Ascorbate-glutathione cycle of mitochondria in osmoprimed soybean cotyledons in response to imbibitional chilling injury

Osmopriming treatment of chilling-sensitive soybean (Glycine max L. cv. Zhonghuang-22) seeds for 72 h at 25 °C with polyethylene glycol (PEG8000) solution at −1.5 MPa strongly improves chilling resistance. The aim of the present work was to investigate whether the beneficial effect of osmopriming is associated with restoration of the ascorbate-glutathione (ASC-GSH) cycle of mitochondria in soybean seeds. Compared with the control, both H₂O₂ and malondialdehyde (MDA) contents in mitochondria of osmoprimed seeds decreased after chilling treatment, and these changes were associated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and l-galactono-γ-lactone dehydrogenase (GLDH). However, the activity of dehydroascorbate reductase (DHAR) showed no obvious change during osmopriming treatment. Increased ASC and GSH contents accompanied prolonged osmopriming, and the reduced/oxidized ratios of ASC and GSH increased differently during osmopriming. These results indicate that osmopriming treatment enhances activity of the ASC-GSH cycle of mitochondria, which raises the chilling tolerance in soybean seeds and protects against H₂O₂ that is generated in mitochondria during imbibition at low temperature.     

How membranes organize during seed germination: three patterns of dynamic lipid remodelling define chilling resistance and affect plastid biogenesis

Imbibitional chilling injury during germination causes agricultural losses, but this can be overcome by osmopriming. It remains unknown how membranes reorganize during germination. Herein, we comparatively profiled changes of membrane lipids during imbibition under normal and chilling temperatures in chilling‐tolerant and ‐sensitive soybean seeds. We found three patterns of dynamic lipid remodelling during the three phases of germination. Pattern 1 involved a gradual increase in plastidic lipids during phases I and II, with an abrupt increase during phase III. This abrupt increase was associated with initiation of photosynthesis. Pattern 3 involved phosphatidic acid (PA) first decreasing, then increasing, and finally decreasing to a low level. Patterns 1 and 3 were interrupted in chilling‐sensitive seeds under low temperature, which lead a block in plastid biogenesis and accumulation of harmful PA, respectively. However, they were rescued and returned to their status under normal temperature after polyethylene glycol osmopriming. We specifically inhibited phospholipase D (PLD)‐mediated PA formation in chilling‐sensitive seeds of soybean, cucumber, and pea, and found their germination under low temperature was significantly improved. These results indicate that membranes undergo specific and functional reorganization of lipid composition during germination and demonstrate that PLD‐mediated PA causes imibibitional chilling injury.     

Terminal drought and seed priming improves drought tolerance in wheat

Plants retain the preceding abiotic stress memory that may aid in attainment of tolerance to subsequent stresses. This study was conducted to evaluate the influence of terminal drought memory (drought priming) and seed priming in improving drought tolerance in wheat (Triticum aestivum L.). During first growing season, wheat was planted in field under optimal (well-watered) and drought stress imposed at reproductive stage (BBCH growth stage 49) until maturity (BBCH growth stage 83). Seeds collected from both sources were subjected to hydropriming or osmopriming (with 1.5% CaCl₂ solution); while, dry seed was taken as control. Treated and control seeds, from both sources, were sown in soil filled pots. After the completion of seedling emergence, pots were maintained at 50% water holding capacity (drought) or 100% water holding capacity (well-watered). Drought stress suppressed the plant growth (2–44%), perturbed water relations (1–18%) and reduced yield (192%); however, osmolytes accumulation (3–14%) and malondialdehyde contents (26–29%) were increased under drought. The crop raised from the seeds collected from terminal drought stressed plants had better growth (5–63%), improved osmolyte accumulation (13–45%), and lower lipid peroxidation (3%) than the progeny of well-watered crop. Seed priming significantly improved the crop performance under drought stress as compared to control. However, osmopriming was more effective than hydropriming in this regard as it improved leaf area (9–43%), tissue water status (2–47%), osmolytes accumulation (6–48%) and grain yield (14–79%). In conclusion, terminal drought induced modifications in seed composition and seed priming improved transgenerational drought tolerance through improvement in tissue water status and osmolytes accumulation, and decrease in lipid peroxidation.     

