12 research outputs found
Effects of climate warming and human activities on the distribution patterns of Fritillaria unibracteata in eastern Qinghai-Tibetan Plateau
Abstract Fritillaria unibracteata is an endangered medicinal material species endemic to the Qinghai Tibet Plateau, and belongs to the national Class III endangered plant. In addition to expelling wind and removing damne, it also warms menstruation and relieves pain in clinic use of tranditional Chinese medicine. In recent years, affected by the destruction of shrubs and climate change, the habitat of F. unibracteata wild resources has been seriously damaged, indicating of great significance to predict its potential suitable habitat using MaxEnt model. The AUC values without human activities were 0.983 ± 0.013–0.988 ± 0.001, while it is 0.982 ± 0.015–0.989 ± 0.000 with human activities, justifying their applications for predicting the potential areas of F. unibracteata. Without human activities, there were 8.47 × 104 km2 of highly suitable habitats in northern Sichuan, southern Gansu and southeastern Qinghai. But the poorly, moderately and highly suitable areas of F. unibracteata have decreased to 33.8 × 104 km2, 9.66 × 104 km2 and 6.64 × 104 km2 due to human activities. Environmental variables affecting F. unibracteata distribution included the minimum temperature in the coldest month (−16.89–−4.96 °C), annual precipitation (416.64–866.96 mm), temperature annual range (24.83–31.97 °C), elevation (2879.69–3981.82 m), human footprint (2.58–23.66) and mean UV-B of highest month (7381.92–8574.27 kJ/m2). In the 2050s and 2090s, human activities would significantly reduce the highly suitable habitats of F. unibracteata. Under SSP1-2.6, SSP2-4.5 and SSP5-8.5 scenarios, the centroid would move to the low latitude area from the current position first, and then to a high latitude area. Wild resources of F. unibracteata in China can be effectively conserved based on our results
Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis
Intron Lariat RNA Inhibits MicroRNA Biogenesis by Sequestering the Dicing Complex in <i>Arabidopsis</i>
<div><p>Lariat RNAs formed as by-products of splicing are quickly degraded by the RNA debranching enzyme 1 (DBR1), leading to their turnover. Null <i>dbr1</i> mutants in both animals and plants are embryo lethal, but the mechanism underlying the lethality remains unclear. Here we characterized a weak mutant allele of <i>DBR1</i> in <i>Arabidopsis</i>, <i>dbr1-2</i>, and showed that a global increase in lariat RNAs was unexpectedly accompanied by a genome-wide reduction in miRNA accumulation. The <i>dbr1-2</i> mutation had no effects on expression of miRNA biogenesis genes or primary miRNAs (pri-miRNAs), but the association of pri-miRNAs with the DCL1/HYL1 dicing complex was impaired. Lariat RNAs were associated with the DCL1/HYL1 dicing complex <i>in vivo</i> and competitively inhibited the binding of HYL1 with pri-miRNA. Consistent with the impacts of lariat RNAs on miRNA biogenesis, over-expression of lariat RNAs reduced miRNA accumulation. Lariat RNAs localized in nuclear bodies, and partially co-localize with HYL1, and both DCL1 and HYL1 were mis-localized in <i>dbr1-2</i>. Together with our findings that nearly four hundred lariat RNAs exist in wild type plants and that these lariat RNAs also associate with the DCL1/HYL1 dicing complex <i>in vivo</i>, we thus propose that lariat RNAs, as decoys, inhibit miRNA processing, suggesting a hitherto unknown layer of regulation in miRNA biogenesis.</p></div
Lariat RNAs competitively inhibit DCL1/HYL1 binding to pri-miRNAs.
