Liver metastasis is the main reason for the poor prognosis of colorectal cancer, and identifying molecules involved in liver metastases of colorectal cancer may provide effective therapeutic targets. Zinc-α2-glycoprotein 1(AZGP1) is a candidate biomarker for diagnosis and prognosis in cancer. However, its function and molecular mechanism in metastatic colorectal cancer remains largely unknown. We previously found that up-regulated AZGP1 promotes proliferation, migration and invasion in colorectal cancer cell line, here we elucidated the mechanism of AZGP1 in regulating metastasis. In this article, we found that AZGP1 was also highly expressed in colorectal cancer tissues with liver metastasis relative to those without metastasis, and abundant expression of AZGP1 was associated with poor prognosis, also, AZGP1 down regulation prevented cell metastasis in vivo and in vitro. We further demonstrated that AZGP1 promotes metastasis by regulating the epithelial-mesenchymal transition (EMT) and associating with molecules involved in the focal adhesion pathway, including the adhesion molecule FLNA, which acts as an important protein interactor. More importantly, AZGP1 down regulation inhibited the phosphorylation of FLNA mediated by the restrain of PAK2 kinase, thereby inducing its proteolysis and subsequently affecting its subcellular localization, where it regulates the EMT and promotes metastasis. Collectively, these results highlight AZGP1 as a new and promising therapeutic molecule for liver metastatic colorectal cancer. Colorectal cancer (CRC) is one of the top five most frequent malignant tumors worldwide. Despite many advances in the diagnosis and therapeutic improvements, the prognosis of CRC patients remains poor. The most important factor is high frequency of metastasis and recurrence Zinc-a2-glycoprotein  is a secreted41-kDa protein that was initially identified and purified in human serum Epithelial-to-mesenchymal trans-differentiation (EMT), which often leads to aggressive cancer progression, is a crucial event in the carcinogenesis of CRC. EMT is characterized by a loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. AZGP1 was also shown to regulate metastasis. The analysis of AZGP1 expression in malignant prostate epithelium in prostatectomy specimens from 228 prostate cancer patients revealed that absent or weak AZGP1 expression was associated with clinical recurrence, including localized recurrence, metastasis, or death. These results suggest that AZGP1 may be an accurate and timely indicator of the risk of metastatic progression In this study, we found that AZGP1 expression was significantly increased in tissues from patients with liver metastasis relative to those without distant metastasis. Additionally, increased expression of AZGP1 indicated a poorer prognosis. Most importantly, our results demonstrated that AZGP1 silencing blocks the EMT and reduces metastasis in vivo and in vitro, which may be mediated by filamin A (FLNA) and the focal adhesion pathway.th edition of the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) TNM classification. Routine chemoradiotherapy had been given postoperatively to patients with advanced-stage disease. Ethical approval was obtained from the Clinical Research Ethics Committee of Zhongshan Hospital of Fudan University. Signed informed consent was obtained from all patients for the acquisition and use of patient tissue samples and anonymized clinical data. The follow-up data were regularly acquired through outpatient visits, telephone calls, or office visits. The median follow-up time was 56.5 months (range 1-144 months). OS was calculated from the day of surgery to the date of death due to CRC or the date of the last follow-up.A total of 280 CRC patients from Zhongshan Hospital of Fudan University  were enrolled between January 2001 and March 2010. Clinicopathologic data were retrieved from our prospectively constructed CRC database. Tumor stage was determined according to the 72.Human HCT116 and SW480 CRC cell lines and human embryonic kidney 293 cells were obtained from the cell bank of the Chinese Academy of Sciences . All cell lines were routinely maintained in DMEM  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified chamber under 5% COFormalin-fixed, paraffin-embedded sections were stained by a non-biotin enzymatic method  for the specific detection of AZGP1 expression  in CRC tissues. The immunohistochemistry (IHC) results were calculated as the product of two independent scores: the proportion of positive tumor cells in the tissues  and the average intensity of positive tumor cells in the tumor tissues . The final score was determined by multiplying the staining intensity score with that of the positive proportion. Scores were determined independently by two senior pathologists. The final score was classified as low- or high-expression using the median value as the cut-off.4 with 100 µl per well. Six hundred microliters of media containing 10% FBS were added to the lower chamber . After 48 h, the chambers were washed twice with PBS, and the cells on the apical side of each insert were scraped off. The invaded cells were fixed with 4% paraformaldehyde and stained with crystal violet. Cells were counted under a microscope.Cells were trypsinized, re-suspended in low serum media (0.1% FBS), and seeded at a density of 1 × 10Cells were cultured on coverslips and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with ice-cold 95% methanol for 30 min. Cells were washed with PBS, blocked with 3% BSA/PBS/0.1% Triton-X for 1 h at room temperature and incubated with the appropriate primary antibody. After overnight incubation at 4°C, cells were washed, incubated with the fluorescence-labeled secondary antibody for 1 h at room temperature and mounted with anti-fade medium including DAPI. Images were captured by fluorescence microscopy . The gain and offset settings were fixed across all samples.4) were injected into the spleens of BALB/c mice. Animals were weighed regularly before the experimental endpoint. Mice were sacrificed 39 days later, and the rate of tumor formation and surface liver nodules were counted. All animal experiments were performed in accordance with NIH guidelines for the use of experimental animals.Liver metastasis assays were performed using an intra-splendid injection CRC mouse model as previously described Cells were lysed in 1ml of chilled RIPA buffer, and the cleared lysate (400 µl) was incubated with 5 µl of the indicated antibody  and 20 µl of protein-A agarose beads . The reaction was brought up to a final volume of 500µl with IP buffer. After constant rotation for 3 hours, beads were resuspended in 2× SDS buffer and subjected to Western blot analysis.Results are expressed as the mean ± SEM of at least 3 independent experiments. A paired Student's t-test was performed for data analysis. All statistical analyses were performed using GraphPad Prism software version 5.02 .Liver is the most frequent site of metastases from CRC. To figure out the liver metastasis of colorectal cancer, we obtained abnormal expressed genes through 6 patients without liver metastasis and 5 patients with liver metastasis by the HG-U133 microarray (Affymetrix), among these, AZGP1 was one of the most significant highly expressed molecules. To explore the role of AZGP1 in liver metastastic colorectal cancer, firstly, we verified the mRNA expression by qPCR, the result showed that AZGP1 was more abundant in primary tumors with liver metastasis than those without liver metastasis, besides, in metachronous metastasis primary tumors, it was higher than simultaneous metastasis tumor, although there was no statistical difference . For patients with high AZGP1 expression, the 3- and 5-year OS rates were 49.8% and 38.8%, respectively. For patients with low AZGP1 expression, the 3- and 5-year OS rates were 75.1% and 72.1%, respectively  or a control scrambled shRNA (shCON), the efficiency of AZGP1 knockdown was verified by Western Blot Fig. A. AZGP1 http://www.uniprot.org) and literature reviews  Fig. A. 75 Posews Fig. B. We fouews Fig. C-D. NumeIn order to verify this conjecture, rescue experiments were conducted. Firstly, lentivirus mediated FLNA shRNA were constructed and the protein expression were verified by Western Blot after infected. Then, transwell assays indicated that over expressed AZGP1 promoted migration, while the migration promotion was damaged when FLNA was down regulated Fig. E. BesideCollectively, these data provide biochemical evidence that AZGP1 interacts with FLNA and facilitates CRC cell metastasis mediated by FLNA.To determine the specific pathway by which AZGP1 promotes metastasis, the gene expression profile was first examined using an Affymetrix microassay following knockdown of AZGP1. Among the genes identified, 801 were upregulated, while 591 were downregulated. KEGG pathway analysis was performed, and it suggested that the focal adhesion pathway is responsible for the regulation of metastasis Fig. A. The coPAK2, one member of p21-activating kinases (PAKs), mediates oncogenic effects by phosphorylating multiple substrates involved in proliferation, migration and cytoskeletal reorganization. As shown in Figure Multiple studies have shown that FLNA exhibited opposing roles in cancer progression depending on its location. In prostate cancer, the cytoplasmic localization of FLNA determines a propensity to metastasize that can be prevented by cleavage and subsequent nuclear translocation Liver metastasis is the main cause of CRC mortality, however, the mechanism remains largely unknown. To identify the genes responsible for liver metastasis, we used a microassay to compare primary tumors without liver metastasis to those with liver metastasis. AZGP1 was identified as a promising molecular regulating liver metastasis. In this study, we demonstrated that AZGP1 is highly expressed in CRC tissues with liver metastasis. Moreover, abundant expression of AZGP1 was associated with a poor prognosis. Additional molecular biology experiments demonstrated that AZGP1 promotes metastasis by mechanistically interacting with FLNA and regulating the adhesion pathway.Recent studies have shown that AZGP1 regulates a variety of tumors. AZGP1 expression has, in general, been reported as downregulated in certain carcinomas, including those in the breast, prostate, liver, and stomach. However, increased expression has been reported in other tumors, such as lung adenocarcinoma. Moreover, AZGP1 was significantly upregulated in a tissue microarray containing 190 samples of primary colon cancer tissue compared with normal colonic tissue. On this basis, we found that AZGP1 is highly expressed in CRC tissue with liver metastasis relative to those without liver metastasis. Logistic regression analysis also revealed that abundant expression of AZGP1 is a risk factor for metastasis in CRC.Currently, abnormal expression of AZGP1 can be used as an important indicator for prognosis, however, the mechanism in cancer progression remains largely unknown. In hepatocellular carcinoma, the underlying molecular mechanism by which AZGP1 inhibits cell migration and invasion is regulating the PTEN/Akt and CD44 pathways FLNA, an actin-binding protein, is important for organogenesis during development. Previous reports have revealed its involvement in cancer development In summary, our results show that AZGP1 is an oncogene that plays an important role in promoting liver metastasis in CRC. AZGP1 knockdown prevented cancer cell EMT, which could contribute to the inhibition of metastasis. The mechanism involved is correlated with the activation of the focal adhesion pathway for example increased expression of CDC24. In addition, FLNA phosphorylation inhibition occurred due to the reduced PAK2 activity and then contributed to its instability. The dissociation of FLNA and its subsequent proteolysis promoted its nuclear localization accordingly. Finally it regulated the EMT marker and prevented the formation of distant metastases. Thus, silencing AZGP1 could be an efficient method to prevent metastasis and improve OS in CRC patients by delaying tumor progression.