Drought resistance in rice seedlings conferred by seed priming

Seed priming is a method by which seeds are subjected to different stress conditions to impart stress adaptation in seedlings germinating and growing under stressful situations. Drought stress is a major reason behind failure of crops. We studied the effects of hydropriming, dehydration priming (induced by PEG), and osmopriming (induced by NaCl and KH₂PO₄) on subsequent germination, growth and anti-oxidant defense mechanisms of 2-week-old rice seedlings under continuing dehydration stress. Unprimed seeds grown in PEG showed significantly lower germination and growth along with significantly higher reactive oxygen species (ROS) and lipid peroxidation levels. Among the priming methods, 5 % PEG priming was found to be the best in terms of germination and growth rate along with the lowest amount of ROS and lipid peroxidation (malondialdehyde [MDA]) values. MDA levels were reduced significantly by all of the priming methods. Hence, reduction of lipid peroxidation may be a key factor underlying the drought tolerance produced by the priming treatments. Glutathione peroxidase (GPX) activity seemed to bear an excellent correlation with oxidative stress resistance through seed priming. The PEG priming produced minimum peroxidative damage and superior germination and growth rate along with efficient GPX activity, overexpressed MnSOD and maintenance of HSP70 expression in normal as well as in drought condition. Therefore, in PEG-primed seeds the existence of robust protective mechanisms is definitely indicated.     

Drought tolerance of Periploca sepium during seed germination: antioxidant defense and compatible solutes accumulation

Periploca sepium Bunge is a native and widespread shrub on the Loess Plateau, an arid and semi-arid region in China. To understand the adaptability of its seed germination to dry environments, we investigated the germination rate, water relations, lipid peroxidation, antioxidant capacity and accumulation of major organic solutes during seed germination under water deficit conditions. Results showed that seeds pre-treated by hydration–dehydration or ?0.9 MPa PEG germinated faster than control seeds, indicating strong resistance of P. sepium to drought condition. The re-dried seeds showed higher proline, total free amino acids (TFAA) and soluble proteins (SP) contents than control dry seeds, indicating the maintenance of physiological advancement when dehydrated. Osmotic stress made seed germination stay on the plateau phase (phase II). However, germinating seeds moved into phase III immediately once transferred into distilled water. Large increases in SP and soluble sugars (SS) of both re-dried and osmotic stressed seeds help themselves to resist drought stress. The re-hydrated seeds showed significantly higher levels of proline, TFAA, SP and SS than control seeds. The largely accumulated SS during osmotic stress declined sharply when transferred into distilled water. Our data demonstrate that P. sepium’s tolerance to drought stresses during germination is associated with enhanced activity of antioxidant enzymes and accumulation of some compatible solutes. Seed physiological advancement progressed slowly under low water conditions and it was maintained when seeds were air dried. This strategy ensures high and more rapid seed germination of P. sepium under drying and wetting conditions in drought-prone regions.     

Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.     

Germination of salt-stressed seeds as related to the ethylene biosynthesis ability in three Stylosanthes species

