Di Zhang

Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings

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 48 h or 72 h 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.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

Key messageOxidative stress and apoptosis-like programmed cell death, induced in part by H2O2, are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability.AbstractWe hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O2·−, H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA–GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85xa0% and 89.91xa0%, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation

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.

A comprehensive analysis of flowering transition in Agapanthus praecox ssp. orientalis (Leighton) Leighton by using transcriptomic and proteomic techniques.

Comprehensive transcriptomic and proteomic analyses were performed to gain further understanding of the molecular mechanisms of floral initiation in Agapanthus praecox ssp. orientalis. Samples of stem apexes were collected at three different time points including the vegetative, induced, and reproductive period. A total of 374 transcript-derived fragments and 72 proteins showed significant differential expression between the samples. The largest proportion of the identified genes and proteins are involved in metabolism, followed by signal transduction, protein fate, cellular transport, and biogenesis of cellular components. A large number of these genes and proteins were upregulated during the induced and reproductive stages. Their expression profiles demonstrate that carbohydrate metabolism provides nutrients foundation for floral initiation in Agapanthus. Furthermore, a transcription factors GAI (GA insensitive protein) that negatively regulates gibberellin signaling, auxin receptor protein TIR1 (Transport inhibitor response 1), a key enzyme of ethylene biosynthesis SAMS (S-adenosylmethionine synthase), and ethylene receptor protein ETR were isolated and identified. Expression patterns of these proteins, in combination with the results of quantitative phytohormone and immunolocalization analyses, indicated that GA, indole-acetic acid (IAA), and ethylene regulate floral morphogenesis and flowering. In conclusion, these data provide novel insight into the early regulatory steps of flowering in Agapanthus.

GA4 and IAA were involved in the morphogenesis and development of flowers in Agapanthus praecox ssp. orientalis.

The transition from vegetative to reproductive growth represents a major phase change in angiosperms. Hormones play important roles in this process. In this study, gibberellic acid (GA), cytokinins (CKs), indoleacetic acid (IAA), and abscisic acid (ABA) were analyzed during the flowering in Agapanthus praecox ssp. orientalis. Eleven types of endogenous gibberellins in addition to GA1 were detected in various organs. GA9 was detected with the highest concentrations, followed by GA5, GA8, and GA19. However, GA4 was the main bioactive GA that was involved in the regulation of flowering. Eight types of endogenous cytokinins were detected in A. praecox ssp. orientalis, and zeatin, zeatin riboside, zeatin-O-glucoside, and N(6)-isopentenyladenosine-5-monophosphate were present at higher levels throughout the study, of which zeatin plays an important role in the development of various organs. IAA increased by 581% in the shoot tips from the vegetative to inflorescence bud stages and had the most significant changes during flowering. Phytohormone immunolocalization analysis suggested that IAA involved in differentiation and development of each floral organs, GA and zeatin play important roles in floret primordia differentiation and ovule development. Using exogenous plant growth regulators proved that GA signaling regulate the scape elongation and stimulate early-flowering, and IAA signaling is involved in the pedicel and corolla elongation and delay flowering slightly.

Transcriptomic profiling revealed the regulatory mechanism of Arabidopsis seedlings response to oxidative stress from cryopreservation

Key messageElevated 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.AbstractCryoprotectants 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.

RNA-Seq-based transcriptome analysis of stem development and dwarfing regulation in Agapanthus praecox ssp. orientalis (Leighton) Leighton