The self-assembly of proteins and peptides into amyloids is a key feature of an increasing number of diseases. Amyloid fibrils display a unique surface reactivity endowing the sequestration of molecules such as MicroRNAs, which can be the active moiety of the toxic action. To test this hypothesis we studied the recognition between a model RNA and two different steric zipper spines using molecular dynamics simulations. We found that the interaction occurs and displays peptide-sequence dependence. Interestingly, interactions with polar zipper surfaces such as the formed by SNQNNF are more stable and favor the formation of β-barrel like complexes resembling the structures of toxic oligomers. These sequence-structure-recognition relationships of the two different assemblies may be exploited for the design of compounds targeting the fibers or competing with RNA-amyloid attachment The self-assembly of peptides and proteins into amyloid aggregates is the common thread of a large number of degenerative diseases including Creutzfeldt-Jakob, Parkinson's, Alzheimer's, Amyotrophic Lateral Sclerosis, type 2 diabetes, among others , miR378a (CUCCUGACUCCAGGUCCUGUGU), and miR344 (AGUCAGGCUCCUGGCUAGAUUCCAGG) released in exosomes of prion-infected cells , followed by a run with conjugate gradient algorithm (7 × 103 steps). After minimization, the production simulations were conducted with isotropic position scaling (ntp = 1) and the temperature was kept constant using a weak-coupling algorithm  method as implemented in Amber 16 . Each basic unit cell was replicated along two directions, while explicit water was added along the third direction providing simulation boxes with the dimensions reported in Table For each of the interaction simulations, four different RNA chains  were randomly placed in the bulk water layer. Interestingly, once docked onto the amyloid the RNA molecules may shift their position on the surface, and undergo conformational changes. In some cases, the formation of dimers was observed Figure . ConsideEach of the representative structures was next used to calculate the energetics of the interaction by means of the MMPBSA approach . Within the limitations of our model  . The most representative conformations of RNA in complex with either peptide sequences are qualitatively similar, and are shown in Figure To verify the potential validity of the observed RNA docking modes on the surfaces, we searched the Protein Data Bank for experimentally characterized structures of RNA-protein complexes. It is interesting to note that our simulation-based observation shown in Figure In terms of drug design, we suggest that pharmacophore models may be designed by keeping into account the relative stereochemical relationships of the functional groups most responsible for binding the amyloid aggregates observed along the trajectories and by using different relative weights to keep into account the information on the persistence, stability and/or variability of the interactions established with the model aggregate. In this case, an ideal pharmacophore could be constituted by a central scaffold decorated with 3 to 4 aromatic polar moieties that mimic the pyrimidine groups Figure . IdeallyWe have studied the recognition between a model RNA stretch and different amyloid fibrils by molecular dynamics simulations. To the best of our knowledge, this is one of the first reports of a possible interference or RNA-trapping mechanism exerted by an ordered amyloid fibril surface. We found that this interaction occurs and that for a common RNA stretch, the recognition is clearly peptide-sequence dependent. Interestingly, in the case of the SNQNNF fibril surface the interactions appear to be more stable and to favor the formation of β-barrel like complexes resembling the structures of toxic oligomers. On the basis of these results, specific amyloid sequences may sequester nucleic acid material on the surfaces their fibrillar and oligomeric assembly and derange the biochemical pathways in which RNA is used as an information carrying molecule. Moreover, we suggest that such initial sequence-structure-recognition relationships of the two different fibers may be a source of information for the design of compounds targeting the fibers or competing with RNA-amyloid attachment.MM designed research, ran simulations, wrote the paper. MG and GC designed research, wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae as well as contributing to the integration of the nuclear export of mature mRNA with preceding steps in the nuclear phase of the gene expression pathway. Nab2 is constructed from an N‐terminal PWI‐fold domain, followed by QQQP and RGG motifs and then seven CCCH Zn fingers. The nuclear pore‐associated proteins Gfd1 and Mlp1 bind to opposite sides of the Nab2 N‐terminal domain and function in the nuclear export of mRNA, whereas the Zn fingers, especially fingers 5–7, bind to A‐rich regions of mature transcripts and function to regulate poly(A) tail length as well as mRNA compaction prior to nuclear export. Nab2 Zn fingers 5–7 have a defined spatial arrangement, with fingers 5 and 7 arranged on one side of the cluster and finger 6 on the other side. This spatial arrangement facilitates the dimerization of Nab2 when bound to adenine‐rich RNAs and regulates both the termination of 3′ polyadenylation and transcript compaction. Nab2 also functions to coordinate steps in the nuclear phase of the gene expression pathway, such as splicing and polyadenylation, with the generation of mature mRNA and its nuclear export. Nab2 orthologues in higher Eukaryotes have similar domain structures and play roles associated with the regulation of splicing and polyadenylation. Importantly, mutations in the gene encoding the human Nab2 orthologue ZC3H14 and cause intellectual disability.The poly(A) RNA binding Zn finger ribonucleoprotein Nab2 functions to control the length of 3′ poly(A) tails in  Saccharomyces cerevisiae Nab2 (ScNab2) accompanies mature transcripts as they move through nuclear pores to the cytoplasm, after which ScNab2 is thought to be removed from the mRNA by the DEAD‐box RNA helicase, Dbp5ScNab2 is then recycled back to the nucleus through nuclear pores using the transport factor, karyopherin β2 .ScNab2 shows genetic interactions with the splicing machineryIn eukaryotes, the separation by the nuclear envelope of transcription from translation enables mRNAs to be modified by capping, splicing, and polyadenylation. These processing steps are mediated by a large number of different proteins that interact with transcripts as they pass through the nuclear phase of the gene expression pathway before they are finally exported to the cytoplasm through nuclear pores.Drosophila orthologue, DmNab2 are also required for proper poly(A) tail length control,ZC3H14 gene have been linked to a nonsyndromic form of autosomal recessive intellectual disability,DmNab2 mutant flies exhibit impaired short‐term memory and defects in neuronal patterning in the learning and memory center (mushroom body) of the fly brain.DmNab2 mutant flies rescues function, indicating that ZC3H14 is a functional orthologue of DmNab2.In higher eukaryotes, the human Nab2 orthologue, ZC3H14, and ScNab2 in S. cerevisiae. ScNab2 contains an N‐terminal domain that has a Proline‐Tryptophan‐Isoleucine (PWI)‐like fold,ScNab2 Zn fingers (ZnFs) are arranged into three groups: ZnF12; ZnF34; and ZnF567. The Nab2 orthologues in other species—S. pombe Nab2 (SpNab2); C. thermophilum Nab2 (CtNab2); D. melanogaster Nab2 (DmNab2); C. elegans SUT‐2 (CeSUT‐2); H. sapiens ZC3H14 (HsZC3H14)—have a similar overall domain architecture  RNA using CtNab2 and ScNab2.ScNab2 Zn fingers 5, 6, and 7 (ZnF567) are critical for function, and studies demonstrate that a nab2 mutant lacking ZnF567 is not functional in budding yeast.ScNab2.Binding to polyadenosine RNA, a primary function of the Nab2 protein family, is mediated by the Zn finger motifs.ScNab2 N‐terminal domain (Nab2‐N)nab2 mutant in which the N‐terminal domain has been deleted (nab2‐ΔN) exhibits severely impaired growth and nuclear accumulation of poly(A) RNA.nab2‐ΔN cells, indicating that the N‐terminal domain might contribute to the control of poly(A) tail length.ScNab2 N‐terminal domain interacts physically with both Mlp1, a component of the nuclear basket that is located on the nuclear face of nuclear pores,ScNab2 Phe73, which is located on a hydrophobic surface patch on the Nab2 N‐terminal domain [Fig. ScNab2 F73A and F73D variants show impaired binding to Mlp1 in yeast lysates and in vitro.The crystal and solution structures of the ScNab2 N‐terminal domain also interacts with Gfd1,dbp5(rat8‐2) RNA helicase mutantgle1‐8 RNA export factor mutant.ScNab2 N‐terminal domain and identified a key contribution made by ScNab2 Tyr34, which is located on the opposite side of the N‐terminal domain to ScNab2 Phe73, which is important for Mlp1 bindingnab2‐Y34A dbp5(rat8‐2) double mutant shows a synthetic slow growth phenotype.in vivo.The ScNab2‐N interacts with the nuclear pore associated proteins, Mlp1 and Gfd1, suggests that Nab2‐N PWI domain could also function as a protein–protein interaction module in higher eukaryotes, although no partners have currently been identified.Although the N‐terminal domain is conserved in Nab2 orthologues [Fig. ScNab2 ZnF567CtNab2ScNab2 ZnF1 and ZnF2 interact with one another as do ScNab2 ZnF3 and ZnF4 to form defined structural units.The structures of several Nab2 Zn finger clusters have been established using both X‐ray crystallography and NMR.ScNab2 ZnF567, NMR chemical shift perturbations associated with binding either AMP or A3 identified a series of basic and aromatic residues associated with RNA bindingScNab2 ZnF567 residues are strongly conserved between Nab2 orthologues [Fig. ScNab2 variants in which these ZnF residues were substituted showed reduced affinity for A9 RNA.nab2 ZnF567 mutants containing substitutions of these basic and aromatic residues had growth rates similar to wild‐type cells, the nab2 ZnF567 mutants generated longer poly(A) tails in vivo and also showed genetic interactions with both Dbp5 and Yra1, consistent with their also influencing the generation of mature mRNPs.ScNab2, structural coherence between ZnF567 was lost in the ScNab2 RNA‐binding mutant, nab2‐C437S, in which a Ser was substituted for the first Zn‐coordinated Cys in ZnF6 [Fig. nab2‐C437S yeast mutant exhibited cold‐sensitive growth and hyperadenylation of bulk poly(A) tails.nab2‐C437S mutant with the dbp5(rat8‐2) RNA helicase mutant suppressed the growth defect of the dbp5(rat8‐2) mutant.nab2 ZnF mutants in combination with the dbp5(rat8‐2) mutant indicated that dbp5(rat8‐2) suppression by nab2 ZnF mutants was more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to the affinity of the mutant Nab2 for poly(A) RNA.ScNab2 has an unanticipated function associated with generating export‐competent mRNPs, and that changes within ZnF567 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.In ScNab2 ZnF567CtNab2 ZnF34511G and A8 RNA, respectively, indicated the basis for selective binding of Nab2 Zn fingers to adenine and identified the importance of H‐bonds formed by adenine N6  RNA in vitro and generated longer bulk poly(A) tails in vivo.The crystal structures of ScNab2 Zn fingers 567 and CtNab2 Zn fingers 345 complexed with poly(A) RNA, the spatial arrangement of the fingers precludes them from all binding to the same RNA chain, so that the first and third Zn fingers of the module were bound to one RNA chain, whereas the middle Zn finger was bound to a second RNA chain. Moreover, the crystal structure of ScNab2 ZnF567 complexed