Revisiting the pericarp as a barrier restricting water entry/loss from cotyledons and embryonic axis of temperate desiccation-sensitive Quercus acorns.

MAIN CONCLUSIONS: Fully mature acorns of Quercus variabilis, Q. aliena, Q. mongolica, and Q. glandulifera are desiccation-sensitive. X-ray computer tomography showed that cotyledons shrink during drying, but embryos are protected. Information available on recalcitrant acorns of tropical and sub-tropical species of Quercus suggests that an impermeable pericarp, which limits the entry and loss of water only through the hilum (scar), is the underlying mechanism that prevents drying of the embryo axis following dispersal until the germination season. However, there is a lack of consensus supporting this proposition across species, and it is not well understood if such mechanisms occur in temperate Quercus species. This study investigated the significance of the acorn pericarp for temperate oak species and presents an ecological framework based on the post-dispersal climatic conditions. Using Quercus variabilis, Q. aliena, Q. mongolica, and Q. glandulifera acorns, the relationship between moisture content (MC) and germination was established, and X-ray computed tomography (X-ray CT) was used to understand the internal structural changes of cotyledons and embryonic axis occurring during desiccation. Water entry and exit routes through the scar, pericarp and apex were determined by imbibition and drying experiments. Climatic data and acorn morphological characteristics and germination were subjected to a principal component analysis (PCA). Freshly dispersed acorns of all species had a moisture content (MC) above 35% fresh weight (FW) basis, but drying to 15-10% MC resulted in complete loss of viability, implying recalcitrance behaviour. X-ray CT images suggested that the pericarp offers some protection to cotyledons and embryonic axis during desiccation, but it is contingent on MC. Extensive drying to a low MC with the scar and apex covered with vaseline resulted in internal tissues shrinkage, corresponding with viability loss. Water could enter or exit through the pericarp, albeit at a much slower rate than through the scar. A combination of factors including acorn anatomy, moisture content at the time of dispersal, microhabitat, the position of acorns in the soil prevent embryo desiccation below the critical MC and thus promotes survival of acorns on/in the soil during winter in temperate regions. Pericarp anatomy, to some extent, prevents excessive drying of the embryonic axis by slowing water movement, but prolonged drying or predatory pressure could result in pericarp cracks, favouring the absorption of water during sporadic rain. In the latter case, the survival of acorns possibly depends extensively on the continuous erratic rainfall, i.e. continuous wet-dry cycle, but in-situ experiments are yet to be performed to test this hypothesis.

Unravelling the paradox in physically dormant species: elucidating the onset of dormancy after dispersal and dormancy-cycling.

BACKGROUND: For species that produce seeds with a water-impermeable coat, i.e. physical dormancy (PY), it has been widely recognized that (1) seeds shed at a permeable state cannot become impermeable after dispersal; and (2) dormancy-cycling, i.e. a permeable ↔ impermeable transition, does not occur. Given a tight relationship between moisture content and onset of seed-coat impermeability, seeds maturing at low relative humidity (RH) and occurring in a high-temperature environment are inferred to produce impermeable coats, and ex situ drying of permeable seeds can lead to the onset of impermeability. SCOPE AND CONCLUSION: It is proposed here that permeable seeds dispersed at low RH and in high-temperature soils might become impermeable due to continuous drying. Similarly, seeds with shallow PY dormancy (with higher moisture content immediately after becoming impermeable) can cycle back to a permeable state or absolute PY (complete dry state) when RH increases or decreases, respectively. A conceptual model is developed to propose that seeds from several genera of 19 angiosperm families at the time of natural dispersal can be (1) impermeable (dormant), i.e. primary dormancy; (2) impermeable (dormant) and become permeable (non-dormant) and then enter a dormant state in the soil, often referred to as secondary dormancy; (3) permeable (non-dormant) and become impermeable (dormant) in the soil, i.e. enforced dormancy; or (4) dormant or non-dormant, but cycle between permeable and non-permeable states depending on the soil conditions, i.e. dormancy-cycling, which is different from sensitivity-cycling occurring during dormancy break. It is suggested that this phenomenon could influence the dormancy-breaking pattern, but detailed studies of this are lacking.

Cryopreservation of human T lymphocytes under fast cooling with controlled ice nucleation in cryoprotective solutions of low toxicity.

