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MicroRNAs (miRNAs) are an emerging class of small RNAs regulating a wide range of biological processes. Soybean cotyledons evolved as sink tissues to synthesize and store seed reserves which directly affect soybean seed yield and quality. However, little is known about miRNAs and their regulatory networks in soybean cotyledons. We sequenced 292 million small RNA reads expressed in soybean cotyledons, and discovered 130 novel miRNA genes and 72 novel miRNA families. The cotyledon miRNAs arose at various stages of land plant evolution. Evolutionary analysis of the miRNA genes in duplicated genome segments from the recent Glycine whole genome duplication revealed that the majority of novel soybean cotyledon miRNAs were young, and likely arose after the duplication event 13 million years ago. We revealed the evolutionary pathway of a soybean cotyledon miRNA family (soy-miR15/49) that evolved from a neutral invertase gene through an inverted duplication and a series of DNA amplification and deletion events. A total of 304 miRNA genes were expressed in soybean cotyledons. The miRNAs were predicted to target 1910 genes, and form complex miRNA networks regulating a wide range of biological pathways in cotyledons. The comprehensive characterization of the miRNAs and their underlying regulatory networks at gene, pathway and system levels provides a foundation for further studies of miRNAs in cotyledons. [ABSTRACT FROM AUTHOR]

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology. [ABSTRACT FROM AUTHOR]

Summary To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues. [ABSTRACT FROM AUTHOR]

Abstract. We have created a DNA construct, TREGED (transposon-and recombinase-mediated genome deletion), that will automatically induce deletions in plant genomes. TREGED contains the maize Ac/Ds transposon, the yeast R-RS site-specific recombination system, the bacterial tetR repression systems, a novel artificial superintron, and the marker genes GUS and Lc. The novelty of TREGED is that only one cross is required to trigger a sequence of events leading to deletion and, simultaneously, to a color assay to detect the deletion. Crossing is done to introduce Ac transposase which activates Ds transposition from TREGED to a nearby chromosome region. Ds transposition, in turn, activates recombination between an engineered RS site on TREGED and an RS site on the transposed Ds fragment, thus deleting the genome segment between TREGED and Ds. The recombination event also deletes Lc or GUS and part of tetR, which triggers expression of GUS or Lc color genes for an upstream or downstream deletion respectively. Each TREGED insertion site will produce multiple kinds of deletions identifiable by inspecting a single F[sub 1] plant and its progeny for colored tissue. The color markers can also be used to differentiate between deletion and other more rare events such as translocation and inversion. We anticipate TREGED will greatly simplify the steps required to obtain useful deletions - eventually allowing the creation of detailed deletion libraries. Such libraries will be particularly useful for analysis of gene and chromatin function in plant species with large genomes. [ABSTRACT FROM AUTHOR]

Summary Cis‐regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower‐specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non‐coding RNAs (ncRNAs), which were processed into 24‐nt small interfering RNAs (siRNAs) that drove RNA‐directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis‐activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE–CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable. [ABSTRACT FROM AUTHOR]

SUMMARY: Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer–promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well‐characterized insulators and binding factor(s). In this study, we isolated a 1.2‐kb RS2‐9 insulator from the Oryza sativa (rice) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ‐specific enhancers in transgenic Arabidopsis and Nicotiana tabacum (tobacco). In the rice genome, the genes flanking RS2‐9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that O. sativa Homeobox 1 (OSH1) bound two regions of RS2‐9, as well as over 50 000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1‐binding sites in RS2‐9 impaired insulation activity by up to 60%, whereas the mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator CCCTC‐binding factor (CTCF) in mammals. Taken together, our findings indicate that OSH1‐RS2‐9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes. Significance Statement: The role of insualtor in the regulation of enhancer‐promoter interaction and topological architecture of genomes has been well characterized in mammals but rarely in plants largely due to a lack of a true insualtor. We report the isolation and characterization of an insualtor from rice genome and its binding protein prominently associated with the boundaries of chromatin loops. Our findings are of significance in understanding the insualtor‐based gene regulation and genome demarcation in plants [ABSTRACT FROM AUTHOR]