with A11GIn vitro binding studiesScNab2 ZnF567 and either A12 or A11G, indicating that it was not a crystallization artifact and also that the 3′ terminal G was not necessary for its formation. Unusually, the dimerization of the ScNab2 protein chains was mediated almost entirely by each RNA chain binding to both protein chains and not by specific interactions between residues on the protein chains themselves, precluding the engineering of ScNab2 variants in which the Nab2‐Nab2 interaction was impaired. However, ScNab2 dimerization was impaired in Nab2 variants in which RNA binding was disturbed, with substitution of Phe450 in ZnF6, that is important for the interaction with the two adenines that bind ZnF6  tail length in vivo.ScNab2 F450A variant exhibited some of the strongest genetic interactions with Yra1 and Dbp5.In the structures of both ScNab2 ZnF567‐A11G heterotetramer, not all the adenines interact directly with the Nab2 protein chains, suggesting that Nab2 could also form analogous dimers with RNA sequences that only contain adenines in key positions [Fig. ScNab2 binds along the coding region of transcripts as well as to the poly(A) tail,ScNab2 ZnF567 in mediating the compaction of GAL1 transcripts in vitro [Fig. ScNab2 ZnF567, which were able to form dimers, resulted in much more compact complexes than those formed by the ScNab2 F450A variant, in which dimerization was impaired.In the S. cerevisiae, the 3′‐end processing machinery comprises cleavage factors IA and 1B and the cleavage and polyadenylation factor (CPF), a complex including the riboendonuclease, Ysh1/Brr5, the poly(A) polymerase, Pap1, and the Pap1 regulation factor, Fip1.S. cerevisiae, the CPF component, Fip1, binds Pap1 directly and tethers it to CPF to stimulate/regulate Pap1 activity.S. cerevisiae poly(A) tails have a length of ~60–80 nucleotides  RNA as it loops between Nab2 chains. Consequently, it is not clear whether generating a single Nab2 dimer is sufficient to terminate polyadenylation or whether instead it is necessary to form two dimers. Further work will be required to address this question.Polyadenylation is the final processing step in the nuclear phase of the gene expression pathway. In ScNab2 and SpNab2 can protect poly(A) RNA from degradation by the RNA exosome in vitro.In addition to terminating polyadenylation by dissociating Pap1 from the CPF, Nab2 may also contribute to regulating poly(A) tail length through interactions with the 3′‐5′ riboexonuclease exosome complex,ScNab2 Zn finger variants has indicated that the ScNab2 ZnF567 interaction with polyadenosine RNA plays a central role in the regulation of poly(A) tail length and mRNA compaction in S. cerevisiae. In this context, the observation that the loss of DmNab2 in Drosophila and ZC3H14 in mice and humans results in longer poly(A) tailsDmNab2 ZnF123 and ZC3H14 ZnF123, which are most similar to ScNab2 ZnF567 [Fig. ScNab2 to contribute to the control of poly(A) tail length. In the future, it will be informative to assess the functional consequences of specific DmNab2/ZC3H14 Zn finger variants, such as DmNab2 C879S and HsZC3H14 C622S in ZnF2 that are equivalent to ScNab2 C437S in ZnF6 [Fig. Overall, extensive analyses of S. cerevisiae, Nab2 also shows genetic interactions with components of the mRNA nuclear export machinery, such as Yra1, Sub2, and Mex67.ScNab2 may also interact directly with Mex67, one possible mechanism for signaling the completion of polyadenylation could be mediated through Pcf11 and the THO complex as a result of the dissociation of poly(A) polymerase Pap1 from the CPF complex.In addition to its function in controlling poly(A) tail length in S. cerevisiae, direct evidence for such a dimer containing full‐length Nab2 or the way in which the protein chains are arranged in the Nab2 dimer has not yet been obtained either in vitro or in vivo. It is also unclear whether one or two Nab2 dimers are needed to terminate polyadenylation. Although CPF components, such as Pcf11, appear to participate in signaling the termination of polyadenylation to the mRNA export machinery (TREX/Yra1/Sub2/Mex67:Mtr2) that generates an export‐competent mRNP, precise details of the signaling pathway and how polyadenylation termination is transmitted to Pcf11 remain to be established. Similarly, although there is clearly crosstalk between Nab2/ZC3H14 and the splicing machinery, most notably involving genetic interactions between ScNab2 and the splicosome component, Mud2,Although Nab2 dimerization following binding to the growing poly(A) tail could provide a mechanism by which Nab2 terminates polyadenylation in S. cerevisiae. Nab2 orthologues also contribute to these functions in higher Eukaryotes, however, the greater complexity of these systems has made establishing precise molecular mechanisms and signaling pathways more difficult. The spatial arrangement of Nab2 Zn fingers facilitates dimerization when bound to adenine‐rich RNAs that is important for the termination of 3′ polyadenylation and transcript compaction, albeit the precise structure of the Nab2 dimers generated in vivo remains to be established. Overall, the wealth of information that has been generated about the structure of Nab2 and its interactions with other components of the nuclear gene expression machinery has laid the foundation for beginning to define the precise ways in which these pathways are coordinated and also provides insight into the contribution made by Nab2 orthologues to these processes in higher Eukaryotes.In summary, a combination of functional and structural studies has provided a wealth of insight into the way in which Nab2 regulates mRNA poly(A) tail length, contributes to mRNA compaction and the integration of nuclear steps in the gene expression pathway with nuclear export in


Leiosporoceros dussii, the sister taxon to all hornworts. The Leiosporoceros organellar genomes show conserved gene structure and order with respect to the other hornworts and other bryophytes. Additionally, using RNA-seq data we quantified the frequency of RNA-editing events  in both organellar genomes. In total, 109 sites were found in the plastome and 108 in the mitogenome, respectively. The proportion of edited sites corresponds to 0.06% of the plastome and 0.05% of the mitogenome , in contrast to 0.58% of edited sites in the plastome of Anthoceros angustus . All edited sites in the plastome and 88 of 108 sites in the mitogenome are C-to-U conversions. Twenty reverse edited sites (U-to-C conversions) were found in the mitogenome (17.8%) and none in the plastome. The low frequency of RNA editing in Leiosporoceros, which is nearly 88% less than in the plastome of Anthoceros and the mitogenome of Nothoceros, indicates that the frequency of RNA editing has fluctuated during hornwort diversification. Hornworts are a pivotal land plant group to unravel the genomic implications of RNA editing and its maintenance despite the evident evolutionary disadvantages.Because hornworts occupy a pivotal position in early land colonization as sister to other bryophytes, sister to tracheophytes, or sister to all other land plants, a renewed interest has arisen in their phylogenetic diversity, morphology, and genomes. To date, only five organellar genome sequences are available for hornworts. We sequenced the plastome  and mitogenome  of the hornwort  Bryophytes—liverworts, mosses and hornworts—have in common a haploid-dominant life cycle. Because of their pivotal evolutionary position, bryophytes have been targets of several phylogenomic analyses to disentangle their interrelationships, resulting in three main competing and equally supported hypotheses. The liverwort-sister hypothesis suggests that bryophytes are paraphyletic with liverworts sister to all other land plants and hornworts sister to tracheophytes . The horAnthoceros  and the monotypic Leiosporoceros (L. dussii), the sister taxon to all hornworts [Leiosporoceros genome project targets L. dussii due to its crucial phylogenetic position, small genome size (approximately 160 Mbp) and its morphological innovations that include a unique symbiotic arrangement of endophytic cyanobacteria, isobilateral tetrad development and spore architecture [From this debate, it has become evident that hornworts have a crucial place in the evolution of early land plants, prompting a renewed interest in their phylogenetic diversity, morphology, and genomes ,6. Threeornworts ,7  and three mitochondrial (mitogenome) genome sequences are available for hornworts. The plastomes of angustus ,9. The mangustus ,12. The The most important characteristic found in organellar genomes of hornworts is the high level of RNA editing relative to other bryophytes ,14,15. RA. angustus plastome is the only hornwort organellar genome for which complete cDNA sequences are known. It shows an elevated rate of RNA editing with up to 942 sites, including 509 C-to-U and 433 U-to-C conversions. This is the highest frequency of reverse editing among land plants [L. dussii [rbcL, rpoC, and atpB genes revealed no edited sites in Leiosporoceros [rbcL alone, a total of 72 edited sites (43 C-to-U and 28 U-to-C conversions) were identified in the other taxa, with 20–34 edited sites per taxon. The mitochondrial nad5 gene of Leiosporoceros displayed only 0.7% of edited sites (8/110), while edited bases accounted for 3.1–4.1% of the sites in other hornworts [N. aenigmaticus mitogenome revealed the presence of 422 edited sites [The d plants ,15,17. I. dussii ,19. Analornworts . A parti sites 8/0, while Physcomitrella patens [Takakia. Among the seed-free tracheophytes ferns and lycophytes, there exist high heterogeneity in the levels of RNA editing [The availability of only a few organellar transcriptomes from early land plants precludes genome-wide analyses of RNA editing, thus hampering the reconstruction of a clear evolutionary trend for this widespread phenomenon. The complete absence of RNA editing in complex thalloid (marchantioids) liverworts is puzzling  and it ma patens  or highe editing . In cont editing .L. dussii within hornworts, we sequenced and assembled the plastome and mitogenome of this species. Using high-coverage transcriptome sequencing, we also studied the extent of RNA editing in both organelles of L. dussii to assess whether the apparent lack of editing in plastids and the low rate of editing in the mitochondrial nad5 gene are organellar-wide phenomena. Our study was thus aimed at elucidating critical information in reconstructing organelle genome evolution in early land plants.Given the critical phylogenetic position of Leiosporoceros dussii (Steph.) Hässel  was collected in Río el Guayabo, El Valle de Antón, Prov. De Coclé, Panamá and brought to the laboratory for immediate DNA extraction.Material of L. dussii using the Power Plant@ ProDNA isolation kit (MoBIo). The preparations were used to produce sequencing libraries with the Nextera DNA Sample Prep Kit . Sequencing of the two libraries was performed at the Smithsonian Tropical Research Institute , using Illumina MiSeq technology. Over 9 million paired-end reads were obtained for the first library and 8 million paired-end reads for the second library. Low-quality reads were trimmed using CLC Genomics Workbench  and de novo assemblies were conducted using A5 in CLC Genomics Workbench . Contigs of the plastome and mitogenome were identified by BlastN similarity searches  against the A. angustus plastome (NC_004543.1) and P. laevis mitogenome (GQ376531.1), respectively. Final assemblies were completed using Sequencer 5.1 .Two separate genomic total DNA preparations were obtained for To annotate the two organelle genomes, we