Cryopreservation of human T lymphocytes has become an essential tool for some cell-based immunotherapy. However, the cryopreservation procedure of the cells has not been systematically studied. In particular, the key factors of ice seeding and cryoprotective agents (CPA) driving the success of cryopreservation remain unclear. We systematically investigated the key factors, including cooling rate, ice-seeding temperature, CPA concentration, and types of CPA, during cryopreservation of human T lymphocytes with controlled ice nucleation. We found that ice seeding at below -10 °C could enable human T lymphocytes to be cooled at 90 °C min-1 with high relative viability and recovery after rewarming, 94.9% and 90.2%, respectively, which are significantly higher than those without ice seeding (P < 0.001). After optimization, the concentration of dimethyl sulphoxide was as low as 2% (v/v) with relative viability and recovery of 95.4% and 100.8%, respectively, at the cooling rate of 90 °C min-1 after ice seeding at -16 °C. The cryopreservation procedure developed in this study could facilitate the understanding of the mechanism for ice seeding and cell injury and offer a promising cryopreservation method with a high cooling rate and extremely low toxicity for extensive clinical application of immunotherapy.

Investigating solution effects injury of human T lymphocytes and its prevention during interrupted slow cooling.

Cooling rate is a critical parameter affecting the success of cell cryopreservation. Fast cooling can result in intracellular ice formation (IIF), while slow cooling can bring solution effects injury, both are detrimental to the cells. Whilst most of the studies have investigated how IIF affects cells, solution effects injury has received little attention. Here, we studied the solution effects injury of human T lymphocytes by cryomicroscopy and tested the osmoprotective ability of some frequently used cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, trehalose, urea and l-proline. We further investigated the relationship between cell volume, latent heat and solution effects cell injury. We found that solution effects injury during interrupted slow cooling was caused by high concentration of the extracellular solution rather than eutectic formation and solutes precipitation. DMSO, glycerol and trehalose can protect cells from solution effects injury, while l-proline and urea cannot under the same condition. The cell volume and latent heat are not crucial for causing solution effects injury in cells. This work confirms that high osmotic pressure, rather than eutectic formation, leads to cell injury. It also suggests that cell volume and latent heat may not be a key factor for explaining solution effects injury and its prevention in the cryopreservation of human T lymphocytes.

Experimental Warming Hastens Physical Dormancy Break and Germination in Tropical Fabaceae.

Climate warming may threaten the germination strategies of many plants that are uniquely adapted to today's climate. For instance, species that employ physical dormancy (PY) - the production of seeds that are impermeable to water until high temperatures break them, consequently synchronizing germination with favorable growing conditions - may find that their seeds germinate during unfavorable or potentially fatal periods if threshold temperatures are reached earlier in the year. To explore this, we subjected the seeds of five species with physical dormancy (from the genera Abrus, Bauhinia, Cassia, Albizia, and Acacia) to "mild" (+2°C) and "extreme" (+4°C) future warming scenarios and documented their germination over 2 years relative to a control treatment. Under current climatic conditions, a proportion of seeds from all five species remained dormant in the soil for 2 years. A mild warming of 2°C had little to no effect on the germination of four of the five study species. Contrastingly, an extreme warming of 4°C dramatically increased germination in all five species within the first year, indicating a reduction in their ability to persist in the soil long-term. Cassia fistula was particularly susceptible to warming, exhibiting a similar increase in germination under both mild and extreme warming relative to control. Our findings suggest that climate warming in the tropics may cause the seeds of species that rely on physical dormancy to stagger the risk of unsuccessful germination across years to leave soil seed banks prematurely - the long-term implications of which remain unknown.

Mechanisms underpinning the onset of seed coat impermeability and dormancy-break in Astragalus adsurgens.

Impermeable seed coats, i.e. physical dormancy (PY) influence the germination ecology of plants from 18 angiosperm families. Astragalus adsurgens (Fabaceae; Papilinoidaae) is a perennial plant widespread in temperate regions that is thought to produce both permeable and impermeable seeds. Why seeds vary in the permeability of their coat, in addition to the mechanisms by which impermeable seeds break dormancy, are not completely understood. However, seeds are often consumed by herbivores; a phenomenon that might facilitate the germination of impermeable seeds. Here, we tested whether: (1) moisture content plays a significant role in the onset of seed coat impermeability (and therefore PY) at similar ranges reported for species from tropical ecosystems; and (2) the presence of impermeable coats offer any benefits for seed survival when consumed by animals. We tested these hypotheses using A. adsurgens seeds collected from Inner Mongolia, China. Freshly collected seeds with a moisture content of 9.7% were permeable to water and therefore not physically dormant. However, seeds became impermeable when dried below a threshold of 6.5% moisture content. Treating impermeable seeds with hydrochloric acid effectively broke dormancy. Scanning electron microscope (SEM) revealed that HCl treated seeds had a narrow opening in the hilum and extra-hilar regions, through which water entered. Seeds with impermeable coats survived significantly better than permeable seeds when consumed by cows. Irrespective of coat permeability, most seeds were egested between 12 and 24 h. In seeds that maintained dormancy after gut passage, this was broken by additional acid scarification. Overall results suggest that: (1) seed coat impermeability is induced by reduced moisture content; (2) imbibition primarily occurs at the hilum and extra-hilar region; and (3) impermeable seeds may benefit from endozoochory.