The potential for pollen-mediated transgene flow into wild or closely related species has provoked unease in terms of transgenic modification of agricultural plant species. One approach to remedy this situation in species whose seeds and fruits are not of particular value is to engineer male sterility into the transgenic lines. In this study, three meiosis-critical genes, namely AHP2, AtRAD51C and SWITCH1 ( SWI), were chosen as silencing targets to test the feasibility of incorporating sterility into plants using an RNAi-based approach. Our results indicated that the silencing of each of these genes via hairpin RNA (termed AHPi, RAD51Ci and SWIi lines) in Arabidopsis thaliana yielded a proportion of transgenic plants exhibiting a similar 'partially sterile' phenotype in which less than 50% of pollen was viable. In addition, a 'sterile' phenotype was also evident in a minority of RAD51Ci and SWIi, but not AHPi, lines in which plants yielded no seeds and either produced inviable pollen ( RAD51Ci lines) or displayed a complete absence of pollen ( SWIi lines). This suggests that AtRAD51C and SWI may function at distinct stages of meiosis. Further analyses of SWIi lines demonstrated that the 'sterile' phenotype was associated with a substantial reduction in the level of targeted gene transcript in floral tissues and resulted from sterility of the male, but not female gametes. This work demonstrates that generating male sterility through the silencing of key genes involved in the regulation of meiosis is feasible, and its advantages and potential applications for transgene containment are discussed. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the AtAGIP promoter derived from the Arabidopsis AGAMOUS ( AG) second intron/enhancer specifies a carpel- and stamen-specific expression in its native host species, but not in heterologous species such as tobacco, which restricts its application in the engineering of male and female sterility. These findings also imply that the AG regulatory mechanism that has evolved in Arabidopsis may, to some extent, have diverged from that of tobacco. To test whether a similar chimeric promoter created using the AG second intron/enhancer can overcome this barrier of evolutionary divergence in closely related species, we generated forward- and reverse-oriented chimeric promoters, fPtAGIP and rPtAGIP, from the petunia AG second intron/enhancer ( PtAGI) fragment and tested them in tobacco, which, like petunia, belongs to the Solanaceae family. Our results demonstrate that both fPtAGIP and rPtAGIP confer similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues in tobacco as revealed by tissue-specific gene expression and tissue ablation. This pattern resembles that driven by the AtAGIP in Arabidopsis and indicates that the AG regulatory mechanism is more conserved between tobacco and petunia than between tobacco and Arabidopsis. The petunia-derived promoters also exhibited petal-specific activity, and their activities in floral organs were substantially influenced by the orientation of the PtAGI enhancer, with reverse-oriented enhancers displaying approximately double the effectiveness of forward-oriented enhancers. These two properties are novel and have not been observed previously with AtAGIP promoters. Furthermore, we found that PtAGIP promoter-driven tissue ablation is effective for engineering complete sterility in plants, and the resulting sterile trait is stable for at least three mitotic generations at various temperature regimes, which is important for the complete containment of seed-, pollen-, and fruit-mediated gene flow in field conditions. [ABSTRACT FROM AUTHOR]

Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to determine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter ( AGIP) fused to the β-glucuronidase ( GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb λ fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1-kb λ insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer–promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments. [ABSTRACT FROM AUTHOR]

Trees use many mechanisms to adapt and respond to stressful conditions. The phenylpropanoid pathway in particular is known to be associated with a diverse suite of plant stress responses. In this study, we explored the relationship between the phenylpropanoid pathway metabolite production, gene expression and adaptive trait variation associated with floral bud reactivation during and following dormancy in Prunus armeniaca L. (apricot). Concentrations of eight phenylpropanoid metabolites were measured during chill accumulation and at developmental stages corresponding to the emergence of sepals and petals in floral buds of varieties that differ phenotypically in bloom date (BD). A significant interaction effect of chill hours and BD phenotype on the concentration of each of the compounds was observed (mixed analysis of variance, P  < 0.05), with the concentration of most phenylpropanoid metabolites dropping precipitously when sepals and petals emerged. While phenylpropanoid biosynthetic gene expression patterns were more variable in general, expression changed over time and was impacted, although to a lesser degree, by BD phenotype. Furthermore, separation of BD phenotypic groups was most pronounced when early and late BD varieties were at different developmental stages, i.e. 800 chill hours. Taken together, these results suggest that the phenylpropanoid pathway is associated with floral bud reactivation in apricot. Furthermore, we show that the phenylpropanoid pathway is also impacted by phenological trait variation associated with dormancy. A better understanding of how apricot and other perennial tree species respond and adapt to environmental perturbations will be critical for improvement programs aimed at identifying and breeding trees more suitable for rapidly changing environments. [ABSTRACT FROM AUTHOR]

