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Ren L.,Shanghai JiaoTong University | Zhang D.,Shanghai JiaoTong University | Chen G.-Q.,Shanghai JiaoTong University | Reed B.M.,National Clonal Germplasm Repository | And 2 more authors.
Plant Cell Reports | Year: 2015

Key message: Elevated antioxidant status and positive abiotic stress response in dehydration enhance cell resistance to cryoinjury, and controlling oxidative damage via reactive oxygen species homeostasis maintenance leads to high survival.Abstract: Cryoprotectants are important for cell survival in cryopreservation, but high concentrations can also cause oxidative stress. Adding vitamin C to the cryoprotectant doubled the survival ratio in Arabidopsis thaliana 60-h seedlings (seedlings after 60-h germination) cryopreservation. In this study, the metabolites and transcriptional profiling of 60-h seedlings were analyzed in both the control cryopreservation procedure (CCP) and an improved cryopreservation procedure (ICP) to reveal the mechanism of plant cell response to oxidative stress from cryopreservation. Reactive oxygen species (ROS) and peroxidation levels reached a peak after rapid cooling–warming in CCP, which were higher than that in ICP. In addition, gene regulation was significantly increased in CCP and decreased in ICP during rapid cooling–warming. Before cryogenic treatment, the number of specifically regulated genes was nearly 10 times higher in ICP dehydration than CCP dehydration. Among these genes, DREBs/CBFs were beneficial to cope with cryoinjury, and calcium-binding protein, OXI1, WRKY and MYB family members as key factors in ROS signal transduction activated the ROS-producing and ROS-scavenging networks including AsA-GSH and GPX cycles involved in scavenging H2O2. Finally, elevated antioxidant status and oxidative stress response in the improved dehydration enhanced seedling resistance to cryogenic treatment, maintained ROS homeostasis and improved cell recovery after cryopreservation. © 2015 Springer-Verlag Berlin Heidelberg Source

Uchendu E.E.,Oregon State University | Uchendu E.E.,University of Guelph | Muminova M.,Tashkent Institute of Chemical Technology | Gupta S.,Tissue Culture and Cryopreservation Unit | Reed B.M.,National Clonal Germplasm Repository
In Vitro Cellular and Developmental Biology - Plant | Year: 2010

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for 'Chehalem' and at all steps for 'Hull Thornless'. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ̃25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols. © 2010 The Society for In Vitro Biology. Source

Chen G.-Q.,Shanghai JiaoTong University | Ren L.,Shanghai JiaoTong University | Zhang J.,Shanghai JiaoTong University | Reed B.M.,National Clonal Germplasm Repository | And 2 more authors.
Cryobiology | Year: 2015

Plant recovery status after cryopreservation by vitrification had a negative relationship to the oxidative stress induced by reactive oxygen species (ROS). Arabidopsis thaliana seedlings germinated for 48h or 72h with different survival tolerances were examined at five steps of cryopreservation, to determine the role of ROS (O2 -, H2O2 and OH) and antioxidant systems (SOD, POD, CAT, AsA and GSH) in cryo-injury. In addition, the effects of the steps on membrane lipid peroxidation were studied using malondialdehyde (MDA) as an indicator. The results indicated that H2O2-induced oxidative stress at the steps of dehydration and rapid warming was the main cause of cryo-injury of 48-h seedlings (high survival rate) and 72-h seedlings (no survival). The H2O2 was mainly generated in cotyledons, shoot tips and roots of seedlings as indicated by Amplex Red staining. Low survival of 72-h seedlings was associated with severe membrane lipid peroxidation, which was caused by increased OH generation activity and decreased SOD activity. The antioxidant-related gene expression by qRT-PCR and physiological assays suggested that the antioxidant system of 48-h seedlings were activated by ROS, and they mounted a defense against oxidative stress. A high level of ROS led to the weakening of the antioxidant system of 72-h seedlings. Correlation analysis indicated that enhanced antioxidant enzymes activities contributed to the high survival rate of 48-h seedlings, which could reflect by cryopreservation of antioxidant mutant seedlings. This model system indicated that elevated CAT activity and AsA content were determinants of cryogenic stress tolerance, whose manipulation could improve the recovery of seedlings after cryopreservation. © 2014 Elsevier Inc. Source

Parchert K.J.,University of New Mexico | Spilde M.N.,University of New Mexico | Porras-Alfaro A.,University of New Mexico | Porras-Alfaro A.,Western Illinois University | And 2 more authors.
Geomicrobiology Journal | Year: 2012

Rock varnish is a darkly pigmented coating rich in manganese oxides. Though microbes inhabit varnish deposits, it is unclear whether they are involved in varnish formation. The fungal communities of rock varnish and adjacent rock sites with no visible varnish deposits were examined. Microcolonial fungi were identified at all sampling sites, and were associated with manganese oxides in patches of incipient varnish at non-varnish sites. Fungi were closely related to manganese-oxidizing genera and seventeen isolates oxidized manganese in culture, producing six distinct manganese-oxide morphologies. Our results indicate that microcolonial fungi may play a crucial role in rock varnish formation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file. © 2012 Copyright Taylor and Francis Group, LLC. Source

Ren L.,Shanghai JiaoTong University | Zhang D.,Shanghai JiaoTong University | Jiang X.-N.,Beijing Forestry University | Gai Y.,Beijing Forestry University | And 3 more authors.
Plant Science | Year: 2013

Cryopreservation can be a safe and cost-effective tool for the long-term storage of plant germplasm. In Arabidopsis, the ability to recover from cryogenic treatment was lost as growth progressed. Growth could be restored in 48-h seedlings, whereas 72-h seedlings died after cryogenic treatment. Why seedling age and survival are negatively correlated is an interesting issue. A comparative transcriptomics was performed to screen differentially expressed genes between 48- and 72-h seedlings after exposure to cryoprotectant. Among differentially expressed genes, oxidative stress response genes played important roles in cryoprotectant treatment, and peroxidation was a key factor related to cell survival. Seedlings underwent more peroxidation at 72-h than at 48-h. A comprehensive analysis indicated that peroxidation injured membrane systems leading to photophosphorylation and oxidative phosphorylation damage. Furthermore, the apoptosis-like events were found in cryogenic treatment of Arabidopsis seedlings. 48- and 72-h seedlings underwent different degrees of membrane lipid peroxidation during cryoprotectant treatment, and reducing the injury of oxidative stress was an important factor to successful cryopreservation. This study provided a novel insight of genetic regulatory mechanisms in cryopreservation, and established an excellent model to test and evaluate the effect of exogenous antioxidants and conventional cryoprotectants in plant cryopreservation. © 2013 Elsevier Ireland Ltd. Source

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