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How does ionizing radiation affect amyloidogenesis in plants?

ABSTRACT: Ionizing radiation is a harsh environmental factor that could induce plant aging. We hypothesized that radiation-related aging remodels proteome, particularly triggering the accumulation of prion-like proteins in tissues. The object of this study, pea (Pisum sativum), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. Pea seeds were irradiated by 50 Gy of X-rays. Afterwards, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to reveal putative prion-like proteins. We showed a reduced germination rate, yet higher plant height and faster appearance of reproductive organs in the irradiated group compared with the control; furthermore, we demonstrated more β-sheets and amyloid aggregates in the leaves of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 62 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, discovery proteomics provided deep data on novel aspects of plant stress biology. Our data hinted that protein accumulation stimulated seedlings' growth and affected the regulatory system, primarily through translation and RNA processing. Increased abundance of primary metabolism-related proteins points to more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Pisum Sativum (garden Pea)

TISSUE(S): Plant Cell, Root

SUBMITTER: Maksym Danchenko

LAB HEAD: Peter Baráth

PROVIDER: PXD044798 | Pride | 2024-03-18

REPOSITORIES: Pride

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Project description:Plant growth is the result of cell proliferation in the meristems, which requires a dynamic balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. There is much that remains unknown about this vital developmental process. In this study, we have reported the generation of 2735 ESTs from three cDNA libraries derived from dissected garden pea (Pisum sativum cv Torsdag) shoot apical meristems. Clustering analysis of the resulting sequences gave rise to 1686 non-redundant ESTs. The unique sequences generated together with 500 other pea transcripts randomly selected from the GenBank pea protein database have been utilized to construct a 2K oligonucleotide array. Using this pea array, we have obtained the transcript profiles of pea shoot apical meristems in comparison to non-apical-meristem tissues. A total of 181 or 174 transcripts were identified to be significantly up- or down-regulated in the pea shoot apical meristem, respectively. As expected, close to 61% of the transcripts down-regulated in the shoot apical mersitem are those retrieved from the public database, whereas sequences from the same source only made up 12% of the genes that were expressed at higher levels in SAMs. This revelation highlights the under-representation of transcripts from the meristmatic tissues in the current public protein database. Manual inspection of the list of up-regulated transcripts reveals the presence of sequences predicted to encode products associated with cell division and proliferation, epigenetic regulation, auxin-mediated responses and miRNA regulation. Similar genes have been implicated in the regulation of plant stem cell activity. Taken together, our EST collection as well as the microarray data provides useful starting points for more in depth analysis of the meristem function and maintenance. Keywords: shoot apical meristem transcript profiling Total RNA was extracted from dissected SAM (approximately 80 SAMs per extraction) or other plant parts (primary stem, primary roots and mature leaves) using Qiagen RNeasy Mini Kit. Four independent tissue collections and RNA extractions (designated A, B, C and D) were performed for each of the microarray hybridization experiment.The Cy5- or Cy3-labelled cDNA was then hybridized to different sector of the chip according to a balanced block design dual label experiment scheme (Cochran & Cox, 1992): Sector 1: Cy3-SAM A vs Cy5-NM A Sector 2: Cy5-SAM B vs Cy3-NM B Sector 3: Cy3-SAM C vs Cy5-NM C Sector 4: Cy5-SAM D vs Cy3-NM D

Project description:Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance; of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a; gamma-irradiated stable mutant, (ii) the M1 generation of a 100-Gy gamma-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. Experiment Overall Design: Plant Materials Experiment Overall Design: Two genetically stable Oryza sativa L. ssp. japonica lines: a gamma-irradiated ricemutant(cv. EstrelaA)and a well characterized transgenic rice line (cv. Bengal) were used as well as controls. The stable mutant was obtained in 1988 by gamma-irradiation, had already gone over 10 generations of self-pollination, and had a mature average height about 45 cm lower than the wild type. The stable transgenic line, which was already in the third generation of self-pollination after transformation, expresses a ScFV antibody (ScFvT84.66) against carcinoembryonic antigen, a well characterized tumor-associated marker antigen. Experiment Overall Design: We have also used two genetically unstable rice lines: the M1 generation of a 100-Gy gamma-irradiated line (98% survival after mutagenesis) and the T1 generation of an Agrobacterium-transformed transgenic line (both cv. Nipponbare) containing one copy of the BCBF1 gene driven by the AtRD29A promoter from Arabidopsis and one copy of the hpt II gene. We used seeds from the same self-pollinated panicle for control and irradiation/transgenesis. The unstable mutant line chosen for this experiment was the one showing a phenotype more similar to that of the nonirradiated control. Experiment Overall Design: In the case of the transgenic lines, stability was based on the stable Experiment Overall Design: inheritance of the introduced transgenes in the homozygous progeny. Regarding the mutagenized plants we have defined as genetically stable plants those that, after mutagenesis, had already gone through several cycles of self-pollination while maintaining the desirable traits. Experiment Overall Design: RNA Extraction and Microarrays Experiment Overall Design: Two pools of six whole seedlings were prepared for each condition under test, and RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) following the manufacturerâ??s instructions. Total RNA was kept at -80Â°C and sent to the Affymetrix core facility (Instituto Gulbenkian de CiÃªncia, Experiment Overall Design: Oeiras, Portugal), where quality-control analysis was carried out before cDNA synthesis from themRNA[with appropriate oligo(dT) primers], labeling (through synthesis of cRNA with incorporation of biotinylated ribonucleotide analogs), and hybridization to the GeneChip Rice Genome Array (Affymetrix). This array contains probes to query 51,279 transcripts representing two rice subspecies (48,564 japonica transcripts and 1,260 transcripts of indica subspecies). Experiment Overall Design: Data Analysis Experiment Overall Design: Microarrays data analysis was performed with Partek Genomics Suite software. Affymetrix CEL files were imported by using the Robust Multichip Average method, which involves four steps: background correction of the perfect match values, quintile normalization across all of the chips in the experiment, Log2 transformation, and median polish summarization. The logged data were used for hierarchical cluster analysis and statistical analysis. Experiment Overall Design: Hierarchical cluster analysis was performed by using Pearsonâ??s dissimilarity product moment correlation coefficient and Wardâ??s algorithm. Experiment Overall Design: For the identification of differentially expressed genes we used ANOVA Experiment Overall Design: and a false discovery rate with a 0.05 threshold.

Project description:Inflorescence architecture contributes to essential plant traits. It determines plant shape, contributing to morphological diversity, also determines position and number of the flowers and fruits produced by the plant, influencing seed yield. Most legumes have compound inflorescences, where flowers are produced in secondary inflorescences (I2), formed at the flanks of the main primary inflorescence (I1), in contrast to simple inflorescences of plants like Arabidopsis, in which flowers are directly formed on the I1. The pea VEGETATIVE1/ FULc (VEG1) gene, and its homologues in other legumes, specify the formation of the I2 meristem, a function apparently restricted to legumes. To understand the control of I2 development it is important to identify the genes working downstream of VEG1. In this study, we adopted a novel strategy to identify genes expressed in the I2 meristem, as potential regulatory targets of VEG1. To identify pea I2-meristem genes we compared the transcriptomes of inflorescence apices from wild type and mutants affected in I2 development, proliferating inflorescence meristems (pim - with more I2 meristems), veg1 and vegetative2 (both without I2 meristems). Analysis of the differentially expressed genes using Arabidopsis genome databases combined with RT-qPCR expression analysis in pea, allowed the selection of genes expressed in the pea inflorescence apex. In situ hybridization of four of these genes showed that all four genes are expressed in the I2 meristem, proving our approach to identify I2-meristem genes successful. Finally, analysis by VIGS in pea identified one gene, PsDAO1, whose silencing lead to small plants and another gene, PsHUP54, whose silencing leads to plants with very large stubs, meaning that this gene controls activity of the I2 meristem. PsHUP54-VIGS plants also are large and, more importantly, produce large pods with almost double of seeds than the control. Our study shows a new useful strategy to isolate I2-meristem genes and identifies a novel gene, PsHUP54, which seems a promising tool to improve yield in pea and another legumes.

			Action	DRS
	211216_PeaC2b.raw	Raw
	211216_PeaC2c.raw	Raw
	211216_PeaC3a.raw	Raw
	211216_PeaC3b.raw	Raw
	211216_PeaR2a.raw	Raw

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How does ionizing radiation affect amyloidogenesis in plants?

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