Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green AlgaKlebsormidium crenulatum

Adaptation strategies in freezing resistance were investigated inKlebsormidium crenulatum, an early branching streptophyte green alga related to higher plants.Klebsormidiumgrows naturally in unfavorable environments like alpine biological soil crusts, exposed to desiccation, high irradiation and cold stress. Here, chilling and freezing induced alterations of the ultrastructure were investigated. Control samples (kept at 20 degrees C) were compared to chilled (4 degrees C) as well as extracellularly frozen algae (-2 and -4 degrees C). A software-controlled laboratory freezer (AFU, automatic freezing unit) was used for algal exposure to various temperatures and freezing was manually induced. Samples were then high pressure frozen and cryo-substituted for electron microscopy. Control cells had a similar appearance in size and ultrastructure as previously reported. While chilling stressed algae only showed minor ultrastructural alterations, such as small inward facing cell wall plugs and minor alterations of organelles, drastic changes of the cell wall and in organelle distribution were found in extracellularly frozen samples (-2 degrees C and -4 degrees C). In frozen samples, the cytoplasm was not retracted from the cell wall, but extensive three-dimensional cell wall layers were formed, most prominently in the corners of the cells, as determined by FIB-SEM and TEM tomography. Similar alterations/adaptations of the cell wall were not reported or visualized inKlebsormidiumbefore, neither in controls, nor during other stress scenarios. This indicates that the cell wall is reinforced by these additional wall layers during freezing stress. Cells allowed to recover from freezing stress (-2 degrees C) for 5 h at 20 degrees C lost these additional cell wall layers, suggesting their dynamic formation. The composition of these cell wall reinforcement areas was investigated by immuno-TEM. In addition, alterations of structure and distribution of mitochondria, dictyosomes and a drastically increased endoplasmic reticulum were observed in frozen cells by TEM and TEM tomography. Measurements of the photosynthetic oxygen production showed an acclimation ofKlebsormidiumto chilling stress, which correlates with our findings on ultrastructural alterations of morphology and distribution of organelles. The cell wall reinforcement areas, together with the observed changes in organelle structure and distribution, are likely to contribute to maintenance of an undisturbed cell physiology and to adaptation to chilling and freezing stress

Drought affects the heat-hardening capacity of alpine plants as indicated by changes in xanthophyll cycle pigments, singlet oxygen scavenging, α-tocopherol and plant hormones

AbstractAlpine environments in Europe are increasingly affected by more erratic precipitation patterns, and more frequent drought and heat waves. Heat-hardening capacity is a key feature for survival of these abiotic stress factors, but it is poorly understood how heat and drought affect plant performance when combined. The main objectives of this study were (1) to determine maximum heat hardening capacity in 14 selected plant species and (2) to study how alpine plants respond to combined heat and drought stress compared to heat alone. (3) For risk assessment maximum leaf temperatures were measured in the field and (4) important methodological aspects of testing heat tolerance were evaluated. Heat hardening capacity was assessed by Tc, the heat threshold of photosystem II (PS II), and by heat tolerance tests based on visual inspection of leaf tissue damage or potential quantum efficiency of PS II (Fv/Fm). A purpose-built Heat Tolerance Testing System (HTTS) was used, which allows for controlled heat exposure of whole plants under nearly natural conditions. Additionally, in two species from contrasting habitats, Senecio incanus and Primula minima, the dynamics of heat hardening was studied during and after 8days exposure to heat (H), or to a combination of heat and severe drought (H+D) within a light-transmissive heat hardening chamber at the alpine field site. In both species, H treatment significantly increased heat tolerance (LT50), determined by the HTTS, to 58.0°C and 54.9°C, respectively, and was accompanied by elevated production of abscisic acid (ABA) and salicylic acid (SA), whereas jasmonic acid (JA) levels decreased. Under H+D the LT50 was only 56.5°C and 51.6°C, respectively, and levels of ABA were higher in S. incanus and SA lower in both species in comparison to H. Changes in xanthophyll cycle pigments, α-tocopherol and carotenoids:chlorophyll ratio were more pronounced in P. minima than in S. incanus. In P. minima both H and H+D significantly increased singlet oxygen (1O2) scavenging capacity, determined by electron paramagnetic resonance spectroscopy (EPR). In the field, the maximum half-hourly mean (HHM) leaf temperature of P. minima (32.2°C) was significantly lower than of S. incanus (46.5°C, a potentially harmful temperature). We conclude that the investigated species are well adapted to the prevailing temperature conditions in the field. They also possess an outstanding heat hardening capacity, but this can be curtailed when heat is combined with drought. As drought further increases leaf temperatures, the risk of suffering lethal heat damage of some species may increase in the future, particularly at south exposed, ruderal alpine sites with uncertain water supply