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co-existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer-promoter and promoter-promoter interactions in transgenic plants and demonstrated that three of four flower-specific enhancer/promoters were capable of distantly activating a pollen- and stigma-specific Pps promoter (fused to the cytotoxic DT-A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen- and carpel-specific DT-A expression, thus resulting in tissue ablation in an orientation-independent manner; this activation was completely abolished by the insertion of an enhancer-blocking insulator ( EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue-specific DT-A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant-derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue-specific engineering of multiple traits using a single-vector stacking approach. Therefore, our work highlights the importance of adopting enhancer-blocking insulators in transformation vectors to minimize promoter-promoter interactions. The practical and fundamental significance of these findings will be discussed. [ABSTRACT FROM AUTHOR]

Germination is a biological process important to plant development and agricultural production. Barley and rice diverged 50 million years ago, but share a similar germination process. To gain insight into the conservation of their underlying gene regulatory programs, we compared transcriptomes of barley and rice at start, middle and end points of germination, and revealed that germination regulated barley and rice genes (BRs) diverged significantly in expression patterns and/or protein sequences. However, BRs with higher protein sequence similarity tended to have more conserved expression patterns. We identified and characterized 316 sets of conserved barley and rice genes (cBRs) with high similarity in both protein sequences and expression patterns, and provided a comprehensive depiction of the transcriptional regulatory program conserved in barley and rice germination at gene, pathway and systems levels. The cBRs encoded proteins involved in a variety of biological pathways and had a wide range of expression patterns. The cBRs encoding key regulatory components in signaling pathways often had diverse expression patterns. Early germination up-regulation of cell wall metabolic pathway and peroxidases, and late germination up-regulation of chromatin structure and remodeling pathways were conserved in both barley and rice. Protein sequence and expression pattern of a gene change quickly if it is not subjected to a functional constraint. Preserving germination-regulated expression patterns and protein sequences of those cBRs for 50 million years strongly suggests that the cBRs are functionally significant and equivalent in germination, and contribute to the ancient characteristics of germination preserved in barley and rice. The functional significance and equivalence of the cBR genes predicted here can serve as a foundation to further characterize their biological functions and facilitate bridging rice and barley germination research with greater confidence. [ABSTRACT FROM AUTHOR]

Trans -acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173- TAS1/2 , produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)- trans -acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR   phasiRNA production is triggered by different 22-nucleotide miRNAs , including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS -like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA – TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173 , implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. [ABSTRACT FROM AUTHOR]

The 3' untranslated regions (UTRs) of RNA1 and RNA2 of Tomato ringspot virus (ToRSV) are 1.3 kb in size and virtually identical. In this study, sequences containing most of the 3' UTRs (1168-1265 bp) were determined from 18 ToRSV isolates collected from fruit trees, small fruits and grapevines in North America. Pairwise nucleotide sequence comparisons with sequences of two isolates available in GenBank showed identities ranging from 95.1 to 100% for the majority of ToRSV isolates. Most genetic variation occurs as point mutations and single insertions and/or deletions. Phylogenetic analysis showed that 17 of the 20 isolates grouped together into a major cluster and three isolates grouped into two minor clusters, with genetic divergence rates among the three clusters of 11.8% to 23.8%. No correlation was found between the 3' UTR sequences and symptom expression on Nicotiana benthamiana, host plant or geographic origin of ToRSV isolates. RT-PCR using primers designed within the highly conserved 3' UTR regions detected all 18 ToRSV isolates as well as two isolates from a vineyard. This assay can serve as a practical methodology for the sensitive detection of varied ToRSV isolates as it is more sensitive than a RT-PCR assay based on previously reported U1/D1 primers. [ABSTRACT FROM AUTHOR]

The UVH6 gene from Arabidopsis thaliana is predicted to function in transcription regulation, based on known activities of its human and yeast homologs. In this study, we show that uvh6-1 mutants are ultra-sensitive to cold and suggest that this defect results from reduced expression of cold-stress genes. Comparison of mRNA levels in cold-treated wild-type and uvh6-1 plants reveals that expression of two cold-stress genes ( Cor6.6 and Cor15a) is impaired in the mutant. In contrast, the mutant shows normal cold induction of three transcription factor genes ( CBF1, 2, 3), which regulate these Cor genes, and normal induction of several additional CBF-targeted genes. Thus, we propose that UVH6 promotes cold resistance by specifically regulating transcription of Cor6.6 and Cor15a genes. We further find features among the regulatory sites in the Cor6.6 and Cor15a promoters which suggest unique regulation of these genes. [ABSTRACT FROM AUTHOR]

Background: 'Rapid Apple Decline' (RAD) is a newly emerging problem of young, dwarf apple trees in the Northeastern USA. The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing. Methods: Illumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5′/3' RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus. Results: Three common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards. Conclusions: A new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role. [ABSTRACT FROM AUTHOR]