used a custom-built suite of bioinformatics tools allowing the automated execution of the following three steps: (1) ORFs were found using GETORF in EMBOSS , (2) theRNA was recovered from the same colony that was used for DNA sequencing. Separate samples of this colony were placed in RNA Later  and shipped to the Beijing Genomic Institute in Shenzhen (BGI-Shenzen) where a rRNA-depleted library was constructed using the Ribo-Zero Plant Leaf rRNA Removal Kit  using random hexamers. A transcriptome library was constructed using a TruSeq mRNA stranded sample preparation kit . Sequencing of this library was performed using a HiSeq instrument, which yielded a total of 45,351,761 reads (over 9 billion bp).Leiosporoceros were compared to those previously reported for the organelles of other hornworts [Nothoceros aenigmaticus was used for comparison. We avoided comparisons with the mitogenome of P. laevis because the analysis was done using an RNA-editing predicting software [Illumina RNA-seq data were mapped to both organellar genomes using CONSED . Within ornworts ,15, 34. Leiosporoceros plastome is about the same size as Anthoceros  and Nothoceros  plastomes  of Anthoceros and a number of green algae is absent from the IRs of both Leiosporoceros and Nothoceros, as well as all other hornworts and other land plants. The absence of this rrl intron in all hornworts examined thus far except Anthoceros, points to a potential horizontal transfer between a green alga and species of Anthoceros [trnS (CGA) and matK in L. dussii, which are absent or occur as pseudogenes in A. angustus and N. aenigmaticus  and N. aenigmaticus  [A. angustus . It also has the highest number of functional genes, including three protein-coding genes  and three tRNA genes (trnS (UCU), trnS (UGA), trnS (GCU) that are either absent or pseudogenized in P. laevis and N. aenigmaticus  , 11, butgmaticus . In tota. laevis .Leiosporoceros mitogenome also contrasts with its P. laevis and N. aenigmaticus relatives at the intron content level. All of the 35 introns are cis-spliced and with the exception of the first intron in the cob gene, belong to the group II class. Only the first (group I) and second introns located in cob, feature an open reading frame. Twenty-six of the Leiosporoceros group II introns are present at identical positions in both the P. laevis and N. aenigmaticus mitogenomes, and three additional ones are shared with P. laevis alone. Of these 29 hornwort introns, a small fraction is shared with other bryophytes and/or vascular plants. These results indicate that most of the introns found in hornworts were acquired early during their evolutionary history and were transmitted by vertical descent.The Leiosporoceros plastome and mitogenome (clpP (A→G) and psaA (T→G) genes and in three intergenic spacers -ftsH and atpH-atpI) of the plastome (trnl(uaa)-nad5 and mttB-rps4), and accounted for 24 to 86% of all reads (RNA editing events were investigated in every transcript derived from the togenome . The RNAtogenome  Tables. plastome . BlastN ll reads . The latThe majority of RNA edited sites in the plastome (102/109) and mitogenome (94/108) affect coding regions of protein-coding genes, most frequently the second codon position . In the A. angustus plastome, where serine codons are the most altered and are mainly converted to leucine and phenylalanine codons [Of the 102 codons edited in the plastome, 57% are serine codons: 43 are changed into leucine and 16 into phenylalanine codons . The sece codons .Leiosporoceros and Anthoceros plastomes show striking differences in RNA editing frequency with 109 edited sites in Leiosporoceros versus 939 sites in Anthoceros [Leiosporoceros contrasts sharply with the abundance of U-to-C conversions (432) observed in Anthoceros. Re-establishment of proper start codons and removal of stop codons are more important in A. angustus (164 codons removed) compared to L. dussii (only one start codon and one stop codon created and no stop codons removed). This may explain the lower number of edited sites in L. dussii, in particular the absence of U-to-C editing. Only 37 edited sites are shared between the L. dussii and Anthoceros plastomes  and mitogenome (108), an unusual case among land plants. The number of edited sites in the mitogenome is higher in Nothoceros than in Leiosporoceros (over 422 vs 108), with much fewer U-to-C conversions in Leiosporoceros than in Nothoceros (19 sites vs 61)   and a lorbcL across the hornwort phylogeny showed up to 72 edited sites, 20 of which were present in A. angustus and 34 in Phaeomegaceros coriaceus [nad5 gene was heavily edited with up to 45 edited sites in Nothoceros fuegiensis [Analysis of the plastid gene oriaceus . Nearly oriaceus . Similaregiensis , out of egiensis . If thisegiensis .A. agrestis identified the PPR domains as the most abundant repeat types in the nuclear genome [Anthoceros is hand-to-hand with the proliferation of the PPR domains [A. angustus is the only reliable data from a hornwort organelle to compare with the transcriptomic data we generated for Leiosporoceros. Leiosporoceros represents an ideal candidate to study the expansion of the editosome across hornworts and land plants. The reduced number of edited sites could facilitate the search for PPR domains and related proteins involved in the organellar editosomes.Elucidating the mechanisms of RNA editing across land plants, in particular the origin and maintenance of reverse editing, remains an active field of research. Regarding the canonical RNA editing, a vast amount of research has detailed the role of the editosome and the pentatricoptide repeat proteins (PPR) . A recenr genome . The lar domains . But at Leiosporoceros with nearly 88% less in the plastome than Anthoceros suggests idiosyncratic and highly variable levels of RNA editing during hornwort diversification, advocating for the need for additional plant organellar RNA-editing studies to further clarify any phylogenetic trend. The position of hornworts in the land plant phylogeny remains ambiguous but pivotal, being either the earliest diverging land plants, sister to tracheophytes or sister to all other bryophytes [L. dussii plastome and mitogenome would indicate that reverse editing evolved independently in hornworts and seedless tracheophytes . The alternative hypothesis would suggest that reverse editing arose in the most recent common ancestor of hornworts and tracheophytes, with subsequent secondary losses in some lycophytes, ferns and seed plants. In either scenario, hornworts remain a central group to unravel the genomic implications of RNA editing in land plants and its intriguing maintenance despite the evident evolutionary disadvantages.We sequenced both organellar genomes of a crucial lineage in the hornwort phylogeny ,7 and pryophytes . ElucidaS1 FigL. dussii plastome.A. Transcriptome read coverage of the  The x-axis shows the gene map of this genome, starting with the LSC region, while the y-axis indicates read depth. B. L. dussii mitogenome.Transcriptome read coverage of the  The x-axis shows the gene map of this genome, while the y-axis indicates read depth. A color-coded scale classifies the genes into functional categories.(EPS)Click here for additional data file.S2 FigThe numbers on the right of the histograms indicate the edited sites that are shared between the two hornworts.(TIFF)Click here for additional data file.S3 FigCoverage goes from 165 times up to 65770 times .(PDF)Click here for additional data file.S4 FigCoverage goes from 102 times up to 44015 times .(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Drosophila suzukii is an invasive fruit pest and represents a potential economic threat to viticulture. After first observations of D. suzukii in Europe in 2008, research mainly focused on the evaluation of the host range and infestation risk for fruit and berry crops. However, the risk assessment of D. suzukii in viticulture has only recently started. Understanding the factors influencing preferences of D. suzukii for host species and varieties as well as offspring performance is essential to improve management strategies. We investigated the field infestation of different grape varieties across Baden-Wuerttemberg, southwestern Germany, between 2015 and 2018. Moreover, we performed dual-choice assays in the laboratory to investigate whether adults show preferences for certain varieties and whether offspring performance differs between varieties. Furthermore, we studied the impact of grape damage on choice behavior. Field monitoring revealed that D. suzukii show preferences for red varieties, whereas almost no oviposition occurred in white varieties. The results of dual-choice assays confirmed that D. suzukii preference and performance are influenced by grape variety and that flies preferred damaged over intact “Pinot Noir”, “Pinot Blanc”, and “Müller-Thurgau” berries. Overall, these findings may have important implications for winegrowers regarding cultivated varieties, grape health, and insecticide reduction. Drosophila suzukii Matsumura  was accidentally introduced into Europe and North America and nowadays it is a well-established economic pest across both continents  [Individuals of D. suzukii preference behavior as well as their performance in laboratory dual-choice assays. These varieties were chosen because they are commonly grown in the viticultural districts of Baden-Wuerttemberg and show different ripening time points  as well as different susceptibility to D. suzukii based on results of the field sampling .Three red grape varieties, “Acolon”, “Regent”, and “Pinot Noir”, and two white varieties, “Müller-Thurgau” and “Pinot Blanc”, were selected to assess Thus, we compared the fly behavior towards meaningful pairs of red vs. white and red vs. red varieties. Two early season, red and more susceptible varieties  were tested against one early season, white and not susceptible variety (“Müller-Thurgau”). One late season, red and not susceptible variety (“Pinot Noir”) was tested against one late-season, white and not susceptible variety (“Pinot Blanc”). Furthermore, two red (“Regent”: early season and susceptible vs. “Pinot Noir”: late season and not susceptible) varieties were tested against each other  at an ovD. suzukii were tested using “Pinot Noir”, “Müller-Thurgau”, and “Pinot Blanc.” Half of the intact grape berries were artificially damaged at the surface with a scalpel. The wounds were approximately 2–3 mm wide.Grape berry clusters of each variety used for insect bioassays were randomly collected from vineyards that received the same cultivation practice and were not treated with insecticides. Grape berries were picked when ripe and stored for a maximum period of 24 h before starting dual-choice assays. Prior to the dual-choice assays, berries were examined under the dissecting microscope  to exclude pre-infestation and to discard visibly damaged and overripe berries. To investigate whether varieties that were less accepted in choice tests may turn more susceptible when being damaged, the effects of berry condition on preferences and performance of D. suzukii under controlled conditions, behavioral preferences (location and oviposition) of D. suzukii, as well as offspring performance (emergence rate), were evaluated in dual-choice assays. Two sets of dual-choice assays were performed: (1) dual-choice tests between intact berries of different varieties and (2) choice tests between intact and damaged berries of the same varieties. In total, seven combinations were tested, and assays were replicated 10–40 times in 2016 and 2017  of ten randomly selected berries of the same grape cluster