Julia Buitink

Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance

A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting α helices and β sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.

Membrane Stabilization in the Dry State

Abstract We discuss current ideas of how membranes in desiccation-tolerant plant organ(ism)s are protected from the deleterious effect of complete water removal. Results of studies with model membranes showed that sugars play a major role in preventing fusion, phase transitions and most likely also phase separations. The sugars ability to form a stable glass and to interact directly with the phosphate of the phospholipid polar headgroup is the requirement for the protection of dry liposomes. Disaccharides alone fulfil these requirements. Dry membranes of desiccation tolerant plants in situ often have elevated phase transition temperatures (Tm) that are readily restored upon rehydration. Elevated Tm may point to insufficient interaction of sucrose with the polar headgroups. Attempts to observe this interaction in situ by analyzing the asymmetric phosphate stretching band failed. Thus, we suggest factors other than sugars in the suppression of Tm in intact cells and provide suggestions concerning potential roles of amphipathic compounds in this regard.

Calorimetric Properties of Dehydrating Pollen (Analysis of a Desiccation-Tolerant and an Intolerant Species)

The physical state of water in the desiccation-tolerant pollen of Typha latifolia L. and the desiccation-sensitive pollen of Zea mays L. was studied using differential scanning calorimetry in an attempt to further unravel the complex mechanisms of desiccation tolerance. Melting transitions of water were not observed at water content (wc) values less than 0.21 (T. latifolia) and 0.26 (Z. mays) g H2O/g dry weight. At moisture levels at which melting transitions were not observable, water properties could be characterized by changes in heat capacity. Three hydration regions could be distinguished with the defining wc values changing as a function of temperature. Shifts in baseline power resembling second-order transitions were observed in both species and were interpreted as glass-to-liquid transitions, the glass-transition temperatures being dependent on wc. Irrespective of the extent of desiccation tolerance, both pollens exhibited similar state diagrams. The viability of maize pollen at room temperature decreased gradually with the removal of the unfrozen water fraction. In maize, viability was completely lost before grains were sufficiently dried to enter into a glassy state. Apparently, the glassy state per se cannot provide desiccation tolerance. From the existing data, we conclude that, although no major differences in the physical behavior of water could be distinguished between desiccation-tolerant and -intolerant pollens, the physiological response to the loss of water varies between the two pollen types.

Characterization of Molecular Mobility in Seed Tissues: An Electron Paramagnetic Resonance Spin Probe Study

The relationship between molecular mobility (tauR) of the polar spin probe 3-carboxy-proxyl and water content and temperature was established in pea axes by electron paramagnetic resonance (EPR) and saturation transfer EPR. At room temperature, tauR increased during drying from 10(-11) s at 2.0 g water/g dry weight to 10(-4) s in the dry state. At water contents below 0.07 g water/g dry weight, tauR remained constant upon further drying. At the glass transition temperature, tauR was constant at approximately 10(-4) s for all water contents studied. Above Tg, isomobility lines were found that were approximately parallel to the Tg curve. The temperature dependence of tauR at all water contents studied followed Arrhenius behavior, with a break at Tg. Above Tg the activation energy for rotational motion was approximately 25 kJ/mol compared to 10 kJ/mol below Tg. The temperature dependence of tauR could also be described by the WLF equation, using constants deviating considerably from the universal constants. The temperature effect on tauR above Tg was much smaller in pea axes, as found previously for sugar and polymer glasses. Thus, although glasses are present in seeds, the melting of the glass by raising the temperature will cause only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.

Molecular mobility in the cytoplasm of lettuce radicles correlates with longevity

Molecular mobility is hypothesised to be a key factor influencing storage stability of seeds because it may control the rate of deteriorative reactions responsible for reduced shelf life. The relationship between the longevity of lettuce seeds and the molecular mobility was investigated in the cytoplasm of lettuce radicles. Longevity of lettuce seeds was predicted using the viability equation of Ellis and Roberts, and the molecular mobility was determined by saturation transfer electron paramagnetic resonance spectroscopy measurements of rotational motion of a polar spin probe inserted into the cytoplasm. Increasing the temperature resulted in faster rotational motion and shorter longevity. There was a linear relationship between the logarithms of rotational motion and estimated seed longevity for temperatures above 5°C. Below 5°C, there was a deviation from linearity. Based on the hypothesis that the molecular mobility in the cytoplasm determines the rate of detrimental reactions, seed longevity at sub-zero temperatures was predicted by extrapolation of the rotational motion using the linear relationship. Predictions of longevity at sub-zero temperatures based on rotational motion indicated longer survival times than those based on the viability equation. A kinetic approach to ageing using molecular mobility measurements is expected to improve our understanding of seed storage stability and might eventually lead to realistic predictions of longevity at low temperatures.

Molecular characterization of the acquisition of longevity during seed maturation in soybean

Seed longevity, defined as the ability to remain alive during storage, is an important agronomic factor. Poor longevity negatively impacts seedling establishment and consequently crop yield. This is particularly problematic for soybean as seeds have a short lifespan. While the economic importance of soybean has fueled a large number of transcriptome studies during embryogenesis and seed filling, the mechanisms regulating seed longevity during late maturation remain poorly understood. Here, a detailed physiological and molecular characterization of late seed maturation was performed in soybean to obtain a comprehensive overview of the regulatory genes that are potentially involved in longevity. Longevity appeared at physiological maturity at the end of seed filling before maturation drying and progressively doubled until the seeds reached the dry state. The increase in longevity was associated with the expression of genes encoding protective chaperones such as heat shock proteins and the repression of nuclear and chloroplast genes involved in a range of chloroplast activities, including photosynthesis. An increase in the raffinose family oligosaccharides (RFO)/sucrose ratio together with changes in RFO metabolism genes was also associated with longevity. A gene co-expression network analysis revealed 27 transcription factors whose expression profiles were highly correlated with longevity. Eight of them were previously identified in the longevity network of Medicago truncatula, including homologues of ERF110, HSF6AB, NFXL1 and members of the DREB2 family. The network also contained several transcription factors associated with auxin and developmental cell fate during flowering, organ growth and differentiation. A transcriptional transition occurred concomitant with seed chlorophyll loss and detachment from the mother plant, suggesting the activation of a post-abscission program. This transition was enriched with AP2/EREBP and WRKY transcription factors and genes associated with growth, germination and post-transcriptional processes, suggesting that this program prepares the seed for the dry quiescent state and germination.

Desiccation Tolerance and Long Term Structural Stability

Suspended life in completely desiccated organisms requires the protection of proteins and phospholipids in the cellular membranes. Desiccation tolerant organisms usually contain large amounts of sugars. Di-, tri- and tetra—saccharides in particular, play a major role in preventing fusion, phase transitions and most likely phase separations in dry membranes. The ability of the sugars to form a glass and to directly interact with the polar headgroups of the phospholipids are the hypothesized mechanisms involved in this protection. Sugars also protect labile proteins from major structural rearrangements during dehydration.

Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigour in Medicago truncatula: Sugar composition links with seed vigour of M. truncatula

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.