Membrane phospholipids remodeling upon imbibition in Brassica napus L. seeds.

Successful seed germination depends on the rapid repair of cell membrane damaged by dry storage. However, little is known about the reorganization of lipids during this process. In this study, the changes of intracellular redox environment, cell membrane integrity, lipid composition, and expression of genes related to phospholipid metabolism were assessed during imbibition of Brassica napus seeds. A total number of 443 lipids belonging to 7 categories were detected by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). In the 24 h-imbibed seeds, the relative content of triacylglycerol was lower than in dry seeds, while the relative content of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), especially PC (36:2, number of carbons in the acyl chains: number of double bonds), PC (36:3), and PE (36:3) were higher than those in dry seeds. Meanwhile, the content and unsaturation levels of phospholipids increased, indicating membrane lipids remodeling during seed imbibition. The plasma membrane integrity, which was measured by the relative electrolyte leakage (REL) of the membrane and FM4-64 fluorescent dye, was improved upon imbibition, confirming that cell membrane was repaired after 24 h-imbibition. The reduction of H2O2 content, redox potential, and malondialdehyde (MDA) content indicated that the degree of membrane lipid peroxidation was significantly decreased upon imbibition. Gene expression analysis showed that the differential expression of genes for key enzymes occurred in the plateau phase of the imbibition curve, i.e. after 8 h-to 24 h-imbibition. Moreover, the differential expression of genes such as those encoding phospholipase C (PLC), phospholipase D (PLD), triacylglycerol lipase (TAG lipase), choline/ethanolamine phosphotransferase (CEPT), and phosphatidylserine synthase (PTDSS2) during imbibition indicated that membrane lipid remodeling was related to complex metabolic pathways, among which the degradation of triacylglycerol and the synthesis of phospholipids using diacylglycerol might play an important role during membrane remodeling.

Oxidative damage and antioxidative indicators in 48 h germinated rice embryos during the vitrification-cryopreservation procedure.

KEY MESSAGES: Cu/Zn SOD and other genes may be critical indicators of a stress response to reactive oxygen species (ROS) accumulation in 48 h germinated rice embryos subjected to vitrification cryopreservation. In the current study, reactive oxygen species (ROS) accumulation was investigated in 48 h germinated rice embryos during the vitrification-cryopreservation process. We found that vitrification-cryopreservation significantly affected ROS levels, especially superoxide anion levels, in 48 h germinated rice embryos. Malonaldehyde content in the apical meristems of germinated embryos was significantly positively correlated with the rate of superoxide anion generation and the highest levels of malonaldehyde content were reached after vitrification treatment. Cell viability in 48 h germinated embryos was significantly negatively correlated with the rate of superoxide anion generation, malonaldehyde content, and electrolyte leakage. Spatial and temporal patterns in ROS accumulation in these embryos existed during the vitrification procedure. Among the vitrification-cryopreservation treatments we assessed, the preculture treatment was found to stimulate superoxide anion generation and to activate the response system in the apical meristems of germinated embryos. Loading treatments motivated the catalase and ascorbate peroxidase activities. During the vitrification-dehydration treatment, oxidative stress reached the highest levels causing an antioxidative response. This response involved antioxidant enzymes promoting detoxification of ROS. Based on a comprehensive correlation analysis involving ROS accumulation, cell viability, the activities of antioxidant enzymes, and gene expression profiles, Cu/Zn SOD, CAT1, APX7, GR2, GR3, MDHAR1, and DHAR1 may be critical indicators of oxidative stress affected by the vitrification-cryopreservation treatments. The investigation of these antioxidative responses in 48 h germinated rice embryos may, therefore, provide useful information with respect to plant vitrification-cryopreservation.

CRYOPRESERVATION OF IN VITRO-GROWN SHOOT TIPS OF CHINESE MEDICINAL PLANT Atractylodes macrocephala KOIDZ. USING A DROPLET-VITRIFICATION METHOD.

BACKGROUND: Atractylodes macrocephala Koidz. is an important medicinal species from China that has been used for thousands of years for its special pharmacological antioxidant, hepatoprotective, anti-inflammatory, anti-allergic, antithrombotic, antiviral, and anticarcinogenic activities. OBJECTIVE: The aim of this research was to develop an efficient droplet-vitrification protocol for A. macrocephala shoot tips which could be used as a strategy for long-term conservation within gene banks. MATERIALS AND METHODS: The duration of preculture, loading, and PVS2 steps, as well as the recovery medium formulation, were optimized to achieve high levels of survival and regrowth for A. macrocephala shoot tips after liquid nitrogen exposure. RESULTS: Survival and regrowth levels after cryopreservation in the cultivar 'Baizhu' were as high as 76% and 62%, respectively. Thermal analysis using differential scanning calorimetry suggested that the PVS2 treatment plays a critical role for successful cryopreservation. CONCLUSION: The droplet-vitrification method established in this study could be used to cryopreserve A. macrocephala.