used in the dual-choice assays was determined for each variety using a handheld, temperature-compensated refractometer . Both infestation and sugar content were determined within 24 h after collection of the berries in the field.Before starting the dual-choice assays a small sample of berries was offered in rearing cages to test for the motivation of Flies were removed from rearing colonies approximately 1–2 h before the start of each assay. To select females only, we used a minimal invasive method for anesthetizing the flies by placing them on ice. After females were released into the center of the arenas , their position was recorded at three time points, after 5 h, 7 h, and 24 h (location preference). After 24 h, flies were removed and eggs deposited on the berries were counted (oviposition preference) under the dissecting microscope, summing up numbers of eggs that were placed on the berry surface as well as eggs that were already dead due to wound healing of the berry. Most females were still alive after the experimental period . In addition, in 2017 all test berries were stored in plastic cups (500 mL), incubated at 24 °C, 60–70% r.h., photoperiod L16:D8, and scored every 5–7 days for adult emergence (emergence rate) as a measure for offspring survival for a period of about 30 days.D. suzukii, separate linear mixed-effects models (LMMs) with a Gaussian error distribution were performed for each test combination .D. suzukii females per choice option as the response, the factor variety or berry condition as fixed effects, and the test arena, as well as date nested within year as random effects.For statistical analyses of the location preference data, the number of flies sitting on or within a radius of <1 cm around the berries of the same variety or berry condition (n = 6) after 24 h was summed up for each test arena. We calculated seven LMMs (one per test combination), which comprised the number of For the oviposition preference data, only the number of successfully deposited eggs  in berries of the same choice option (variety or berry condition) was calculated per arena. We computed seven LMMs (one per test combination), which comprised the number of eggs per choice option as the response, the factor variety or berry condition as fixed effects, and the test arena, as well as date nested within year as random effects.p-values for the effects of all predictor variables [To choose the appropriate error distribution (“family”) for LMMs, it was assured that residuals exhibited variance homogeneity and normal distribution of residuals by means of visual inspection . We applariables .For statistical analyses of the adult emergence data of 2017, we calculated the proportion of emerged adults from deposited eggs per choice option and arena. A non-parametric Mann–Whitney U test was performed to determine statistical differences between choice options.D. suzukii flies showed varietal preferences. All other red varieties were infested except “Cabernet Cantor”, “Cabernet Jura”, “Cabernet Mitos”, and “Saint Laurent”. In every year of observation, samples of the varieties “Trollinger”, “Dornfelder”, “Lemberger”, “Portugieser”, “Rotel Gutedel”, “Schwarzriesling”, “Regent”, and “Pinot Noir” displayed infestation with D. suzukii eggs  similarly displayed the highest level of oviposition, followed by “Pinot Noir” which displayed an infestation severity of less than 2% . SamplesD. suzukii test females had made a choice for one of the two offered grape varieties. The number of flies sitting on berries (= location preference) was significantly affected by the grape variety in all four dual-choice assays after 24 h   . There wp < 0.1) .There were no differences in adult emergence rate from eggs deposited in berries of “Regent” vs. “Müller-Thurgau” tested in 2017, whereas from infested “Regent” berries about ten times more adults emerged than from infested “Pinot Noir” berries .D. suzukii test females had made a choice for one of the two offered berry conditions. When given the choice between intact and damaged berries of “Pinot Noir” or “Müller-Thurgau”, flies significantly preferred to reside on damaged over intact berries (p < 0.1)  .D. suzukii showed significant oviposition preferences for damaged berries in all three dual-choice assays  in choice tests. These white varieties may have other traits that turn them highly susceptible or alternatively, fly populations in these regions may respond differently. Overall, the field infestation severity of grapes in the present study appeared to vary strongly within and across years, which might be explained by seasonally changing environmental conditions, such as temperature, humidity, composition of the surrounding landscape and microclimatic conditions of sampling sites, known to potentially influence D. suzukii abundance [Based on our four-year field monitoring data, we compiled a ranking of grape infestation severity indicating that the three main economically important grape varieties “Pinot Noir”, “Müller-Thurgau” and “Pinot Gris” of the Baden wine region are only at very low risk of infestation by  suzukii . Only soposition . Previouposition ,14,27. Hposition , and Swiposition , in whicposition  reportedposition  found thbundance ,45,46,47D. suzukii females show similar behavioral preferences for certain varieties in the laboratory as those found in the field. We observed significant location preferences of D. suzukii for red over white varieties. Our approach also revealed a slightly higher preference of D. suzukii to rest on “Regent” compared to “Pinot Noir” berries, although both varieties are red-colored  [D. suzukii do not match on two Prunus species. Olazcuaga et al. (2019) [To our knowledge, the combination of location, oviposition preferences, and offspring performance has rarely been studied in . (2019)  who obse. (2019)  found no. (2019) ,50. This. (2019) ,51.D. suzukii infestation, by turning initially less accepted fruits more susceptible. Location and oviposition preference behavior were affected by berry condition particularly in the red grape variety with more flies resting and higher egg numbers oviposited on damaged compared to intact berries  insect species. Our results show that D. suzukii oviposition on intact berries of certain grape varieties is possible but overall very low infestation and adult emergence rates suggest that intact grape berries are not very suitable hosts for D. suzukii. Moreover, the physical presence of D. suzukii on grape varieties such as Pinot Noir does not necessarily result in grape damage, which is of particular interest for winegrowers who observe D. suzukii in their vineyards. Preventive strategies for reducing pest pressure and pre-damage of grapes, such as the selection of less susceptible varieties, sanitation, and agronomic practices improving plant health, are alternative methods to currently recommended insecticides applications.Understanding the factors driving host use of


Transcriptomic studies have demonstrated that the vast majority of the genomes of mammals and other complex organisms is expressed in highly dynamic and cell-specific patterns to produce large numbers of intergenic, antisense and intronic long non-protein-coding RNAs (lncRNAs). Despite well characterized examples, their scaling with developmental complexity, and many demonstrations of their association with cellular processes, development and diseases, lncRNAs are still to be widely accepted as major players in gene regulation. This may reflect an underappreciation of the extent and precision of the epigenetic control of differentiation and development, where lncRNAs appear to have a central role, likely as organizational and guide molecules: most lncRNAs are nuclear-localized and chromatin-associated, with some involved in the formation of specialized subcellular domains. I suggest that a reassessment of the conceptual framework of genetic information and gene expression in the 4-dimensional ontogeny of spatially organized multicellular organisms is required. Together with this and further studies on their biology, the key challenges now are to determine the structure–function relationships of lncRNAs, which may be aided by emerging evidence of their modular structure, the role of RNA editing and modification in enabling epigenetic plasticity, and the role of RNA signaling in transgenerational inheritance of experience. A role for RNA as regulatory molecules, rather than just as templates (mRNAs) and components of the machinery  for the production of proteins, was established in the 1990s with the surprising discovery of the RNA interference (RNAi) and related pathways, which utilize small RNA guides to regulate mRNA stability and translation, and to control transposons ,4,5,6,7.At the same time, seminal long non-protein-coding RNAs (lncRNAs), involved notably in X inactivation (Xist) ,9,10,11 The subsequent revelation from high-throughput cDNA sequencing (RNAseq) in the 2000s that tens of thousands of long intronic, intergenic and antisense lncRNAs are transcribed from the genomes of mammals ,25,26,27However, despite the RNAi precedent, and with few exceptions ,38,39, tThe possibility that lncRNAs might be functional also contradicted the widely held belief, dating from the late 1970s, that the intronic and ‘intergenic’ sequences from which they are transcribed, and which dominate the real estate of the mammalian genome, are largely evolutionary debris (‘junk’) , comprisThe idea that the most non-coding RNAs are noise from biologically inert regions of the genome was superficially bolstered by the observation that most are expressed at low levels and are generally less conserved than protein-coding sequences  similarThe (lack of a high) conservation argument also fails to take into account the fact that adaptive radiation occurs mainly by the relatively rapid evolution of the regulatory sequences under positive selection, that such sequences have quite different structure–function constraints to proteins, and that they are subject to rapid turnover ,50,51. TAt this point it is important to remember that the metazoan proteome is remarkably static. Both the nematode and human genomes contain ~20,000 protein-coding genes, most of which are functionally orthologous, despite orders of magnitude difference in their developmental (and cognitive) complexity. By contrast, the proportion of the genome that is non-protein-coding, and the number and range of non-coding RNAs expressed therefrom, increases with developmental complexity ,52, raisIndeed, there are many different rate classes of sequence evolution in mammals, indicating that at least 45% of the alignable regions of mammalian genomes are not evolving neutrally , with atThere are also many indices of lncRNA functionality ,56, inclThe case for lncRNA functionality is also supported by their dynamic expression patterns in differentiating cells and their highly specific spatial (including subcellular) localization ,60,61,62http://www.lncrnadb.org [https://lncipedia.org [http://www.noncode.org [There have been many studies examining lncRNA biology over the past decade  linkingcode.org ,73,74, ccode.org , regulatcode.org ,77, enhacode.org ,79,80, acode.org ,83,84,85code.org  and Dnmtcode.org ,87. Somecode.org ,89,90,91code.org ,93, and code.org ,94,95, scode.org  and canccode.org ,97,98). Knockdown of lncRNAs by small interfering RNA (siRNA)-related methodologies frequently results in observable changes in cellular behavior or characteristics in culture . On the In this context, it is worth noting that deletion of a subset of the most highly conserved sequences in the mammalian genome, ultraconserved elements (UCEs) ,104, whiWhile skeptics remain, the most likely interpretation is that the documented functional examples are emblematic of an army of regulatory RNAs that guide epigenetic trajectories and specify cell state during a very complex and precise developmental ontogeny—from a single fertilized cell to a mobile, cognizant adult—and that most of the human genome is devoted to this purpose ,111,112.Indeed, the growing body of evidence is now leading to a general acceptance of the relevance of (many or most) lncRNAs to cell and developmental biology , and incIf so, the current protein-centric framework for understanding the genetic programming of differentiation and development is incomplete, a legacy of the mechanical worldview that held sway at the birth of molecular biology. Reconsideration of this framework to incorporate not only proteins but also structural and regulatory RNAs ,118,119 The most pressing challenges now are to determine the structure–function relationships in lncRNAs and to parse their functional repertoire. This should resolve lingering questions and place lncRNAs into an integrated conceptual framework, together with small regulatory RNAs, transcription factors and signaling pathways, among others, for understanding the decisional hierarchies that control the 4-dimensional ontogeny of complex multicellular organisms .There is logic and experimental evidence to suggest that lncRNAs have a modular architecture, given their likely role as scaffolds and epigenetic guide molecules ,82,120. If this is correct, the establishment of the exon as the primary unit of lncRNA structure–function, combined with the observation of conservation of lncRNA structure  and the This framework should also allow parsing of the different types and roles of lncRNAs in establishing chromatin territories, enhancer looping, guidance of epigenetic modifier proteins that impose DNA and histone modifications, and the formation of subcellular domains, among others. In addition, while most lncRNAs are nuclear and associated with chromatin, some are cytoplasmically localized ,63 with A new and rapidly emerging frontier is the role of RNA editing ,127 and There is still much to do to understand the role of small RNAs, especially the ti/spliRNAs that are derived from transcription start sites and exonic borders ,132, andFinally, and most intriguingly, is the role of RNA in intercellular and transgenerational inheritance (soft-wired inheritance), for which there is not only evolutionary logic ,141,142


Chrysanthemum morifolium and their original cultivars. The mutant chrysanthemum cultivars were generated by treatment with various doses of 60Co gamma irradiation of stem cuttings of three commercial chrysanthemum cultivars as follows: ‘ARTI-Dark Chocolate’ (50Gy), ‘ARTI-Purple Lady’ (30 Gy), and ‘ARTI-Yellow Star’ (50 Gy) derived from ‘Noble Wine’; ‘ARTI-Red Star’ (50 Gy) and ‘ARTI-Rising Sun’ (30 Gy) from ‘Pinky’; ‘ARTI-Purple’ (40 Gy) and ‘ARTI-Queen’ (30 Gy) from ‘Argus’; and ‘ARTI-Rollypop’ (70 Gy) from ‘Plaisir d’amour’. Quantitative analysis of flavonoids, phenolic acids, anthocyanins, and carotenoids in the flowers of the 12 chrysanthemum cultivars was performed using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS). Essential oils from the flowers of these cultivars were analyzed by gas chromatography–mass spectrometry (GC-MS). The mutant cultivars, ‘ARTI-Dark Chocolate’, ‘ARTI-Purple Lady’, ‘ARTI-Purple’, and ‘ARTI-Queen’ showed higher total amounts of flavonoid and phenolic acid compared with those of the respective original cultivars. The mutant cultivars, ‘ARTI-Dark Chocolate’, ‘ARTI-Purple Lady’ and ‘ARTI-Purple’, which produce purple to pink petals, contained more than two-times higher amounts of anthocyanins compared with those of their original cultivars. Of the mutant cultivars, ‘ARTI-Yellow Star’ in which petal color was changed to yellow, showed the greatest accumulation of carotenoids. Ninety-nine volatile compounds were detected, of which hydrocarbons and terpenoids were abundant in all cultivars analyzed. This is the first report that demonstrated the phytochemical analysis of novel chrysanthemum cultivars derived from C. morifolium hydrid using HPLC-DAD-ESIMS and GC-MS. These findings suggest that the selected mutant chrysanthemum cultivars show potential as a functional source of phytochemicals associated with the abundance of health-beneficial components, as well as good source for horticulture and pigment industries.The flowers of chrysanthemum species are used as a herbal tea and in traditional medicine. In addition, members of the genus have been selected to develop horticultural cultivars of diverse floral colors and capitulum forms. In this research, we investigated the phytochemical composition of eight gamma-irradiation mutant cultivars of  Chrysanthemum species belong to the Asteraceae family and comprise about 40 species distributed in Asia and Eastern Europe . ChrysanC. morifolium, the original cultivar, ‘Novel Wine’, and the mutant cultivar, ‘ARTI-Dark Chocolate’ which is one of novel chrysanthemum cultivars developed by our research team +) . These cM + H]+) . In groutR 2−4 min in their chromatograms showed maximum absorbance at 230 and 270 nm from DAD, indicating that they were not carotenoids. All peaks after tR 5 min in their chromatograms confirmed to be carotenoid compounds, all of which had a maximum absorbance at 420, 450, or 470 nm from DAD. Therefore, the sum of area values of these peaks was substituted into the calibration of lutein to calculate the total carotenoid contents. As a result, ‘ARTI-Yellow Star’ with yellow petals showed much higher content of carotenoids  than the control plant with white petals and purple stripes, ‘Noble Wine’ . Among its other gamma irradiated mutant cultivars, ‘ARTI-Dark Chocolate’ with dark purple petal  had a slightly low level of carotenoids and ‘ARTI-Purple Lady’  with bright purple petals exhibited a slightly high content of carotenoids, compared with that of ‘Noble Wine’. In group II, the gamma-irradiated mutant cultivar with bright red petals, ‘ARTI-Red Star’  contained more carotenoids contents than the control plant, ‘Pinky’ , which in contrast to another mutant cultivar with white/light pink petals, ‘ARTI-Rising Sun’, showed a low level of carotenoids . In group III, the control plant, ‘Argus’ and its mutant cultivars, ‘ARTI-Queen’ and ‘ARTI-Purple’ showed almost the same carotenoids contents, 0.055 ± 0.002 mg/g, dry weight, 0.054 ± 0.003 mg/g, dry weight and 0.056 ± 0.003 mg/g, dry weight, respectively. The mutant cultivar of ‘Plaisir d’amour’, ‘ARTI-Rollypop’ has yellow/red petals, and its carotenoids content  was slightly higher than that of ‘Plaisir d’amour’ . As a comprehensive result, the mutant cultivar that changed into yellow petals, ‘ARTI-Yellow Star’ had the most accumulation of carotenoids, and ‘ARTI-Rollypop’ in which part of petal changed to yellow/red and ‘ARTI-Red Star’ with red petals showed increased carotenoids compared to each control plant. In addition, in the chromatograms of the yellow flowers, ‘ARTI-Yellow Star’ and ‘ARTI-Rollypop’, a specific peak at tR 6−8 min was shown, however this peak, corresponding to certain carotenoid, was not identified. ‘ARTI-Yellow Star’ with yellow petals accumulated carotenoids twice as much as other chrysanthemum cultivars. In the previous study on functional analysis of genes related with the accumulation of carotenoid in the biosynthesis pathway [CmCCD4a) may be responsible for the development of yellow petals of chrysanthemum.The carotenoids extracted from the flowers of the chrysanthemum cultivars were analyzed using HPLC-DAD-ESIMS. However, due to unsatisfactory identification of each peaks for carotenoids detected at a wavelength of 450 nm by ESIMS, the total content of carotenoids in the chrysanthemum cultivars was calculated according to the total peak area of HPLC chromatograms at 450 nm. The large peaks at  pathway ,27,28, tThe volatile compounds identified in the flowers of the 12 chrysanthemum cultivars, together with their retention time, molecular formula, and contents (%) are shown in DL-α-tocopherol, and squalene—known as bioactive ingredients [dl-α-Tocopherol was also found in all chrysanthemum cultivars except for ‘Argus’, with content range of 2.57% to 0.48%  was used for gamma-irradiation. The standard compounds apigenin, luteolin, acacetin-7-O-β-glucoside, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, and linarin (acacetin-7-O-rutinoside) were isolated from the flowers of ‘ARTI-Dark Chocolate’ as described previously [O- , and lutein  were purchased. All other chemicals and solvents used in the study were of analytical grade.Analytical HPLC-DAD-ESIMS was carried out using an Agilent 1200 series system and an Agilent 6120 quadrupole MS system  equipped with a YMC-Triart C18 column  for analysis of flavonoids and phenolic compounds, a YMC-Hydrosphere C18 column  for analysis of anthocyanins, and a YMC-Carotenoid column  for analysis of carotenoids. Data acquisition and processing were performed using the ChemStation software . Volatile compounds in the flowers of chrysanthemum cultivars were analyzed using a GC-MS  instrument equipped with a DB-5MS column . A [Chrysanthemum morifolium were generated by the treatment of stem cuttings of the original cultivars with various doses of gamma (60Co) irradiation as follows: ‘ARTI-Dark Chocolate’ (50 Gy), ‘ARTI-Purple Lady’ (30 Gy), and ‘ARTI-Yellow Star’ (50 Gy) derived from ‘Noble Wine’; ‘ARTI-Red Star’ (50 Gy) and ‘ARTI-Rising Sun’ (30 Gy) derived from ‘Pinky’; ‘ARTI-Purple’ (40 Gy) and ‘ARTI-Queen’ (30 Gy) derived from ‘Argus’; and ‘ARTI-Rollypop’ (70 Gy) derived from ‘Plaisir d’amour’. These mutants were selected from among petal-color variants and exhibited stable inheritance of the petal-color phenotype for four years. The radiation mutant cultivars have been grown by the Radiation Breeding Research Team, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute and have been registered as new plant varieties in the Korea Seed and Variety Service. These flowers were handpicked and randomly collected at the stage of fully open-flowering in the same plantation. The flowers were freeze-dried, ground to powder, and stored at −20 °C in polyethylene plastic bags until further analysis. Voucher specimens were deposited at the Radiation Breeding Research Center, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute.The radiation mutant cultivars of +); scan range = m/z 100−1000; scan rate = 1.06 s/cycle; capillary voltage = 4000 V; drying gas flow = 10 L/min (N2); nebulizer pressure = 30 psi; drying gas temperature = 350 °C. The GC-MS analytical conditions for volatiles in the flowers were set as follows: ionization voltage, 70 eV; mass scan range, 50–450 mass units; injector temperature, 250 °C; detector temperature, 230 °C; inject volume, 1 μL; splitless; carrier gas, helium; and flow rate, 4.4 mL/min.The LC-MS analytical conditions for flavonoids, phenolic compounds, anthocyanins, and carotenoids in the flowers were set as follows: positive ionization mode  and 0.1% formic acid in acetonitrile  was performed as follows: 0–60 min, 15%–35% B; 60–70 min, 35%–60% B; 70–71 min, 60%–95% B; 71–80 min, 95% B; 80–81 min, 95%–15% B; 81–90 min, 15% B. The total flow rate was maintained at 0.8 mL/min and the injection volume was 10 μL. Chromatograms were acquired at 280 nm by the DAD detector. The standard compounds (six flavonoids and two phenolic acids) were weighed accurately and dissolved in methanol at 1.0 mg/mL. For quantification, calibration curves of these standards were