From seed longevity to passive defense against pathogens: co-evolution of two traits to remain alive in the dry state?

In orthodox seeds, longevity is gradually acquired at the later stages of seed maturation. In our laboratory, we use seeds of Medicago truncatula, a model species for legumes, to unravel the mechanisms and regulatory pathways implicated in the acquisition of longevity. In this species, longevity increases progressively over 30-fold after seed filling is terminated and desiccation tolerance is acquired, allowing the separation of genes related to the different developmental  processes. In order to further discriminate other developmental programs from those related to longevity, an extensive physiological and molecular analysis was undertaken in developing seeds from  plants that were grown under different environmental conditions after seed set. This way, the timing and extent to which longevity is acquired was modulated. Using 104 transcriptomes acquired at different time points during seed maturation for five different parental environments, a network-based approach was applied to isolate a co-expression module related to longevity. Functional analysis in Arabidopsis confirmed the predictability and the conserved nature of this module. Interestingly, the longevity module is enriched with genes playing a crucial role in defense against biotic stress. Here, we will present evidence supporting a link between mechanisms implicated in defense against pathogens and survival in the dry state. We will discuss how seeds activate a developmentally regulated defense response during maturation that is also beneficial to long-term survival in the dry state

Simulation of radio emission from air showers in atmospheric electric fields

We study the effect of atmospheric electric fields on the radio pulse emitted by cosmic ray air showers. Under fair weather conditions the dominant part of the radio emission is driven by the geomagnetic field. When the shower charges are accelerated and deflected in an electric field additional radiation is emitted. We simulate this effect with the Monte Carlo code REAS2, using CORSIKA-simulated showers as input. In both codes a routine has been implemented that treats the effect of the electric field on the shower particles. We find that the radio pulse is significantly altered in background fields of the order of ~100 V/cm and higher. Practically, this means that air showers passing through thunderstorms emit radio pulses that are not a reliable measure for the shower energy. Under other weather circumstances significant electric field effects are expected to occur rarely, but nimbostratus clouds can harbor fields that are large enough. In general, the contribution of the electric field to the radio pulse has polarization properties that are different from the geomagnetic pulse. In order to filter out radio pulses that have been affected by electric field effects, radio air shower experiments should keep weather information and perform full polarization measurements of the radio signal.Comment: 26 pages, 12 figures, accepted for publication in Astroparticle Physic

Monte Carlo simulations of air showers in atmospheric electric fields

The development of cosmic ray air showers can be influenced by atmospheric electric fields. Under fair weather conditions these fields are small, but the strong fields inside thunderstorms can have a significant effect on the electromagnetic component of a shower. Understanding this effect is particularly important for radio detection of air showers, since the radio emission is produced by the shower electrons and positrons. We perform Monte Carlo simulations to calculate the effects of different electric field configurations on the shower development. We find that the electric field becomes important for values of the order of 1 kV/cm. Not only can the energy distribution of electrons and positrons change significantly for such field strengths, it is also possible that runaway electron breakdown occurs at high altitudes, which is an important effect in lightning initiation.Comment: 24 pages, 19 figures, accepted for publication in Astroparticle Physic

Desiccation tolerance: From genomics to the field

Desiccation tolerance is defined as the ability to survive the removal of all, or almost all the cellular water without irreversible damage. It confers to dried organisms the ability to survive extreme conditions of the environment and to stay alive in a suspended animation for long periods of time. The biotechnological potential of anhydrous biology has been recognized for more than 60 years. With the fast development of “omics” technologies, it is now possible to better appreciate the biotechnological promises that can be made from the understanding of desiccation tolerance. This review will discuss the impact of post-genomics tools on identifying genes or gene products, and will give a comprehensive overview of the agronomical and biotechnological applications. We propose the term desiccomics to define the approach consisting of combining “omics” approaches to address the specific issues associated with the dry state

Cosmic-ray energy spectrum and composition up to the ankle - the case for a second Galactic component

We have carried out a detailed study to understand the observed energy spectrum and composition of cosmic rays with energies up to ~10^18 eV. Our study shows that a single Galactic component with subsequent energy cut-offs in the individual spectra of different elements, optimised to explain the observed spectra below ~10^14 eV and the knee in the all-particle spectrum, cannot explain the observed all-particle spectrum above ~2x10^16 eV. We discuss two approaches for a second component of Galactic cosmic rays -- re-acceleration at a Galactic wind termination shock, and supernova explosions of Wolf-Rayet stars, and show that the latter scenario can explain almost all observed features in the all-particle spectrum and the composition up to ~10^18 eV, when combined with a canonical extra-galactic spectrum expected from strong radio galaxies or a source population with similar cosmological evolution. In this two-component Galactic model, the knee at ~ 3x10^15 eV and the second knee at ~10^17 eV in the all-particle spectrum are due to the cut-offs in the first and second components, respectively. We also discuss several variations of the extra-galactic component, from a minimal contribution to scenarios with a significant component below the ankle (at ~4x10^18 eV), and find that extra-galactic contributions in excess of regular source evolution are neither indicated nor in conflict with the existing data. Our main result is that the second Galactic component predicts a composition of Galactic cosmic rays at and above the second knee that largely consists of helium or a mixture of helium and CNO nuclei, with a weak or essentially vanishing iron fraction, in contrast to most common assumptions. This prediction is in agreement with new measurements from LOFAR and the Pierre Auger Observatory which indicate a strong light component and a rather low iron fraction between ~10^17 and 10^18 eV.Comment: Added Table 4; Published in A&A, 595 (2016) A33 (Highlight paper