Stylosanthes, a genus of tropical forage legume, is known to exhibit good persistence in saline soils, yet mechanisms for regulation of seed germination under salt stress are poorly understood. This study was carried out to evaluate the mode of action of salt stress on seed germination of Stylosanthes. 1-Aminocyclopropane-1-carboxylic acid (ACC) increased ethylene biosynthesis and germination of NaCl-inhibited seeds in a dose-dependent manner. Contents of ACC and germination of Stylosanthes humilis seeds increased following transfer from NaCl solution to deionised water, but not after transfer to l-α-(2-aminoethoxyvinyl)-glycine (AVG) solution, an inhibitor of ethylene biosynthesis. Ethylene biosynthesis was much larger in NaCl-treated seeds of Stylosanthes guianensis than in seeds of S. humilis and Stylosanthes capitata, a fact which was reflected in higher germination rates. S. guianensis seedlings also displayed higher growth and survival rates than S. humilis and S. capitata under salt stress. Moreover, smaller ACC levels, as well as reduced ethylene biosynthesis of S. capitata seeds were accompanied by lower germination under salt stress. In addition, S. capitata seedlings treated with NaCl solutions exhibited relatively lower growth and survival rates in comparison with S. humilis and S. guianensis. Thus, different abilities to synthesize ethylene by S. guianensis, S. humilis and S. capitata seeds explain the differences in tolerance to salt stress of the three species.     

Understanding the cellular mechanism of recovery from freeze–thaw injury in spinach: possible role of aquaporins,heat shock proteins,dehydrin and antioxidant system

Recovery from reversible freeze–thaw injury in plants is a critical component of ultimate frost survival. However, little is known about this aspect at the cellular level. To explore possible cellular mechanism(s) for post‐thaw recovery (REC), we used Spinacia oleracea L. cv. Bloomsdale leaves to first determine the reversible freeze–thaw injury point. Freeze (–4.5°C)–thaw‐injured tissues (32% injury vs <3% in unfrozen control) fully recovered during post‐thaw, as assessed by an ion leakage‐based method. Our data indicate that photosystem II efficiency (Fv/Fm) was compromised in injured tissues but recovered during post‐thaw. Similarly, the reactive oxygen species (O₂^?? and H₂O₂) accumulated in injured tissues but dissipated during recovery, paralleled by the repression and restoration, respectively, of activities of antioxidant enzymes, superoxide dismutase (SOD) (EC. 1.14.1.1), and catalase (CAT) (EC.1.11.1.6) and ascorbate peroxidase (APX) (EC.1.11.1.11). Restoration of CAT and APX activities during recovery was slower than SOD, concomitant with a slower depletion of H₂O₂ compared to O₂^??. A hypothesis was also tested that the REC is accompanied by changes in the expression of water channels [aquaporines (AQPs)] likely needed for re‐absorption of thawed extracellular water. Indeed, the expression of two spinach AQPs, SoPIP2;1 and SoδTIP, was downregulated in injured tissues and restored during recovery. Additionally, a notion that molecular chaperones [heat shock protein of 70 kDa (HSP70s)] and putative membrane stabilizers [dehydrins (DHNs)] are recruited during recovery to restore cellular homeostasis was also tested. We noted that, after an initial repression in injured tissues, the expression of three HSP70s (cytosolic, endoplasmic reticulum and mitochondrial) and a spinach DHN (CAP85) was significantly restored during the REC.     

Increased Chilling Tolerance Following Transfer of a betA Gene Enhancing Glycinebetaine Synthesis in Cotton (Gossypium hirsutum L.)

A betA gene encoding choline dehydrogenase from Escherichia coli was transformed into cotton (Gossypium hirsutum L.) via Agrobacterium-mediated transformation. Transgenic cotton plants exhibited improved tolerance to chilling due to accumulation of glycinebetaine (GB). The results of our experiment showed that GB contents of leaves of transgenic lines 1, 3, 4, and 5, both before and after chilling stress, were significantly higher than those of wild-type (WT) plants. At 15°C, transgenic lines 1, 3, 4, and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage and, displayed less inhibition in seedling shoot growth rate than WT plants. Under chilling stress, transgenic lines 4 and 5 maintained higher relative water content, upper carbon dioxide (CO₂) fixation capacity and PSII electron transfer rate, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation when compared with WT plants. Chilling resistance of the transgenic lines was demonstrated to be positively correlated with GB content under chilling stress. The high levels of GB in transgenic cotton plants might not only protect the integrity of cell membrane from chilling damage, but also be involved in OA which alleviated chilling induced water stress. Moreover, under chilling-stressed conditions, transgenic cotton plants enhanced stomatal conductance, PSII electron transport rate, and further leaf photosynthesis through accumulating high levels of GB.     