prepared by measurement at four concentrations . The linear equations for chlorogenic acid, luteolin-7-O-β-glucoside, 3,5-dicaffeoylquinic acid, apigenin-7-O-β-glucoside, linarin (acacetin-7-O-rutinoside), acacetin-7-O-β-glucoside, luteolin, and apigenin were y = 18.675x − 17.907, y = 14.105x − 16.180, y = 22.766x − 25.32, y = 33.317x − 9.170, y = 21.849x − 21.340, y = 14.277x + 20.120, y = 23.094x + 5.220, and y = 36.953x − 46.980, respectively. The calibration curves were linear with high correlation coefficients (r2 = 0.9991–0.9996).Dried flowers of the chrysanthemum cultivars were individually ground to powder, and a sample of the powder (1 g for each cultivar) was extracted with 95% ethanol (30 mL) for 24 h at room temperature. The extracted solutions were filtered through a polyvinylidene fluoride syringe filter (0.45 μm) for HPLC analysis. Each extraction was repeated in triplicate. The standard compounds (six flavonoids and two phenolic acids) were separated using a YMC-Triart C18 column  equipped with the Agilent 1200 series LC system. Binary gradient elution with 0.1% formic acid in water  and sonicated for 30 min. Each extracted solution was filtered through a polytetrafluoro-ethylene hydrophilic syringe filter (0.45 μm) for HPLC analysis. Each extraction was repeated in triplicate. Cyanidin chloride, cyanidin-3-glucoside chloride, and cyanidin-3- were separated using a YMC-Hydrosphere C18 column  equipped with the Agilent 1200 series LC system. Binary gradient elution with 5% formic acid in water  and 5% formic acid in acetonitrile  was performed as follows: 0–40 min, 5%–50% B; 40–41 min, 50%–100% B; 41–45 min, 100% B; 45–46 min, 100%–5% B; 46–50 mi and 5% B. The oven temperature was set to 40 °C. The total flow rate was maintained at 0.8 mL/min and the injection volume was 10 μL. Chromatograms were acquired at 520 nm by the DAD detector. The standard compounds ) were weighed accurately and dissolved in methanol at 1.0 mg/mL. For quantification, calibration curves of these standards were prepared from measurements at four concentrations . The linear equations for cyanidin chloride, cyanidin-3-glucoside chloride, and cyanidin-3- were y = 44.603x−381.8, y = 44.087x−134.39, and y = 32.221x−120.13, respectively. The calibration curves were linear with high correlation coefficients (r2 = 0.993–0.996).Samples (each 1 g) of the powder from dried flowers of each chrysanthemum cultivar were extracted with 5% formic acid in water (w/v) and the sample tubes were mixed using vortex for 20 s and placed in a water bath at 85 °C for 5 min. The carotenoid extracts were saponified with potassium hydroxide  at 85 °C for 10 min. After saponification, the sample tubes were placed on ice, and cold deionized water (15 mL) was added. The extracted solutions were filtered through a polyvinylidene fluoride syringe filter (0.45 μm) for HPLC analysis. Each extraction was repeated in triplicate. The standard compounds (lutein and β-carotene) were separated using a YMC-Carotenoid column  equipped with the Agilent 1200 series LC system. Binary gradient elution with 8% water containing 10 mM ammonium acetate in methanol  and 100% methyl tert-butyl ether (solvent B) was performed as follows: 0–20 min, 10%–17% B; 20–30 min, 17%–25% B; 30–35 min, 25%–70% B; 35–45 min, 70%–75% B; 45–50 min, 75%–10% B; 50–60 min and 10% B. The total flow rate was maintained at 0.8 mL/min and the injection volume was 10 μL. Chromatograms were acquired at 450 nm by the DAD detector. The standard compound, lutein, was weighed accurately and dissolved in 50% dichloromethane in methanol (v/v) at 1.0 mg/mL. For quantification, calibration curves of these standards were prepared from measurements at four concentrations . The linear equation for lutein was y = 89.289x −221.26. The calibration curves were linear with a high correlation coefficient (r2 = 0.988). The total content of carotenoids in the 12 chrysanthemum cultivars was calculated from the calibration curve for lutein.To extract carotenoids from the flowers of the chrysanthemum cultivars, the powdered samples (each 1 g) were extracted with ethanol (20 mL) containing 0.1% ascorbic acid . The chemical analysis data were subjected to analysis of variance using a multiple comparisons method with the SPSS version 12 statistical software package . Differences were determined to be significant at α = 0.05. When the treatment effect was significant, means were separated using Duncan’s Multiple Range Test. The resulting matrix of data for volatile compounds was used to construct a dendrogram using the average linkage method with the SPSS version 12 statistical software package.In the present study, we obtained abundant information on the phytochemical composition, such as flavonoids, phenolic acids, anthocyanins, carotenoids, and volatile compounds, in the flowers of novel chrysanthemum mutant cultivars. Significant differences in phytochemical composition between the original chrysanthemum cultivars and their γ-irradiated mutant cultivars were observed, depending on the phenotype, such as a change in petal color. The chrysanthemum mutant cultivar which were changed to purple or pink in the color of petals and/or petaloid discs, ‘ARTI-Dark Chocolate’, ‘ARTI-Purple Lady’, ‘ARTI-Purple’, and ‘ARTI-Queen’ had higher total content of flavonoids and phenolic acids than their original cultivars, ‘Noble Wine’ and ‘Argus’. In relation to the phenomenon of petal color variation , ‘ARTI-Dark Chocolate’, ‘ARTI-Purple Lady’, ‘ARTI-Red Star’, ‘ARTI-Purple’, ‘ARTI-Queen’, and ‘ARTI-Rollypop’ showed the increase in anthocyanin content. In addition, the total carotenoid content of ‘ARTI-Yellow Star’ with yellow petals was about two-times higher than those of the other cultivars. In the GC analysis of the 12 chrysanthemum cultivars, 57 volatile compounds were identified from five functional groups: hydrocarbons, terpenoids, alcohols, esters, and others. Cluster analysis revealed that the original cultivars clustered closely with the mutant cultivars based on data for the 57 volatile compounds. Thus, we interpret that these volatile compounds are useful for classification and identification of chrysanthemum mutant cultivars. Also, our findings suggest that the novel chrysanthemum mutant cultivars with enhanced content of certain bioactive phytochemicals by radiation breeding may be provided as good source for functional food, pharmaceutical, and horticulture applications.


Falcataria moluccana), formerly known as Albizia falcataria or Paraserianthes falcataria, is an essential tree species for the development of community-based timber plantation, especially in Indonesia. The plantations nowadays are facing a significant disease threat, namely infection of gall-rust fungi . However, a clear understanding of the molecular mechanisms of the tree response against the disease is still unknown. We carried out transcriptome assembly using BGISEQ-500 technology to provide assembled de novo transcriptome dataset generated from gall-rust infected and non-infected trees. The construction of assembled transcriptome was conducted using Trinity v.2.3.2 The raw reads are available in the DDBJ platform with accession number, DRA007983.Sengon ( The Sengon tree is cultivated widely in Indonesia as a material source for veneer, light construction, and pellet in bioenergy. However, the plantations are facing the most damaging threat that damaging the wood and lowering the productivity, gall-rust  fungi infection. Altough the fungi could infect any tissue, the wood or stem, which is the main product of the plantation, was the part that damaged badly. After the fungi entered the bark, the gall-rust fungi will wounded the cell in cambium, causing death in some cells and grow itself in cambium tissues by blocking the nutrient. The fungi also secretes growth hormones that caused the undifferentiated cell in cambium to grow rapidly and created wood deformity. The transcriptome of Sengon was derived from the RNA collected from the cambium part of the stem. The sequencing process was generated using BGISEQ-500 platform. The properties of the reads and the assembled sequences are shown in This data presented the de novo transcriptome assembly of 2The cambiums of Sengon tree were collected from a private plantation in Bogor, West Java, Indonesia (6°32′38.9″S 106°44′24.5″E). The RNA was extracted from the cambium part originated from gall-rust infected and gall-rust free tree, each type of tree consist of one individual and no replication. The RNA extracted by BGI from 80 mg tissue sample using established CTAB-pBIOZOL method. The integrity and quantity of isolated-RNA were monitored and quantified by a NanoDrop ND-1000 spectrophotometer and Agilent 2100 Bioanalyzer. The non-infected sample was treated with Ribozero due to ribosomal RNA contamination. The RNA sequencing was carried out using the BGISEQ-500  and produced pre-processing reads to discard the adaptors and low-quality reads. The pre-processed reads were de novo assembled by Trinity v.2.3.2


Yet, efficient delivery of such RNA molecules to plants and particularly to trees poses major technical challenges. Here, we describe simple methods for efficient delivery of hairpin RNAs (hpRNAs) and small interfering RNAs (siRNAs) to Malus domestica, Vitis vinifera, and Nicotiana benthamiana that are based on trunk injection and/or petiole absorption. The applied RNA molecules were efficiently taken up and systemically transported. In apical leaves, the RNA was already detectable 1 day post-application (dpa) and could be detected at least up to 10 dpa, depending on the method of application. Confocal microscopy revealed that the uptaken and systemically transported RNA molecules were strictly restricted to the xylem and apoplast which may illustrate why the applied hpRNAs were not processed into siRNAs by plant DICER-LIKE (DCL) endonucleases. These innovative methods may have great impact in pest management against chewing and/or xylem sap-feeding vectors and eukaryotic pathogens that reside in the xylem.Since its discovery, RNA interference has been widely used in crop protection. Recently, transgene-free procedures that were based on exogenous application of RNA molecules having the capacity to trigger RNAi  In plants, RNA interference (RNAi) is triggered by double stranded RNAs (dsRNAs) which are processed by DICER-LIKE endonucleases (DCLs) into 21–24 nucleotide (nt) small interfering RNAs (siRNAs). SiRNAs are incorporated into the RNA-induced silencing complex (RISC) that contains an ARGONAUTE (AGO) protein. In general, RISC-loaded 21-nt siRNAs recognize by Watson:Crick hybridization complementary single stranded RNA transcripts which are then cleaved by RISC . ImportaAlthough RNAi regulates normal plant development and genome stability, its role is also instrumental in the defense against invading nucleic acids and hostile organisms. Thus, since its breakthrough discovery 20 years ago by Nicotiana benthamiana, against Pepper mild mottle virus (PMMoV)  (Tobacco mosaic virus (TMV) (Cucumber mosaic virus (CMV) (Zucchini yellow mosaic virus (ZYMV)  transgene in N. benthamiana  (Tuta absoluta and Diabrotica spp.)  , in Zea s (SCMV) , in tobaus (TMV)  and Cucuus (CMV)  and in cs (ZYMV) . In addithamiana . Moreovegraminae  and in t cinerea . Yet, pr cinerea . In term cinerea , to pota beetle) , and to ca spp.) .in vitro transcribed dsRNA by root drenching and trunk injections, dsRNA was transported to the apical parts of the plant. However, dsRNA detection was only achieved by PCR-based methods, known to be extremely sensitive and also prone to false positives. Moreover, no information about the accumulation and processing of the applied dsRNA was provided. Here, we present novel and simple methods for efficient  RNA delivery  to Malus domestica, Vitis vinifera and N. benthamiana. The details of the application methods, the processing of the applied RNAs, their localization and function, as well as the implications of our findings in crop protection are discussed.Nevertheless, all exogenous RNA delivery methods that have been described above, referred to herbaceous plants. Woody plants are far more recalcitrant to such procedure, which may be also indicated by the scarcity of relevant reports. Yet, in a pioneering work, M. domestica and V. vinifera. Several M. domestica and V. vinifera endogenous micro RNAs (miRNAs) have been identified  and V. vinifera . It has to be noted here that in the case of V. vinifera, since it is propagated by grafting, the hole was drilled into the woody rootstock . Through these holes, the hpRNA was gently applied with the help of a 1 ml insulin syringe (without needle). The plants were transferred back to the glasshouse and kept at 25°C, 16/8 h light/dark. After this single hpRNA application, pooled leaves (distant from the site of application but not necessarily confined to young expanding leaves) from the upper parts of each plant were collected 1, 3, and 10 days post-application (dpa). Thus, from these leaves total RNA was extracted and subjected to Northern blot analysis. The data obtained for both, M. domestica and V. vinifera revealed high accumulation of hpRNA already 1 dpa but decreased at 3 and 10 dpa. . Thus, the trunk drilling method resulted in the rapid uptake and systemic transport of the exogenous RNA, but the overall effect was not lasting more than 3 or 10 days . To be noted, this transport was not affected by the grafting junction in V. vinifera, reminiscent of previous reports where the mobility of RNAi signals was not inhibited by graft junctions , indicating that that the full-length hpRNA was, if at all, only poorly processed by DCLs into few siRNAs below the detection limit of our method. In addition to the full-length hpRNA, RNA molecules smaller than full-length were detected, but these molecules were more likely hydrolytic degradation products rather than DCL-mediated processing products.As RNA input, a 500-nt unctions . ImportaV. vinifera  and N. benthamiana  revealed efficient uptake and transport of the applied hpRNA already 1 dpa . In contrast to delivery of hpRNA by trunk drilling and injection, the hpRNA levels increased from day 1 up to 10 dpa . This was a rather unexpected observation, given the fast vascular flow that normally occurs plants. It should be noted that the hpRNA solution was totally absorbed from the tube 1 dpa. Therefore, the gradual increase of hpRNA accumulation from 1 to 10 dpa was not due to the gradual uptake of the exogenously provided hpRNA, but rather reflected a peculiar physiological mechanism whose details are not very clear to us. Whatever the reasons underlying this observation may be, compared to trunk drilling, the petiole absorption method leads to slower but more long-lasting delivery of RNA molecules. Yet, similar to the trunk drilling method, the hpRNA was also largely intact and no siRNAs were detected when the petiole absorption method was applied .Since wounds generated by trunk drilling of RNA may offer entrance to pathogenic microorganisms, we sought to establish alternative and less abrasive methods for RNA delivery. We reasoned that, removing a leaf and applying a solution on the protruding cut stump of the petiole that is still attached to the plant, would lead to efficient uptake of the solution by the plant, most likely through capillary forces and vasculature flow. Indeed, by attaching tubes that contained hpRNA solutions to protruding petioles of N. benthamiana (Nb-16C) , and although efficiently uptaken, failed to target GFP mRNA for silencing . Corroborating the Northern blot data, ultraviolet monitoring of Nb-16C plants that had uptaken the 22-nt GFP siRNA through their petioles exhibited no visible RNAi phenotype . Taken together, the inability of hpRNA to be processed by DCLs into siRNAs and the failure of the delivered siRNAs to trigger silencing strongly suggested that the exogenously provided RNA (either hpRNA or siRNA) was not given access to the interior of the cell where these processes take place. In order to get detailed information about the transport and localization of the delivered RNA, an in vitro synthesized and HPLC-purified CY3-labeled 22-nt siRNA was applied by petiole absorption to N. benthamiana wild type plants (Nb-WT) . Examination of the abaxial surface of a systemic leaf 1 dpa revealed the presence of the labeled siRNA in class I (midrib), class II, and class III veins , according to the previously described vein classification system of N. benthamiana . Ultrastructural and histochemical studies on guard cells of various plant species revealed that stomata guard cells are symplastically isolated and lack plasmodesmata that would physically connect them with neighboring epidermal and mesophyll cells . Similar results suggesting exclusive xylem transport were obtained when the labeled siRNA was applied through petiole absorption in M. domestica plants , suggesting that there is virtually no difference in the transport of the exogenously delivered RNAs between herbaceous and woody plants. In order to test whether there are any differences between the transport of the siRNA and the much larger hpRNA, CY3-labeled hpRNA was applied through the petiole in N. benthamiana plants . However, the hpRNA was also detected exclusively in the xylem . In contrast, the free CY3 dye (control experiment) was detected in both xylem and phloem , suggesting that, RNA molecules (siRNA or hpRNA) are too big to be transported into phloem cells. In general, phloem is the living tissue through which metabolites, but also RNA molecules, are transported throughout the plant according to a source-to-sink direction (We have previously shown that high pressure spraying of a 22-nt GFP siRNAs into GFP-expressing (Nb-16C)  triggers(Nb-16C) . The samthamiana . Confocall cells . Howeverll cells . Thus, tirection . While pirection . In contHelicoverpa armigera) was enhanced in an Arabidopsis dcl2 dcl3 dcl4 triple mutant (Diabrotica ssp.) were sensitive to RNAi upon feeding of dsRNAs but not siRNAs  and a drill bit of 4 mm a hole was drilled into the stem approximately up to the tree’s pith. Into this hole, the hpRNA solution was gently applied  using a 1 ml insulin syringe (without the needle). After the application the hole was not sealed and trees were grown in the glasshouse applying 25°C and a 16/8 h light/dark period. Sixteen pooled leaves (eight from each of the two plants) were harvested 1, 3, and 10 dpa and subjected to Northern blot analysis. The pooled material for each dpa was non-homogeneous and represented all possible leaf stages, from young and not fully expanded to mature and fully expanded leaves. For V. vinifera, three approximately 0.5 m tall plants with a 1.5 cm trunk diameter (rootstock) were used. Trunk drilling of the rootstock was performed as described above. Three pooled leaves (one from each of the three plants) were harvested 1, 3, and 10 dpa. The pooled material for each dpa was non-homogeneous and represented all possible leaf stages, from young not fully expanded to mature fully expanded leaves. Total RNA was extracted from the leaf material and subjected to Northern blot analysis.For V. vinifera, three approximately 0.5 m tall plants were used. The first leaf was detached and a 0.5 ml tube whose bottom was cut off and which contained the hpRNA solution  was attached to the protruding petiole. Three leaves (one from each of the three plants) were harvested and pooled 1, 3, and 10 dpa, respectively. The pooled material for each dpa was non-homogeneous and represented all possible leaf stages, from young not fully expanded to mature fully expanded leaves . Only leaves above the site of RNA application were sampled. Total RNA was extracted from the leaf material and subjected to Northern blot analysis. For N. benthamiana, six approximately 10 cm tall plants were used. RNA delivery was performed as described above. Six leaves (one from each of the six plants) were harvested and pooled 1, 3, and 10 dpa, respectively. The pooled material for each dpa was non-homogeneous and represented different leaf stages, from young not yet expanded to almost fully expanded leaves (at that stage the plants were still quite young and fully expanded leaves were not yet available). Only leaves above the site of RNA application were sampled. Total RNA was extracted from the leaf material and subjected to Northern blot analysis.For 1 and at a pressure of 7–8 bar provided by the METABO Elektra Beckum Classic 250 compressor 2.One hundred microliters of an aqueous siRNA solution (8 μM) was sprayed from a 2–4 cm distance at the abaxial surface of leaves with the CONRAD air brush gun AFC-250A 3 according to the manufacturer’s instruction. For Northern blot analysis of large RNA molecules, 5 μg of total RNA was separated on a 1.2% agarose formaldehyde gel, capillary transferred onto the positively charged nylon membrane Bright Star4 and UV312nm-cross-linked (300 mJ/cm2). For the detection of GFP hpRNA and GFP mRNA, the PerfectHyb Plus 1x (see footnote 3) was used for overnight hybridization at 64°C with the random-primed αα-32P-dCTP labeled GFP cDNA fragment (792 bp). Membranes were washed at 64°C with buffer 1  for 30 min and with buffer 2  for 15 min. Membranes were then exposed to FujiFilm Imaging Plates5 for 24 h and scanned using PharosFX Plus PhosphorImager6. For Northern blot analysis of small RNA molecules, 20 μg were separated on a 15% TBE-urea gel7 at 120 V for 4 h. Gels were blotted onto the positively charged nylon membranes Bright Star (see footnote 4) by electro-blotting at 300 mA for 1 h and membranes were UV312nm-cross-linked (300 mJ/cm2). For detection of GFP siRNAs, the random-primed α-32P-dCTP labeled GFP cDNA fragment (792 bp) was used as a hybridization probe. For detection of U6 snRNA, the DNA oligo 5′-AGG GGC CAT GCT AAT CTT CTC-3′ was used in an end-labeling reaction using γ-32P ATP8 and T4 polynucleotide kinase9 as a hybridization probe. The hybridization temperature was 42°C and membranes were washed twice with buffer 1  at 42°C for 30 min.Total RNA was extracted with Spectrum Plant Total RNA Kitin vitro transcribed GFP hpRNA was provided by BASF (Limburgerhof). The CY3-labeling of the hpRNA was performed with the Silencer siRNA labeling kit10 according to the manufacturer’s instructions. The 22-nt siRNA was in vitro synthesized by Metabion11. The HPLC-purified CY3-labeled 22-nt siRNA was in vitro synthesized by Sigma-Aldrich (see footnote 3).The 500-nt For confocal laser microscopy, sections were imaged using a Zeiss LSM 510 confocal laser setup with an Axio Observer Z1 inverted microscope. Cy3 dye was excited at 514 nm with a diode pumped solid state laser and its fluorescence was detected using a 575–615 nm red bandpass filter. Overview images of entire stem and root sections were taken with a Zeiss FLUAR 5x objective, while for detailed images of leaf cells we used a 40x Zeiss C-Apochromat with water immersion. For fluorescence stereomicroscopy, the abaxial surface of a leaf was imaged under CY3 filter with a Zeiss SteREO Lumar.V12 stereomicroscope with a 0.8× objective at 80× magnification.AD, GK, and MW conceived the experiments. AD, WJ, GB, AB, MB, and TM performed the experiments. AD and MW wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.