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1.  Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR 
BMC Plant Biology  2009;9:87.
Background
MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. In higher plants, miRNAs are involved in developmental processes and stress responses. Sexual reproduction in flowering plants relies on pollen, the male gametophyte, to deliver sperm cells to fertilize the egg cell hidden in the embryo sac. Studies indicated that post-transcriptional processes are important for regulating gene expression during pollen function. However, we still have very limited knowledge on the involved gene regulatory mechanisms. Especially, the function of miRNAs in pollen remains unknown.
Results
Using miRCURY LNA array technology, we have profiled the expression of 70 known miRNAs (representing 121 miRBase IDs) in Arabidopsis mature pollen, and compared the expression of these miRNAs in pollen and young inflorescence. Thirty-seven probes on the array were identified using RNAs isolated from mature pollen, 26 of which showed significant differences in expression between mature pollen and inflorescence. Real-time PCR based on TaqMan miRNA assays confirmed the expression of 22 miRNAs in mature pollen, and identified 8 additional miRNAs that were expressed at low level in mature pollen. However, the expression of 11 miRNA that were identified on the array could not be confirmed by the Taqman miRNA assays. Analyses of transcriptome data for some miRNA target genes indicated that miRNAs are functional in pollen.
Conclusion
In summary, our results showed that some known miRNAs were expressed in Arabidopsis mature pollen, with most of them being low abundant. The results can be utilized in future research to study post-transcriptional gene regulation in pollen function.
doi:10.1186/1471-2229-9-87
PMCID: PMC2715406  PMID: 19591667
2.  Global small RNA analysis in fast-growing Arabidopsis thaliana with elevated concentrations of ATP and sugars 
BMC Genomics  2014;15:116.
Background
In higher eukaryotes, small RNAs play a role in regulating gene expression. Overexpression (OE) lines of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) were shown to grow faster and exhibit higher ATP and sugar contents. Leaf microarray studies showed that many genes involved in microRNAs (miRNAs) and trans-acting siRNAs (tasiRNAs) biogenesis were significantly changed in the fast-growing lines. In this study, the sRNA profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on sRNA expression were analyzed.
Results
9-13 million reads from each library were mapped to genome. miRNAs, tasiRNAs and natural antisense transcripts-generated small interfering RNAs (natsiRNAs) were identified and compared between libraries. In the leaf of OE lines, 15 known miRNAs increased in abundance and 9 miRNAs decreased in abundance, whereas in the root of OE lines, 2 known miRNAs increased in abundance and 9 miRNAs decreased in abundance. miRNAs with increased abundance in the leaf and root samples of both OE lines (miR158b and miR172a/b) were predicted to target mRNAs coding for Dof zinc finger protein and Apetala 2 (AP2) proteins, respectively. Furthermore, a significant change in the miR173-tasiRNAs-PPR/TPR network was observed in the leaves of both OE lines.
Conclusion
In this study, the impact of high energy content on the sRNA profiles of Arabidopsis is reported. While the abundance of many stress-induced miRNAs is unaltered, the abundance of some miRNAs related to plant growth and development (miR172 and miR319) is elevated in the fast-growing lines. An induction of miR173-tasiRNAs-PPR/TPR network was also observed in the OE lines. In contrast, only few cis- and trans-natsiRNAs are altered in the fast-growing lines.
doi:10.1186/1471-2164-15-116
PMCID: PMC3925372  PMID: 24507710
Chloroplasts; Mitochondria; miRNAs; MORF; PPR; tasiRNAs and natsiRNAs
3.  Transcriptome analysis of haploid male gametophyte development in Arabidopsis 
Genome Biology  2004;5(11):R85.
A transcriptome analysis of male gametophyte development in Arabidopsis uncovers distinct temporal classes of gene expression and opens the door to detailed studies of the regulatory pathways involved.
Background
The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development.
Results
We have developed staged spore isolation procedures for Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth.
Conclusions
The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development.
doi:10.1186/gb-2004-5-11-r85
PMCID: PMC545776  PMID: 15535861
4.  Physcomitrella patens DCL3 Is Required for 22–24 nt siRNA Accumulation, Suppression of Retrotransposon-Derived Transcripts, and Normal Development 
PLoS Genetics  2008;4(12):e1000314.
Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants.
Author Summary
Very small RNAs (between ∼21 and ∼30 single-stranded bases) are a ubiquitous component of gene regulation in nearly all eukaryotic organisms. The small RNA repertoire of angiosperms (the flowering plants) is exceptionally diverse and includes conspicuous populations of 21 nt microRNAs, as well a diverse set of 24 nt short, interfering RNAs (siRNAs). The 24 nt siRNAs have well-documented roles in enforcing the silence of parasitic regions of the genome, but are not readily apparent in the small RNA populations of several lineages of ancient, non-flowering plants. We found numerous “hotspots” of small RNA production from the genome of the moss P. patens that produced a mix of 21–24 nt siRNAs. Except for their broad mix of sizes, these hotspots were reminiscent of the 24 nt siRNA loci of angiosperms: they tended to associate with decayed transposons, to avoid annotated genes, and to be densely modified with the epigenetic mark 5-methyl cytosine. Deletion of a P. patens Dicer gene abolished production of 22–24 nt siRNAs both from these loci and transcriptome-wide, especially from repetitive regions. We conclude that both microRNAs and intergenic/repeat-associated siRNAs are ancient small RNA regulators in plants, but that the sizes of the siRNAs themselves have drifted over time.
doi:10.1371/journal.pgen.1000314
PMCID: PMC2600652  PMID: 19096705
5.  Endogenous TasiRNAs Mediate Non-Cell Autonomous Effects on Gene Regulation in Arabidopsis thaliana 
PLoS ONE  2009;4(6):e5980.
Background
Different classes of small RNAs (sRNAs) refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs), which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs), and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells.
Principal Findings
We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF)-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper) side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower) side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities.
Conclusions/Significance
Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.
doi:10.1371/journal.pone.0005980
PMCID: PMC2694355  PMID: 19543387
6.  Identification of Novel miRNAs and miRNA Dependent Developmental Shifts of Gene Expression in Arabidopsis thaliana 
PLoS ONE  2010;5(4):e10157.
microRNAs (miRNAs) are small, endogenous RNAs of 20∼25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA) deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.
doi:10.1371/journal.pone.0010157
PMCID: PMC2854152  PMID: 20405016
7.  Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing 
BMC Plant Biology  2012;12:146.
Background
Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date.
Results
To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated.
Conclusions
We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.
doi:10.1186/1471-2229-12-146
PMCID: PMC3462689  PMID: 22894611
Chinese fir; miRNA; rasiRNA; tasiRNA; Cunninghamia lanceolata
8.  Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1 
PLoS Biology  2010;8(6):e1000388.
Species-preferential osmotic pollen tube burst and sperm discharge in maize involve induced opening of the pollen tube-expressed potassium channel KZM1 by the egg apparatus-derived defensin-like protein ZmES4.
In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K+-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K+ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K+ influx. We further suggest that K+ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.
Author Summary
Sperm cells of animals and lower plants are mobile and can swim to the oocyte or egg cell. In contrast, flowering plants generate immobile sperm encased in a pollen coat to protect them from drying out and are transported via the pollen tube cell towards the egg apparatus to achieve double fertilization. Upon arrival the pollen tube tip bursts to deliver two sperm cells, one fusing with the egg cell to generate the embryo and the other fusing with the central cell to generate the endosperm. Here, we report the mechanisms leading to pollen tube burst and sperm discharge in maize. We found that before fertilization the defensin-like protein ZmES1-4 is stored in the secretory zone of the egg apparatus cells and that pollen tubes cannot discharge sperm in ZmES1-4 knock-down plants. Application of chemically synthesized ZmES4 leads to pollen tube burst within seconds in maize, but not in other plant species, suggesting this mechanism may be species specific. Finally, we identified the pollen tube-expressed potassium channel KZM1 as a target of ZmES4, which opens after ZmES4 treatment and probably leads to K+ influx and sperm release after osmotic burst.
doi:10.1371/journal.pbio.1000388
PMCID: PMC2879413  PMID: 20532241
9.  Gene Expression and Stress Response Mediated by the Epigenetic Regulation of a Transposable Element Small RNA 
PLoS Genetics  2012;8(2):e1002474.