Inducing salt tolerance in maize (Zea mays L.) through seed priming with chloride salts: Growth and ion transport at early growth stages

The study was conducted to determine whether salt tolerance could be induced in maize at germination stage by soaking of seeds for 8 h in distilled water or in 200 meq·L⁻¹ of NaCl, KCl, CaCl₂·2H₂O. Both primed and un-primed seeds were subjected for 14 days to 0, 100 or 200 mol·m⁻³ NaCl under controlled conditions. Although all priming agents were effective in alleviating adverse effects of salt stress on maize at germination stage, CaCl₂·2H₂O proved to be more effective since the seeds primed with this salt had significantly higher final germination, rate of germination and fresh and dry weights of plumules and radicles than those treated with other salts or distilled water. Concentration of Na⁺, K⁺ and Ca²⁺ increased significantly in all parts of germinating seeds of maize seeds primed with NaCl, KCl, or CaCl₂·2H₂O, respectively. In addition, seeds primed with CaCl₂·2H₂O were the highest in Cl⁻ accumulation in all parts of the germinating seeds, followed by seeds treated with NaCl and KCl. Most of the Ca²⁺ was retained in seeds and mesocotyl, because of which, transport of this ion to plumules and radicles was low.     

Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea

Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration status.     

Temperature and water requirements for germination and effects of discontinuous hydration on germinated seed survival in Tillandsia recurvata L.

Tillandsia recurvata is an epiphytic bromeliad with a wide distribution in the Americas; however, little is known about the development of its post-seminal adaptations for survival in epiphytic environments. The purpose of this study was to define the temperature and water requirements for the germination of T. recurvata seeds. The absence of radicle emergence in T. recurvata seeds resulted in 2?stages of germination: swollen with broken seed coat (stage-1) and chlorophyllic embryos (stage-2). The effects of partial or discontinuous hydration on germinated seed survival were also assessed. The seeds were collected in a semi-arid shrubland of Mexico City. We explored: (1) whether water vapour can provide a sufficient water source for germination; (2) the temperature required for germination stage-1 and the optimal and critical osmotic potentials for germination in both germination stages; (3) the effect of seed incubation at different osmotic potentials that undergo subsequent dehydration on their survival in stage-2; and (4) the loss of dehydration tolerance during early post-seminal development. In addition, an image of T. recurvata seed anatomy was obtained to illustrate its structures. Germination stage-1 of T. recurvata seeds is rather similar across the tested temperature range. The seeds required to be in contact with liquid water to germinate. The interval of osmotic potential facilitating both germination stages was from 0 to ?0.6?MPa. Although germinated seeds displayed dehydration tolerance, this tolerance decreased in germination stage-2. The osmotic potential during germination affected the tolerance of the chlorophyllic embryos (stage-2) to subsequent dehydration.     

Differential antioxidation activities in two alfalfa cultivars under chilling stress

To understand the adaptability of alfalfa (Medicago sativa L.) to chilling stress, we analyzed the antioxidative mechanism during seed germination. The germination rates of six alfalfa cultivars were studied comparatively at 10°C. Xinmu No. 1 and Northstar were selected as chilling stress-tolerant and stress-sensitive cultivars for further characterization. After chilling treatment, Xinmu No. 1 showed higher seedling growth than Northstar. Xinmu No. 1 exhibited low levels of hydrogen peroxide and lipid peroxidation compared with Northstar. In addition, shoots in Xinmu No. 1 treated with chilling showed higher activities of the superoxide dismutase, ascorbate peroxidase (APX), and catalase than those of Northstar, whereas Xinmu No. 1 showed higher APX activity in roots that Northstar. These results indicated that high antioxidation activity in Xinmu No. 1 under chilling stress is well associated with tolerance to chilling condition during germination.     