Agapanthus praecox is a monocotyledonous ornamental bulb plant. Generally, the scape (inflorescence stem) length can develop more than 1m, however application 400 mg·L(-1) paclobutrazol can shorten the length beyond 70%. To get a deeper insight into its dwarfism mechanism, de novo RNA-Seq technology has been employed, for the first time, to describe the scape transcriptome of A. praecox. We got 71,258 assembled unigenes, and 45,597 unigenes obtained protein functional annotation. Take the above sequencing results as a reference gene set, using RNA-seq (quantification) technology analyzed gene expression profiles between the control and paclobutrazol-treated samples, and screened 2838 differentially expressed genes. GO, KEGG and MapMan pathway analyses indicated that these differentially expressed genes were significantly enriched in response to stimulus, hormonal signaling, carbohydrate metabolism, cell wall, cell size, and cell cycle related biological process. To validate the expression profiles obtained by RNA-Seq, real-time qPCR was performed on 24 genes selected from key significantly enriched pathways. Comprehensive analysis suggested that paclobutrazol blocks GA signal that can effectively inhibit scape elongation; the GA signal interact with other hormonal signals including auxin, ethylene, brassinosteroid and cytokinins, and trigger downstream signaling cascades leading to metabolism, cell wall biosynthesis, cell division and the cycle decreased obviously, and finally induced dwarfism trait. Furthermore, AP2/EREBP, bHLH, C2H2, ARR, WRKY and ARF familys transcription factors were involved in the regulation of scape development in A. praecox. This transcriptome dataset will serve as an important public information platform to accelerate research on the gene expression and functional genomics of Agapanthus.

Gibberellin and auxin signals control scape cell elongation and proliferation in Agapanthus praecox ssp. orientalis

Plant height is determined by the processes of cell proliferation and elongation. Plant hormones play key roles in a species-dependent manner in these processes. We used paclobutrazol (PAC) at 400 mg·L-1 in this study to spray Agapanthus praecox ssp. orientalis plants in order to induce dwarf scape (inflorescence stem). Morphological examination showed that PAC reduced scape height by inhibiting the cell elongation by 54.56% and reducing cell proliferation by 10.45% compared to the control. Quantification and immunolocalization of endogenous gibberellins (GAs) and indole-3-acetic acid (IAA) showed that the GA1, GA3, and GA4 levels and the IAA gradient were reduced. Among these hormones, GA4 was the key component of GAs, which decreased 59.51-92.01% compared to the control in scape. The expression of cell wall synthesis related genes cellulose synthase (CESA) and UDP-glucuronic acid decarboxylase (UXS) were upregulated significantly, whereas cell wall loosening gene xyloglucan endotransglucosylase 2 (XET2) was downregulated by 99.99% surprisingly. Correlation analysis suggested GA regulated cell elongation and auxin modulated cell proliferation in Agapanthus scape. Additionally, the accumulation of sugars played roles in cell wall synthesis and cell expansion. These results indicated GA and IAA signals triggered a downstream signaling cascade, controlled cell expansion and proliferation during scape elongation.

Cloning and characterization of ApCystatin , a plant cystatin gene from Agapanthus praecox ssp. orientalis responds to abiotic stress

Plant cystatins are involved in the regulation of protein turnover and play important roles in defense mechanisms. We cloned the ApCystatin gene from Agapanthus praecox ssp. orientalis, a famous ornamental and medical plant. The complete cDNA sequence of ApCystatin is comprised of 1439 nucleotides with a 423 bp ORF encoding 140 amino acids. The mRNA level of ApCystatin was significantly up-regulated under various abiotic stress, such as salt, osmosis, oxidative and cold stresses, which suggested that ApCystatin participated in the plants resistance to stress. The recombinant ApCystatin fusion protein expressed in E. coli transetta (DE3) cells was approximate 18u202fkDa. 25u202fμg of ApCystatin inhibited more than 95% activity of papain, suggesting ApCystatin as a papain-like protease inhibitor. As an exogenous substance, 1.60u202fμg/mL ApCystatin protein improved the regrowth percentage of Arabidopsis 60-h seedlings after cryopreservation from 30% to 47%. In addition, the relative survival rate of A. praecox embryogenic callus after cryopreservation also increased for 30% with addition of 1.20u202fμg/mL ApCystatin protein. This indicated that ApCystatin performed protective property against cryoinjury to Arabidopsis 60-h seedlings and A. praecox embryogenic callus during cryopreservation. Under various abiotic stress conditions, the recombinant ApCystatin protein showed significant advantage in growth rates at NaCl, mannitol, PEG6000, cold, acidic and alkaline conditions, compared to control. In conclusion, ApCystatin as a new member of plant cystatins exhibited protective property against cryoinjury in plant cryopreservation and abiotic stress in E. coli.