Biological glasses : nature's way to preserve life

As a result of drying, the cytoplasm of desiccation-tolerant organisms, such as seed and pollen, enters into a highly viscous, solid-like, semi-equilibrium state: the glassy state. The work in this dissertation is focussed on the function and characteristics of intracellular glasses in these organisms.It was established that intracellular glasses are formed in both desiccation-tolerant and -intolerant pollen (chapter 1). However, desiccation-intolerant pollen loses its viability during drying before intracellular glasses are formed. This indicates that desiccation tolerance is not related with the formation of glasses during drying. Storage of cattail ( Typha latifolia ) pollen under different water contents and temperatures revealed the existence of an optimum water content for survival at a constant relative humidity (11-15%) (chapter 2). The water content corresponding to this relative humidity shifted to higher values with lower storage temperatures, and was found to be associated with the Brunauer, Emmet, and Teller monolayer value. Drying of the pollen below these water contents had detrimental effects on longevity. The water content-temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. There was a change in ageing kinetics of cattail pollen associated with the melting of the intracellular glass. Above the glass transition temperature (T g ) the activation energy of the ageing rates increased two to three times. This suggests that the presence of glasses in the dry state improves storage stability by decreasing viscosity and, thus, ageing rate. It was concluded that T g curves might be useful for predictions of storage longevity above optimum water contents. However, they cannot be used solely to predict the precise conditions of optimum storage. Subsequently, we sought for a more direct measurement to assess the viscosity of the cytoplasm of tissues.For this purpose, we used electron paramagnetic resonance (EPR) spectroscopy to study the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl (CP) that was incorporated into embryonic axes of pea seeds and cattail pollen. Using the distance between the outer extrema of the EPR spectrum (2 A zz ) as a measure of molecular mobility, a sharp increase in mobility was observed at a definite temperature during heating (chapter 3). This temperature increased with decreasing water content of the samples, and was found to be associated with the melting of the glassy state as measured by differential scanning calorimetry (DSC). Molecular mobility was found to be inversely correlated with storage stability: the higher the molecular mobility, the shorter the longevity: with decreasing water content, the molecular mobility reached a minimum, in concert with ageing rates. At very low water contents, both molecular mobility and ageing rates increased again. Minimum mobility and maximum storage stability occurred at similar water contents, suggesting that storage stability might be partially controlled by molecular mobility. To understand the nature of the changes in 2 A zz in spectra of CP in the tissues, echo detected (ED) EPR spectroscopy was employed (chapter 4). The shape of the ED EPR spectrum revealed the presence of librational motion of the spin probe, a motion typically present in glassy materials. The change in 2 A zz appeared to be the result of librational motion of the spin probe.With the use of saturation transfer (ST) EPR spectroscopy, a more quantitative measure of molecular mobility was acquired: the rotational correlation time (τ R ), which corresponds to the time it takes for the spin probe to rotate a radian around its axis (chapter 5). At room temperature,τ R of CP in pea embryonic axes increased during drying from 10 -11 s in de hydrated state to 10 -4 s in the dry state. At T g ,τ R was constant at10 -4 s for all water contents studied. The temperature dependence ofτ R at all water contents studied followed Arrhenius behaviour with a break at T g . The temperature effect onτ R above T g was much smaller in pea axes as found previously for sugar and polymer glasses. Thus, the melting of the intracellular glass by raising the temperature caused only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.The application of saturation transfer EPR spectroscopy to biological tissues enabled a quantitative comparison between storage stability and molecular mobility in different tissues (section III). The temperature and moisture dependence of ageing rates of seeds and pollen was found to correlate with the rotational motion of CP in the cytoplasm (chapter 6-8). An increase in the temperature resulted in a faster rotational motion in the cytoplasm of cattail pollen, analogous to faster ageing rates (chapter 6). Decreasing the water content of the pollen resulted in a decrease in rotational motion until a minimum was reached, after which rotational motion slightly increased again. The water content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm of the pollen.We also investigated the relationship between the longevity of lettuce seeds and the molecular mobility in the cytoplasm of their radicles (chapter 7). Longevity of lettuce seeds was predicted using the viability equation of Ellis and Roberts. Increasing the temperature resulted in faster rotational motion and shorter longevity. There was a linear relationship between the logarithms of rotational motion and estimated longevity for temperatures above 5°C, which is the same temperature range for which experimental data were used to obtain the viability constants of the viability equation. Below 5°C, there was a deviation from linearity, which might stem from inaccurate predictions by the viability equation at low temperatures.Chapter 8 further demonstrates that there is a linear relationship between the logarithms of rotational motion in the cytoplasm of seed and pollen of several plant species and their ageing rates or longevities. This linearity suggests that cytoplasmic mobility might be an important controlling factor of ageing rates. The linear relationship between the two parameters could be used to predict lifespan of germplasm at low temperatures (at which experimental determination of longevity is practically impossible) by simply measuring theτ R values at these low temperatures (chapter 7 and 8). Based on the predictions using the linear regression between ageing rate and rotational motion of CP in pea embryonic axes, an optimum water content of storage was found. This optimum water content shifted to higher values with lowering the storage temperature, as was found previously for cattail pollen based on experimental data (chapter 2). It was predicted that the longevity of seeds at high (0.12 to 0.16 g/g) water content is much higher than previously suggested on the basis of the viability equation. The predictions show that drying germplasm too far leads to decreased longevity compared to storage of germplasm at higher water contents, suggesting that current storage protocols might have to be re-examined.Desiccation-tolerant organisms contain large amounts of soluble sugars. This, and the fact that sugars are excellent glass-formers has led to the suggestion that sugars play an important role in intracellular glass formation. The presence and amounts of oligosaccharides have been found to correlate with longevity. Furthermore, oligosaccharide glasses are known to increase the T g and viscosity of model sucrose glasses. This suggests that oligosaccharides might enhance the stability of intracellular glasses (chapter 9 and 10). Osmo-priming, i.e. pre-imbibition of seeds in an osmotic solution, can result in a decrease in oligosaccharide content and longevity. Priming pea seeds decreased the total oligosaccharide content in the embryonic axes (chapter 9). Despite the change in oligosaccharide:sucrose ratio, no differences in T g values were detected in the dry axes before and after priming as determined by DSC. Also no difference was found between the rotational mobility of CP in dry untreated axes and that of dry primed axes. Chapter 10 demonstrates that osmo-priming of impatiens and bell pepper seeds resulted in considerable decreases in longevity and oligosaccharide contents, while sucrose contents increased. Again, no differences in the T g curves were found between control and primed impatiens seeds. Similarly, there was no difference in rotational motion of CP in the cytoplasm between control and primed impatiens seeds and between control and primed bell pepper embryonic axes. It was concluded that oligosaccharides in seeds do not appear to affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.To understand the nature and composition of biological glasses we investigated the molecular mobility around T g in sugars, poly-L-lysine and dry desiccation-tolerant biological systems, using ST-EPR, 1 H-NMR and FTIR spectroscopy. Two distinct changes in the temperature dependence of molecular mobility were detected in sugars and poly-L-lysine. With heating, the first change was associated with the melting of the glassy state (T g ). The second change, at which the molecular mobility abruptly increased over several orders of magnitude, was found to correspond with a critical temperature (T c ) where the dynamics of the system changed from solid-like to liquid-like. The temperature interval between T g and T c increased with increasing molecular weight of the sugars. The interval between T g and T c in biological tissues was over 50°C, implying that the stability remained high even at temperatures far above T g . A comparably high T c -T g interval was found for the molecular mobility of poly-L-lysine, suggesting that proteins rather than sugars play an important role in the intracellular glass formation. The exceptionally high T c of intracellular glasses is expected to provide excellent long-term stability to dry organisms, maintaining a slow molecular motion in the cytoplasm even at temperatures far above T g .</p