Ectopic overexpression of vacuolar and apoplastic <Emphasis Type="Italic">Catharanthus roseus</Emphasis> peroxidases confers differential tolerance to salt and dehydration stress in transgenic tobacco

CrPrx and CrPrx1 are class III peroxidases previously cloned and characterized from Catharanthus roseus. CrPrx is known to be apoplastic in nature, while CrPrx1 is targeted to vacuoles. In order to study their role in planta, these two peroxidases were expressed in Nicotiana tabacum. The transformed plants exhibited increased peroxidase activity. Increased oxidative stress tolerance was also observed in transgenics when treated with H₂O₂ under strong light conditions. However, differential tolerance to salt and dehydration stress was observed during germination of T1 transgenic seeds. Under these stresses, the seed germination of CrPrx-transformed plants and wild-type plants was clearly suppressed, whereas CrPrx1 transgenic lines showed improved germination. CrPrx-transformed lines exhibited better cold tolerance than CrPrx1-transformed lines. These results indicate that vacuolar peroxidase plays an important role in salt and dehydration stress over cell wall-targeted peroxidase, while cell wall-targeted peroxidase renders cold stress tolerance.     

Ethylene antagonizes the inhibition of germination in Arabidopsis induced by salinity by modulating the concentration of hydrogen peroxide

The capacity of plants to achieve successful germination and early seedling establishment under high salinity is crucial for tolerance of plants to salt. The gaseous hormone ethylene has been implicated in modulating salt tolerance, but the detailed role of how ethylene modulates the response of early seedling establishment to salt is unclear. To better understand the role of the ethylene signal transduction pathway during germination and seedling establishment, an ethylene insensitive mutation (ein2-5) and an ethylene sensitive mutation (ctr1-1) of Arabidopsis were analyzed under saline conditions and compared with the wild type plant (Col-0) as control. High salinity (>100?mM NaCl) inhibited and delayed germination. These effects were more severe in the ethylene insensitive mutants (ein2-5) and less severe in the constitutive ethylene sensitive plants (ctr1-1) compared with Col-0 plants. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or inhibitors of ethylene action implied that ethylene was essential for early seedling establishment under normal and saline conditions. Salt stress increased the endogenous concentration of hydrogen peroxide (H₂O₂) in germinating seeds and ACC reduced its concentration. Our results suggest that ethylene promotes germination under salinity by modulating the endogenous concentration of H₂O₂ in germinating seeds. These findings demonstrate that ethylene is involved in regulating germination as an initiator of the process rather than consequence, and that ethylene promotes germination by modulating the endogenous concentration of H₂O₂ in germinating seeds under salinity.     

Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses

To understand the adaptability of alfalfa (Medicago sativa L.) to environmental stresses, we analyzed the activity of several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT), in alfalfa shoots and roots subjected to salt and drought stresses during germination. The germination rate of six alfalfa cultivars was comparatively studied under 200 mM NaCl or 35% PEG treatment. Alfalfa Xinmu No. 1 and Northstar varieties were selected as stress-tolerant and -sensitive cultivars, respectively, and were used for further characterization. After NaCl or PEG treatment, Xinmu No. 1 showed enhanced seedling growth, compared with Northstar. Xinmu No. 1 also exhibited low levels of hydrogen peroxide (H₂O₂) production and lipid peroxidation, compared with Northstar. In addition, Xinmu No. 1 showed higher enzymatic activity of SOD, APX, CAT, and POD in its shoots and roots than Northstar. These results seem to indicate that Xinmu No. 1 cultivar's tolerance to salt or drought stresses during germination is associated with enhanced activity of antioxidant enzymes. This study highlights the importance of antioxidant enzymes in the establishment of alfalfa seedlings under drought and salinity conditions typical of desertification.