Ageing kinetics of fern chlorophyllous spores during dry storage is determined by its antioxidant potential and likely induced by photosynthetic machinery.

Ageing in dry chlorophyllous propagules is leaded by photooxidation through the photosynthetic machinery, but why species differ in longevity and the ageing mechanisms of when light and oxygen are absent are unknown. We hypothesize that the cellular antioxidant capacity is key for the inter- and intra-specific differences in the ageing process. We have tested this hypothesis in chlorophyllous spores of two ferns. They were subjected to four different storage regimes resulting from light/dark and normoxia/hypoxia combinations. Lipophilic and hydrophilic antioxidants, reactive oxygen species (ROS), and photosynthetic pigments were analysed in parallel to germination and the recovery of Fv/Fm over a storage period of up to 22-months. We show that light and oxygen accelerate the ageing process, but their mechanisms (ROS, increase, antioxidant capacity decrease, loss of efficiency of the photosystem II, pigment degradation) appear the same under all conditions tested. The end of the asymptomatic phase of longevity, when a sudden drop of germination occurs, seems to be determined by a threshold in the depletion of antioxidants. Our results support the hypothesis that ageing kinetics in dry plant propagules is determined by the antioxidant system, but also suggests an active role of the photosynthetic machinery during ageing, even in darkness and hypoxia.

Unraveling metabolic mechanisms behind chloroplast desiccation tolerance: Chlorophyllous fern spore as a new promising unicellular model.

Fern spores are unicellular structures produced by the sporophyte generation that give rise to the haploid gametophyte. When released from the sporangium, spores are desiccation tolerant (DT) in the royal fern (Osmunda regalis) and contain fully developed chloroplasts. As a consequence, this type of spores is called chlorophyllous spores (CS). Upon transfer to germination conditions, CS initiate a process of imbibition that suppresses DT in 72 h, before the germination starts. In parallel to such change in DT, thylakoids undergo a profound remodelling in composition and function. Firstly, sustained quenching of chlorophyll fluorescence is relaxed, giving rise to photochemically active CS, while lipid composition shifts from that of a resting structure to a metabolically active cell. Basically trigalactolipids decreased in favour of monogalactolipids, with a parallel desaturation of fatty acids. Storage lipids such as triacylglycerol were quickly depleted. These results highlight the importance of the structure of thylakoids lipid as a key to protect membrane integrity during desiccation, together with the saturation of fatty acids and the constitutive chlorophyll quenching to prevent oxidative damage. The CS used here, in which the same cell shifts from DT to sensitive strategy in 72 h, reveal their potential as unicellular models for future studies on DT.

Does plant colour matter? Wax accumulation as an indicator of decline in Juniperus thurifera.

The photosynthesis in evergreen trees living in Mediterranean ecosystems is subjected to multiple climatic stresses due to water shortage and high temperatures during the summer and to low temperatures during the winter. Mediterranean perennials deploy different photoprotective mechanisms to prevent damage to the photosynthetic system. Wax accumulation in leaves is a primary response which by enhancing light scattering in the leaf surface reduces incident radiation in the mesophyll. The existence of high variability in wax accumulation levels between coexisting individuals of a species has a visual effect on colour that provides distinguishable green and glaucous phenotypes. We explored this variability in a Mediterranean evergreen tree Juniperus thurifera (L.) to evaluate the impact of epicuticular wax on optical and ecophysiological properties and on the abundance of photoprotective pigments throughout an annual cycle. Because of light attenuation by waxes, we expected that glaucous phenotypes would lower the need for photoprotective pigments. We evaluated the effect of phenotype and season on reflectance, defoliation levels, photochemical efficiency and photoprotective pigment contents in 20 green and 20 glaucous junipers. Contrary to our expectations, the results showed that glaucous trees suffered from a diminution in photochemical efficiency, but there was no reduction in photoprotective pigments. Differences between glaucous and green phenotypes were greater in winter, which is the most stressful season for this species. Glaucous individuals also showed the highest levels of leaf defoliation. The lower photochemical efficiency of glaucous trees, together with higher defoliation rates and equal or greater number of physiological photoprotective mechanisms, suggests that in spite of wax accumulation, glaucous trees suffer from more severe stress than green ones. This result suggests that changes in colouration in Mediterranean evergreens may be a decline indicator.

Dehydration-mediated activation of the xanthophyll cycle in darkness: is it related to desiccation tolerance?

The development of desiccation tolerance by vegetative tissues was an important step in the plants' conquest of land. To counteract the oxidative stress generated under these conditions the xanthophyll cycle plays a key role. Recent reports have shown that desiccation itself induces de-epoxidation of xanthophyll cycle pigments, even in darkness. The aim of the present work was to study whether this trait is a common response of all desiccation-tolerant plants. The xanthophyll cycle activity and the maximal photochemical efficiency of PS II (F(v)/F(m)) as well as ß-carotene and α-tocopherol contents were compared during slow and rapid desiccation and subsequent rehydration in six species pairs (with one desiccation-sensitive and one desiccation-tolerant species each) belonging to different taxa. Xanthophyll cycle pigments were de-epoxidised in darkness concomitantly with a decrease in F(v)/F(m) during slow dehydration in all the desiccation-tolerant species and in most of the desiccation-sensitive ones. De-epoxidation was reverted in darkness by re-watering in parallel with the recovery of the initial F(v)/F(m). The stability of the ß-carotene pool confirmed that its hydroxylation did not contribute to zeaxanthin formation. The α-tocopherol content of most of the species did not change during dehydration. Because it is a common mechanism present in all the desiccation-tolerant taxa and in some desiccation-sensitive species, and considering its role in antioxidant processes and in excess energy dissipation, the induction of the de-epoxidation of xanthophyll cycle pigments upon dehydration in the dark could be understood as a desiccation tolerance-related response maintained from the ancestral clades in the initial steps of land occupation by plants.

Unravelling the roles of desiccation-induced xanthophyll cycle activity in darkness: a case study in Lobaria pulmonaria.

Desiccation-tolerance ability in photosynthetic organisms is largely based on a battery of photoprotective mechanisms. Xanthophyll cycle operation induced by desiccation in the absence of light has been previously proven in the desiccation-tolerant fern Ceterach officinarum. To understand the physiological function of xanthophyll cycle induction in darkness and its implication in the desiccation tolerance in more detail, we studied its triggering factors and its photochemical effects in the lichen Lobaria pulmonaria. We found that both the drying rate and the degree of desiccation play a crucial role in the violaxanthin de-epoxidase activation. De-epoxidation of violaxanthin to zeaxanthin (Z) occurs when the tissue has lost most of its water and only after slow dehydration, suggesting that a minimum period of time is required for the enzyme activity induction. Fluorescence analysis showed that Z, synthesised during tissue dehydration in the absence of light, prevents photoinhibition when rewatered tissues are illuminated. This is probably due to Z implication in both non-photochemical quenching and/or antioxidative responses.