Search

SUMMARY: Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co‐expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage‐specific manner. Members of the miR482/miR2118 superfamily and their 21‐nucleotide phasiRNAs originating from resistance genes show a robust co‐expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage‐specific manner, related to modulated hormonal responses and transcription factor expression. Significance Statement: Small RNAs in coffee floral development: unveiling stage‐specific accumulation patterns and regulatory networks. [ABSTRACT FROM AUTHOR]

SUMMARY: The anther‐enriched phased, small interfering RNAs (phasiRNAs) play vital roles in sustaining male fertility in grass species. Their long non‐coding precursors are synthesized by RNA polymerase II and are likely regulated by transcription factors (TFs). A few putative transcriptional regulators of the 21‐ or 24‐nucleotide phasiRNA loci (referred to as 21‐ or 24‐PHAS loci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations suffice to activate any PHAS locus is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the 21‐ or 24‐PHAS loci. Comparisons of these coexpression modules and gene sets dysregulated in several reported male sterile TF mutants provided insights into TF timing with regard to phasiRNA biogenesis, including antagonistic roles for OUTER CELL LAYER4 and MALE STERILE23. Trans‐activation assays in maize protoplasts of individual TFs using bulk‐protoplast RNA‐sequencing showed that two of the TFs coexpressed with 21‐PHAS loci could activate several 21‐nucleotide phasiRNA pathway genes but not transcription of 21‐PHAS loci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of 24‐PHAS loci using single‐cell (protoplast) RNA‐sequencing, did not detect reproducible activation of either 21‐PHAS or 24‐PHAS loci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive PHAS loci. Significance Statement: This work identifies the temporal gene coexpression networks associated with maize anther development, including two modules highly enriched for the 21‐ or 24‐nucleotide reproductive phased, small interfering RNA (phasiRNA) loci. Trans‐activation assays of putative transcriptional regulators of the phasiRNA loci using bulk and single‐cell RNA‐sequencing suggest that endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive phasiRNA loci. [ABSTRACT FROM AUTHOR]

SUMMARY: Small RNAs play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: (i) pre‐meiotic 21‐nucleotide (nt) phasiRNAs triggered by miR2118 and (ii) meiotic 24‐nt phasiRNAs triggered by miR2275. Here, we describe conserved and non‐conserved triggers of 24‐nt phasiRNAs in several eudicots. We found that the abundant 24‐nt phasiRNAs in the basal eudicot columbine (Aquilegia coerulea) are produced by the canonical trigger miR2275, as well as by other non‐canonical triggers, miR482/2118 and miR14051. These triggering microRNAs (miRNAs) are localized in microspore mother cells and tapetal cells of meiotic and post‐meiotic stage anthers. Furthermore, we identified a lineage‐specific trigger (miR11308) of 24‐nt phasiRNAs and an expanded number of 24‐PHAS loci in wild strawberry (Fragaria vesca). We validated the presence of the miR2275‐derived 24‐nt phasiRNA pathway in rose (Rosa chinensis). Finally, we evaluated all eudicots that have been validated for the presence of 24‐nt phasiRNAs as possible model systems in which to study the biogenesis and function of 24‐nt phasiRNAs. We conclude that columbine (Aquilegia coerulea) would be a strong model because of its extensive number of 24‐PHAS loci and its diversity of trigger miRNAs. Significance Statement: We show that the 24‐nucleotide (nt) reproductive phased small interfering RNA (phasiRNA) pathway is conserved in a diverse eudicot, Aquilegia. We validated this pathway in rose. We also identified novel conserved (e.g., miR2118) and lineage‐specific triggers involved in initiating expression of 24‐nt phasiRNAs in eudicots. Our results detail the diversification of this pathway in several eudicots and the cellular localization of conserved and non‐conserved triggers. We combine these data to propose Aquilegia as a model eudicot in which to study reproductive phasiRNAs. [ABSTRACT FROM AUTHOR]

SUMMARY: Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear‐encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1‐dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development. Significance Statement: Accumulation of an undefined apocarotenoid, ACS1, derived from cis‐carotenoids leads to important defects in leaf development via a previously unknown molecular mechanism. In this work we show that accumulation of ACS1 initiates a retrograde signal cascade resulting in the transcriptional modulation of diverse genes and define the molecular events involved in ACS1's regulation of leaf development. Our work illustrates how two sequential plastidial retrograde signaling cascades modulate leaf development during a particular developmental window. These results will be instrumental in efforts to biochemically define ACS1. [ABSTRACT FROM AUTHOR]

