Multiple RNA Processing Defects and Impaired Chloroplast Function in Plants Deficient in the Organellar Protein-Only RNase P Enzyme

<div><p>Transfer RNA (tRNA) precursors undergo endoribonucleolytic processing of their 5’ and 3’ ends. 5’ cleavage of the precursor transcript is performed by ribonuclease P (RNase P). While in most organisms RNase P is a ribonucleoprotein that harbors a catalytically active RNA component, human mitochondria and the chloroplasts (plastids) and mitochondria of seed plants possess protein-only RNase P enzymes (PRORPs). The plant organellar PRORP (PRORP1) has been characterized to some extent <i>in vitro</i> and by transient gene silencing, but the molecular, phenotypic and physiological consequences of its down-regulation in stable transgenic plants have not been assessed. Here we have addressed the function of the dually targeted organellar PRORP enzyme <i>in vivo</i> by generating stably transformed <i>Arabidopsis</i> plants in which expression of the <i>PRORP1</i> gene was suppressed by RNA interference (RNAi). <i>PRORP1</i> knock-down lines show defects in photosynthesis, while mitochondrial respiration is not appreciably affected. In both plastids and mitochondria, the effects of <i>PRORP1</i> knock-down on the processing of individual tRNA species are highly variable. The drastic reduction in the levels of mature plastid tRNA-Phe(GAA) and tRNA-Arg(ACG) suggests that these two tRNA species limit plastid gene expression in the <i>PRORP1</i> mutants and, hence, are causally responsible for the mutant phenotype.</p></div

Transmission electron microscopic images of chloroplasts and mitochondria in 15-day-old leaves from <i>PRORP1</i> RNAi mutants and wild-type plants.

<p>(<b>A, B</b>) Ultrastructure of chloroplasts and mitochondria in wild-type cells. For easy organelle identification, a chloroplast (P) and a mitochondrion (M) are labeled. (<b>C-E</b>) Ultrastructure of chloroplasts and mitochondria in cells of the strong <i>PRORP1</i> RNAi mutant line RNAi-2. Note smaller chloroplasts with more pronounced grana stacking and bigger, more elongated mitochondria. (<b>F</b>) Ultrastructure of chloroplasts and mitochondria in line RNAi-5. (<b>G</b>) Chloroplast ultrastructure in the weakest RNAi line (RNAi-12). Scale bars: 1 μm.</p

RNA gel blot analyses to assess accumulation and processing of chloroplast and mitochondrial mRNAs and rRNAs in <i>PRORP1</i> mutants and wild-type plants.

<p>The 25S rRNA band of the ethidium bromide-stained gel prior to blotting is shown as a loading control for all blots. Transcript sizes are indicated in kb. (<b>A</b>) Accumulation and processing patterns of chloroplast mRNAs and rRNAs as determined by northern blotting. (<b>B</b>) Accumulation and processing patterns of mitochondrial mRNAs. The <i>cox1</i> mRNA harbors a tRNA-like structure in its 5’ UTR, a so-called t-element, that potentially could be processed by RNase P [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.ref049" target="_blank">49</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.ref007" target="_blank">7</a>].</p

Accumulation of chloroplast and mitochondrial proteins in the wild type and the <i>PRORP1</i> mutant line RNAi-2.

<p>(<b>A</b>) Immunoblot analysis of selected chloroplast and mitochondria proteins. While PsbD and AtpB are chloroplast-encoded proteins, the three light harvesting complex proteins (Lhcb2, Lhcb4 and Lhca2) are nucleus-encoded and post-translationally imported into the chloroplast. Note that the two chloroplast-encoded proteins are strongly reduced in the <i>PRORP1</i> mutant, whereas the nucleus-encoded proteins accumulate to higher levels than in the wild type. Cox2, a mitochondrial genome-encoded protein, also accumulates to lower levels in the RNAi-2 mutant plants. Immunoblot analyses were conducted with samples of total cellular protein (20 μg) extracted from leaves and probed with specific antibodies against PsbD (the photosystem II reaction center protein D2), AtpB (the β-subunit of the chloroplast ATP synthase), Lhcb2 and Lhcb4 (light-harvesting proteins of the photosystem II antenna), Lhca2 (a light-harvesting protein of the photosystem I antenna), Cox2 (subunit II of the mitochondrial cytochrome c oxidase), and MnSOD (the nucleus-encoded mitochondrial superoxide dismutase). (<b>B</b>) As a control for equal loading, a replicate gel was stained with Coomassie brilliant blue.</p

Investigation of the relationship between tRNA editing and 5’ end maturation.

<p>(<b>A</b>) Analysis of the editing status of tRNA-Arg(ACG) in the chloroplast. The A-to-I editing event changes the adenosine in the wobble position (position 34) of the ACG anticodon to inosine, which is read as guanosine (boxed) by reverse transcriptases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.ref005" target="_blank">5</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.ref006" target="_blank">6</a>]. Editing efficiencies are shown for the wild type (WT) and the most affected RNAi line (RNAi-2). (<b>B</b>) Assessment of the efficiency of tRNA-Arg(ACG) processing by RNase P in the absence of A-to-I editing. <i>tada-1</i> is a knock-out allele of the specific adenosine deaminase that edits the anticodon of the plastid tRNA-Arg(ACG) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.ref005" target="_blank">5</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.ref006" target="_blank">6</a>]. Due to the weak exposure of the blot, the mature tRNA in the RNAi line is hardly visible.</p

Flexible Electronic Circuits - Vertical Transistors and Passive Devices

<p>Poster shown on the European Conference on Molecular Electronics ECME 2017 in Dresden.<br></p><p>Organic large area electronics have the potential to enable fully flexible applications. This requires efficient transistors on flexible substrates as well as capacitors, inductors, and resistors to complete an electrical circuit.</p><p>We operate Organic Permeable Base Transistors (OPBT) on polymer substrates to combine impressive transistor characteristics, facile manufacturing techniques and mechanical flexibility. Large current densities and on/off-ratios are achieved with simple shadow mask structuring well known from OLED technology. Currently our flexible devices reach on/off-ratios exceeding 10⁶ and current densities above 1 A/cm².<br></p>Thin-film capacitors and inductors are produced to fit the needs of a transmitter circuit that we aim to use for indoor wireless localization applications

Phenotypic and molecular analysis of <i>PRORP1</i> RNAi mutant lines generated in <i>Arabidopsis</i>.

<p>(<b>A</b>) Phenotypes of three independently generated <i>PRORP1</i> RNAi mutants (RNAi-2, RNAi-5 and RNAi-12) in comparison to wild-type plants (WT). Seven-day-old seedlings raised on synthetic medium were transferred to soil and grown under long-day conditions for 21 days. (<b>B</b>) Phenotypes of the same plants after 35 days under long-day conditions. (<b>C</b>) Down-regulation of <i>PRORP1</i> expression in the three independently generated RNAi lines as determined by qRT-PCR. Error bars indicate the standard deviation (n = 3). (<b>D</b>) PRORP1 protein accumulation in RNAi mutants and wild-type plants. Total protein was extracted from 25 day-old plants grown under long day conditions, and the PRORP1 protein was detected with a specific antibody (kindly provided by Dr. Philippe Giegé). For quantitative assessment of protein accumulation in the RNAi mutants, a dilution series of the wild-type sample (100%, 50% and 25%) was loaded. The Coomassie-stained RbcL protein band is shown as a loading control. (<b>E</b>) Pigment accumulation in 20-day-old RNAi mutants and WT plants. Error bars indicate the standard deviation (n = 3). Chl: chlorophyll. (<b>F</b>) Phenotypes of five-day-old etiolated seedlings. Scale bar: 1mm. (<b>G</b>) Hypocotyl length of 5-day-old etiolated seedlings. Error bars indicate the standard deviation (n = 15).</p

Analysis of photosynthetic activity and mitochondrial respiration in 30-day-old <i>PRORP1</i> mutants and wild-type plants grown under short day conditions.

<p>(<b>A</b>) Minimum fluorescence (<i>F</i>₀). (<b>B</b>) Maximum quantum efficiency of PSII (<i>F</i>_v/<i>F</i>_m). (<b>C</b>) Light saturation curve of linear electron flux as calculated from the PSII yield. (<b>D</b>) Non-photochemical quenching (qN). (<b>E</b>) 77K chlorophyll <i>a</i> fluorescence emission spectra. Note that the fluorescence emission maxima of PSII (688 nm) and PSI (733 nm in the wild type) are slightly shifted towards shorter wavelengths in the RNAi mutants. (<b>F</b>) Measurement of total leaf respiration in the dark (n = 4). FW: fresh weight.</p

Leaf anatomy in wild-type plants and three independently generated <i>PRORP1</i> RNAi mutants (RNAi-2, RNAi-5 and RNAi-12).

<p>Cross sections of 15-day-old leaves stained with toluidine blue O are shown. Note reduced spongy mesophyll cell layers and increased size of cylindrical palisade cells in the RNAi plants. E: epidermis; P: palisade parenchyma; S: spongy mesophyll. Scale bars: 200 μm.</p

Accumulation and processing patterns of chloroplast and mitochondrial tRNAs in <i>PRORP1</i> RNAi mutants and wild-type plants as determined by northern blotting.

<p>Detectable precursor RNA species are marked with asterisks. (<b>A</b>) Northern blot analysis of chloroplast tRNAs. Weaker exposures of the northern blots for tRNA-Glu(UUC), tRNA-Gln(UUG) and tRNA-Asp(GUC) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120533#pone.0120533.s004" target="_blank">S4 Fig</a>. (<b>B</b>) Northern blot analysis of mitochondrial tRNAs. (<b>C</b>) An ethidium bromide (EtBr)-stained agarose gel photographed prior to blotting as a control for equal loading. Fragment sizes of the RNA size marker are indicated in kilobases (kb).</p