Dynamic Changes in Membrane Lipid Metabolism and Antioxidant Defense During Soybean (Glycine max L. Merr.) Seed Aging.

Seed viability depends upon the maintenance of functional lipids; however, how membrane lipid components dynamically change during the seed aging process remains obscure. Seed storage is accompanied by the oxidation of membrane lipids and loss of seed viability. Understanding membrane lipid changes and their effect on the cell membrane during seed aging can contribute to revealing the mechanism of seed longevity. In this study, the potential relationship between oxidative stress and membrane lipid metabolism was evaluated by using a non-targeted lipidomics approach during artificial aging of Glycine max L. Merr. Zhongdou No. 27 seeds. We determined changes in reactive oxygen species, malondialdehyde content, and membrane permeability and assessed antioxidant system activity. We found that decreased non-enzymatic antioxidant contents and catalase activity might lead to reactive oxygen species accumulation, resulting in higher electrolyte leakage and lipid peroxidation. The significantly decreased phospholipids and increased glycerolipids and lysophospholipids suggested that hydrolysis of phospholipids to form glycerolipids and lysophospholipids could be the primary pathway of membrane metabolism during seed aging. Moreover, the ratio of phosphatidylcholine to phosphatidylethanolamine, double bond index, and acyl chain length of phospholipids were found to jointly regulate membrane function. In addition, the observed changes in lipid metabolism suggest novel potential hallmarks of soybean seed aging, such as diacylglycerol 36:4; phosphatidylcholine 34:2, 36:2, and 36:4; and phosphatidylethanolamine 34:2. This knowledge can be of great significance for elucidating the molecular mechanism underlying seed aging and germplasm conservation.

Proteome analysis of maize seeds: the effect of artificial ageing.

Previous understanding of the mechanism of seed ageing is largely based on observations on imbibed seeds rather than dry seeds. The present research was conducted to investigate whether seed ageing has effects on the dry seeds through proteome analysis. Maize (Zea mays cv. Dabaitou) seeds were artificially aged at 50°C (13.58% moisture content) for 5 or 13 days, and the total protein was extracted from embryos of the dry seeds. Two-dimensional electrophoresis was performed and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. A total of 40 proteins were identified, in which 16 proteins were upregulated, indicating that artificial ageing affected the proteome of the dry seeds. Proteomic studies revealed that the signal transduction and transcription were disturbed by artificial ageing, which might lead to reduced protection against ageing. Artificial ageing also increased proteases and broke down stored proteins, impaired metabolism and energy supply, and ultimately resulted in seed deterioration. Proteins involved in metabolism and energy were the largest downregulated protein group, with regard to glycolysis, tricarboxylic acid cycle, the electron transport chain and oxidative phosphorylation. The downregulation of these proteins, together with reduction in the specific activity of glucose-6-phosphate dehydrogenase, and the content of glucose 6-phosphate, pyruvic acid and ATP in aged seeds, suggested the important roles of the mobilization of stored carbohydrates and energy supply in seed ageing and seed vigor. The present work provides new information about the proteomic changes during seed ageing and provides a possible mechanism for seed deterioration.

Timing for antioxidant-priming against rice seed ageing: optimal only in non-resistant stage.

Seed deterioration due to ageing strongly affects both germplasm preservation and agricultural production. Decelerating seed deterioration and boosting seed viability become increasingly urgent. The loss of seed viability is inevitable even under cold storage. For species with short-lived seed or for regions with poor preservation infrastructure where cold storage is not readily available, seed enhancement is more reliable to increase seed viability and longevity. Antioxidant priming as a way of seed enhancement usually improves seed germination. As for post-priming survival, however, significant uncertainty exists. The controversy lies particularly on seeds of high germination percentage (GP > 95%) whose viability is hardly improvable and the benefits of priming depend on prolonging seed longevity. Therefore, this study timed antioxidant priming to prolong the longevity of high-viability seeds under artificially accelerated ageing (AAA). Rice (Nipponbare) seeds (GP > 97%) under room-temperature-storage (RTS) for 6 months. were resistant to AAA first with little viability loss for a certain period, the resistant stage. This resistance gradually vanished without GP change, during a prolonged RTS period which was named the vulnerable stage. According to the results, although antioxidant priming severely curtailed the resistant stage for seeds with a long plateau in the survival curve, it decelerated viability loss for seeds in the vulnerable stage. In complement to seed storage, priming potentially retains high seed GP which would decrease without seed enhancement. To maximize the benefits of priming for high-GP seeds, two time points are advised as the start of a time window for priming: (1) just at the end of the resistant stage without notable viability loss, which is hard to grasp by GP monitoring; (2) slight but identifiable GP decline.