The epigenetic activity of transposable elements (TEs) can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs) in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs). Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA–binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21–22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3′ untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3′ untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs the lines between TE and gene-regulating small RNAs.
Author Summary
The portion of the genome that does not encode for genes is often overlooked as a source of cellular regulatory information. Here, we demonstrate that regulatory information controlling expression and protein production from a gene called UBP1b is coming from a distant non-gene transposable element (TE). TEs are fragments of DNA that, unlike genes, are capable of duplicating themselves from one location in the genome to another, and occupy nearly half of the human genome. TEs are often referred to as “junk DNA,” as the study of cellular regulation and function is focused on genes. The regulation of TEs is distinct from genes, as a process termed epigenetic silencing heritably represses TE expression and activity. We have demonstrated that the epigenetic status (active versus silenced) of the Athila TE family regulates the UBP1b gene through the activity of a TE small RNA. The function of the UPB1b gene is to respond to and regulate cellular stress, and the epigenetic regulatory status of the Athila TE therefore modulates this stress response. This demonstrates that the epigenetic regulation of TEs can be a source of gene regulatory information, influencing a basic cellular function such as the stress response.
doi:10.1371/journal.pgen.1002474
PMCID: PMC3276544  PMID: 22346759
10.  Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max 
BMC Plant Biology  2012;12:177.
Background
Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes. Soybean (Glycine max) now has many genomics resources available including a complete genome sequence and predicted gene models. Relatively little is known, however, about the full complement of its endogenous smRNAs populations and the silenced genes.
Results
Using Illumina sequencing and computational analysis, we characterized eight smRNA populations from multiple tissues and organs of soybean including developing seed and vegetative tissues. A total of 41 million raw sequence reads collapsed into 135,055 unique reads were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatic analyses were used to distinguish miRNAs and siRNAs and to determine their genomic origins and potential target genes. In addition, we identified two soybean TAS3 gene homologs, the miRNAs that putatively guide cleavage of their transcripts, and the derived tasiRNAs that could target soybean genes annotated as auxin response factors. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. Our global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive elements including transposons. Detailed analysis of the size classes associated with ribosomal RNAs and transposable element families showed greater diversity of smRNAs in the 22- and 24-nt size classes.
Conclusions
The flux of endogenous smRNAs within multiple stages and tissues of seed development was contrasted with vegetative tissues of soybean, one of the dominant sources of protein and oil in world markets. The smRNAs varied in size class, complexity of origins, and possible targets. Sequencing revealed tissue-preferential expression for certain smRNAs and expression differences among closely related miRNA family members.
doi:10.1186/1471-2229-12-177
PMCID: PMC3534067  PMID: 23031057
11.  SINE RNA Induces Severe Developmental Defects in Arabidopsis thaliana and Interacts with HYL1 (DRB1), a Key Member of the DCL1 Complex 
PLoS Genetics  2008;4(6):e1000096.
The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.
Author Summary
Short interspersed elements (SINEs) are transposable elements in eukaryotic genomes that mobilize through an RNA intermediate. Recently, mammalian SINE RNAs were shown to have roles as noncoding riboregulators in stress situations or in specific tissues. Mammalian SINE RNAs modulate the level of mRNAs and proteins by interacting with key proteins involved in gene transcription and translation. Here we show that constitutive production of a plant SINE RNA induces developmental defects in Arabidopsis thaliana and that this SINE RNA interacts with HYL1, a double-stranded RNA-binding protein required for the production of microRNA and trans-acting small interfering (tasi)RNA. We mapped the binding site of HYL1 to a SINE RNA region that mimics the hairpin structure of microRNA precursors. We also found that HYL1 induces conformational changes upon binding to RNA substrates. These data suggest that SINE RNAs modulate the activity of RNAi pathways in Arabidopsis.
doi:10.1371/journal.pgen.1000096
PMCID: PMC2408557  PMID: 18551175
12.  Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata 
BMC Plant Biology  2012;12:209.
Background
Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies.
