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Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

ABSTRACT: We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant-DSBs versus alkaline-DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

SUBMITTER: Rybaczek D

PROVIDER: S-EPMC4636323 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

Rybaczek Dorota D Musiałek Marcelina Weronika MW Balcerczyk Aneta A

PloS one 20151106 11

We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chrom ...[more]

PMID: 26545248

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Project description:Cell death (CD) may be induced by endogenous or exogenous factors and contributes to all the steps of plant development. This paper presents results related to the mechanism of CD regulation induced by kinetin (Kin) in the root cortex of Vicia faba ssp. minor. To explain the process, 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55), adenine (Ad), 5'-amine-5'-deoxyadenosine (Ado) and N-(2-chloro-4-piridylo)-N'-phenylurea (CPPU) were applied to (i) block cytokinin receptors (CKs) and inhibit the activities of enzymes of CK metabolism, i.e., (ii) phosphoribosyltransferase, (iii) kinases, and (iv) oxidases, respectively. Moreover, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), lanthanum chloride (LaCl3), ruthenium red (RRed) and cyclosporine A (CS-A) were applied to (i) chelate extracellular calcium ions (Ca2+) as well as blocks of (ii) plasma-, (iii) endoplasmic reticulum- (ER) membrane Ca2+ ion channels and (iv) mitochondria- (MIT) Ca2+ ions release by permeability transition por (PTP), respectively. The measured physiological effectiveness of these factors was the number of living and dying cortex cells estimated with orange acridine (OA) and ethidium bromide (EB), the amounts of cytosolic Ca2+ ions with chlortetracycline (CTC) staining and the intensity of chromatin and Ca2+-CTC complex fluorescence, respectively. Moreover, the role of sorafenib, an inhibitor of RAF kinase, on the vitality of cortex cells and ethylene levels as well as the activities of RAF-like kinase and MEK2 with Syntide-2 and Mek2 as substrates were studied. The results clarified the previously presented suggestion that Kin is converted to appropriate ribotides (5'-monophosphate ribonucleotides), which cooperate with the ethylene and Ca2+ ion signalling pathways to transduce the signal of kinetin-programmed cell death (Kin-PCD). Based on the present and previously published results related to Kin-PCD, the crosstalk between ethylene and MAP kinase signalling, as well as inhibitors of CK receptors and enzymes of their metabolism, is proposed.

Project description:Drought and salinity are the major factors that limit the faba bean (Vicia faba L.) production worldwide. The aim of this study is to identify the water stress differentially expressed genes (DEGs) through the root transcriptome analyses of the drought-tolerant Hassawi 2 genotype at vegetative and flowering stages. A total of 624.8 M high-quality Illumina reads were generated and assembled into 198,155 all-unigenes with a mean length of 738 bp and an N50 length of 1347 bp. Among all-unigenes, 78,262 were assigned to non-redundant (Nr), 66,254 to nucleotide (Nt), 54,034 to KEGG, and 43,913 to gene ontology (GO) annotations. A total of 36,834 and 35,510 unigenes were differentially expressed at the vegetative and flowering stages of Hassawi 2 under drought stress, respectively. The majority of unigenes were down-regulated at both developmental stages. However, the number of genes up-regulated (15,366) at the flowering stage exceeded the number of those up-regulated (14,097) at the vegetative stage, and the number of genes down-regulated (20,144) at the flowering stage was smaller than the number of those down-regulated (22,737) at the vegetative stage. The drought stress-responsive differentially expressed unigenes coded for various regulatory proteins, including protein kinases and phosphatases, transcription factors and plant hormones and functional proteins including enzymes for osmoprotectant, detoxification and transporters were differentially expressed, most of which were largely up-regulated. Moreover, a substantial proportion of the DEGs identified in this study were novel, most exhibited a significant change in their expression levels under water stress, making them an unexploited resource that might control specific responses to drought stress in the faba bean. Finally, qRT-PCR results were found almost consistent with the results of next-generation sequencing. Our data will help in understanding the drought tolerance mechanisms in plants and will provide resources for functional genomics.

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Project description:BackgroundSoil moisture deficiency causes yield reduction and instability in faba bean (Vicia faba L.) production. The extent of sensitivity to drought stress varies across accessions originating from diverse moisture regimes of the world. Hence, we conducted successive greenhouse experiments in pots and rhizotrons to explore diversity in root responses to soil water deficit.MethodsA set of 89 accessions from wet and dry growing regions of the world was defined according to the Focused Identification of Germplasm Strategy and screened in a perlite-sand medium under well watered conditions in a greenhouse experiment. Stomatal conductance, canopy temperature, chlorophyll concentration, and root and shoot dry weights were recorded during the fifth week of growth. Eight accessions representing the range of responses were selected for further investigation. Starting five days after germination, they were subjected to a root phenotyping experiment using the automated phenotyping platform GROWSCREEN-Rhizo. The rhizotrons were filled with peat-soil under well watered and water limited conditions. Root architectural traits were recorded five, 12, and 19 days after the treatment (DAT) began.ResultsIn the germplasm survey, accessions from dry regions showed significantly higher values of chlorophyll concentration, shoot and root dry weights than those from wet regions. Root and shoot dry weight as well as seed weight, and chlorophyll concentration were positively correlated with each other. Accession DS70622 combined higher values of root and shoot dry weight than the rest. The experiment in GROWSCREEN-Rhizo showed large differences in root response to water deficit. The accession by treatment interactions in taproot and second order lateral root lengths were significant at 12 and 19 DAT, and the taproot length was reduced up to 57% by drought. The longest and deepest root systems under both treatment conditions were recorded by DS70622 and DS11320, and total root length of DS70622 was three times longer than that of WS99501, the shortest rooted accession. The maximum horizontal distribution of a root system and root surface coverage were positively correlated with taproot and total root lengths and root system depth. DS70622 and WS99501 combined maximum and minimum values of these traits, respectively. Thus, roots of DS70622 and DS11320, from dry regions, showed drought-avoidance characteristics whereas those of WS99501 and Mèlodie/2, from wet regions, showed the opposite.DiscussionThe combination of the germplasm survey and use of GROWSCREEN-Rhizo allowed exploring of adaptive traits and detection of root phenotypic markers for potential drought avoidance. The greater root system depth and root surface coverage, exemplified by DS70622 and DS11320, can now be tested as new sources of drought tolerance.

Dataset Information

Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

Publications

Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

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