<p>(A) Lariat RNAs associate with DCL1 using a RIP assay performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006422#pgen.1006422.g002" target="_blank">Fig 2B</a>. Immunoprecipitated RNAs were analyzed by qRT-PCR with divergent primers to detect the indicated lariat RNAs. <i>UBQ5</i> was used as the loading and negative control. Error bars show SE calculated from three biological replicates. (B) Lariat RNAs associate with HYL1 using a RIP assay performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006422#pgen.1006422.g002" target="_blank">Fig 2C</a>. qRT-PCR was performed according to (A). <i>hyl1-2</i> was used as the negative control. <i>UBQ5</i> was used as the loading and negative control. Error bars show SE calculated from three biological replicates. (C) R-EMSA to determine HYL1 binding to <i>pri-miR167b</i> in the presence of circular RNAs from Col-0 or <i>dbr1-2</i> plants. Recombinant MBP-HYL1-D1D2 (MBP-HYL1) was incubated with a 5’_biotin_labeled <i>pri-miR167b</i> probe after addition of different amounts of circular RNAs isolated from Col-0 or <i>dbr1</i>-2 inflorescences, respectively. The arrow indicates the HYL1-<i>pri-miR167b</i> complex. (D) Hybridization intensities were quantified and normalized to the controls (lane 2 in C), and are shown in the line graph. Bars represent the average normalized intensity of three biological replicates.</p
Subcellular localization of DBR1 and lariat RNA.
<p>(A) Subcellular localization of <i>Arabidopsis</i> DBR1. 35S::DBR1-RFP was transiently expression in tobacco leaves and RFP signal was observed after 48 hr. Roots from 35S::DBR1-GFP transgenic plants were observed under the GFP channel. (B) Strategy to visualize lariat RNAs in live cells. The MS2 sequence (indicated as stem-loops) was inserted into a lariat24a-located intron. A co-expressed GFP-tagged MS2-CP protein was used to visualize lariat RNAs. Grey boxes indicate exons, and lines indicate the intron. (C) Genomic DNA of lariat24a and lariat41 was fused to 6XMS2 repeats and co-infiltrated into tobacco leaves with MS2-CP-GFP and HYL1-RFP. The genomic DNA of pri-miR163 was used as the positive control. Scale bars = 10 μm.</p
DBR1 is required for miRNA accumulation.
<p>(A) Morphological phenotypes of Col-0 and <i>dbr1-2</i> plants. DBR1-GFP <i>dbr1-2</i>, DBR1-RFP <i>dbr1-2</i>, and DBR1-Flag <i>dbr1-2</i> represent rescued lines of <i>dbr1-2</i> with different protein fusion constructs driven by the native <i>DBR1</i> promoter. (B) miRNA northern blot analysis in Col-0, <i>dbr1-2</i>, and a <i>dbr1-2</i> transgenic line containing the <i>pDBR1</i>::<i>DBR1-RFP</i> transgene (Compl). The numbers indicate the relative abundance of miRNAs among the three genotypes and represent the mean of three repeats (P < 0.05). U6 was used as a loading control. (C) Deep sequencing analysis of miRNAs in Col-0 and <i>dbr1-2</i>. Small RNA libraries were generated from inflorescences of two biological replicates. The normalized abundances of miRNAs were calculated as reads per ten million (RPTM), and log2-transformed ratios of <i>dbr1-2</i>/Col-0 were plotted. Each circle represents one miRNA. Thick lines indicate mean values. Rep1 and Rep2 denote replicate 1 and replicate 2, respectively.</p
A Model for lariat RNA inhibits miRNA biogenesis in plants.
<p>Pri-miRNAs are specifically recruited to the DCL1/HYL1 complex (blue indicated) for processing. In wild type plants, most lariat RNAs (indicated in purple) are quickly debranched by DBR1, only a small portion of lariat RNAs (indicated in red) naturally escapes debranching, also associates with the DCL1/HYL1 complex. In contrast, in <i>dbr1-2</i>, over-accumulated lariat RNAs might be recognized by the DCL1/HYL1 complex, and thus attenuate the association between the DCL1/HYL1 complex and pri-miRNAs, producing less miRNA.</p
DCL1 and HYL1 are mis-localized in <i>dbr1-2</i> mutants.