Results
We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs.
Conclusions
We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata’s responses to herbivore attack.
doi:10.1186/1471-2229-12-209
PMCID: PMC3502350  PMID: 23134682
Anti-herbivore defense; Jasmonate; Manduca sexta; miRNA; Nicotiana attenuata; tasiRNA
13.  Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes 
Genome Biology  2014;15(7):414.
Background
Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life cycle is that gene expression is required in the haploid gametophytes. Consequently, many mutant phenotypes are expressed in this phase.
Results
We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. Transposon-related transcripts are present in high levels in both gametophytes, suggesting a connection between gamete production and transposon expression in maize not previously identified in any female gametophytes. Two classes of small signaling proteins and several transcription factor gene families are enriched in gametophyte transcriptomes. Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2 indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than subgenome 2 genes with other expression patterns. Analysis of available insertion mutant collections shows a statistically significant deficit in insertions in gametophyte-expressed genes.
Conclusions
This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies maize gametophyte functions, gametophyte expression of transposon-related sequences, and unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed genes is supporting evidence for their function in the gametophytes. Expression patterns of extant, duplicated maize genes reveals that selective pressures based on male gametophytic function have likely had a disproportionate effect on plant genomes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0414-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0414-2
PMCID: PMC4309534  PMID: 25084966
14.  Auxin regulation of the microRNA390-dependent transacting small interfering RNA pathway in Arabidopsis lateral root development 
Nucleic Acids Research  2009;38(4):1382-1391.
MicroRNA (miR)390 cleaves the non-coding TAS3 precursor RNA for the production of tasiRNA-ARF, a group of an endogenous trans-acting small-interfering RNAs which cleave the transcripts of auxin response factor (ARF) 3/4. miR390-cleaved TAS3 RNA is polymerized and diced into tasiRNA-ARF by RNA-dependent RNA polymerase6 (RDR6) and Dicer-like4 (DCL4), respectively. tasiRNA-ARF-dependent post-transcriptional gene silencing (PTGS) of ARF3/4 is involved in auxin-mediated polarity establishment in the development of aerial lateral organs, such as leaf and flower. To understand how auxin regulates ARF4 expression, we examined auxin responsiveness of miR390 expression, which comprises a regulatory step for the biogenesis pathway of tasiRNA-ARF (the tasiRNA-ARF pathway), in Arabidopsis thaliana lateral root (LR) development. The results of this study provide evidence that miR390 expression is sensitive to TIR1-dependent transcriptional regulation and auxin concentration, and also that mutual negative-regulation between the tasiRNA-ARF pathway and ARF4 modulates the spatiotemporal expression of ARF4. We propose that, together with auxin concentration sensing through miR390 transcription, the tasiRNA-ARF pathway mediates the auxin response and ARF4-mediated LR developmental processes.
doi:10.1093/nar/gkp1128
PMCID: PMC2831332  PMID: 19969544
15.  Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins 
PLoS Genetics  2007;3(10):e171.
The female gametophyte of flowering plants, the embryo sac, develops within the diploid (sporophytic) tissue of the ovule. While embryo sac–expressed genes are known to be required at multiple stages of the fertilization process, the set of embryo sac–expressed genes has remained poorly defined. In particular, the set of genes responsible for mediating intracellular communication between the embryo sac and the male gametophyte, the pollen grain, is unknown. We used high-throughput cDNA sequencing and whole-genome tiling arrays to compare gene expression in wild-type ovules to that in dif1 ovules, which entirely lack embryo sacs, and myb98 ovules, which are impaired in pollen tube attraction. We identified nearly 400 genes that are downregulated in dif1 ovules. Seventy-eight percent of these embryo sac–dependent genes were predicted to encode for secreted proteins, and 60% belonged to multigenic families. Our results define a large number of candidate extracellular signaling molecules that may act during embryo sac development or fertilization; less than half of these are represented on the widely used ATH1 expression array. In particular, we found that 37 out of 40 genes encoding Domain of Unknown Function 784 (DUF784) domains require the synergid-specific transcription factor MYB98 for expression. Several DUF784 genes were transcribed in synergid cells of the embryo sac, implicating the DUF784 gene family in mediating late stages of embryo sac development or interactions with pollen tubes. The coexpression of highly similar proteins suggests a high degree of functional redundancy among embryo sac genes.
