protein translation (Ambros, 2001;Carrington and Ambros, 2003;Bartel, 2004). The plant miRNAs play a crucial role in diverse biological phenomenon, such as development of leaf (Rhoades et al., 2002;Chen, 2005;Guo et al., 2005;Lauter et al., 2005), nutrient homeostasis (Chiou, 2007) and various stress responses (Chapman et al., 2004;Jones-Rhoades and Bartel, 2004). Understanding the differential expression pattern of miRNAs and their role during the virus infection in papaya will provide the clues to design advanced strategies to control the PRSV infection in papaya and other high value horticultural crops. ...
Papaya ringspot virus (PRSV) is one of the most devastating viruses of papaya that has significantly hampered papaya production across the globe. Although PRSV resistance is known in some of its wild relatives, such as Vasconcellea cauliflora and in some of the improved papaya genotypes, the molecular basis of this resistance mechanism has not been studied and understood. Plant microRNAs are an important class of small RNAs that regulate the gene expression in several plant species against the invading plant pathogens. These miRNAs are known to manifest the expression of genes involved in resistance against plant pathogens, through modulation of the plant's biochemistry and physiology. In this study we made an attempt to study the overall expression pattern of small RNAs and more specifically the miRNAs in different papaya genotypes from India, that exhibit varying levels of tolerance or resistance to PRSV. Our study found that the expression of some of the miRNAs was differentially regulated in these papaya genotypes and they had entirely different miRNA expression profile in healthy and PRSV infected symptomatic plants. This data may help in improvement of papaya cultivars for resistance against PRSV through new breeding initiatives or biotechnological approaches such as genome editing.
... SCL6 can be negatively regulated by microRNA171 (miR171) [12]. miR171 specifically recognizes and binds to the SCL6 mRNA to mediate its cleavage [13,14]. The miR171-SCL6 Plants 2024, 13, 1232 2 of 11 module participates in various developmental and physiological processes, including shoot branching [7,12,15,16], phase transition [1,17], root growth [12,18], inflorescence axis elongation [17,[19][20][21], trichome initiation [22], silique production [20,21,23], and meristem development [9,18,[24][25][26]. ...
... SCL6 can be negatively regulated by microRNA171 (miR171) [12]. miR171 specifically recognizes and binds to the SCL6 mRNA to mediate its cleavage [13,14]. The miR171-SCL6 module participates in various developmental and physiological processes, including shoot branching [7,12,15,16], phase transition [1,17], root growth [12,18], inflorescence axis elongation [17,[19][20][21], trichome initiation [22], silique production [20,21,23], and meristem development [9,18,[24][25][26]. ...
SCARECROW-LIKE6 (SCL6) plays a role in the formation and maintenance of the meristem. In Larix kaempferi (Lamb.) Carr., an important afforestation tree species in China, SCL6 (LaSCL6) has two alternative splicing variants—LaSCL6-var1 and LaSCL6-var2—which are regulated by microRNA171. However, their roles are still unclear. In this study, LaSCL6-var1 and LaSCL6-var2 were transformed into the Arabidopsis thaliana (L.) Heynh. genome, and the phenotypic characteristics of transgenic A. thaliana, including the germination percentage, root length, bolting time, flower and silique formation times, inflorescence axis length, and branch and silique numbers, were analyzed to reveal their functions. It was found that LaSCL6-var1 and LaSCL6-var2 overexpression shortened the root length by 41% and 31%, respectively, and increased the inflorescence axis length. Compared with the wild type, the bolting time in transgenic plants was delayed by approximately 2–3 days, the first flower and silique formation times were delayed by approximately 3–4 days, and the last flower and silique formation times were delayed by about 5 days. Overall, the life cycle in transgenic plants was prolonged by approximately 5 days. These results show that LaSCL6 overexpression inhibited the transitions from the vegetative meristem to inflorescence meristem and from the flower meristem to meristem arrest in A. thaliana, revealing the roles of LaSCL6-var1 and LaSCL6-var2 in the fate transition and maintenance of the meristem.
... Consistent with prior studies in maize and rice (Xu et al. 2017), the analysis of potential targets for both known and candidate novel miRNAs did not reveal any direct binding of miRNAs to C 4 genes in this study. Given that a significant proportion of miRNA targets are TFs that can further regulate the expression of other genes (Rhoades et al. 2002). In this study, TFs targeted by miRNAs exhibited co-expression patterns with C 4 genes, suggesting that miRNAs may indirectly regulate C 4 genes through these TFs. ...
... The plant-specific TF family GRAS has roles in diverse biological processes. For instance, miR171targeted SCL TFs, such as SCL6/SCL6-IV, SCL22/SCL6-III, and SCL27/SCL6-II [also known as Hairy Meristems (HAM) and Lost Meristems (LOM)], have been established to play crucial roles in meristematic cell proliferation, polar organization, and chlorophyll synthesis (Llave et al. 2002;Rhoades et al. 2002;Bolle 2004;Schulze et al. 2010;Wang et al. 2010;Curaba et al. 2013). Consequently, miR171y/am may play a role in targeting SsHAM3a in sugarcane. ...
Main Conclusion
This study reveals miRNA indirect regulation of C4 genes in sugarcane through transcription factors, highlighting potential key regulators like SsHAM3a.
Abstract
C4 photosynthesis is crucial for the high productivity and biomass of sugarcane, however, the miRNA regulation of C4 genes in sugarcane remains elusive. We have identified 384 miRNAs along the leaf gradients, including 293 known miRNAs and 91 novel miRNAs. Among these, 86 unique miRNAs exhibited differential expression patterns, and we identified 3511 potential expressed targets of these differentially expressed miRNAs (DEmiRNAs). Analyses using Pearson correlation coefficient (PCC) and Gene Ontology (GO) enrichment revealed that targets of miRNAs with positive correlations are integral to chlorophyll-related photosynthetic processes. In contrast, negatively correlated pairs are primarily associated with metabolic functions. It is worth noting that no C4 genes were predicted as targets of DEmiRNAs. Our application of weighted gene co-expression network analysis (WGCNA) led to a gene regulatory network (GRN) suggesting miRNAs might indirectly regulate C4 genes via transcription factors (TFs). The GRAS TF SsHAM3a emerged as a potential regulator of C4 genes, targeted by miR171y and miR171am, and exhibiting a negative correlation with miRNA expression along the leaf gradient. This study sheds light on the complex involvement of miRNAs in regulating C4 genes, offering a foundation for future research into enhancing sugarcane's photosynthetic efficiency.
... In angiosperms, miRNAs generally target coding sequence regions [77][78][79][80]. However, in A. thaliana, there are several examples of cleavage site localization in the 3' UTR of mRNA targets: miR156/157-At1g53160, miR156-At2g33810, miR169-At1g17590/At1g54160 and miR319b.2-At1g53910 ...
... However, in A. thaliana, there are several examples of cleavage site localization in the 3' UTR of mRNA targets: miR156/157-At1g53160, miR156-At2g33810, miR169-At1g17590/At1g54160 and miR319b.2-At1g53910 modules [77,81,82]. ...
MicroRNAs regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that the differential expression of conserved miRNAs among land plants and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.
... The results of this study show that certain miRNAs are transferred from resistant melon varieties to broomrape. One of the screened transfer miRNAs, cme-miR156a, is a very conserved small non-coding RNAs in plants, and its target gene family is the SPL (SQUAOSA promoter binding like) family (Matthew et al., 2002;Bonnet et al., 2010). The miR156-SPL pathway plays a regulatory role in several plant growth and developmental processes, including phytohormone signaling and stress response (Wang, 2014;Lei et al., 2016;Miao et al., 2019). ...
Bidirectional trans-kingdom RNA silencing, a pivotal factor in plant-pathogen interactions, remains less explored in plant host-parasite dynamics. Here, using small RNA sequencing in melon root systems, we investigated microRNA (miRNA) expression variation in resistant and susceptible cultivars pre-and post-infection by the parasitic plant, broomrape. This approach revealed 979 known miRNAs and 110 novel miRNAs across 110 families. When comparing susceptible (F0) and resistant (R0) melon lines with broomrape infection (F25 and R25), 39 significantly differentially expressed miRNAs were observed in F25 vs. F0, 35 in R25 vs. R0, and 5 in R25 vs. F25. Notably, two miRNAs consistently exhibited differential expression across all comparisons, targeting genes linked to plant disease resistance. This suggests their pivotal role in melon’s defense against broomrape. The target genes of these miRNAs were confirmed via degradome sequencing and validated by qRT-PCR, ensuring reliable sequencing outcomes. GO and KEGG analyses shed light on the molecular functions and pathways of these differential miRNAs. Furthermore, our study unveiled four trans-kingdom miRNAs, forming a foundation for exploring melon’s resistance to broomrape.
... Changes in their expression indicate early responses triggered by external stimuli because miRNA amounts in cells can change rapidly [22]. A growing body of evidence illustrated that a single miRNA can regulate over 200 genes, and conversely, multiple miRNAs can collaboratively regulate one gene [23,24]. MiRNAs are well studied to target 3′ untranslated regions (3′ UTRs) and non-3' UTR regions in mRNA transctipts [25], thereby regulating protein synthesis. ...
The study of neuroimmune crosstalk and the involvement of neurotransmitters in inflammation and bone health has illustrated their significance in joint-related conditions. One important mode of cell-to-cell communication in the synovial fluid (SF) is through extracellular vesicles (EVs) carrying microRNAs (miRNAs). The role of neurotransmitter receptors in the pathogenesis of inflammatory joint diseases, and whether there are specific miRNAs regulating differentially expressed HTR2A, contributing to the inflammatory processes and bone metabolism is unclear. Expression of neurotransmitter receptors and their correlated inflammatory molecules were identified in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium from a scRNA-seq dataset. Immunohistochemistry staining of synovial tissue (ST) from RA and OA patients was performed for validation. Expression of miRNAs targeting HTR2A carried by SF EVs was screened in low- and high-grade inflammation RA from a public dataset and validated by qPCR. HTR2A reduction by target miRNAs was verified by miRNAs mimics transfection into RA fibroblasts. HTR2A was found to be highly expressed in fibroblasts derived from RA synovial tissue. Its expression showed a positive correlation with the degree of inflammation observed. 5 miRNAs targeting HTR2A were decreased in RA SF EVs compared to OA, three of which, miR-214-3p, miR-3120-5p and miR-615-3p, mainly derived from monocytes in the SF, were validated as regulators of HTR2A expression. The findings suggest that fibroblast HTR2A may play a contributory role in inflammation and the pathogenesis of RA. Additionally, targeting miRNAs that act upon HTR2A could present novel therapeutic strategies for alleviating inflammation in RA.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10238-024-01352-w.
... Although miR-430 also regulates genes in specific tissues for proper organogenesis, presumably through targeting purely zygotic genes [35,36], our results underscore its function during earlier developmental stages. This supports a previously alluded role for microRNAs on regulating mRNAs of genes that are concurrently and continuously de novo transcribed [32,61,62]: Keeping zygotic mRNAs accumulation at bay in time and/or space during MZT. As an example, left2 and stq, which regulate embryo left-right asymmetry and are targeted by miR-430 [34], are also pure zygotic genes (Additional file 6: Table S2). ...
Background
Early embryonic developmental programs are guided by the coordinated interplay between maternally inherited and zygotically manufactured RNAs and proteins. Although these processes happen concomitantly and affecting gene function during this period is bound to affect both pools of mRNAs, it has been challenging to study their expression dynamics separately.
Results
By employing SLAM-seq, a nascent mRNA labeling transcriptomic approach, in a developmental time series we observe that over half of the early zebrafish embryo transcriptome consists of maternal-zygotic genes, emphasizing their pivotal role in early embryogenesis. We provide an hourly resolution of de novo transcriptional activation events and follow nascent mRNA trajectories, finding that most de novo transcriptional events are stable throughout this period. Additionally, by blocking microRNA-430 function, a key post transcriptional regulator during zebrafish embryogenesis, we directly show that it destabilizes hundreds of de novo transcribed mRNAs from pure zygotic as well as maternal-zygotic genes. This unveils a novel miR-430 function during embryogenesis, fine-tuning zygotic gene expression.
Conclusion
These insights into zebrafish early embryo transcriptome dynamics emphasize the significance of post-transcriptional regulators in zygotic genome activation. The findings pave the way for future investigations into the coordinated interplay between transcriptional and post-transcriptional landscapes required for the establishment of animal cell identities and functions.
... In plants, microRNAs have sprung up as important players in plant growth and react against the "biotic and abiotic stresses", including micronutrient deficiency [5]. These miRNAs contribute to the "post-transcriptional regulation of gene expression by down-regulating the amounts of their complementary target transcripts" (mostly transcription factors) either by transcript cleavage or translational inhibition [6]. All the miRNAs of plants are not responsible for particular micronutrient stress which means the challenge remains to uncover (if any) that are directly relevant to the micronutrient [5]. ...
... The miR159 and miR164 are key players in the hormone-responsive signalling cascade. miR159 regulated the abscisic acid (ABA) and gibberellin signalling via MYC transcription factor, while miRNA164 regulated the auxin signalling process via ARF transcription factor [84][85][86]. The interaction of TaTPKs with these miRNAs, and transcription factor MYC and ARF revealed the regulation of TaTPKs through hormone-related signalling processes. The interacting tae-miR395a is reported to be A. Kaur et al. significantly upregulated in K + deficient conditions which indicated the ′role of TaTPKs in plant survival at the time of K + ion deficit [87]. ...
Two-pore K⁺ (TPK) channels are voltage-independent and involved in stress response in plants. Herein, we identified 12 TaTPK genes located on nine chromosomes in the Triticum aestivum genome. The majority of TaTPK genes comprised two exons. Each TaTPK channel comprised four transmembrane (TM) helices, N- and C-terminal ion-channel domains, two EF-hand domains and one 14-3-3 binding site. Additionally, highly conserved ‘GYGD’ motif responsible for K⁺ ion specificity, was found in between the TMs in both the ion-channel domains. Nine TaTPK channels were predicted to be localised at the plasma membrane, while three were vacuolar. The protein-protein and protein-chemical interactions indicated the coordinated functioning of the TaTPK channels with the other K⁺ transporters and their possible interaction with the Ca²⁺-signaling pathway. Expression studies suggested their importance in both vegetative and reproductive tissues development. Significantly modulated expression of various TaTPK genes during heat, drought, combined heat and drought and salt stresses, and after fungal infestation, depicted their function in stress responses. The miRNAs and transcription factors interaction analyses suggested their role in the hormone, light, growth and development-related, and stress-responsive signaling cascades. The current study suggested vital functions of various TaTPK genes, especially in stress response, and would provide an opportunity for their detailed characterization in future studies.
... AP1 is not only the A-class gene that specifies sepal and petal identity but also an important f loral meristem (FM) maintenance gene, regulated by the upstream LEAFY (LFY) gene activating f loral meristem initiation [34][35][36][37]. SEPALLATA (SEP) in Arabidopsis is highly redundant in its function to determine f loral organ identity, but RCD, the ortholog of the SEP in plants of the Asteraceae family, undergoes significant expansion and functional divergence. ...
Chrysanthemum ×morifolium has great ornamental and economic value on account of its exquisite capitulum. However, previous studies have mainly focused on the corolla morphology of the capitulum. Such an approach cannot explain the variable inflorescence architecture of the chrysanthemum. Previous research from our group has shown that NO APICAL MERISTEM (ClNAM) is likely to function as a hub gene in capitulum architecture in the early development stage. In the present study, ClNAM was used to investigate the function of these boundary genes in the capitulum architecture of C. lavandulifolium, a closely related species of C. ×morifolium in the genus. Modified of ClNAM in C. lavandulifolium resulted in an advanced initiation of the floral primordium at the capitulum. As a result, the receptacle morphology was altered and the number of florets decreased. The ray floret corolla was shortened, but the disc floret was elongated. The number of capitula increased significantly, arranged in more densely compound corymbose synflorescences. The yeast and luciferase reporter system revealed that ClAP1, ClRCD2 and ClLBD18 target and activate ClNAM. Subsequently, ClNAM targets and activates ClCUC2a/c, which regulates the initiation of floral and inflorescence in C. lavandulifolium. ClNAM was also targeted and cleaved by cla-miR164 in this process. In conclusion, this study established a boundary gene regulatory network with cla-miR164-ClNAM as the hub. This network not only influence the architecture of capitulum, but also affect compound corymbose synflorescences of the C. lavandulifolium. These results provide new insights into the mechanisms regulating inflorescence architecture in chrysanthemum.
... The sequences of miR156s and miR157s are highly similar, with only 1/2 different nucleotides (Zhou et al. 2021). Posttranscriptional mRNAs of SPLs with miRNA response elements (MREs) complementary to miR156 were cleaved and/or translationally repressed by miR156 (Rhoades et al. 2002). The key role of the miR156/157-SPL regulatory module in plant responses to abiotic stress has become increasingly clear. ...
SQUAMOSA PROMOTER BINDING PROTEIN-LIKEs (SPLs) encode plant-specific transcription factors that regulate plant growth and development, stress response, and metabolite accumulation. However, there is limited information on Scutellaria baicalensis SPLs. In this study, 14 SbSPLs were identified and divided into 8 groups based on phylogenetic relationships. SbSPLs in the same group had similar structures. Abscisic acid-responsive (ABRE) and MYB binding site (MBS) cis-acting elements were found in the promoters of 8 and 6 SbSPLs. Segmental duplications and transposable duplications were the main causes of SbSPL expansion. Expression analysis based on transcriptional profiling showed that SbSPL1, SbSPL10, and SbSPL13 were highly expressed in roots, stems, and flowers, respectively. Expression analysis based on quantitative real-time polymerase chain reaction (RT‒qPCR) showed that most SbSPLs responded to low temperature, drought, abscisic acid (ABA) and salicylic acid (SA), among which the expression levels of SbSPL7/9/10/12 were significantly upregulated in response to abiotic stress. These results indicate that SbSPLs are involved in the growth, development and stress response of S. baicalensis. In addition, 8 Sba-miR156/157 s were identified, and SbSPL1-5 was a potential target of Sba-miR156/157 s. The results of target gene prediction and coexpression analysis together indicated that SbSPLs may be involved in the regulation of L-phenylalanine (L-Phe), lignin and jasmonic acid (JA) biosynthesis. In summary, the identification and characterization of the SbSPL gene family lays the foundation for functional research and provides a reference for improved breeding of S. baicalensis stress resistance and quality traits.
... miRNAs regulate cellular homeostasis by serving as nodes of signaling networks (5). Culminating evidence from research illustrated that even a single miRNA can regulate multiple (>200) genes, and conversely, multiple miRNAs can regulate a single gene (6,7). This dynamic yet highly complex interaction of miRNAs and their target genes and how it is impacted by the abundance of miRNAs and the affinity and access to target mRNAs make miRNA a hot topic in biomedical research. ...
MicroRNAs (miRNAs) are small non-coding RNAs that alter the expression of target genes at the post-transcriptional level, influencing diverse outcomes in metabolism, cell differentiation, proliferation, cell survival, and cell death. Dysregulated miRNA expression is implicated in various rheumatic conditions, including ankylosing spondylitis (AS), gout, juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis, rheumatoid arthritis (RA), Sjogren’s syndrome, systemic lupus erythematosus (SLE) and systemic sclerosis. For this review, we used an open-source programming language- PowerShell, to scan the massive number of existing primary research publications on PubMed on miRNAs in these nine diseases to identify and count unique co-occurrences of individual miRNAs and the disease name. These counts were used to rank the top seven most relevant immuno-miRs based on their research volume in each rheumatic disease. Individual miRNAs were also screened for publication with the names of immune cells, cytokines, and pathological processes involved in rheumatic diseases. These occurrences were tabulated into matrices to identify hotspots for research relevance. Based on this information, we summarize the basic and clinical findings for the top three miRNAs — miR-146, miR-155, and miR-21 — whose relevance spans across multiple rheumatic diseases. Furthermore, we highlight some unique miRNAs for each disease and why some rheumatic conditions lack research in this emerging epigenetics field. With the overwhelming number of publications on miRNAs in rheumatic diseases, this review serves as a ‘relevance finder’ to guide researchers in selecting miRNAs based on the compiled existing knowledge of their involvement in disease pathogenesis. This approach applies to other disease contexts with the end goal of developing miRNA-based therapeutics.
... miR156, and the closely related miRNAs, miR157 and miR529, act by repressing the expression of an ancient plant-specific family of transcription factors, known as SQUAMOSA PRO-MOTER BINDING PROTEINS (SBP/SPL). [69][70][71] As their name indicates, SBP family members were first identified by their ability to bind to the promoter of the snapdragon floral identity gene, SQUAMOSA, the ortholog of APETALA1 (AP1) in Arabidopsis. 72 SPL genes were subsequently identified in Arabidopsis, 73,74 the alga Chlamydomonas reinhardtii, 75 in the moss Physcomitrium patens, 76,77 and in all other surveyed land plants. ...
... A similar result was claimed (Huo et al., 2015) for miR158b down and non-canonical predicted target fucosyl transferase1/ At2g03220/FT1 concordantly down in response to dark-induced senescence, a process of carbon re-allocation. Consistent with that result, we observe concordant changes but in the opposite direction in response to high sucrose stress: both miR158 and PPR/ AT1G64100 target (Rhoades et al., 2002) plus FT1 target were concordantly upregulated (Table 1, Supplementary Dataset S6). On the other hand, we observed the majority of validated PPR targets of miR173 and miR161 (which manifested non-significant trend of upregulation in response to sucrose; Supplementary Dataset S3), namely, At1g62910, At1g63130, At1g62930, At1g63400, At1g63150, including PHAS loci AT1G62914, AT1G63130, and AT1G63400 (Supplementary Dataset S4) , were significantly anti-concordantly downregulated by sucrose (Supplementary Dataset S6), supporting a hypothesized function in ROS regulation. ...
In plants, sucrose is the main transported disaccharide that is the primary product of photosynthesis and controls a multitude of aspects of the plant life cycle including structure, growth, development, and stress response. Sucrose is a signaling molecule facilitating various stress adaptations by crosstalk with other hormones, but the molecular mechanisms are not well understood. Accumulation of high sucrose concentrations is a hallmark of many abiotic and biotic stresses, resulting in the accumulation of reactive oxygen species and secondary metabolite anthocyanins that have antioxidant properties. Previous studies have shown that several MYeloBlastosis family/MYB transcription factors are positive and negative regulators of sucrose-induced anthocyanin accumulation and subject to microRNA (miRNA)–mediated post-transcriptional silencing, consistent with the notion that miRNAs may be “nodes” in crosstalk signaling by virtue of their sequence-guided targeting of different homologous family members. In this study, we endeavored to uncover by deep sequencing small RNA and mRNA transcriptomes the effects of exogenous high sucrose stress on miRNA abundances and their validated target transcripts in Arabidopsis. We focused on genotype-by-treatment effects of high sucrose stress in Production of Anthocyanin Pigment 1-Dominant/pap1-D, an activation-tagged dominant allele of MYB75 transcription factor, a positive effector of secondary metabolite anthocyanin pathway. In the process, we discovered links to reactive oxygen species signaling through miR158/161/173-targeted Pentatrico Peptide Repeat genes and two novel non-canonical targets of high sucrose-induced miR408 and miR398b*(star), relevant to carbon metabolic fluxes: Flavonoid 3’-Hydroxlase (F3’H), an important enzyme in determining the B-ring hydroxylation pattern of flavonoids, and ORANGE a post-translational regulator of Phytoene Synthase expression, respectively. Taken together, our results contribute to understanding the molecular mechanisms of carbon flux shifts from primary to secondary metabolites in response to high sugar stress.
... The FMIR with the greatest number of potential targets was FMIR5139, with 11 possible targets, including EXPANSIN1 and βE1,3, which were classified as gene targets for this FMIR in rose petals during flowering [68] and are also related to the softening of the fruit [52]; the rest of the target genes were derived from multiple alignments made between the miRNA sequence and the guava genome assembly. Next was FMIR164, with eight target genes, including OMTN 1-6 in Oriza sativa [69] and the CUC1 and CUC2 proteins in A. thaliana [70]. The remaining families had five or fewer target genes ( Figure 3A). ...
The guava (Psidium guajava L.) is a climacteric fruit with an accelerated post-harvest overripening. miRNAs are small RNA sequences that function as gene regulators in eukaryotes and are essential for their survival and development. In this study, miRNA libraries were constructed, sequenced and analyzed from the breaker and ripe stages of guava fruit cv. Siglo XXI. One hundred and seventy-four mature miRNA sequences from 28 miRNA families were identified. The taxonomic distribution of the guava miRNAs showed a high level of conservation among the dicotyledonous plants. Most of the predicted miRNA target genes were transcription factors and genes involved in the metabolism of phytohormones such as abscisic acid, auxins, and ethylene, as revealed through an ontology enrichment analysis. The miRNA families miR168, miR169, miR396, miR397, and miR482 were classified as being directly associated with maturation, whereas the miRNA families miR160, miR165, miR167, miR3930, miR395, miR398, and miR535 were classified as being indirectly associated. With this study, we intended to increase our knowledge and understanding of the regulatory process involved in the ripening process, thereby providing valuable information for future research on the ripening of guava fruit.
... SQUAMOSA promoter binding protein-like (SPL) transcription factors, which are specific to plants, mediate various aspects of plant development, including branching [32]. Different members of the SPL gene family in Arabidopsis are post-transcriptionally regulated by miR156 [73]. Overaccumulation of miR156 leads to a considerably bushy phenotype [74,75]. ...
Shoot branching is a complex and tightly regulated developmental process that is essential for determining plant architecture and crop yields. The outgrowth of tiller buds is a crucial step in shoot branching, and it is influenced by a variety of internal and external cues. This review provides an extensive overview of the genetic, plant hormonal, and environmental factors that regulate shoot branching in several plant species, including rice, Arabidopsis, tomato, and wheat. We especially highlight the central role of TEOSINTE BRANCHED 1 (TB1), a key gene in orchestrating bud outgrowth. In addition, we discuss how the phytohormones cytokinins, strigolactones, and auxin interact to regulate tillering/branching. We also shed light on the involvement of sugar, an integral component of plant development, which can impact bud outgrowth in both trophic and signaling ways. Finally, we emphasize the substantial influence of environmental factors, such as light, temperature, water availability, biotic stresses, and nutrients, on shoot branching. In summary, this review offers a comprehensive evaluation of the multifaced regulatory mechanisms that underpin shoot branching and highlights the adaptable nature of plants to survive and persist in fluctuating environmental conditions.
... After AGO2 is loaded with a miRNA 'driver' (AGO2:miRNA) it can use partial complementarity between the 'driver' to identify 'target' sequences on other RNA molecules. Unlike plant miRNAs that have fully complementary targets [5], in mammals most known AGO2:miRNA binding sites show partial complementarity focused on a 'seed' region located at the 5'end of the 'driver' sequence. A 'canonical seed' sequence denotes a fully Watson-Crick complementary stretch of at least 6 nucleotides starting at the second position from the 5' end of the miRNA 'driver'. ...
Argonaute proteins are instrumental in regulating RNA stability and translation. AGO2, the major mammalian Argonaute protein, is known to primarily associate with microRNAs, a family of small RNA ‘driver’ sequences, and identifies its targets primarily via a ‘seed’ mediated partial complementarity process Despite numerous studies, a definitive experimental dataset of AGO2 'driver'-'target' interactions remains elusive. Our study employs two experimental methods - AGO2 CLASH and AGO2 eCLIP, to generate thousands of AGO2 target sites verified by chimeric reads. These chimeric reads contain both the AGO2 loaded small RNA 'driver' and the target sequence, providing a robust resource for modeling AGO2 binding preferences. Our novel analysis pipeline reveals thousands of AGO2 target sites driven by microRNAs and a significant number of AGO2 'drivers' derived from fragments of other small RNAs such as tRNAs, YRNAs, snoRNAs, rRNAs, and more. We utilize convolutional neural networks to train machine learning models that accurately predict the binding potential for each 'driver' class and experimentally validate several interactions. In conclusion, our comprehensive analysis of the AGO2 targetome broadens our understanding of its 'driver' repertoire and potential function in development and disease. Moreover, we offer practical bioinformatic tools for future experiments and the prediction of AGO2 targets. All data and code from this study are freely available at https://github.com/ML-Bioinfo-CEITEC/HybriDetector/
... Conserved motif analysis revealed the occurrence of 10 motifs in oil palm SPL genes as reported in other plants including foxtail millet [14], alfalfa [13], and quinoa [44]. The post-transcriptional regulation of SPLs by miR156 determines finetuning functions of SPLs [52]. In our study, a total of 14 oil palm SPLs contained the miR156 sites, mostly in the coding regions and lesser in UTR regions. ...
The SPL (SQUAMOSA-promoter binding protein-like) gene family is one of the largest plant transcription factors and is known to be involved in the regulation of plant growth, development, and stress responses. The genome-wide analysis of SPL gene members in a diverse range of crops has been elucidated. However, none of the genome-wide studies on the SPL gene family have been carried out for oil palm, an important oil-yielding plant. In this research, a total of 24 EgSPL genes were identified via a genome-wide approach. Phylogenetic analysis revealed that most of the EgSPLs are closely related to the Arabidopsis and rice SPL gene members. EgSPL genes were mapped onto the only nine chromosomes of the oil palm genome. Motif analysis revealed conservation of the SBP domain and the occurrence of 1–10 motifs in EgSPL gene members. Gene duplication analysis demonstrated the tandem duplication of SPL members in the oil palm genome. Heatmap analysis indicated the significant expression of SPL genes in shoot and flower organs of oil palm plants. Among the identified EgSPL genes, a total 14 EgSPLs were shown to be targets of miR156. Real-time PCR analysis of 14 SPL genes showed that most of the EgSPL genes were more highly expressed in female and male inflorescences of oil palm plants than in vegetative tissues. Altogether, the present study revealed the significant role of EgSPL genes in inflorescence development.
... This is obviously not the case. For example, in Arabidopsis Thaliana the precursors of miR-165a encode polypeptides (miPEP165a) that regulate root development [3], while miR-165a is thought to target mRNAs coding for HD-Zip transcription factors [4]. Although miR-165a of course overlaps with a portion of its microRNA precursor sequence, they have different functions. ...
... It is now well-known that miRNAs are not only found in mammals and other animals, but also in plants [12,13]. Systematic analysis has revealed a remarkable number of plant miRNAs, of which miR-168-5p (miR-168) may be one of the most abundant. ...
Introduction:
Diet is one of the most important factors contributing to the multistep process of carcinogenesis. The clinical relevance of exogenous food-derived xeno-microRNAs (miRNAs) in human diseases is poorly understood. In this study, we aimed to evaluate the potential clinical relevance of the xeno-miRNA miR-168 in the gastric mucosa along the preneoplastic conditions and gastric carcinogenesis.
Methods:
For a systematic analysis, we included stomach tissues from patients with different pathologies, including normal mucosa (N), chronic non-atrophic (CNAG) and atrophic gastritis (CAG) and intestinal metaplasia (IM) (n = 72), matched non-tumorous (NT) and tumorous (T) gastric cancer (GC) tissues (n = 81), matched colorectal cancer (CRC) tissues (n = 40), and colon mucosa and faeces from controls and IBD patients.
Results:
miR-168 was reproducibly detectable in all samples studied, with the highest levels in the proximal upper GI and in non-tumorous compared to tumorous tissues in both GC and CRC. There was no difference related to H. pylori positivity or inflammation grade, while higher miR-168 levels were observed in patients with moderate or severe AG/IM or OLGIM3/4. Survival analysis showed only a small, non-significant trend towards worse overall survival for patients with the highest to lowest miR-168 levels, while no differences were related to Lauren's classification.
Conclusions:
Food-derived xeno miRNAs are reproducibly detectable in the gastric and colonic mucosa. Although the clinically relevant function remains to be elucidated, higher levels of miR-168 in patients with moderate and severe IM merit further investigation.
This chapter aims to provide an understanding of the regulatory and functional roles of noncoding RNAs (ncRNAs) in plant stress responses. This study explores various types of noncoding RNAs, including circular RNAs (circRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs). It investigates their regulatory interactions by utilizing the PncStress database. This chapter emphasizes the significant contributions of these noncoding RNAs to signaling pathways, gene expression modulation, and stress tolerance enhancement. This study specifically focuses on microRNAs and their precise regulation of stress-responsive gene expression. It also examines their roles in enhancing plant stress tolerance. By establishing the foundation for innovative strategies, this chapter aims to improve crop productivity and resilience against both biotic and abiotic stresses through the exploration of noncoding RNAs. Ultimately, it provides valuable insights and contributes to the advancement of the field of noncoding RNAs in plant stress biology.
A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.
Flavonoids are secondary metabolites found in plants that possess antioxidant properties. MicroRNAs are small non-coding RNAs that play a crucial role in regulating gene expression and are involved in both developmental and metabolic pathways. The study analysed the differences in indices of lettuce with different leaf colours, a STTM-miR858 silencing vector was constructed using molecular biology techniques, and genetically modified lettuce plants with reduced expression of Las-miR858 were obtained. The interaction mechanism between miR858 and MYB111 was predicted and verified. The study found that reduced expression of miR858 regulated its target MYB111, which subsequently stimulated the expression of key enzyme genes in the flavonoid biosynthesis pathway. This resulted in the accumulation of flavonoids in lettuce, enhancing its antioxidant capacity. This study is the first to explain the regulation of flavonoids by the LasmiR858 network, providing a theoretical and empirical basis and genetic resources for the study of lettuce flavonoid mechanisms.
BACKGROUND
MicroRNA-1 (miR1), encoded by the genes miR1-1 and miR1-2 , is the most abundant microRNA in the heart and plays a critical role in heart development and physiology. Dysregulation of miR1 has been associated with various heart diseases, where a significant reduction (>75%) in miR1 expression has been observed in patient hearts with atrial fibrillation or acute myocardial infarction. However, it remains uncertain whether miR1-deficiency acts as a primary etiological factor of cardiac remodeling.
METHODS
miR1-1 or miR1-2 knockout mice were crossbred to produce 75%-miR1-knockdown (75%KD; miR1-1 +/− :miR1-2 −/− or miR1-1 −/− :miR1-2 +/− ) mice. Cardiac pathology of 75%KD cardiomyocytes/hearts was investigated by ECG, patch clamping, optical mapping, transcriptomic, and proteomic assays.
RESULTS
In adult 75%KD hearts, the overall miR1 expression was reduced to ≈25% of the normal wild-type level. These adult 75%KD hearts displayed decreased ejection fraction and fractional shortening, prolonged QRS and QT intervals, and high susceptibility to arrhythmias. Adult 75%KD cardiomyocytes exhibited prolonged action potentials with impaired repolarization and excitation-contraction coupling. Comparatively, 75%KD cardiomyocytes showcased reduced Na ⁺ current and transient outward potassium current, coupled with elevated L-type Ca ²⁺ current, as opposed to wild-type cells. RNA sequencing and proteomics assays indicated negative regulation of cardiac muscle contraction and ion channel activities, along with a positive enrichment of smooth muscle contraction genes in 75%KD cardiomyocytes/hearts. miR1 deficiency led to dysregulation of a wide gene network, with miR1’s RNAi–direct targets influencing many indirectly regulated genes. Furthermore, after 6 weeks of bi-weekly intravenous tail-vein injection of miR1 mimics, the ejection fraction and fractional shortening of 75%KD hearts showed significant improvement but remained susceptible to arrhythmias.
CONCLUSIONS
miR1 deficiency acts as a primary etiological factor in inducing cardiac remodeling via disrupting heart regulatory homeostasis. Achieving stable and appropriate microRNA expression levels in the heart is critical for effective microRNA-based therapy in cardiovascular diseases.
Argonaute proteins are instrumental in regulating RNA stability and translation. AGO2, the major mammalian Argonaute protein, is known to primarily associate with microRNAs, a family of small RNA ‘guide’ sequences, and identifies its targets primarily via a ‘seed’ mediated partial complementarity process. Despite numerous studies, a definitive experimental dataset of AGO2 ‘guide’–’target’ interactions remains elusive. Our study employs two experimental methods—AGO2 CLASH and AGO2 eCLIP, to generate thousands of AGO2 target sites verified by chimeric reads. These chimeric reads contain both the AGO2 loaded small RNA ‘guide’ and the target sequence, providing a robust resource for modeling AGO2 binding preferences. Our novel analysis pipeline reveals thousands of AGO2 target sites driven by microRNAs and a significant number of AGO2 ‘guides’ derived from fragments of other small RNAs such as tRNAs, YRNAs, snoRNAs, rRNAs, and more. We utilize convolutional neural networks to train machine learning models that accurately predict the binding potential for each ‘guide’ class and experimentally validate several interactions. In conclusion, our comprehensive analysis of the AGO2 targetome broadens our understanding of its ‘guide’ repertoire and potential function in development and disease. Moreover, we offer practical bioinformatic tools for future experiments and the prediction of AGO2 targets. All data and code from this study are freely available at https://github.com/ML-Bioinfo-CEITEC/HybriDetector/.
MicroRNAs (miRNAs) are a class of non-protein-coding short transcripts that provide a layer of post-transcriptional regulation essential to many plant biological processes. MiR858, which targets the transcripts of MYB transcription factors, can affect a range of secondary metabolic processes. Although miR858 and its 187-nt precursor have been well studied in Arabidopsis (Arabidopsis thaliana), a systematic investigation of miR858 precursors and their functions across plant species is lacking due to a problem in identifying the transcripts that generate this sub-class. By re-evaluating the transcript of miR858 and relaxing the length cut-off for identifying hairpins, we found in kiwifruit (Actinidia chinensis) that miR858 has long-loop hairpins (1,100-2,100-nt), whose intervening sequences between miRNA generating complementary sites were longer than all previously reported miRNA hairpins. Importantly, these precursors of miR858 containing long-loop hairpins (termed MIR858L) are widespread in seed plants including Arabidopsis, varying between 350- and 5,500-nt. Moreover, we showed that MIR858L has a greater impact on proanthocyanidin and flavonol levels in both Arabidopsis and kiwifruit. We suggest that an active MIR858L-MYB regulatory module appeared in the transition of early land plants to large upright flowering plants, making a key contribution to plant secondary metabolism.
Leaf growth initiates in the peripheral region of the meristem at the apex of the stem, eventually forming flat structures. Leaves are pivotal organs in plants, serving as the primary sites for photosynthesis, respiration, and transpiration. Their development is intricately governed by complex regulatory networks. Leaf development encompasses five processes: the leaf primordium initiation, the leaf polarity establishment, leaf size expansion, shaping of leaf, and leaf senescence. The leaf primordia starts from the side of the growth cone at the apex of the stem. Under the precise regulation of a series of genes, the leaf primordia establishes adaxial-abaxial axes, proximal-distal axes and medio-lateral axes polarity, guides the primordia cells to divide and differentiate in a specific direction, and finally develops into leaves of a certain shape and size. Leaf senescence is a kind of programmed cell death that occurs in plants, and as it is the last stage of leaf development. Each of these processes is meticulously coordinated through the intricate interplay among transcriptional regulatory factors, microRNAs, and plant hormones. This review is dedicated to examining the regulatory influences of major regulatory factors and plant hormones on these five developmental aspects of leaves.
Tobacco black shank induced by Phytophthora nicotianae causes significant yield losses in tobacco plants. MicroRNAs (miRNAs) play a pivotal role in plant biotic stress responses and have great potential in tobacco breeding for disease resistance. However, the roles of miRNAs in tobacco plants in response to P. nicotianae infection has not been well characterized. In this study, we found that Nta-miR6155, a miRNA specific to Solanaceae crops, was significantly induced in P. nicotianae infected tobacco. Some of predicted target genes of Nta-miR6155 were also observed to be involved in disease resistance. To further investigate the function of miR6155 in tobacco during P. nicotianae infection, Nta-miR6155 overexpression plants (miR6155-OE) were generated in the Honghua Dajinyuan tobacco variety (HD, the main cultivated tobacco variety in China). We found that the Nta-miR6155 overexpression enhanced the resistance in tobacco towards P. nicotianae infections. The level of reactive oxygen species (ROS) was significantly lower and antioxidant enzyme activities were significantly higher in miR6155-OE plants than those in control HD plants during P. nicotianae infection. In addition, we found that the accumulation of salicylic acid and the expression of salicylic acid biosynthesis and signal transduction-related genes is significantly higher in miR6155-OE plants in comparison to the control HD plants. Furthermore, we found that Nta-miR6155 cleaved target genes NtCIPK18 to modulate resistance towards P. nicotianae in tobacco plants. Additionally, phenotypic analysis of miR6155-OE plants showed that Nta-miR6155 could inhibit the growth of tobacco by suppressing nitrogen uptake and photosynthesis. In conclusion, our findings indicated that miR6155 plays a crucial role in the regulation of growth and resistance against P. nicotianae infections in tobacco plants.
Plants are confronted with various environmental stresses and develop sophisticated adaptive mechanisms. Our previous work demonstrated that the crosstalk of flg22 and ultraviolet (UV)‐B‐induced signalling cascades reprograms the expression of flavonol pathway genes (FPGs), benefiting plant defence responses. Although several transcription factors have been identified to be involved in this crosstalk, the underlying mechanism is largely unclear. Here, we analyzed microRNAs (miRNAs) and identified 126, 129 and 113 miRNAs with altered abundances compared to untreated control in flg22‐, UV‐B‐ and flg22/UV‐B‐treated seedlings, respectively. Two distinct modules were identified: The first consists of 10 miRNAs repressed by UV‐B but up‐regulated by flg22, and the second with five miRNAs repressed by flg22 but up‐regulated by UV‐B. In Arabidopsis , the knockdown of miR858a, a representative of module I, increased the abundance of CHS (a marker gene for FPGs), whereas its overexpression reduced CHS . Conversely, knockout of miR164b from module II decreased CHS and its overexpression increased CHS transcript levels. These data suggest a decisive role of miRNAs in the crosstalk. In the next, we described the interaction between miR858a and its target MYB111 (a positive regulator of FPGs) from module I in detail. We showed that MYB111 was profoundly post‐transcriptionally regulated by miR858a during the crosstalk, whose expression was specifically but antagonistically controlled by UVR8‐ and FLS2‐mediated signallings. Moreover, transcriptional monitoring using the GUS reporter gene demonstrates that miRNA‐mediated posttranscriptional regulation is the main driving force in reprogramming the expression of FPGs and regulates plant adaptation to multiple concurrent environmental stresses.
A wide variety of functional regulatory non-coding RNAs (ncRNAs) have been identified as essential regulators of plant growth and development. Depending on their category, ncRNAs are not only involved in modulating target gene expression at the transcriptional and post-transcriptional levels but also are involved in processes like RNA splicing and RNA-directed DNA methylation. To fulfill their molecular roles properly, ncRNAs must be precisely processed by multiprotein complexes. In the case of small RNAs, DICER-LIKE (DCL) proteins play critical roles in the production of mature molecules. Land plant genomes contain at least four distinct classes of DCL family proteins (DCL1–DCL4), of which DCL1, DCL3 and DCL4 are also present in the genomes of bryophytes, indicating the early divergence of these genes. The liverwort Marchantia polymorpha has become an attractive model species for investigating the evolutionary history of regulatory ncRNAs and proteins that are responsible for ncRNA biogenesis. Recent studies on Marchantia have started to uncover the similarities and differences in ncRNA production and function between the basal lineage of bryophytes and other land plants. In this review, we summarize findings on the essential role of regulatory ncRNAs in Marchantia development. We provide a comprehensive overview of conserved ncRNA–target modules among M. polymorpha, the moss Physcomitrium patens and the dicot Arabidopsis thaliana, as well as Marchantia-specific modules. Based on functional studies and data from the literature, we propose new connections between regulatory pathways involved in Marchantia’s vegetative and reproductive development and emphasize the need for further functional studies to understand the molecular mechanisms that control ncRNA-directed developmental processes.
Flowering is an indicator of plant transformation from vegetative to reproductive growth. miR160 has been shown to have a significant effect on the growth and development of fruits, leaves, and roots of plants or their stress response to environment, but the participation of miR160 in regulating flowering time in plants is unclear. In this study, we found that two FvemiR160s (FvemiR160a/FvemiR160b) mature sequences in strawberry ( Fragaria vesca ) were consistent. It was displayed that the miR160 mature sequence is highly conserved in various species, and the miR160 mature sequence formed by the 5′ arm of the MIR160 precursor was more conserved. Three FveARFs in woodland strawberry were negatively regulated by FvemiR160a, among which FveARF18A was the most significant. Phylogenetic analysis indicated that FvemiR160 is closely related to apple ( Malus domestica ), grape ( Vitis vinifera ), and Arabidopsis thaliana , while FveARF18A is closely related to RcARF18. Subsequently, we demonstrated that FvemiR160a can target cutting FveARF18A to negatively regulate its expression by RLM‐5′ RACE, cleavage site mutation, and GFP fluorescence assay. Moreover, we observed that FveMIR160a overexpressed plants have advanced flowering, while mFveARF18A overexpressed plants have delayed flowering. We also verified that FveARF18A negatively regulates the expression of FveAP1 and FveFUL by binding their promoters by yeast one‐hybrid, LUC, and GUS assay, and FveAP1 and FveFUL transgenic Arabidopsis showed early flowering phenotype. In addition, the expression level of FvemiR160a was decreased obviously while that of FveARF18A was increased obviously by MeJA, GA and IAA. In conclusion, our study reveals the important role of the FvemiR160‐FveARF18A‐FveAP1/FveFUL module in the flowering process of woodland strawberry and provides a new pathway for studying flowering.
Glycolate oxidase (GLO) is an FMN-containing enzyme localized in peroxisomes and performs in various molecular and biochemical mechanisms. It is a key player in plant glycolate and glyoxylate accumulation pathways. The role of GLO in disease and stress resistance is well-documented in various plant species. Although studies have been conducted regarding the role of GLO genes from spinach on a microbial level, the direct response of GLO genes to various stresses in short-season and leafy plants like lettuce has not been published yet. The genome of Lactuca sativa cultivar ‘Salinas’ (v8) was used to identify GLO gene members in lettuce by performing various computational analysis. Dual synteny, protein–protein interactions, and targeted miRNA analyses were conducted to understand the function of GLO genes. The identified GLO genes showed further clustering into two groups i.e., glycolate oxidase (GOX) and hydroxyacid oxidase (HAOX). Genes were observed to be distributed unevenly on three chromosomes, and syntenic analysis revealed that segmental duplication was prevalent. Thus, it might be the main reason for GLO gene diversity in lettuce. Almost all LsGLO genes showed syntenic blocks in respective plant genomes under study. Protein–protein interactions of LsGLO genes revealed various functional enrichments, mainly photorespiration, and lactate oxidation, and among biological processes oxidative photosynthetic carbon pathway was highly significant. Results of in-depth analyses disclosed the interaction of GLO genes with other members of the glycolate pathway and the activity of GLO genes in various organs and developmental stages in lettuce. The extensive genome evaluation of GLO gene family in garden lettuce is believed to be a reference for cloning and studying functional analyses of GLO genes and characterizing other members of glycolate/glyoxylate biosynthesis pathway in various plant species.