It is not over until it is dry: molecular aspects of late seed maturation.

The maturation phase is described as a developmental program encompassing the accumulation of storage reserves, drying and abscission from the mother plant. Besides regulatory pathways involved in seed filling,  additional pathways must be activated to confer to orthodox seeds its dispersal characteristics, namely germination capacity, dormancy and the ability to survive in the dry state. The molecular pathways underlying the acquisition of longevity during late seed maturation and its interaction with the environment have received little attention. Yet, this trait is an important factor in the preservation of seed viability and quality during dry storage and an essential parameter to ensure fast and homogenous seedling establishment. In this presentation, we will focus on the physiological, biochemical and molecular events that occur during late seed maturation of Medicago truncatula using transcriptomic and metabolomic profiling together with a conditional-dependent network of global transcriptional interactions. Using an integrative biology approach linking phenotype with these molecular data, we will demonstrate how to identify key genes that govern the acquisition of longevity. The role of the transcriptional regulator ABSCISIC ACID INSENSITIVE 3 (ABI3) in late seed maturation will be discussed, based on molecular analysis of Mtabi3 mutants. We will show that the long maturation phase of Medicago makes it particularly adapted model to apply network-based approaches to unravel regulatory pathways underlying the preparation for the dry and quiescent state

LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance

In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4g H2O g DW–1. Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds