Genotoxic effects and proteomic analysis on Allium cepa var. agrogarum L. root cells under Pb stress.

Ionic lead (Pb) in the environment has accumulated due to anthropogenic activities, causing a potential threat to plants and plant consumers. We conducted this study to reveal the molecular mechanism of Pb stress response in plants. The effects of Pb (5.0 and 15.0 µM) on mitosis, DNA replication, gene expression and proteins in root-tip cells of Allium cepa var. agrogarum L. were addressed. The results indicated that root growth was inhibited dramatically in Pb treatment groups. Chromosomal aberrations were observed and the mitotic index decreased during Pb treatments at different concentrations. The accumulation of reactive oxygen species (ROS) in onion roots was induced by Pb stress. Pb increased DNA damage and suppressed cell cycle progression. The above toxic effects got more serious with increasing Pb concentration and prolonging exposure time. A total of 17 proteins were expressed differentially between control and Pb exposure groups. Under Pb treatment, the decreased expression of Anx D1 indicated decreased defensive response; the decreased expression of SHMT1 indicated decreased respiration; the decreased expression of COMT2 indicated decreased response of other funtions; the increased expression of NDPK indicated increased transcription and protein synthesis; the increased expression of PR1 and CHI1 indicated increased pathogen invasion; the increased expression of ORC5 and MPK5 indicated the reduced DNA replicating activity; the decreased expression of POLD1 indicated the reduced DNA repair activity. Our results provide new insights at the proteomic level into the Pb-induced responses, defensive responses and toxic effects, and provide new molecular markers of the early events of plant responses to Pb toxicity.

Quantitative Proteomic Analyses Identify STO/BBX24 -Related Proteins Induced by UV-B.

Plants use solar radiation for photosynthesis and are inevitably exposed to UV-B. To adapt to UV-B radiation, plants have evolved a sophisticated strategy, but the mechanism is not well understood. We have previously reported that STO (salt tolerance)/BBX24 is a negative regulator of UV-B-induced photomorphogenesis. However, there is limited knowledge of the regulatory network of STO in UV-B signaling. Here, we report the identification of proteins differentially expressed in the wild type (WT) and sto mutant after UV-B radiation by iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomic analysis to explore differential proteins that depend on STO and UV-B signaling. A total of 8212 proteins were successfully identified, 221 of them were STO-dependent proteins in UV-B irradiated plants. The abundances of STO-dependent PSB and LHC (light-harvesting complex) proteins in sto mutants decreased under UV-B radiation, suggesting that STO is necessary to maintain the normal accumulation of photosynthetic system complex under UV-B radiation to facilitate photosynthesis photon capture. The abundance of phenylalanine lyase-1 (PAL1), chalcone synthetase (CHS), and flavonoid synthetase (FLS) increased significantly after UV-B irradiation, suggesting that the accumulation of flavonoids do not require STO, but UV-B is needed. Under UV-B radiation, STO stabilizes the structure of antenna protein complex by maintaining the accumulation of PSBs and LHCs, thereby enhancing the non-photochemical quenching (NPQ) ability, releasing extra energy, protecting photosynthesis, and ultimately promoting the elongation of hypocotyl. The accumulation of flavonoid synthesis key proteins is independent of STO under UV-B radiation. Overall, our results provide a comprehensive regulatory network of STO in UV-B signaling.

Effects of Ca addition on the uptake, translocation, and distribution of Cd in Arabidopsis thaliana.

Cadmium (Cd) pollution poses a risk to human health for its accumulation in soil and crops, but this can be alleviated by calcium (Ca) addition. However, its mechanism remains unclear yet. In this study, Arabidopsis thaliana was used to explore the alleviating effects of Ca on Cd toxicity and its specific function during uptake, upward-translocation, and distribution of Cd. Supplementing plants with 5mM CaCl2 alleviated the intoxication symptoms caused by 50µM CdCl2, such as smaller leaves, early bolting and root browning. Ca addition decreased uptake of Cd, possibly by reducing the physical adsorption of Cd since the root cell membrane was well maintained and lignin deposition was decreased as well, and by decreasing symplastic Cd transport. Expression of the genes involved (AtZIP2 and AtZIP4) was also decreased. In addition, Ca accumulated in the plant shoot to help facilitating the upward-translocation of Cd, with evidence of higher translocation factor and expression of genes that were involved in Ca transport (AtPCR1) and Cd xylem loading (AtHMA2 and AtHMA4). Dithizone-staining of Cd in leaves showed that in Cd+Ca-treated plants, Ca addition initially protected the leaf stomata by preventing Cd from entering guard cells, but with prolonged Cd treatment facilitated the Cd accumulation around trichomes and maybe its excretion. We conclude that Ca promotes the upward-translocation of Cd and changes its distribution in leaves. The results may have relevance for bioremediation.

Application of in-house virtual protein database performed in genomic-proteomic combined research on heavy-metal stressed onion roots.

OBJECTIVES: To establish an in-house virtual protein database that can be employed in proteomic research on non-model plants. RESULTS: A total of 87,430 unigenes were obtained through transcriptome sequencing from onion roots. Of these, 24,305 unigenes were annotated and their nucleotide sequences of coding regions were translated into amino acid sequences. The corresponding 24,305 amino acid sequences were considered as an in-house virtual protein database. Thirty-two protein spots with significant differential abundance were selected. Their MS data were submitted to a restriction enzyme map which was converted from the in-house virtual protein database. A total of 27 proteins were finally matched. CONCLUSIONS: The in-house protein database is a feasible and innovative strategy for proteomic research on non-model plants.

Proteomics research on the effects of applying selenium to apple leaves on photosynthesis.

Untreated and Se-enriched apple leaves (Malus domestica Borkh. cv. 'Red Fuji') were used as the experimental materials. Proteomes of the differentially prepared tissues were compared through two-dimensional electrophoresis analysis and mass spectrum identification. There were 505 more protein spots in the proteome of the Se-enriched leaves than in the control leaves. Forty-seven protein spots were significantly differentially expressed (P < 0.05), among those, 32 protein spots were up-regulated while 12 protein points were down-regulated, and three new protein spots were found with the relative molecular masses of 31, 29, 26 kDa. Twenty-three protein spots with good shape and significant expression were selected for mass spectrometry analysis. These spots were excised from the gel and analyzed by a matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Peptide mass fingerprints (PMF) of all the proteins were submitted to NCBInr for protein identification, and 10 differential proteins were positively identified. Biological information of the identified proteins was found via http://www.uniprot.org/. There were three (1475, 1479, 1527) ribulose-1,5-bisphosphate carboxylase/oxygenase large subunits (Rubisco), two ribulose-1,5-bisphosphate carboxylases (346, 486) belonging to the Rubisco large chain family, one photosystem I reaction center subunit II (297), one chloroplast oxygen-evolving enhancer protein 1 (619), one Os12g0127100 protein whose function was unknown (927), one monodehydroascorbate reductase (1451), and one polyphenol oxidase V (1596). The major subcellular location for these proteins was the chloroplast, and they play important roles in photosynthesis and stress resistance for plants.