Summary: The Lemnaceae family comprises aquatic plants of angiosperms gaining attention due to their utility in wastewater treatment, and rapid production of biomass that can be used as feed, fuel, or food. Moreover, it can serve as a model species for neotenous growth and environmental adaptation. The latter properties are subject to post‐transcriptional regulation of gene expression, meriting investigation of how miRNAs in Spirodela polyrhiza, the most basal and most thoroughly sequenced member of the family, are expressed under different growth conditions. To further scientific understanding of its capacity to adapt to environmental cues, we measured miRNA expression and processing of their target sequences under different temperatures, and in the presence of abscisic acid, copper, kinetin, nitrate, and sucrose. Using two small RNA sequencing experiments and one degradome sequencing experiment, we provide evidence for 108 miRNAs. Sequencing cleaved mRNAs validated 42 conserved miRNAs with 83 targets and 24 novel miRNAs regulating 66 targets and created a list of 575 predicted and verified targets. These analyses revealed condition‐induced changes in miRNA expression and cleavage activity, and resulted in the addition of stringently reviewed miRNAs to miRBase. This combination of small RNA and degradome sequencing provided not only high confidence predictions of conserved and novel miRNAs and targets, but also a view of the post‐transcriptional regulation of adaptations. A unique aspect is the role of miR156 and miR172 expression and activity in its clonal propagation and neoteny. Additionally, low levels of 24 nt sRNAs were observed, despite the lack of recent retrotransposition. Significance Statement: Taking advantage of the sequenced genome of an aquatic species, the expression of miRNA and processing of its targets sites in response to environmental adaptation was investigated, providing an enhanced miRNA database. [ABSTRACT FROM AUTHOR]

Summary: Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level. Significance Statement: Fluorescence‐based microscopy methods are challenging in plants due to the broad range of interfering autofluorescence. Here, we provide a detailed protocol to detect small RNAs by fluorescence in situ hybridization that can specifically detect multiple targets and is compatible with advanced imaging technologies such as super‐resolution microscopy. [ABSTRACT FROM AUTHOR]

The altered carbon assimilation pathway of crassulacean acid metabolism ( CAM) photosynthesis results in an up to 80% higher water-use efficiency than C3 photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and micro RNA (mi RNA) expression patterns in the obligate CAM plant pineapple [ Ananas comosus (L.) Merr.]. The high-resolution transcriptome atlas allowed us to distinguish between CAM-related and non- CAM gene copies. A differential gene co-expression network across green and white leaf diel datasets identified genes with circadian oscillation, CAM-related functions, and source-sink relations. Gene co-expression clusters containing CAM pathway genes are enriched with clock-associated cis-elements, suggesting circadian regulation of CAM. About 20% of pineapple micro RNAs have diel expression patterns, with several that target key CAM-related genes. Expression and physiology data provide a model for CAM-specific carbohydrate flux and long-distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering of CAM into C3 photosynthesis crop species. [ABSTRACT FROM AUTHOR]

Small RNAs are a class of non-coding RNAs that are of great importance in gene expression regulatory networks. Different families of small RNAs are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary si RNAs (phasi RNAs); these require RDR6, DCL4, and (typically) a micro RNA (mi RNA) trigger for their biogenesis. Protein-encoding genes are an important source of phasi- RNAs. The model legume Medicago truncatula generates phasi RNAs from many PHAS loci, and we aimed to investigate their biogenesis and mechanism by which mi RNAs trigger these molecules. We modulated mi RNA abundances in transgenic tissues showing that the abundance of phasi RNAs correlates with the levels of both mi RNA triggers and the target, precursor transcripts. We identified sets of phasi RNAs or PHAS loci that predominantly and substantially increase in response to mi RNA overexpression. In the process of validating targets from mi RNA overexpression tissues, we found that in the mi RNA- mRNA target pairing, the 3′ terminal nucleotide (the 22nd position), but not the 10th position, is important for phasi RNA production. Mutating the single 3′ terminal nucleotide dramatically diminishes phasi RNA production. Ectopic expression of Medicago NB- LRR-targeting mi RNAs in Arabidopsis showed that only a few NB- LRRs are capable of phasi RNA production; our data indicate that this might be due to target inaccessibility determined by sequences flanking target sites. Our results suggest that target accessibility is an important component in mi RNA-target interactions that could be utilized in target prediction, and the evolution of mRNA sequences flanking mi RNA-target sites may be impacted. [ABSTRACT FROM AUTHOR]