<p>(A-B) Subnuclear localization of DCL1 (A) and HYL1 (B) in Col-0 and <i>dbr1-2</i> root cells. Right panels show the percentage distribution of dicing bodies per cell. The X-axis represents the number of dicing bodies per cell, and the Y-axis represents the percentage of cells with the corresponding numbers. “n” represents the numbers of analyzed cells. (C) Morphological phenotypes of the <i>hyl1-2</i> and the <i>dbr1-2 hyl1-2</i> double mutant. Pictures were taken of 5-week-old plants. (D) Expression levels of indicated pri-miRNAs in Col-0, <i>dbr1-2</i>, <i>hyl1-2</i>, and <i>dbr1-2 hyl1-2</i> plants by qRT-PCR. <i>UBQ5</i> was used as an internal control and for normalization of the data. Standard deviations were calculated from three technical replicates. The results shown were reproduced with three biological replicates. Scale bars = 10 μm.</p
Over-expression of lariat41 caused reduced miRNA accumulation.
<p><b>(A)</b> Strategy to over-express lariat41 in <i>Arabidopsis</i>. Full length genomic sequence (indicated as lariat41-OE/gDNA-OE) or full length coding sequence (indicated as local41-OE/CDS-OE) was driven by the 35S promoter and fused by an YFP tag, and introduced into plants. Grey boxes indicate exons, and lines indicate the intron. (B) Morphological phenotypes of transgenic plants over-expressing lariat 41 (lariat41-OE) and the corresponding gene of lariat41 (local41-OE). Curly leaves, late flowering, altered phyllotaxy, and reduced fertility were shown in lariat41-OE. More than 10 independent transgenic lines were obtained for each. (C) RT-PCR and western blot analysis to detect expression of the corresponding gene of lariat41. Total RNA from inflorescences of a representative T3 transgenic line for each was used for cDNA synthesis, and At5g37720 was amplified to indicate the mRNA level of the corresponding gene, <i>UBQ5</i> as the loading control. In similar, YFP was detected using total protein from inflorescences by western blot, Hsc70 as the loading control. Two additional biological replicates were performed, and similar results were obtained. (D) RT-PCR to detect expression of lariats. Total RNA from inflorescences of a representative T3 transgenic line for each was used for cDNA synthesis, and lariat41 was amplified, lariat28 was used as the negative control, and <i>UBQ5</i> as the loading control. Two additional biological replicates were performed, and similar results were obtained. (E) miR159 and miR167 northern blot analysis in Col-0, local41-OE, and lariat41-OE. U6 was used as a loading control. Another biological replicate was performed, and similar results were obtained.</p
Pri-miRNA binding to DCL1 and HYL1 is reduced in <i>dbr1-2</i>.
<p>(A) Detection of pri-miRNAs in Col-0, <i>dbr1-2</i> and <i>hyl1-2</i> by qRT-PCR. <i>UBQ5</i> was the loading control. Standard deviations were calculated from three biological replicates. (B and C) Association between pri-miRNAs and the DCL1/HYL1 complex by RIP analysis. RNA was immunoprecipitated from inflorescences or seedlings of Col-0 and <i>dbr1-2</i> using DCL1 or HYL1 antibodies, respectively. The amount of pri-miRNAs was determined by qRT-PCR and normalized to the input. <i>hyl1-2</i> was used as the negative control for HYL1 antibody. <i>UBQ5</i> was used as a negative control. Error bars show SE calculated from three biological replicates. (D) The occupancy of DCL1 and HYL1 at <i>MIR164a</i>, <i>MIR169e</i>, and <i>MIR171c</i>. The coverage regions are shown as normalized peaks. The <i>x</i> axis indicates the relative position of miRNA and miRNA* location. The <i>y</i> axis indicates normalized peaks from the genomic region. Reads counts were normalized to tag per 10 million (TP10M) to adjust for sequencing depth differences of the two RIP-seq libraries.</p