SPL (SQUAMOSA promoter binding proteins-like) are plant-specific transcription factors that play essential roles in a variety of developmental processes as well as the ability to withstand biotic and abiotic stresses. To date, numerous species have been investigated for the SPL gene family, but so far, no SPL family genes have been thoroughly identified and characterized in the sunflower (Helianthus annuus). In this study, 25 SPL genes were identified in the sunflower genome and were unevenly distributed on 11 chromosomes. According to phylogeny analysis, 59 SPL genes from H. annuus, O. sativa, and A. thaliana were clustered into seven groups. Furthermore, the SPL genes in groups-I and II were demonstrated to be potential targets of miR156. Synteny analysis showed that 7 paralogous gene pairs exist in HaSPL genes and 26 orthologous gene pairs exist between sunflower and rice, whereas 21 orthologous gene pairs were found between sunflower and Arabidopsis. Segmental duplication appears to have played a vital role in the expansion processes of sunflower SPL genes, and because of selection pressure, all duplicated genes have undergone purifying selection. Tissue-specific gene expression analysis of the HaSBP genes proved their diverse spatiotemporal expression patterns, which were predominantly expressed in floral organs and differentially expressed in stem, axil, and root tissues. The expression pattern of HaSPL genes under water stress showed broad involvement of HaSPLs in the response to flood and drought stresses. This genome-wide identification investigation provides detailed information on the sunflower SPL transcription factor gene family and establishes a strong platform for future research on sunflower responses to abiotic stress tolerance.
Plant development and reproduction are complex processes during which an individual fulfills its life cycle, starting from germination and the elaboration of new organs and growth, leading to the formation of reproductive structures and ultimately terminating in the production of the next generation. These mechanisms are the result of a long evolutionary history that has led to sophisticated regulatory mechanisms involving multiple levels of regulators. MicroRNAs (miRNAs) are a class of small regulatory molecules that play a pivotal role in regulatory networks by negatively controlling target genes. Since miRNA very first identification twenty years ago, they have attracted much interest for their role as essential regulators of plant development. In this review, we propose a comprehensive and critical analysis of the importance of miRNAs during plant development and reproduction. We begin by presenting the current understanding of miRNAs’ evolutionary history, biogenesis, mode of action, position in regulatory networks, and their potential as mobile molecules, exploring how these aspects contribute to their functions in plant development and reproduction. Then, we explore the genetic strategies employed to effectively analyze their roles, with an emphasis on recent advancements resulting from genome editing techniques. Next, we focus on miRNA contributions to four crucial processes: growth, organ patterning and identity, life cycle progression and reproduction. Through this analysis, the importance of miRNAs during plant development and reproduction emerges, which we finally discuss in light of the current view miRNAs’ roles during animal development.
MicroRNA(miRNA), a small non-coding class of RNA that regulates the gene expression, is conserved among several plant species. In the present study, an in-silico approach was adopted to identify miRNA from the known expressed sequences tags (ESTs) of Citrus reticulata L. A total of 17 miRNAs from 23 different ESTs along with their secondary structures and targets were predicted. The identified 63 targets include several transcription factors, proteins that regulate plant growth, development, flowering, and seed development together with stress response. The cis-regulatory element present at the promoter region of the MIR genes of C. reticulata describes their role during light responsiveness, auxin, gibberellins, abscisic acid (ABA), anthocyanin responsiveness, salicylic acid responsiveness, anaerobic induction, circadian control, nitrate dependent regulation of the cell cycle and DNA replication, defense, and stress responsiveness. The present study identifies the miRNAs along with their regulatory elements in C. reticulate. The study will also be useful for the research on identification from the genomic data of different plants and prediction of expression probability of identified MIR genes based on the presence of upstream promoter and other regulatory elements.
Neuropathic pain (NP) is a kind of chronic pain caused by direct injury to the peripheral or central nervous system (CNS). microRNAs (miRNAs) are small noncoding RNAs that mostly interact with the 3 untranslated region of messenger RNAs (mRNAs) to regulate the expression of multiple genes. NP is characterized by changes in the expression of receptors and mediators, and there is evidence that miRNAs may contribute to some of these alterations. In this review, we aimed to fully comprehend the connection between NP and miRNA; and also, to establish a link between neurology, biology, and dentistry. Studies have shown that targeting miRNAs may be an effective therapeutic strategy for the treatment of chronic pain and potential target for the prevention of NP.
Bidirectional trans-kingdom RNA silencing, a pivotal factor in plant-pathogen interactions, remains less explored in plant host-parasite dynamics. Here, using small RNA sequencing in melon root systems, we investigated miRNA expression variation in resistant and susceptible cultivars pre- and post-infection by the parasitic plant, broomrape. This approach revealed 979 known miRNAs and 110 novel miRNAs across 110 families. When comparing susceptible (F0) and resistant (R0) melon lines with broomrape infection (F25 and R25), 39 significantly differentially expressed miRNAs were observed in F25 vs. F0, 35 in R25 vs. R0, and 5 in R25 vs. F25. Notably, two miRNAs consistently exhibited differential expression across all comparisons, targeting genes linked to plant disease resistance. This suggests their pivotal role in melon's defense against broomrape. The target genes of these miRNAs were confirmed via degradome sequencing and validated by qRT-PCR, ensuring reliable sequencing outcomes. GO and KEGG analyses shed light on the molecular functions and pathways of these differential miRNAs. Furthermore, our study unveiled four trans-kingdom miRNAs, forming a foundation for exploring melon's resistance to broomrape.
Maize has widely been studied as a model of plant-growth promoting rhizobacteria (PGPR). Here, the genome sequences of 9P. The strains, together with 26 other sequenced Maize were comparatively studied. Phylogenetic analysis of the concatenated 244 single-copy core genes suggests that the 9P. The strains and 5 other Paenibacillus spp., isolated from diverse geographic regions and ecological niches, formed a closely related clade (here it is called Poly-clade). Analysis of single nucleotide polymorphisms (SNPs) reveals local diversification of the 14 Poly-clade genomes. SNPs were not evenly distributed throughout the 14 genomes and the regions with high SNP density contain the genes related to secondary metabolism, including genes coding for polyketide. Recombination played an important role in the genetic diversity of this clade, although the rate of recombination was clearly lower than mutation. The distinction among people and different creatures can be gotten by relative examinations. This study reveals that both maize and its closely related species have plant growth promoting traits and they have great potential uses in agriculture and horticulture as PGPR.
Drought stress is one of the most serious threats to sustainable agriculture and is predicted to be further intensified in the coming decades. Therefore, understanding the mechanism of drought stress tolerance and the development of drought-resilient crops are the major goals at present. In recent years, noncoding microRNAs (miRNAs) have emerged as key regulators of gene expressions under drought stress conditions and are turning out to be the potential candidates that can be targeted to develop drought-resilient crops in the future. miRNAs are known to target and decrease the expression of various genes to govern the drought stress response in plants. In addition, emerging evidence also suggests a regulatory role of long non-coding RNAs (lncRNAs) in the regulation of miRNAs and the expression of their target genes by a process referred as miRNA sponging. In this review, we present the regulatory roles of miRNAs in the modulation of drought-responsive genes along with discussing their biosynthesis and action mechanisms. Additionally, the interactive roles of miRNAs with phytohormone signaling components have also been highlighted to present the global view of miRNA functioning under drought-stress conditions.
The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans--the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.
Maternal gene products deposited in an animal egg determine the polarity of embryonic axes and regulate embryonic cell-cell communication important for morphogenesis. Here we report the first maternal-effect embryo-defective mutation discovered in a plant. Recessive mutations in the SHORT INTEGUMENT (SIN1) gene in Arabidopsis were previously shown to influence ovule development and flowering time. Here we show that a sin1 mutation has a pronounced maternal effect on zygotic embryo development. A homozygous sin1 mutant embryo is normal when nursed by a sin1/+ heterozygous maternal sporophyte. Strikingly, a sin1 or a sin1/+ embryo that is nursed by a sin1 homozygous maternal sporophyte develops morphogenetic defects in the apical-basal and radial axes. The defects resemble those seen in some zygotic-effect embryonic pattern formation mutants. These results imply that in maternal cells the SIN1 gene either codes for or controls the production of a diffusible morphogen necessary for proper zygotic embryogenesis.
In the Arabidopsis root meristem, initial cells undergo asymmetric divisions to generate the cell lineages of the root. The scarecrow mutation results in roots that are missing one cell layer owing to the disruption of an asymmetric division that normally generates cortex and endodermis. Tissue-specific markers indicate that a heterogeneous cell type is formed in the mutant. The deduced amino acid sequence of SCARECROW (SCR) suggests that it is a member of a novel family of putative transcription factors. SCR is expressed in the cortex/endodermal initial cells and in the endodermal cell lineage. Tissue-specific expression is regulated at the transcriptional level. These results indicate a key role for SCR in regulating the radial organization of the root.
The short integument (sin1) mutation causes a female-specific infertility, and a defect in the control of time to flowering in Arabidopsis. Female sterility of Sin- plants is due to abnormal ovule integument development and aberrant differentiation of the megagametophyte in a subset of ovules. An additional defect of sin1 mutants is the production of an increased number of vegetative leaf and inflorescence primordia leading to delayed flowering. The delayed flowering phenotype of sin1-1 is not due to a defect in the perception of day length periodicity or in gibberellic acid metabolism. Phenotypes of double mutant combinations of sin1 with terminalflower (tfl1) indicate that SIN1 activity is required for precocious floral induction typical in a tfl1 mutant. Unexpectedly, sin1-1 tfl1-1 plants do not make pollen, thus revealing a novel role for TFL1 in the anther. Early flowers of sin1-1 ap1-1 double mutants are transformed to long inflorescence-like shoots. A genetic model for the role of SIN1 in flowering time control is proposed.
The Caenorhabditis elegans heterochronic gene lin-14 generates a temporal gradient of the LIN-14 proteins to control stage-specific patterns of cell lineage during development. Down-regulation of LIN-14 is mediated by the lin-14 3' untranslated region (UTR), which bears seven sites that are complementary to the regulatory lin-4 RNA. Here we report molecular and genetic evidence that RNA duplexes between the lin-4 and lin-14 RNAs form in vivo and are necessary for LIN-14 temporal gradient generation. lin-4 RNA binds in vitro to a lin-14 mRNA bearing the seven lin-4 complementary sites but not to a lin-14 mRNA bearing point mutations in these sites. In vivo, the lin-4 complementary regions are necessary for lin-14 3' UTR-mediated temporal gradient formation. Based on lin-14 3' UTR sequence comparisons between C. elegans and C. briggsae, four of the seven lin-4/lin-14 RNA duplexes are predicted to bulge a lin-4 C residue, and three sites are predicted to form nonbulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 binding sites show almost wild-type temporal gradient formation, whereas those bearing multimerized nonbulged lin-4 binding sites do not form a temporal gradient. Paradoxically, lin-4 RNA binds in vitro to nonbulged lin-14 RNA more avidly than to the bulged lin-14 RNA. This suggests that a specific secondary structure of lin-4/lin-14 RNA duplex that may be recognized by an accessory protein, rather than an RNA duplex per se, is required in vivo for the generation of the LIN-14 temporal gradient.
Mutations in the heterochronic gene lin-28 of C. elegans cause precocious development where diverse events specific to the second larval stage are skipped. lin-28 encodes a cytoplasmic protein with a cold shock domain and retroviral-type (CCHC) zinc finger motifs, consistent with a role for LIN-28 in posttranscriptional regulation. The 3'UTR of lin-28 contains a conserved element that is complementary to the 22 nt regulatory RNA product of lin-4 and that resembles seven such elements in the 3'UTR of the heterochronic gene lin-14. Both lin-4 activity and the lin-4-complementary element (LCE) are necessary for stage-specific regulation of lin-28. Deleting the LCE produces a dominant gain-of-function allele that causes a retarded phenotype, indicating that lin-28 activity is a switch that controls choices of stage-specific fates.
Mutations in CUC1 and CUC2 (for CUP-SHAPED COTYLEDON), which are newly identified genes of Arabidopsis, caused defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of shoot apical meristems. These defects were most apparent in the double mutant. Phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. We cloned the CUC2 gene and found that the encoded protein was homologous to the petunia NO APICAL MERISTEM (NAM) protein, which is thought to act in the development of embryos and flowers.
The Arabidopsis gai mutant allele confers a reduction in gibberellin (GA) responsiveness. Here we report the molecular cloning of GAI and a closely related gene GRS. The predicted GAI (wild-type) and gai (mutant) proteins differ only by the deletion of a 17-amino-acid segment from within the amino-terminal region. GAI and GRS contain nuclear localization signals, a region of homology to a putative transcription factor, and motifs characteristic of transcriptional coactivators. Genetic analysis indicates that GAI is a repressor of GA responses, that GA can release this repression, and that gai is a mutant repressor that is relatively resistant to the effects of GA. Mutations at SPY and GAR2 suppress the gai phenotype, indicating the involvement of GAI, SPY, and GAR2 in a signaling pathway that regulates GA responses negatively. The existence of this pathway suggests that GA modulates plant growth through derepression rather than through simple stimulation.
An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA resulted in a partial mutant phenotype. Sense expression caused some transgenic lines to develop goblet-like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms.
Arabidopsis thaliana floral meristems are determinate structures that produce a defined number of organs, after which cell division ceases. A new recessive mutant, carpel factory (caf), converts the floral meristems to an indeterminate state. They produce extra whorls of stamens, and an indefinite number of carpels. Thus, CAF appears to suppress cell division in floral meristems. The function of CAF is partially redundant with the function of the CLAVATA (CLV) and SUPERMAN (SUP) genes, as caf clv and caf sup double mutants show dramatically enhanced floral meristem over-proliferation. caf mutant plants also show other defects, including absence of axillary inflorescence meristems, and abnormally shaped leaves and floral organs. The CAF gene was cloned and found to encode a putative protein of 1909 amino acids containing an N-terminal DExH/DEAD-box type RNA helicase domain attached to a C-terminal RNaseIII-like domain. A very similar protein of unknown function is encoded by a fungal and an animal genome. Helicase proteins are involved in a number of processes, including specific mRNA localization and mRNA splicing. RNase III proteins are involved in the processing of rRNA and some mRNA molecules. Thus CAF may act through some type of RNA processing event(s). CAF gives rise to two major transcripts of 2.5 and 6.2 kb. In situ hybridization experiments show that CAF RNA is expressed throughout all shoot tissues.
Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of approximately 65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An approximately 88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.
lin-4 encodes a small RNA that is complementary to sequences in the 3' untranslated region (UTR) of lin-14 mRNA and that acts to developmentally repress the accumulation of LIN-14 protein. This repression is essential for the proper timing of numerous events of Caenorhabditis elegans larval development. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results indicate that the rate of synthesis of lin-14 mRNA, its state of polyadenylation, its abundance in the cytoplasmic fraction, and its polysomal sedimentation profile do not change in response to the accumulation of lin-4 RNA. Our results indicate that association of lin-4 RNA with the 3' UTR of lin-14 mRNA permits normal biogenesis of lin-14 mRNA, and normal translational initiation, but inhibits step(s) thereafter, such as translational elongation and/or the release of stable LIN-14 protein.
Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsis mutant, pat (phytochrome A signal transduction)1-1, which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1 mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncated PAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.
Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-413'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. We suggest that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29.
The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific
to plants.Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of
the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to
each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.
The 21-nucleotide small temporal RNA (stRNA) let-7regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA
interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer
to produce the active small-RNA component that represses gene expression.
Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains approximately 22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. Here we characterize the Caenorhabditis elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41).
Two small temporal RNAs (stRNAs), lin-4 andlet-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs
(miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed withlet-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively
throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply
that, as a class, miRNAs have broad regulatory functions in animals.
Over the past several years, RNAi and its related phenomena have emerged not only as a powerful experimental tool but also as a new mode of gene regulation. Through a combination of genetic and biochemical approaches we have learned much about the mechanisms underlying dsRNA responses. However, many of the most intriguing aspects of dsRNA-induced gene silencing have yet to be illuminated. What has become abundantly clear is that the complex and highly conserved biology underlying RNA interference is critical both for genome maintenance and for the development of complex organisms. However, it seems probable that we have only begun to reveal the diversity of biological roles played by RNAi-related processes.
We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.
Micro RNAs are a large family of noncoding RNAs of 21-22 nucleotides whose functions are generally unknown. Here a large subset of Drosophila micro RNAs is shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation. These findings suggest a more general role for micro RNAs in gene regulation through the formation of RNA duplexes.
Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsis mutant, pat ( p hytochrome A signal t ransduction) 1-1 , which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1 mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncated PAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.
We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB1O1, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley α-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA1 by 11-fold and GA4 by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.
The MYB family of proteins is large, functionally diverse and represented in all eukaryotes. Most MYB proteins function as transcription factors with varying numbers of MYB domain repeats conferring their ability to bind DNA. In plants, the MYB family has selectively expanded, particularly through the large family of R2R3-MYB. Members of this family function in a variety of plant-specific processes, as evidenced by their extensive functional characterization in Arabidopsis (Arabidopsis thaliana). MYB proteins are key factors in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses. The elucidation of MYB protein function and regulation that is possible in Arabidopsis will provide the foundation for predicting the contributions of MYB proteins to the biology of plants in general.
A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 sequences identified, the vast majority (90%) arose from intergenic regions, although small RNAs corresponding to predicted protein-coding genes, transposon-like sequences, and a structural RNA gene also were identified. Evidence consistent with the derivation of small RNAs of both polarities, and from highly base-paired precursors, was obtained through the identification and analysis of clusters of small RNA loci. The accumulation of specific small RNAs was regulated developmentally. We propose that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.
The recessive rga mutation is able to partially suppress phenotypic defects of the Arabidopsis gibberellin (GA) biosynthetic mutant ga1-3. Defects in stem elongation, flowering time, and leaf abaxial trichome initiation are suppressed by rga. This indicates that RGA is a negative regulator of the GA signal transduction pathway. We have identified 10 additional alleles of rga from a fast-neutron mutagenized ga1-3 population and used them to isolate the RGA gene by genomic subtraction. Our data suggest that RGA may be functioning as a transcriptional regulator. RGA was found to be a member of the VHIID regulatory family, which includes the radial root organizing gene SCARECROW and another GA signal transduction repressor, GAI. RGA and GAI proteins share a high degree of homology, but their N termini are more divergent. The presence of several structural features, including homopolymeric serine and threonine residues, a putative nuclear localization signal, leucine heptad repeats, and an LXXLL motif, indicates that the RGA protein may be a transcriptional regulator that represses the GA response. In support of the putative nuclear localization signal, we demonstrated that a transiently expressed green fluorescent protein–RGA fusion protein is localized to the nucleus in onion epidermal cells. Because the rga mutation abolished the high level of expression of the GA biosynthetic gene GA4 in the ga1-3 mutant background, we conclude that RGA may also play a role in controlling GA biosynthesis.
Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of ∼65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An ∼88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.
Several sites of nuclear protein interaction within the promoter region of theAntirrhinum majus floral meristem identity geneSQUAMOSA were detected using an electrophoretic mobility shift assay. One of these sites displayed a particularly clear interaction with nuclear protein extracted from inflorescences but not with nuclear protein extracted from young, non-flowering plants. This site could thus represent a binding motif for a transcriptional activator. A South-western screen of an inflorescence cDNA expression library resulted in the isolation of several cDNAs representing two different genes namedSBP1 andSBP2 (forSQUAMOSA-pROMOTERBINDINGP
ROTEIN gene 1 and 2). Both genes encode highly similar protein domains which were found to be necessary and sufficient for binding DNA in a sequence-specific manner. This DNA-binding domain showed no similarity to known proteins in the databases. However, it is characteristic for a small family of gene products inA. majus and other plant species. Expression ofSBP1 and2 is developmentally regulated and their transcriptional activation precedes that ofSQUAMOSA. The data presented support the idea that members of the newly identifiedSBP gene family function as transcription factors involved in the control of early flower development.
The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans— the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.
Thesis (Ph. D., Division of Medical Sciences (Genetics))--Harvard University, 1999. Includes bibliographical references.
During C. elegans development, the temporal pattern of many cell lineages is specified by graded activity of the heterochronic gene Lin-14. Here we demonstrate that a temporal gradient in Lin-14 protein is generated posttranscriptionally by multiple elements in the lin-14 3'UTR that are regulated by the heterochronic gene Lin-4. The lin-14 3'UTR is both necessary and sufficient to confer lin-4-mediated posttranscriptional temporal regulation. The function of the lin-14 3'UTR is conserved between C. elegans and C. briggsae. Among the conserved sequences are seven elements that are each complementary to the lin-4 RNAs. A reporter gene bearing three of these elements shows partial temporal gradient activity. These data suggest a molecular mechanism for Lin-14p temporal gradient formation: the lin-4 RNAs base pair to sites in the lin-14 3'UTR to form multiple RNA duplexes that down-regulate lin-14 translation.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.
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The recessive rga mutation is able to partially suppress phenotypic defects of the Arabidopsis gibberellin (GA) biosynthetic mutant ga1-3. Defects in stem elongation, flowering time, and leaf abaxial trichome initiation are suppressed by rga. This indicates that RGA is a negative regulator of the GA signal transduction pathway. We have identified 10 additional alleles of rga from a fast-neutron mutagenized ga1-3 population and used them to isolate the RGA gene by genomic subtraction. Our data suggest that RGA may be functioning as a transcriptional regulator. RGA was found to be a member of the VHIID regulatory family, which includes the radial root organizing gene SCARECROW and another GA signal transduction repressor, GAI. RGA and GAI proteins share a high degree of homology, but their N termini are more divergent. The presence of several structural features, including homopolymeric serine and threonine residues, a putative nuclear localization signal, leucine heptad repeats, and an LXXLL motif, indicates that the RGA protein may be a transcriptional regulator that represses the GA response. In support of the putative nuclear localization signal, we demonstrated that a transiently expressed green fluorescent protein-RGA fusion protein is localized to the nucleus in onion epidermal cells. Because the rga mutation abolished the high level of expression of the GA biosynthetic gene GA4 in the ga1-3 mutant background, we conclude that RGA may also play a role in controlling GA biosynthesis.
Auxin response factors (ARFs) are transcription factors that bind with specificity to TGTCTC auxin response elements (AuxREs) found in promoters of primary/early auxin response genes. ARFs are encoded by a multi-gene family, consisting of more than 10 genes. Ten ARFs have been analyzed by Northern analysis and were found to be expressed in all major plant organs and suspension culture cells of Arabidopsis. The predicted amino acid sequences indicate that the 10 ARFs contain a novel amino-terminal DNA binding domain and a carboxyl-terminal dimerization domain, with the exception of ARF3 which lacks this dimerization domain. All ARFs tested bind with specificity to the TGTCTC AuxRE, but there are subtle variations in the sequence requirements at positions 5 (T) and 6 (C) of the AuxRE. While the amino-terminal domain of about 350 amino acids is sufficient for binding ARF1 to TGTCTC AuxREs, this domain is not sufficient for the binding of some other ARFs to palindromic AuxREs. Our results suggest that ARFs must form dimers on palindromic TGTCTC AuxREs to bind stably, and this dimerization may be facilitated by conserved motifs found in ARF carboxyl-terminal domains. Dimerization in at least some cases may dictate which ARF(s) are targeted to AuxREs.
Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.
The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.
Asymmetric cell divisions play an important role in the establishment and propagation of the cellular pattern of plant tissues. The SHORT-ROOT (SHR) gene is required for the asymmetric cell division responsible for formation of ground tissue (endodermis and cortex) as well as specification of endodermis in the Arabidopsis root. We show that SHR encodes a putative transcription factor with homology to SCARECROW (SCR). From analyses of gene expression and cell identity in genetically stable and unstable alleles of shr, we conclude that SHR functions upstream of SCR and participates in a radial signaling pathway. Consistent with a regulatory role in radial patterning, ectopic expression of SHR results in supernumerary cell divisions and abnormal cell specification in the root meristem.
In plants, double-stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms, which require common proteins in diverse organisms, and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing.
With the human genome sequence approaching completion, a major challenge is to identify the locations and encoded protein sequences of all human genes. To address this problem we have developed a new gene identification algorithm, GenomeScan, which combines exon-intron and splice signal models with similarity to known protein sequences in an integrated model. Extensive testing shows that GenomeScan can accurately identify the exon-intron structures of genes in finished or draft human genome sequence with a low rate of false-positives. Application of GenomeScan to 2.7 billion bases of human genomic DNA identified at least 20,000-25,000 human genes out of an estimated 30,000-40,000 present in the genome. The results show an accurate and efficient automated approach for identifying genes in higher eukaryotic genomes and provide a first-level annotation of the draft human genome.
The upper side of the angiosperm leaf is specialized for efficient capture of sunlight whereas the lower side is specialized for gas exchange. In Arabidopsis, the establishment of polarity in the leaf probably requires the generation and perception of positional information along the radial (adaxial versus abaxial or central versus peripheral) dimension of the plant. This is because the future upper (adaxial) side of the leaf develops from cells closer to the centre of the shoot, whereas the future under (abaxial) side develops from cells located more peripherally. Here we implicate the Arabidopsis PHABULOSA and PHAVOLUTA genes in the perception of radial positional information in the leaf primordium. Dominant phabulosa (phb) and phavoluta (phv) mutations cause a dramatic transformation of abaxial leaf fates into adaxial leaf fates. They do so by altering the predicted sterol/lipid-binding domains of ATHB14 and ATHB9, proteins of previously unknown function that also contain DNA-binding motifs. This change probably renders the protein constitutively active, implicating this domain as a central regulator of protein function and the PHB and PHV proteins as receptors for an adaxializing signal.
RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.
An early event in RNA interference (RNAi) is the cleavage of the initiating double-stranded RNA (dsRNA) to short pieces, 21 to 23 nucleotides in length. Here we describe a null mutation in dicer-1 (dcr-1), a gene proposed to encode the enzyme that generates these short RNAs. We find that dcr-1(-/-) animals have defects in RNAi under some, but not all, conditions. Mutant animals have germ line defects that lead to sterility, suggesting that cleavage of dsRNA to short pieces is a requisite event in normal development.
The lin-4 and let-7 antisense RNAs are temporal regulators that control the timing of developmental events inCaenorhabditis elegans by inhibiting translation of target mRNAs. let-7 RNA is conserved among bilaterian animals, suggesting that this class of small RNAs [microRNAs (miRNAs)] is evolutionarily
ancient. Using bioinformatics and cDNA cloning, we found 15 new miRNA genes in C. elegans. Several of these genes express small transcripts that vary in abundance during C. elegans larval development, and three of them have apparent homologs in mammals and/or insects. Small noncoding RNAs of the miRNA
class appear to be numerous and diverse.
In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotide (nt) RNAs, respectively, which function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as small temporal RNAs (stRNAs). We show that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs, similar to let-7 stRNA, are highly conserved. This suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.
Auxins are a class of phytohormones implicated in virtually every aspect of plant growth and development. Many early plant responses to auxin are apparently mediated by members of a family of Aux/IAA proteins that dimerize with and inhibit members of the auxin response factor (ARF) family of transcription factors. Aux/IAA proteins are unstable, and their degradation is triggered by a ubiquitin-protein ligase that is regulated by modification with a ubiquitin-related protein. Recent genetic and biochemical evidence indicates that auxin accelerates the degradation of the already short-lived Aux/IAA proteins to derepress transcription by ARF proteins. Several pieces of the auxin-signaling puzzle remain to be assembled, including the proteins that initially bind auxin, the proteins that convey this signal to the protein degradation machinery, and the targets of the transcriptional derepression.
Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.
In organisms as diverse as nematodes, trypanosomes, plants, and fungi, double-stranded RNA triggers the destruction of homologous mRNAs, a phenomenon known as RNA interference. RNA interference begins with the transformation of the double-stranded RNA into small RNAs that then guide a protein nuclease to destroy their mRNA targets.