Determination of the water gap and the germination ecology of Adenanthera pavonina (Fabaceae, Mimosoideae); the adaptive role of physical dormancy in mimetic seeds.

Dormancy caused by impermeable seed coats, i.e. physical dormancy (PY), regulates the timing of seed germination in species of several genera belonging to 18 angiosperm families. Physical dormancy also occurs in some mimetic species whose seeds mimic brightly coloured, fleshy fruits or arilled seeds. However, the conditions that break dormancy, as well as the location of water gaps in mimetic seeds, remain unclear. Here, we investigated the adaptive role of impermeable coats in the mimetic seeds of Adenanthera pavonina (Fabaceae: Mimosoideae). Specifically, we explored: (i) the conditions that break PY; (ii) the location of the primary water gap that forms during dormancy break; and (iii) the effect of seasonal temperature regimes on seed germination. Seeds were subjected to hot-water treatment, rapid temperature fluctuations and storage at temperatures mimicking summer and autumn conditions. Seed coat anatomy and water-gap regions were characterized using scanning electron microscopy (SEM) and light microscopy. Seeds were artificially buried in the field at 3 and 7 cm depths and exhumed every 6 months for 2 years to monitor germination. Adenanthera pavonina had impermeable seed coats, and thus PY. Seeds treated with hot water and exposed to summer-autumn temperature regimes broke dormancy. Water entered only through the lens (Type-II simple) due to dislodgement of the palisade layer. Seeds buried at 3 cm depth had significantly higher germination than those buried at 7 cm depth, with germination primarily occurring in autumn. Seeds required high summer temperatures followed by moderate autumn temperatures to become permeable to water and germinate in the field during the wet season. We conclude that the impermeable seed coat of A. pavonina is an adaptation that synchronizes germination with the growing season.

Physiological Mechanisms Only Tell Half Story: Multiple Biological Processes are involved in Regulating Freezing Tolerance of Imbibed Lactuca sativa Seeds.

The physiological mechanisms by which imbibed seeds survive freezing temperatures in their natural environment have been categorized as freezing avoidance by supercooling and freezing tolerance by extracellular freeze-desiccation, but the biochemical and molecular mechanisms conferring seed freezing tolerance is unexplored. In this study, using imbibed Lactuca sativa seeds we show that fast cooled seeds (60 °C h-1) suffered significantly higher membrane damage at temperature between -20 °C and -10 °C than slow cooled (3 °Ch-1) seeds (P < 0.05), presumably explaining viability loss during fast cooling when temperature approaches -20 °C. Total soluble sugars increase in low temperature environment, but did not differ significantly between two cooling rates (P > 0.05). However, both SOD activity and accumulation of free proline were induced significantly after slow cooling to -20 °C compared with fast cooling. RNA-seq demonstrated that multiple pathways were differentially regulated between slow and fast cooling. Real-time verification of some differentially expressed genes (DEGs) revealed that fast cooling caused mRNA level changes of plant hormone and ubiquitionation pathways at higher sub-zero temperature, whilst slow cooling caused mRNA level change of those pathways at lower sub-zero ttemperatures. Thus, we conclude that imbibed seed tolerate low temperature not only by physiological mechanisms but also by biochemical and molecular changes.

Devitrification and recrystallization of nanoparticle-containing glycerol and PEG-600 solutions.

Nanoparticles in solution offer unique electrical, mechanical and thermal properties due to their physical presence and interaction with the state of dispersion. This work is aimed to study the effects of hydroxyapatite (HA) nanoparticles on the devitrification and recrystallization events of two important cryoprotective solutions used in cell and tissue preservation namely glycerol (60%w/w) and PEG-600 (50%w/w). HA nanoparticles (20, 40 or 60 nm) were incorporated into solutions at the content of 0.1% or 0.5%(w/w), and were studied by differential scanning calorimeter (DSC) and cryomicroscopy. The presence of nanoparticles does not change the glass transition temperatures and melting temperatures of quenched solutions, but significantly affects the behavior of devitrification and recrystallization upon warming. Cryomicroscopic investigation showed the complex interactions among solution type, nanoparticle size and nanoparticle content, which apparently influence ice crystal growth or recrystallization in the quenched dispersions. These findings have significant implications for biomaterial cryopreservation, cryosurgery, and food manufacturing. The complexity of ice crystal growth kinetics in nanoparticle-containing dispersions remains to be poorly understood at the moment.