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat-inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding-leucine rich repeat proteins and genes encoding pentatricopeptide repeat-containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense-associated genes in soybean during Phytophthora infection. [ABSTRACT FROM AUTHOR]

Short interfering RNAs (si RNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation ( Rd DM) and transcriptional gene silencing ( TGS) of target genes. In our system, si RNAs are produced by transcribing an inverted DNA repeat ( IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into si RNAs of 21-24 nt. Primarily 24-nt si RNAs trigger Rd DM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed si RNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II ( Pol II)- and Pol IV-dependent pathways of si RNA biogenesis. At the original silencer locus, Pol- II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant si RNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt si RNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. si RNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing si RNAs revealed the preferential accumulation of si RNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and si RNA accumulation, our results highlight the complexity of si RNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template. [ABSTRACT FROM AUTHOR]

Summary Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice ( Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5′ rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice. [ABSTRACT FROM AUTHOR]

Disease resistance ( R) genes are often clustered in plant genomes and may exhibit heterogeneous rates of evolution. Some (type I R genes) have evolved rapidly through frequent sequence exchanges, while others (type II R genes) have evolved independently and tend to be conserved in different genotypes or related species. The RPP8 resistance gene in Arabidopsis thaliana is located at a complex locus that also harbors the sequence-related resistance genes HRT and RCY1 in different ecotypes. We sequenced 98 homologs of RPP8 from A. thaliana, Arabidopsis arenosa and Arabidopsis lyrata. Three lineages of type II and one lineage of type I RPP8 homologs were identified. Two of the three lineages of type II genes are each represented by a single-copy locus on either chromosomes I or V. Chromosome V contains two small clusters of RPP8 paralogs. One cluster contains both type I and type II genes and the other comprises only type I genes. These multi-copy loci have expanded and contracted through unequal crossovers, which have generated chimeric genes as well as variations in copy number. Sequence exchanges, most likely gene conversions, were detected between RPP8 homologs that are spatially separated by 2.2 Mb and 12 cM. The sequence exchanges between type I homologs within a locus have been more frequent than sequence exchanges between homologs from two different loci, indicating the influence of chromosomal position on the evolution of these R genes. However, physical distance was not the only factor determining the frequency of sequence exchange, because some closely linked paralogs exhibited little sequence exchange. At least two distinct lineages of type II RPP8 homologs were identified in different species, with obvious allelic/orthologous relationships within each lineage. Therefore, the differentiation of type I and type II RPP8 homologs seems to have occurred before speciation of A. thaliana, A. arenosa and A. lyrata. [ABSTRACT FROM AUTHOR]

Summary: The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins. [ABSTRACT FROM AUTHOR]

Summary The Toll/interleukin-1 receptor (TIR) domain is found in one of the two large families of homologues of plant disease resistance proteins (R proteins) in Arabidopsis and other dicotyledonous plants. In addition to these TIR-NBS-LRR (TNL) R proteins, we identified two families of TIR-containing proteins encoded in the Arabidopsis Col-0 genome. The TIR-X (TX) family of proteins lacks both the nucleotide-binding site (NBS) and the leucine rich repeats (LRRs) that are characteristic of the R proteins, while the TIR-NBS (TN) proteins contain much of the NBS, but lack the LRR. In Col-0, the TX family is encoded by 27 genes and three pseudogenes; the TN family is encoded by 20 genes and one pseudogene. Using massively parallel signature sequencing (MPSS), expression was detected at low levels for approximately 85% of the TN-encoding genes. Expression was detected for only approximately 40% of the TX-encoding genes, again at low levels. Physical map data and phylogenetic analysis indicated that multiple genomic duplication events have increased the numbers of TX and TN genes in Arabidopsis . Genes encoding TX, TN and TNL proteins were demonstrated in conifers; TX and TN genes are present in very low numbers in grass genomes. The expression, prevalence, and diversity of TX and TN genes suggests that these genes encode functional proteins rather than resulting from degradation or deletions of TNL genes. These TX and TN proteins could be plant analogues of small TIR-adapter proteins that function in mammalian innate immune responses such as MyD88 and Mal. [ABSTRACT FROM AUTHOR]