Durum wheat roots adapt to salinity remodeling the cellular content of nitrogen metabolites and sucrose

Plants are currently experiencing increasing salinity problems due to irrigation with brackish water. Moreover, in fields, roots can grow in soils which show spatial variation in water content and salt concentration, also because of the type of irrigation. Salinity impairs crop growth and productivity by inhibiting many physiological and metabolic processes, in particular nitrate uptake, translocation, and assimilation. Salinity determines an increase of sap osmolality from about 305 mOsmol kg-1 in control roots to about 530 mOsmol kg-1 in roots under salinity. Root cells adapt to salinity by sequestering sodium in the vacuole, as a cheap osmoticum, and showing a rearrangement of few nitrogencontaining metabolites and sucrose in the cytosol, both for osmotic adjustment and oxidative stress protection, thus providing plant viability even at low nitrate levels. Mainly glycine betaine and sucrose at low nitrate concentration, and glycine betaine, asparagine and proline at high nitrate levels can be assumed responsible for the osmotic adjustment of the cytosol, the assimilation of the excess of ammonium and the scavenging of ROS under salinity. High nitrate plants with half of the root system under salinity accumulate proline and glutamine in both control and salt stressed split roots, revealing that osmotic adjustment is not a regional effect in plants. The expression level and enzymatic activities of asparagine synthetase and δ1-pyrroline-5-carboxylate synthetase, as well as other enzymatic activities of nitrogen and carbon metabolism, are analyzed

Комплексная МР-ангиографическая и МР-томографическая диагностика атеросклеротических поражений сонных артерий с парамагнитным контрастированием у больных с распространенным атеросклерозом

Aim. To evaluate the possibility of simultaneous magnetic resonance angiography of carotid arteries and contrast-enhanced magnetic resonance imaging of carotid atherosclerotic plaques. Material and methods. 24 persons entered in research: 16 (66.7%) patients with extensive atherosclerosis of aorta and large arteries and in 8 (33.3%) control persons. Using four-channel quadratur phase-array coil for head studies the brain MRI, MR-angiography of carotid arteries and MR-tomography of carotid atherosclerotic plaques were carried out using contrast enhancement with 0,5M cyclomang (Mn-diaminocyclohexanetetraacetate) solution. The angiography employed 3D GR FFE fast gradient echo protocol (TR/TE/FA/ST = 10 ms / 2.7ms / 20°/ 1.5 mm). MRI of carotid arteries used the T1-w.spin-echo scanning with TR = 500-700 ms, TE = 10 ms, with slices as thin as 1-3 mm, matrix 256 x 256, and voxel as small as 0.2 x x 0.2 x 2 mm. Results. The mean transit time for the paramagnetic contrast passage through brain haemispheres was in healthy control persons as short as MTT = 4.23 ± 0.14 s for the left and MTT = 4.27 ± 0.15 s for the right. The MTT in patients with single-side stenosis was on the involved side as long as 4.89 ± 0.23 s, whereas on the intact side 4.56 ± 0.19 s (p > 0.05). In bilateral stenosis the MTT was 4.98 ± 0.21 s and 5.01 ± 0.16 s (p > 0.05) for the left and right sides respectively. In all cases of aherosclerotic stenoses the contrast-enhanced MRA with cyclomang provided the correct diagnosis of both location and extent of the stenosis. The degree of stenosis calculated for the MR-angiography correlated significantly with the data of ultrasonic study calculated using ECST technique both for monolateral (r = 0.87, p 0,05). При двустороннем поражении эти показатели для левого и правого полушарий составляли 4,98 ± 0,21 с и 5,01 ± 0,16 с. (р > 0,05). МРА с цикломангом позволила во всех случаях визуализировать локализацию и характер стеноза. Величины степени стеноза, рассчитанные для МР-ангиограммы, высокодостоверно коррелировали с данными ультразвукового исследования, выполненного по методике ECSt, для случаев как одностороннего (r = 0,87, р < 0,05), так и двустороннего стенотического поражения (r = 0,85, р < 0,05). Неоднородные рыхлые бляшки с высоким содержанием липидов имели высокие показатели индекса усиления при контрастировании - 1,26 ± 0,07, тогда как плотные фиброзные аваскулярные бляшки - 1,09 ± 0,04 (р < 0,05). Полное время исследования составляло 41 ± 5 мин при выполнении времяпролетной МРА и 29 ± 5 мин без нее. Заключение. Одновременное проведение МРА и МРТ сонных артерий с парамагнитным контрастированием цикломангом возможно и целесообразно в рамках единого исследования с использованием квадратурной катушки для головы

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

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

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

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