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam–scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress

Non-invasive diagnosis of viability in seeds and lichens by infrared thermography under controlled environmental conditions

Background: Non-invasive procedures for the diagnosis of viability of plant or fungal tissues would be valuable for scientific, industrial and biomonitoring purposes. Previous studies showed that infrared thermography (IRT) enables non-invasive assessment of the viability of individual "orthodox" (i.e. desiccation tolerant) seeds upon water uptake. However, this method was not tested for rehydrating tissues of other desiccation tolerant life forms. Furthermore, evaporative cooling could obscure the effects of metabolic processes that contribute to heating and cooling, but its effects on the shape of the "thermal fingerprints" have not been explored. Here, we further adapted this method using a purpose-built chamber to control relative humidity (RH) and gaseous atmosphere. This enabled us to test (i) the influence of relative humidity on the thermal fingerprints during the imbibition of Pisum sativum (Garden pea) seeds, (ii) whether thermal fingerprints can be correlated with viability in lichens, and (iii) to assess the potential influence of aerobic metabolism on thermal fingerprints by controlling the oxygen concentration in the gaseous atmosphere around the samples. Finally, we developed a method to artificially "age" lichens and validated the IRT-based method to assess lichen viability in three lichen species. Results: Using either 30% or 100% RH during imbibition of pea seeds, we showed that "live" and "dead" seeds produced clearly discernible "thermal fingerprints", which significantly differed by > vertical bar 0.15 vertical bar degrees C in defined time windows, and that RH affected the shape of these thermal fingerprints. We demonstrated that IRT can also be used to assess the viability of the lichens Lobaria pulmonaria, Pseudevernia furfuracea and Peltigera leucophlebia. No clear relationship between aerobic metabolism and the shape of thermal fingerprints was found. Conclusions: Infrared thermography appears to be a promising method for the diagnosis of viability of desiccation-tolerant tissues at early stages of water uptake. For seeds, it is possible to diagnose viability within the first hours of rehydration, after which time they can still be re-dried and stored until further use. We envisage our work as a baseline study for the use of IR imaging techniques to investigate physiological heterogeneity of desiccation tolerant life forms such as lichens, which can be used for biomonitoring, and for sorting live and dead seeds, which is potentially useful for the seed trade.We gratefully acknowledge financial support by the EU to BFM and IK (Marie Curie Action FP7-PEOPLE-2012-IEF 328370 "MELISSA") and the University of Innsbruck to JIGP for a visiting professorship. Support by the Spanish Ministry of Education to JIGP for a Salvador de Madariaga fellowship and Basque Government to JIGP and BFM (UPV/EHU-GV IT-1018-16) are also acknowledged

Temporal and spatial trade-offs between resistance and performance traits in herbaceous plant species

Frost resistance (FR) is a highly adaptive trait and important for plant performance, survival and distribution. While overall seasonal changes in the frost resistance of herbaceous species are well documented, knowledge of the variability during the growth period is scarce. Responses could be expected due to differences in temperature yet investment in frost resistance might be at the expense of plant performance. To analyse temporal and spatial (i.e. same date but differing temperatures) variability, FR of leaves of six herbaceous species on five sampling dates were assessed along an elevational gradient in the northern limestone Alps. We used chlorophyll fluorescence techniques to calculate the lethal temperature of 90% of the population (LT90) thereof. To test the association with plant performance, we measured eco-morphological leaf traits (specific leaf area (SLA), leaf dry matter content, leaf phosphorous and magnesium content as well as stomatal pore area index (SPI)) in parallel. We found that FR as well as leaf traits exhibit a strong temporal variation whereas spatial variability was low. When analysing the relationship of FR to leaf traits we found that SLA as a proxy of growth rate was negatively associated with FR indicating a trade-off between growth and resistance, whereas SPI showed a positive relationship to FR. This finding gives further insight into the variability of traits and will help to improve predictions concerning plant performance and distribution under changing climate regimes