Author Summary
During the sexual reproduction of flowering plants, a pollen tube delivers sperm cells to a specialized group of cells known as the embryo sac, which contains the egg cell. It is known that embryo sacs are active participants in guiding the growth of pollen tubes, in facilitating fertilization, and in initiating seed development. However, the genes responsible for the complex biology of embryo sacs are poorly understood. The authors use two recently developed technologies, whole-genome tiling microarrays and high-throughput cDNA sequencing, to identify hundreds of genes expressed in embryo sacs of Arabidopsis thaliana. Most embryo sac–dependent genes have no known function, and include entire families of related genes that are only expressed in embryo sacs. Furthermore, most embryo sac–dependent genes encode small proteins that are potentially secreted from their cells of origin, suggesting that they may act as intracellular signals or to modify the extracellular matrix during fertilization or embryo sac development. These results illustrate the extent to which our understanding of plant sexual reproduction is limited and identifies hundreds of candidate genes for future studies investigating the molecular biology of the embryo sac.
doi:10.1371/journal.pgen.0030171
PMCID: PMC2014789  PMID: 17937500
16.  A Species-Specific Cluster of Defensin-Like Genes Encodes Diffusible Pollen Tube Attractants in Arabidopsis 
PLoS Biology  2012;10(12):e1001449.
AtLURE1 defensin-like peptides, which show species-specific evolution, are essential in Arabidopsis for attracting pollen tubes and can function in the breakdown of reproductive isolation barriers.
Genes directly involved in male/female and host/parasite interactions are believed to be under positive selection. The flowering plant Arabidopsis thaliana has more than 300 defensin-like (DEFL) genes, which are likely to be involved in both natural immunity and cell-to-cell communication including pollen–pistil interactions. However, little is known of the relationship between the molecular evolution of DEFL genes and their functions. Here, we identified a recently evolved cluster of DEFL genes in A. thaliana and demonstrated that these DEFL (cysteine-rich peptide [CRP810_1]) peptides, named AtLURE1 peptides, are pollen tube attractants guiding pollen tubes to the ovular micropyle. The AtLURE1 genes formed the sole species-specific cluster among DEFL genes compared to its close relative, A. lyrata. No evidence for positive selection was detected in AtLURE1 genes and their orthologs, implying neutral evolution of AtLURE1 genes. AtLURE1 peptides were specifically expressed in egg-accompanying synergid cells and secreted toward the funicular surface through the micropyle. Genetic analyses showed that gametophytic mutants defective in micropylar guidance (myb98, magatama3, and central cell guidance) do not express AtLURE1 peptides. Downregulation of the expression of these peptides impaired precise pollen tube attraction to the micropylar opening of some populations of ovules. Recombinant AtLURE1 peptides attracted A. thaliana pollen tubes at a higher frequency compared to A. lyrata pollen tubes, suggesting that these peptides are species-preferential attractants in micropylar guidance. In support of this idea, the heterologous expression of a single AtLURE1 peptide in the synergid cell of Torenia fournieri was sufficient to guide A. thaliana pollen tubes to the T. fournieri embryo sac and to permit entry into it. Our results suggest the unique evolution of AtLURE1 genes, which are directly involved in male–female interaction among the DEFL multigene family, and furthermore suggest that these peptides are sufficient to overcome interspecific barriers in gametophytic attraction and penetration.
Author Summary
Defensin-like (DEFL) peptides commonly function as effector peptides and are involved in male-female and host-parasite interactions in eukaryotes. In higher plants, DEFL genes belong to a large multigene family and are highly variable between species. However, little is known about the relationship between the molecular evolution of DEFL genes and their functions. By comparing multiply duplicated DEFL genes between A. thaliana and its close relative A. lyrata, we have now identified pollen tube attractant peptides called AtLURE1 peptides, in A. thaliana. We find that AtLURE1 genes form a species-specific gene cluster and that the AtLURE1 peptides these genes encode are specifically expressed in the synergid (egg-accompanying) cells and are secreted along the path down which the pollen tube elongates to reach the female gametophyte. AtLURE1 peptides attract pollen tubes in a species-preferential manner and their downregulation impairs pollen tube guidance. Interestingly, the genetic introduction of a single AtLURE1 gene from A. thaliana into another plant, T. fournieri, is sufficient to breakdown reproductive isolation barriers in pollen tube guidance and penetration. These results suggest that AtLURE1 peptides, which show species-specific evolution, are key molecules that attract pollen tubes from a plant's own species to the embryo sac to enable successful reproduction.
doi:10.1371/journal.pbio.1001449
PMCID: PMC3525529  PMID: 23271953
17.  DRB2 Is Required for MicroRNA Biogenesis in Arabidopsis thaliana 
PLoS ONE  2012;7(4):e35933.
Background
The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants).
Principal Findings
Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants.
Conclusions/Significance
Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue.
doi:10.1371/journal.pone.0035933
PMCID: PMC3335824  PMID: 22545148
18.  Comparative analysis of non-autonomous effects of tasiRNAs and miRNAs in Arabidopsis thaliana 
Nucleic Acids Research  2010;39(7):2880-2889.
In plants, small interfering RNAs (siRNAs) can trigger a silencing signal that may spread within a tissue to adjacent cells or even systemically to other organs. Movement of the signal is initially limited to a few cells, but in some cases the signal can be amplified and travel over larger distances. How far silencing initiated by other classes of plant small RNAs (sRNAs) than siRNAs can extend has been less clear. Using a system based on the silencing of the CH42 gene, we have tracked the mobility of silencing signals initiated in phloem companion cells by artificial microRNAs (miRNA) and trans-acting siRNA (tasiRNA) that have the same primary sequence. In this system, both the ta-siRNA and the miRNA act at a distance. Non-autonomous effects of the miRNA can be triggered by several different miRNA precursors deployed as backbones. While the tasiRNA also acts non-autonomously, it has a much greater range than the miRNA or hairpin-derived siRNAs directed against CH42, indicating that biogenesis can determine the non-autonomous effects of sRNAs. In agreement with this hypothesis, the silencing signals initiated by different sRNAs differ in their genetic requirements.
doi:10.1093/nar/gkq1240
PMCID: PMC3074149  PMID: 21134910
19.  Analysis of Antisense Expression by Whole Genome Tiling Microarrays and siRNAs Suggests Mis-Annotation of Arabidopsis Orphan Protein-Coding Genes 
PLoS ONE  2010;5(5):e10710.
Background
MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20–22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery.
Principal Findings
We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis ‘orphan’ hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the “ancient” (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for “new” rapidly-evolving MIRNA genes.
Conclusions
Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other kingdoms, which can provide insight into antisense transcription, miRNA evolution, and post-transcriptional gene regulation.
doi:10.1371/journal.pone.0010710
PMCID: PMC2877095  PMID: 20520764
20.  Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth—Application to the Eif3 Complex 
PLoS ONE  2011;6(3):e17640.
Background
Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.
Methodology/Principal Findings
Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation.
Conclusion/Significance
eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.
doi:10.1371/journal.pone.0017640
PMCID: PMC3049774  PMID: 21408229
21.  DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana 
Plant Signaling & Behavior  2012;7(10):1224-1229.
DOUBLE-STRANDED RNA BINDING (DRB) proteins have been functionally characterized in viruses, prokaryotes and eukaryotes and are involved in all aspects of RNA biology. Arabidopsis thaliana (Arabidopsis) encodes five closely related DRB proteins, DRB1 to DRB5. DRB1 and DRB4 are required by DICER-LIKE (DCL) proteins DCL1 and DCL4 to accurately and efficiently process structurally distinct double-stranded RNA (dsRNA) precursor substrates in the microRNA (miRNA) and trans-acting small-interfering RNA (tasiRNA) biogenesis pathways respectively. We recently reported that DRB2 is also involved in the biogenesis of specific miRNA subsets.1 Furthermore, the severity of the developmental phenotype displayed by the drb235 triple mutant plant, compared with those expressed by either drb2, drb3 and drb5 single mutants, or double mutant combinations thereof, indicates that DRB3 and DRB5 function in the same non-canonical miRNA pathway as DRB2. Through the use of our artificial miRNA (amiRNA) plant expression vector, pBlueGreen2,3 we demonstrate here that unlike DRB2, DRB3 and DRB5 are not involved in the dsRNA processing stages of the miRNA biogenesis pathway, but are required to mediate RNA silencing of target genes of DRB2-associated miRNAs.
doi:10.4161/psb.21518
PMCID: PMC3493401  PMID: 22902697
Arabidopsis; artificial miRNA; double-stranded RNA; double-stranded RNA binding protein; microRNA; non-canonical; RNA silencing
22.  GAMETOPHYTE DEFECTIVE 1, a Putative Subunit of RNases P/MRP, Is Essential for Female Gametogenesis and Male Competence in Arabidopsis 
PLoS ONE  2012;7(4):e33595.
RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing) are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1), a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants.
doi:10.1371/journal.pone.0033595
PMCID: PMC3324470  PMID: 22509260
23.  Apple miRNAs and tasiRNAs with novel regulatory networks 
Genome Biology  2012;13(6):R47.
Background
MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown.
Results
We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis.
Conclusions
Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family.
doi:10.1186/gb-2012-13-6-r47
PMCID: PMC3446319  PMID: 22704043
24.  Regulation of Small RNA Accumulation in the Maize Shoot Apex 
PLoS Genetics  2009;5(1):e1000320.
MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial–abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.
Author Summary
Small RNAs regulate many key developmental processes. Consistent with a prominent role in development, miRNAs exhibit complex and distinctive expression patterns. In this study, we identify regulatory mechanisms that allow for the precise spatial accumulation of developmentally important small RNAs in plants. Plants generate new leaves throughout their lifetime. These arise on the flank of a specialized stem cell niche, termed meristem, at the plant's growing tip. Each newly formed leaf becomes polarized and develops distinct adaxial (top) and abaxial (bottom) sides. The establishment of adaxial–abaxial polarity requires a complex genetic network, including miRNAs and trans-acting siRNAs. We used a focused laser to microdissect regions of the shoot apical meristem and developing leaves of maize to analyze the expression profiles of the small RNA precursor molecules. By comparing these expression profiles to the accumulation patterns of the mature small RNAs, we show that precursor genes are subject to tissue-specific regulation and exhibit diverse expression patterns during leaf development. Our findings suggest that mechanisms exist to regulate the biogenesis, stability, and possibly even the intercellular movement of small RNAs. Such regulation should be considered when designing artificial miRNAs and has implications for the roles miRNAs play during plant and animal development.
doi:10.1371/journal.pgen.1000320
PMCID: PMC2602737  PMID: 19119413
25.  Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa 
BMC Genomics  2010;11:338.
Background
Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to quickly germinate, produce a polar-growth pollen tube derived from the vegetative cell, and deliver two sperm cells into the embryo sac for double fertilization. The gene expression profiles of developing and germinated pollen have been characterised by use of the eudicot model plant Arabidopsis. Rice, one of the most important cereal crops, has been used as an excellent monocot model. A comprehensive analysis of transcriptome profiles of developing and germinated pollen in rice is important to understand the conserved and diverse mechanism underlying pollen development and germination in eudicots and monocots.
Results
We used Affymetrix GeneChip® Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stage-enriched transcripts displayed a "U-type" change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each.
Conclusions
Our results demonstrated that rice pollen expressed a set of reduced but specific transcripts in comparison with vegetative tissues, and the number of stage-enriched transcripts displayed a "U-type" change during pollen development, with the lowest at the bicellular pollen stage. These features are conserved in rice and Arabidopsis. The shift in gene expression program at the bicellular pollen stage may be important to the transition from earlier cell division to later pollen maturity. Pollen at maturity pre-synthesized transcripts needed for germination and early pollen tube growth. The transcription regulation associated with pollen development would have divergence between the two species. Our results also provide novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination.
doi:10.1186/1471-2164-11-338
PMCID: PMC2895629  PMID: 20507633

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